ArgP’s role in osmoregulationshodhganga.inflibnet.ac.in/bitstream/10603/8551/12/12...ArgP’s role in osmoregulation Chapter 3 64 Table 3.1 Regulation of gdhA-lac expression by ArgP

ArgP’s role in osmoregulation Chapter 3

62

Introduction

Studies on the ArgP protein were initiated in this laboratory because of the

finding that in a Glu synthase (GOGAT encoded by gltBD)- deficient strain (defective in

one of the two pathways of ammonium assimilation and Glu synthesis, the other being

that through Glu dehydrogenase [GDH], see Figure 1.6 in Chapter 1), the argP mutant is

crippled for ammonium assimilation and is osmosensitive. This gltBD argP strain

showed reduced intracellular levels of Glu and GDH compared to a gltBD single mutant

in both low and high osmolarity conditions (Nandineni et al., 2004). Accordingly, it was

hypothesized that (i) the osmosensitivity of the gltBD argP strain is because of limited

Glu as a compatible solute and counter-ion to the potassium that accumulates on

hyperosmotic stress, (ii) ArgP has a role in control of ammonium assimilation and Glu

synthesis pathway in a strain wherein only the GDH pathway was functional. This

control is probably through its direct or indirect regulation of expression of gdhA, the

gene encoding GDH (Nandineni et al., 2004).

Another finding that supports this hypothesis is that by Bender and coworkers

who have shown that gdhA transcription in Klebsiella aerogenes is repressed about 3-

fold upon Lys supplementation; to explain this, these authors postulated that an

unidentified regulator protein which is Lys sensitive activates gdhA in the strain (Goss et

al., 2002). Since E. coli ArgP at its well-studied target gene argO is known to repress

transcription in the presence of Lys, it is likely that this regulator protein is the

orthologous ArgP.

This chapter describes in vivo promoter-lac fusion and in vitro EMSA

experiments performed to investigate the role of ArgP in the transcriptional regulation of

gdhA. Subsequently, the combined effect of ArgP, GltBD and hyperosmotic stress on

gdhA expression was determined. Evidence of increased expression of gdhA relieving the

osmosensitive phenotype of a gltBD argP is provided. The ArgP regulated promoter was

mapped by primer extension and verified by site directed mutagenesis.

Results

3.1 Construction of transcriptional lac fusion to cis regulatory region of gdhA

To enable studies on transcriptional regulation of gdhA, 435-bp of its upstream

regulatory region from 322 to +113 (w.r.t. transcription start site taken as +1) was PCR-

amplified from E. coli genomic DNA using primers 5’-


63

ATTTTGATCCTGCAGAACGCAGCACTG-3’ and 5’-

GTTTGATTCGGATCCCGCTTTTGGACATG-3’(restriction sites in italics) and cloned

at the PstI and BamHI sites of plasmid vector pCU22 (Ueguchi and Mizuno, 1993) which

is a derivative of pUC19 with strong, tandem, phage fd transcription terminators flanking

the MCS useful for preparing supercoiled DNA template for in vitro transcription

reactions. This plasmid was designated as pHYD2601. Subsequently sub-cloning at the

PstI and BamHI sites in pMU575 (Andrews et al., 1991), which is a single copy number

plasmid vector for generating lacZ transcriptional fusions that encodes a trimethoprim

resistance marker, was achieved. The plasmid construct was designated as pHYD2602.

3.2 In vivo expression of gdhA-lac fusion in argP+, argP

d and argP strains

To test whether gdhA expression is under ArgP control, pHYD2602 was

introduced into argP strain derivatives carrying argP+ or the argP

d alleles on pCL1920

(pSC101-based, spectinomycin resistance) vector backbone (Lerner and Inouye, 1990).

-Galactosidase assays were performed after growing strains in minimal A (MA)-glucose

media without or with supplementation of previously reported co-effectors, Arg or Lys at

a concentration of 1 mM. The results from these experiments are presented in Table 3.1.

The data in the following table demonstrated that, (i) ArgP activates gdhA

expression by about 4-fold (compare values of argP+

[Nil] and argP [Nil], see

Variant/ ratio for argP+) (ii) Unlike at argO, Arg addition did not cause further

activation of expression (compare values of Nil and Arg for argP+) but there was an

ArgP-dependent Lys repression of about 3-fold (compare values of Nil and Lys for

argP+, see second row of Nil/Lys ratio). (iii) ArgP

d variants -A68V, -S94L, -V144M and

-P217L behaved more or less like wild-type ArgP (compare ratios Nil/Lys and Variant/

of each of these variants with those of argP+). On the other hand, ArgP

d variants -P108S

and -R295C showed much higher activation than argP+

(about 7-fold, see Variant/ ratio

for these variants) but ArgPd-P274S acted like argP itself in rendering gdhA expression

very low and non-repressible by Lys (see ratios Nil/Lys and Variant/ for ArgPd-P274S

and argP).


64

Table 3.1 Regulation of gdhA-lac expression by ArgP and its variants a

-gal sp. act. Ratio

ArgP

variant Nil Arg Lys Nil/ Lys Variant/Δ

argP 211 198 192 1.0 1.0

argP+ 801 711 307 2.6 3.7

-A68V 743 749 313 2.3 3.5

-S94L 700 687 314 2.2 3.3

-P108S 1502 1401 383 3.9 7.1

-V144M 614 555 278 2.2 2.9

-P217L 609 612 314 1.9 2.8

-P274S 253 219 177 1.4 1.1

-R295C 1512 1360 331 4.5 7.1

a Values shown are of -Galactosidase specific activity (Miller units) after growth in glucose-MA without

(Nil) or with supplementation of Arg (Arg) or Lys (Lys) at 1 mM concentration. The ratios Nil/Lys and

Variant/ indicate, respectively, the degree of Lys repression for the concerned strain, and the degree of

regulation imposed by the concerned ArgP variant (or the wild type ArgP) relative to that in the argP.

3.3 In vitro binding of ArgP to upstream regulatory region of gdhA

The above results indicate that ArgP is a transcriptional regulator of gdhA.

Consequently, the binding ability of ArgP to the upstream regulatory region of gdhA was

checked by EMSA. Varying concentrations of purified, His6-tagged ArgP protein

(Laishram and Gowrishankar, 2007) were incubated with radiolabelled DNA fragment

encompassing the gdhA cis regulatory region (same as that used for generating the lac

fusion construct), before examining the DNA-protein complexes by gel electophoresis

and phosporimaging. The dissociation constants (Kd) for this binding were estimated as

equivalent to the protein concentration (nM) at which half maximal binding of the DNA

was observed by considering the following equation:


65

Kd= [ArgP][DNA] / [ArgP-DNA]

When [ArgP-DNA] equals [DNA] (that is when 50% of DNA fraction is protein bound),

and when [ArgP] >> [ArgP-DNA]

Kd= the concentration of ArgP present in the EMSA reaction…………...Equation 3.1

Where [ArgP] is the concentration of unbound ArgP protein, [DNA] is the concentration

of unbound DNA and [ArgP-DNA] is the concentration of the ArgP protein-DNA

complex.

Figure 3.1 shows that ArgP binds to cis elements at gdhA with an apparent Kd of

80 nM; the binding is inhibited on addition of Lys to the binding reaction and the Kd

increased to 150 nM. This is in contrast to the previous binding profile of ArgP at argO,

where binding is not inhibited by Lys.

Figure 3.1 EMSA with ArgP and cis regulatory regions of gdhA in absence or presence of the co-

effector Lys. ArgP monomer concentrations are indicated for each lane. Bands corresponding to free DNA

and to DNA in binary complex with ArgP are marked by open and filled arrowheads, respectively.

3.4 The combined effect of ArgP, GltBD and high osmolarity on in vivo expression

of gdhA-lac fusion

The next objective in this study was the determination of gdhA expression in the

gltBD argP strain under high osmolarity conditions and evaluation of a probable

correlation of these expression levels with the osmosensitive phenotype of the strain. For

this pHYD2602 (gdhA-lac fusion) was introduced into wild-type (MC4100), argP

(GJ9602), gltBD (GJ4652) and argP gltBD (GJ9611) strains and lac expression was

measured after growing the strains in glucose-MA without ( NaCl) and with (+ NaCl)

supplementation of 0.4 M NaCl. (The plates were also supplemented with 1 mM glycine


66

Table 3.2 Regulation of gdhA expression by ArgP, GltBD and high osmolarity a

Strain genotype NaCl + NaCl

gltBD+

argP+ 1016 331

gltBD+ argP 340 102

gltBD argP+ 1307 998

gltBD argP 540 424

a Values reported are the specific activities of -Galactosidase in derivatives of the indicated strains

carrying gdhA-lac on plasmid pHYD2602, after growth in glucose-MA without ( NaCl) and with (+

NaCl) supplementation of 0.4 M NaCl and 1 mM glycine betaine.

betaine as an osmoprotectant to partially alleviate the growth-inhibitory effects of NaCl).

The results are presented in Table 3.2.

The results obtained suggest that:

(i) ArgP increases gdhA transcription by about 3-fold in both low and high-osmolar

growth media, and in both gltBD+

and gltBD backgrounds (compare column-wise

values in gltBD+

argP+

with gltBD+ argP and values in gltBD argP

+ with gltBD

argP).

(ii) GltBD loss increases gdhA transcription by about 4-fold only at high osmolarity,

in both argP+ and argP backgrounds (consider values in + NaCl column and compare

values of gltBD argP+

with gltBD+

argP+ and values of gltBD argP with gltBD

+

argP).

(iii) Independent of the argP status, high osmolarity decreases gdhA transcription by

about 3-fold in gltBD+ but not in the gltBD background (see rows gltBD

+ argP

+ and

gltBD+ argP and compare across each row, values obtained without and with NaCl).

(iv) At high osmolarity, the activating effects of presence of ArgP and absence of

GltBD are independent and additive, resulting in as high as 10-fold increase in gdhA-lac

transcription (compare values of gltBD argP+

and gltBD

+ argP on NaCl addition).

Taking into account the above mentioned gdhA expression dynamics, as also the

facts that GltBD serves as the primary enzyme for Glu synthesis and that gltBD argP+

is not osmosensitive, it may be concluded that the gltBD argP mutant is osmosensitive

because of loss of the all too important activation of gdhA by ArgP. In a gltBD argP


67

strain, gdhA expression (540 units) is apparently sufficient to meet cellular Glu

requirements under ordinary growth conditions; under hyperosmotic (+ NaCl) conditions,

gdhA expression (424 units) is not sufficient to meet the combined requirements of Glu

both for cellular processes and for serving as a counter-ion for the accumulated

potassium.

3.5 Direct correlation between the ArgPd-effected gdhA expression and

osmosensitivity of gltBD argP

Based on the expression levels effected at the gdhA promoter (Table 3.1), the

ArgPd variants can be classified into three classes namely (i) similar to wild-type (an

example being -A68V), (ii) higher expression levels than wild-type (examples being

-P108S and -R295C), and (iii) constitutively low and argP-like (an example being

-P274S). Following this observation, an attempt was made to determine if there is a

correlation between different extents of gdhA expression and the osmosensitivity of the

gltBD argP mutant strain. Accordingly, the wild-type argP+

or the argPd

alleles, -A68V,

-P108S, -R295C and -P274S on the pSC101-based plasmid vector pCL1920 were

introduced into the gltBD argP (GJ9611) strain. These derivatives were then streaked

on MA-glucose plates containing 0.5 M NaCl without or with 1 mM Lys. (The plates

were also supplemented with 1 mM glycine betaine as an osmoprotectant to partially

alleviate the growth-inhibitory effects of NaCl). The pictures of these plates are shown in

Figure 3.2 and results tabulated in Table 3.3.

The results indicate that strains with high gdhA-lac expression (argP+, -P108S,

-R295C, -A68V) are osmotolerant (see plate [A] and rows Nil and + NaCl corresponding

to these alleles); whereas strains with a low gdhA-lac expression (argP and -P274S) are

osmosensitive (see plate [A] and rows Nil and + NaCl corresponding to this genotype).

Lys addition decreases gdhA-lac expression and this is reflected in the decreased growth

of strains bearing argP+, -P108S, -R295C, -A68V alleles on a plate containing NaCl and

Lys (compare values Nil and Lys, + NaCl and + NaCl + Lys and growth on plate [A] to

growth on plate [B] of these alleles); growth of argP and -P274S strains is more or less

indistinguishable from the previous condition.

Thus there exists a direct correlation between the level of gdhA expression and

the osmosensitivity of the different argPd bearing strains. The most noteworthy result

was for strain derivatives of argPd-P274S, which like argP, showed both low gdhA


68

expression and inability to grow on medium with NaCl (compare values for -P274S and

argP).

Figure 3.2 and Table 3.3 Correlation between the ArgPd-effected gdhA expression and

osmosensitivity of gltBD argP mutant strain b

-gal sp. act.a Growth score

b

argP genotype Nil Lys +NaCl +NaCl +Lys

argP 216 201 + +

argP+ 801 291 ++++ ++

-A68V 765 311 ++++ ++

-P108S 1502 383 ++++ ++

-R295C 1571 314 ++++ ++

-P274S 216 165 + +

a Values indicated are -Galactosidase specific activity as previously described for gdhA-lac in table 3.1.

b The gltBD argP strain carrying argP

+ or the specified argP

d alleles were streaked on MA-glucose plates

with 0.5 M NaCl + 1 mM glycine betaine without (+ NaCl) or with 1 mM Lys (+ NaCl + Lys) and

incubated for 42-48 hrs at 37oC. Growth was scored on the following qualitative 5-point scale (in

increasing order): (no growth), +, ++, +++ and ++++ (full growth).


69

3.6 Multicopy gdhA is able to relieve osmosensitivity of gltBD argP strain

As a corollary of the above result, it was next examined if multicopy gdhA could

relieve the osmosensitivity of gltBD argP strain.

Figure 3.3 and Table 3.4 Effect of multicopy gdhA on osmosensitivity of gltBD argP

strain a

Strain Plasmid

– NaCl

+ NaCl + NaCl

+ IPTG

wild-type

pTrc99A

(vector)

++++ ++++ ++++

pHYD2685

(m.c. gdhA)

++++ ++++ ++++

gltBD argP

pTrc99A

(vector)

++++ + +

pHYD2685

(m.c. gdhA)

++++ ++++ ++++

a The indicated strain derivatives were streaked on MA-glucose plates without (– NaCl) or with 0.5 M

NaCl + 1 mM glycine betaine without (+ NaCl) or with 0.5 mM IPTG (+ NaCl + IPTG) and incubated for

48 hrs at 37oC. Growth was scored on the following qualitative 5-point scale (in increasing order): (no

growth), +, ++, +++ and ++++ (full growth).

The gdhA coding region was PCR-amplified using forward primer JGJgdhAfullF

(5’-ACAAGAATTCCTGCAAAAGCACATG-3’) and reverse primer JGJgdhAfullR (5’-

AACAAAGCTTTGTAGGCCTGATAAG- 3’) to give a 1468-bp DNA fragment.

Utilising the restriction sites that were incorporated in the primers, the fragment was

cloned downstream of the trc promoter at the EcoRI and HindIII site of pTrc99A vector


70

(Amersham Pharmacia) which is a ColE1 based plasmid conferring ampicillin resistance

and with an isopropyl--D-1-thiogalactopyranoside (IPTG)-inducible trc promoter. The

clone was designated as pHYD2685. This plasmid or the pTrc99A vector (serving as

negative control) was transformed into wild-type and gltBD argP strains. The resultant

strain derivatives were streaked on MA-glucose media without or with (i) 0.5 M NaCl, or

(ii) 0.5 M NaCl with 0.5 mM IPTG, and scored for growth after 48 hrs incubation at

37oC (Figure 3.3 and Table 3.4).

It was observed that the gltBD argP strain with pTrc99A was unable to grow

on medium with NaCl (without or with addition of IPTG) whereas pTrc99A bearing

gdhA (pHYD2685) restores growth of the strain on the same medium (without or with

addition of IPTG) (compare rows corresponding to gltBD argP [pTrc99A] and

gltBD argP [pHYD2685] and growth of same strains in panel [B] and [C]). The wild-

type strain showed growth under all tested conditions.

These results show that expression of gdhA from the multicopy pHYD2685,

irrespective of presence or absence of IPTG in the medium alleviates the osmosensitive

phenotype of a gltBD argP strain.

3.7 Mapping of gdhA transcription start site by primer extension analysis

One gdhA transcription start site has previously been mapped to a position 63-bp

upstream of the translation start site. DNA sequence analysis has suggested the existence

of three possible promoters (Valle et al., 1983). Results in the previous section showed

that there are multiple factors (ArgP, GltBD and osmolarity) affecting gdhA

transcription. Hence, it was of interest to find out if gdhA transcriptional regulation

involved a system of multiple promoters, where each promoter is responsive to a specific

factor or growth condition.

To examine this possibility, primer extension assays for mapping probable

multiple transcription start sites of gdhA were performed. Three primers namely

JGJgdhA1 (5’-AGAGAATATGTCTGATCCAT-3’), JGJgdhA2 (5’-

CATGTGCTTTTGCAGTTTTC-3’) and JGJgdhA3 (5’-

ATAACGAGAGTAATCTCATA-3’) end labelled with 32

P-γ-ATP were used; the 3’

ends of these primers were complementary, respectively to +1, 70 and 150 bp w.r.t.

the translation start site of gdhA. These primers were annealed to total cellular RNA

extracted from strains gltBD+

argP+

grown in MA-glucose and ΔgltBD argP+

grown in


71

MA-glucose supplemented with 0.4 M NaCl and 1 mM glycine betaine (high level gdhA

expression was observed for these strains under these conditions, see Table 3.2) followed

by a primer extension reaction with reverse transcriptase. The reaction products were

resolved by electrophoresis on a 6 % urea denaturing polyacrylamide gel and visualized

by phosphorimaging.

Figure 3.4 Primer extension analyses to map transcription start site of gdhA. Strains gltBD

+ argP

+ grown

in MA-glucose and gltBD argP+

grown in MA-glucose with 0.4 M NaCl were used. Of the three primers,

namely JGJgdhA1(1), JGJgdhA2 (2) and JGJgdhA3 (3) used, a transcription start site in both strains and

conditions was mapped only by primer JGJgdhA1 to +63-bp w.r.t. translation start site (shown by arrows).

Lanes A, C, G, T represent sequence ladder with dideoxy ATP or CTP or GTP or TTP in the sequencing

ladder reaction mixture. The sequencing ladder on the left hand side of the gel was generated by using

pUC19 as template and universal M13 forward primer, while the one at the right hand was generated using

pHYD2601 as template and JGJgdhA1.


72

As depicted in the Figure 3.4, a prominent extension product was visible with

primer JGJgdhA1 in RNA preparations from gltBD+

argP+

grown without NaCl and

ΔgltBD argP+ strains grown with NaCl. This was equivalent to the previously reported

transcription start site at 63-bp w.r.t. translation start site. No other extension products

were seen in the case of this primer or of the other two primers in either strains i.e. no

alternative transcription start sites were detected.

3.8 Determination of ArgP-regulated gdhA promoter by site directed mutagenesis

Figure 3.5 Upstream regulatory sequences of gdhA. The mapped transcription start site at –63-bp and the

putative 10 and 35 sequences are shown (A). The mutated 10 sequences in the constructs pHYD2653

(B) and pHYD2654 (C) are also shown.

Figure 3.5 shows that in the sequence upstream of the mapped gdhA transcription

start site, a putative 10 hexamer motif TATAGT was observed (the consensus hexamer

sequence being TATAAT, with more than 90% conservation for second A and last T

McClure, 1985; Browning and Busby, 2004). Situated 19 nucleotides upstream is a

putative 35 hexamer motif TTGCTT (the consensus for which is TTGACA, the first

three residues TTG being more conserved). To determine if this is the ArgP-regulated

promoter, the consensus residues of the putative 10 hexamer motifs were changed to Cs

by a site directed mutagenesis approach. The corresponding nucleotides were modified

on plasmid pHYD2601 template with the aid of primers in which the alternative

nucleotides have been incorporated (shown in primer sequence below) using

QuikChange Site Directed Mutagenesis kit (Stratagene) as described in Materials and

Methods. Thus, the putative 10 sequence was changed at the first and second positions

from TATAGT to CCTAGT (plasmid pHYD2649) with primers JGJgdhAqc1 (5’-

TTCTTGATGGCCTAGTCGAAAAC-3’) and JGJgdhAqc2 (5’-

TTTTCGACTAGGCCATCAAGAATG-3’), and at the last position to TATAGC with


73

primers JGJgdhAqc3 (5’-TGATGGTATAGCCGAAAACTGC-3’) and JGJgdhAqc4 (5’-

GCAGTTTTCGGCTATACCATC-3’). The fragments with mutations incorporated were

then sub-cloned in the PstI and BamHI sites of pMU575 to measure the promoter

activity. The resultant plasmids were designated as pHYD2653 (bearing the TATAGT to

CCTAGT change) and pHYD2654 (bearing the TATAGT to TATAGC change).

The plasmids pHYD2653 and pHYD2654 were transformed into wild-type

(MC4100) and argP (GJ9602) and promoter activity was judged by -Galactosidase

enzyme activity after growth of the derivatives in MA-glucose without and with

supplementation of either 1 mM Lys or 0.4 M NaCl (Table 3.4).

Table 3.4 Determination of ArgP regulated promoter of gdhA a

Strain Plasmid Nil Lys + NaCl

wild-type

pHYD2602 (wt promoter)

1095 402 423

pHYD2653 (mutant 1

promoter)

34 38 35

pHYD2654 (mutant 2

promoter)

43 48 48

argP

pHYD2602 (wt promoter)

399

380 110

pHYD2653 (mutant 1

promoter)

36 33 29

pHYD2654 (mutant 2

promoter)

46 40 42

a Values reported are the specific activities of -Galactosidase of the indicated strains carrying the indicated

plasmids, after growth in MA-glucose without (Nil) and with supplementation of 1 mM Lys (Lys) or 0.4

M NaCl (+ NaCl) and 1 mM glycine betaine.

The above experiment revealed that mutations in the conserved residues of the

putative 10 motif upstream of the mapped transcription site at 63, results in significant

loss of promoter activity both in the wild-type and argP strains in all growth conditions

(compare activity of wild-type promoter on pHYD2602 with that of mutant promoters on


74

pHYD2653 and pHYD2654 in both strains). These observations lead to the conclusion

that this hexamer motif indeed constitutes the authentic 10 element of the functional

gdhA promoter and that ArgP exerts its regulation through this promoter itself.

Discussion

In this study it was shown that ArgP activates gdhA, the gene encoding Glu

dehydrogenase by about 4-fold in a Lys sensitive manner; this clearly correlated with the

manner of ArgP binding to the cis regulatory region. At the time that this study was

being carried out, similar findings were also reported for gdhA in Klebsiella aerogenes

by Goss (2008).

Ensuing work found that gdhA-lac expression is activated 3-fold upon loss of

gltBD. The activating effects of presence of ArgP and loss of gltBD were additive and

resulted in about 10-fold activation. Two lines of evidence clearly implicate a role for

ArgP in osmoregulation through regulation of GDH and hence Glu levels in the cell: (i)

osmosensitive phenotype of the two strains namely, of gltBD argP+ on supplementation

with Lys and of gltBD argP-P274S (harboring the argPd allele that is argP-like w.r.t.

gdhA-lac expression); and (ii) the ability of multicopy gdhA to relieve the osmosensitive

phenotype of the gltBD argP double mutant strain. Primer extension analysis and site

directed mutagenesis also enabled the precise location of the gdhA functional and ArgP

regulated promoter at hexamer motifs located immediately upstream of the sole

transcription start site mapped at 63-bp w.r.t. start site of gdhA translation.

An interesting observation made in this study is that a gene involved in Glu

biosynthesis is regulated by a Lys-sensitive regulator. This suggests a close inter-

dependence between regulation of cellular pools of these two amino acids. In enteric

bacteria, Glu serves the important role of being the primary donor of amino groups in the

biosynthesis of other nitrogenous compounds (Magasanik, 1993) and as the potassium

counter-ion in osmotic homeostasis (Csonka and Epstein, 1996). Of the 20 common

amino acids, Lys is noteworthy in that, despite its relatively high nitrogen content, enteric

bacteria do not readily use Lys as a source of nitrogen or even carbon (Brenchley et al.,

1973; Gutnick et al., 1969; Tyler 1978). Further, Lys can be decarboxylated to

cadaverine (Charlier and Glansdorff, 2004), which is either used as a polyamine or

excreted as a means of neutralizing an acidic environment (Slonczewski and Foster,

1996). Additionally, the size of the intracellular pool of Lys is likely to be related to that


75

of its immediate precursor, diaminopimelate (Park, 1996), which is required for cell wall

biosynthesis.

A related observation is that E. coli permits an exception to the rule of one

synthetase per amino acid for Lys such that it has two distinct genes encoding lysyl

tRNA synthetase activity, namely the constitutively expressed lysS and the inducible

(under high temperature, low pH, anaerobiosis or presence of leucine) lysU (Hirshfield et

al., 1977; Matthews and Neidhardt, 1988; Clark and Neidhardt, 1990; Leveque et al.,

1991; Brevet et al., 1995).These unique roles and features of Lys could account for its

possible use as a sensor for various physiological states of the cell. In fact, Goss (2008)

has proposed that Lys may serve as a stand-in for Glu in signaling the extent of Glu

overflow from osmotic pressure homeostasis into the biosynthetic pathways for other

nitrogenous compounds.

It is also to be noted that, under low pH conditions, E. coli sequentially employs

three amino acid decarboxylases namely the Lys, Arg and Glu decarboxylases

(Slonczewski and Foster, 1996). It may therefore not be coincidental that it is these three

amino acids that are in some way implicated in the ArgP regulon. This is an additional

clue that implies a close relationship between Glu and Lys intracellular pools.

Microarray profiling and ArgP-mediated regulation of lysine permease Chapter 4

76

Introduction

As previously mentioned, the functional role of the ArgP or IciA protein in E. coli

is an enigma. It has been variously described as an inhibitor of chromosome replication

initiation, a transcriptional activator, and a nucleoid-associated protein.

First identified as a protein that binds specific sequences in oriC to inhibit

chromosomal initiation of replication (Hwang and Kornberg, 1990, 1992; Hwang et al.,

1992), ArgP was subsequently described as a transcriptional regulator belonging to the

LTTR family (Thony et al., 1991). Studies from this laboratory has indicated it to be

involved in osmoregulation (Nandineni et al., 2004) and to be essential both in vivo and

in vitro for transcription of the gene encoding the Arg exporter, ArgO (Nandineni and

Gowrishankar, 2004; Laishram and Gowrishankar, 2007). Furthermore, ArgP-regulated

transcription in vivo has been demonstrated for the genes dapB of E. coli (Bouvier et al.,

2008) and gdhA of Klebsiella aerogenes (Goss, 2008). As described in Chapter 3, ArgP

is a transcriptional regulator of gdhA in E. coli also, which likely explains its role in

osmoregulation. In addition, in vitro studies have suggested that ArgP activates the

transcription of the dnaA and nrdA genes involved in DNA replication and metabolism

(Lee et al., 1996, 1997; Han et al., 1998).

As mentioned above and in Chapter 1 (Section 1.2.7 c), the argO gene, coding for

an Arg exporter, is one of the well-characterized targets for transcriptional regulation by

ArgP (Nandineni and Gowrishankar, 2004; Laishram and Gowrishankar, 2007; Peeters et

al., 2009). Transcription of argO in vivo is activated by both Arg as well as its toxic

analog CAN, both effects being mediated by ArgP. On the other hand, argO transcription

is drastically reduced in medium supplemented with Lys, to a level equivalent to that

observed in ΔargP mutants. Dominant, gain-of-function mutations in argP have been

identified (designated argPd) that confer a CAN

r phenotype and act in trans to

considerably increase argO transcription in vivo over that obtained in the argP+ strain

(Nandineni and Gowrishankar, 2004).

In an attempt to get a better insight into the role of ArgP as a transcriptional

regulator in E. coli, a whole genome differential gene expression microarray was

performed. This chapter describes the methodology used for short-listing and identifying

from the microarray data, genes activated by ArgP and their subsequent validation. It

also describes in vivo and in vitro experiments to demonstrate and describe the features

of ArgP transcriptional regulation of the Lys-specific permease, lysP.


