A global response to sulfur starvation in Pseudomonas putida and its relationship to the expression of low-sulfur-content proteins

Aglobal response to sulfur starvation inPseudomonasputida andits relationship to the expressionof low-sulfur-content proteinsColin Scott, Margaret E. Hilton, Christopher W. Coppin, Robyn J. Russell, John G. Oakeshott &Tara D. Sutherland

CSIRO, Entomology, Canberra, ACT, Australia

Correspondence: Colin Scott, CSIRO,

Entomology, GPO Box 1700, Canberra, ACT

2601, Australia. Tel.: 161 2 6246 4090;

fax: 161 2 6246 4173;

e-mail: [email protected]

Received 18 October 2006; revised 2 November

2006; accepted 6 November 2006.

First published online 20 December 2006.

DOI:10.1111/j.1574-6968.2006.00575.x

Editor: Christiane Dahl

Keywords

sulfur assimilation; sulfur starvation;

environmental adaptation.

Abstract

Sulfur is essential for life on Earth, but its availability is limited in many

environments. Here the sulfur-starvation response of the model soil bacterium

Pseudomonas putida KT2440 is shown to be associated with an approximately

fivefold reduction in the total soluble thiol content of the cell. A bioinformatic

survey of the P. putida KT2440 genome identified 646 genes encoding proteins

with a significantly lower than average sulfur content (low sulfur-content proteins,

LSPs), the expression of which may have a role in the global reduction of cellular

thiol content during sulfur starvation. Analysis of the genetic organization of the

LSP-encoding genes showed that 31% were potentially transcriptionally associated

with at least one other gene encoding a protein defined as an LSP. In particular, 55

LSP genes were located in three large clusters, termed low-sulfur islands (LSIs)

here. The predicted identities of the proteins encoded by the LSIs strongly suggest

that the LSIs have a role in acquiring sulfur from organic sulfur sources during

sulfur starvation. This hypothesis was supported by transcription fusion studies on

a limited number of LSP promoters under low-sulfur conditions. In a wider survey

of bacterial species, LSIs were found to be more prevalent in free-living, Gram-

negative bacteria than in Gram-positive or obligately intracellular bacteria.

Introduction

Sulfur is ubiquitously required for life, fulfilling a large and

varied number of roles (Beinert, 2000). It is primarily used

as a component of the amino acids cysteine and methionine

as well as of cellular cofactors including biotin, coenzyme A,

s-adenosylmethionine, thiamine, lipoic acid, molybdopter-

ins and iron–sulfur clusters. Sulfur is also critical for

processes such as redox homeostasis via glutathione and

thioredoxin (Ritz & Beckwith, 2001), transcriptional regula-

tion (Green & Paget, 2004) and translation initiation (via

formyl methionine; Laursen et al., 2005). In addition, the

oxidation of sulfur plays a pivotal role in energy transduc-

tion in many eubacteria and archeae (Friedrich et al., 2005).

In general, the preferred source of sulfur is inorganic

sulfate (Kertesz & Weitek, 2001). However, inorganic sulfur

constitutes only a small proportion (3–6%) of total soil

sulfur reserves (Mclaren et al., 1985). The remainder is

largely in the form of carbon-bound sulfur and ester sulfates

(Fitzgerald, 1976; Scherer, 2001). Soil-dwelling bacteria have

evolved a number of systems that allow the use of these

organic sulfur sources (Kertesz, 1999; Mirleau et al., 2005).

Previous attempts to elucidate the proteins or genes in-

volved in these systems have used both proteomic and

transposon-mutagenesis approaches. Although these have

been somewhat successful, many proteins are below the

limits of detection by 2D gel electrophoresis (Beil et al.,

1995) and substrate redundancy has restricted the usefulness

of transposon mutagenesis. For example, the msu, ssu and

tau operons act on an overlapping range of sulfonates (van

der Ploeg et al., 1996; Kertesz et al., 1999; van der Ploeg et al.,

1999; Kahnert et al., 2000). Therefore, there is limited

genetic information regarding many of the systems involved

in the metabolism of organosulfur compounds. For in-

stance, sulfatase activity has been used for many years as a

diagnostic tool for Mycobacterium identification (Mougous

et al., 2002), yet the genes involved are unknown.

Sulfur-acquisition genes in the soil-dwelling bacterium

Pseudomonas putida have been identified either directly or

by homology with known genes from other organisms.

Characterized directly are the ssu operon, which encodes

proteins involved in transport and desulfurization of aryl

sulfates and sulfate esters (Kahnert et al., 2000), and the asf

operon responsible for the import and liberation of sulfur

FEMS Microbiol Lett 267 (2007) 184–193Journal compilation c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. No claim to original Australian government works

from the aryl sulfonates (Vermeij & Kertesz, 1999). Homo-

logs of the Pseudomonas aeruginosa msu (Kertesz et al.,

1999) and Rhodococcus erythropolis dsz operons (Denome

et al., 1994; Piddington et al., 1995) have been detected.

These operons encode monooxygenases and ABC transport

systems that allow the liberation and use of sulfur from

methanesulfonate (msu) and dibenzothiophene (dsz). Also

implicated are the snfECR and snfFG gene products, which

allow the utilization of dimethylsulfone as a sulfur source

(Endoh et al., 2003a, b, 2005), and proteins encoded by the

tau operon, which confers the ability to release sulfur from

taurine, a naturally occurring sulfonate (Eichhorn et al.,

1997; Vermeij et al., 1999).

Here the response of P. putida to sulfur deprivation was

shown to involve a significant reduction in overall thiol

content, possibly involving protein thiols. Along with ob-

servations by other investigators that the proteins required

for sulfur acquisition from nonpreferred sulfur sources often

contain lower than average proportions of sulfur-containing

amino acids (Kertesz, 2001; van der Ploeg et al., 2001), this

result suggests that the sulfur content of gene products could

be used to identify genes and proteins that might be

involved in the sulfur-starvation response in bacteria. The

validation of such an in silico approach in P. putida and the

results in other bacteria from a range of different environ-

ments are described here.

Materials and methods

Bacterial strains, media and plasmids

Pseudomonas putida KT2440 and Escherichia coli JM109

were routinely grown in L medium (Lennox, 1955) at 28

and 37 1C, respectively. Sulfur-free medium (SFM) with

0.2% glucose as carbon source was produced as described

in Sutherland et al. (2000). Media were supplemented with

200 mg mL�1 ampicillin and sodium sulfate as appropriate.

