Cloning, characterization and expression analysis of nucleotide metabolism-related genes of mycobacteriophage L5

R E S E A R C H L E T T E R

Cloning, characterizationand expressionanalysis of nucleotidemetabolism-relatedgenesofmycobacteriophageL5Bidisha Bhattacharya, Nabanita Giri, Mahasweta Mitra & Sujoy K. Das Gupta

Department of Microbiology, Bose Institute, Kolkata, India

Correspondence: Sujoy K. Das Gupta,

Department of Microbiology, Bose Institute,

P1/12 C.I.T. Scheme VIIM, Kolkata 700054,

India. Tel.: 191 33 23559416; fax: 191 33

2553886; e-mail: [email protected]

Received 24 August 2007; accepted

25 November 2007.

First published online February 2008.

DOI:10.1111/j.1574-6968.2007.01047.x

Editor: Dieter Jahn

Keywords

multienzyme complex; flavin-dependent

thymidylate synthase; phage infection.

Abstract

The genomes of mycobacteriophages of the L5 family, which includes the lytic phage

D29, contain several genes putatively linked to nucleotide-metabolizing functions.

Two such genes, 48 and 50, encoding thymidylate synthase and ribonucleotide

reductase (RNR), respectively, were overexpressed in Escherichia coli and the

recombinant proteins were biochemically characterized. It was established that

Gp50 was a class II RNR having properties similar to that of the corresponding

enzyme from Lactobacillus leichmanni, whereas Gp48 was a flavin-dependent

thymidylate synthase (ThyX) that resembled the Paramecium bursaria chlorella

virus-1 ThyX enzyme in its properties. That both these proteins play a role in phage

development was evident from the observation that they were detectable soon after

the lytic phase of growth commenced. Gp48 and 50 were also found to coimmuno-

precipitate, which indicates the possible existence of an L5 thymidylate synthase

complex. Thymidylate synthase assays revealed that during the intracellular stage of

phage growth, a significant decrease in the host thymidylate synthase (ThyA) activity

occurred. It appears that synthesis of the viral enzyme (ThyX) is necessary to

compensate for this loss in activity. In general, the results suggest that phage-

encoded nucleotide metabolism-related functions play an important role in the lytic

propagation of L5 and related mycobacteriophages.

Introduction

Mycobacteriophages have been considered to be corner-

stones of mycobacterial research (Hatfull & Jacobs, 1994).

Among the mycobacteriophages that have been well studied

are the closely related members of the L5 family, which

includes the lysogenic phages L1 (Doke, 1960; Lee et al.,

1991), L5 (Snapper et al., 1988; Hatfull & Sarkis, 1993) and

BxB1 (Jain & Hatfull, 2000) and the lytic phage D29 (Ford

et al., 1998). Although the L5 family of phages is considered

to be lysogenic or potentially lysogenic (D29) (Ribeiro et al.,

1997), they have features that strongly resemble lytic phages

(Hatfull & Sarkis, 1993). One such feature is the ability of

these phages to shut down host protein synthesis immedi-

ately following infection.

A large number of mycobacteriophage genome sequences

are currently available in the databases (Pedulla et al., 2003).

Comparative analysis of these sequences revealed that at

least half of the mycobacteriophage genes have no database

matches (NDMs), indicating thereby the possibility of the

existence of novel genes, whose functions are unknown.

Although several mycobacteriophage genome sequences are

available, only a limited number of genes have been char-

acterized, most of which belong to the L5 family. These are:

gene 71, the phage repressor (Donnelly-Wu et al., 1993); gene

33, integrase (Lee & Hatfull, 1993); and gene 36, excisionase

(Lewis & Hatfull, 2000). In addition, several other myco-

bacteriophage genes have been characterized, such as those

encoding a tape measure protein from TM4 (Piuri &

Hatfull, 2006), a polynucleotide kinase from Omega and

Cjw1 (Zhu et al., 2004) and a lysis protein from Ms6 (Garcia

et al., 2002).

In silico analysis (Hatfull & Sarkis, 1993; Ford et al., 1998)

of L5 and D29 genome sequences revealed the presence of

several genes that putatively code for enzymes involved in

DNA synthesis. One such gene (44) encodes a viral DNA

polymerase. Other DNA synthesis-related genes are 58, 59

and 65, which putatively encode primase, exonuclease and

FEMS Microbiol Lett 280 (2008) 64–72c� 2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

helicase, respectively. Apart from genes encoding enzymes

related to DNA replication, there are several genes that

appear to code for nucleotide metabolism-related functions.

