| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Department of Microbiology and Infectious Diseases, University of Calgary, Calgary AB T2N 4N1, Canada
2 Department of Biochemistry and Molecular Biology, University of Calgary, Calgary AB T2N 4N1, Canada
Correspondence
Michael G. Surette
surette{at}ucalgary.ca
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
cys strains could be restored to wild-type levels by the addition of cysteine to swarm medium. Two regulatory mutants, cysB and cysE, failed to swarm unless cysteine was supplemented to the medium. We show that all CysB-responsive operons involved in cysteine biosynthesis are upregulated in the swarm state, even though swarm cells are cultivated on a medium that represses cysteine biosynthesis in the swim state. While swarm medium has sufficient cysteine for growth of S. typhimurium, it does not contain enough for swarm-cell differentiation. We hypothesize that in these cells, the additional cysteine requirement is for use in pathways not directly related to cell growth.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). Free swimming behaviour is most commonly associated with flagellated bacteria; however, many of these bacteria are also capable of moving over a surface. Swarming is a form of coordinated surface motility performed by some flagellated bacteria when grown on semi-solid media. Vegetative cells undergo a differentiation programme to produce swarm cells, which are typically elongated, hyper-flagellated and multi-nucleate (Fraser & Hughes, 1999; Harshey, 2003). While motility is required for cells to swarm, chemotaxis is not essential (Burkart et al., 1998). Swarming was first described, and has been most studied, in Proteus mirabilis (Harshey, 2003; Henrichsen, 1972). In this species, swarm cells align along their long axis and migrate in multi-cellular rafts on agar concentrations up to 2 % (Fraser & Hughes, 1999). Salmonella enterica serovar Typhimurium (S. typhimurium) can swarm on nutrient-rich media supplemented with a rich carbon source, and having an agar concentration ranging from 0.5 % to 0.8 % (Burkart et al., 1998; Kim & Surette, 2003; Toguchi et al., 2000).
In addition to the motility phenotype, swarming has been associated with important physiological changes in numerous bacterial species. Virulence factor expression, notably urease, protease and haemolysin production, is increased in Proteus mirabilis in the swarm state, as is the ability to invade urinary epithelial cells (Allison et al., 1992a, b; Liaw et al., 2003). In S. typhimurium, actively migrating swarm cells have increased antibiotic resistance (Kim et al., 2003), a switch in central metabolism from catabolic to anabolic growth (Kim & Surette, 2004), and increased ability to sense acylhomoserine lactones (Kim & Surette, 2006). The increased resistance of S. typhimurium swarm cells has been observed to many classes of antibiotics with a broad range of cellular targets and is not due to a genetic change (Kim et al., 2003). Swarm cells transferred to liquid media reacquire the antibiotic sensitivity of vegetative cells (Kim & Surette, 2004). Furthermore, since swarm cells are growing exponentially, increased resistance is not due to slow growth or the presence of persister cells (Balaban et al., 2004; Kim & Surette, 2004). Rather, swarm populations are an example of an induced intrinsic antibiotic resistance whereby a genetically identical population that is normally sensitive to the action of an antibiotic becomes transiently more resistant (Levin & Rozen, 2006). To be induced, this phenotype does not require the presence of antibiotics (Kim & Surette, 2004).
S. typhimurium can assimilate inorganic sulfur into sulfide for the incorporation of reduced sulfur into cysteine (Fig. 1) (Peck, 1961). The final reaction of cysteine biosynthesis is catalysed by one of the isozymes encoded by cysK or cysM. CysK is produced at high levels and is thought to be the primary enzyme responsible for this reaction (Kredich, 1996); however, CysM is the predominant enzyme catalysing the reaction in anaerobic conditions, and when thiosulfate is present to react with O-acetyl-L-serine (OAS) (Kredich, 1996). The enzyme encoded by cysQ is thought to degrade a toxic intermediate of this pathway (Neuwald et al., 1992). Three separate transport systems have been identified for cysteine transport: CTS-1, CTS-2 and CTS-3 (Baptist & Kredich, 1977). CTS-1, a part of the CysB regulon, accounts for the majority of cysteine uptake, and is repressed by growth on cysteine and other reduced sulfur sources. CTS-2 and CTS-3 are lower-capacity transporters of cysteine than CTS-1, although CTS-2 has a higher affinity for cysteine (Baptist & Kredich, 1977). Sulfate and thiosulfate are imported by the same permease as encoded by the genes cysU-cysW-cysA but having different periplasmic binding proteins, sbp and cysP, respectively (Hryniewicz & Kredich, 1991).