Ion chromatography was used to quantify the residual

sulfur/sulfate content of SFM, and indicated that 4.6 mM

sulfate was present. A Waters 2695 separations module and

Waters IC-Pack Anion HR 4.6� 75 mm column were used

to separate the anions in the medium with a lithium borate/

gluconate mobile phase. Anions were detected using a

Waters 432 conductivity detector. The data were analysed

using Waters EMPOWER 2 chromatography software.

Putative sulfur responsive promoter regions from P.

putida KT2440 (upstream of the PP5106, PP0173, PP3217,

PP2763, PP0169, PP0174, PP3213, PP2762, PP3223, PP5904

and lsfA genes; see below), corresponding to approx. 800 bp

upstream and 200 bp downstream from predicted initiator

methionine codons, were amplified by PCR and fragments

were cloned into the P. putida/E. coli shuttle plasmid pQF50

(Farinha & Kropinski, 1990; kindly provided by Dr Max

Schobert, Technische Universitat Braunschweig) to produce

b-galactosidase reporter plasmids. The lsfA promoter was

also cloned into pQF50 as a lacZ : fusion as a positive

control, as expression of the lsfA gene is known to be up-

regulated by sulfur starvation (Kahnert et al., 2000). Pseu-

domonas putida was transformed by electroporation (Cho

et al., 1995).

b-Galactosidase, protein and thiol assays

The b-galactosidase assays were carried out according to the

method of Miller (1972) with three independent biological

replicates for each sample. Recombinant P. putida cultures

were grown in L medium, and cells were washed in SFM and

used to inoculate (equivalent to 1 : 200 inoculum) SFM

supplemented with 10 mM or 10 mM sodium sulfate. Cul-

tures were grown to an OD600 nm of 0.2–0.3. Cell-free

extracts were obtained by sonication using a Branson

Sonifier 250, and cell debris was removed by centrifugation.

Protein concentrations were estimated using the Bio-Rad

protein assay dye (Bio-Rad). Total soluble cell sulfhydryl

concentrations (including protein thiols, glutathione, cy-

steine, homocysteine and all other free SH-groups) were

estimated according to Thelander (1973).

Bioinformatics

The percentage and distribution of sulfur in proteins

predicted from the genome sequences of the following 31

bacterial species (27 genera and 19 families; GenBank) were

calculated: Agrobacterium tumefaciens str. C58 (Wood et al.,

2001), Bacillus anthracis str. Ames (Read et al., 2003),

Bacillus subtilis ssp. subtilis 168 (Kunst et al., 1997), Bifido-

bacterium longum NCC2705 (Schell et al., 2002), Buchnera

aphidicola str. Bp (Baizongia pistaciae) (Van Ham et al.,

2003), Campylobacter jejuni ssp. jejuni NCTC 11168 (Park-

hill et al., 2000), Chlamydophila pneumoniae AR39 (Read

et al., 2000), Corynebacterium efficiens YS-314 (Nishio et al.,

2003), E. coli O157 : H7 EDL933 (Perna et al., 2001),

Helicobacter hepaticus ATCC 51449 (Suerbaum et al., 2003),

Lactobacillus plantarum WCFS1 (Kleerebezem et al., 2003),

Mycobacterium tuberculosis CDC1551 (Fleischmann et al.,

2002), Mesorhizobium loti (Kaneko et al., 2000), Mycoplasma

genitalium (Fraser et al., 1995), P. aeruginosa PA01 (Stover

et al., 2000), P. putida KT2440 (Nelson et al., 2002),

Pseudomonas syringae pv. tomato str. DC3000 (Buell et al.,

2003), Ralstonia solanacearum (Salanoubat et al., 2002),

Salmonella enterica serovar Typhi (Deng et al., 2003),

Shigella flexneri 2a str. 301 (Jin et al., 2002), Sinorhizobium

meliloti (Galibert et al., 2001), Staphylococcus aureus subsp

aureus MW2 (Baba et al., 2002), Streptomyces avermitilis

MA-4680 (Omura et al., 2001), Tropheryma whipplei str

twist (Raoult et al., 2002), Vibrio parahaemolyticus RIMD

2210633 (Makino et al., 2003), Vibrio vulnificus CMCP6

FEMS Microbiol Lett 267 (2007) 184–193 Journal compilation c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. No claim to original Australian government works

185Sulfur content of Pseudomonas putida proteins

(Kim et al., 2003), Wigglesworthia glossinidia (endosymbiont

of Glossina brevipalpis; Akman et al., 2002), Wolinella

succinogenes (Baar et al., 2003), Xanthomonas campestris pv.

campestris str. ATCC 33913 (de Silva et al., 2002), Xylella

fastidiosa strain Temecula1 (Van Sluys et al., 2003) and

Yersinia pestis KIM (Deng et al., 2002). Analysis of P. putida

gene function was conducted using the comprehensive

microbial resource (CMR) database at the Institute for

Genomic Research (TIGR) (Peterson et al., 2001).

Results and discussion

Pseudomonas putida responds to sulfurstarvation by reducing cellular thiolconcentration

To explore the relationship between sulfur availability and

free-thiol concentration on a whole organism scale, P. putida

was grown in a medium replete or restricted in inorganic

sulfate; the total soluble thiol concentration of each sample

was then determined. The cellular thiol concentration was

corrected for cell density by comparing it with the cellular

protein concentration. The [SH] : [protein] ratio differed by

approximately fivefold in medium that had been supple-

mented with between 1mM and 10 mM sulfate (Fig. 1). This

suggests that part of the response to reduced sulfate avail-

ability involves a reduction in total cellular thiol concentra-

tion. Although changes to the concentrations of other

cellular thiols (e.g. glutathione) undoubtedly influence the

adaptive reduction in total thiol concentration, it seems

likely that the sulfur content of the proteome plays a

significant role in this response to sulfur starvation. Indeed,

it has been observed that proteins involved in the acquisition

and utilization of low-preference sulfur sources generally

contain disproportionately low levels of sulfur-containing

amino acids (Kertesz, 2001; van der Ploeg et al., 2001).

The P. putida genome encodes many low-sulfurproteins

Low-sulfur proteins (LSPs) encoded by P. putida genes were

sought by in silico analysis of the P. putida KT2440 genome.

LSPs were defined as proteins that have a sulfur-containing

amino acid content one SD or more lower than the average

after removal of the initiating methionine (as the vast

majority of encoded proteins contain a formylmethionine

initiation codon, it was considered that it was not subject to

the same selective pressures as noninitiating methionines).