Two such genes are 48 (thymidylate synthase) and 50

[ribonucleotide reductase (RNR)]. In the case of D29, but

apparently not in the case of L5, there may be a gene for

dCMP deaminase also. All of these genes are clustered in the

central region of the phage genome, indicating that their

products have some related functions. If the entire scenario is

considered in its totality, it seems that, as in case of the lytic

phages such as T4, L5/D29 phage-encoded DNA polymeriza-

tion and deoxy-ribonucleotide synthesis-related enzymes act

in concert to meet the replicative demands of the viral DNA,

particularly during the early stage of infection.

In nature, RNR (Poole et al., 2002) accounts for the

acquisition of three (dCTP, dGTP and dATP) of the four

deoxyribonucleotides needed for DNA biosynthesis. The

fourth nucleotide, dTTP, is produced by two well-known

processes. Exogenous thymidine can be directly salvaged by

thymidine kinase or dTMP can be synthesized de novo from

dUMP, a reaction catalyzed by thymidylate synthase. Thy-

midylate synthases may be of two types (Myllykallio et al.,

2002) – the classical thymidylate synthase designated as

ThyA and the alternative class of thymidylate synthases

known as ThyX. Both these enzymes catalyze the conversion

of dUMP to dTMP, but the reductive mechanisms are

different. In ThyA-catalyzed reactions, CH2H4folate serves

both as a reductant and as a CH2– donor whereas in a ThyX-

catalyzed reaction CH2H4folate acts only as a CH2– donor,

reducing equivalents being donated by NADPH through the

intermediate reduction of the enzyme-bound FAD. The

protein encoded by L5 gene 48 contains the highly conserved

ThyX motif RHRX7S (Myllykallio et al., 2002). ThyX

proteins in general are found in many pathogenic bacteria

and it has been proposed that this protein is an attractive target

for developing drugs against such bacteria (Graziani et al.,

2004). Interestingly, mycobacteria possess both the versions,

ThyX as well as ThyA, but the significance of this is not clearly

understood (Myllykallio et al., 2002; Sampathkumar et al.,

2005).

Mycobacteriophage L5 gene 50 putatively codes for a class

II RNR (RNR II). RNRs of the class II family typically use

adenosylcobalamin as the cofactor for the generation of the

free radicals (Poole et al., 2002). It is generally considered

that Class II enzymes function under micro-aerophillic

conditions, whereas class I and III enzymes function under

aerobic and anaerobic conditions, respectively.

Enzymes catalyzing sequential metabolic reactions are

often organized in the form of multienzyme complexes.

Such multienzyme complexes also exist in case of dNTP

biosynthesis. Although the dNTP-synthesizing complexes

exist in several prokaryotic and eukaryotic systems, the

best-characterized system is the complex encoded by the

Escherichia coli lytic phage T4 (Tomich et al., 1974; Reddy

et al., 1977; Prem veer Reddy & Pardee, 1980; Allen et al.,

1983; Mathews, 1993). In case of T4, it has been demon-

strated that several phage-encoded nucleotide metabolism-

related proteins such as thymidylate synthase, dihydrofolate

reductase, ribonucleotide reductase, deoxycytidylate deami-

nase, dC/UTPase and deoxycytidylate hydroxymethylase form

a functional aggregate. It is believed that such an aggregate

acts as a ‘metabolome’, which plays an important role in

maintaining an adequate supply of deoxy-nucleotide precur-

sors for the rapid synthesis of viral DNA (Mathews, 1993).

The presence of genes for ThyX and RNR II in the

genome of L5 and related mycobacteriophages has been

predicted purely on the basis of in silico analysis. No

functional characterization of these proteins has been car-

ried out so far. The present work was thus initiated with the

dual objective of characterizing the translated products of

genes 48 and 50 and analyzing their expression status during

infection. The results, apart from confirming bio-chemically

the in silico predictions, shed significant light on the nature

of these enzymes, their expression status and their ability to

associate with each other.

Materials and methods

Phages, bacterial strains and plasmids

Escherichia coli XL1Blue was used for routine purposes.