|
). Serine transacetylase (CysE) is feedback inhibited by cysteine, providing kinetic control of the production of cysteine through OAS. Sulfide and thiosulfate are potent anti-inducers of the CysB regulon, by antagonizing binding of NAS to CysB (Hryniewicz & Kredich, 1991; Ostrowski & Kredich, 1990). Operons involved in the cysteine biosynthetic pathway in the S. typhimurium CysB regulon include cysPUWAM, cysDNC, cysJIH, cysQ and cysK (Fig. 1
). CysB binds to NAS and activates transcription at each operon. For maximal expression of the CysB regulon, sulfur limitation is required to relieve end-product inhibition of CysE by cysteine and allow the synthesis of OAS (Kredich, 1996). This study was initiated by asking whether the swarm phenotypes of metabolic differentiation and elevated antibiotic resistance were coupled. To block metabolic differentiation, amino acid auxotrophs were generated. These mutants were subsequently screened in the swarm state for the presence of increased antibiotic resistance phenotype. From this initial screen, it became apparent that cysteine biosynthesis was crucial for complete swarm-cell differentiation, as only cysteine auxotrophs did not have increased antibiotic resistance in the swarm state. This led us to further study cysteine biosynthesis in swarm cells. We investigated expression of operons encoding cysteine biosynthetic enzymes using transcriptional fusions. Mutants in each cys locus were generated to allow study of antibiotic resistance and swarm colony morphologies in cysteine auxotroph swarm colonies. We investigated the regulatory mechanism by which swarm cells were able to induce the cys pathway despite growing on medium containing sufficient reduced sulfur, which usually represses cysteine biosynthesis in swim cells. The mutant studies were confirmed using a chemical genetics approach to induce cysteine auxotrophy in wild-type cells. As swarm medium contains sufficient cysteine to support growth and swarming of cysteine auxotrophs, we hypothesize that the increased cysteine requirement is a prerequisite for complete swarm-cell differentiation in S. typhimurium.
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
. For routine culture, strains were grown in Lennox's Luria–Bertani broth (LB) at 37 °C, with shaking (200 r.p.m.). Swarm medium NBG (Nutrient Broth, Difco, with 0.5 % glucose, 0.5 % agar) and swim medium NBG (Nutrient Broth with 0.5 % glucose, 0.25 % agar) were used in swarm and swim assays, respectively, as previously described (Kim & Surette, 2003). M9 minimal medium was from Difco. Kanamycin was used at 50 µg ml–1, and chloramphenicol was used at 12.5 µg ml–1. Sulfate, sulfite, sulfide, thiosulfate, L-cysteine and 1,2,4-triazole (Sigma-Aldrich) were added as indicated.
|
). The site of the chloramphenicol resistance gene, cat, insertion was determined using arbitrary-primed PCR. Mutants obtained from this technique were cysD : : Tn, cysN : : Tn, cysH : : Tn, cysQ : : Tn and cysP : : Tn.
Mutant strains cysC, cysIJ, cysE, cysK, cysK cysM and cysB were constructed using the 
Red system (Datsenko & Wanner, 2000). Primers used for the generation of PCR products are listed in Table 2. The antibiotic resistance gene was resolved from both sites in the cysK cysM double mutant as described by Datsenko & Wanner (2000).
|
. All promoters were cloned from S. typhimurium 14028 genomic DNA by PCR. Each pair of PCR primers for amplifying a promoter contained an XhoI and a BamHI site and amplified products were cloned into pCS26, a low-copy vector containing a promoterless luciferase operon (luxCDABE), as described previously (Bjarnason et al., 2003). All enzymes used in cloning were purchased from Invitrogen and molecular biology techniques were conducted using standard techniques (Sambrook & Russell, 2001). Cloned promoters were verified by DNA sequencing.
Luciferase assays.
Cells were harvested from the periphery of swarm or swim colonies using sterile toothpicks and transferred to 400 µl volumes of PBS. One hundred microlitres of cells in PBS were measured in triplicate, with a 1 s read time, using a Trilux Scintillation Counter (Wallec). Each strain was repeated in triplicate from a separate overnight culture, used to inoculate three swarm plates for each condition. Remaining cells in PBS were serially diluted and plated on Luria–Bertani (LB) agar with kanamycin to determine c.f.u. in each sample. Luciferase activity, in counts per second (c.p.s.), was normalized to 107 c.f.u. ml–1. This value is referred to as relative light units (RLU), with 1 RLU equalling 1 c.p.s. per 107 c.f.u.
Luminescent images of swarm and swim plates were taken using an Alpha Imager FluorChem 8900 CCD camera (Alpha Innotech) with no exogenous light and using an exposure of 10 s to 5 min to capture light emitted by the luciferase reporter.