77

Results

Section A: Differential gene expression microarray to identify ArgP-

regulated genes

4.1.1 Details of strains used for microarray profiling

The strains provided for the microarray-based comparison of whole-genome

mRNA abundances were an isogenic pair of strains that was either ΔargP (GJ9602) or

expressing the dominant gain of function ArgPd-S94L variant in the argP

+ strain

(MC4100/pHYD926). It was expected that the ArgPd variant would activate expression

of all ArgP-regulated genes irrespective of their need for other co-effectors in the

medium, whereas these genes would remain un-activated in the ∆argP mutant. The

strains were grown to exponential phase in Arg-supplemented MA-glucose medium. The

RNA extraction, microarray hybridization and initial data processing were performed by

Genotypic Technology Pvt. Ltd., Bengaluru.

4.1.2 Methodology for short-listing candidate genes and validation of microarray

data to identify genes activated by ArgP

In order to consolidate the data from the microarray experiment, genes with high-

level mRNA expression in MC4100/pHYD926 compared to GJ9602 and showing log2-

fold difference ≥ 1.0 were further considered. This derived list comprising a total of 344

genes (Appendix I) represented the probable genes activated, at least 2-fold, by ArgP.

Similarly, a second list of genes with high-level mRNA expression in GJ9602 compared

to MC4100/pHYD926 and showing log2-fold difference ≥ 1.0 was obtained. This

comprised a total of 738 entries (Appendix II) and represented probable genes repressed

at least 2-fold by ArgP. However, since ArgP has been previously described as a

transcriptional activator, it was decided to initially explore its regulation of the genes in

the first (activated genes) list.

Of the genes previously reported to be ArgP regulated, dnaA and nrdA did not

feature in the list of activated genes, but argO, dapB and gdhA showed log2-fold

differences of 2.3, 2.7, and 1.3 respectively. A common feature of regulation among

these three genes is that their expression is repressed in the presence of Lys. Hence, other

genes were then short-listed in this list that were previously reported to be Lys-

repressed, as candidates for activation by ArgP and for further validation experiments.

These genes comprised of (gene function and log2-fold microarray expression difference


78

between argPd

and ΔargP in parentheses) lysP (Lys-specific permease, 2.3), lysC (Lys-

sensitive aspartokinase, 1.9) and asd (aspartate semialdehyde dehydrogenase, 1.1). The

proposed strategy for validation was (i) to construct promoter-lac fusions of these genes

followed by β-Galactosidase assays in argP+, argP

d and argP strain backgrounds; and

(ii) for promoters showing in vivo ArgP activation in the lac fusion experiments, to

undertake EMSA of regulatory regions in vitro. This strategy is schematically depicted in

Figure 4.1.

Figure 4.1 Methodology for short-listing candidate genes and validation of microarray data

Section B: ArgP-mediated regulation of the Lys-specific permease lysP

Using the above-described methodology and as described in detail below, it was

found that the Lys-specific permease lysP had the highest fold activation in argPd

compared to argP strain backgound. Accordingly, the following systematic in vivo and

in vitro studies were carried out to examine the features of lysP regulation by ArgP.

4.2.1 Thialysine resistance test of argP strain

As described in Chapter 1, two systems for the transport of Lys exist in E. coli

(Patte, 1996): (i) the Lys-Arg-ornithine (LAO) system, transports Lys and ornithine and

is inhibited by Arg, although Arg is not a substrate (Rosen, 1971, 1973) (ii) the Lys-


79

specific permease (LysP) system, is inhibited by the Lys analog S-(-aminoethyl)-L-

cysteine (thialysine or thiosine) but not by ornithine or Arg (Rosen, 1971). lysP mutation

confers resistance to thialysine such that a lysP strain grows on a plate containing up to

100 g/ml thialysine, whereas the wild-type strain fails to grow (Steffes et al., 1992).

It was reasoned that if lysP is regulated by ArgP, then a argP mutant would also

be thialysine-resistant since lysP will not be expressed in the strain. Hence, preliminary

phenotypic tests to check for thialysine resistance (Tlr) of argP strain were performed.

On a MA-glucose plate containing 100 g/ml of thialysine, it was found that incubation

for 24 hours at 37C allowed comparable growth of lysP, argP and arg lysP but

not of the wild-type strain (Figure 4.2). Thus, the argP strain is Tlr i.e. it phenocopied

the lysP strain suggesting that ArgP may regulate lysP. The observation that argP

lysP is not more Tlr

than lysP suggests that lysP and argP perhaps act in a single

pathway to mediate thialysine resistance.

Figure 4.2 Thialysine resistance test of argP strain. Wild-type (wt), lysP, argP and argP lysP

strains were streaked on MA-glucose plates without and with 100 g/ml thialysine and growth was

observed after incubation for 24 hours at 37C.

4.2.2 Construction of lysP-lac fusion and in vivo expression in argP+, argP

d and

argP strains

Next, the promoter-lac fusion of the lysP gene was constructed. A 263-bp

amplicon comprising the lysP regulatory region from 219 to +43 relative to

transcription start site taken as +1, and expected to encompass all the cis regulatory

sequences, was PCR-amplified using forward primer JGJlysPF (5’ -

GCGCTTTCTGCAGTATTGCGATCC-3’) and reverse primer JGJlysPR (5’-


80

TAGTTTCGGATCCCATACAAAAATGC-3’). The primers were designed to

incorporate PstI and BamHI sites at the upstream and downstream ends respectively, of

the amplicon (shown in italics in the sequences in parentheses). From the 263-bp

amplicon, a 238-bp fragment extending from –206 to +32 of lysP promoter region was

obtained after digestion with PstI and BamHI and was cloned at the same sites of the

plasmid vector pMU575 (Andrews et al. 1991). The resultant plasmid was designated as

pHYD2636. This plasmid was used in all the subsequent in vivo β-Galactosidase assays

to report for lysP promoter activity in different strain backgrounds.

Plasmid pHYD2636 (lysP-lac) was transformed into argP (GJ9602) strain

carrying either argP+, or any of the argP

d alleles on the pSC101-based plasmid vector

pCL1920; similar derivatives of argP with vector pCL1920 alone served as negative

controls in these experiments. The β-Galactosidase specific activities of these derived

strains are shown in Table 4.1.

The results in Table 4.1 indicated that:

(i) lysP-lac expression was very low in the argP strain in all media tested but it

was substantially elevated in the argP+

or argPd

bearing strains. In fact the fold activation

of lysP transcription by wild-type ArgP was as high as 34-fold (compare Variant/Δ value

of argP+

and argP).

(ii) Transcription from the lysP promoter in the strains bearing argP+

or argPd

variants showed no further activation on Arg supplementation (compare columns Nil and

Arg for each of these strains). This indicated that the co-effector Arg has no effect on lysP-

lac expression.

(iii) lysP-lac expression exhibited repression upon Lys supplementation in the argP+

or argPd variants but not the argP strain background (compare Nil/Lys ratios of argP

+ or

argPd variants with that of argP), suggesting that Lys repression of this promoter is

ArgP-mediated.

(iv) Among the argPd alleles, -A68V, -S94L, -V144M, -P217L showed similar behavior to

wild-type i.e. about 20- to 30-fold activation and 3- to 5-fold Lys repression; -P108S and

-R295C were somewhat different by showing about 30-fold activation but only 2-fold Lys

repression (compare Variant/ and Nil/Lys ratios of argP+

with that of these argPd

variants). However, the most striking difference was presented by -P274S that showed

much lower activation (of about 10-fold) compared to argP+ and 3-fold Lys repression

(compare Variant/ and Nil/Lys ratios of argP+

with that of -P274S).


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Table 4.1 Regulation of lysP-lac expression by ArgP and its variants a

-gal sp. act. Ratio

ArgP


argP 51 50 48 1.0 1.0

argP+ 1731 1592 325 5.3 34

-A68V 1558 1455 587 2.6 31

-S94L 1561 1653 473 3.3 30

-P108S 1378 1434 922 1.4 27

-V144M 1174 1136 251 4.6 23

-P217L 881 858 166 5.3 17

-P274S 499 465 169 2.9 10

-R295C 1938 1883 987 1.9 38


(Nil) or with supplementation of Arg (Arg) or Lys (Lys) at 1 mM concentration. The ratios Nil/Lys and

Variant/ indicate, respectively, the degree of Lys repression for the concerned strain, and the degree of

regulation imposed by the concerned ArgP variant (or the wild-type ArgP) relative to that in the argP.

4.2.3 In vitro binding of ArgP to the lysP upstream regulatory region

The simplest explanation to account for the above in vivo observation on lysP

regulation is that of direct regulation of the lysP promoter by ArgP. To examine this

possibility, the purified His6-tagged ArgP protein (Laishram and Gowrishankar, 2007)

was tested for its ability to bind DNA of the lysP regulatory region. An EMSA was

undertaken using the 263-bp amplicon (–219 to +43) described above.


82

Figure 4.3 Binding of His6-ArgP protein to 263-bp (219 to +43) lysP fragment in absence (A) or

presence of co-effectors, Arg (B) or Lys (C) shown by EMSA. ArgP monomer concentrations are indicated

for each lane. In Figure 4.3 (A), lanes CS and NS depict results of addition of 100-fold excess of unlabelled

specific (263-bp lysP fragment) or non-specific (253-bp fragment from the ilvG locus) competitor DNA,

respectively, to the EMSA reactions undertaken with 160 nM ArgP. The previously described 427-bp argO

DNA fragment (Laishram and Gowrishankar, 2007) was used as positive control for ArgP binding. Bands

corresponding to free DNA and to DNA in binary complex with ArgP, are marked by open and filled

arrowheads respectively.

The fragment was radiolabelled as described in Chapter 2, Section 2.2.4.5, and it

was incubated with varying concentrations of purified ArgP (from 0 to 160 nM) in

absence (Figure 4.3 A) or presence of Arg (Figure 4.3 B) or Lys (Figure 4.3 C), before

being subjected to electrophoresis on a 5% non-denaturing polyacrylamide gel and then

being visualized by phosphorimaging.

It was observed that ArgP binds the lysP regulatory region in absence of any


83

amino acids and that the addition of Arg (at a concentration of 0.1 mM) did not hinder

the binding. However, the presence of Lys (at a concentration of 0.1 mM) in the reaction

considerably reduced ArgP binding. The results in Figure 4.3 suggest that ArgP binds the

lysP regulatory DNA specifically, such that, it is competed by excess (100-fold) of the

cognate unlabelled fragment (CS) but not so by non-specific unlabelled DNA fragment

(NS: NS was a 253-bp PCR amplicon from the ilvG coding region obtained using

primers JGBPMAILVGFP1, 5’- CAGCAACACTGCGCGCAGCTGCGTGATC-3’ and

JGBPMAILVGRP1 5’-CTTGTGCGCCAACCGCCGCCGGTAAAC-3’).

4.2.4 Binding curves and dissociation constants for ArgP and lysP interaction in

absence and presence of Arg and Lys

To obtain a quantitative estimate of the strength of the above-described binding,

the distribution of radioactivity of unbound DNA in Figure 4.3 A, B and C was

quantitated by densitometry of the autoradiograms. Taking the value of radioactivity in

the lane without ArgP protein as total or hundred percent radioactivity, ‘% unbound

fraction’ for each lane was calculated. From this ‘% bound fraction’ (which is the

fraction of the total radioactivity of lysP DNA that is shifted upon ArgP binding) was

derived as ‘100 % unbound fraction’. Binding curves were obtained by plotting the

values of ‘% bound fraction’ as a function of the added ArgP concentration (Figure 4.4).

A hyperbolic curve was obtained in the absence of Arg or Lys, suggesting that the

binding of ArgP to lysP DNA is non-cooperative. In presence of Arg, there was no

significant change in the nature of protein binding, whereas when Lys was added, a sharp

decrease in the slope of the curve was seen. By considering Equation 3.1, previously

described in Chapter 3, the dissociation constants (Kd) were calculated from the various

binding curves obtained in Figure 4.4 as equivalent to the protein concentration at which

half maximal binding was observed.

The calculations revealed that the Kd of ArgP binding to lysP DNA was about 40

nM in absence of Arg or Lys, 50 nM in presence of Arg and >160 nM in the presence of

Lys. It can be concluded that whereas Arg addition does not cause any significant change

in the binding affinity, Lys causes at least a 5-fold decrease in affinity of binding of ArgP

to lysP.


84

Figure 4.4 Binding curves for ArgP binding to lysP DNA in the absence (Nil) or presence of co-effectors

Arg or Lys. These curves were established from the EMSA experiment shown in Figure 4.3.

4.2.5 Multiple sequence alignment of upstream region of lysP with those of

previously identified ArgP targets (argO and dapB)

To further investigate the extent of ArgP binding on the lysP upstream region, a

multiple sequence alignment of lysP upstream regulatory region (extending from its

intervening intergenic region with yeiE to the 10 sequence), with DNase I footprinted

region of ArgP at argO (Laishram and Gowrishankar, 2007) and the ArgP binding

sequence at dapB (Bouvier et al., 2008), was executed using Clustal 2.1 available on

www.genome.jp/tools/clustalw (Figure 4.5).

Considerable sequence identity of the three compared sequences (denoted by

asterisks), extending till –98 (w.r.t. transcription start site taken as +1) of lysP, was seen.

This observation indicates of a probable common ArgP binding site within the upstream

region of these genes. Further, it suggests that sequences up to –98 of lysP may be

sufficient for ArgP regulation. In fact, an examination of dapB (93 to 48) and lysP

(91 to 47) sequences reveals a pair of closely resembling imperfect inverted repeats in

each. As with other LTTRs (Maddocks and Oyston, 2008) the promoter distal and

proximal sequences were designated as the Recognition Binding Site (RBS) and

Activation Binding Site (ABS), respectively (Figure 4.6).


85

Figure 4.5 Clustal 2.1 alignment of lysP regulatory region (encompassing its intervening intergenic

region with yeiE to 10 sequence) with argO (80 to 25) and dapB (104 to 10 sequence). Considerable

sequence identity was seen till 98 of lysP. Underlined are the 35 sequence of argO and the 35 and 10

sequence of dapB and lysP. Asterisks indicate sequence identity.

Figure 4.6 Clustal 2.1 alignment of regulatory regions of lysP

(91 to 47) and dapB (93 to 48) revealing ~ 57% identity (A). Probable ArgP Recognition Binding Site

(RBS) and Activation Binding Site (ABS) boxed in the dapB and lysP sequences based on the closely

resembling imperfect inverted repeats in each (B).

4.2.6 In vivo lac-fusion assays of nested deletion constructs of lysP regulatory region

Based on the sequence alignment described in the previous section, it was

decided to construct two nested deletion products of the 238-bp lysP DNA fragment.

Both these fragments extended to the same base (+32) as the 238-bp fragment at the

downstream end but were different and truncated to various extents at the upstream end.

The first truncated fragment extended from 114 to +32 and was generated by PCR

amplification, using forward primer JGJlysPFP1 (5’-

ACAACTGCAGTTCGCCAGAAAA-3’) and reverse primer JGJlysPR (5’-


86

TAGTTTCGGATCCCATACAAAAATGC-3’). The second truncated fragment extended

from 76 to +32 and was generated by PCR amplification, using forward primer

JGJlysPFP2 (5’-ACAACTGCAGCTGGGCGATCAT-3’) and reverse primer JGJlysPR

(5’-TAGTTTCGGATCCCATACAAAAATGC-3’). The fragments were cloned into the

PstI and BamHI site of plasmid vector pMU575 (Andrews et al., 1991). The respective

plasmids were designated as pHYD2647 and pHYD2648. Either of these plasmids or

pHYD2636 (bearing the 238-bp lysP fragment and serving as a control plasmid) were

introduced by transformation into argP+

(MC4100) and argP (GJ9602) strains. -

Galactosidase assay of the resultant strains was done in MA-glucose media without and

with supplementation of Lys. The results are as shown in Table 4.2.

Table 4.2 ArgP regulation of lac fusions to nested deletions of lysP a

____________argP+__________ ___________argP__________

Plasmid

(lysP extent) Nil Lys Nil Lys

pHYD2636

(219 to +32) 802 150 31 30

pHYD2647

(114 to +32) 852 122 52 50

pHYD2648

(76 to +32) 43 47 41 42

a Values reported are the specific activities of -Galactosidase in derivatives of MC4100 (argP

+) or

GJ9602 (ΔargP) carrying the indicated lysP-lac fusion plasmids, after growth in glucose-MA without (Nil)

or with (Lys) Lys supplementation at 1 mM. Extents of lysP shown are in bp relative to start-site of

transcription.

It was observed from values in Table 4.2 that, the lysP(114 to +32)-lac

expression (like the previously described lysP[219 to +32]-lac) was activated

tremendously (17-fold) in the presence of ArgP (compare Nil values of argP+

and

argP strains for the two lac fusions); an 8-fold, ArgP-dependent Lys repression was

also evident (compare Nil, Lys values in argP+

and argP strains for the two lac

fusions). On the other hand, the lysP(76 to +32)-lac fusion showed low levels of lac


87

expression in both argP+

and argP strain backgrounds (see values of argP+

and argP

strains under both Nil, Lys conditions for this lac fusion) and did not behave like

lysP(219 to +32)-lac. This suggests that the lysP(76 to +32)-lac fusion no longer bears

the ArgP responsive cis element. Thus, the lysP upstream region between 114 and 76

carries critical sequence determinants for regulation of lysP by ArgP.

4.2.7 In vitro EMSA experiments with nested deletion fragments of lysP regulatory

region

To determine if the above in vivo observations could be supported by in vitro

ArgP binding data, EMSA experiments using each of the DNA fragments, i.e. lysP (114

to +32) and lysP (76 to +32) and varying concentrations of the ArgP protein, were

performed. As can be seen from Figure 4.7, lysP (114 to +32) showed similar binding

affinity and Lys sensitive ArgP binding as that described in Section 4.2.3 for the 263-bp

(219 to +43) fragment. In contrast, the lysP (76 to +32) fragment showed insignificant

ArgP binding even at high ArgP concentrations used, irrespective of the absence or

presence of Lys (Figure 4.8). These findings corroborate the previous in vivo

observations and suggest that sequences up to 114 are sufficient for ArgP binding and

regulation, and that the sequences between 114 to 76 house an important determinant

for ArgP binding and ArgP mediated regulation of lysP.

Figure 4.7 ArgP binding to lysP (114 to +32) DNA in the absence (A) or presence of 0.1 mM Lys (B).

ArgP monomer concentrations are indicated for each lane. Bands corresponding to free DNA and DNA in

binary complex with ArgP are marked by open and closed arrowheads respectively.


88

Figure 4.8 ArgP binding to lysP (76 to +32) DNA in the absence (A) or presence of 0.1 mM Lys (B).

ArgP monomer concentrations are indicated for each lane. Bands corresponding to free DNA are marked

by open arrowheads.

Section C: Role of ArgP in cadBA regulation

As discussed in Chapter 1 (Section 1.1.5), the lysP gene, which encodes the Lys-

specific permease, is also described in literature as cadR, because it has been implicated

in transcriptional regulation of the Lys decarboxylation system encoded by the two gene

operon, cadBA (cadB codes for Lys decarboxylase and cadA codes for the Lys-

cadaverine antiporter). cadBA expression is induced only in conditions of low pH,

anaerobiosis and in the presence of Lys and is dependent on a membrane bound

activator, CadC (Neely et al., 1994; Tetsch et al., 2008). In lysP defective mutants,

cadBA is induced at low pH even in absence of Lys, and the model is that LysP

negatively regulates cadBA in absence of Lys by sequestering CadC (Neely et al., 1994;

Tetsch et al., 2008). Since, it was found that ArgP is needed for activation of lysP

transcription, it was pertinent to determine if cadBA expression would be rendered Lys

independent even in the argP strain. Accordingly, the following experiments were

performed.

4.3.1 Construction of cadBA-lac promoter fusion

A cadBA-lac fusion was first constructed by PCR-amplifying cadBA upstream

regulatory region using forward primer JGJcadBF (5’-

TTTAATTTACCTGCAGGGGCAAAC-3’) and reverse primer JGJcadBR (5’-

TGCAATACCGGATCCCATCATATTA-3’); PstI and BamHI restriction sites were

incorporated into the forward and reverse primer respectively and are shown in italics in

the primer sequences. Following restriction digestion of the PCR amplicon with the same


89

enzymes, a 527-bp fragment extending from 387 to +140 with respect to transcription

start site of cadB, taken as +1, was cloned at the PstI and BamHI sites of vector pMU575

(Andrews et al., 1991). The resultant plasmid was designated as pHYD2674.

4.3.2 Effects of CadC, LysP, and ArgP on cadBA-lac expression

Next, the plasmid pHYD2674 bearing cadBA-lac fusion was introduced by

transformation into the following strains: wild-type (MC4100), lysP (GJ9623), cadC

(GJ9647), argP (GJ9602), lysP cadC (GJ9649), argP cadC (GJ9648), argP

lysP (GJ9624). -Galactosidase assays were performed after growth in MA-glucose

based media of pH 7.4 or pH 5.8, supplemented either with 19 amino acids other than

Lys (Nil) or with 19 amino acids and 10 mM Lys (Lys). The results of this assay are

shown in Table 4.3.

Table 4.3 Effects of CadC, LysP, and ArgP on cadBA-lac expression a

________ pH 7.4___________ _________pH 5.8____________

Strain Nil Lys Nil Lys

wild-type 25 28 24 98

lysP 27 27 94 96

cadC 25 26 26 24

argP 23 22 26 96

lysP cadC 21 28 24 26

argP cadC 25 26 24 28

argP lysP 27 24 87 99

a Values reported are the specific activities of -Galactosidase in the indicated strain derivatives carrying

the cadBA-lac fusion plasmid pHYD2674, after growth in MA-glucose based medium of pH 7.4 or pH 5.8

supplemented with: Nil, 19 amino acids (other than Lys); or Lys, 19 amino acids and 10 mM Lys.

The results show that, as previously reported, cadBA expression was induced only

at low pH, in the presence of Lys (compare Nil, Lys values derived at pH 7.4 for all


90

strains with Lys value of wild-type at pH 5.8) and the CadC transcriptional activator

(compare Lys values of wild-type at pH 5.8 with Lys values of cadC at pH 5.8); lysP

mutation rendered its expression constitutive only in the presence of CadC (compare Nil

values at pH 5.8 of wild-type, lysP, cadC and lysP cadC). However the argP

mutant failed to phenocopy the lysP mutant (compare Nil, Lys values of lysP, argP

and argP lysP strains at pH 5.8) in that cadBA expression continued to be Lys-

dependent in the former. Hence, ArgP does not seem to have a role in the regulation of

the cadBA operon.

Section D: In vitro binding studies of the ArgPd proteins -S94L, -P108S

and -P274S with the lysP regulatory region

4.4.1 Cloning of argPd alleles -S94L, -P108S and -P274S in protein expression vector

pET21b

As can be seen in Table 4.1 and as described in section 4.2.2, the argPd alleles

can be classified into three categories depending on the effects on lysP transcription (i)

alleles (-A68V, -S94L, -V144M, -P217L) that were more or less like wild-type with 20-

to 30-fold activation and 3- to 5-fold Lys repression; (ii) alleles (-P108S and -R295C)

that showed about 30-fold activation but only 2-fold Lys repression; and (iii) allele

-P274S that showed much less activation of about 10-fold and 3-fold Lys repression. To

find out if these in vivo regulatory differences exhibited by the argPd alleles compared to

the wild-type, were an outcome of differences in native ArgP and ArgPd binding patterns

at this locus, the ArgPd

proteins -S94L, -P108S and -P274S, each being representative of

one of the above three mentioned categories, respectively, were overexpressed and

purified.

As templates for PCR amplification of the argPd

alleles -S94L, -P108S and

-P274S, plasmids pHYD926, pHYD927 and pHYD2606 were respectively used. These

plasmids are derived from the low-copy-number cloning vector pCL1920 and contain a

1.86 kb SalI fragment from the E. coli chromosome which encompasses the argP locus

bearing the mutant alleles -S94L, -P108S and -P274S along with the native promoter

(Nandineni and Gowrishankar, 2004). The primers used for PCR amplification of the

920-bp protein coding region of each of the three argPd variants were JGARGP1r (5΄-

AGCAGACAACACATATGAAACGCCCGGA-3΄) as forward primer and JGARGP3r

(5΄-ATTATTTGATCTCGAGATCCTGACGAAG-3΄) as reverse primer; restriction


91

enzyme sites pertaining to NdeI and XhoI were respectively incorporated in the forward

and the reverse primers which are shown in the primer sequences in italics. The ATG

sequence of the NdeI site corresponds to the initiation codon of ArgPd proteins, and the

CGA sequence of the XhoI site on the reverse primer, eliminates the termination codon

of ArgPd proteins. The three resultant amplicons encoding the full length ArgP

d proteins

-S94L, -P108S and -P274S were then digested with NdeI and XhoI and cloned at the

same sites in plasmid pET21b (a medium-copy-number ColE1-based vector, with a

strong T7 promoter and an efficient ribosome binding site lying upstream of the NdeI site

in the multiple cloning site region) (Novagen, EMD Biosciences). The plasmids thereby

derived for argPd alleles -S94L, -P108S and -P274S were designated as pHYD2678,

pHYD2679, pHYD2680 respectively. The sequence of each of the three plasmids was

verified by DNA sequencing. Since the stop codon was abolished by incorporating XhoI

restriction enzyme site in the reverse primer, the reading frame of the alleles has been

extended in the plasmids to include, and to terminate just beyond, the His6-codons

situated after the multiple cloning site region of the vector. The proteins thus have a His6-

tag at the C-terminal end to enable protein purification with the aid of Ni-NTA affinity

chromatography.

4.4.2 Functional complementation studies with pHYD2678, pHYD2679 and

pHYD2680 encoding His6-ArgPdS94L, His6-ArgP

dP108S and His6-ArgP

dP274S

respectively

For functional complementation studies of the His6-tagged ArgPd

constructs, test

for CAN tolerance was done. It has been previously reported (Nandineni and

Gowrishankar, 2004) that a strain with wild-type argP allele cannot grow on medium

with CAN above a concentration of 40 g/ml i.e. it is CANs. However, a strain with the

argPd alleles can grow on medium supplemented with 65-100 g/ml of CAN i.e. it is

CANr. This is correlated with the ability of the argP

d alleles to activate the Arg exporter,

argO, to a much higher degree than wild-type argP. CAN resistance is also exhibited by

an argR

strain, in which there is derepression of the Arg biosynthetic pathway and CAN

resistance is achieved because of inability of CAN to compete with the excess cytosolic

Arg. For the CAN tolerance test, plasmids pHYD2678, pHYD2679 and pHYD2680 were

transformed into argP strain (GJ9602) and the ampicillin resistant colonies were

streaked on 0.2% glucose-MA agar plates containing 65 μg/ml CAN. Strains argR


92

(GJ4748) harboring plasmid vector pET21b and argP (GJ9602) carrying pHYD1705

(wild-type ArgP cloned in pET21b) served as positive and negative controls respectively.

After 36 hours incubation at 37C, as expected, it was found that the positive control and

the strains harboring the argPd plasmids exhibited CAN resistance whereas the strain

carrying wild-type ArgP failed to grow. This indicates that pHYD2678, pHYD2679 and

pHYD2680 can recapitulate the previously reported argPd phenotype and that the C-

terminal His6-tag does not affect function of the ArgPd proteins.

4.4.3 Overexpression and purification of ArgPd proteins -S94L, -P108S and

-P274S

Strain BL21 (DE3) was used for overexpression of ArgPd proteins -S94L,

-P108S and -P274S. In this strain, the T7 RNA polymerase gene is under the control of

the lac repressor-operator system that is induced by IPTG (Studier and Moffatt, 1986).

The plasmids, pHYD2678, pHYD2679 and pHYD2680 were transformed into BL21

(DE3) and protein overexpression and purification was achieved by the procedure

Figure 4.9 Overexpression and purification of His6-ArgP

dS94L, His6-ArgP

dP108S, His6-ArgP

dP274S,

demonstrated by Coomassie blue staining following sodium dodecyl sulfate-polyacrylamide gel (12%)

electrophoresis. Lanes 1, 2 indicate cell lysates of strain BL21 (DE3) carrying plasmid vector, pET21B,

without (U) and with induction (I) with 1mM IPTG respectively. Similarly, lanes 3 to 11 indicate

uninduced (U), induced (I) cultures and purified (P) proteins pertaining to ArgPdS94L, ArgP

dP108S,

ArgPdP274S respectively. Lane 12 shows the profile of protein molecular weight marker (M) of sizes in

kilodaltons as indicated alongside.


93

described in the Material and Methods Section 2.2.3.1. Briefly, the strains were grown in

LB to an A600 of around 0.6, were then induced with 1 mM IPTG and harvested after 4-

hrs of induction. Bacterial cells were lysed by sonication and protein purification was

achieved by passing the lysate through Ni-NTA (Qiagen) chromatographic column.