The expression vector pQE 30 (Qiagen) was used for over-

expression of the phage genes. For phage induction experi-

ments, the thermoinducible lysogenic strain Mycobacterium

smegmatis L1c1ts (obtained from Dr N.C. Mandal) was used

(Chaudhuri et al., 1993; Chatterjee et al., 2000). This strain

harbors a thermo-inducible lysogenic mutant of mycobac-

teriophage L1 as a prophage. Although the complete gen-

ome sequence of L1 phage is not available, restriction maps

(Lee et al., 1991) and DNA sequences of several L1 genes

(Datta & Mandal, 1998; Chattopadhyay et al., 2003; Sau

et al., 2004; Ganguly et al., 2007) indicate that L1 is only a

minor variant of L5. Hence, the use of an L1 lysogen to study

the L5 developmental pathways is justified. Cloning and

expression of mycobacteriophage genes 48 and 50 were

performed using genomic DNA derived from mycobac-

teiophage L5 (Hatfull & Sarkis, 1993). In case of Gp50, both

enzymatic and immunological experiments were conducted

using the L5 protein. In case of Gp48, immunological

experiments were conducted using antisera raised against

the L5 protein, but for enzymological studies Gp48 of D29

(Ford et al., 1998) was used, as the L5 protein could not be

recovered in the soluble fraction. D29 is a lytic phage

belonging to the L5 family. Many of the polypeptides

encoded by D29 share extensive homology with those

encoded by L5. In case of Gp48, the homology is 470%.

FEMS Microbiol Lett 280 (2008) 64–72 c� 2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

65Mycobacteriophage ThyX and RNR

Cloning of mycobacteriophage genes inexpression vectors

Mycobacteriophage DNA were isolated from L5 or D29 as

per standard methods using CsCl purified phages (Chatter-

jee et al., 2000). The phage DNA was then used for PCR

amplification of desired genes. For L5 gene 50, the primers

used were P1, forward primer (50-GGGGTACCTTGACTG

ACGAAATCC-30) and P2, reverse primer (50-CCCAAGCTT

CACTTAATGGGGCATGC-30), (the KpnI and HindIII sites

are underlined). The PCR product was digested with KpnI

and HindIII for cloning in the expression vector pQE 30

(Qiagen). The L5 gene 48 was amplified using primers P3,

forward primer (50-GAAGATCTCATATGAAAGCCAAACT

GATC-30), and P4, reverse primer (50-CCCAAGCTTCA

TATGTCAGTAGCTGTAG-30) (the BglII and HindIII sites

are underlined). The PCR product was digested with BglII

and HindIII for cloning in the expression vector pQE 30. D29

gene 48 was amplified using the forward primer (50-CG

GGATCCATGAAAGTCCAACTGATC-30) and the reverse

primer (50-CCCAAGCTTTCAGCCTCCGTAGCTG-30) (the

BamHI and HindIII sites are underlined). The amplicon was

cloned at the BamHI–HindIII site of pQE-30.

Expression and purification of recombinantproteins

Hexa-histidine-tagged recombinant protein purification

was performed using Ni-nitrilotriacetic acid (NTA) agarose

chromatography either under denaturing conditions or

under native conditions as per standard protocols (Qiagen).