The sulfur-containing amino acid contents of predicted

P. putida KT2440 proteins (of greater than 50 amino acid

residues) are distributed normally about a mean of 3.13%

sulfur-containing amino acids per protein, with a SD of

1.34% (supplementary Appendix S1). Of the 5421 predicted

proteins, 11.9% (646 predicted proteins, fully listed in

Appendix S1) were defined as LSPs.

Over 50% of the LSPs had no known functions (Table 1),

in comparison with 30% for the whole P. putida KT2440

proteome (www.tigr.org). Thirty-four of the LSPs were

among the 88 predicted proteins so far annotated as being

involved in sulfur metabolism. Among these LSPs were the

0

5

10

15

20

25

30

[Sulphate] (M)

[SH

]:[P

rote

in] (

µmol

µg−1

)

10−7 10−6 10−5 10−4 10−3 10−2 10−1

Fig. 1. The sulfhydryl : protein ratio of Pseudomonas putida total soluble

cell-free extract grown in SFM supplemented with 1 mM–10 mM sodium

sulfate. These results are the average of three independent samples;

error bars indicate SDs.

Table 1. Functional classification of LSP genes of Pseudomonas putida

Cellular function

No. of

genes

Putative/unknown function 339 (52.4)

Transport 98 (15.1)

Transcription/transcription regulation 47 (7.3)

Central metabolism/energy

transduction/cofactor biosynthesis

39 (6.0)

Cell envelope 34 (5.3)

DNA/nucleotide metabolism 28 (4.3)

Protein fate 11 (1.7)

Taxis/mobility 11 (1.7)

Cellular processes 16 (2.5)

Amino acid metabolism 10 (1.5)

Protein synthesis 8 (1.2)

Phage 5 (0.8)

Total 646 (99.8)

The percentage of genes in each functional category is indicated in

parentheses. Functional categories are based on the annotations in the

TIGR-CMR database.


186 C. Scott et al.

products of msu, ssu, tau and dsz gene operons, cystine-

specific ABC transporters, GST transferase, and sulfate and

thiosulfate transporters (Table 2). Interestingly, five of the

predicted LSPs are annotated as uncharacterized monoox-

ygenases (PP1929, 3218, 3219, 3529 and 3573), which could

potentially be involved in currently undescribed organosul-

fur utilization systems.

Known sulfur-metabolizing proteins that are not LSPs

include genes involved in cysteine and methionine biosynth-

esis and several previously characterized sulfatases. Many of

these are expected to have roles outside the sulfur-starvation

response. Biosynthesis of the sulfur-containing amino acids

is not regulated by sulfur abundance per se, but rather by the

abundance of those amino acids (Sekowska et al., 2000).

Sulfatases play many roles aside from scavenging of sulfur

during sulfur starvation. For example, there is a high-sulfur-

content sulfatase from a Pseudomonas species involved in

carbon assimilation from the sulfate ester SDS (Davison

et al., 1992). Sulfatases have also been proposed to act in

regulatory roles. In particular, sulfatases from Rhizobia and

Mycobacteria are thought to be involved in the modulation

of extracellular processes such as cell adhesion and receptor/

ligand binding that mediate host–pathogen interactions

(Mougous et al., 2006).

Although the low cysteine and methionine content of

LSPs is consistent with a role of the proteins during sulfur

starvation, there could be unrelated functional constraints

upon their sulfur contents. For example, a quarter of the

LSPs identified as transporters imported metal ions includ-

ing iron, manganese, nickel, zinc, cadmium and cobalt, and

Table 2. Genes encoding proteins with a known role in sulfur metabolism in Pseudomonas putida identified as LSPs in this study

Gene Encoded protein

PP0169� Dioxygenase, TauD/TfdA family (Pseudomonas entomophila, 98%)

PP0222� Monooxygenase, DszA family (Pseudomonas entomophila, 89%)

PP0223� Monooxygenase, DszC family (Pseudomonas entomophila, 84%)

PP0224� Monooxygenase, DszC family (Pseudomonas fluorescens Pf-5, 78%)

PP0225� Cystine ABC transporter, ATP-binding protein, putative (Pseudomonas entomophila L48, 96%)

PP0227� Cystine ABC transporter, periplasmic cysteine-binding protein, putative (Pseudomonas entomophila L48, 94%)

PP0228� Serine O-acetyltransferase, putative (Pseudomonas syringae pv. phaseolicola 1448A, 89%)

tauD Alpha-ketoglutarate-dependent taurine dioxygenase (P. putida DS1, 98%)

tauC Taurine ABC transporter, permease protein (P. putida DS1, 93%)

tauB Taurine ABC transporter, ATP-binding protein (P. putida DS1, 98%)

tauA Taurine ABC transporter, periplasmic taurine-binding protein (Pseudomonas fluorescens Pf-5, 85%)

lsfA Antioxidant protein LsfA (Pseudomonas entomophila L48, 97%)

ssuA Sulfonate ABC transporter, periplasmic sulfonate-binding protein SsuA (P. putida DS1, 97%)

ssuE FMN reductase (P. putida DS1, 93%)

ssuD Organosulfonate monooxygenase (P. putida DS1, 98%)

ssuC Sulfonate ABC transporter, permease protein SsuC (P. putida DS1, 98%)

ssuB Sulfonate ABC transporter, ATP-binding subunit SsuB (P. putida DS1, 94%)

selB Selenocysteine-specific translation elongation factor (Pseudomonas entomophila L48, 86%)

PP1162� Glutathione S-transferase family protein (Pseudomonas entomophila L48, 87%)

csdA� Cysteine sulfinate desulfinase (Pseudomonas entomophila L48, 89%)

PP2474� Glutathione S-transferase family protein (Erwinia carotovora ssp. atroseptica SCRI1043, 60%)

msuE� NADH-dependent FMN reductase MsuE (P. putida F1, 99%)

msuD� Sulfonate monooxygenase MsuD, putative (P. putida F1, 99%)

PP2933� Glutathione S-transferase family protein (P. putida F1, 98%)

PP3136� Serine O-acetyltransferase (P. putida F1, 98%)

PP3217� Periplasmic aliphatic sulfonate-binding protein, putative

PP3219� Alkansulfonate monooxygenase, putative (Azotobacter vinelandii AvOP, 72%)

PP3228� Periplasmic aliphatic sulfonate-binding protein, putative (Azotobacter vinelandii AvOP, 72%)