Antibiotic resistance measurements.
Antibiotic resistance on swarm and swim media was determined by placing an E-Test strip (AB Biodisk) in the centre of the plate as described previously (Kim et al., 2003; Kim & Surette, 2003). Briefly, 1 µl of overnight culture was spotted on either side of the strip adjacent to the strip. Plates were incubated at 37 °C and the antibiotic resistance was read according to the manufacturer's instructions after the swarm colony covered the plate. The conventional method of streaking cells onto a plate, resulting in a confluent layer of growth, is not suitable for measuring antibiotic resistance in swarm cells. Broth-grown cells spread plated on swarm medium cannot actively swarm, thus not allowing measurement of antibiotic resistance in this population of cells.
Composition analysis of Nutrient Broth (NB).
HPLC analysis was performed by the Biochemical Genetics Laboratory at the Alberta Children's Hospital under the direction of Dr Floyd F. Snyder. Samples were reduced with 0.5 M DTT. Proteinase K digestion was performed on NB (40 g l–1). After autoclaving, filter-sterilized proteinase K (Invitrogen) was added to a final concentration of 400 mg l–1 to one flask. To the control flask, the same volume of sterile water was added. These flasks were incubated with stirring for 12 h at 37 °C, after which time both flasks were autoclaved again to inactivate the proteinase K. This sample was then analysed by HPLC.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). If these two phenotypes are physiologically coupled, we reasoned that blocking biosynthetic growth may interfere with the antibiotic resistance phenotype. We hypothesized that auxotrophs would be forced to take up an amino acid from the medium and therefore would be incapable of undergoing full metabolic differentiation. Amino acid auxotrophs were generated using P22 transposon mutagenesis and were examined for motility and the elevated antibiotic resistance swarm phenotypes. In the initial screen, 23 auxotrophs in nine different biosynthetic pathways were examined (data not shown). All mutants displayed wild-type swim motility, chemotactic behaviour and swarm motility (data not shown). This is consistent with previous genetic screens that did not identify any auxotrophs in screens for swarm motility mutants (Kim et al., 2003). The auxotrophs were tested for resistance to gentamicin and ciprofloxacin under swim and swarm conditions. All auxotrophs displayed antibiotic resistance similar to wild-type swarm cells, with the exception of cysteine auxotrophs. Each cysteine auxotroph examined showed decreased antibiotic resistance in the swarm state. Although this screen was not exhaustive of all amino acids, it demonstrated the importance of cysteine in swarm-cell differentiation. An auxotroph for methionine, another sulfur-containing amino acid, behaved like the wild-type, showing the characteristic elevated resistance to antibiotics in the swarm state (data not shown). In the vegetative (swim) state, the cysteine auxotrophs showed similar susceptibility to antibiotics as wild-type cells. However, under swarm growth conditions, while the cysteine auxotrophs retained swarm motility, the ability to induce elevated antibiotic resistance was lost.
Cysteine auxotrophs have decreased antibiotic resistance in the swarm state
To further explore the link between cysteine auxotrophs and induced antibiotic resistance, mutants were generated in all cys operons. To test the relationship between the cysteine biosynthetic pathway and the induced antibiotic resistance of swarm cells, each auxotrophic strain was tested for susceptibility to gentamicin and ciprofloxacin using E-Test strips (Table 3). All mutants retained their ability to swarm with the exception of the regulatory mutants cysE and cysB. For the cys mutants that retained swarm motility, each had increased susceptibility to at least one antibiotic tested. Ciprofloxacin MIC values for cysteine auxotrophs were 188- to 255-fold lower than wild-type (Table 3). The pattern of resistance to gentamicin was similar, with most cysteine auxotrophs demonstrating >3- to >8-fold decreased MIC compared to wild-type (Table 3). Obtaining absolute ratios between auxotrophs and wild-type was not possible for gentamicin because the value for the wild-type exceeded the maximum MIC value on the strip (256 µg ml–1).
|
). However, this cysK strain is not a cysteine auxotroph since CysK and CysM are functionally redundant, although this strain grows poorly on intermediates of the sulfate assimilation pathway, such as sulfide. In the absence of CysK, CysM can produce cysteine from sulfide or thiosulfate and OAS (Kredich, 1996). Having one functional O-acetylserine thiolase may allow an intermediate level of swarm-cell differentiation between the full differentiation observed in wild-type cells and the less differentiated cysteine auxotrophs. A cysP deletion mutant is also not an auxotroph; however, the cysP strain used in this study is polar on downstream genes in the operon, causing auxotrophy due to lack of a functional ABC transporter for sulfate and thiosulfate (Sirko et al., 1995).