Cells were harvested by centrifugation and sonicated in

buffer A (50 mM sodium phosphate pH 8.0, 300 mM NaCl,

20 mM imidazole). After centrifugation at 14 000 g, the

clear supernatant was loaded onto a 2-mL Ni21-NTA

agarose column, pre-equilibrated with buffer A. The

column was washed with 10 column volumes of buffer B

(50 mM sodium phosphate pH 8.0, 300 mM NaCl, 50 mM

imidazole) and the bound protein was eluted with buffer C

(50 mM sodium phosphate pH 8.0, 300 mM NaCl, 250 mM

imidazole) and analyzed by 12% sodium dodecyl sulfate-

polyacrylamide gel electrophoresis (SDS-PAGE). Fractions

containing pure protein were pooled and dialyzed over-

night at 4 1C in the dark against 50 mM Tris-HCl, pH 7.4,

containing 10% glycerol. For isolating proteins under

denaturing conditions, pellet was dissolved in lysis buffer D

(10 mM Tris, 50 mM sodium phosphate, 300 mM NaCl,

8 M urea, pH 8.0), sonicated, centrifuged and loaded onto

a Ni-NTA column. After washing with 10 column volume of

buffer E (10 mM Tris, 50 mM sodium phosphate, 300 mM

NaCl, 8 M urea, pH 6.3), protein was eluted in buffer F

(10 mM Tris, 50 mM sodium phosphate, 300 mM NaCl,

8 M urea, pH 4.5).

Induction of lysogen

The thermoinducible lysogen, M. smegmatis L1c1ts, was

grown to an OD of 0.8 at 28 1C in Middlebrook 7H9

(MB7H9) broth as described earlier (Chaudhuri et al.,

1993) and induced at 42 1C for 30 min, followed by growth

at 37 1C for 3 h. Aliquots were removed at definite intervals

(15, 30 and 60 min) after the flasks were immersed in the

42 1C water bath and processed for immunological and

enzymatic assays.

Immunodetection

Recombinant mycobacteriophage proteins were purified to

near homogeneity by affinity chromatography on Ni-NTA

agarose under denaturing conditions. The purified protein

was further separated from any contaminant proteins by

SDS-PAGE. The bands were excised and crushed before

injecting into rabbits according to previously standardized

protocols (Basu et al., 2002). Both immune and preimmune

sera were collected. The specificities of the derived antisera

were confirmed by Western blotting (1 : 1000 dilution) using

purified proteins. For immunodetection of phage-derived

proteins, cell-free extracts of thermo-induced M. smegmatis

L1c1ts lysogens were subjected to Western blotting using

specific antibodies (1 : 1000 dilution) according to standard

protocols (Sambrook et al., 1989).

Immunoprecipitation

Mycobacterium smegmatis L1c1ts lysogenic cells were grown

at 28 1C and then the temperature was shifted to 42 1C. After

15 min of induction, the proteins expressed were pulse label-

ed with 35S methionine (specific activity 3000 Ci mmol�1),

obtained from BRIT, Mumbai, India), for 3 min. Extracts

were prepared by sonicating the labeled cells in immuno-

precipitation buffer (50 mM Tris pH 7.4, 500 mM NaCl,

1 mM EDTA, 0.1% Nonidet P-40) and then incubated with

immune or preimmune sera (1 : 1000 dilution), overnight at

4 1C in the same buffer. Antibody-target complexes were

then pulled down using 25 mL of Protein-A Sepharose beads

(Amersham GE) (pre-equilibrated with the immunopreci-

pitation buffer containing 1% bovine serum albumin to

block nonspecific interactions) by centrifugation at 1000 g.

After washing five times with the immunoprecipitation

buffer, the beads were suspended in 1� SDS sample buffer,

boiled for 5 min and resolved by 13.5% SDS-PAGE. Bands

were visualized by autoradiography.

Determination of ThyX activity

The NADPH oxidase activity of ThyX was measured in a

total volume of 1 mL. The basal reaction components were

50 mM Tris-HCl, pH 7.4, 1 mM MgCl2, 10% glycerol. In

addition, dUMP, NADPH and FAD were added either alone


66 B. Bhattacharya et al.

or in specific combinations at desired concentrations.

Activity was monitored by net decrease of A340 nm using a

CARY 50 spectrophotometer (Varian). An extinction coeffi-

cient of 6400 cm�1 at 340 nm (e340) was used to quantify

absorption changes.

Thymidylate-synthesizing activity of ThyX was deter-

mined by monitoring the amount of tritium transferred to

water using [5-3H] dUMP (Movarek Biochemicals) as a

substrate as described previously (Griffin et al., 2005). The

standard reaction mixture (100mL) contained 50 mM

Tris-HCl, pH 7.4, 200 mM 5, 10-CH2H4folate, 500 mM

NADPH, 60mM FAD, 1 mM MgCl2 and 10% glycerol. The

specific activity of the [5-3H]dUMP stock used in the

experiments was 14.3 Ci mmol�1. The reaction was started

with the addition of 9 mg of purified ThyX. After 3 min, the

reaction was terminated by the addition of 300 mL of a

100 mg mL�1 activated charcoal suspension containing 2%

tricarboxylic acid, to remove the unused radiolabeled sub-

strate. Samples were mixed at room temperature for 1 h and

centrifuged at 12 000 g for 10 min to pellet the charcoal.