PP3229� Periplasmic aliphatic sulfonate-binding protein, putative (Pseudomonas aeruginosa PACS2, 73%)

tpx Thiol peroxidase (P. putida F1, 99%)

PP3637� Sulfonate ABC transporter, ATP-binding protein (P. putida F1, 98%)

PP4305� Periplasmic thiosulfate-binding protein (P. putida F1, 98%)

PP4637� 5-Methyltetrahydropteroyltriglutamate-homocysteine S-methyltransferase family protein (P. putida F1, 96%)

rhdA-2 Thiosulfate sulfurtransferase (P. putida F1, 96%)

serB Phosphoserine phosphatase (P. putida F1, 98%)

PP5118� Thiosulfate sulfurtransferase, putative (P. putida F1, 97%)

cysP Sulfate ABC transporter, periplasmic sulfate-binding protein (P. putida F1, 98%)

�Annotation is based on homology only. The closest homolog identified using a BLAST search, and its identity score are indicated.



a low cysteine content may be essential for correctly co-

ordinating the relevant ions. Equally, there are 40 LSP

transcriptional regulators and 10 antioxidant LSPs (the

majority of the 16 LSPs annotated as having a role in

‘cellular processes’; Table 1) that may require a lower than

average cysteine content to prevent miscoordination of

ligands or oxidative inactivation of the proteins. However,

these arguments fail to explain the lower than average

methionine content of these proteins, or the presence of

functionally equivalent proteins in E. coli that do not fulfil

the low sulfur requirements of an LSP (the zinc transporter

ZntA and the peroxide stress regulator OxyR, for example;

Rensing et al., 1997, Paget & Buttner, 2003). This suggests

that many of the LSPs may be expressed during sulfur

starvation, and that the sulfur-starvation response impacts

upon many cellular processes.

Many of the LSPs in P. putida are geneticallyassociated

Overall, 201 LSPs (31%) could potentially be transcribed

with at least one neighboring LSP gene encoded on the same

strand and therefore could belong to the same operon

(Appendix S1). Three larger clusters of genes encoding LSPs

were also found. These three ‘low-sulfur islands’ (LSIs),

defined as runs of at least eight out of 10 genes encoding

proteins with low-sulfur content, account for 8.8% of the P.

putida LSPs (57 genes). The composition of the LSIs is

consistent with a direct involvement in acquisition of sulfur

from nonpreferred sulfur sources. They are comprised of

transporters and catalytic enzymes – 21 of which have

previously been implicated in the assimilation of sulfur from

organosulfate/sulfonates (Table 3). The clustering of genes

encoding proteins involved in the metabolism of low-

preference sulfur sources could facilitate a coordinated

response to sulfur starvation.

LSI1 is homologous to the tauD-ssuF region of P. putida

DS1 which encodes proteins of the tau, dsz and ssu operons

(Endoh et al, 2003a, b). LSI2 encodes proteins with homol-

ogy to the msu operon of P. aeruginosa PAO1 (Kertesz et al.,

1999) in addition to a number of other genes with functions

consistent with organosulfur utilization. LSI3 is yet to be

characterized, but is predicted to encode proteins consistent

with the liberation of sulfur from organic sulfonates, in-

cluding transporter proteins with homology to sulfonate

transporters (PP3217, 3220, 3221, 3222, 3223, 3228 and

3229), a monooxygenase of the NtaA/SnaA/SoxA family

(PP3218), a predicted alkanesulfonate monooxygenase

(PP3219), an acyl-coA dehydrogenase somewhat related to

the P. putida DS1 DszC protein (28%) and a LysR-type

regulator (PP3227). Islands of genes involved in sulfur

metabolism have previously been noted in E. coli (Rocha

et al., 2000), although the proteins encoded by those ‘sulfur

islands’ were not observed to deviate significantly in sulfur

composition from the average for E. coli. It seems likely then

that LSIs are a subset of these sulfur islands that have a

specialized role in obtaining sulfur from nonpreferred

sources.

LSPs are generally expressed in response tosulfur starvation

To test the hypothesis that LSPs identified by the in silico

analysis of the P. putida KT2440 genome were subject to

sulfur-mediated regulation, 10 LSP promoters were selected

randomly (without prior reference to gene names or anno-

tations) and tested for activity during sulfur starvation. The

promoter regions were cloned into the broad-host range

reporter plasmid pQF50 (Farinha & Kropinski, 1990) to

drive expression of b-galactosidase. As a positive control, the

lsfA promoter (Kahnert et al., 2000) was cloned into the

same vector. The activity of the promoters was then assessed

at replete and limited sulfate concentration (Fig. 2). Un-

supplemented SFM was unable to support sufficient growth

of P. putida (data not shown); therefore, SFM was supple-

mented with 10 mM sodium sulfate to produce sulfate-

limited medium and 10 mM for sulfate-replete medium.

Three of the promoters were inactive under the conditions

tested. Of the seven other promoters, six demonstrated a

differential response to the concentration of sulfate, all with

greater activity at the lower sulfate concentration. As ex-

pected, the lsfA promoter also showed such a differential

response dependent upon the sulfate content of the med-

ium.

The promoters that exhibited a sulfate concentration-

dependent response were associated with three hypothetical

proteins (PP5106, PP2763 and PP0174; www.tigr.org), a

putative transcriptional regulator (PP0173), a putative sul-

fonate-binding protein (PP3217) and a predicted TauD-like

dioxygenase (PP0169) (Fig. 2). Only PP3217 and PP0169

have previously been implicated as having a function related

to sulfur metabolism. PP3213, the one which was not sulfate

responsive, was predicted to drive the expression of a

sulfonate-specific ABC-type transport protein.

Two of the six sulfate-responsive promoters were asso-

ciated with LSIs; promoter PP2763 with LSI2 and promoter

PP3217 with LSI3.