Cysteine auxotrophs have altered swarm colony morphology
Closer analysis of swarm colonies of the cysteine auxotroph mutants revealed altered morphologies that were distinct from wild-type (Fig. 2). The mutant swarm morphologies fell into three categories. cysP, cysH and cysIJ mutants have a migrating swarm population that forms tendrils that dichotomously branch and terminate without intercepting, and also lack the complex circular structure surrounding the site of inoculation (Fig. 2b).
|
). This group is typified by thick growth at the site of inoculation and complete lack of a circular structure surrounding the site of inoculation. This structure in wild-type cells consists of a mixture of swim and differentiating swarm cells, and the site of inoculation has only a thin layer of bacterial growth.
As noted above, two mutants in the regulatory genes, cysE and cysB, do not swarm using the standard swarm medium (Fig. 2d). After 24 h, when wild-type cells have covered a Petri dish, these strains are the size of a swarm colony after approximately 5 h.
Swarm phenotypes of cysteine auxotrophs could be complemented by addition of 0.2 mM cysteine to the swarm medium, making the swarm colony morphologies of all cysteine auxotrophs indistinguishable from wild-type (Fig. 2). The addition of cysteine also allowed cysB and cysE mutants to swarm. Furthermore, the addition of 0.2 mM cysteine to the swarm medium restored antibiotic resistance of each auxotrophic strain to wild-type levels (data not shown), indicating that antibiotic resistance of cysteine auxotrophic swarm cells can be attributed to cysteine starvation.
The CysB regulon is induced in swarm cells, and these cells remain responsive to environmental inorganic sulfur source
To examine the different requirements for cysteine in swarm and swim cells, we measured transcription of genes in the CysB regulon in both cellular states using promoter–luxCDABE fusions. Gene expression was monitored by imaging lux expression on swim and swarm plates. This also produced spatial information on gene expression (Fig. 3a). With the exception of cysK and sbp, mutants in all other CysB-regulated genes showed little or no activity on swim plates. However, on swarm plates expression of all cys operons was induced.
|
70 promoter (Kim & Surette, 2006) was used as a control in the gene expression experiments to demonstrate that overall transcriptional activity in the cell did not vary between the swarm and swim states. This reporter had a swarm-to-swim ratio of 0.9 (Fig. 3b). cysB was expressed at low constitutive levels in both the swim and swarm states; however, expression was consistently slightly higher in swarm cells. As cysB encodes a transcriptional regulator, a large increase in expression is not expected, since a small change in expression may have large consequences on transcription of its regulon. Consistent with results above, only sbp and cysK were expressed above background levels. sbp was expressed at similar levels in swarm and swim cells; the ratio of expression between the two states was 1.2 (Fig. 3b). cysK was expressed at a level 68-fold greater in the swarm state. In this state all operons in the CysB regulon were induced between 14- and 68-fold (Fig. 3).
To test whether environmental sulfur availability affected induction of the CysB regulon in swarm cells, cysJ expression was used to represent CysB regulon activity, as the cells were grown in NBG swarm and swim media with sulfur sources added. There was no expression of cysJ in swim cells with any of the sulfur sources tested (Fig. 4). In the swarm state, cysJ expression was detected in unsupplemented medium and with all exogenous sulfur sources except cysteine. Growth on sulfite, thiosulfate or sulfide reduced cysJ expression to a similar level. Expression of cysJ was the greatest on unsupplemented medium and medium supplemented with sulfate, which modestly reduced expression levels (2.5-fold).
|
). The motility and antibiotic resistance phenotypes of wild-type S. typhimurium were examined in the presence of 1,2,4-triazole. At concentrations up to 100 mM, swim and swarm motility were unaffected (data not shown). In medium supplemented with 75 mM and 100 mM 1,2,4-triazole, the MIC of gentamicin decreased to 12 µg ml–1 and 8 µg ml–1, respectively, in swarm cells, from >256 µg ml–1 in untreated swarm cells. These values are comparable to MICs observed for swim cells of either cysteine auxotrophs or wild-type cells. The MIC of ciprofloxacin dropped from 12 µg ml–1 in untreated cells to 0.064 µg ml–1 in medium containing 75 mM and 100 mM 1,2,4-triazole, similar to the level obtained with most cysteine auxotrophs on standard swarm medium.