Figure 4.9 shows overexpression and purification of ArgPd proteins -S94L, -P108S and

-P274S. It can be estimated that the extent of protein purification achieved in each case

was about 95 %.

4.4.4 EMSA studies of the ArgPd proteins -S94L, -P108S and -P274S with the lysP

regulatory region

To determine the binding pattern of ArgPd proteins -S94L, -P108S and -P274S at

lysP, EMSA was performed in which varying concentrations of ArgPd proteins (0 to 300

nM) were incubated with the radiolabelled, 263-bp long, lysP DNA. -S94L, -P108S and

-P274S binding at lysP was more or less comparable to native ArgP: firstly, in terms of

binding affinities, approximate Kds being 60 nM, 100 nM and 60 nM respectively, the

previously determined Kd for native ArgP being about 40 nM (Figure 4.10 A) and

secondly with respect to reduced affinity of binding to the lysP regulatory region in the

presence of Lys (Figure 4.10 B).

Figure 4.10 EMSA of 263-bp lysP fragment with ArgP (WT) or its variants -S94L, -P274S and

-P108S at indicated protein concentrations in the absence (A) or presence (B) of Lys (0.1 mM). Bands

corresponding to free DNA and to DNA in binary complex with each of the ArgP proteins are marked by

open and filled arrows respectively.

These observations suggest that the differences in in vivo regulation seen

between strains harboring argP or the argPd alleles cannot be simply correlated with


94

binding affinities. This is particularly true for -P274S, which is least effective for lysP

activation but shows proficient binding to the regulatory element.

A striking difference that was seen between native ArgP and the ArgPd variants in

these EMSA experiments was that the DNA-protein complexes formed by all the three

ArgPd variants migrated faster than those formed by native ArgP. It has been previously

reported that such migration differences of DNA-protein complexes are a result of the

differences in DNA bending such that less bent DNA produces faster migrating

complexes than highly bent DNA (Wu and Crothers, 1984). For a number of LTTRs

(Colyer and Kredich, 1996; Parsek et al., 1995; Akakura and Winans, 2002; Dangel et

al., 2005) less bent DNA in the DNA-protein complex is associated with higher

activation. Figure 4.10 reveals that the fastest mobility was shown by the DNA-protein

complex formed by -P274S, followed by -P108S and -S94L; this suggests that -P274S

induces the least DNA bending. However this observation was inconsistent with the

finding that -P274S exhibited the least extent of lysP activation.

SECTION E: Interplay of other transcription factors in regulation of

lysP

4.5.1 Role of LysR in lysP regulation

ArgP belongs to the LTTR family of which LysR is the prototypic member

(Maddocks and Oyston, 2008). Since LysR has also been reported to mediate the

repression by Lys at lysA (Stragier et al. 1983a, 1983b), we tested for the possibility of

an interplay between the two transcription factors at lysP in vivo.

4.5.1.1 Construction of a Para-lysA derivative and its functional complementation

in a lysR strain

A lysR strain fails to express lysA and hence is a Lys auxotroph (Stragier et.

al., 1983a, 1983b). Therefore to test for LysR regulation of lysP in the absence of Lys,

it was decided to construct a plasmid derivative in which lysA was ectopically

expressed from the Para promoter on pBAD18 plasmid (Guzman et al. 1995). For this,

the lysA coding region was PCR amplified from E. coli genomic DNA with primer

pairs JGJlysAF (5’-ACAAGGTACCTTTTATGATGTGGCGT-3’) and JGJlysAR (5’-

ACAATCTAGAAGTCATCATGCAACC-3’); KpnI and XbaI sites were incorporated

in the primers and are shown in italics in the sequences. The 1341-bp sized, KpnI-


95

XbaI digested product was cloned into the corresponding sites of pBAD18. The

plasmid was designated as pHYD2673. For verification of this construct, its ability to

alleviate auxotrophy of a lysR strain on arabinose induction was examined. Plasmid

pHYD2673 was hence transformed into lysR (GJ9652); while to serve as positive

and negative control, respectively, pBAD18 was transformed into wild-type (GJ9650)

and lysR strain. These three strains were streaked on MA ampicillin plates with

glycerol as carbon source and arabinose added at 0.2% concentration, for induction.

After overnight incubation at 37 C, it was found that the positive control and lysR

bearing pHYD2673 strain showed healthy growth while the negative control failed to

grow (since the plate was not supplemented with Lys). Thus it was concluded that,

pHYD2673 encodes a functional LysA.

4.5.1.2 Effect of LysR in lysP-lac expression

To determine if LysR has any role in lysP regulation, we transformed plasmids

pHYD2673 (Para-lysA) and pHYD2636 (lysP-lac) into the following strains: wild-type

(GJ9650), argP (GJ9651), lysR (GJ9652) and argP lysR (GJ9653). The resultant

strains were grown in MA supplemented with 0.2% each of glycerol and L-arabinose

before performing β-Galactosidase assay as described in Section 2.2.1.5 of Chapter 2.

The results are as listed in Table 4.4.

Table 4.4 Effect of LysR on lysP-lac expression a

Strain lysP-lac

wild-type 1004

lysR 1052

argP 49

argP lysR 49

a Values shown are of -Galactosidase specific activity (Miller units) after growth in glycerol-arabinose-

MA without co-effector(s).

It was noted from the results above that, as expected, lysP-lac expression was

activated in the presence of ArgP (compare values of wild-type and argP strains).


96

However, there was no difference in expression levels in the wild-type and lysR strains,

suggesting that LysR has no role in lysP regulation. Also, the argP or argP lysR

strains showed identical regulatory outcomes indicating that there is no cross-regulation

between LysR and ArgP at this locus.

4.5.2 Role of Lrp in lysP regulation

The transcriptional regulator Lrp has previously been implicated in regulation of

one of the ArgP targets, argO (Peeters et al., 2009). Hence, it was checked if Lrp had any

effect on lysP transcription. Accordingly, pHYD2636 (lysP-lac) was transformed into

wild-type (MC4100), argP (GJ9602), lrp (GJ9625), and argP lrp (GJ9626). -

Galactosidase assays were performed after growth of these strains in MA-glucose

medium, without or with supplementation of Arg, Lys or leucine (known to be a co-

effector of Lrp at some loci) at a concentration of 1 mM. The results are tabulated in

Table 4.5.

Table 4.5 Effect of Lrp on lysP-lac expression a

Strain Nil Leu Arg Lys

wild-type 1029 1099 985 158

lrp 468 323 456 141

argP 49 45 47 47

argP lrp 57 38 54 54


(Nil) or with supplementation of leucine (Leu), Arg (Arg) or Lys (Lys) at a concentration of 1 mM.

Results as shown in Table 4.5 demonstrated that Lrp activates lysP by about 2-

fold (compare Nil values of wild-type and lrp) and leucine has no effect on this

activation (compare Nil, Leu values of wild-type and lrp). Unlike at argO, Arg

supplementation did not enhance lysP-lac expression in a lrp strain (Peeters et al.,

[2009] reported that argO-lac expression in a lrp strain is induced much further than in

the wild-type strain on Arg supplementation. This is because Lrp competes for ArgP

binding and hence activation at argO). Furthermore, Lys repression of expression was


97

independent of Lrp (compare Lys values of wild-type and lrp). Also, the previously

described activation of lysP by ArgP is much more pronounced than Lrp, such that

argP and argP lrp strains show more or less the same values for β-Galactosidase

activity.

Discussion

To examine ArgP’s role as a transcriptional regulator in E. coli, a microarray-

based comparison of whole genome mRNA abundances between an argPd and argP

strain was done. Data obtained from a microarray experiment do not represent the

regulation targets under the direct control of the test transcription factor alone but instead

also include large numbers of genes, which are affected indirectly due to the changes in

the expression levels of direct target genes. Generally, therefore the direct targets

represent only a minor fraction of the differentially expressed genes detected by

microarray analysis. A bottom up approach was therefore taken in this study and genes

were short-listed from the microarray, that showed (i) at least 2-fold activation in argPd

compared to argP, strain and (ii) whose expression (like previously identified ArgP

targets) was repressed in the presence of Lys. Three genes, namely, lysP (Lys-specific

permease), lysC (Lys-sensitive aspartokinase) and asd (aspartate semialdehyde

dehydrogenase) were arrived at and considered for validation experiments. The primary

aim of the approach taken was to be able to identify as many direct targets as possible

from the vast list identified from the microarray experiment. However, it is to be noted

that this approach may have created a bias and may have allowed identification of only a

subset of genes under direct regulation of ArgP.

Further detailed experiments with lysP, showed that ArgP activates lysP by about

35-fold and that there is about 5-fold ArgP dependent repression, in the presence of Lys.

A argP mutant phenocopies a lysP mutant for resistance to the Lys analog, thialysine.

In vitro, ArgP binds the lysP regulatory region with a Kd of around 40nM and the binding

affinity is diminished upon Lys addition (to a Kd>160nM) suggesting that Lys represses

lysP in vivo by engendering the loss of ArgP binding to the lysP operator region. This is

unlike the argO locus, where ArgP binding to the cis regulatory region is not Lys

sensitive. The sequences upto 114 of the lysP regulatory region are sufficient for in vivo

regulation and in vitro binding; also sequences between 114 and 76 carry important

determinants for ArgP regulation and binding of lysP.


98

In addition to its role in active uptake of Lys, LysP also participates in Lys

dependent transcriptional regulation of the cadBA operon (encoding genes for Lys

decarboxylation), by sequestering the CadC activator in the absence of Lys but not in its

presence (Neely et al., 1994; Tetsch et al., 2008). We found in this study that the argP

mutant does not phenocopy a lysP mutant for cadBA expression, suggesting that the

basal level of LysP in a argP strain is sufficient for the negative regulation of cadBA in

absence of Lys. Also, the regulation of cadBA by LysP is not through regulation of its

(LysP) abundance but predominantly through the presence or absence of its interaction

with CadC. ArgP thus does not seem to have a role in the regulation of the cadBA

operon.

The argPd

alleles, based on their in vivo effect on lysP-lac, could be classified

into three categories, namely, (i) alleles -A68V, -S94L, -V144M, -P217L that were more

or less like wild-type with 20- to 30-fold activation and 3- to 5-fold Lys repression, (ii)

alleles -P108S and -R295C that showed about 30-fold activation but only 2-fold Lys

repression, and (iii) allele -P274S that showed much less activation of about 10-fold and

3-fold Lys repression. To further analyse the basis of these differences, the ArgPd

proteins -S94L, -P108S, -P274S, each protein being representative of one of the three

categories described above were cloned, overexpressed and purified. Subsequent EMSA

of the lysP regulatory region and native ArgP or ArgPd proteins did not reveal any

significant differences in binding affinities. On the other hand, differences in mobility of

the protein-DNA complexes formed by native ArgP and the ArgPd variants were seen,

such that the ArgPd-DNA complexes migrated faster than native ArgP-DNA complex, in

the order (fastest to slowest) -P274S, -P108S and -S94L. Such differences in mobility of

protein-DNA complexes have been reported to be due to differences in DNA bending

such that less bent DNA migrates faster.

Numerous transcriptional regulators have been reported to bend the DNA of their

target sequences but the effect caused by DNA bending is different depending on the

type of regulator. Extensive DNA bending studies have been done for catabolite activator

protein (CAP) also known as cAMP receptor protein (CRP) (Gartenberg and Crothers,

1988; Schultz et al., 1991), p4 of Bacillus subtilis phage 29 (Rojo and Salas, 1991;

Salas, 1998), factor for inversion stimulation (FIS) (Finkel and Johnson, 1992; Betermier

et al., 1993) and integration host factor (IHF) (Friedman, 1988; Sugimura and Crothers,

2006). These proteins generally increase DNA bending and activation is as a result of the


99

simultaneous achievement of favorable promoter architecture and a stabilized regulator-

RNAP contact to form a productive nucleoprotein transcription initiation complex

(Perez-Martin et al., 1994). However, certain LTTRs (OccR, CysB, ClcR, CbbR) that

activate only in the presence of an inducing co-effector, apparently do so through a

decrease (rather than increase) in DNA bending (Akakura and Winans, 2002; Colyer and

Kredich, 1996; Parsek et al., 1995; Dangel et al., 2005).

Contrary to these LTTR studies, ArgPd-P274S, even though showed least DNA

bending, it was also the most deficient in lysP activation. A probable explanation can

perhaps be drawn from in vivo experiments that have shown that ArgP does not require

an inducing co-effector at lysP, suggesting that lysP promoter architecture or ArgP

mechanism of activation at lysP may be different from the prototypic LTTRs described.

There are also reports which indicate that any event that bends promoter sequences in a

direction unfavorable for RNA polymerase binding can effect transcription repression

(Rojo and Salas, 1991). It may be speculated that ArgPd-P274S bending at lysP may be

unfavorable for proper RNA polymerase contacts and hence least activation is seen with

this ArgPd variant.

We also examined the effect of the regulators LysR and Lrp on lysP transcription.

LysR was found not to have any role in lysP expression; Lrp on the other hand activated

lysP by about 2-fold. However, Lrp was not a competitive activator of ArgP as at argO

(Peeters et al., 2009). In comparison with Lrp, ArgP was found to be the predominant

and determining transcriptional regulator at the lysP locus. Lrp is known to be a global

transcriptional regulator that is involved in regulation of many loci associated with amino

acid metabolism (Calvo and Matthews, 1994; Seshasayee et al., 2008). It is thus, not

completely surprising to find its involvement in lysP regulation, although this may be

only to the extent of fine-tuning of its expression.

ArgP regulation of enzymes of the Lys biosynthetic pathway Chapter 5

100

Introduction

As described in Chapter 4, three genes namely lysP, lysC and asd were shortlisted

for validation from the microarray data, as candidate targets of the LTTR ArgP. Ensuing

work showed that lysP is indeed a transcriptional target of ArgP. The present chapter

begins with a description of experiments to test whether ArgP regulates transcription of

the other two candidate genes lysC and asd, both of which are part of the Lys

biosynthetic pathway. Since the results from these experiments indicated that ArgP does

mediate Lys repression of lysC and asd, additional experiments were then undertaken to

test whether ArgP also regulates two other genes, dapD and lysA that did not feature in

the microarray, but from literature are known to be Lys-repressed and which also

function as part of the Lys biosynthetic pathway.

As described in Chapter 1 (Section 1.1.4 and Figure 1.4), in E. coli Lys is

synthesized from aspartate and involves nine successive enzymatic reactions, eight being

common with DAP and the first two being common with methionine and threonine

(Patte, 1996). The specific biosynthetic branch leading to DAP and eventually Lys

includes enzymes (coding gene names in parentheses), aspartokinase III (lysC), aspartate

semialdehyde dehydrogenase (asd) dihydrodipicolinate synthase (dapA),

dihydrodipicolinate reductase (dapB), tetrahydrodipicolinate succinylase (dapD), N-

succinyl diaminopimelate aminotransferase (dapC or argD), N-succinyl diaminopimelate

desuccinylase (dapE), diaminopimelate epimerase (dapF) and diaminopimelate

decarboxylase (lysA). The penultimate step catalysed by diaminopimelate epimerase

(dapF) results in the formation of DAP, which is then decarboxylated by

diaminopimelate decarboxylase (lysA) to Lys.

Of all the genes of this pathway, lysC, asd, dapA, dapB, dapD and lysA are

reported to be Lys repressed. For LysC and DapA this is achieved through feedback

inhibition (Patte, 1996); additional regulation through a Lys-responsive riboswitch

mechanism has also been described for lysC (Rodionov et al., 2003; Sudarshan et al.,

2003). During the course of this study, Bouvier et al. (2008) reported that transcription of

dapB is Lys repressed through ArgP. Also, lysA expression is strictly dependent on the

regulatory protein LysR, a transcriptional activator responding to the internal

concentrations of DAP and Lys (Stragier et al., 1983a, 1983b). However, the mechanism

of Lys repression of asd and dapD have not been elucidated. Also, no common trans-


101

acting transcriptional regulatory factor that could effect repression of these genes in

response to increasing cellular Lys concentration has been identified.

This Chapter describes in vivo studies using argP+, argP

d and argP strains to

conclusively show ArgP regulation of lysC, asd, dapD and lysA. In vitro EMSAs show

that direct association with the upstream regulatory region of these genes, mediates ArgP

regulation. Studies investigating inter-relationships between ArgP regulation and the

previously described regulatory elements of this pathway are also discussed.

Results

Section A: ArgP regulation of lysC, asd, dapD and lysA

5.1.1 Construction of transcriptional lac fusions to cis regulatory regions of lysC,

asd, dapD and lysA

For in vivo studies of ArgP-mediated regulation of lysC, asd, dapD and lysA,

promoter-lac fusions were constructed by cloning the corresponding upstream regulatory

regions in pMU575, which is a single-copy number plasmid vector for generating

promoter-lacZ transcriptional fusions that encodes a trimethoprim resistance marker.

Four plasmids, namely, pHYD2664 (lysC-lac), pHYD2668 (asd-lac), pHYD2610 (dapD-

lac) and pHYD2670 (lysA-lac) were consequently obtained. The sequences of resultant

clones of each lac fusion were verified by sequencing. A detailed description of the

construction of each plasmid is provided below:

1) pHYD2664 (lysC-lac) : 615-bp of the lysC upstream regulatory region extending

from 244 to +371 was PCR-amplified from E. coli genomic DNA using primers

(restriction sites shown in italics), JGJlysCF (5’-ACAACTGCAGGTCTGCGTTGGATT-

3’) and JGJlysCR (5’-ACAATCTAGACGGTTCATGGCGT-3’). The PCR product was

digested with restriction enzymes, PstI and XbaI and cloned at the same sites in

pMU575.

2) pHYD2668 (asd-lac): Primers (restriction sites shown in italics) JGJasdF (5’-

GTATGTTTCAGTGTCGACATGAAAATAG-3’) and JGJasdR (5’-

GGCGTCGAAGCTTCGCTCTTCAACCA-3’) were used to amplify 366-bp of the asd

regulatory region. Digestion with SalI and HindIII resulted in a 343-bp DNA fragment,

encompassing the region from 207 to +136 that was cloned at the corresponding sites of

pMU575.


102

3) pHYD2610 (dapD-lac): 401-bp of the dapD regulatory region was PCR-

amplified with primers (restriction sites shown in italics) JGJdapDF (5’-

CGCCATTTTACTGCAGAAACCGAAG-3’) and JGJdapDR (5’-

CGGGTAACGGATCCTGCATTGGCT-3’). The resulting amplicon was digested with

PstI and BamHI, to obtain a 373-bp (273 to +100) DNA fragment, which was cloned at

the same sites in pMU575.

4) pHYD2670 (lysA-lac): Primers JGJlysAFnew (5’-

ACAACTGCAGCCTCAGTCAGGCTTC-3’), and JGJlysARnew (5’-

ACAAGGATCCCAGCCAAATTCAGC-3’) were used for PCR amplification of lysA

promoter region extending from 139 to +129. Restriction sites, PstI and BamHI were

incorporated in the primer sequence allowing digestion and subsequent cloning of the

same fragment at the corresponding sites of pMU575.

5.1.2 In vivo expression of lysC-lac, asd-lac, dapD-lac and lysA-lac fusions in argP+,

argPd

and argP strains

To ascertain the effect of ArgP and the ArgPd variants on the lysC-lac, asd-lac,

dapD-lac and lysA-lac, derivatives of the argP strain (GJ9602) each carrying two

plasmids namely, (i) plasmid vector pCL1920 or its derivatives with argP+ or the

different argPd variants and (ii) one of the following lac fusion plasmids, pHYD2664

(lysC-lac), pHYD2668 (asd-lac), pHYD2610 (dapD-lac) or pHYD2670 (lysA-lac) were

contructed. As control, strains with pHYD1723 (argO-lac) were also employed. The

strains were grown in glucose-MA supplemented with 18 amino acids other than Arg or

Lys, 18 amino acids with 1 mM Arg, or 18 amino acids with 1 mM Lys. β-Galactosidase

assays were then performed for each lac fusion set of strains.

The data from these experiments as documented in Table 5.1, permitted the

following interpretations:

(i) As has already been reported earlier (Nandineni and Gowrishankar, 2004; Laishram

and Gowrishankar, 2007; Peeters et al., 2009), argO-lac expression was stimulated 4-

fold in the argP+

strain relative to argP, but only in Arg-supplemented medium.

(ii) Transcription from promoters of the genes lysC, asd, dapD and lysA, in cultures of

the argP+ strains grown in Arg- or Lys-unsupplemented medium was higher than that in

the corresponding argP derivatives, by factors of approximately 4, 2.5, 4 and 2


103

TABLE 5.1 Expression of lac fusions in different argP variant derivatives a

_____________________________________________________________________________________________________________

argO lysC asd

------------------------------------------------ ---------------------------------------------- ------------------------------------------------

-gal sp. act. Ratio -gal sp. act. Ratio -gal sp. act. Ratio

argP ----------------------- -------------------- ------------------------ -------------------- ------------------------ --------------------

genotype Nil Arg Lys Nil/ Variant/Δ Nil Arg Lys Nil/ Variant/Δ Nil Arg Lys Nil/ Variant/Δ

Lys Lys Lys

______________________________________________________________________________________________________________

ΔargP 38 31 32 1.0 1.0 553 52 107 5.1 1.0 265 284 302 0.8 1.0

argP+ 41 106 25 4.2 3.4 2095 197 214 10 3.7 608 632 281 2.1 2.2

-A68V 2248 2613 1036 2.5 84 1477 1436 197 7.4 2.6 447 443 209 2.1 1.6

-S94L 3615 3227 3776 0.9 104 1806 1719 214 8.4 3.2 478 450 265 1.8 1.8

-P108S 1621 1633 755 2.2 53 2928 2736 250 12 5.2 871 760 283 3.0 3.2

-V144M 737 930 176 5.3 30 1264 1268 158 8.0 2.2 331 344 229 1.4 1.2

-P217L 2126 2436 623 3.9 79 1677 1610 186 9.0 3.0 352 283 157 2.2 1.3

-P274S 7884 8246 8392 1.0 266 678 646 165 4.1 1.2 195 207 186 1.0 0.7

-R295C 115 256 688 27 8.3 2399 2282 236 10 4.3 662 675 295 2.2 2.4

____________________________________________________________________________________________________________________________________________________

a Values shown are of -Galactosidase specific activity (Miller units) in glucose-MA supplemented with the 18 amino acids other than Arg or Lys (Nil), 18 amino acids

and 1 mM Arg (Arg) or 18 amino acids and 1 mM Lys (Lys). The ratios Nil/Lys and Variant/ indicate, respectively, the degree of Lys repression for the concerned

strain, and the degree of regulation imposed by the concerned ArgP variant (or the wild-type ArgP) relative to that in the argP.


104

TABLE 5.1 (-continued) Expression of lac fusions in different argP variant derivatives

a

_____________________________________________________________________________

dapD lysA

------------------------------------------------ ----------------------------------------------

-gal sp. act. Ratio -gal sp. act. Ratio

argP ----------------------- -------------------- ------------------------ --------------------

genotype Nil Arg Lys Nil/ Variant/Δ Nil Arg Lys Nil/ Variant/Δ

Lys Lys

_____________________________________________________________________________

ΔargP 161 152 162 0.9 1.0 493 535 80 6.1 1.0

argP+ 642 572 213 3.0 3.9 1004 980 77 13 2.0

-A68V 437 322 130 3.3 2.7 984 980 71 14 1.9

-S94L 672 567 249 2.6 4.1 910 1300 71 13 1.8

-P108S 659 757 238 2.7 4.0 1075 1038 68 16 2.1

-V144M 229 278 134 1.7 1.4 782 836 77 10 1.5

-P217L 262 216 120 2.1 1.6 841 820 64 13 1.7

-P274S 160 139 144 1.1 0.9 720 511 92 7.8 1.4

-R295C 301 230 152 1.9 1.8 893 892 69 13 1.8

_______________________________________________________________________________________________________





105

respectively (see Variant/Δ ratio of argP+ for each lac fusion). For each of these lac-

fusions, similar β-Galactosidase values were obtained for strains grown in cultures

without and with Arg supplementation (compare Nil and Arg values of argP and its

variants for all these lac fusions), suggesting that Arg supplementation was without effect

for any of these genes. This implies that ArgP activates these genes and, unlike at argO,

activation is not dependent on Arg as a co-effector.

(iii) All the genes above also exhibited much higher repression upon Lys

supplementation in the argP+ than the argP strain, suggesting that Lys repression of

their promoters is ArgP-mediated (see Nil/Lys ratio of argP+ for each lac fusion).

(iv) The argPd variants used in this study had been selected to confer L-canavanine-

resistance (that is, for enhanced expression of ArgO which is the exporter of Arg and L-

canavanine) (Celis, 1999; Nandineni and Gowrishankar, 2004). As expected, all of them

activated argO-lac to a much greater extent than argP+

(see Variant/∆ ratio for argO-

lac) and this was observed both without and with Arg or Lys supplementation. The

-P274S variant in particular was the most effective for constitutive argO expression

(with 270-fold degree of activation).

(v) In the case of lysC, asd, dapD and lysA, many of the argPd variants behaved much

like the argP+ allele itself for both activation as well as Lys-repression, although a few

combinations exhibited differences. For example, the levels of activation obtained with

the -P108S variant at lysC (5-fold) and asd (4-fold) were higher than that with argP+ at

these promoters; on the other hand, both -V144M and -P217L were ineffective for

activation of asd and dapD (compare Variant/ ratios of argP+ and these argP

d alleles

for asd-lac and dapD-lac fusions). However, the most conspicuous discrepancy was that

seen with the -P274S variant; although it was the most proficient of all the ArgPd mutants

for constitutive argO expression, this variant was amongst the least effective for

activation of expression from all the other ArgP-regulated promoters, and indeed

behaved like ΔargP at three of them, namely lysC, asd and dapD (compare Variant/

ratios of argP+ and argP

d-P274S for these lac fusions).

5.1.3 In vitro binding studies of ArgP to upstream regulatory region of lysC, asd,

dapD and lysA

To determine whether the in vivo expression differences between argP+ and argP

mutant strains for the various promoter-lac fusion constructs was associated with ArgP


106

Figure 5.2 EMSAs with ArgP and cis regulatory regions of different genes in absence or presence of the

co-effector Lys. ArgP monomer concentrations are indicated for each lane. Bands corresponding to free

DNA and to DNA in binary complex with ArgP are marked by open and filled arrowheads, respectively.

binding in vitro to the corresponding cis regulatory regions, EMSAs were performed.

Earlier studies have shown that ArgP-mediated Lys repression can occur by two

alternative mechanisms at different promoters. At argO, Lys-liganded ArgP binds the

regulatory region (Laishram and Gowrishankar, 2007; Bouvier et al., 2008; Peeters et al.,

2009) but it then inhibits productive transcription at a step further downstream by

trapping RNAP at the promoter (Laishram and Gowrishankar, 2007). On the other hand,


107

at dapB (Bouvier et al., 2008), gdhA of K. aerogenes (Goss, 2008), or at the gdhA and

lysP loci previously described in this thesis, ArgP binding to the regulatory region is

diminished upon Lys addition. Accordingly in this study, EMSA with His6-ArgP were

done in the absence or presence of 0.1 mM Lys, and the cis regulatory regions of lysC,

asd, dapD, lysA, argO, dapB and lysP were employed. The data from these experiments

are shown in Figure 5.2.

The apparent Kds for binding of ArgP to the different templates in the presence or

absence of Lys were estimated as equivalent to the protein concentration at which half

maximal binding was seen (from equation 3.1 in Chapter 3) and the values are given in

Table 5.2.

TABLE 5.2 Apparent Kds (nM) of ArgP binding to cis regulatory regions of different

genes

cis regulatory region Lys + Lys

argO 15 15

dapB 120 >150

lysP 55 >140

lysC 70 110

asd 170 >200

dapD 70 120

lysA 150 210

Kd values listed in Table 5.2, show that ArgP exhibits high-affinity binding to

argO (Kd = 15 nM) which is not diminished in presence of Lys, which is consistent with

the data from earlier reports (Bouvier et al. 2008, Laishram and Gowrishankar, 2007;

Peeters et al., 2009). Under identical experimental conditions as for argO, the regulatory

regions of the other ArgP-regulated genes (dapB, lysP, lysC, asd, dapD and lysA) were

also bound by ArgP, with apparent Kds ranging from 55 nM to 170 nM (monomeric

concentrations). In all these cases (unlike the situation with argO), Lys addition was

associated with increase in the apparent Kds, indicating that ArgP binding to the DNA


108

regions in these instances is Lys-sensitive.