LSIs in other species

A survey of LSPs in an additional 30 bacterial species

revealed LSIs: of similar composition, but diverse genetic

organization, in six of the 24 free-living bacterial species

(Agrobacterium tumefaciens, P. aeruginosa, P. syringae, Ral-

stonia solanacearum, Mesorhizobium loti and Sinorhizobium

meliloti; Table 3). Two other genomes from free-living

species (Bacillus subtilis and Salmonella enterica) contain an


188 C. Scott et al.

Table 3. Organization of LSIs in seven bacterial species

Bacterium LSI

Pseudomonas putida (LSI1)w PP_0220(� , ABC transporter), 0221(� , ABC transporter), 0222(� , DszA�), 0223(� , acyl-coA dehydrogenase�),

0224(� , acyl-coA dehydrogenase�), 0225(� , ABC transporter�), 0226(� , ABC transporter�), 0227(� , ABC

transporter�), 0228(1, serine O-acetyltransferase), 0229(1, transport protein), 0230(� , dioxygenase�), 0231

(� , TauC�), 0232(� , TauB�), 0233(� , TauA�), 0234(1, porin), 0235(1, LsfA), 0236(1, oxidoreductase�), 0237

(1, SsuA�), 0238(1, SsuD�), 0239(1, SsuC�), 0240(1, SsuB�)

Pseudomonas putida (LSI2) PP_2751(1, ABC transporter), 2752(1, ABC transporter), 2753(1, ABC transporter), 2754(1, porin), 2755

(1, acyl-coA dehydrogenase), 2756(1, unknown), 2757(1, ABC transporter), 2758(1, ABC transporter), 2759

(1, ABC transporter), 2760(1, ABC transporter), 2761(1, ABC transporter), 2762(� , acyl-coA dehydrogenase),

2763(1, unknown), 2764(1, MsuE�), 2765(1, MsuD�), 2766(1, long-chain-fatty-acid–CoA ligase), 2767

(1, ABC transporter), 2768(1, ABC transporter), 2769(1, ABC transporter), 2770(1, ABC transporter), 2771

(� , transcriptional regulator), 2772(� , monooxygenase)

Pseudomonas putida (LSI3) PP_3216(� , unkown), 3217(1, periplasmic binding-protein�), 3218(1, monooxygenase�), 3219(� ,

monooxygenase�), 3220(� , ABC transporter), 3221(� , ABC transporter), 3222(� , ABC transporter), 3223

(� , ABC transporter), 3224(� , aldolase), 3225(� , dehydrogenase), 3226(� , acyl-coA dehydrogenase), 3227

(� , transcriptional regulator�), 3228(1, periplasmic-binding protein�), 3229(1, periplasmic-binding protein�)

Pseudomonas aeruginosa PA2594(� , ABC-transpoter�), 2595(� , ABC-transpoter�), 2596(� , ABC-transpoter�), 2597(� , acyl-CoA

dehydrogenase), 2598(� , monooxygenase�), 2599(� , monooxygenase�), 2600(� , unkown�), 2601

(� , transcriptional regulator�), 2602(1, dioxygenase), 2603(1, thiosulfate sulfurtransferase�)

Sinorhizobium meliloti

(plasmid pSymA)

Sma2049(1, transcriptional regulator), 2051(1, lipid desaturase), 2053(1, MocE), 2055(1, unknown), 2067

(� , sulfonate-binding protein), 2069(� , ABC transporter), 2071(� , unknown), 2073(� , acyl-coA dehydrogenase ),

2075(� , unknown), 2077(� , oxidoreductase), 2079(� , ABC transporter), 2081(� , ABC transporter), 2082

(� , ABC transporter�), 2085(� , ABC transporter�), 2087(1, TRAP transporter�), 2089(1, acyl-CoA

dehydrogenase�), 2091(1,acyl-CoA dehydrogenase�), 2093(1, ABC transporter), 2095(1, isoflavone reductase),

2097(1, monooxygenase), 2099(1, NADH-flavin reductase�), 2101(1, monoxygenase)

Mesorhizobium loti Mlr5216(1, MsuD�), 5217(1, unknown), 5217(1, unkown�), 5218(1, monooxygenase), 5219(1, ABC transporter),

5220(1, ABC transporter), 5222(1, ABC transporter), 5223(1, ABC transporter), 5224(1, ABC transporter), 5225

(1, acyl-coA dehydrogenase�), 5227(1, MsuE�)

Ralstonia solanacearum Rsc1336(1, ABC transporter), 1337(1, oxidoreductase), 1338(1, periplasmic-binding protein), 1339(1,

monooxygenase) 1340(1, ABC transporter), 1341(1, ABC transporter), 1342(1, SsuF), 1343(1, unknown), 1344

(1, esterase), 1345(1, ABC transporter), 1346(1, ABC transporter), 1347(1, ABC transporter), 1348(1, cysB)

Pseudomonas syringae PSPTO5178(� , serine O-acetyltransferase), 5179(1, deaminase), 5180(1, ABC transporter�), 5181(1, ABC

transporter), 5182(1, ABC transporter), 5183(1, unknown), 5184(1, acyl-CoA dehydrogenase), 5185(1, acyl-CoA

dehydrogenase�), 5186(1, monooxygenase�), 5187(1, ABC transporter), 5188(1, ABC transporter), P5189(1, ABC

transporter), 5190(1, unknown), 5191(� , RND transporter), 5192(� , RND transporter), 5193(� , RND transporter),

5194(� , unknown), 5195(� , ABC transporter�), 5196(� , ABC transporter�), 5197(� , ABC transporter�), 5918

(� , TauD�)

Agrobacterium tumefaciens Atu3415(� , ABC transporter), 3416(� , ABC transporter), 3417(� , ABC transporter), 3418(� , acetyltransferase),

3419(� , hydrolase), 3420(� , monooyxygenase�), 3421(� , monooxygenase), 3422(1, unknown), 3423(1,

unkown), 3424(� , acyl-coA dehydrogenase ), 3425(� , monooxygenase), 3426(� , SsuD�), 3427(1, unknown),

3428(� , unkown), A3429(1, unknown), 3430(1, SsuD�), 3431(� , acyl-coA dehydrogenase), 3432(� , TauA�),

3433(� , ABC transporter), 3434(1, ABC transporter), 3435(1, ABC transporter), 3436(1, ABC transporter), 3437

(1, ABC transporter), 3438(1, oxidoreductase�), 3439(1, acyl-coA dehydrogenase�), 3440(1, transcriptional

regulator), 3441(� , ABC transporter), 3442(� , ABC transporter), 3443(1, unknown), 3444(� , ABC transporter),

3445(� , hydrolase), 3446(� , monooxygenase), 3447(� , isoflavone reductase), 3448(� , dszB�), 3449

(� , unknown), 3450(1, monooxygenase�), 3451(� , monooxygenase)

Agrobacterium tumefaciens

(plasmid AT)

Atu5518(� , monooxygenase), 5519(1, unkown), 5520(� , acyl-coA dehydrogenase), 5521(� , ABC transporter),

5522(� , transport protein), 5523(� , ABC transporter), 5524(� , ABC transporter), 5525(� , ABC transporter),

5526(� , monooxygenase�), 5527(� , oxidoreductase�)

Agrobacterium tumefaciens

(plasmid Ti)

Atu6077(� , monooxygenase�), 6078(� , monooxygenase�), 6079(� , monooxygenase�), 6080(1, transcriptional

regulator�), 6081(1, unknown), 6082(1, AAA-ATPase), 6083(1, transcriptional regulator�), 6084

(1, monooxygenase�),6085(1, ABC transporter�)

�Gene products with implied function in sulfur assimilation from organosulfate/sulfonates.wHomologous to the tauD-ssuF region of Pseudomonas putida DS1.