Radioactivity in the supernatant was determined by liquid

scintillation counting. Enzymatic activity of the protein was

expressed as nmol of dTMP formed min�1 mg�1.

Thymidylate synthase activity in cell extracts of induced

M. smegmatis L1c1ts cells was performed by inducing

200 mL of M. smegmatis L1c1ts culture at 42 1C. Fifty

milliliters of aliquots were removed at specified time points

and the harvested cells were sonicated. After removal of

debris by centrifugation, the extracts (60mL) were used in

the assays. The protein concentrations in the extracts were

nearly the same. If necessary, the activity data were normal-

ized with respect to the protein concentration. An assay for

ThyX was performed as described above. For ThyA activity,

the same buffer was used, except that NADPH and FAD

were omitted. The results were expressed as relative activity

taking the activity of the uninduced extract as unity.

Spectrophotometric detection of protein-boundFAD

Detection of FAD bound to protein was performed as

described earlier (Myllykallio et al., 2002) by incubating the

protein at 95 1C for 10 min in the dark. Precipitated protein

were pelleted by centrifugation at 10 000 g for 10 min. The

absorption spectra of the released flavin present in the

supernatant was determined spectrophotometrically from

250 to 750 nm in a 1-cm quartz cuvette.

RNR assay

RNR activity was determined by the diphenylamine proce-

dure of Blakley (1966) using ATP as the substrate. A

complete reaction mixture (0.5 mL) contained 0.2 mM ATP,

20 mM dithiothreitol, 10 mM 50-deoxyadenosylcobalamine

(coenzyme B12), 1 mM EDTA, 50 mM potassium phosphate

buffer, pH 7.3, and an appropriate amount of enzyme. The

reaction mixture was incubated for 1 h at 37 1C and the

reaction was terminated by placing the tubes in ice. The

reaction mixtures were treated with chloroacetamide and

subsequently with diphenylamine. Conversion of ATP to

dATP was monitored by recording the A595 nm. In some

cases, deoxyribonucleotides were incorporated in the assay

at desired concentrations to study stimulatory/inhibitory

effects.

Results

Immunodetection experiments

To test whether Gp48 and 50 were at all produced during

phage growth, the total protein extracted from M. smegmatis

L1c1ts cells induced for varying lengths of time was sub-

jected to Western blotting experiments using respective

antisera. The results (Fig. 1a and b) showed that Gp48 or

50 are present at detectable levels in the induced, but not

uninduced, extracts.

Following the demonstration of the presence of these

proteins in extracts of induced lysogenic cells by Western

blotting, immunoprecipitation of 35S-methionine pulse-

labeled proteins was attempted using the respective anti-

bodies. The rationale for this was twofold – first, a positive

CtrlkDa97664329

21

14

Antisera

kDa

kDa

976643292114

9766

43

29

21

0′ 15′ 30′ 60′ Ctrl 0′ 15′ 30′ 60′

Gp48

Gp48

Gp48

Gp49

Gp49

Gp50

Gp50

Gp50

I P I P I P

(a) (b)

(c)

Fig. 1. (a and b) Immunodetection of polypeptides synthesized from

genes 48 and 50 (a and b, respectively) using the corresponding antisera.

(c) Immunoprecipitation using antisera against Gp48, 50 and 49 as

indicated. P and I are abbreviated forms of preimmune and immune,

respectively. The identities of the immuno-precipitated proteins are

indicated by solid arrows. Bands representing unidentified proteins are

indicated by broken arrows. L5 Gp49 was expressed and processed for

the antibody essentially as described in the case of Gp48 and Gp50. In

each blot, the purified protein was incorporated as a control (Ctrl). The

time periods (mins) for which the inductions were performed are shown

on the top. The positions of molecular weight standards are shown.