5.1.4 In vivo expression of dapB-lac in argPd

and argP strains

It has previously been shown that ArgP activates expression of dapB (coding for

dihydropicolinate reductase), by 4-fold in a Lys-sensitive manner (Bouvier et al., 2008).

To document the effect of the various argPd alleles on dapB expression, a dapB-lac

fusion was constructed. For this, the dapB regulatory region was PCR-amplified using

primers (restriction sites in italics) JGJdapBF (5’- CCCTGTTTTGCTGCAGTGGAAAC-

3’) and JGJdapBR (5’-AATGCCAGCGGATCCTGAATCAAC-3’). The resulting 411-bp

amplicon was digested with restriction enzymes PstI and BamHI to generate a 386-bp

fragment extending from 285 to +101, which was subsequently cloned in pMU575 at

the same sites. This plasmid clone was designated pHYD2669. Similar strain

backgrounds and conditions as described in Section 5.1.2 were used to set up β-

Galactosidase assays using pHYD2669. The result of these β-Galactosidase assays

demonstrating the effect of the ArgPds on dapB-lac is shown in Table 5.3.

It was observed that in media without supplementation, dapB-lac expression in

argP+ was 25-fold higher than in ∆argP indicating that ArgP activates dapB expression

by this extent. Arg supplementation did not result in further activation of dapB-lac, but

Lys supplementation caused about a 6-fold decrease in expression only in argP+

(see

Nil/Lys ratio pertaining to argP+) confirming previously reported findings of ArgP-

mediated Lys repression. It was also noted that the fold activation of dapB-lac by ArgP,

seen in the above experiments, is much higher than the 4-fold activation reported by

Bouvier et al. and may be due to differences in the medium used; while MA-glucose

supplemented with 18 amino acids other than Arg and Lys were used in the above

experiments, MA-glucose was used by Bouvier et al. (2008). The MA-glucose medium

supplemented with 18 amino acids is in principle similar to the Arg free (AF) rich

medium that has been used in studies of Arg biosynthesis and transport to obtain

derepression by the repressor protein ArgR which uses Arg as a corepressor (Novick and

Maas, 1961; Celis 1977; Celis et al., 1998). It has been reported that growth in AF

medium is twice as fast as in minimal medium and causes depletion of the limiting

amounts of intracellular Arg, thereby resulting in derepression of ArgR targets. Similarly

in the above experiments, it may be reasoned that growth in MA-glucose medium


109

supplemented with 18 amino acids (unlike growth in MA-glucose medium) causes

depletion of intracellular Lys and hence higher expression levels of dapB-lac.

Table 5.3 Effect of the ArgPds on dapB-lac expression

a

-gal sp. act. Ratio

ArgP


argP 100 108 140 0.7 1.0

argP+ 2344 2380 401 5.8 23

-A68V 1074 1199 289 3.7 11

-S94L 1561 1468 299 5.2 16

-P108S 2891 2882 421 6.8 28

-V144M 1692 1592 305 5.5 17

-P217L 1143 1024 275 4.1 11

-P274S 190 173 127 1.4 1.9

-R295C 3717 3071 572 6.4 37

a Values shown are of -Galactosidase specific activity (Miller units) in glucose-MA supplemented with

the 18 amino acids other than Arg or Lys (Nil), 18 amino acids and 1 mM Arg (Arg) or 18 amino acids and

1 mM Lys (Lys). The ratios Nil/Lys and Variant/ indicate, respectively, the degree of Lys repression for

the concerned strain, and the degree of regulation imposed by the concerned ArgP variant (or the wild-type

ArgP) relative to that in the argP.

It was also found that ArgPd variants -P108S, -R295C were as potent in activation

of dapB-lac as the wild-type allele (see Variant/∆ ratio of the respective alleles showing

28-, 37- and 23-fold activation respectively), variants -A68V, -S94L, -V144M, -P217L

were not as effective in activation (see Variant/∆ ratio showing 11-, 16-, 17- and 11-fold

activation respectively). The most drastic difference, as previously described for lysC,

asd and dapD, was exhibited by -P274S, which failed to activate and phenocopied

argP. In case of all the argPd

variants, β-Galactosidase values obtained on Arg


110

supplementation were comparable to those without such supplementation (compare Nil

and Arg values for each argPd), suggesting that Arg does not have an effect on dapB-lac

expression. On the other hand, Lys addition (other than in variant -P274S), resulted in 4-

to 7-fold decrease in expression (see Nil/Lys ratios for each argPd), allowing the

conclusion that argPd variants like wild-type argP cause Lys-repression of dap-lac

expression.

Section B: Inter-relationships between ArgP regulation and the

previously described regulatory elements for different genes

5.2.1 Lysine repression mediated by riboswitch and ArgP at lysC

As previously mentioned, lysC expression is regulated at a post-transcriptional

level through a Lys-responsive riboswitch that is proposed to exist in the leader region of

the lysC mRNA (Rodionov et al., 2003; Sudarshan et al., 2003). In vivo lysC-lac fusion

studies (Table 5.1) showed that there is a 5-fold Lys-repression in the argP strain that is

ArgP-independent (see Nil/Lys ratio of argP for lysC-lac,); this presumably represents

the regulation imposed by the riboswitch, but there is additional repression to 10-fold in

an argP+ strain suggestive of an additional component of control that was ArgP

dependent.

5.2.2 LysR, ArgP regulation at lysA

lysA coding for diaminopimelate decarboxylase, the enzyme that decarboxylates

DAP into Lys, is strictly dependent on the regulatory protein LysR, a transcriptional

activator responding to the internal concentrations of DAP and Lys (Stragier et al.,

1983b). lysR is divergently transcribed from and shares a common regulatory region with

lysA. Also, LysR expression is negatively autoregulated. As both LysR and ArgP are

reported to be involved in regulation at lysA, it was of interest to find out if ArgP directly

regulated lysA or mediated its effect indirectly through regulation of lysR. Further an

examination of the existence of any crosstalk between the two regulators at this locus

was planned. For achieving these objectives, the following experiments were performed:

5.2.2.1 Construction of lysR-lac

The lysR regulatory region (268-bp in length and extending from –127 to +141)

was amplified using primers JGJlysRF (5’- ACAACTGCAGCAGCCAAATTCAGC-3’)


111

and JGJlysRR (5’- ACAAGGATCCCCTCAGTCAGGCTTC-3’), digested with PstI and

BamHI and cloned at the same sites in the single copy, promoter expression vector,

pMU575, to obtain pHYD2675.

5.2.2.2 Combined effect of LysR and ArgP on lysA-lac and lysR-lac expression

To check the effect of ArgP and LysR (along with co-effectors) at lysA-lac or

lysR-lac, a ΔlysR strain was purposed to be constructed. However, a ΔlysR strain fails to

express LysA and hence is a Lys auxotroph. Plasmid pHYD2673, in which lysA was

ectopically expressed from the Para promoter (details of construction having been

described in Chapter 4, Section 4.5.1.1) was thus transformed into strains: wild-type

(GJ9650), ΔlysR (GJ9652), ΔargP (GJ9651), ΔargP ΔlysR (GJ9653), already carrying

lysA-lac (pHYD2670) or lysR-lac (pHYD2675). β-Galactosidase assays were performed

after growing strains in MA-glucose supplemented with 0.2% each of glycerol and L-

arabinose along with 18 (other than Arg and Lys) amino acids; or 18 amino acids and

1 mM Arg or 18 amino acids and 1 mM Lys.

The results tabulated for lysA-lac expression as tabulated in Table 5.4 allowed the

following conclusions:

(i) In the wild-type strain, lysA expression was about 4-fold lower on Lys

supplementation than without or with Arg supplementation. This shows that lysA

expression is Lys-sensitive

(ii) Comparison of β-Galactosidase values of wild-type and ΔlysR show that lysA-lac

expression is about 10-fold lower in ΔlysR. Also, in ΔlysR, Lys addition did not result in

further decrease in lysA-lac expression. These observations substantiate previous reports

of involvement of LysR in the activation and Lys-repression at lysA.

(iii) lysA-lac expression was about 3-fold lower in ∆argP compared to wild-type in all

media tested, suggesting that ArgP activates lysA. However, this ability to activate

occurred only in a lysR+ background such that a ΔlysR and ΔargP ΔlysR showed

identical values.


112

Table 5.4 Cross-regulation by LysR and ArgP at lysA-laca

Strain Nil Arg Lys

wild-type 215 300 68

ΔlysR 23 26 22

ΔargP 72 75 29

ΔargP ΔlysR 24 23 23

a Values shown are of -Galactosidase specific activity (Miller units) obtained on growing strains in

glycerol arabinose-MA supplemented with the 18 amino acids other than Arg or Lys (Nil), 18 amino acids

and 1 mM Arg (Arg) or 18 amino acids and 1 mM Lys (Lys).

Table 5.5 Cross-regulation by LysR and ArgP at lysR-lac a

Strain Nil Arg Lys

wild-type 30 33 33

ΔlysR 59 65 63

ΔargP 29 33 34

ΔargP ΔlysR 66 66 65

a Values shown are of -Galactosidase specific activity (Miller units) obtained on growing strains in

glycerol arabinose-MA supplemented with the 18 amino acids other than Arg or Lys (Nil), 18 amino acids

and 1 mM Arg (Arg) or 18 amino acids and 1 mM Lys (Lys).

Likewise, data for lysR-lac expression (Table 5.5) showed that:

(i) Expression of lysR-lac is increased two-fold in ΔlysR in comparison to wild-type,

irrespective of amino acid supplementation, indicative of negative autoregulation that has

been described earlier (Stragier et al., 1983a, 1983b).

(ii) Expression levels in wild-type and ∆argP or ΔlysR and ΔargP ΔlysR were similar

indicating that ArgP does not have an effect on lysR-lac expression and that ArgP

regulation of lysA is not through regulation of lysR.


113

Discussion

In the results described in this chapter, the genes lysC, asd, dapD and lysA were

found to be activated by ArgP to varying extents (2- to 4-fold), this activation being

independent of the co-effector Arg but being Lys-sensitive. Several of the various argd

variants used in this study behaved much like the argP+ allele both for activation and Lys

repression at these four loci and at dapB. However, allele -P274S was odd in showing

ΔargP behaviour; this being strikingly different from its effect at argO where it activates

by 270-fold. EMSAs showed Lys-sensitive binding of ArgP to the regulatory regions of

lysC, asd, dapD and lysA suggesting that Lys repression is due to inability of the

activator to bind to the operator.

Three lines of evidence thereby suggest that the mechanism of ArgP regulation at

argO is fundamentally different from that at the loci lysC, asd, dapD, lysA and dapB.

First, argO is the only gene that requires Arg as co-effector for its activation. The second

is the differential response to the various argPd mutations (although it must be noted that

these mutations were selected on the basis of their ability to confer greatly increased

argO expression). Of these the most prominent distinction is that obtained with the

-P274S variant of ArgP; this mutant is by far the most effective for constitutive argO

expression, whereas it exhibits diametrically opposite effects and behaves much like

ΔargP for a majority of other target genes. Finally, argO is the only example in which

ArgP’s binding to the cis regulatory region is not Lys-sensitive, so that repression by Lys

is achieved by an RNA polymerase-trapping mechanism at the argO promoter. Also, it is

to be mentioned here that while ArgP is essential for argO transcription (Nandineni and

Gowrishankar, 2004; Laishram and Gowrishankar, 2007), just as LysR itself is essential

for lysA transcription (Stragier et al., 1983a, 1983b; Maddocks and Oyston, 2008), ArgP

only plays an auxiliary role in transcription of genes of the Lys biosynthetic pathway.

Of the ArgP-regulated genes, lysC and lysA exhibit additional mechanisms for

Lys-repression involving, respectively, a postulated riboswitch (Rodionov et al., 2003,

Sudarshan et al., 2003) and the LysR regulator protein (Stragier et al., 1983; Maddocks

and Oyston, 2008). Our results indicate that ArgP-mediated regulation of lysC is

independent of and additive to the putative riboswitch regulatory mechanism. On the

other hand ArgP activates lysA transcription 2-fold, but only in cells that are also

proficient for LysR. These results lend support to the notion that multi-target regulators

and multi-factor promoters represent the norm in bacterial gene regulation (Ishihama,


114

2010).

Of the ArgP-regulated and Lys-repressed genes identified earlier and in this

study, five encode enzymes for biosynthesis of DAP and Lys: two in the pathway that is

common for Lys, threonine and methionine biosynthesis (lysC, asd), and three in the

Lys-specific pathway (dapB, dapD, lysA). One may speculate that Lys-dependent

modulation by ArgP of multiple genes of the Lys pathway provides a fine-tuning effect

in controlling the flux of intermediates serving the biosynthesis of three different amino

acids for protein synthesis as also of DAP for peptidoglycan assembly.

Our results also allow us to suggest that all Lys-liganded repression in E. coli is

mediated by ArgP. LysR also mediates Lys-repression (of lysA), but in this case it has

been suggested that Lys repression is indirect and that the co-effector ligand is actually

DAP (which is the substrate for lysA), and that the latter’s binding to LysR converts it

into an activator conformation (Maddocks and Oyston, 2008).

ArgP as a non-canonical transcriptional regulator Chapter 6

115

Introduction

As described in Chapter 1 (Sections 1.2.7 a), there are previous reports of ArgP

(IciA) binding the three AT-rich 13-mers at oriC, preventing opening of these regions

and thereby blocking the initiation of replication (Hwang and Kornberg, 1990; 1992;

Thony et al., 1991). ArgP (IciA) has also been described as a transcriptional activator of

dnaA and nrdA, which are genes involved in DNA replication and metabolism (see

Section 1.2.7 a and 1.2.7 b in Chapter 1). Lee et al. (1997) showed that IciA binds the

dnaA 1P promoter and that in vivo overproduction of the protein enhances transcription

from the same promoter. EMSA and DNase I footprinting assays also indicated that IciA

binds the AT-rich sites in the upstream region of nrdA. RNase protection assays

demonstrated that in vivo overexpression of IciA enhances transcription from the nrd

promoter, but purified IciA protein did not affect in vitro transcription from the same

promoter (Han et al., 1998).

Despite these reports of IciA regulating crucial genes in DNA replication and

metabolism, an iciA mutant or an overproducing strain is not compromised for DNA

replication or growth rate (Thony et al., 1991).

In other studies, the argP genetic locus was described independently by Celis et

al., (1973) and Rosen (1973) as mutations conferring canavanine resistance (CANr) and

it was postulated to be a regulator of Arg uptake. Although subsequent work (Nandineni

and Gowrishankar, 2004; Laishram and Gowrishankar, 2007) conclusively showed that

ArgP is a regulator of the Arg exporter, argO, it has never been determined if ArgP has

any role in transcriptional regulation of the genes involved in Arg uptake or import.

As described in Chapter 1 (Section 1.1.3), three uptake systems for Arg have been

described in E. coli: (i) the LAO (Lysine, Arg, Ornithine) system for basic amino acids

(ii) the AO (Arg, Ornithine) system and (iii) the Arg specific system. The LAO system is

encoded by argT-hisJQMP with two transcription units of the same polarity. The AO and

Arg specific system are encoded by the artPIQM- artJ locus that are organized into two

transcriptional units of the same polarity (see Figure 1.3 in Chapter 1).

At the same time, ArgP (IciA) has also been reported as a nucleoid-associated

protein that shows apparently sequence-nonspecific DNA binding activity (Azam and

Ishihama, 1999). The protein exhibits affinity for AT-rich and curved DNA sequences as

determined in vitro by DNase I footprinting or EMSAs (Wei and Bernander, 1996; Azam

and Ishihama, 1999).


116

This chapter describes in vivo lac fusion studies performed in argP, argP+,

argPd strain backgrounds to verify and analyse the previously reported involvement of

ArgP in transcriptional regulation of DNA replication genes (dnaA, nrdA) and Arg

uptake genes (argT, hisJ, artP, artJ). In vitro EMSAs with ArgP and regulatory regions

of dnaA, nrdA, argT and a DNA fragment from the coding region of E. coli lacZ are also

reported.

Results

Section A: In vivo experiments to examine the role of ArgP in the

regulation of dnaA, nrdA, argT, hisJ, artJ and artP

6.1.1 Construction of transcriptional lac fusions to cis regulatory regions of dnaA,

nrdA, argT, hisJ, artJ, artP

With the objective to examine the role of ArgP in the regulation of dnaA, nrdA,

argT, hisJ, artJ and artP, promoter-lac fusions were constructed. The respective

upstream regulatory regions were cloned in promoter expression vector pMU575. The

plasmids pHYD2671 (dnaA-lac), pHYD2672 (nrdA-lac), pHYD2660 (argT-lac),

pHYD2661 (hisJ-lac), pHYD2658 (artJ-lac) and pHYD2659 (artP-lac) were thereby

obtained. The sequences of resultant clones of each lac fusion were verified by

sequencing. A detailed description of the construction of each plasmid is provided below

(restriction sites incorporated in primers being indicated in italics):

1) pHYD2671 (dnaA-lac) : 668-bp of the dnaA upstream regulatory region extending

from 460 to +208 and inclusive of the previously described ArgP-regulated dnaA 1P

and 2P promoters was PCR-amplified from E. coli genomic DNA using primers

JGJdnaAF (5’-ACAACTGCAGTTTCATGGCGATT-3’) and JGJdnaAR (5’-

ACAAGGATCCGCTGGTAACTCAT-3’). The PCR product was digested with

restriction enzymes, PstI and BamHI and cloned at the same sites in pMU575.

2) pHYD2672 (nrdA-lac): Primers JGJnrdAF (5’-

ACAACTGCAGCGCCATCAACAAT-3’) and JGJnrdAR (5’-

ACAAGGATCCGTCGAGATTGATGC-3’) were used to amplify 481-bp of the ArgP-

regulated nrdA regulatory region which have beforehand been defined (Han et al., 1998).

Digestion with PstI and BamHI was followed by cloning at the same sites in pMU575.


117

3) pHYD2660 (argT-lac): The argT regulatory region was PCR-amplified with

primers JGJargTF (5’-ACAACTGCAGATCTCTTTGCCCGC-3’) and JGJargTR (5’-

ACAAGGATCCGTAGCGCCGCATA-3’). The resulting 367-bp amplicon was digested

with PstI and BamHI and cloned at the same sites in pMU575.

4) pHYD2661 (hisJ-lac): Primers JGJhisJF (5’-

ACAACTGCAGTGTCTACGGTGACTG-3’) and JGJhisJR (5’-

ACAAGGATCCTCGCAGCAAACG-3’) were used for PCR-amplification of hisJ

promoter region extending from 187 to +119. Restriction sites, PstI and BamHI were

incorporated in the primer sequence allowing digestion and subsequent cloning of the

same fragment at the corresponding sites of pMU575.

5) pHYD2658 (artJ-lac): The artJ-lac fusion was constructed by cloning the 416-bp

region from 270 to +146 at the PstI and BamHI sites of pMU575. The primers used for

amplification of this region were JGJartJF (5’-ACAACTGCAGCAAAGCGCTGGCA-

3’) and JGJartJR (5’-ACAAGGATCCGGATAGGTGGCTGAAA-3’).

6) pHYD2659 (artP-lac): PCR using genomic DNA as template and primers

JGJartPF (5’-ACAACTGCAGGAATCGCTAACGCC-3’) and JGJartPR (5’-

ACAAGGATCCTATCGAACAGCGCC-3’) resulted in an amplicon of 288-bp

containing the artP regulatory region from 192 to 196. Following digestion with PstI

and BamHI, this DNA fragment was cloned at the corresponding sites in pMU575.

6.1.2 In vivo expression of dnaA-lac, nrdA-lac, argT-lac, hisJ-lac, artJ-lac and artP-

lac fusions in argP+, argP

d and argP strains

The effect of ArgP and the ArgPd variants on dnaA-lac, nrdA-lac, argT-lac, hisJ-

lac, artJ-lac and artP-lac expression was evaluated by transforming plasmids

pHYD2671 (dnaA-lac) or pHYD2672 (nrdA-lac) or pHYD2660 (argT-lac) or

pHYD2661 (hisJ-lac) or pHYD2658 (artJ-lac) or pHYD2659 (artP-lac) into a argP

(GJ9602) strain bearing the argP or argPd alleles on the plasmid pCL1920 vector

backbone, or the vector alone. Plasmid pHYD1723 (previously described argO-lac) was

also transformed into this strain to serve as a positive control for argP and argPd

activation. β-Galactosidase assays were performed after growing the strains in glucose-

MA supplemented with the 18 amino acids other than Arg or Lys, along with (i) 1 mM

Arg, (ii) 1 mM Lys, or (iii) neither of the above. The outcomes of these experiments are

recorded in Table 6.1.


118

TABLE 6.1 Expression of lac fusions in different argP variant derivatives a

________________________________________________________________________________________________________________

dnaA nrdA argT

-------------------------------------------------- -------------------------------------------------- ----------------------------------------------


argP ----------------------- -------------------- ---------------------- -------------------- ---------------------- --------------------

genotype Nil Arg Lys Nil/ Variant/Δ Nil Arg Lys Nil/ Variant/Δ Nil Arg Lys Nil/ Variant/

Lys Lys Lys

________________________________________________________________________________________________________________

ΔargP 88 89 95 0.9 1.0 338 348 377 0.8 1.0 208 218 204 1.0 1.0

argP+ 85 94 90 0.8 0.8 377 357 400 0.9 1.1 214 194 205 1.0 1.0

-A68V 87 87 82 1.0 0.9 419 399 391 1.0 1.2 211 197 211 1.0 1.0

-S94L 72 71 85 0.8 0.8 361 331 409 0.8 1.0 227 221 224 1.0 1.0

-P108S 78 78 91 0.8 0.8 386 398 426 0.9 1.1 196 200 194 1.0 0.9

-V144M 98 85 85 1.1 1.1 421 413 429 0.9 1.2 184 187 189 0.9 0.8

-P217L 92 90 95 0.9 1.0 340 418 377 0.9 1.0 216 215 211 1.0 1.0

-P274S 87 85 83 1.0 0.9 374 369 399 0.9 1.1 187 197 206 0.9 0.8

-R295C 93 87 90 1.0 1.0 389 417 387 1.0 1.1 191 197 196 0.9 0.9

________________________________________________________________________________________________________________________________________________________





119

TABLE 6.1 (-continued) Expression of lac fusions in different argP variant derivatives a

___________________________________________________________________________________________________________________________

hisJ artJ artP

-------------------------------------------------- -------------------------------------------------- -----------------------------------------------


argP ----------------------- -------------------- ---------------------- -------------------- ---------------------- -------------------

genotype Nil Arg Lys Nil/ Variant/Δ Nil Arg Lys Nil/ Variant/Δ Nil Arg Lys Nil/ Variant/Δ

Lys Lys Lys

________________________________________________________________________________________________________________

____

ΔargP 244 114 296 0.8 1.0 1256 231 1252 1.0 1.0 184 122 210 0.9 1.0

argP+ 227 115 262 0.9 0.9 1353 286 1480 0.9 1.0 170 105 197 0.9 0.9

-A68V 211 98 208 1.0 0.9 1419 299 1345 1.0 1.1 190 107 211 1.0 1.0

-S94L 200 91 221 0.9 0.8 1361 331 1409 1.0 1.0 207 121 211 1.0 1.1

-P108S 237 98 219 1.0 1.0 1387 328 1406 1.0 1.1 196 100 194 1.0 1.0

-V144M 228 105 209 1.0 0.9 1421 313 1429 0.9 1.1 184 107 189 1.0 1.0

-P217L 211 109 213 0.9 0.9 1340 318 1371 0.9 1.0 186 115 198 1.0 1.0

-P274S 227 95 222 1.0 0.9 1364 269 1394 0.9 1.0 197 117 206 1.0 1.0

-R295C 241 118 234 1.0 1.0 1412 317 1387 1.0 1.1 191 107 196 1.0 1.0

________________________________________________________________________________________________________________________________________________________ a Values shown are of β-Galactosidase specific activity (Miller units) in glucose-MA supplemented with the 18 amino acids other than Arg or Lys (Nil), 18 amino acids




120

The above results indicate that each of the promoter-lac fusions were unaltered

for expression in the argP+or argP

d variants compared to that in the argP strain (see

ratio Variant/ which is 1.0 in each case). Also, for all promoter-lac fusions and in each

strain, expression under both conditions that is without or with Lys supplementation, was

nearly identical (see ratio Nil/ Lys which is 1.0 in each case). The expression of argO-

lac, under the same conditions as that employed for these promoter-lac fusions showed

ArgP-dependent activation and Lys repression as previously described in Table 5.1.

Thus, the data in Table 6.1 indicates that, ArgP does not activate transcription of DNA

replication associated genes, dnaA and nrdA in vivo nor does it regulate the genes of Arg

uptake system, contrary to previous reports. In the case of hisJ-lac, artJ-lac and artP-lac,

2-fold, 6-fold and 2-fold repression of expression in cultures grown with Arg

supplementation was observed, respectively (compare columns Nil and Arg for

respective lac fusions). Arg-dependent repression of these Arg uptake genes has been

previously reported to be ArgR-mediated (Caldara et al., 2007).

Section B: ArgP as a non-canonical transcriptional regulator

6.2.1 In vitro binding of ArgP to upstream regulatory region of dnaA and nrdA

Contrary to previous reports (Lee et al., 1996, 1997; Han et al., 1998), results in

the previous section show that neither ArgP nor the ArgPd variants activate dnaA or nrdA

in vivo. To investigate the in vitro finding from the same reports, of ArgP binding to

upstream regulatory region of dnaA and nrdA, EMSAs were performed. This involved

using radiolabelled DNA fragments encompassing the dnaA and nrdA cis regulatory

regions (same as that used for generating the lac fusion constructs) and varying

concentrations of purified His6-ArgP. The binding reactions were set up in the absence

and presence of 0.1 mM Lys.

Figure 6.1 shows that like previously reported, ArgP does bind to the upstream

regulatory region of dnaA and nrdA, with an apparent Kd of 150 nM in each case. The

binding was unchanged in presence or absence of Lys.


121

Figure 6.1 EMSAs with ArgP and cis regulatory regions of dnaA and nrdA in absence or presence of the

co-effector Lys. ArgP monomer concentrations are indicated for each lane. Bands corresponding to free

DNA and to DNA in binary complex with ArgP are marked by open and filled arrowheads, respectively.

Lanes 10-12 in each panel represent control EMSA reactions with lysP template.

6.2.2 In vitro binding studies of ArgP to upstream regulatory region of argT and to

the coding region of lacZ

Since ArgP showed inability to activate dnaA and nrdA in vivo but showed

proficient binding of their regulatory elements in vitro, it was pertinent to find out if in

vitro binding in the absence of in vivo regulation was also observed for the genes

involved in Arg uptake. EMSA reactions were set up using argT regulatory region (with

the same genomic co-ordinates as that used for generating the lac fusion construct) and

varying concentrations of purified His6-ArgP, in the absence and presence of 0.1 mM

Lys. It was found that the upstream regulatory region of argT revealed a very low affinity

of binding by ArgP (Kd>400nM) that was Lys-independent (Figure 6.2 A).

Based on these results that showed ArgP binding to several DNA templates in

vitro without a corresponding regulatory effect in vivo, it was next determined whether at

high concentrations it would exhibit binding to a completely non-specific control DNA

fragment. When tested by EMSA (Figure 6.2 B), very negligible binding of ArgP was

observed even at 400 nM to an internal 295-bp DNA sequence from lacZ (obtained by

PCR-amplification from genomic DNA using primer pair 5’-

GTGGTGCAACGGGCGCTGGGTCGGTTAC-3’ and 5’-

CAACTCGCCGCACATCTGAACTTCAG-3’). These findings are consistent with


122

Figure 6.2 EMSAs with ArgP and cis regulatory region of argT and internal lacZ DNA fragment in

absence and presence of co-effector Lys. ArgP monomer concentrations are indicated for each lane. Bands

corresponding to free DNA and to DNA in binary complex with ArgP (latter not observed for lacZ) are

marked by open and filled arrowheads, respectively. Lanes 10-12 in panel B represent control EMSA

reactions with dapD template.

other reports that ArgP does not bind DNA non-specifically (Hwang et al., 1992; Ruiz et

al., 2011).

Table 6.2 is a comprehensive tabulation of the apparent Kds of ArgP binding to

cis regulatory regions of the in vivo regulated genes namely, argO, gdhA, lysP, dapB,

lysC, asd, dapD, lysA (as previously described in Chapters 3, 4 and 5) and the in vivo

non-regulated genes namely, dnaA, nrdA, argT and coding region of lacZ, in the absence

and presence of 0.1 mM Lys.