The direction of transcription and predicted function of the gene product for each gene are given in parentheses. LSPs are denoted in italics.



LSI; but, unlike the LSIs in other bacteria, these appear to

encode phage-related proteins rather than sulfur acquisition

proteins. The remaining 18 genomes, including all of the

obligately intracellular bacteria (Wigglesworthia glossinidia,

Buchnera aphidicola, Chlamydophila pneumoniae, Mycoplas-

ma genitalium, T. whipplei and Wolinella succinogenes),

along with Campylobacter jejuni, Shigella flexneri, Coryne-

bacterium efficiens, L. plantarum, H. hepaticus, Bifidobacter-

ium longum, V. vulnificus, Xanthomonas campestris, Y. pestis,

Mycobacterium tuberculosis, E. coli, Bacillus anthracis, Vibrio

parahaemolyticus, Streptomyces avermitilis and Staphylococ-

cus aureus contain no LSIs at all.

That the obligate endosymbiotes/pathogens contain no

LSIs may be a result of the smaller genomes of these

intracellular organisms, the largest analysed here being the

Wolinella succinogenes genome at 2.11 Mbp, as compared to

the 3.72 Mbp of the smallest genome with an associated LSI

(Ralstonia solanacearum). Alternatively, the absence of LSIs

in obligately intracellular bacteria could suggest that the

presence of LSIs is dependent upon the lifestyle of the

bacterial species. Soil-borne bacteria are frequently required

to acquire sulfur from a diverse range of recalcitrant sources,

such as organic sulfonates, which necessitate the presence of

a large number of tightly regulated genes whose expression

is sulfur-source specific. As intracellular organisms are

exposed to a small and less variable set of sulfur sources

they would not require the large array of highly regulated

sulfur-acquisition mechanisms used by soil-associated bac-

teria. Although the sample of organisms analysed is limited,

it is tempting to speculate that the presence of LSIs in a

genome is dependent on the lifestyle of the organism in

question. It is also intriguing that LSIs involved with sulfur

acquisition are not found in the Gram-positive soil bacteria

investigated here. Although the sample size here is small, this

observation suggests that genetic organization of sulfur

acquisition genes into LSIs may be restricted to Gram-

negative soil bacteria, although the reason for this difference

is not clear.

In summary, it is apparent that the data-mining approach

described here has revealed not only a number of potentially

novel sulfur-metabolism-related genes but also a high level

of organization of some of those genes involved in sulfur

acquisition into LSIs. The presence of LSIs containing

sulfur-acquisition genes is not unique to P. putida, and there

0

500

1000

1500

2000

2500

lsfA

0

200

400

600

PP

0169

PP

0173

PP

0174

PP

2762

PP

2763

PP

3213

PP

3217

PP

3223

PP

5094

PP

5106

Pla

smid

onl

y

β- g

alac

tosi

dase

act

ivity

(M

iller

uni

ts)

Fig. 2. Response of predicted sulfur-responsive Pseudomonas putida KT2440 promoters to sulfate concentration. The b-galactosidase activity from P.

putida KT2440 expressing the lacZ gene from the putative promoters of 10 predicted sulfur-responsive genes (gene designations indicated) grown in

defined, SFM supplemented with 10 mM (white) and 10 mM (light gray) sodium sulfate. A plasmid-only control and positive control (lsfA) have been

included. These results are the average of three independent samples; error bars indicate SDs.


190 C. Scott et al.

is evidence to suggest that the presence of LSIs in a genome

may depend on the lifestyle and phylogeny of the bacterium

in question.

Acknowledgements

We thank Dr Max Schobert of Institut fuer Mikrobiologie at

the Technische Universitaet, Braunschweig for kindly pro-

viding the pQF50 reporter plasmid and Dr Anthony Herlt of

the Research School of Chemistry at the Australian National

University, Canberra for his help with the ion chromatogra-

phy of the SFM.

References

Akman L, Yamashita A, Watanabe H et al. (2002) Genome

sequence of the endocellular obligate symbiont of tsetse flies,

Wigglesworthia glossinidia. Nature Genet 32: 402–407.

Baar C, Eppinger M, Raddatz G et al. (2003) Complete genome

sequence and analysis of Wolinella succino genes. Proc Natl

Acad Sci USA 100: 11690–11695.

Baba T, Takeuchi F, Kuroda M et al. (2002) Genome and virulence

determinants of high virulence community-acquired MRSA.

Lancet 359: 1819–1827.

Beil S, Kehrli H, James P, Staudenmann W, Cook AM, Leisinger T

& Kertesz MA (1995) Purification and characterization of the

arylsulfatase synthesized by Pseudomonas aeruginosa PAO

during growth in sulfate-free medium and cloning of the

arylsulfatase gene (atsA). Eur J Biochem 229: 385–394.

Beinert H (2000) A tribute to sulfur. Eur J Biochem 267:

5657–5664.

Buell CR, Joardar V, Lindeberg M et al. (2003) The complete

genome sequence of the Arabidopsis and tomato pathogen

Pseudomonas syringae pv. tomato DC3000. Proc Natl Acad Sci

USA 100: 10181–10186.

Cho JH, Kim EK & So JS (1995) Improved transformation of

Pseudomonas putida KT2440 by electroporation. Biotechnol

Tech 9: 41–44.

Davison J, Brunel F, Phanopoulos A, Prozzi D & Terpstra P (1992)

Cloning and sequencing of Pseudomonas genes determining

sodium dodecyl sulfate biodegradation. Gene 114: 19–24.

Deng W, Burland V, Plunkett G III et al. (2002) Genome sequence

of Yersinia pestis KIM. J Bacteriol 184: 4601–4611.

Deng W, Liou SR, Plunkett G III et al. (2003) Comparative

genomics of Salmonella enterica serovar Typhi strains Ty2 and

CT18. J Bacteriol 185: 2330–2337.

