1 Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, UK
2 Centre d'Immunologie de Marseille Luminy, Case 906, 13288 Marseille-Cedex 9, France
Correspondence
George P. C. Salmond
gpcs{at}mole.bio.cam.ac.uk
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ABSTRACT |
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The GenBank accession numbers for the sequences reported in this paper are AJ628150, AJ628151 and AJ628152.
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INTRODUCTION |
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It has now been established that diverse species of bacteria, both Gram-positive and Gram-negative, produce AI-2 activity and/or possess a luxS homologue, leading to the suggestion that AI-2 might be a universal (non-species-specific) and/or interspecies signalling molecule (Schauder et al., 2001). Inactivation of luxS in a variety of bacteria has produced a range of effects, from no observable phenotype (e.g. in Helicobacter pylori and Proteus mirabilis), through to altered production of virulence determinants (e.g. in Porphyromonas gingivalis, Streptococcus pyogenes and Clostridium perfringens), and decreased virulence (in Neisseria meningitidis, Streptococcus pneumoniae and Vibrio vulnificus) (Burgess et al., 2002
; Joyce et al., 2000
; Kim et al., 2003
; Lyon et al., 2001
; Ohtani et al., 2002
; Schneider et al., 2002
; Stroeher et al., 2003
; Winzer et al., 2002b
). However, the elucidation of the biosynthetic pathway of AI-2 has revealed a metabolic role for LuxS, in the S-adenosylmethionine-utilization pathway (Schauder et al., 2001
; Winzer et al., 2002a
). LuxS converts S-ribosylhomocysteine (produced by the detoxification of S-adenosylhomocysteine) to homocysteine (which is recycled back to methionine) and AI-2. Therefore some phenotypes of luxS mutants may, in fact, be due to a metabolic defect caused by the loss of function of LuxS in the activated methyl cycle, rather than being due to a genuine signalling defect (Winzer et al., 2002a
). Outside of the Vibrio spp., where components of AI-2-dependent signalling cascades similar to those in V. harveyi are being identified (e.g. controlling virulence in Vibrio cholerae (Miller et al., 2002
), it is currently unclear what proportion of the phenotypes described for luxS mutants is actually due to a genuine signalling defect rather than a metabolic defect.
Serratia marcescens is a Gram-negative, enteric bacterium that is able to inhabit a wide variety of ecological niches and causes disease in plant, vertebrate and invertebrate hosts (Grimont & Grimont, 1978). It is an opportunistic human pathogen and is responsible for an increasing number of serious nosocomial infections, a problem exacerbated by the resistance of many strains to multiple antibiotics (Hejazi & Falkiner, 1997
). S. marcescens strains produce a range of secreted products, including proteases, nucleases, lipases, chitinases and haemolysin (Braun et al., 1993
; Hejazi & Falkiner, 1997
). Many strains also produce the red pigment prodigiosin, a tripyrrole antibiotic reported to have antibacterial, antifungal, antiprotozoan and immunosuppressant activities (Han et al., 1998
; Slater et al., 2003
). Prodigiosin is regarded as a classical secondary metabolite and its production is regulated by a range of environmental signals (Slater et al., 2003
).
S. marcescens ATCC 274 (S. marcescens 274) is a pigmented strain which does not possess a detectable aHSL QS system (unpublished results). Serratia ATCC 39006 (Serratia 39006), a taxonomically ill-defined Serratia, produces prodigiosin and the -lactam antibiotic 1-carbapen-2-em-3-carboxylic acid (carbapenem), with both secondary metabolites being under aHSL-mediated QS control (Slater et al., 2003
). Serratia 39006 has many characteristics in common with the enteric phytopathogen Erwinia carotovora subsp. carotovora (Ecc), for example production of extracellular cellulase and pectate lyase activities, as well as aHSL-controlled carbapenem production (Slater et al., 2003
; Whitehead et al., 2001
). S. marcescens has been shown to be pathogenic in the Caenorhabditis elegans virulence assay, which is proving to be an attractive model system for the in vivo identification of bacterial virulence factors (Kurz et al., 2003
).
The aim of this work was to determine whether Serratia has a luxS/AI-2-dependent signalling system. More specifically, we intended to assess whether several strains of Serratia produce AI-2 activity, and then, if so, to inactivate the luxS gene in order to investigate which phenotypes are regulated by luxS and therefore potentially by an AI-2-dependent signalling system. In this study, we describe the detection of AI-2 activity and the sequencing of luxS in two strains of Serratia, the construction of defined luxS mutants, and the phenotypic analysis of these mutants. We report that the phenotypes of the luxS mutants are strain-dependent and include decreased prodigiosin, haemolysin and carbapenem production and modulated virulence. Finally we show that the supernatant of one wild-type strain (but not its isogenic luxS mutant derivative) contains a signal, presumably AI-2, capable of complementing the pigment defect of the luxS mutant of another strain, even when substantially diluted.
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METHODS |
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DNA manipulations and sequencing of luxS.
All molecular biological techniques, unless otherwise stated, were performed by standard procedures (Sambrook et al., 1989). Enzymes for DNA manipulations were used according to the manufacturer's instructions. Oligonucleotide primers were obtained from Sigma Genosys; primer sequences are given in Table 2
. DNA sequencing was performed by the DNA sequencing facility, University of Cambridge. Nucleotide sequence data were analysed using the GCG package (Genetics Computer Group, University of Wisconsin); sequences were compared to protein and nucleotide databases using the BLAST suite of programs (Altschul et al., 1990
) at GenBank (http://www.ncbi.nlm.nih.gov/blast/). Plasmids are described in Table 1
.