Induction of the CysB regulon was estimated by measuring the amount of expression from the cysJ promoter–luciferase fusion. Addition of 1,2,4-triazole decreased cysJ expression in a dose-dependent manner (Fig. 5). Compared to the standard swarm medium, cysJ expression decreased 4-fold and 7.5-fold on NBG supplemented with 5 mM or 10 mM 1,2,4-triazole, respectively. When cells were grown on NBG supplemented with 100 mM 1,2,4-triazole, cysJ expression was below detectable levels.
|
Chemical composition of swarm medium
Since Nutrient Broth (NB) supported the growth of all cysteine auxotrophs, it is clear that the medium contains sufficient cysteine and reduced sulfur to support growth of S. typhimurium. However, actively migrating swarm cells induced the genes for cysteine biosynthesis, and cysteine biosynthesis is required for the antibiotic resistance phenotype. The concentration of cysteine in NB was determined by HPLC. DTT was added to reduce disulfide bonds between free cysteine and thiol groups in peptides, allowing the quantification of total free cysteine in the medium. The concentration of cysteine in NB and after reduction with DTT was found to be 5.0±2.1 µM and 21.3±1.5 µM, respectively.
NB containing oligopeptides may provide a source of cysteine to S. typhimurium and would not be reflected in our amino acid analysis. To measure the cysteine in peptides, HPLC analysis was repeated on NB digested with proteinase K. This increased concentrations of the most abundant amino acids; however, the concentration of cysteine increased only modestly to 29.0±4.3 µM.
Cysteine at a concentration of 30 µM as the only sulfur source in minimal medium allowed weak growth of wild-type and was insufficient to support growth of auxotrophs. In M9 minimal medium broth or agar plates, a cysE mutant requires 50 µM cysteine to grow (data not shown).
From these results, it is evident that NB contains sufficient cysteine for growth but not enough for swarm-cell differentiation, either free or in peptides. Other reduced sulfur sources, such as thiosulfate or sulfide, may be present at a concentration to permit the production of cysteine without inducing the CysB regulon in wild-type swim cells.
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Kim & Surette, 2004, 2006). In this paper, we show that cysteine auxotrophs are impaired in the antibiotic resistance phenotype and that regulatory mutants of cysteine biosynthesis are incapable of swarm motility. All cys mutants in the sulfate assimilation pathway were still capable of swarm motility; however, these auxotrophs did not exhibit the increased antibiotic resistance observed in wild-type swarm cells. These auxotrophs are probably still capable of synthesizing cysteine from reduced sulfur sources in the medium, since CysK is expressed abundantly at basal levels without CysB such that sulfide can support the growth of a cysB strain (Kredich, 1971).
The only mutants that were incapable of swarming on the standard swarm medium were the regulatory mutants cysB and cysE. To our knowledge, this is the first report of a cysteine auxotroph that is unable to swarm. Previously, mutants in the histidine biosynthetic pathway were identified in a screen of mutants unable to swarm on Difco agar; however, these mutants were not investigated further (Toguchi et al., 2000). Other screens for swarm-defective mutants in S. typhimurium identified genes in LPS biosynthesis and chemotaxis (Harshey & Matsuyama, 1994; Kim et al., 2003; Toguchi et al., 2000). Induction of the cys biosynthetic genes would not be possible in either cysB or cysE mutants, indicating that these mutants would rely on imported cysteine for growth (Baptist & Kredich, 1977). Another strain entirely reliant on imported cysteine for survival, a cysK cysM double mutant, is still capable of swarming. One difference between the biosynthetic mutants and the regulatory mutants lies in the CTS-1 cysteine permease that is induced by CysB. CTS-1 has the highest capacity for cysteine of the three cysteine permeases and is thought to be responsible for rapid cysteine transport during times of cysteine starvation (Baptist & Kredich, 1977). Interestingly, cysB and cysE are still motile and are not impaired in swimming or chemotaxis behaviour. Thus, the two lower-capacity cysteine permeases, CTS-2 and CTS-3, may fulfil the cysteine requirements of swim cells but not swarm cells.
An increased requirement for cysteine in swarm cells may explain why cells grown on swim or swarm plates have different profiles of cys gene expression. Swim cells have no detectable expression of cysteine biosynthetic genes except for cysK and sbp. In Escherichia coli, sbp is positively regulated by Cbl, a transcriptional regulator with 60 % amino acid similarity to CysB, involved in the regulation of cysteine biosynthesis from organic sulfur sources (Kredich, 1996; Stec et al., 2006; Van Der Ploeg et al., 1997). S. typhimurium does not contain a Cbl homologue. cysK has a significant level of basal expression in the absence of CysB activation as observed in swim cells. All other cys operons had very low activity on swim plates and were induced in swarm conditions, consistent with CysB activation.