immunoprecipitation would indicate that these proteins are

synthesized actively during the induced phase of phage

growth, and second, such an experiment might lead to

important associated proteins. The pulse-labeled proteins

obtained from cells induced for 15 min were thus subjected

to immunoprecipitation using either immune or preim-

mune sera against Gp48 and 50, respectively (Fig. 1c). As a

control experiment, immunoprecipitation of a third protein

Gp49 expressed from gene 49 was performed using the

corresponding antisera. The results showed that antisera

against Gp48 immunoprecipitated the corresponding

28 kDa polypeptide (Fig. 1c marked by an arrow). No

immunoprecipitation of this polypeptide, or for that matter

any other polypeptide, was observed using the preimmune

sera. In case of anti-Gp50, similar immunoprecipitation of

the specific target was observed. Interestingly, anti-Gp48

and 50 cross-immunoprecipitated each other’s targets and

also a couple of other proteins (Fig. 1c, marked by broken

arrows). This result indicates that in vivo, Gp48, 50 and

several other unidentified proteins associate to form a

complex. The fact that none of these bands were precipitated

in case of Gp49 gives additional support to the claim that the

precipitation is not artifactual. Moreover, it is unlikely that

coimmunoprecipitation is due to epitope sharing because in

the Western blots, performed at the same dilution of antisera

(Fig. 1a and b) no evidence of aberrant reactivity was found.

Gp48-a mycobacteriophage flavin-dependentthymidylate synthase (FDTS)

In order to characterize Gp48 biochemically, attempts were

first made to express the L5 gene in E. coli. Although a high

expression level was obtained, the protein could not be

recovered in the soluble state. In order to overcome this

difficulty, the D29 gene 48 that shares extensive homology

with the L5 counterpart was expressed in E. coli (Fig. 2a).

Suspecting that the purified protein is an FDTS, the possi-

bility of FAD being tightly bound to the enzyme was

investigated spectrophotometrically. The enzyme prepara-

tion was first heated to release the bound FAD if any and

then the absorption spectra of the supernatant were re-

corded. The resulting spectra revealed discrete peaks that

were characteristic of oxidized FAD, (Fig. 2b). This proved

that the enzyme contained bound FAD confirming that it is

a flavoprotein. ThyX enzymes are known to function

through two different mechanisms: ping-pong and sequen-

tial. In the ping-pong mechanism, the enzyme reduces FAD

in the presence of NADPH in a dUMP-independent manner.

Fig. 2. Characterization of D29 Gp48 ThyX activity: (a) Expression and purification of Hexa Histidine-tagged D29 Gp48 protein, – (sup) represents the

soluble supernatant, while (E) denotes the protein eluted under native conditions. (b) Spectroscopic analysis of the purified protein following

denaturation (solid line). The dotted line represents the typical spectra for FAD. The arrows represent characteristic oxidized flavin peaks at 260, 375 and

450 nm, respectively. (c) NADPH oxidation assay in the presence of varying concentrations of dUMP. The experiment was performed using 2 mM ThyX, in

the presence of 60 mM FAD and 200mM NADPH. The initial velocities were plotted against the concentration of dUMP. (d) Effect of CH2H4folate, the co-

substrate for ThyX. The experiment was performed as in (c), except that the dUMP concentration was fixed at 100 mM (e) Conversion of dUMP to dTMP

by Gp48. Enzyme activity was measured by monitoring the release of tritium from the radiolabeled substrate [5-3H]dUMP. The activities of the enzyme at

different substrate concentrations were plotted according to Michaelis–Menten. Km and Vmax were determined from the corresponding double-

reciprocal plot (not shown). All the enzymatic assays (c, d and e) were run in triplicate and the mean values� SE are shown. In some cases, error bars are

not visible as they are shorter than the height of the symbol.



In the sequential mechanism, however, both dUMP and

NADPH must bind to the enzyme for the reduction of FAD.

In other words, the enzyme is expected to have dUMP-

dependent NADPH oxidase activity. In order to obtain an

insight into which of these mechanisms operates in case of

D29 ThyX-catalyzed reactions, the NADPH oxidase activity

of the protein was assessed in the absence or in the presence

of increasing concentrations of dUMP, maintaining NADPH

at a saturating concentration of 200mM. The results show

that the enzyme is a dUMP-dependent NADPH oxidase

(Fig. 2c). From the corresponding double-reciprocal plot,

the Km could be determined to be about 1mM for dUMP.