Denome SA, Oldfield C, Nash LJ & Young KD (1994)

Characterisation of the desulfurization genes from

Rhodococcus sp. strain IGTS8. J Bacteriol 176: 6707–6716.

de Silva AC, Ferro JA, Reinach FC et al. (2002) Comparison of the

genomes of two Xanthomonas pathogens with differing host

specificities. Nature 417: 459–463.

Eichhorn E, van der Ploeg JR, Kertesz MA & Leisinger T (1997)

Characterization of alpha-ketoglutarate-dependent taurine

dioxygenase from Escherichia coli. J Biol Chem 272:

23031–23036.

Endoh T, Kasuga K, Horinouchi M, Yoshida T, Habe H, Nojiri H

& Omori T (2003a) Characterization and identification of

genes essential for dimethyl sulfide utilization in Pseudomonas

putida strain DS1. Appl Microbiol Biotechnol 62: 83–91.

Endoh T, Habe H, Yoshida T, Noriji H & Omori T (2003b) A

CysB-regulated and s54-dependent regulator, SnfR, is essential

for dimethyl sulfone metabolism of Pseudomonas putida strain

DS1. Microbiology 149: 991–1000.

Endoh T, Habe H, Noriji H, Yamane H & Omori T (2005) The

s54-dependent transcriptional activator SfnR regulates the

expressoin of the Pseudomonas putida sfnFG operon

responsible for dimethyl sulfone utilization. Mol Microbiol 55:

897–911.

Farinha MA & Kropinski AM (1990) Construction of broad-host

range plasmid vectors for easy visible selection and analysis of

promoters. J Bacteriol 172: 3496–3499.

Fitzgerald JW (1976) Sulfate ester formation and hydrolysis: a

potentially important yet often ignored aspect of the sulfur

cycle of aerobic soils. Bacteriol Rev 40: 698–721.

Fleischmann RD, Alland D, Eisen JA et al. (2002) Whole genome

comparison of Mycobacterium tuberculosis clinical and

laboratory strains. J Bacteriol 184: 5479–5490.

Fraser CM, Gocayne JD, White O et al. (1995) The minimal gene

complement of Mycoplasma genitalium. Science 270: 397–403.

Friedrich CG, Bardischewsky F, Rother D, Quentmeier A &

Fischer J (2005) Prokaryotic sulfur oxidation. Curr Opin

Microbiol 8: 253–259.

Galibert F, Finan TM, Long SR et al. (2001) The composite

genome of the legume symbiont Sinorhizobium meliloti.

Science 293: 668–672.

Green J & Paget MS (2004) Bacterial redox sensors. Nat Rev


Jin Q, Yuan Z, Xu J et al. (2002) Genome sequence of Shigella

flexneri 2a: insights into pathogenicity through comparison

with genomes of Escherichia coli K12 and O157. Nucleic Acids

Res 30: 4432–4441.

Kahnert A, Vermeij P, Weitek C, James P, Leisinger T & Kertesz

MA (2000) The ssu locus plays a key role in organosulfur

metabolism in Pseudomonas putida S-313. J Bacteriol 182:

2869–2878.

Kaneko T, Nakamura Y, Sato S et al. (2000) Complete genome

sequence of the nitrogen fixing symbiotic bacterium

Mesorhizobium loti. DNA Res 6: 331–338.

Kertesz MA (1999) Riding the sulfur cycle-metabolism of

sulfonates and sulfate esters in gram-negative bacteria. FEMS

Microbiol Rev 24: 135–175.

Kertesz MA, Schmidt-Larbig K & Wuest T (1999) A novel

reduced flavin mononucleotide-dependent methanesulfonate

sulfonatase encoded by the sulfur-regulated msu operon of

Pseudomonas aeruginosa. J Bacteriol 181: 1464–1473.

Kertesz MA (2001) Bacterial transporters for sulfate and

organosulfur compounds. Res Microbiol 152: 279–290.



Kertesz MA & Weitek C (2001) Desulfurization and

desulfonation: applications of sulfur-controlled gene

expression in bacteria. Appl Microbiol Biotechnol 57: 460–466.

Kim YR, Lee SE, Kim CM et al. (2003) Characterisation and

pathogenic significance of Vibrio vulnificus antigens

preferentially expressed in sepinemic patients. Infect Immun

71: 5461–5471.

Kleerebezem M, Boekhorst J, van Krannenburg R et al. (2003)

Complete genome sequence of Lactobacillus plantarum

WCFS1. Proc Natl Acad Sci USA 100: 1990–1995.

Kunst F, Ogasawara N, Moszer I et al. (1997) The complete

genome sequence of the Gram-positive bacterium Bacillus

subtilis. Nature 390: 249–256.

Laursen BS, S�rensen HP, Mortensen KK & Sperling-Petersen HU

(2005) Initiation of protein synthesis in bacteria. Microbiol

Mol Biol Rev 69: 101–123.

Lennox ES (1955) Transduction of linked genetic characters by of

host by bacteriophage P1. Virology 1: 190–206.

Makino K, Oshima K, Kurokawa K et al. (2003) Genome

sequence of Vibrio parahaemolyticus: a pathogenic mechanism

distinct from V. cholerae. Lancet 361: 743–749.

McLaren RG, Keer JI & Swift RS (1985) Sulfur transformations in

soils using sulfur-35 labelling. Soil Biol Biochem 17: 73–79.

Miller JH (1972) Experiments in Molecular Genetics, Cold Spring

Harbor Laboratory Press, Cold Spring Harbor, NY.

Mirleau P, Wogelius R, Smith A & Kertesz MA (2005) Importance

of organosulfur utilization for the survival of Pseudomonas

putida in soil and rhizosphere. Appl Environ Microbiol 71:

6571–6577.

Mougous JD, Green RE, Williams SJ, Brenner SE & Bertozzi CR

(2002) Sulfotransferases and sulfatases in Mycobacteria. Chem

Biol 9: 767–776.

Mougous JD, Senaratne RH, Petzold CJ et al. (2006) A sulfated

metabolite produced by stf3 negatively regulates the virulence

of Mycobacterium tuberculosis. Proc Natl Acad Sci USA 103:

4258–4263.

Nelson KE, Weinel C, Paulsen IT et al. (2002) Complete genome

sequence and comparative analysis of the metabolically

versatile Pseudomonas putida KT2440. Environ Microbiol 4:

799–808.