From this data, it may be concluded that the genes exhibiting ArgP regulation in

vivo can be divided into two categories based on the Kd of binding of ArgP to their cis

regulatory region in absence and presence of Lys: (i) gene (argO) exhibiting high

affinity, Lys-insensitive binding and (ii) genes (gdhA, lysP, dapB, lysC, asd, dapD, lysA)

exhibiting moderate affinity, Lys-sensitive binding. At the cis regulatory region of genes

that did not display regulation in vivo (dnaA, nrdA and argT), ArgP effected low affinity

binding but unlike for the regulated genes of the second category, binding was Lys-

insensitive.


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Table 6.2 Apparent Kds (in nM) of ArgP binding to cis regulatory regions of different

genes

cis regulatory region Lys + Lys

argO 15 15

gdhA 80 130

lysP 55 >140

dapB 120 >150

lysC 70 110

asd 170 >200

dapD 70 120

lysA 150 210

dnaA 150 150

nrdA 150 150

argT >400 >400

lacZ >400 >400

Discussion

In its description as IciA, ArgP has been reported to bind oriC in E. coli (Hwang

and Kornberg, 1990; 1992; Hwang et al., 1992) and M. tuberculosis (Kumar et al.,

2009), and to regulate transcription from promoters of the dnaA and nrdA genes (Lee et

al., 1996, 1997; Lee and Hwang 1997; Han et al., 1998).Contrary to these reports, in this

study, dnaA-lac or nrdA-lac expression was unaffected between argP+, argP

d and argP

strains, suggesting that ArgP does not regulate their transcription in vivo. These results

also allow the conclusion that this regulator protein does not play a role in transactions

involving DNA metabolism or replication in E. coli, a conclusion that is supported by the

facts that neither a ΔargP mutant nor an ArgP-overproducing strain is compromised for

DNA replication or growth rate (Thony et al., 1991). Additional confidence in the above

results showing lack of regulation was offered by use of the argPd

alleles, which showed

variable activation of the ArgP-regulated genes argO, gdhA, lysP, dapB, lysC, asd, dapD

and lysA, but consistently showed absence of activation of dnaA and nrdA.

Since ArgP is required for transcription of the Arg exporter ArgO, ArgP


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dependence of expression of other genes known to be involved in Arg transport (artP,

artJ, argT, and the hisJ) was determined. None of them was affected by either the ΔargP

mutation or Lys supplementation, indicating that ArgP does not regulate Arg import in E.

coli.

On revisiting the findings of in vitro binding of ArgP at dnaA and nrdA (Lee et

al., 1996, 1997; Lee and Hwang 1997; Han et al., 1998), it was found that ArgP does

bind to the regulatory regions of dnaA and nrdA with moderate affinity and in a Lys-

insensitive manner. On checking for binding at argT regulatory region, a similar weak

affinity, Lys-insensitive binding was observed. Nonetheless, on using an internal lacZ

DNA fragment in EMSA reactions, negligible binding was noticed. With the exception

of argO (which showed Lys-insensitive, high affinity binding), a distinguishing feature

between in vivo ArgP regulated and non-regulated genes was Lys-sensitive binding in

vitro.

As described in Chapter 4, probable ArgP binding sequences were detected in the

regulatory regions of lysP and dapB (genes with highest fold activation by ArgP).

However, an attempt to determine similar sites in the regulatory regions of lysC, asd,

dapD and lysA was unsuccessful. This observation, combined with the fact that ArgP

shows binding affinity for AT-rich regions (oriC [as described in Chapter 1], upstream

regions of dnaA, nrdA and argT) which are known to have higher bending and twisting

flexibilities (Zhang et al., 2004) suggests that DNA structure may also determine binding

site recognition. High specificity of binding despite a relaxed sequence dependency has

also been reported to be a common feature for IHF and CRP in E. coli (Steffen, 2002;

Chen et al., 2001).

Although a genome wide analysis was not undertaken, the above findings suggest

that the ArgP or IciA protein is a non-canonical transcriptional regulator, in that it does

exhibit specific DNA binding to mediate the transcriptional regulation of particular genes

in vivo but in addition it also binds at other sites in the genome that are not associated

with regulatory outcomes. Genome-wide chromatin immunoprecipitation studies reveal

that DNA binding by the activator protein CRP is also apparently non-canonical since

there are about 70 sites where it binds strongly and exerts its transcriptional regulatory

function, and a further approximately 104 sites of low-affinity binding (Grainger et al.,

2005). Another example of non-canonical binding is RutR, many of whose binding sites

fall within gene coding regions (Shimada et al., 2008). LexA is described as binding to


125

targets containing degenerate consensus sequences (Wade et al., 2005). On the other

hand, ChIp-chip studies of transcriptional regulators such as MelR (Grainger et al.,

2004), MntR (Yamamoto etal., 2011), NsrR (Partridge et al., 2009), and PurR (Cho et

al., 2011) reveal that they behave canonically in that each exhibits binding only to those

sites where it serves to control transcription of the adjacent genes or operons. Lrp also

appears to be a canonical transcriptional regulator, although it binds nearly 140 sites in

the genome (Cho et al., 2008). CRP’s closely related paralog FNR binds canonically at

around 65 locations without any noise of low-affinity binding (Grainger et al. 2007). In

this context, transcriptional regulators in E. coli can be imagined as a spectrum with non-

canonical regulators such as CRP, RutR and ArgP at its one end and canonical regulators

such as MelR, MntR, NsrR, PurR, FNR and Lrp at the other.

The purpose of non-canonical binding by ArgP remains to be determined. It has

been speculated in the case of CRP that by bending the DNA at its non-canonical binding

sites, the protein may contribute to chromosome shaping and chromatin compaction

(Grainger et al., 2005; Wade et al., 2007). It is also possible that lack of in vivo

regulation of genes to which in vitro binding is demonstrated may be because the CRP

transcription factor functions at these loci only in specific conditions (Wade et al., 2007).

Like CRP, ArgP too may similarly participate in shaping the nucleoid architecture, given

its reported propensity to bind curved DNA (Wei and Bernander, 1996; Azam and

Ishihama, 1999). In the case of RutR it is proposed that evolution has been slow to

eliminate non-functional DNA sites because they do not have an adverse effect on cell

fitness (Shimada et al., 2008).

It is also possible that the putative dual functions of ArgP, in transcriptional

regulation and in chromosome organization, are in some way related to its ability to

dimerize in two different modes (as deduced from the crystal structure of the M.

tuberculosis ortholog [Zhou et al., 2010]). It is possible that the two dimer interfaces on

ArgP engage in sequential alternating interactions to assemble as a polymeric scaffold,

analogous to that which has been described for another nucleoid protein H-NS (Arold et

al., 2010). Ruangprasert et al., (2010) had deduced from structural studies a similar

infinite polymer arrangement for the LTTR BenM (Figure 1.15 in Chapter 1). In this

context, a ChIp-chip analysis would greatly facilitate the exact measurement of the

chromosome-wide DNA-binding profile of ArgP in vivo.

Transcriptional cross-regulation between ArgP-argO and LysG-lysE Chapter 7

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Introduction

The arginine exporter argO is presently the most extensively studied, bona fide

transcriptional target of ArgP. As detailed in Chapter 1, it was previously designated as

yggA and was proposed to be an amino acid exporter primarily based on its sequence

homology to LysE, the Lys and Arg exporter in the Gram-positive bacterium C.

glutamicum (Nandineni and Gowrishankar, 2004). lysE is under the transcriptional

control of LysG, the ArgP ortholog in C. glutamicum (Bellmann et al. 2001). Thus, the

pairs ArgP-argO of E. coli and LysG-lysE of C. glutamicum are orthologous, with the

first member of each pair being a LTTR and the second its target encoding a basic amino

acid exporter.

The ArgP and LysG proteins show 35% identity and 53% similarity. Further, a

sequence alignment of the DNase I footprinted region of argO (85 to 20) with the

previously described upstream regulatory region of lysE (Bellmann et al., 2001) shows

about 44% identity. However, whereas LysE is an exporter of Arg and Lys whose

expression is induced by LysG in presence of co-effectors Arg, Lys or His, ArgO exports

Arg alone and ArgP activates expression in presence of co-effectors Arg but not Lys or

His. Based on the similarities between these orthologous transcriptional pairs and in an

attempt to address the differences in their features of regulation, transcriptional cross-

regulation studies between ArgP-argO of E. coli and LysG-lysE of C. glutamicum were

carried out.

This chapter describes experiments to examine the cross-regulation ability of

ArgP on lysE and LysG on argO. The effect of the argPd alleles on lysE-lac expression

was also determined; in vitro EMSA experiments to analyse the probable causes for the

differences in regulation exerted by wild-type argP and the argPd

alleles at argO and

lysE are described. Subsequently, the combined effects of ArgP, ArgPds and LysG on

lysE-lac expression are discussed.


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Results

Section A: In vivo transriptional cross-regulation studies in E. coli of

ArgP on lysE and LysG on argO

7.1.1 Construction of Para-lysG derivative and lysE-lac fusion

The lysG coding region was derived from C. glutamicum ATCC 13032 genomic

DNA as a 939-bp PCR-amplicon using primer pair (restriction sites indicated in italics)

5´-ACAAGAATTCGGTTCTTAACATGGT-3´ and 5´-

ACAAAAGCTTGCGAAGAAGTGAAA-3’. This DNA fragment was cloned at the

EcoRI and HindIII sites downstream of Para in plasmid pBAD18 (pMB9 replicon for

Ara-induced expression of target genes, ampicillin) to enable arabinose-inducible

expression of LysG in E. coli. This plasmid was designated as pHYD2676.

Similarly, a 334-bp PstI-BamHI fragment with the C. glutamicum lysE regulatory

region (from 289 bp to +45 bp with respect to start site of transcription taken as +1) was

obtained with the primer pair 5´-TAGTTTCTGCAGGCAGCAACAC-3´ and 5´-

GTCCGATGGATCCTAAAAGACTGG-3´ from C. glutamicum ATCC 13032 genomic

DNA. Plasmid pHYD2677 was derived after cloning this fragment at the PstI-BamHI

sites of plasmid pMU575 (IncW single-copy-number replicon with promoter-less lacZYA

operon, trimethoprim) and represented a lysE-lacZ transcriptional fusion.

7.1.2 In vivo β-Galactosidase assays to determine effects of C. glutamicum LysG and

E. coli ArgP on respective non-cognate targets argO and lysE

As mentioned above, ArgP has previously been shown to activate argO in

presence of Arg or citrulline and to repress it in presence of Lys (Nandineni and

Gowrishankar, 2004; Laishram and Gowrishankar, 2007; Peeters et al., 2009). On the

other hand, LysG activates lysE expression in the presence of any of the co-effectors Arg,

Lys, citrulline, or His (Bellmann et al., 2001).

To determine the effect of LysG on argO-lac expression, plasmid pHYD2676

(Para-lysG) or pBAD18 (as negative control) was transformed into a argP (GJ9651)

strain carrying pHYD1723 (argO-lac fusion). The resultant strains were grown in MA

with 0.2% each of glycerol and arabinose in absence and presence of co-effectors Arg (or

its intermediate citrulline), Lys or His or their corresponding dipeptides (with Ala); β-

Galactosidase assays were performed to determine the extent of argO-lac expression in

each case. Table 7.1 shows the results of these assays.


128

Comparison of -Galactosidase specific activity units in strains without or with

expression of LysG (and in the presence of any co-effector) indicates that LysG is unable

to activate expression of argO-lac under any growth condition. Also, ectopic LysG

expression in an argP+

(GJ9650) strain did not interfere with the ability of ArgP to

regulate argO-lac.

Table 7.1 Absence of LysG effect on argO-lac expression a

∆argP argP+

Supplement LysG + LysG LysG + LysG

Nil 25 29 29 33

Lys 30 27 12 14

His 26 30 16 17

Arg 24 30 150 (563) 174 (655)

Citrulline 28 27 135 198

a Values shown are -Galactosidase specific activity (Miller units) in MA-glycerol arabinose without (Nil)

or with supplementation of Arg or Arg-Ala dipeptide (Arg) or Lys or Lys-Ala dipeptide (Lys), His or His-

Ala dipeptide (His), and citrulline (as free amino acid only) at 1 mM. Values obtained using free amino

acids or dipeptides were identical (within error) except in the case of argP+ strain grown in presence of

Arg-Ala, where there was a 4-fold higher induction than that obtained on use of Arg as a free amino acid;

the latter values are indicated in parentheses.

Similarly, the effect of ArgP on lysE-lac expression was ascertained by using

derivatives of argP+

(MC4100) and argP (GJ9602) strains carrying pHYD2677 (lysE-

lac fusion) in β-Galactosidase assays. The strains were grown in MA-glucose

supplemented either with Arg, Lys, His or with their corresponding dipeptides (with

Ala). Table 7.2 shows the results of these assays.

It was observed that the lysE-lac fusion directed a low level of -Galactosidase

expression which was not different between ΔargP and argP +strains in all media tested,

suggesting that native ArgP also cannot cross-activate lysE.


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Table 7.2 Absence of ArgP effect on lysE-lac expression a

Supplement argP argP+

Nil 25 25

Lys 24 27

His 28 25

Arg 28 27

a Values shown are -Galactosidase specific activity (Miller units) in MA-glucose without (Nil) or with

supplementation of Arg or Arg-Ala dipeptide (Arg) or Lys or Lys-Ala dipeptide (Lys), His or His-Ala

dipeptide (His) at 1 mM. Values obtained using free amino acids or dipeptides were identical (within

error).

7.1.3 Reconstitution of C. glutamicum LysG-lysE regulation in E. coli

A possible reason for the lack of activation of argO by LysG could be the

inability of the Gram-positive C. glutamicum transcriptional regulator to recruit Gram-

negative E. coli RNAP. To examine this possibility, reconstitution of C. glutamicum

LysG-lysE regulation in E. coli was attempted. As previously described, in C.

glutamicum, LysG activates lysE expression by about twenty-fold in the presence of co-

effectors Arg, Lys or His (Bellmann et al., 2001).

Table 7.3 shows that in a ΔargP strain with Ara-induced LysG, lysE-lac

expression was significantly up-regulated (4- to 5- fold) in presence of Arg, Lys or His,

in comparison with the level of expression observed in absence of any co-effector. For

Lys and His, activation was observed upon their supplementation either as free amino

acids or as dipeptides (with Ala). For Arg however, it was only the dipeptide and not the

free amino acid that activated lysE expression.

Although the possibility exists that it is the Arg-containing dipeptide which is the

co-effector for LysG-mediated activation of lysE, it is more likely for the following

reasons that Arg is indeed the co-effector and that uptake of exogenously provided Arg is

not as efficient as that of the dipeptide in generating the intracellular concentrations of

amino acid needed for lysE induction: (i) it is known that the E. coli Arg uptake systems

are feedback repressed in presence of Arg (Caldara et al., 2007); (ii) since the data

indicate that the Lys-Ala and His-Ala effects of lysE are mediated by the free amino

acids Lys and His, respectively, it is reasonable to assume that the Arg-Ala effect is also


130

mediated by Arg; (iii) LysG’s ortholog ArgP possesses a single binding pocket for

competitive binding of the co-effectors Arg and Lys ( Laishram and Gowrishankar, 2007;

Zhou et al., 2010); and (iv) ArgP-mediated argO-lac induction was also determined to be

4-fold higher with Arg-Ala than with Arg (data shown in Table 7.1).

Table 7.3 lysE-lac activation by LysG in E. coli a

Supplement Free amino acid Dipeptide (with Ala)

Nil 22 18

Lys 110 95

His 95 89

Arg 22 111

Citrulline 21 n.d.

a Values shown are -galactosidase specific activity (Miller units). A derivative of strain ΔargP (GJ9651)

with plasmids pHYD2676 and pHYD2677 bearing Para-lysG and lysE-lac, respectively, were grown for -

galactosidase assays in MA medium with 0.2% each of glycerol and Ara without (Nil) or with addition at 1

mM of Lys, His, Arg or citrulline either as free amino acid or as dipeptide with Ala (for all but citrulline;

that is, Lys-Ala, His-Ala or Arg-Ala). n.d. implies not determined.

Thus, the results recapitulate in E. coli, at least qualitatively, the LysG regulation

of lysE as described in C. glutamicum.

7.2 Section B: Transcriptional cross-regulation studies of ArgPd

variants on lysE

7.2.1 In vivo -Galactosidase assays to show the effect of ArgPd

variants on lysE-lac

expression

As mentioned in the previous chapters, several gain-of-function single amino acid

substitutions in ArgP are known (Nandineni and Gowrishankar, 2004) that mediate high

and constitutive expression of argO in E. coli (values for argO-lac expression effected

by these argP variants are shown in chapter 5, Table 5.1). These are designated as the

argPds and seven such ArgP

d variants were tested for their ability to cross-regulate lysE.


131

The argP strain derivatives harboring two plasmids namely pHYD2677 (lysE-

lac) and any one of the seven argPd variants cloned in vector pCL1920 (pSC101

replicon, streptomycin and spectinomycin), were employed in these experiments. -

Galactosidase assays were performed after growth in MA-glucose with or without

supplementation of amino acids Arg and Lys.

Table 7.4 demonstrates that while four of the ArgP variants namely -A68V,

-V144M, -P217L, and -R295C were unable to activate lysE-lac in E. coli, the remaining

three namely -S94L, -P108S, and -P274S were able to do so to different extents. ArgPds

-P274S and -S94L were the most effective with a 5- to 6-fold activation of lysE

expression, which in both cases was independent of co-effector addition and comparable

to the maximal activation obtained with LysG itself upon co-effector supplementation

(see Table 7.3). ArgPd-P108S activated mildly by 2-fold and this was Lys-sensitive.

Table 7.4 lysE-lac activation by certain ArgPd

variants a

argP genotype Nil Arg Lys

∆argP 15 15 14

argP+ 15 19 18

-A68V 25 24 19

-S94L 84 85 67

-P108S 35 36 18

-V144M 15 16 14

-P217L 16 19 14

-P274S 126 138 118

-R295C 11 12 14

a Values shown are -galactosidase specific activity (Miller units) obtained after growth of strains in MA-

glucose without (Nil) or with supplementation of amino acids Arg and Lys at 1 mM.


132

7.2.2 In vitro binding studies of the ArgPd

proteins -S94L, -P274S and -P108S with

the argO and lysE regulatory regions

To determine if the increased activation at argO and lysE by the ArgPd variants

-S94L, -P274S and -P108S (relative to that by native ArgP) was due to alterations in

either affinity or pattern of binding of the proteins to the corresponding regulatory

regions, EMSAs were first performed with the purified (His6-tagged) proteins and

upstream regulatory regions of argO (427-bp DNA fragment from 293 to +109 bp) or

lysE (334-bp DNA fragment from 289 to +45 bp). Attempts to use purified LysG as

well in these experiments were unsuccessful, since the protein could not be recovered in

active form from insoluble inclusion bodies following its overexpression.

Figure 7.1 EMSAs with native ArgP (WT) or its variants -S94L, -P274S and -P108S at the indicated

protein concentrations and cis regulatory region of argO (427-bp fragment) in the absence of co-effector

(Nil) or in the presence of Arg or Lys at 0.1 mM concentration. Bands corresponding to free DNA and to

DNA in binary complex with each of the ArgP proteins are marked by open and filled arrowheads,

respectively.


133

Figure 7.1 A shows that at argO, the Kds of binding of ArgP and its variants

-S94L, -P274S and -P108S were not significantly different from one another (around 10

nM in all cases); however, significant differences were observed in migration rates of the

DNA-protein complexes, in decreasing order of -P274S, -P108S, -S94L and native ArgP.

Neither the Kd of binding nor migration rate of the DNA-protein complexes at argO was

affected by Arg or Lys addition (Figure 7.1 B and 7.1 C).

Figure 7.2 EMSAs with native ArgP (WT) or its variants -S94L, -P274S and -P108S at the indicated

protein concentrations and cis regulatory region of lysE (334-bp fragment) in the absence of co-effector

(Nil) and in the presence of Arg or Lys at 0.1 mM concentration. Bands corresponding to free DNA and to

DNA in binary complex with each of the ArgP proteins are marked by open and filled arrowheads,

respectively.

At lysE by contrast (Figure 7.2 A), native ArgP and the ArgPd

variants showed

significant differences in their binding affinities; whereas native ArgP displayed

negligible binding even at 300 nM, the -S94L, -P274S and -P108S variants exhibited Kds


134

of ~300 nM, 60 nM, and >300 nM, respectively. As with argO, the complexes of lysE

DNA with ArgPd variants also migrated faster than the complex of lysE with wild-type

ArgP. The EMSA features above were unaltered by Arg or Lys supplementation (Figure

7.2 B, Figure 7.2 C respectively).

In the case of other LTTRs, differences in degree of DNA bending upon protein

binding (Akakura and Winans, 2002; Colyer and Kriedrich, 1996; Dangel et al., 2005;

Kreusch et al., 1995; Kullik et al, 1995, McFall et al., 1997; Parsek et al., 1995; Wang et

al., 1992) or in protein pIs (Leveau et al., 1996) have earlier been invoked to explain

differences in migration rates of the protein-DNA complexes in EMSA experiments.

Since native ArgP is a homodimer (Azam and Ishihama, 1999; Laishram and

Gowrishankar, 2007; Zhou et al., 2010), a third possibility that the ArgPd proteins may

function as monomers to therefore exhibit faster migration after binding to DNA could

also be considered. These different possibilities were tested in the experiments described

in the following sections.

7.2.3 pI measurements of ArgP and the ArgPd proteins

It was reasoned that the difference in mobilty of the DNA-protein complexes of

native ArgP and ArgPd variants could be due to altered (lower) pI compared to the wild-

type protein (predicted pI being 6.8). Hence, in a native EMSA gel of pH 8.3, the ArgPd

variants would have more negative charge and show enhanced mobility.

To test this possibility, native isoelectric focusing was done (Figure 7.3) using

Pharmacia Phast Gel Apparatus and precast IEF gel (pH 3-9) from GE healthcare. The

results indicated that the pI of the ArgPd proteins (~6.6) was not lower than that of native

ArgP itself (~6.3), thereby excluding pI changes as the explanation for the observed

EMSA mobility differences.

7.2.4 Gel filtration chromatography to determine the oligomeric state of the ArgPd

proteins -S94L, -P274S and -P108S

To determine the oligomeric state of the ArgPd proteins -S94L, -P274S and -

P108S, the purified proteins were passed through Sephadex G100 gel filtration column,

along with native ArgP as control. Each of the four proteins -S94L, -P274S and -P108S

eluted as a single peak at similar elution volumes (Figure 7.4 A). Molecular weights

corresponding to the peaks, as estimated from the calibration curve derived from running


135

Figure 7.3 Native isoelectric focusing of ArgP and its variants -S94L, -P274S -P108S. Lane at right

depicts protein markers of pIs as indicated alongside. Bands corresponding to native ArgP and the variants

are indicated by arrows.

Figure 7.4 Elution profiles of wild-type ArgP, -S94L,-P274S and -P108S on gel filtration

chromatography through sephadex G-100 (A). Subunit composition was determined from a plot of log10

molecular masses in kilodaltons (MW) of protein standards (solid squares denoting in descending order,

bovine serum albumin, ovalbumin, chymotrypsinogen A and ribonuclease A) against the fraction number

representing their peaks of elution; shown by the interrupted lines are the fraction number representing the

elution peak for each of the four proteins (WT, -S94L,-P274S and -P108S) and the corresponding log10MW

value as intercepts of the X- and Y- axes, respectively (B).


136

molecular mass markers approximated for each protein, to a dimeric state (Figure 7.4 B).

Thus, the ArgPd proteins, like native ArgP, exist as homodimers in solution and an

altered oligomeric state of the ArgPd proteins is not an explanation for their altered

electrophoretic mobility.

7.2.5 DNA bending studies with native ArgP and ArgPd proteins -S94L, -P274S,

-P108S at argO

The degree of electrophoretic mobility retardation of a bent DNA fragment

depends on position of the bend with respect to fragment ends, so that retardation is most

pronounced when the bend is located near the centre of the fragment (Wu and Crothers,

1984; Wang et al., 1992). Accordingly, migration rates of argO DNA complexes were

determined using (i) 100 nM each of purified ArgP or ArgPd proteins and (ii) three PCR-

generated, radiolabelled DNA fragments of nearly identical size but in which the 85 to

20 argO segment (which is the region footprinted by ArgP) was located near the

downstream end (D), the middle (M), or the upstream end (U) of the fragment. The

fragments (length and extent with respect to argO transcription start site, indicated in

parentheses): D (335-bp, 305 to +30), M (333-bp, from 212 to +121) and U (332-bp,

from 105 to +227) were generated using primer pairs 5’-

GTGCGCCTGAACGAACTTGGTG-3’ and 5’-CACGTTGGATATTCCGAATT-3’, 5’-

CTGGAGCGTATTAAACGTGA-3’ and 5’-GTATGCCCTGATTCATCACAAAAG-3’,

5’-CGCTGAGGCCAGATAATACT-3’ and 5’-CACGGCGACTGCATCAATAA-3’,

respectively. Although the fragment sequences were not permuted with respect to one

another, it was reasoned that the different neighbouring sequences in the three fragments

would not affect their mobility under the experimental conditions.

Figure 7.5 shows the results of these EMSAs which indicated the following: (i) In

the absence of any co-effector (Figure 7.5 A), the migration for the standard argO

fragment (with ArgP binding site at middle) in complexes with the different ArgP

proteins were reproduced, so that the complexes with native ArgP and ArgPd-P274S

migrated the slowest and fastest, respectively (compare encircled bands in the different

lanes labelled M). (ii) These migration differences between the different ArgP proteins

were largely abolished for argO fragments in which the protein binding sites were

located near the downstream or upstream ends, so that all complexes now migrated at the

faster rate (see lanes labelled D and U, respectively). These data therefore strongly


137

suggest that argO DNA is bent upon native ArgP binding, and that bending is

considerably less in case of binding by the three ArgPd variant proteins.

Figure 7.5 B and Figure 7.5 C shows similar binding assays performed in the

presence of co-effector Arg and Lys at 0.1 mM concentration. These figures reveal that

(i) unlike for other reported LTTRs, addition of the positive co-effector Arg did not cause

decreased DNA bending by native ArgP (compare lanes M in Figure 7.5 A and Figure

7.5 B for WT); (ii) neither did addition of the negative co-effector Lys result in increased

DNA bending by native ArgP (compare lanes M in Figure 7.5 A and Figure 7.5 C for

WT); and (iii) among the ArgPd-DNA complexes, the migration profiles were more or

less similar to that in the condition without co-effector. However, one significant finding

was that there was a speeding up of the argO complex with the -S94L variant (compare

lanes M in Figure 7.5 A and Figure 7.5 B for -S94L) but not that with native

Figure 7.5 EMSAs with native ArgP (WT) or its variants -S94L, -P274S and -P108S at 100 nM and the

argO templates D, M and U (with LTTR-binding site at the downstream end, middle and upstream end of

the fragments, respectively and as illustrated adjoining panel A). The experiments were done in absence

(Nil) or presence of co-effectors Arg or Lys at 0.1 mM concentration. Open arrowhead in each panel

denotes the unbound DNA probe, and bands corresponding to binary complexes of protein with template M

are circled.


138

ArgP in presence of Arg, suggesting that the bend induced only by the former is reversed

upon Arg addition (at least at the concentration used in the experiments).

7.2.6 DNA bending studies with native ArgP and ArgPd proteins -S94L, -P274S,

-P108S at lysE

Figure 7.6 EMSAs with native ArgP (WT) or its variants -S94L, -P274S and -P108S at 400 nM and the

lysE templates D, M and U (with LTTR-binding site at the downstream end, middle and upstream end of

the fragments, respectively). The experiments were done in absence (Nil) or presence of co-effectors Arg

or Lys at 0.1 mM concentration. Open arrowhead in each panel denotes the unbound DNA probe, and

bands corresponding to binary complexes of protein with template M are circled.

Similar binding experiments were undertaken with respect to lysE using 400 nM

of purified ArgP and ArgPd

proteins; the three fragments (extent with respect to lysE

transcription start site, indicated in parentheses) D (268 to +21), M (188 to +101) and

U (88 to +201) were each 289-bp in length. They were generated by PCR amplication

from C. glutamicum ATCC 13032 genomic DNA using the respective primer pairs: 5’-

CTGCTTGCACAAGGACTTCACC-3’ and 5’-ACCTGTAATGAAGATTTCCAT-3’,


139

5’-TCGAGAGCTTTAACGCGCTGAC-3’ and 5’-CCTTCGCGCTTAATTCCTTGTT-

3’, 5’-CCAGTTGAATGGGGTTCATGA-3’ and 5’-

CACGATCGGCGCGGCATTGGAC-3’.