The ability of a ThyX to act as a dUMP-dependent NADPH

oxidase has been reported in case of the Paramecium

bursaria chlorella virus (PBCV)-I enzyme. In the above,

assay CH2H4folate was absent. If the same NADPH oxida-

tion assay was performed in the presence of CH2H4folate, a

significant dose-dependent inhibition of the NADPH oxi-

dase activity was observed (Fig. 2d). This indicates that as in

the case of the PBCV-1 enzyme, the CH2H4folate-binding

site of D29 ThyX overlaps with that of the NADPH-binding

site. The ability of the enzyme to convert dUMP to dTMP in

the presence of NADPH and CH2H4folate (200mM each)

was then assayed by the tritium release assay. From the

resulting curve (as shown in Fig. 2e), the Km for dUMP was

estimated to be about 3 mM, which is comparable to the

1 mM value obtained using the NADPH assay. The results,

taken together, indicate that Gp48 acts as an FDTS by the

sequential mechanism as proposed in case of the PBCV-1

enzyme (Graziani et al., 2004).

These investigations were performed with the D29 en-

zyme. However, a soluble glutatione-S-transferase-tagged

version of the L5 protein was studied. The properties of this

fusion protein were similar to that of the D29 protein.

Gp50- a Class II ribonucleotide reductase

In order to characterize Gp50, biochemically the L5 gene

was overexpressed in E. coli and purified (Fig. 3a). To test

whether this enzyme is capable of reducing ribonucleotides

to their deoxycounterparts in a coenzyme B12-dependent

manner, assays were performed in the presence of this

cofactor, using ATP as the substrate. The results show that

this enzyme is capable of reducing ATP to dATP. The activity

was absolutely dependent on coenzyme B12 for its activity.

Omitting this coenzyme resulted in complete loss of activity

(data not shown). This confirms that the reductase belongs

to the class II type. The enzyme was also found to reduce

only triphosphates and not diphosphates. The Km for the

enzymatic reduction of ATP by Gp50 was found to be

0.57 mM (Fig. 3b). Ribonucleotide reductases are known to

be allosterically modulated by the activity of various nucleo-

tides. The effect of various deoxy-nucleotide substrates on

ATP reduction was studied. Only dGTP was found to

stimulate the reduction of ATP (Fig. 3c). The remaining

nucleotides had very little effect. The results suggest that the

protein has properties similar to that of the reductase from

Lactobacillus leichmanii (Booker & Stubbe, 1993).

Thymidylate synthase activity in thermallyinduced M. smegmatis L1c1ts lysogens

In order to investigate the level of thymidylate synthase

activity (ThyA and/or ThyX) during induction of phage,

Fig. 3. Characterization of the RNR activity of Gp50: (a) Expression and

purification of Gp50; – (sup) represents the soluble supernatant and

(E) represents the eluted protein. (b) Lineweaver–Burk plot for the

enzymatic reduction of ATP to dATP. Assays were carried out using 2 mM

enzyme in the presence of 20 mM dithiothreitol, 10mM Coenzyme B12

and varying concentrations of ATP from 0 to 2 mM. Reduction to dATP

was determined by monitoring the absorbance at 595 nm following

treatment with diphenylamine. Initial velocities (D OD595 nm min�1) were

measured at different concentrations of the substrate ATP. (c) Effect of

various deoxyribonucleotides. RNR assays were performed at different

ATP concentrations in the absence (�) or in the presence of nucleotides –

dATP (�), dCTP (&), dGTP (’) and dTTP (D). The nucleotides were used

at a concentration of 0.1 mM.



cell-free extracts of induced M. smegmatis L1c1ts lysogenic

cells were assayed separately for both. To determine ThyA,

activity assays were performed in the absence of FAD and

NADPH. The results (Fig. 4) showed that ThyA activity in

induced extracts decreased steadily relative to the uninduced

extracts (line marked by open circles) whereas the ThyX

activity increased moderately (squares). Both ThyX and

ThyA assays share the same cosubstrate CH2H4folate; there-

fore, in the ThyX assay, ThyA is also expected to contribute

proportionately. In other words, the ThyX assay should give

the total thymidylate synthase activity. Now, because ThyA

activity reduces it may be inferred that the contribution

from ThyX must have increased, as a result of which the

overall activity not only did not decrease but actually

increased to a certain extent. The results indicate that gene

48 expression may be critical for the maintenance of an

adequate supply of thymidylate in a situation where the host

enzyme loses activity.