Interestingly, cysE expression was also induced in the swarm state. Transcriptional regulation of cysE is not known to occur and it is not thought to be part of the CysB regulon (Kredich, 1996). In E. coli, serine acetyltransferase is implicated in having a role in biofilm formation since cysE mutants formed biofilms more quickly than wild-type cells (Sturgill et al., 2004). The authors postulated that serine acetyltransferase may be producing an extracellular signalling molecule that is neither OAS nor NAS. Unexpectedly, the addition of cysteine decreased the rate of biofilm formation. If CysE produces an extracellular signal required for swarm motility, it is not likely to account for why cysE did not swarm unless cysteine was provided since the cysB strain also did not swarm, and in this strain serine acetyltransferase and OAS would be produced.
CysB is known to activate transcription of genes that do not have a direct role in cysteine biosynthesis. Arginine decarboxylase expression is increased by CysB binding its promoter, and cysB mutants do not ferment many carbohydrates as well as the parent strains of E. coli and S. typhimurium (Quan et al., 2002; Shi & Bennett, 1994). Since CysB is the regulator of sulfur metabolism in the cells, a central metabolic pathway, it could be considered a global regulator in S. typhimurium. Cross-talk between metabolic pathways could help the bacterium coordinate utilization of a metabolite in one metabolic pathway with those in another (Quan et al., 2002). In addition, cysB and cysE mutants of S. typhimurium and E. coli have been reported to have increased resistance to the β-lactam antibiotic mecillinam and the quinolone novobiocin (Anton, 2000; Costa & Anton, 2006; Lilic et al., 2003; Oppezzo & Anton, 1995; Rakonjac et al., 1991). Since swarming motility in both cysB and cysE is restored by the inclusion of cysteine in the swarm medium, a CysB-regulated gene outside of the cysteine biosynthetic genes is not the reason that these strains are incapable of swarming on standard swarm medium. A cysB, but not a cysE strain can swarm in media supplemented with sulfide, indicating that sulfide is also limiting in swarm medium (data not shown). Exogenous cysteine also restores the antibiotic resistance phenotype, showing that cysteine and not those genes outside of cysteine biosynthesis in the CysB regulon are important.
It is apparent that differentiated swarm cells either cannot access the cysteine in NB or require more cysteine than swim cells. Swarm cells may be using the reduced sulfur for reasons other than growth, to maintain disulfide bond formation in extracellular and cytosolic proteins by the synthesis of glutathione and other cellular reductants (Bjur et al., 2006). The sulfur in cysteine is used as the reduced sulfur source for the synthesis of sulfur-containing molecules in the cells in S. typhimurium. Swarm cells, by relying on oxidative phosphorylation for energy generation more than swim cells, may generate more reactive oxygen species (González-Flecha & Demple, 1995; Kim & Surette, 2004). This may induce oxidative stress pathways to prevent damage to cytoplasmic proteins. The glutathione- and thioredoxin-reduction systems are critical for maintaining the reduction potential of the cystoplasm; these reductants and their respective reductases in most cases contain cysteine residues (Carmel-Harel & Storz, 2000). Thus, cysteine has a central role in maintaining the reducing environment of the cytoplasm. If cys auxotrophs are oxidatively stressed, the added stress of an antibiotic may lower the MIC for a particular antibiotic. Alternatively, an antibiotic may cause further oxidative stress and if auxotrophs have impaired responses to oxidative stress, they may have a lower MIC. Variable soxS transcript levels were observed in efflux pump mutants of S. typhimurium (Eaves et al., 2004).
This research indicates an important role for cysteine in S. typhimurium swarm cells, as cysteine biosynthetic regulatory mutants fail to swarm, cysteine auxotrophs exhibit decreased MICs for two unrelated antibiotics in the swarm state, and wild-type cells induce the cysteine biosynthetic operon in the swarm state. Use of chemical genetics to target the cysteine biosynthetic pathway and reduced antibiotic resistance in the swarm state provides potential for therapeutic use of compounds to prevent the induction of intrinsic antibiotic resistance pathways. Our current hypothesis is that swarm cells require additional cysteine for reasons other than growth, perhaps to counter oxidative stress generated by oxidative phosphorylation. We are currently testing this hypothesis.
|
ACKNOWLEDGEMENTS |
|---|
Edited by: P. H. Everest
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Allison, C., Coleman, N., Jones, P. L. & Hughes, C. (1992b). Ability of Proteus mirabilis to invade human urothelial cells is coupled to motility and swarming differentiation. Infect Immun 60, 4740–4746.