Figure 7.6 demonstrates that except under the condition of Arg addition, native

ArgP binding to lysE is associated with more pronounced DNA bending than is binding

of either of the ArgPd variants (compare lanes M of WT and of the ArgP

d variants). Arg

addition led to faster mobility of the complex of wild-type ArgP with the M DNA

fragment (compared to that in the absence of Arg), so that the M fragment complexes

with ArgP and the different ArgPd

proteins now exhibited similar mobilities.

7.3 Section C: Combined effect of LysG and ArgPd variants on lysE

expression

Since the typical LTTR is dimeric in solution and assembles on DNA as a dimer

of dimers or as higher-order oligomers to activate transcription (Maddocks and Oyston

2008, Momany and Neidle, 2012), we tested the effects of the combined presence of

LysG and ArgP or its variants on lysE regulation in E. coli.

For this, -Galactosidase assays were performed in argP (GJ9651) strain

derivatives carrying three plasmids namely, (i) pHYD2677 (lysE-lac fusion), (ii)

pHYD2676 (Para-lysG) or pBAD18 vector and (iii) argP+ or argP

d derivatives on

pCL1920 vector. The strains were grown in MA-glycerol arabinose and co-effectors Arg,

Lys and His were added at 1 mM concentration as free amino acids or as dipeptides.

Table 7.5 shows the results of these experiments.

It was found that activation of lysE transcription in presence of LysG and its co-

effectors Arg, Lys and His (added as their respective dipeptides with Ala) was unaffected

by native ArgP (that is, in the argP+

strain in comparison with that in argP).

Interestingly, however, strains in which LysG was co-expressed with any of the lysE

activation-proficient ArgPd

variants (-S94L, -P274S, or -P108S) displayed significantly

lower lysE expression than those with only one of the LTTRs present in certain growth

media (compare values of -S94L, -P274S, or -P108S in LysG and + LysG sections),

indicative of a mutual antagonism or reciprocal dominant negative effect. For example,

such mutual interference was observed for LysG with either ArgPd-S94L or ArgP

d-

P274S in cultures supplemented with either Arg-Ala or His-Ala, and LysG was also

dominant-negative over all three ArgPd variants in cultures without co-effector


140

Table 7.5 Antagonism between ArgPd variants and LysG for lysE-lac activation

a

LysG + LysG

argP

genotype

Nil Arg Arg-

Ala

Lys Lys-

Ala

His His-

Ala

Nil Arg Arg-

Ala

Lys Lys-

Ala

His His-

Ala

∆argP 25 27 27 24 24 29 24 21 24 155 100 115 96 116

argP+ 25 28 22 27 23 28 21 19 25 118 106 131 108 111

-S94L 95 111 97 106 109 120 93 21 21 19 91 110 55 56

-P274S 181 162 185 188 175 174 179 23 23 20 96 103 45 43

-P108S 45 51 40 31 20 46 39 19 23 106 114 104 111 105

a Values shown are -galactosidase specific activity (Miller units) obtained after growth of strains in MA-glycerol arabinose without (Nil) or with supplementation of

amino acids Arg, Lys, His or the corresponding dipeptides (with Ala) at 1 mM. Values indicative of dominant-negative effect of LysG are in bold, and those indicative

of mutual dominant negativity of LysG and ArgPd in bold italics.


141

supplementation. On the other hand, in cultures supplemented with Lys-Ala, lysE

expression in the combined presence of LysG and ArgPd

was similar to that with either

LTTR alone, and no antagonistic regulation was observed.

When free amino acids were used, His behaved like His-Ala in provoking the

mutual antagonistic effect, and likewise Lys behaved like Lys-Ala in activating lysE-lac

in presence of both LTTRs. With Arg supplementation, the results were indistinguishable

from those obtained in the unsupplemented cultures, which may be consistent with the

data described in Section 7.1.2 of this chapter and as shown in Table 7.3 that it is only the

Arg-containing dipeptide (and not Arg as free amino acid) which is proficient for

activating LysG in E. coli.

Discussion

The main results described in this chapter are the following: (i) C. glutamicum

LysG can activate its cognate target lysE in the heterologous milieu of E. coli; (ii) some

ArgPd variants of E. coli can activate C. glutamicum lysE whereas native LysG and ArgP

do not cross-regulate their non-cognate targets (argO and lysE, respectively) in vivo; and

(iii) binding of the ArgPd variant proteins to the regulatory regions of argO or lysE is

associated with a reduced angle of DNA bending compared to that obtained with native

ArgP. Each of these is further discussed below.

a) Reconstitution of C. glutamicum LysG-lysE regulation in E. coli

In the classical model for gene activation (Ptashne and Gann, 1997),

transcription factor binding to a target gene regulatory region is followed by RNAP

recruitment to enable productive transcription. The result showing the ability of C.

glutamicum LysG to activate its cognate target lysE in E. coli in presence of Lys, His or

Arg is remarkable for two reasons. Firstly, it demonstrates that a Gram-positive

transcription factor is successful in recruiting the Gram-negative RNA polymerase

enzyme to make appropriate promoter contacts for transcriptional activation. There have

been no earlier reports of in vivo transcriptional activation across the divide between

Gram-positive and Gram-negative bacteria which some estimates indicate were

established about two billion years ago (Feng et al., 1997; Pace 1997). Since

transcriptional activation requires high-fidelity interactions across macromolecular

interfaces, our results imply that such contacts (between LysG and RNAP) have been

conserved across a vast evolutionary distance. The LTTRs CatR and OccR from,


142

respectively, Pseudomonas putida and Agrobacterium tumefaciens have earlier been

shown to activate transcription with E. coli RNAP in vitro, that is, within the Gram-

negative kingdom itself (McFall et al., 1997; Wang et al., 1992). In this context, the

alternative explanation that ArgP and LysG are not orthologs that have diverged from a

single gene in the ancient common ancestor, but instead represent an example of

horizontal gene transfer (HGT) that occurred more recently in evolution was explored.

One way to distinguish between these possibilities is to compare an organism’s overall

GC content with that of the gene in question, since it is the discordance between the two

that is the often taken as a signature of HGT (Gogarten et al., 2002) Data presented in

Table 7.6 shows that the GC content of the gene encoding the closest relative of E. coli

ArgP in each of seven different bacteria more or less matches that for the entire genome

of the corresponding bacterium, suggesting that the genes are indeed orthologs rather

than outcomes of HGT.

Table 7.6 Percent GC content of genomes and argP orthologs of different bacteriaa

Organism Ortholog IDa %GC for gene %GC for genome

E. coli NP_417391.1 54.4 50.8

C. glutamicum EGV41297.1 55.2 53.8

M. tuberculosis NP_216501.1 65.0 65.6

Streptomyces

coelicolor

NP_631362.1 75.8 72.0

Klebsiella

pneumoniae

YP_002236622.1 59.7 56.9

Haemophilus

influenzae

ZP_01783604.1 33.4 38.0

Pseudomonas

aeruginosa

YP_001350270.1 71.5 64.4

a The ortholog ID is as listed in the NCBI protein database.

A few other instances have been reported of conserved interactions between

Gram-positive and Gram-negative bacteria, that is manifested as successful cross-

complementation of genes whose products (proteins or RNA) function within

macromolecular complexes; the examples include F0-F1 ATPase between Bacillus

megaterium and E. coli (Scarpetta et al., 1991), protein complexes involving YidC


143

between Streptococcus mutans and E. coli (Dong et al., 2008) or Era/Bex between

Bacillus subtilis and E. coli (Minkovsky et al., 2002), and the protein-RNA components

of RNase P between B. subtilis and E. coli (Wegscheid et al., 2006). In vitro

reconstitution of the 30S ribosome complex with protein subunits and rRNA inter-mixed

between E. coli and Bacillus stearothermophilus has also been described (Higo et al.,

1973; Nomura et al., 1968). However, several proteins of the E. coli and B. subtilis

divisome complexes do not productively interact with one another although they are

orthologous (Robichon et al., 2008), nor is the SecA homolog from either B. subtilis

(McNicholas et al., 1995) or Streptomyces lividans (Blanco et al., 1996) able to

participate in protein translocation in E. coli.

Secondly, the result showing the ability of C. glutamicum LysG to activate its

cognate target lysE in E. coli indicates that E. coli RNAP recognizes the C. glutamicum

lysE promoter. A previous study had found that Corynebacterial promoters of varying

similarity to the consensus E. coli promoter sequences and all recognized by the sigA

gene encoded principal sigma factor, are correctly recognized in E. coli (Patek et al.,

2003). The canonical 35 and 10 hexamers found in E. coli and Bacillus sp. are

TTGACA and TATAAT with a 14 to 17 bp intervening region. In the consensus C.

glutamicum promoters, the prominent feature is a conserved extended 10 region

tgngnTA(c/t)aaTgg, while the 35 region is much less conserved (Patek et al., 2003).

Analysis of the upstream regulatory region of lysE revealed CACGAT as a motif

analogous by position and sequence to 10, also 17 bp upstream to this sequence is a

putative 35 bearing the sequence TTTACT. While this 10 is 50% identical, the 35 is

67% identical to the E. coli consensus. Thus considering that the lysE promoter

sequences bear significant identity with those in E. coli and is also recognized by the

principal sigma factor (Patek et al., 2003), we had assumed this promoter may be

functional in E. coli.

b) Cross-regulation between ArgP-argO and LysG-lysE

Several examples of successful cross-regulation between different transcription

factors have been described earlier, including those between BenR-XylS of the AraC

family (Cowles et al., 2000; Jeffrey et al., 1992) XylR-DmpR of the NtrC family

(Fernandez et al., 1994); and CatR-ClcR and TfdR-TfdT-TcbR-ClcR of the LTTR family

of proteins (Parsek et al., 1994; Parsek et al., 1995; Leveau and van der Meer, 1996). In


144

all these cases, the target genes are involved in catabolism of aromatic compounds in

Pseudomonas species or related Gram-negative bacteria and are most often plasmid-

borne; hence it is unclear whether the different regulator proteins are paralogs or merely

orthologs that have been re-united through horizontal gene transfer (Gogarten et al.,

2002). Amongst other LTTRs, limited or no cross-regulation is observed between NocR

and OccR involved in catabolism of nopaline and octopine respectively in A. tumefaciens

(Kreusch et al., 1995); however, overexpressed GcvA (that regulates glycine cleavage) of

E. coli is able apparently to partially substitute for Citrobacter freundii AmpR in

regulation of the ampC gene encoding β-lactamase (Everett et al., 1995).

In this study, no cross-regulation between the native ArgP and LysG proteins for

their respective non-cognate targets lysE and argO was observed. For the LysG-argO

combination, the failure to activate is not because of an inability of LysG to recruit E.

coli RNAP, given that LysG activates lysE in E. coli. Hence it can be suggested that this

failure reflects a decreased binding affinity of LysG to the argO regulatory region.

Likewise, ArgP’s inability to cross-regulate lysE is at least in part explained by the high

Kd of protein binding to lysE, since the two ArgPd variants that were most effective for

activation (-P274S and -S94L) also exhibited improved binding affinities. The fact that

neither native protein exerts a dominant negative effect on the other’s activating ability in

E. coli (that is, LysG on lysE and ArgP on argO) would also suggest that inability to

cross-regulate is likely due to a defect in an early rather than late step in the process.

The ArgPd single amino acid substitution variants used here had originally been

selected to confer elevated argO expression (Celis 1999; Nandineni and Gowrishakar,

2004); the finding that some of them have also become proficient for cross-regulation of

lysE in vivo serves to strengthen the notion that ArgP-argO and LysG-lysE are indeed

orthologous transcription factor-target pairs which are both functionally and

evolutionarily related. The two ArgPd derivatives that were most effective for lysE

activation (-P274S, -S94L) are also those that conferred the highest constitutive levels of

argO expression, but whether this is just a coincidence remains to be determined.

Furthermore, an important distinction between native ArgP and LysG is that the former

fails to activate its target in presence of Lys, suggesting that the -P274S and -S94L ArgPd

variants are more akin to LysG in this regard.

The crystal structure of M. tuberculosis ArgP has been determined (Zhou et al.,

2010) and may be described with the structure of the LTTR CbnR of Ralstonia eutropha

(Muraoka et al., 2003) as being comprised of an N-terminal DNA binding domain (DBD)


145

Figure 7.7 Amino acid sequence alignment of CbnR of R. eutropha, ArgP of E. coli and LTTRs: AmpR

of C. freundi, NodD of R. leguminosarum, OxyR of E. coli and AphB of V. cholerae. The colored lines

below the sequence alignment table represent the domains in CbnR; the coloring scheme is as follows: red,

yellow, blue and green represent DNA-binding domain, the α4 linker helix, and RD-I and RD-II,

respectively. The hinge regions (hinges 1,2 and 3) are indicated below the colored line. Hinge 3 is

composed of two segments, hinge 3(a) and hinge 3(b). Numbering of residues is as those of CbnR. The

residues corresponding to the position of mutation in ArgP variants described in this study are shown in

red. Mutations described in the text with respect to AmpR, NodD, OxyR and AphB are indicated in blue in

the respective sequences.


146

followed sequentially by a linker helix and the C-terminal regulatory domain (RD)-I and

-II (with hinge regions 1, 2 and 3 located between (i) DBD and linker helix, (ii) linker

helix and RD-I, and (iii) RD-I and RD-II, respectively). Upon mapping of the seven

ArgPd substitutions (that render E. coli constitutive for argO expression) it was found

that none is in the DBD, A68V maps in the linker helix which is proposed to be involved

in establishing intra-dimer contacts, S94L, P108S, V144M, P274S, R295C map to RD-I

and P217L was in RD-II. Specifically, the three mutants shown to activate lysE localized

next to hinge 2 (ArgPd-S94L and ArgP

d-P108S) and hinge 3 (ArgP

d-P274S). Hinge 2 is

responsible for the conformational change between the compact and the extended forms

of the monomer, while hinge 3 is the crossover region between RD-I and RD-II that is

postulated to accommodate the regulatory ligand and make associated conformational

changes. Mutation in residues around hinge 2 with LTTRs NodD (residue 95), AmpR

(residue 102), AphB (residue 100) and OxyR (residue 100) also result in co-effector

independent phenotype in each case (Burn et al., 1989; McIver et al., 1989; Kullik et al.,

1995; Bartowsky and Normark, 1991; Taylor et al., 2012) (Figure 7.7). Thus these hinge

regions appear to be crucial for conferring the flexibility needed for a single LTTR to

recognize both the argO and lysE transcriptional targets.

c) Mutual dominant negativity between LysG and ArgPd variants for lysE

regulation

The data from experiments examining the effects of combined presence of LysG

and ArgPd variants on lysE expression in vivo indicate that both LysG on the one hand,

and ArgPd-S94L or -P274S on the other, mutually antagonize each other’s ability to

activate lysE in the Arg- or His-supplemented cultures. Furthermore, LysG prevents lysE

activation by the ArgPd proteins in media not supplemented with any co-effector. Our

findings suggest that mixed oligomers between ArgPd and LysG are being generated

under these conditions, at the level either of the dimer itself or of dimer of dimers upon

DNA binding, which are inactive for appropriate RNAP recruitment and gene activation.

The implication is that the homo-oligomers of LysG and ArgPd themselves activate lysE

by subtly different mechanisms that cannot be successfully integrated in the mixed

oligomers. In cultures with Lys supplementation, however, lysE activation was more or

less the same when LysG and the ArgPd variants were present either alone or together,

indicating that in this case gene regulation is mediated by a common or shared

mechanism; an alternative explanation is that the LysG and ArgPd protomers in their Lys-


147

bound states exclude one another and assemble only as homo-oligomers both in solution

and on DNA. Thus, although LysG mediates activation by all three amino acids of lysE

expression, it appears to do so by at least two different mechanisms that can be

distinguished by their interference or otherwise by the ArgPd proteins.

d) Target DNA bending upon binding by ArgP and its variants

As described in Chapter 1, for a number of LTTRs reduced DNA bending is

correlated with gene activation (Colyer and Kredich, 1996; Kullik et al., 1995; Akakura

and Winans, 2002; Kreusch et al., 1995; Wang et al., 1992; Kreusch et al., 1995; Parsek

et al., 1995; McFall et al., 1997; Dangel et al., 2005). Binding of the activating co-

effector causes a pronounced decrease in DNA bending and in the length of DNAse I

footprinted region. In this context, a sliding dimer model has been proposed (see Chapter

1 Section 1.2.6 and Figure 1.14). In this model, a non-activated LTTR tetramer binds to

the high affinity RBS site and to the proximal ABS’ subsite of the promoter with a high

angle bend. Activating co-effector induced conformational changes in the regulator

causes the regulator to slide to a more distal ABS’’ subsite, while still maintaining

binding to the RBS site, at the same time relaxing the angle of bending of the bound

DNA (Porrua et al., 2007; Monferrer et al., 2010).

However, in a few cases, such as MetR (Lorenz and Stauffer, 1995), DntR

(Smirnova et al., 2004) and TrpI (Pineiro et al., 1997), co-effector addition does not

affect protein induced DNA bending, and for NodD (Chen et al., 2005) the bending is in

fact increased in presence of the co-efffector. Studies with LTTR mutants such as those

for OxyR (Kullik et al., 1995), CysB (Colyer and Kredich, 1996), OccR (Akakura and

Winans, 2002) and CbbR (Dangel et al., 2005) indicate that constitutive activation can be

achieved by different mechanisms even for a single protein-target pair. Some variants

confer less DNA bending than their native counterparts, but others confer unchanged or

even increased bending. For OxyR, improved RNAP recruitment and a role for protein

conformational changes following its binding to DNA have also been suggested to

distinguish the constitutive mutants from the corresponding wild-type proteins (Kullik et

al., 1995). Binding to DNA can also dictate changes in LTTR oligomerization leading to

gene activation, as has been demonstrated for some mutants of the NAC protein (Rosario

et al., 2010).


148

In this context, the present studies indicate that native ArgP binding induces DNA

bending at both argO and lysE, and that the three active ArgPd variants tested confer

relaxation of bending (relative to native ArgP) at these target loci. Thus, the results are

broadly consistent with those for other LTTRs on DNA bending induced by native

protein binding and its alteration with substitutions conferring constitutivity. These

results may be corroborated by more direct read-outs such as AFM-imaging or single

molecule FRET. At lysE, the ArgPd variants also show increased affinity of binding that

is proportionate to the magnitude by which they activate expression in vivo, suggestive of

an induced fit mechanism.

One possible discrepancy that remains to be explained is the finding that bending

by native ArgP at argO is unaffected by the activating co-effector Arg. An examination

of the ArgP bound argO regulatory region does reveal probable ABS’’ and ABS’

sequences at sites previously described for other LTTRs (Figure 7.8).

Figure 7.8 Probable RBS, ABS’’ and ABS’ sites on the ArgP DNase I footprinted region at argO. These

sites are proposed on the basis of sequence comparison with ArgP binding sequences at lysP and dapB

(Chapter 4, figure 4.6) and on the co-ordinates of the ABS’ sites in other LTTR targets. The –35 sequence

is underlined.

However, previous studies have shown that the DNase I footprint of ArgP at

argO is also unaffected by co-effector (Arg or Lys) addition (Laishram and

Gowrishankar, 2007), thus contradicting a probable explanation of activation through the

‘sliding dimer’ hypothesis. This suggests that either (i) the activating co-effector Arg acts

by mechanisms other than (or in addition to) that related to ArgP-induced DNA bending;

or (ii) although the concentration of Arg used (0.1 mM) is in accordance with the highest

concentration of co-effectors used in the case of other LTTRs for similar experiments,

this concentration may not be sufficient to elicit sustained, lower angle DNA bends by

the native ArgP at argO.

Genes activated at least 2-fold by ArgP Appendix I

151

Appendix I: Genes activated at least 2-fold in argP+/argP

d compared to ΔargP

Gene

name

Fold

difference Description

ygiL 7.6 Predicted fimbrial-like adhesin protein

glpB 6.5 sn-glycerol-3-phosphate dehydrogenase (anaerobic), membrane

anchor subunit

glpQ 6.1 Periplasmic glycerophosphodiester phosphodiesterase

glpC 5.9 sn-glycerol-3-phosphate dehydrogenase (anaerobic), small

subunit

glpA 5.9 sn-glycerol-3-phosphate dehydrogenase (anaerobic), large

subunit, FAD/NAD(P)-binding

argP 5.8 DNA-binding transcriptional activator, replication initiation

inhibitor

ibpB 5.2 heat shock chaperone

glpD 4.9 sn-glycerol-3-phosphate dehydrogenase, aerobic, FAD/NAD(P)-

binding

tnaA 4.9 tryptophanase/L-cysteine desulfhydrase, PLP-dependent

atoS 4.7 Sensory histidine kinase in two-component regulatory system

with AtoC

glpK 4.6 Glycerol kinase

ytfQ 4.5 predicted sugar transporter subunit: periplasmic-binding

component of ABC superfamily

ygeV 4.3 predicted DNA-binding transcriptional regulator

dctA 3.9 C4-dicarboxylic acid, orotate and citrate transporter

ytfR 3.8 predicted sugar transporter subunit: ATP-binding component of

ABC superfamily

glpT 3.8 sn-glycerol-3-phosphate transporter

yqeB 3.8 conserved protein with NAD(P)-binding Rossman fold

sgcR 3.8 KpLE2 phage-like element; predicted DNA-binding

transcriptional regulator

cstA 3.7 carbon starvation protein

garR 3.7 Tartronate semialdehyde reductase

yghW 3.6 predicted protein


152

Gene

name

Fold


mglA 3.6 fused methyl-galactoside transporter subunits of ABC

superfamily:ATP-binding components

tdcB 3.5 Catabolic threonine dehydratase, PLP-dependent

ibpA 3.4 heat shock chaperone

garL 3.3 alpha-dehydro-beta-deoxy-D-glucarate aldolase

garK 3.2 Glyceratekinase I

fucO 3.2 L-1,2-propanediol oxidoreductase

ygfJ 3.2 conserved protein

ycgB 3.2 conserved protein

yniA 3.2 Predicted phosphotransferase/kinase

garP 3.2 predicted (D)-galactarate transporter

ydcS 3.1 Predicted spermidine/putrescine transporter subunit

clpB 3.1 Protein disaggregation chaperone

bssR 3.0 conserved protein

lsrK 2.9 autoinducer-2 (AI-2) kinase

atoC 2.9 fused response regulator of ato operon, in two-component system

with AtoS: response regulator/sigma54 interaction protein

ptsA 2.9 fused predicted PTS enzymes: Hpr component/enzyme I

component/enzyme IIA component

yeiT 2.9 Predicted oxidoreductase

hyaC 2.9 hydrogenase 1, b-type cytochrome subunit

fumC 2.8 Fumarate hydratase (fumarase C),aerobic Class II

fumA 2.8 Fumarate hydratase (fumarase A), aerobic Class I

glgC 2.8 glucose-1-phosphate adenylyl transferase

yjfF 2.8 predicted sugar transporter subunit

araG 2.8 fused L-arabinose transporter subunits of ABC superfamily:

ATP-binding components

ytfT 2.7 predicted sugar transporter: membrane component of ABC family


153

Gene

name

Fold


dapB 2.7 Dihydrodipicolinate reductase

hyaD 2.7 protein involved in processing of HyaA and HyaB proteins

glgS 2.7 predicted glycogen synthesis protein

lsrB 2.7 AI2 transporter

ydiR 2.6 predicted electron transfer flavoprotein, FAD-binding

ybaE 2.6 predicted transporter subunit: periplasmic-binding component of

ABC superfamily

pck 2.6 Phosphoenol pyruvate carboxykinase

yccJ 2.6 predicted protein

wrbA 2.6 Predicted flavoprotein in Trp regulation

yraK 2.6 Predicted fimbrial-like adhesin protein

araF 2.5 L-arabinose transporter subunit

ymgF 2.5 predicted protein

yeiA 2.5 Predicted oxidoreductase

ygcB 2.5 Conserved protein, member of DEAD box family

araB 2.4 L-ribulokinase

rhsA 2.4 rhsA element core protein RshA

lsrG 2.4 autoinducer-2 (AI-2) modifying protein LsrG

hha 2.4 Modulator of gene expression, with H-NS

ybaJ 2.4 predicted protein

cpxP 2.4 periplasmic protein combats stress

melR 2.4 DNA-binding transcriptional dual regulator

aldA 2.4 Aldehydedehydrogenase A, NAD-linked

hyaE 2.4 protein involved in processing of HyaA and HyaB proteins

yejG 2.4 predicted protein

yfaW 2.4 Predicted enolase

argO 2.3 arginine transporter


154

Gene

name

Fold


lysP 2.3 lysine transporter

glgB 2.3 1,4-alpha-glucan branching enzyme

yfiQ 2.3 fused predicted acyl-CoA synthetase: NAD(P)-binding

subunit/ATP-binding subunit

yeaH 2.3 conserved protein

uspF 2.3 stress-induced protein, ATP-binding protein

sdhC 2.3 Succinate dehydrogenase, membrane subunit, binds cytochrome

b556

ydcJ 2.3 Conserved protein

tnaC 2.2 tryptophanase leader peptide

ftnB 2.2 Predictedferritin-like protein

lsrF 2.2 putative autoinducer-2 (AI-2) aldolase

fadI 2.2 beta-ketoacyl-CoA thiolase, anaerobic, subunit

yqeC 2.2 conserved protein

yjfN 2.2 predicted protein

ybdD 2.2 conserved protein

glgA 2.1 Glycogen synthase

ypfM 2.1 hypothetical protein b4606

ydcH 2.1 predicted protein

frdD 2.1 Fumarate reductase (anaerobic), membrane anchor subunit

modA 2.1 Molybdate transporter subunit

yggE 2.1 Conserved protein

yqeF 2.0 Predicted acyl transferase

yjfO 2.0 Conserved protein

srlE 2.0 glucitol/sorbitol-specific enzyme IIB component of PTS

ygdH 2.0 conserved protein

ycbJ 1.9 conserved protein


155

Gene

name

Fold


tdcC 1.9 L-threonine/L-serine transporter

hyaB 1.9 hydrogenase 1, large subunit

glpF 1.9 glycerol facilitator

sgcQ 1.9 KpLE2 phage-like element; predicted nucleoside triphosphatase

yfcH 1.9 conserved protein with NAD(P)-binding Rossmann-fold domain

mglB 1.9 methyl-galactoside transporter subunit

sgcC 1.9 KpLE2 phage-like element; predicted phospho-transferase

enzyme IIC component

ydhV 1.9 Predicted oxidoreductase

hsdR 1.9 endonuclease R

bssS 1.9 predicted protein

ydcA 1.9 predicted protein

rihB 1.9 Ribonucleoside hydrolase 2

gudD 1.9 (D)-glucarate dehydratase 1

lysC 1.9 aspartokinase III

ybeL 1.9 conserved protein

lsrR 1.9 lsr operon transcriptional repressor

srlB 1.9 glucitol/sorbitol-specific enzyme IIA component of PTS

lsrA 1.9 fused AI2 transporter subunits of ABC superfamily: ATP-

bindingcomponents

yaiY 1.9 predicted inner membrane protein

ugpB 1.9 Glycerol-3-phosphate transporter subunit

sdhD 1.9 Succinatedehydrogenase, membrane subunit, binds cytochrome

b556

glcC 1.9 DNA-binding transcriptional dual regulator, glycolate-binding

yiaY 1.8 predicted Fe-containing alcohol dehydrogenase

uspB 1.8 predicted universal stress (ethanol tolerance) protein B

sgcE 1.8 KpLE2 phage-like element; predicted epimerase


156

Gene

name

Fold


acs 1.8 acetyl-CoA synthetase

ylaC 1.8 predicted inner membrane protein

glgX 1.8 Glycogen debranching enzyme

hyaA 1.8 hydrogenase 1, small subunit

rbsB 1.8 D-ribose transporter subunit

ompW 1.8 outer membrane protein W

ucpA 1.8 Predicted oxidoredutase, sulfate metabolism protein

glgP 1.8 Glycogen phosphorylase

garD 1.8 (D)-galactarate dehydrogenase

sgcA 1.8 KpLE2 phage-like element; predicted phospho transferase

enzyme IIA component

ydeN 1.8 conserved protein

yhhA 1.8 conserved protein

bdm 1.8 Biofilm-dependent modulation protein

ydhX 1.8 predicted 4Fe-4S ferridoxin-type protein

hycF 1.8 Formate hydrogen lyase complex iron-sulfur protein

galS 1.8 DNA-binding transcriptional repressor

alpA 1.7 CP4-57 prophage; DNA-binding transcriptional activator

frc 1.7 Formyl-CoA transferase, NAD(P)-binding

yrhD 1.7 hypothetical protein b4612

fxsA 1.7 inner membrane protein

ydcT 1.7 Predicted spermidine/putrescine transporter subunit

dnaK 1.7 Chaperone Hsp70, co-chaperone with DnaJ

xylF 1.7 D-xylose transporter subunit

ydjF 1.7 predicted DNA-binding transcriptional regulator

frdC 1.7 Fumarate reductase (anaerobic), membrane anchor subunit

ydhW 1.7 predicted protein


157

Gene

name

Fold


ygcO 1.7 predicted 4Fe-4S cluster-containing protein

macB 1.7 Fused macrolide transporter subunits of ABC superfamily: ATP-

binding component/membrane component

rbsA 1.7 fused D-ribose transporter subunits of ABC superfamily: ATP-

binding components

yidE 1.7 predicted transporter

sgcB 1.7 predicted enzyme IIB component of PTS

allS 1.7 DNA-binding transcriptional activator of the all Doperon

caiF 1.7 DNA-binding transcriptional activator

zraP 1.7 Zn-binding periplasmic protein

arnT 1.7 4-amino-4-deoxy-L-arabinose transferase

bolA 1.6 regulator of penicillin binding proteins and beta-lactamase

transcription (morphogene)