Discussion

Despite being lysogenic by nature, mycobacteriophage L5 is

known to inactivate its host in a manner reminiscent of lytic

phages (Hatfull & Sarkis, 1993). Although the mechanisms

are not known, it follows that following host inactivation the

phage takes control of the cellular metabolism for its

successful propagation. The central region of the genomes

of mycobacteriophage L5 and the related phage D29 con-

tains several genes that putatively encode enzymes required

for DNA synthesis as well as nucleotide metabolism (Ford

et al., 1998). It is not known as yet whether these proteins do

indeed possess the predicted activities and whether they are

at all expressed. This investigation was initiated to address

these issues in the context of the two genes 48 and 50. It is for

the first time that these genes have been expressed in E. coli

and their enzymatic activities have been confirmed. It has

also been demonstrated that they were detectable only

following induction of the lysogen. This indicates that their

expression is closely linked to the onset of the lytic cycle.

A novel observation presented here is that these proteins

coimmunoprecipitate and are therefore possibly associated

in vivo. Such associations involving thymidylate synthase

and RNR have been reported in case of phage T4 (Allen

et al., 1980), but this is the first time that a similar

phenomenon has been indicated to occur in the context of

mycobacteriophages. Moreover, it may be noted that in case

of T4, the corresponding enzymes are a ThyA-type thymi-

dylate synthase and an aerobic RNR, whereas in this case the

thymidylate synthase is of ThyX type and the RNR is of the

microaerophillic variety. Although the polypeptides Gp48

and 50 coimmunoprecipitated from cell-free extracts, a

similar phenomenon could not be demonstrated using the

purified proteins. This indicates that perhaps these proteins

do not interact directly within the multiprotein complex,

but only through the involvement of other proteins (Kim

et al., 2005).

The ThyX activity reported for the L5/D29 Gp48 protein

appears to be mechanistically distinct from that reported in

case of several other bacterial systems including M. tuberculosis

(Sampathkumar et al., 2005). The M. tuberculosis enzyme

follows a ping-pong mechanism whereas L5/D29 ThyX, like

the PBCV-1 enzyme, functions by the sequential mechan-

ism. To the best of the authors’ knowledge; these are the only

two examples of ThyX operating through the sequential

mechanism. The fact that both are viruses may be coin-

cidental; alternatively, there could be some specific reason

why these unrelated viruses use this type of ThyX.

The timing of the synthesis of the respective proteins

strongly suggests that they have a role to play in the early

stages of the lytic cycle. Evidence that supports such a

possibility has also been presented in the form of thymidy-

late synthase assays using cell-free extracts of induced

lysogens. One of the conclusions of these assays is that ThyA

activity reduces following the onset of the lytic pathway.

This in itself is a novel observation. Understanding how this

occurs could in the long run give clues regarding how

mycobacteriophages inactivate their host. In the ThyX assays

that followed, a marginal increase in activity following

induction was detectable. However, as ThyA is also likely to

make a contribution in the ThyX assay and because ThyA

activity decreases with time, it follows therefore that ThyX

activity increases far more than is evident. The results

strongly indicate that a shift from a ThyA to a ThyX mode

Fig. 4. Thymidylate synthase (TS) activity monitored in cell-free extracts

of Mycobacterium smegmatis L1c1ts cells. Aliquots from extracts of

M. smegmatis L1c1ts induced or uninduced cells having the same

protein concentration were used in the dUMP to dTMP conversion assay

under conditions that yield either ThyA (FAD and NADPH absent) or ThyX

activities (all cosubstrates present). The fold increase (or decrease) in

activity in the case of induced cells relative to the uninduced was plotted

against the induction time. Each data point represents the mean� SE of

at least four independent experiments.



of synthesis occurs as phage growth commences. Whatever

is true for ThyX is also probably true for the phage-encoded

RNR II, although this has not been verified. The results

of this investigation indicate that like other well-known

lytic phages, L5 (and related phages) manipulate host

nucleotide metabolism in such a way as to benefit itself at

the cost of the host.

Acknowledgements

The authors thank N.C. Mandal, A.K. Tyagi and R. McNerney

for the phages L1, L5 and D29, respectively. The project was

funded by a grant from DBT, Government of India. B.B.

and N.G. are grateful to CSIR and UGC, Government of

India for their fellowships. The authors thank P. Halder for

technical assistance.

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Cloning, characterization and expression analysis of nucleotide metabolism-related genes of mycobacteriophage L5