Anton, D. N. (2000). Induction of the cysteine regulon of Salmonella typhimurium in LB medium affects the response of cysB mutants to mecillinam. Curr Microbiol 40, 72–77.[CrossRef][Medline]
Balaban, N. Q., Merrin, J., Chait, R., Kowalik, L. & Leibler, S. (2004). Bacterial persistence as a phenotypic switch. Science 305, 1622–1625.
Baptist, E. W. & Kredich, N. M. (1977). Regulation of L-cysteine transport in Salmonella typhimurium. J Bacteriol 131, 111–118.
Bjarnason, J., Southward, C. M. & Surette, M. G. (2003). Genomic profiling of iron-responsive genes in Salmonella enterica serovar Typhimurium by high-throughput screening of a random promoter library. J Bacteriol 185, 4973–4982.
Bjur, E., Eriksson-Ygberg, S., Fredrik Aslund, F. & Rhen, M. (2006). Thioredoxin 1 promotes intracellular replication and virulence of Salmonella enterica serovar Typhimurium. Infect Immun 74, 5140–5151.
Burkart, M., Toguchi, A. & Harshey, R. M. (1998). The chemotaxis system, but not chemotaxis, is essential for swarming motility in Escherichia coli. Proc Natl Acad Sci U S A 95, 2568–2573.
Carmel-Harel, O. & Storz, G. (2000). Roles of the glutathione- and thioredoxin-dependent reduction systems in the Escherichia coli and Saccharomyces cerevisiae responses to oxidative stress. Annu Rev Microbiol 54, 439–461.[CrossRef][Medline]
Costa, C. S. & Anton, D. N. (2006). High-level resistance to mecillinam produced by inactivation of soluble lytic transglycosylase in Salmonella enterica serovar Typhimurium. FEMS Microbiol Lett 256, 311–317.[CrossRef][Medline]
Datsenko, K. A. & Wanner, B. L. (2000). One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97, 6640–6645.
Eaves, D. J., Ricci, V. & Piddock, L. J. V. (2004). Expression of acrB, acrF, acrD, marA, and soxS in Salmonella enterica serovar Typhimurium: role in multiple antibiotic resistance. Antimicrob Agents Chemother 48, 1145–1150.
Fraser, G. M. & Hughes, C. (1999). Swarming motility. Curr Opin Microbiol 2, 630–635.[CrossRef][Medline]
González-Flecha, B. & Demple, B. (1995). Metabolic sources of hydrogen peroxide in aerobically growing Escherichia coli. J Biol Chem 270, 13681–13687.
Harshey, R. M. (2003). Bacterial motility on a surface: many ways to a common goal. Annu Rev Microbiol 57, 249–273.[CrossRef][Medline]
Harshey, R. M. & Matsuyama, T. (1994). Dimorphic transition in Escherichia coli and Salmonella typhimurium: surface-induced differentiation into hyperflagellate swarmer cells. Proc Natl Acad Sci U S A 91, 8631–8635.
Henrichsen, J. (1972). Bacterial surface translocation: a survey and a classification. Bacteriol Rev 36, 478–503.
Hryniewicz, M. M. & Kredich, N. M. (1991). The cysP promoter of Salmonella typhimurium: characterization of two binding sites for CysB protein, studies of in vivo transcription initiation, and demonstration of the anti-inducer effects of thiosulfate. J Bacteriol 173, 5876–5886.
Hulanicka, D., Klopotowski, T. & Smith, D. A. (1972). The effect of triazole on cysteine biosynthesis in Salmonella typhimurium. J Gen Microbiol 72, 291–301.
Kim, W. & Surette, M. G. (2003). Swarming populations of Salmonella represent a unique physiological state coupled to multiple mechanisms of antibiotic resistance. Biol Proced Online 5, 189–196.[CrossRef][Medline]
Kim, W. & Surette, M. G. (2004). Metabolic differentiation in actively migrating swarming Salmonella. Mol Microbiol 54, 702–714.[CrossRef][Medline]
Kim, W. & Surette, M. G. (2006). Coordinated regulation of two independent cell-cell signalling systems and swarmer differentiation in Salmonella enterica serovar Typhimurium. J Bacteriol 188, 431–440.
Kim, W., Killam, T., Sood, V. & Surette, M. G. (2003). Swarm-cell differentiation in Salmonella enterica serovar Typhimurium results in elevated resistance to multiple antibiotics. J Bacteriol 185, 3111–3117.
Kredich, N. M. (1971). Regulation of L-cysteine biosynthesis in Salmonella typhimurium. I. Effects of growth on varying sulfur sources and O-acetyl-L-serine on gene expression. J Biol Chem 246, 3474–3484.