Nishio Y, Nakamura Y, Kawarabayasi Y et al. (2003) Comparative

complete genome sequence analysis of the amino acid

replacements responsible for the thermostability of

Corynebacterium efficiens. Genome Res 13: 1572–1579.

Omura S, Ikeda H, Ishikwa J et al. (2001) Genome sequence of an

industrial microorganism Streptomyced avermitilis: dedcing

the ability of producing secondary metabolites. Proc Natl Acad

Sci USA 98: 12215–12220.

Paget MSB & Buttner MJ (2003) Thiol-based regulatory switches.

Ann Rev Genet 37: 91–121.

Parkhill J, Wren BW, Mungall K et al. (2000) The genome

sequence of the food-borne pathogen Campylobacter jejuni

reveals hypervariable sequences. Nature 403: 665–668.

Perna NT, Plunkett G III, Burland V et al. (2001) Genome

sequence of the enterohaemorrhagic Escherichia coli

O157 : H7. 409: 529–533.

Peterson JD, Umayam LA, Dickinson TM, Hickey EK & White O

(2001) The comprehensive microbial resource. Nucleic Acids

Res 29: 123–125.

Piddington CS, Kovacevich BR & Rambosek J (1995) Sequence

and molecular characterization of a DNA region encoding the

dibenzothiophene desulfurization operon of Rhodococcus sp.

strain IGTS8. Appl Environ Microbiol 61: 468–475.

Raoult D, Audic S, Robert C et al. Tropheryma whipplei illustrates

the diversity of gene loss patterns in small genome bacterial

pathogens. Unpublished GeneBank submission (as of 31 July

2002).

Read TD, Peterson SN, Tourasse N et al. (2003) The genome

sequence of Bacillus anthracis Ames and comparison to closely

related bacteria. Nature 423: 81–86.

Read TD, Brunham RC, Shen C et al. (2000) Genome sequences

of Chlamydia trachomatis MoPn and Chlamydia pneumoniae

AR39. Nucleic Acids Res 28: 1397–1406.

Rensing C, Mitra B & Rosen BP (1997) The zntA gene of

Escherichia coli encodes a Zn(II)-translocating P-typeATPase.

Proc Natl Acad Sci USA 23: 14326–14331.

Ritz D & Beckwith J (2001) Roles of thiol-redox pathways in

bacteria. Ann Rev Microbiol 55: 21–48.

Rocha EPC, Sekowska A & Danchin A (2000) Sulphur islands in

the Escherichia coli genome: markers of the cell’s architecture?

FEBS Lett 476: 8–11.

Salanoubat M, Genin S, Ariguenave F et al. (2002) Genome

sequence of the plant pathogen Ralstonia solanacearum.

Nature 415: 497–502.

Schell MA, Karmirantzou M, Snel B et al. (2002) The genome

sequence of Bifidobacterium longum reflects its adaptation to

the human gastrointestinal tract. Proc Natl Acad Sci USA 99:

14422–14427.

Scherer HW (2001) Sulfur in crop production – invited paper.

Eur J Agro 14: 81–111.

Sekowska A, Kung HF & Danchin A (2000) Sulfur metabolism in

Escherichia coli and related bacteria: facts and fiction. J Mol

Microbiol Biotechnol 2: 145–177.

Stover CK, Pham XQ, Erwin AL et al. (2000) Complete genome

sequence of Pseudomonas aeruginosa PA01, an opportunistic

pathogen. Nature 406: 959–964.

Suerbaum S, Josenhaus C, Sterzbach T et al. (2003) The complete

genome sequence of the carcinogenic bacterium Helicobacter

hepaticus. Proc Natl Acad Sci USA 100: 7901–7906.

Sutherland TD, Horne I, Lacey MJ, Harcourt RL, Russell RJ &

Oakeshott JG (2000) Enrichment of an endosulfan-degrading

mixed bacterial culture. Appl Environ Microbiol 66: 2822–2828.

Thelander L (1973) Physiochemical characterisation of

ribonucleotide diphosphate reductase from Escherichia coli. J

Biol Chem 248: 4591–4601.

van der Ploeg JR, Weiss MA, Saller E, Nashimoto H, Saito N,

Kertesz MA & Leisinger T (1996) Identification of sulfur

starvation-regulated genes in Escherichia coli: a gene cluster


192 C. Scott et al.

involved in the utilization of taurine as a sulfur source. J

Bacteriol 178: 5438–5446.

van der Ploeg JR, Iwanicka-Nowicka R, Bykowski T, Hryniewicz

MM & Leisinger T (1999) The Escherichia coli ssuEADCB gene

cluster is required for the utilization of sulfur from aliphatic

sulfonates and is regulated by the transcriptional activator Cbl.

J Biol Chem 274: 29358–29365.

van der Ploeg JR, Eichhorn E & Leisinger T (2001) Sulfonate-

sulfur metabolism and its regulation in Escherichia coli. Arch


Van Ham RC, Kamerbeek J, Palacios C et al. (2003) Reductive

genome evolution in Buchnera aphidicola. Proc Natl Acad Sci

USA 100: 581–586.

Van Sluys MA, de Oliveira MC, Monteiro-Vitorello CB et al.

(2003) Comparative analyses of the complete genome

sequences of Pierce’s disease and citrus variegated chlorosis

strains of Xylella fastidiosa. J Bacteriol 185: 1018–1026.

Vermeij P & Kertesz MA (1999) Pathways of assimilative sulfur

metabolism in Pseudomonas putida. J Bacteriol 181:

5833–5837.

Vermeij P, Weitek C, Kahnert A, Wuest T & Kertesz MA (1999)

Genetic organization of sulfur-controlled aryl desulfonation in

Pseudomonas putida S-313. Mol Microbiol 32: 913–926.

Wood DW, Setubal JC, Kaul R et al. (2001) The genome of the

natural genetic engineer Agrobacterium tumefaciens C58.

Science 294: 2317–2323.

Supplementarymaterial

The following supplementary material is available for this

article:

Appendix S1. Bioinformatic analysis of the P. putida

KT2440 proteome.

This material is available as part of the online article

from: http://www.blackwell-synergy.com/doi/abs/10.1111/

j.1574.6968.2006.00575.x (This link will take you to the

article abstract).

Please note: Blackwell Publishing are not responsible

for the content or functionality of any supplementary

materials supplied by the authors. Any queries (other than

missing material) should be directed to the corresponding

author for the article.



Documents

A global response to sulfur starvation in Pseudomonas putida and its relationship to the expression of low-sulfur-content proteins