fumB 1.6 anaerobic class I fumarate hydratase (fumarase B)

malF 1.6 maltose transporter subunit

frdB 1.6 Fumarate reductase (anaerobic), Fe-S subunit

ldrD 1.6 toxic polypeptide, small

yagX 1.6 predicted aromatic compound dioxygenase

malM 1.6 Maltose regulon periplasmic protein

fadD 1.6 acyl-CoA synthetase (long-chain-fatty-acid—CoAligase)

idnK 1.6 D-gluconate kinase, thermosensitive

hspQ 1.6 DNA-binding protein, hemimethylated

tdcD 1.6 Propionatekinase/acetate kinase C, anaerobic

ybjD 1.6 conserved protein with nucleoside triphosphatehydrolase domain

yqeG 1.5 predicted transporter

kdpF 1.5 Potassium ion accessory

transporter subunit

rbsC 1.5 D-ribose transporter subunit


158

Gene

name

Fold


adiY 1.5 DNA-binding transcriptional activator

cysQ 1.5 PAPS (adenosine 3'-phosphate 5'-phosphosulfate) 3'(2'),5'-

bisphosphate nucleotidase

gutM 1.5 DNA-binding transcriptional activator of glucitol operon

yjjM 1.5 predicted DNA-binding transcriptional regulator

feaR 1.5 DNA-binding transcriptional dual regulator

yebE 1.5 conserved protein

hyaF 1.5 Protein involved in nickel incorporation into hydrogenase-1

proteins

ydaS 1.5 Rac prophage; predicted DNA-binding transcriptional regulator

aldB 1.5 Aldehydedehydrogenase B

mhpR 1.5 DNA-binding transcriptional activator, 3HPP-binding

mtlA 1.5 Fused mannitol-specific PTS enzymes: IIA components/IIB

components/IIC components

sgcX 1.5 KpLE2 phage-like element; predicted endoglucanase with Zn-

dependent exopeptidase domain

uspE 1.5 stress-induced protein

hybA 1.5 hydrogenase 2 4Fe-4S ferredoxin-type component

yibA 1.5 lyase containing HEAT-repeat

ydhM 1.4 predicted DNA-binding transcriptional regulator

deoC 1.4 2-deoxyribose-5-phosphate aldolase, NAD(P)-linked

yaaW 1.4 conserved protein

tnaB 1.4 tryptophan transporter of low affinity

yeaC 1.4 conserved protein

yneM 1.4 hypothetical protein b4599

ydiL 1.4 conserved protein

ybiI 1.4 conserved protein

hycG 1.4 hydrogenase 3 and formate hydrogenase complex, HycG subunit


159

Gene

name

Fold


araH 1.4 fused L-arabinose transporter subunits of ABC superfamily:

membrane components

aspA 1.4 aspartate ammonia-lyase

yjhI 1.4 KpLE2 phage-like element; predicted DNA-binding

transcriptional regulator

yhcO 1.4 Predicted barnase inhibitor

hypB 1.4 GTP hydrolase involved in nickel liganding into hydrogenases

ybdJ 1.4 predicted inner membrane protein

ivbL 1.4 ilvB operon leader peptide

sufB 1.4 component of SufBCD complex

yecT 1.4 predicted protein

mtlD 1.4 mannitol-1-phosphate dehydrogenase, NAD(P)-binding

dadA 1.4 D-amino acid dehydrogenase

ybdK 1.4 gamma-glutamyl:cysteineligase

yeaG 1.4 conserved protein with nucleoside triphosphatehydrolase domain

mtlR 1.4 DNA-binding repressor

citC 1.4 Citrate lyase synthetase

ybiA 1.3 conserved protein

mokC 1.3 regulatory protein for HokC, overlaps CDS of hokC

lsrC 1.3 AI2 transporter

yrhB 1.3 predicted protein

ygcL 1.3 predicted protein

hcaR 1.3 DNA-binding transcriptional activator of 3-phenylpropionic acid

catabolism

modB 1.3 molybdate transporter subunit

maeB 1.3 Fused malic enzyme predicted oxidoreductase/predicted

phosphotransacetylase


160

Gene

name

Fold

difference

Description

msrB 1.3 Methionine sulfoxide reductase B

eptB 1.3 predicted metal dependent hydrolase

yibT 1.3 predicted protein

ygcN 1.3 Predicted oxidoreductase with FAD/NAD(P)-binding domain

ybaT 1.3 predicted transporter

hycE 1.3 Hydrogenase 3, large subunit

dcuB 1.3 C4-dicarboxylate antiporter

smf 1.3 conserved protein

uspC 1.3 universal stress protein

yeeN 1.3 conserved protein

gdhA 1.3 Glutamatedehydrogenase, NADP-specific

hypC 1.3 protein required for maturation of hydrogenases 1 and 3

glcB 1.3 Malatesynthase G

frdA 1.3 Fumarate reductase (anaerobic) catalytic and NAD/flavoprotein

subunit

tisB 1.3 lexA-regulated toxic peptide

lamB 1.3 maltose outer membrane porin (maltoporin)

gspG 1.3 Pseudopilin, cryptic, general secretion pathway

gudX 1.3 Predicted glucarate dehydratase

aidB 1.2 Isovaleryl CoA dehydrogenase

yfjY 1.2 CP4-57 prophage; predicted DNA repair protein

yjgL 1.2 predicted protein

ycgY 1.2 predicted protein

yacL 1.2 conserved protein

lsrD 1.2 AI2 transporter

raiA 1.2 cold shock protein associated with 30S ribosomal subunit

fre 1.2 Flavin reductase


161

Gene

name

Fold


ydcU 1.2 Predicted spermidine/putrescine transporter subunit

yojO 1.2 hypothetical protein b4604

yqiA 1.2 predicted esterase

yehA 1.2 Predicted fimbrial-like adhesin protein

tdcA 1.2 DNA-binding transcriptional activator

gltS 1.2 Glutamate transporter

ybhF 1.2 fused predicted transporter subunits of ABC superfamily: ATP-

binding components

pdhR 1.2 DNA-binding transcriptional dual regulator

yraJ 1.2 predicted outer membrane protein

yfgF 1.2 predicted inner membrane protein

ymfL 1.2 e14 prophage; predicted DNA-binding transcriptional regulator

gspD 1.2 General secretory pathway component, cryptic

ygcP 1.2 predicted anti-terminator regulatory protein

ade 1.2 cryptic adenine deaminase

wcaF 1.2 Predicted acyltransferase

rhaB 1.2 rhamnulokinase

dedD 1.2 conserved protein

yibI 1.2 predicted inner membrane protein

rcsA 1.2 DNA-binding transcriptional activator, co-regulator with RcsB

ydgD 1.2 predicted peptidase

wcaD 1.2 Predicted colanic acid polymerase

ydiB 1.2 quinate/shikimate 5-dehydrogenase, NAD(P)-binding

ydaU 1.2 Rac prophage; conserved protein

ubiD 1.2 3-octaprenyl-4-hydroxybenzoate decarboxylase

ydcI 1.2 predicted DNA-binding transcriptional regulator

aslA 1.2 Acryl sulfatase-like enzyme


162

Gene

name

Fold


trxC 1.2 thioredoxin 2

hokC 1.2 toxic membrane protein, small

malG 1.2 maltose transporter subunit

clpA 1.2 ATPase and specificity subunit of ClpA-ClpP ATP-dependent

serine protease, chaperone activity

yieE 1.1 Predicted phosphopantetheinyl transferase

yihT 1.1 Predicted aldolase

yebW 1.1 predicted protein

hybO 1.1 hydrogenase 2, small subunit

rhsB 1.1 rhsB element core protein RshB

arcA 1.1 DNA-binding response regulator in two-component regulatory

system with ArcB or CpxA

ygjR 1.1 predicted NAD(P)-binding dehydrogenase

ykfM 1.1 hypothetical protein, no homologs

ychH 1.1 predicted inner membrane protein

hybD 1.1 predicted maturation element for hydrogenase 2

uspG 1.1 universal stress protein UP12

asd 1.1 aspartate-semialdehyde dehydrogenase, NAD(P)-binding

zraS 1.1 Sensory histidine kinase in two-component regulatory system

with ZraR

ybhH 1.1 conserved protein

yfdP 1.1 CPS-53 (KpLE1) prophage; predicted protein

ybbC 1.1 Predicted protein

yhcF 1.1 predicted transcriptional regulator

ycaP 1.1 Conserved inner membrane protein

citD 1.1 Citrate lyase, acyl carrier (gamma) subunit

ydaM 1.1 Predicted diguanylate cyclase, GGDEF domain signalling protein

hycH 1.1 protein required for maturation of hydrogenase 3


163

Gene

name

Fold


malQ 1.1 4-alpha-glucanotransferase (amylomaltase)

gltI 1.1 Glutamate and aspartate transporter subunit

lyx 1.1 L-xylulose kinase

sdhA 1.1 Succinate dehydrogenase, flavoprotein subunit

yicJ 1.1 predicted transporter

ycbB 1.1 Predicted carboxypeptidase

yadI 1.1 predicted PTS Enzyme IIA

yadM 1.1 Predicted fimbrial-like adhesin protein

abgT 1.1 predicted cryptic aminobenzoyl-glutamate transporter

yedP 1.1 Conserved protein

tdcR 1.1 DNA-binding transcriptional activator

sfmA 1.1 Predicted fimbrial-like adhesin protein

yggG 1.1 predicted peptidase

dgsA 1.1 DNA-binding transcriptional repressor

ymgE 1.1 predicted inner membrane protein

yodD 1.1 predicted protein

ygcG 1.1 predicted protein

rhaD 1.1 rhamnulose-1-phosphate aldolase

yqgD 1.1 predicted inner membrane protein

ybhM 1.1 Conserved inner membrane protein

ldrC 1.1 toxic polypeptide, small

citX 1.0 apo-citrate lyase phosphoribosyl-dephospho-CoA transferase

ydhT 1.0 conserved protein

yhfL 1.0 conserved secreted peptide

srlA 1.0 glucitol/sorbitol-specific enzyme IIC component of PTS

fliC 1.0 flagellar filament structural protein (flagellin)


164

Gene

name

Fold


uspD 1.0 stress-induced protein

cspD 1.0 cold shock protein homolog

folC 1.0 Bifunctional folyl polyglutamate synthase/ dihydrofolate synthase

argT 1.0 lysine/arginine/ornithine transporter subunit

ydhI 1.0 Predicted inner membrane protein

yfgG 1.0 Predicted protein

fdoG 1.0 Formate dehydrogenase-O, large subunit

yohC 1.0 Predicted inner membrane protein

ychN 1.0 conserved protein

yejA 1.0 Predicted oligopeptide transporter subunit

sfsA 1.0 Predicted DNA-binding transcriptional regulator

ulaG 1.0 predicted L-ascorbate 6-phosphate lactonase

yliI 1.0 Predicteddehydrogenase

fsaB 1.0 fructose-6-phosphate aldolase 2

yjiS 1.0 Conserved protein

nagE 1.0 fused N-acetyl glucosamine specific PTS enzyme: IIC, IIB , and

IIA components

fruR 1.0 DNA-binding transcriptional dual regulator

yabI 1.0 Conserved inner membrane protein

malP 1.0 Maltodextrin phosphorylase

ydaE 1.0 Rac prophage; conserved protein

ansP 1.0 L-asparagine transporter

Genes activated at least 2-fold in ΔargP Appendix II

165

Appendix II: Genes activated at least 2-fold in ΔargP compared to argP+/argP

d

Gene

name

Fold


glnK 4.5 nitrogen assimilation regulatory protein for GlnL, GlnE, and

AmtB

yhjX 3.4 predicted transporter

metF 3.3 5,10-methylenetetrahydrofolate reductase

tppB 3.3 predicted transporter

pyrC 3.3 dihydro-orotase

narK 3.2 nitrate/nitrite transporter

metN 3 DL-methionine transporter subunit

metI 2.9 DL-methionine transporter subunit

ydjN 2.8 predicted transporter

yeeD 2.8 conserved protein

yciW 2.8 predicted oxidoreductase

cysP 2.7 thiosulfate transporter subunit

metA 2.6 homoserine O-transsuccinylase

mhpF 2.6 acetaldehyde-CoA dehydrogenase II, NAD-binding

deaD 2.6 ATP-dependent RNA helicase

upp 2.6 uracil phosphoribosyltransferase

metK 2.6 methionine adenosyltransferase 1

moeB 2.5 molybdopterin synthase sulfurylase

glnQ 2.5 glutamine transporter subunit

asnA 2.5 asparagine synthetase A

napB 2.4 nitrate reductase, small, cytochrome C550 subunit,

periplasmic

cysD 2.4 sulfate adenylyltransferase, subunit 2

prs 2.4 phosphoribosylpyrophosphate synthase

focA 2.4 formate transporter

purU 2.4 Formyltetrahydrofolate hydrolase


166

Gene

name

Fold


guaA 2.3 GMP synthetase (glutamine aminotransferase)

cysJ 2.2 sulfite reductase, alpha subunit, flavoprotein

ilvA 2.2 threonine deaminase

cysB 2.2 DNA-binding transcriptional dual regulator, O-acetyl-L-

serine-binding

napA 2.2 nitrate reductase, periplasmic, large subunit

yfgB 2.2 predicted enzyme

narL 2.2 DNA-binding response regulator in two-component regulatory

system with NarX (or NarQ)

aroH 2.2 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase,

tryptophan repressible

pyrD 2.2 Dihydro-orotate oxidase, FMN-linked

metR 2.2 DNA-binding transcriptional activator, homocysteine-binding

lpxA 2.2 UDP-N-acetylglucosamine acetyltransferase

ycaD 2.2 predicted transporter

lnt 2.2 apolipoprotein N-acyltransferase

glnA 2.2 glutamine synthetase

ybjX 2.2 conserved protein

glnP 2.1 glutamine transporter subunit

rimM 2.1 16S rRNA processing protein

purK 2.1 N5-carboxyaminoimidazole ribonucleotide synthase

cysS 2.1 cysteinyl-tRNA synthetase

cysH 2.1 3'-phosphoadenosine 5'-phosphosulfate reductase

purM 2.1 Phosphoribosyl amino imidazole synthetase

ybiS 2.1 conserved protein

cysU 2.1 sulfate/thiosulfate transporter subunit

metB 2.1 cystathionine gamma-synthase, PLP-dependent

prmC 2.1 N5-glutamine methyltransferase, modifies release factors RF-

1 and RF-2


167

Gene

name

Fold


rhlE 2.1 RNA helicase

yohK 2 predicted inner membrane protein

lipB 2 lipoyl-protein ligase

cysI 2 sulfite reductase, beta subunit, NAD(P)-binding, heme-

binding

yohJ 2 conserved inner membrane protein

thiI 2 Sulfur transferase required for thiamine and 4-thiouridine

biosynthesis

yebZ 2 predicted inner membrane protein

yciT 2 predicted DNA-binding transcriptional regulator

gltP 2 glutamate/aspartate:proton symporter

dinI 2 DNA damage-inducible protein I

prmB 2 N5-glutamine methyltransferase

narJ 2 molybdenum-cofactor-assembly chaperone subunit (delta

subunit) of nitrate reductase 1

ycdO 2 conserved protein

ychF 1.9 predicted GTP-binding protein

gltX 1.9 glutamyl-tRNA synthetase

yggT 1.9 predicted inner membrane protein

carB 1.9 carbamoyl-phosphate synthase large subunit

msbA 1.9 fused lipid transporter subunits of ABC superfamily:

membrane component/ATP-binding component

yfaY 1.9 conserved protein

ybhC 1.9 predicted pectinesterase

ybgF 1.9 predicted protein

purE 1.9 N5-carboxyaminoimidazole ribonucleotide mutase

folD 1.9 bifunctional 5,10-methylene-tetrahydrofolate dehydrogenase/

5,10-methylene-tetrahydrofolate cyclohydrolase

ycdY 1.9 conserved protein


168

Gene

name

Fold


amiC 1.9 N-acetylmuramoyl-L-alanine amidase

yagJ 1.9 CP4-6 prophage; predicted protein

cysC 1.9 adenosine 5'-phosphosulfate kinase

shiA 1.9 shikimate transporter

mtr 1.9 tryptophan transporter of high affinity

ubiG 1.9 bifunctional 3-demethylubiquinone-9 3-methyltransferase/ 2-

octaprenyl-6-hydroxy phenol methylase

sbp 1.9 sulfate transporter subunit

exoX 1.9 DNA exonuclease X

purR 1.9 DNA-binding transcriptional repressor, hypoxanthine-binding

ddlA 1.9 D-alanine-D-alanine ligase A

mprA 1.9 DNA-binding transcriptional repressor of microcin B17

synthesis and multidrug efflux

napC 1.9 nitrate reductase, cytochrome c-type, periplasmic

sohB 1.9 predicted inner membrane peptidase

yggW 1.9 predicted oxidoreductase

rpsJ 1.9 30S ribosomal subunit protein S10

rfbD 1.9 dTDP-4-dehydrorhamnose reductase subunit, NAD(P)-

binding, of dTDP-L-rhamnose synthase

fabF 1.8 3-oxoacyl-ACP-synthii-monomer

cspA 1.8 major cold shock protein

yejM 1.8 predicted hydrolase, inner membrane

narI 1.8 nitrate reductase 1, gamma (cytochrome b(NR)) subunit

mnmA 1.8 tRNA (5-methylaminomethyl-2-thiouridylate)-

methyltransferase

ilvC 1.8 ketol-acid reductoisomerase, NAD(P)-binding

lysS 1.8 lysine tRNA synthetase, constitutive

ycgM 1.8 predicted isomerase/hydrolase

glyA 1.8 serine hydroxymethyltransferase

dapA 1.8 dihydrodipicolinate synthase

http://biocyc.org/ECOLI/substring-search?type=NIL&object=3-OXOACYL-ACP-SYNTHII-MONOMER


169

Gene

name

Fold


dfp 1.8 fused 4'-phosphopantothenoylcysteine decarboxylase/phospho

pantothenoyl cysteine synthetase, FMN-binding

aroC 1.8 chorismate synthase

apt 1.8 Adenine phosphoribosyl transferase

rplS 1.8 50S ribosomal subunit protein L19

lolB 1.8 chaperone for lipoproteins

trmD 1.8 tRNA (guanine-1-)-methyltransferase

cysA 1.8 sulfate/thiosulfate transporter subunit

mhpD 1.8 2-keto-4-pentenoate hydratase

tyrB 1.8 Tyrosine aminotransferase, tyrosine-repressible, PLP-

dependent

ispF 1.8 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase

chaA 1.8 Calcium/sodium:proton antiporter

guaC 1.8 GMP reductase

cysN 1.8 sulfate adenylyltransferase, subunit 1

purB 1.8 adenylosuccinate lyase

glnH 1.8 glutamine transporter subunit

hemA 1.8 glutamyl tRNA reductase

proS 1.8 prolyl-tRNA synthetase

ilvD 1.8 dihydroxyacid dehydratase

pncB 1.8 nicotinate phosphoribosyl transferase

rho 1.8 transcription termination factor

modF 1.8 fused molybdate transporter subunits of ABC superfamily:


asnB 1.8 asparagine synthetase B

ycdU 1.8 predicted inner membrane protein

ispB 1.8 octaprenyl diphosphate synthase

ycbX 1.8 predicted 2Fe-2S cluster-containing protein

narX 1.8 Sensory histidine kinase in two-component regulatory system

with NarL


170

Gene

name

Fold


ynfM 1.8 predicted transporter

prfB 1.8 peptide chain release factor RF-2

rnb 1.8 ribonuclease II

rstB 1.7 Sensory histidine kinase in two-component regulatory system

with RstA

potA 1.7 polyamine transporter subunit

purN 1.7 Phosphoribosyl glycinamide formyl transferase 1

rplC 1.7 50S ribosomal subunit protein L3

adk 1.7 adenylate kinase

miaB 1.7 isopentenyl-adenosine A37 tRNA methylthiolase

rfbB 1.7 dTDP-glucose 4,6 dehydratase, NAD(P)-binding

rhtC 1.7 threonine efflux system

mdoB 1.7 phosphoglycerol transferase I

yedD 1.7 predicted protein

kdsB 1.7 3-deoxy-manno-octulosonate cytidylyl transferase

prfA 1.7 peptide chain release factor RF-1

rhlB 1.7 ATP-dependent RNA helicase

nupC 1.7 nucleoside (except guanosine) transporter

pyrB 1.7 aspartate carbamoyl transferase, catalytic subunit

metQ 1.7 DL-methionine transporter subunit

ribC 1.7 riboflavin synthase, alpha subunit

glnL 1.7 Sensory histidine kinase in two-component regulatory system

with GlnG

glnB 1.7 regulatory protein P-II for glutamine synthetase

rluF 1.7 23S rRNA pseudouridine synthase

dnaX 1.7 DNA polymerase III/DNA elongation factor III, tau and

gamma subunits

gnsB 1.7 Qin prophage; predicted protein

moeA 1.7 molybdopterin biosynthesis protein

ilvM 1.7 acetolactate synthase II, small subunit


171

Gene

name

Fold


purF 1.7 Amido phosphoribosyl transferase

dppB 1.7 dipeptide transporter

ompX 1.7 outer membrane protein

cysK 1.7 Cysteine synthase A, O-acetylserine sulfhydrolase A subunit

yciH 1.7 conserved protein

glnG 1.7

fused DNA-binding response regulator in two-component

regulatory system with GlnL: response regulator/sigma54

interaction protein

dcm 1.7 DNA cytosine methylase

yjcB 1.7 predicted inner membrane protein

nikE 1.7 nickel transporter subunit

ygaD 1.7 conserved protein

hydN 1.7 Formate dehydrogenase-H,

ansA 1.7 cytoplasmic L-asparaginase I

ybjT 1.7 conserved protein with NAD(P)-binding Rossmann-fold

domain

ypaA 1.7 predicted protein

ccmA 1.7 heme exporter subunit

aroA 1.7 5-enolpyruvylshikimate-3-phosphate synthetase

yicC 1.7 conserved protein

thrA 1.7 fused aspartokinase I and homoserine dehydrogenase I

yedJ 1.7 predicted phosphohydrolase

nhaB 1.7 sodium:proton antiporter

tyrS 1.7 tyrosyl-tRNA synthetase

yicE 1.6 predicted transporter

ppiD 1.6 Peptidyl-prolyl cis-trans isomerase (rotamase D)

dpiA 1.6 DNA-binding response regulator in two-component regulatory

system with citA

yoaE 1.6 fused predicted membrane protein/conserved protein

malY 1.6 bifunctional beta-cystathionase, PLP-dependent/ regulator of


172

maltose regulon

Gene

name

Fold


rplD 1.6 50S ribosomal subunit protein L4

apaG 1.6 protein associated with Co2+

and Mg2+

efflux

ychM 1.6 predicted transporter

yccA 1.6 inner membrane protein

nusA 1.6 transcription termination/antitermination L factor

ymgD 1.6 predicted protein

cyoB 1.6 cytochrome o ubiquinol oxidase subunit I

manA 1.6 mannose-6-phosphate isomerase

mukF 1.6 Involved in chromosome partioning, Ca2+

binding protein

yigB 1.6 predicted hydrolase

sdaC 1.6 predicted serine transporter

ybaL 1.6 predicted transporter with NAD(P)-binding Rossmann-fold

domain

mltB 1.6 membrane-bound lytic murein transglycosylase B

ybfK 1.6 hypothetical protein b4590

ybjM 1.6 predicted inner membrane protein

fis 1.6 global DNA-binding transcriptional dual regulator

nrdH 1.6 glutaredoxin-like protein

dppD 1.6 dipeptide transporter

ispE 1.6 4-diphosphocytidyl-2-C-methylerythritol kinase

nrfG 1.6 heme lyase (NrfEFG) for insertion of heme into c552, subunit

NrfG

rdgC 1.6 DNA-binding protein

greA 1.6 transcription elongation factor

nadE 1.6 NAD synthetase, NH3/glutamine-dependent

leuB 1.6 3-isopropylmalate dehydrogenase

hemE 1.6 uroporphyrinogen decarboxylase

potB 1.6 polyamine transporter subunit

dusB 1.6 tRNA-dihydrouridine synthase B


173

Gene

name

Fold


tolQ 1.6 membrane spanning protein in TolA-TolQ-TolR complex

aroG 1.6 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase,

phenylalanine repressible

cysW 1.6 sulfate/thiosulfate transporter subunit

oppD 1.6 oligopeptide transporter subunit

ybcJ 1.6 predicted RNA-binding protein

thyA 1.6 thymidylate synthetase

hflD 1.6 predicted lysogenization regulator

rpsA 1.6 30S ribosomal subunit protein S1

torS 1.6 hybrid sensory histidine kinase in two-component regulatory

system with TorR

rsgA 1.6 ribosome small subunit-dependent GTPase A

ygfB 1.6 predicted protein

mhpE 1.6 4-hyroxy-2-oxovalerate/4-hydroxy-2-oxopentanoic acid

aldolase, class I

ybiT 1.6 fused predicted transporter subunits of ABC superfamily:


amtB 1.6 ammonium transporter

yadS 1.6 conserved inner membrane protein

mdtK 1.6 multidrug efflux system transporter

hscB 1.6 DnaJ-like molecular chaperone specific for IscU

rpmA 1.6 50S ribosomal subunit protein L27

dnaC 1.6 DNA biosynthesis protein

mqo 1.6 malate dehydrogenase, FAD/NAD(P)-binding domain

suhB 1.6 inositol monophosphatase

tig 1.6 Peptidyl-prolyl cis/trans isomerase (trigger factor)

kdsD 1.6 D-arabinose 5-phosphate isomerase

queA 1.6 S-adenosylmethionine:tRNA ribosyltransferase-isomerase

purD 1.6 Phosphoribosyl glycinamide synthetase phosphoribosylamine-

glycine ligase


174

Gene

name

Fold


yrbD 1.6 predicted ABC-type organic solvent transporter

mukE 1.6 protein involved in chromosome partitioning

rnhA 1.6 ribonuclease HI, degrades RNA of DNA-RNA hybrids

metE 1.5 5-methyltetrahydropteroyltriglutamate-homocysteine S-

methyltransferase

rnt 1.5 ribonuclease T (RNase T)

yfhA 1.5 predicted DNA-binding response regulator in two-component

system

secD 1.5 SecYEG protein translocase auxillary subunit

yraL 1.5 predicted methyltransferase

speC 1.5 ornithine decarboxylase, constitutive

ydcP 1.5 predicted peptidase

tsf 1.5 protein chain elongation factor EF-Ts

cld 1.5 regulator of length of O-antigen component of

lipopolysaccharide chains

yfcD 1.5 predicted NUDIX hydrolase

rpsB 1.5 30S ribosomal subunit protein S2

holA 1.5 DNA polymerase III, delta subunit

yjbJ 1.5 predicted stress response protein

lepB 1.5 leader peptidase (signal peptidase I)