Kredich, N. M. (1996). Biosynthesis of cysteine. In Escherichia coli and Salmonella, pp. 514–527. Edited by R. Curtiss, III, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter & H. E. Umbarger. Washington: ASM Press.
Levin, B. R. & Rozen, D. E. (2006). Non-inherited antibiotic resistance. Nat Rev Microbiol 4, 556–562.[CrossRef][Medline]
Liaw, S.-J., Lai, H.-C., Ho, S.-W., Luh, K.-T. & Wang, W.-B. (2003). Role of RsmA in the regulation of swarming motility and virulence factor expression in Proteus mirabilis. J Med Microbiol 52, 19–28.
Lilic, M., Jovanovic, M., Jovanovic, G. & Savic, D. J. (2003). Identification of the CysB-regulated gene, hslJ, related to the Escherichia coli novobiocin resistance phenotype. FEMS Microbiol Lett 224, 239–246.[CrossRef][Medline]
Maloy, S. R. (1990). Phage P22. In Experimental Techniques in Bacterial Genetics, pp. 11–16. Boston, MA: Jones & Bartlett Publishers.
Neuwald, A. F., Krishnan, B. R., Brikun, I., Kulakauskas, S., Suziedelis, K., Tomcsanyi, T., Leyh, T. S. & Berg, D. E. (1992). cysQ, a gene needed for cysteine synthesis in Escherichia coli K-12 only during aerobic growth. J Bacteriol 174, 415–425.
Oppezzo, O. J. & Anton, D. N. (1995). Involvement of cysB and cysE genes in the sensitivity of Salmonella typhimurium to mecillinam. J Bacteriol 177, 4524–4527.
Ostrowski, J. & Kredich, N. M. (1990). In vitro interactions of CysB protein with the cysJIH promoter of Salmonella typhimurium: inhibitory effects of sulfide. J Bacteriol 172, 779–785.
Peck, H. D. (1961). Enzymatic basis for assimilatory and dissimilatory sulfate reduction. J Bacteriol 82, 933–939.
Quan, J. A., Schneider, B. L., Paulsen, I. T., Yamada, M., Kredich, N. M. & Saier, M. H., Jr (2002). Regulation of carbon utilization by sulfur availability in Escherichia coli and Salmonella typhimurium. Microbiology 148, 123–131.
Rakonjac, J., Milic, M. & Savic, D. J. (1991). cysB and cysE mutants of Escherichia coli K12 show increased resistance to novobiocin. Mol Gen Genet 228, 307–311.[CrossRef][Medline]
Sambrook, J. & Russell, D. W. (2001). Molecular Cloning: a Laboratory Manual, 3rd edn. New York, NY: Cold Spring Harbor Laboratory Press.
Shi, X. & Bennett, G. N. (1994). Effects of rpoA and cysB mutations on acid induction of biodegradative arginine decarboxylase in Escherichia coli. J Bacteriol 176, 7017–7023.
Sirko, A., Zatyka, M., Sadowy, E. & Hulanicka, D. (1995). Sulfate and thiosulfate transport in Escherichia coli K-12: evidence for a functional overlapping of sulfate- and thiosulfate-binding proteins. J Bacteriol 177, 4134–4136.
Stec, E., Witkowska-Zimny, M., Hryniewicz, M. M., Neumann, P., Wilkinson, A. J., Brzozowski, A. M., Verma, C. S., Zaim, J., Wysocki, S. & Bujacz, G. D. (2006). Structural basis of the sulphate starvation response in Escherichia coli: crystal structure and mutational analysis of the cofactor-binding domain of the Cbl transcriptional regulator. J Mol Biol 364, 309–322.[CrossRef][Medline]
Sturgill, G., Toutain, C. M., Komperda, J., O'Toole, G. A. & Rather, P. N. (2004). Role of CysE in production of an extracellular signaling molecule in Providencia stuartii and Escherichia coli: loss of cysE enhances biofilm formation in Escherichia coli. J Bacteriol 186, 7610–7617.
Toguchi, A., Siano, M., Burkart, M. & Harshey, R. M. (2000). Genetics of swarming motility in Salmonella enterica serovar Typhimurium: critical role for lipopolysaccharide. J Bacteriol 182, 6308–6321.
Van Der Ploeg, J. R., Iwanicka-Nowicka, R., Kertesz, M. A., Leisinger, T. & Hryniewicz, M. M. (1997). Involvement of CysB and Cbl regulatory proteins in expression of the tauABCD operon and other sulfate starvation-inducible genes in Escherichia coli. J Bacteriol 179, 7671–7678.
Received 7 May 2008;
revised 10 July 2008;
accepted 16 July 2008.
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