ybeA 1.5 conserved protein

yieG 1.5 predicted inner membrane protein

nusG 1.5 transcription termination factor

yqiC 1.5 conserved protein

mgtA 1.5 magnesium transporter

rlmL 1.5 predicted methyltransferase

fecB 1.5 KpLE2 phage-like element; iron-dicitrate transporter

slmA 1.5 Division inhibitor

speD 1.5 S-adenosylmethionine decarboxylase

bioD 1.5 dethiobiotin synthetase


175

Gene

name

Fold


guaB 1.5 IMP dehydrogenase

leuA 1.5 2-isopropylmalate synthase

tyrP 1.5 Tyrosine transporter

metJ 1.5 DNA-binding transcriptional repressor, S-

adenosylmethionine-binding

murA 1.5 UDP-N-acetylglucosamine 1-carboxyvinyltransferase

dsbG 1.5 periplasmic disulfide isomerase/thiol-disulphide oxidase

cysZ 1.5 predicted inner membrane protein

kdgK 1.5 Keto deoxy gluconokinase

gcvH 1.5 glycine cleavage complex lipoyl protein

rplW 1.5 50S ribosomal subunit protein L23

panD 1.5 aspartate 1-decarboxylase

mltD 1.5 predicted membrane-bound lytic murein transglycosylase D

yajC 1.5 SecYEG protein translocase auxillary subunit

alaS 1.5 alanyl-tRNA synthetase

ydiK 1.5 predicted inner membrane protein

folE 1.5 GTP cyclohydrolase I

rplM 1.5 50S ribosomal subunit protein L13

typA 1.5 GTP-binding protein

ydeA 1.5 predicted arabinose transporter

yeiR 1.5 predicted enzyme

fkpB 1.5 FKBP-type peptidyl-prolyl cis-trans isomerase (rotamase)

iaaA 1.5 L-asparaginase

kdsC 1.5 3-deoxy-D-manno-octulosonate 8-phosphate phosphatase

gyrB 1.5 DNA gyrase, subunit B

aroK 1.5 shikimate kinase I

speA 1.5 biosynthetic arginine decarboxylase, PLP-binding

putA 1.5 fused DNA-binding transcriptional regulator/proline

dehydrogenase/pyrroline-5-carboxylate dehydrogenase

rpsP 1.5 30S ribosomal subunit protein S16


176

Gene

name

Fold


acrA 1.5 multidrug efflux system

ybbB 1.5 tRNA 2-selenouridine synthase, selenophosphate-dependent

pepQ 1.5 proline dipeptidase

appY 1.5 DLP12 prophage; DNA-binding transcriptional activator

fdx 1.5 [2Fe-2S] ferredoxin [Escherichia coli K12]

bcp 1.5 thiol peroxidase, thioredoxin-dependent

btuF

1.5

vitamin B12 transporter subunit: periplasmic-binding

component of ABC superfamily

era 1.5 membrane-associated, 16S rRNA-binding GTPase

gyrA 1.5 DNA gyrase (type II topoisomerase), subunit A

rrmA 1.5 23S rRNA m1G745 methyltransferase

truB 1.4 tRNA pseudouridine synthase

rpsH 1.4 30S ribosomal subunit protein S8

pyrF 1.4 orotidine-5'-phosphate decarboxylase

bcr 1.4 bicyclomycin/multidrug efflux system

yebC 1.4 conserved protein

glpR 1.4 DNA-binding transcriptional repressor

marB 1.4 predicted protein

ruvC 1.4 component of RuvABC resolvasome, endonuclease

yidR 1.4 conserved protein

rplB 1.4 50S ribosomal subunit protein L2

psd 1.4 Phosphatidyl serine decarboxylase

yjcE 1.4 predicted cation/proton antiporter

hemN 1.4 coproporphyrinogen III oxidase, SAM and NAD(P)H

dependent, oxygen-independent

dut 1.4 Deoxyuridine triphosphatase

metC 1.4 cystathionine beta-lyase, PLP-dependent

csrA 1.4 pleiotropic regulatory protein for carbon source metabolism

menC 1.4 o-succinylbenzoyl-CoA synthase

yfbQ 1.4 predicted aminotransferase


177

Gene

name

Fold


rdgB 1.4 dITP/XTP pyrophosphatase

serU 1.4 Ser tRNA

cyoD 1.4 cytochrome ubiquinol oxidase subunit IV

rplQ 1.4 50S ribosomal subunit protein L17

map 1.4 methionine aminopeptidase

rsmC 1.4 16S rRNA m2G1207 methylase

dsbA 1.4 periplasmic protein disulfide isomerase I

glyQ 1.4 glycine tRNA synthetase, alpha subunit

ygdE 1.4 predicted methyltransferase

ribF 1.4 bifunctional riboflavin kinase/FAD synthetase

pth 1.4 peptidyl-tRNA hydrolase

yjaG 1.4 conserved protein

trpD 1.4 fused glutamine amidotransferase (component II) of

anthranilate synthase/anthranilate phosphoribosyl transferase

fmt 1.4 10-formyltetrahydrofolate:L-methionyl-tRNA(fMet) N-

formyltransferase

recJ 1.4 ssDNA exonuclease, 5' --> 3'-specific

lolD 1.4 outer membrane-specific lipoprotein transporter subunit

yqfB 1.4 conserved protein

spoT 1.4 bifunctional (p)ppGpp synthetase II/ guanosine-3',5'-bis

pyrophosphate 3'-pyrophosphohydrolase

plsX 1.4 fatty acid/phospholipid synthesis protein

allR 1.4 DNA-binding transcriptional repressor

yheS 1.4 fused predicted transporter subunits of ABC superfamily:


motA 1.4 proton conductor component of flagella motor

rsxG 1.4 predicted oxidoreductase

pyrI 1.4 aspartate carbamoyltransferase, regulatory subunit

ycdX 1.4 predicted zinc-binding hydrolase

pnp 1.4 polynucleotide phosphorylase/polyadenylase


178

Gene

name

Fold


thrC 1.4 threonine synthase

rpsI 1.4 30S ribosomal subunit protein S9

glmU 1.4 fused N-acetyl glucosamine-1-phosphate

uridyltransferase/glucosamine-1-phosphate acetyl transferase

pdxB 1.4 erythronate-4-phosphate dehydrogenase

rsmB 1.4 16S rRNA m5C967 methyltransferase, S-adenosyl-L-

methionine-dependent

ydiJ 1.4 predicted FAD-linked oxidoreductase

yheT 1.4 predicted hydrolase

hslU 1.4 molecular chaperone and ATPase component of HslUV

protease

nemA 1.4 N-ethylmaleimide reductase, FMN-linked

nlpB 1.4 lipoprotein

rpsS 1.4 30S ribosomal subunit protein S19

hrpB 1.4 predicted ATP-dependent helicase

tgt 1.4 tRNA-guanine transglycosylase

ackA 1.4 acetate kinase A and propionate kinase 2

yceB 1.4 predicted lipoprotein

ycaR 1.4 conserved protein

bcsA 1.4 cellulose synthase, catalytic subunit

rplX 1.4 50S ribosomal subunit protein L24

pldA 1.4 outer membrane phospholipase A

mdoG 1.4 glucan biosynthesis protein, periplasmic

mdtE 1.4 multidrug resistance efflux transporter

argS 1.4 arginyl-tRNA synthetase

yadG 1.4 predicted transporter subunit: ATP-binding component of

ABC superfamily

glmM 1.4 phosphoglucosamine mutase

hpt 1.4 hypoxanthine phosphoribosyltransferase

rplU 1.4 50S ribosomal subunit protein L21


179

Gene

name

Fold


grxC 1.4 glutaredoxin 3

ybbO 1.4 predicted oxidoreductase with NAD(P)-binding Rossmann-

fold domain

yobA 1.4 conserved protein

gpp 1.4 guanosine pentaphosphatase/exopolyphosphatase

ftsY 1.4 fused Signal Recognition Particle (SRP) receptor: membrane

binding protein/conserved protein

lepA 1.3 GTP-binding membrane protein

rplN 1.3 50S ribosomal subunit protein L14

yjeP 1.3 predicted mechanosensitive channel

trpE 1.3 component I of anthranilate synthase

thrS 1.3 threonyl-tRNA synthetase

oxyR 1.3 DNA-binding transcriptional dual regulator

dhaK 1.3 dihydroxyacetone kinase, N-terminal domain

metG 1.3 methionyl-tRNA synthetase

mreB 1.3 cell wall structural complex MreBCD, actin-like component

MreB

ccmD 1.3 cytochrome c biogenesis protein

ygfZ 1.3 predicted folate-dependent regulatory protein

ndh 1.3 respiratory NADH dehydrogenase 2/cupric reductase

ydfO 1.3 Qin prophage; predicted protein

tdk 1.3 thymidine kinase/deoxyuridine kinase

napG 1.3 ferredoxin-type protein essential for electron transfer from

ubiquinol to periplasmic nitrate reductase (NapAB)

atpH 1.3 F1 sector of membrane-bound ATP synthase, delta subunit

dnaB 1.3 replicative DNA helicase

dsbC 1.3 protein disulfide isomerase II

yjjK 1.3 fused predicted transporter subunits of ABC superfamily:


csgE 1.3 predicted transport protein


180

Gene

name

Fold


yggS 1.3 predicted enzyme

ydgH 1.3 predicted protein

yadH 1.3 predicted transporter subunit: membrane component of ABC

superfamily

gcd 1.3 glucose dehydrogenase

atpE 1.3 F0 sector of membrane-bound ATP synthase, subunit c

dacB 1.3 D-alanyl-D-alanine carboxypeptidase

yacG 1.3 conserved protein

rpe 1.3 D-ribulose-5-phosphate 3-epimerase

ycdZ 1.3 predicted inner membrane protein

iscX 1.3 conserved protein

panB 1.3 3-methyl-2-oxobutanoate hydroxymethyl transferase

hda 1.3 ATPase regulatory factor involved in DnaA inactivation

narH 1.3 nitrate reductase 1, beta (Fe-S) subunit

cca 1.3 fused tRNA nucleotidyl transferase/2'3'-cyclic

phosphodiesterase/2'nucleotidase and phosphatase

ycfS 1.3 conserved protein

rpsF 1.3 30S ribosomal subunit protein S6

rarA 1.3 recombination protein

pyrH 1.3 uridylate kinase

apaH 1.3 diadenosine tetraphosphatase

rluB 1.3 23S rRNA pseudouridylate synthase

ydiA 1.3 conserved protein

yneH 1.3 predicted glutaminase

yieH 1.3 predicted hydrolase

tehB 1.3 predicted S-adenosyl-L-methionine-dependent

methyltransferase

solA 1.3 N-methyltryptophan oxidase, FAD-binding

udk 1.3 uridine/cytidine kinase

gnsA 1.3 predicted regulator of phosphatidylethanolamine synthesis


181

Gene

name

Fold


racR 1.3 Rac prophage; predicted DNA-binding transcriptional

regulator

mrdA 1.3 transpeptidase involved in peptidoglycan synthesis (penicillin-

binding protein 2)

yciB 1.3 predicted inner membrane protein

priB 1.3 primosomal protein N

selA 1.3 selenocysteine synthase

mreC 1.3 cell wall structural complex MreBCD transmembrane

component MreC

yrbK 1.3 conserved protein

yddW 1.3 predicted liprotein

purL 1.3 phosphoribosylformyl-glycineamide synthetase

yggX 1.3 protein that protects iron-sulfur proteins against oxidative

damage

hemF 1.3 coproporphyrinogen III oxidase

ruvA 1.3 component of RuvABC resolvasome, regulatory subunit

cyaY 1.3 frataxin, iron-binding and oxidizing protein

rffD 1.3 UDP-N-acetyl-D-mannosaminuronic acid dehydrogenase

gfcA 1.3 predicted protein

nadC 1.3 quinolinate phosphoribosyltransferase

napD 1.3 assembly protein for periplasmic nitrate reductase

hcaB 1.3 2,3-dihydroxy-2,3-dihydrophenylpropionate dehydrogenase

ppx 1.3 exopolyphosphatase

yacF 1.3 conserved protein

glnS 1.3 glutamyl-tRNA synthetase

emrA 1.3 multidrug efflux system

yfiH 1.3 conserved protein

proQ 1.3 predicted structural transport element

cydC 1.3 fused cysteine transporter subunits of ABC superfamily:



182

Gene

name

Fold


creC 1.3 sensory histidine kinase in two-component regulatory system

with CreB or PhoB, regulator of the CreBC regulon

hscA 1.3 DnaK-like molecular chaperone specific for IscU

cydD 1.3 fused cysteine transporter subunits of ABC superfamily:


ybjS 1.3 predicted NAD(P)H-binding oxidoreductase with NAD(P)-

binding Rossmann-fold domain

bioC 1.3 predicted methltransferase, enzyme of biotin synthesis

pdxA 1.3 4-hydroxy-L-threonine phosphate dehydrogenase, NAD-

dependent

ribB 1.3 3,4-dihydroxy-2-butanone-4-phosphate synthase

nudJ 1.3 bifunctional thiamin pyrimidine pyrophosphate hydrolase/

thiamin pyrophosphate hydrolase

rpoA 1.3 RNA polymerase, alpha subunit

rfaG 1.3 glucosyltransferase I

ptsG 1.3 fused glucose-specific PTS enzymes: IIB component/IIC

component

lgt 1.3 phosphatidylglycerol-prolipoprotein diacylglyceryl transferase

gph 1.3 phosphoglycolate phosphatase

recA 1.3 DNA strand exchange and recombination protein with

protease and nuclease activity

dacA 1.3 D-alanyl-D-alanine carboxypeptidase (penicillin-binding

protein 5)

trxB 1.3 thioredoxin reductase, FAD/NAD(P)-binding

diaA 1.3 DnaA initiator-associating factor for replication initiation

proA 1.3 gamma-glutamylphosphate reductase

yeaE 1.3 predicted oxidoreductase

ydhJ 1.3 undecaprenyl pyrophosphate phosphatase

ygiF 1.3 predicted adenylate cyclase

proB 1.3 gamma-glutamate kinase


183

Gene

name

Fold


lpxK 1.3 lipid A 4'kinase

hisS 1.2 histidyl tRNA synthetase

ybeB 1.2 predicted protein

yhbY 1.2 predicted RNA-binding protein

yaiL 1.2 nucleoprotein/polynucleotide-associated enzyme

purH 1.2 fused IMP cyclohydrolase/phosphoribosyl amino imidazole

carboxamide formyl transferase

sppA 1.2 protease IV (signal peptide peptidase)

kgtP 1.2 alpha-ketoglutarate transporter

ybhA 1.2 predicted hydrolase

minD 1.2 membrane ATPase of the MinC-MinD-MinE system

rstA 1.2 DNA-binding response regulator in two-component regulatory

system with RstB

yaeB 1.2 conserved protein

yiaF 1.2 conserved protein

ytjB 1.2 conserved protein

rluD 1.2 23S rRNA pseudouridine synthase

yigL 1.2 predicted hydrolase

yciN 1.2 predicted protein

hsdM 1.2 DNA methylase M

yjgP 1.2 conserved inner membrane protein

folM 1.2 dihydrofolate reductase isozyme

uup 1.2 fused predicted transporter subunits of ABC superfamily:


ybdG 1.2 predicted mechanosensitive channel

rluE 1.2 23S rRNA pseudouridine synthase

pcnB 1.2 poly(A) polymerase I

yegQ 1.2 predicted peptidase

rplE 1.2 50S ribosomal subunit protein L5

ycgL 1.2 conserved protein


184

Gene

name

Fold


ybiV 1.2 predicted hydrolase

yfgM 1.2 conserved protein

essD 1.2 DLP12 prophage; predicted phage lysis protein

mrcA 1.2 fused penicillin-binding protein 1a: murein

transglycosylase/murein transpeptidase

yceD 1.2 conserved protein

lspA 1.2 prolipoprotein signal peptidase (signal peptidase II)

rfaQ 1.2 lipopolysaccharide core biosynthesis protein

serC 1.2 3-phosphoserine/phosphohydroxythreonine aminotransferase

cyoE 1.2 protoheme IX farnesyltransferase

cueO 1.2 multicopper oxidase (laccase)

mnmC 1.2 fused 5-methylaminomethyl-2-thiouridine-forming enzyme

methyltransferase/FAD-dependent demodification enzyme

ydiI 1.2 conserved protein

serA 1.2 D-3-phosphoglycerate dehydrogenase

yebY 1.2 predicted protein

yiaD 1.2 predicted outer membrane lipoprotein

menB 1.2 dihydroxynaphthoic acid synthetase

yfiO 1.2 predicted lipoprotein

ribA 1.2 GTP cyclohydrolase II

yhcC 1.2 predicted Fe-S oxidoreductase

napH 1.2 ferredoxin-type protein essential for electron transfer from

ubiquinol to periplasmic nitrate reductase (NapAB)

hflC 1.2 modulator for HflB protease specific for phage lambda cII

repressor

surA 1.2 peptidyl-prolyl cis-trans isomerase (PPIase)

dcyD 1.2 D-cysteine desulfhydrase, PLP-dependent

metL 1.2 fused aspartokinase II/homoserine dehydrogenase II

ksgA 1.2 S-adenosylmethionine-6-N',N'-adenosyl (rRNA)

dimethyltransferase


185

Gene

name

Fold


avtA 1.2 valine-pyruvate aminotransferase

holC 1.2 DNA polymerase III, chi subunit

focB 1.2 predicted formate transporter

yajG 1.2 predicted lipoprotein

fnr 1.2 DNA-binding transcriptional dual regulator, global regulator

of anaerobic growth

mpl 1.2 UDP-N-acetylmuramate:L-alanyl-gamma-D-glutamyl-meso-

diaminopimelate ligase

plsC 1.2 1-acyl-sn-glycerol-3-phosphate acyltransferase

yfdH 1.2 CPS-53 (KpLE1) prophage; bactoprenol glucosyl transferase

yiiX 1.2 predicted peptidoglycan peptidase

cysM 1.2 cysteine synthase B (O-acetylserine sulfhydrolase B)

thrB 1.2 homoserine kinase

pykF 1.2 pyruvate kinase I

fabA 1.2 beta-hydroxydecanoyl thioester dehydrase

rlpB 1.2 minor lipoprotein

asnS 1.2 asparaginyl tRNA synthetase

sieB 1.2 Rac prophage; phage superinfection exclusion protein

cpxA 1.2 sensory histidine kinase in two-component regulatory system

with CpxR

proC 1.2 pyrroline-5-carboxylate reductase, NAD(P)-binding

uraA 1.2 uracil transporter

rffC 1.2 TDP-fucosamine acetyltransferase

rnlA 1.2 CP4-57 prophage; RNase LS

gshA 1.2 gamma-glutamate-cysteine ligase

ttcA 1.2 predicted C32 tRNA thiolase

xseA 1.2 exonuclease VII, large subunit

fabD 1.2 malonyl-CoA

tolA 1.2 membrane anchored protein in TolA-TolQ-TolR complex

btuE 1.2 predicted glutathione peroxidase


186

Gene

name

Fold


tatD 1.2 DNase, magnesium-dependent

galR 1.2 DNA-binding transcriptional repressor

yhbE 1.2 conserved inner membrane protein

narP 1.2 DNA-binding response regulator in two-component regulatory

system with NarQ or NarX

recO 1.2 gap repair protein

amn 1.2 AMP nucleosidase

ygaH 1.2 predicted inner membrane protein

dnaA 1.2 chromosomal replication initiator protein DnaA, DNA-binding

transcriptional dual regulator

bioA 1.2 7,8-diaminopelargonic acid synthase, PLP-dependent

yraN 1.2 conserved protein

brnQ 1.2 predicted branched chain amino acid transporter (LIV-II)

ribE 1.2 riboflavin synthase beta chain

fklB 1.2 FKBP-type peptidyl-prolyl cis-trans isomerase (rotamase)

bisC 1.2 biotin sulfoxide reductase

ydiH 1.2 predicted protein

ygiQ 1.2 conserved protein

yecE 1.2 conserved protein

rffT 1.2 TDP-Fuc4NAc:lipidIIFuc4NAc transferase

yjjU 1.1 predicted esterase

racC 1.1 Rac prophage; predicted protein

oppF 1.1 oligopeptide transporter subunit

nudK 1.1 predicted NUDIX hydrolase

fabB 1.1 3-oxoacyl-ACP-synthase I

crcB 1.1 predicted inner membrane protein associated with

chromosome condensation

gloA 1.1 glyoxalase I, Ni-dependent

fldA 1.1 flavodoxin 1

lptB 1.1 predicted transporter subunit: ATP-binding component of


187

ABC superfamily

Gene

name

Fold


tolC 1.1 transport channel

nusB 1.1 transcription antitermination protein

minE 1.1 cell division topological specificity factor

rdoA 1.1 Thr/Ser kinase implicated in Cpx stress response

yfiP 1.1 conserved protein

yghG 1.1 predicted protein

aroL 1.1 shikimate kinase II

rpsG 1.1 30S ribosomal subunit protein S7

fecE 1.1 KpLE2 phage-like element; iron-dicitrate transporter subunit

fusA 1.1 protein chain elongation factor EF-G, GTP-binding

fabH 1.1 3-oxoacyl-ACP synthase III

moaE 1.1 molybdopterin synthase, large subunit

ynaI 1.1 conserved inner membrane protein

dam 1.1 DNA adenine methylase

speE 1.1 spermidine synthase (putrescine aminopropyltransferase)

rplV 1.1 50S ribosomal subunit protein L22

malZ 1.1 maltodextrin glucosidase

yqgE 1.1 predicted protein

rpsK 1.1 30S ribosomal subunit protein S11

marA 1.1 DNA-binding transcriptional dual activator of multiple

antibiotic resistance

glyS 1.1 glycine tRNA synthetase, beta subunit

panC 1.1 pantothenate synthetase

menE 1.1 o-succinylbenzoate-CoA ligase

yegD 1.1 predicted chaperone

yfeX 1.1 conserved protein

lon 1.1 DNA-binding ATP-dependent protease La

ptsP 1.1

fused PTS enzyme: PEP-protein phosphotransferase (enzyme

I)/GAF domain containing protein


188

Gene

name

Fold


dnaE 1.1 DNA polymerase III alpha subunit

speB 1.1 agmatinase

fabI 1.1 enoyl-ACP reductase

yfgD 1.1 predicted oxidoreductase

apbE 1.1 predicted thiamine biosynthesis lipoprotein

trpB 1.1 tryptophan synthase, beta subunit

gadW 1.1 DNA-binding transcriptional activator

psiE 1 predicted phosphate starvation inducible protein

yqaB 1 predicted hydrolase

yceA 1 conserved protein

secE 1 preprotein translocase membrane subunit

yqgF 1 predicted Holliday junction resolvase

rplL 1 50S ribosomal subunit protein L7/L12

gcvT 1 Amino methyl transferase, tetrahydrofolate-dependent, subunit

(T protein) of glycine cleavage complex

alsE 1 allulose-6-phosphate 3-epimerase

cdsA 1 CDP-diglyceride synthase

hcaF 1 3-phenylpropionate dioxygenase, small (beta) subunit

cho 1 endonuclease of nucleotide excision repair

yjiH 1 conserved inner membrane protein

nac 1 DNA-binding transcriptional dual regulator of nitrogen

assimilation

yhhQ 1 conserved inner membrane protein

hslJ 1 heat-inducible protein

ybaB 1 conserved protein

mrp 1 antiporter inner membrane protein

yfhG 1 conserved protein

yfcZ 1 conserved protein

yheU 1 conserved protein


189

Gene

name

Fold


bglF 1 fused beta-glucoside-specific PTS enzymes: IIA

component/IIB component/IIC component

ilvE 1 branched-chain amino-acid aminotransferase

ycbL 1 predicted metal-binding enzyme

pepE 1 (alpha)-aspartyl dipeptidase

osmF 1 predicted transporter subunit: periplasmic-binding component

of ABC superfamily

rpsT 1 30S ribosomal subunit protein S20

ygbE 1 conserved inner membrane protein

yfgC 1 predicted peptidase

rbn 1 binuclear zinc phosphodiesterase

rpsC 1 30S ribosomal subunit protein S3

tyrR 1 DNA-binding transcriptional dual regulator, tyrosine-binding

xthA 1 exonuclease III

yraM 1 conserved protein

ynjE 1 predicted thiosulfate sulfur transferase

yccS 1 predicted inner membrane protein

gpmM 1 phosphoglycero mutase III, cofactor-independent

yeaZ 1 predicted peptidase

emtA 1 lytic murein endotransglycosylase E

uof 1 ryhB-regulated fur leader peptide

grxD 1 conserved protein

rpsM 1 30S ribosomal subunit protein S13

hycA 1 regulator of the transcriptional regulator FhlA

cvpA 1 membrane protein required for colicin V production

rlpA 1 minor lipoprotein

cobS 1 cobalamin 5'-phosphate synthase

poxB 1 pyruvate dehydrogenase (pyruvate oxidase), thiamin-

dependent, FAD-binding

fabZ 1 (3R)-hydroxymyristol acyl carrier protein dehydratase


190

Gene

name

Fold


atpB 1 F0 sector of membrane-bound ATP synthase

adhP 1 ethanol-active dehydrogenase/acetaldehyde-active reductase

trpT 1 Trp tRNA

dusA 1 tRNA-dihydrouridine synthase A

ogt 1 O-6-alkylguanine-DNA:cysteine-protein methyltransferase

dppF 1 dipeptide transporter

creB 1 DNA-binding response regulator in two-component regulatory

system with CreC

paaY 1 predicted hexapeptide repeat acetyltransferase

ybjL 1 predicted transporter

zwf 1 glucose-6-phosphate dehydrogenase

rlmB 1 23S rRNA (Gm2251)-methyltransferase

sgrR 1 DNA-binding transcriptional regulator

yibN 1 predicted rhodanese-related sulfurtransferase

pmbA 1 predicted peptidase required for the maturation and secretion

of the antibiotic peptide MccB17

mnmG 1 glucose-inhibited cell-division protein

ruvB 1 ATP-dependent DNA helicase, component of RuvABC

resolvasome

rplJ 1 50S ribosomal subunit protein L10

aroB 1 3-dehydroquinate synthase

yliG 1 predicted SAM-dependent methyltransferase

yfcC 1 predicted inner membrane protein

yigZ 1 predicted elongation factor

bioB 1 biotin synthase

yliJ 1 predicted glutathione S-transferase

cmoB 1 predicted S-adenosyl-L-methionine-dependent

methyltransferase

yecM 1 predicted metal-binding enzyme

tatA 1 TatABCE protein translocation system subunit


191

Gene

name

Fold


menD 1 bifunctional 2-oxoglutarate decarboxylase/ SHCHC synthase

yfiF 1 predicted methyltransferase

gsk 1 inosine/guanosine kinase

hisF 1 imidazole glycerol phosphate synthase, catalytic subunit with

HisH

otsB 1 trehalose-6-phosphate phosphatase, biosynthetic

aroD 1 3-dehydroquinate dehydratase

ybjO 1 predicted inner membrane protein

lpcA 1 D-sedoheptulose 7-phosphate isomerase

nagA 1 N-acetylglucosamine-6-phosphate deacetylase

dppC 1 dipeptide transporter

yrbB 1 predicted protein

serB 1 3-phosphoserine phosphatase

dnaQ 1 DNA polymerase III epsilon subunit

ygiC 1 predicted enzyme

yfgL 1 protein assembly complex, lipoprotein component

ccmE 1 periplasmic heme chaperone

ycbK 1 conserved protein

yhgF 1 predicted transcriptional accessory protein

uvrY 1

DNA-binding response regulator in two-component regulatory

system with

BarA

yafJ 1 predicted amidotransfease

sapD 1 predicted antimicrobial peptide transporter subunit

wbbI 1 conserved protein

lptA 1 predicted transporter subunit: periplasmic-binding component

of ABC superfamily

topB 1 DNA topoisomerase III

rsxE 1 predicted inner membrane NADH-quinone reductase

cusR 1 DNA-binding response regulator in two-component regulatory


192

system with CusS

Gene

name

Fold


kefA 1 fused conserved protein

hemL 1 glutamate-1-semialdehyde aminotransferase (aminomutase)

emrY 1 predicted multidrug efflux system

yffS 1 CPZ-55 prophage; predicted protein

ycgV 1 predicted adhesin

tsx 1 nucleoside channel, receptor of phage T6 and colicin K

Documents

ArgP’s role in osmoregulationshodhganga.inflibnet.ac.in/bitstream/10603/8551/12/12...ArgP’s role in osmoregulation Chapter 3 64 Table 3.1 Regulation of gdhA-lac expression by ArgP