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Construction of luxS mutants.
luxS and flanking sequence from S. marcescens 274 (671 bp in total) were PCR-amplified using primers SC21 and SC32 and cloned into pBluescript-II KS+, generating pSJC4. Similarly, 857 bp of luxS and flanking sequence were cloned from Serratia 39006 using primers SC43 and SC44, generating pSJC13. The Kn-resistance (KnR) cassette from pACYC177 was cloned into the BtrI site in the middle of luxSSma and into the BsiWI site in the middle of luxS39006, generating plasmids pSJC5 and pSJC14 respectively. The luxS : : KnR fragments were excised from pSJC5 and pSJC14 and cloned into the suicide vector pKNG101, generating the marker-exchange plasmids pSJC6 and pSJC15 respectively. Marker-exchange with pSJC6 and pSJC15 was carried out using a similar protocol to that of Kaniga et al. (1991). Transconjugants were selected on minimal medium containing 0·2 % glucose+Sm. Resolvants, in which resolution of the plasmid from the chromosome had occurred, leaving only the disrupted allele, were selected on minimal medium containing 10 % sucrose+Kn. For each strain, the disruption of the locus was confirmed by PCR analysis using primers complementary to the KnR cassette and to the luxS locus outside of the region used in the marker-exchange. Where sequence from the up- or downstream gene was included in the marker-exchange construct, this region was sequenced to ensure that no errors had been introduced. Mutations were also reintroduced into a wild-type genetic background by generalized transduction to confirm association between mutation and phenotype.
Construction of plasmids for the expression of luxS in trans.
Since S. marcescens 274 is Ap-resistant, the Cm-resistance (CmR) cassette from pACYC184 was cloned into the XbaI site of pBluescript-II KS+ to generate the vector control plasmid pSJC20. luxS and flanking sequence (817 bp in total) was PCR-amplified from S. marcescens 274 using primers SC60 and SC61 and cloned into pSJC20 to generate pSJC27 (luxSSma in trans). Similarly, 914 bp of luxS and flanking sequence was amplified from Ecc ATTn10 using primers SC34 and SC20 and cloned into pBluescript-II KS+ to generate pSJC10. The CmR cassette from pACYC184 was cloned into the XbaI site of pSJC10 to generate pSJC16 (luxSEcc in trans).
Measurement of prodigiosin production.
Cells were harvested from 1 ml samples of liquid culture by centrifugation and the pellet was resuspended in 1 ml acidified ethanol (4 % HCl) to extract prodigiosin from the cells. Following a second centrifugation step, the A534 of the supernatant was measured (Slater et al., 2003).
Haemolysin assay.
Blood agar was prepared by adding 5 % washed erythrocytes to LB agar. Defibrinated horse blood was obtained from TCS Biosciences and the erythrocytes were washed in cold 0·9 % NaCl.
C. elegans virulence assay.
Assays of Serratia killing were based on those used by Kurz et al. (2003). NGM plates were inoculated with the Serratia strain to be tested and then incubated at 37 °C for 810 h. For each test, 50 L4 stage hermaphrodite N2 worms per bacterial strain were used. The worms had been previously fed on E. coli OP50 until they reached the N2 stage and were then transferred to the new plates containing the bacterial strain to be tested (ten worms per plate). Plates were incubated at 20 °C and scored for live worms every 24 h. Worms were considered dead when no longer responsive to touch and were transferred to new plates daily. One-sided rank log tests [within the PRISM (Graphpad) software package] were used to assess the similarity between two groups (i.e. worms grown on wild-type and on mutant Serratia, where n=50 for each). P values <0·05 were considered statistically significant.
Complementation with conditioned medium.
Conditioned medium (CM) from Serratia 39006 and SCC6 was prepared as follows. Cultures (100 ml) were grown for 10 h and cell-free supernatant was harvested by centrifugation followed by passage of the supernatant through a 0·22 µm sterile filter (Millipore) and stored at 80 °C. CM was added to the culture at the start of growth, at the stated final concentration.
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RESULTS |
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Analysis of the sequence of the luxS locus in S. marcescens 274 identified a 516 bp ORF encoding a LuxS homologue (luxSSma). The predicted protein, LuxSSma, shows 84 % and 77 % identity to LuxS from Escherichia coli and V. harveyi respectively. Upstream of luxS is a -glutamate cysteine ligase (gshA) gene. Downstream of luxS is a convergently transcribed ORF with sequence similarity to a conserved hypothetical putative membrane protein known as CorB or YfjD (Fig. 2
). Similarly, in Ecc ATTn10, a 516 bp ORF encoding a LuxS homologue was identified (luxSEcc), with LuxSEcc showing 88 % identity to LuxSSma. Upstream of luxS, again, is a gshA gene. The downstream ORF, however, shows sequence similarity to a different conserved hypothetical putative membrane protein known as CorE (Fig. 2
).
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Growth and AI-2 production of luxS mutants of S. marcescens 274 and Serratia 39006
In order to confirm that luxS is indeed responsible for the production of AI-2 activity in S. marcescens 274 and Serratia 39006, the chromosomal copy of the gene was inactivated in each strain using a marker-exchange strategy based on the suicide vector pKNG101 (Kaniga et al., 1991). The correct disruption of the luxS gene was confirmed by PCR analysis and sequencing (data not shown). The luxS mutants of S. marcescens 274 and Serratia 39006 were named SCC4 and SCC6 respectively. AI-2 production throughout growth by SCC4 and SCC6 was determined. As shown in Fig. 1
, SCC4 and SCC6 were unable to produce AI-2 activity (indicated by negligible induction of light production in the BB170 bioassay). Therefore, as expected, luxS is responsible for the production of AI-2 in S. marcescens 274 and Serratia 39006.
As can be seen in Fig. 1 and Fig. 3
(a), the growth rate of the luxS mutants SCC4 and SCC6 in LB is the same as that of the corresponding wild-type strains. The growth of SCC4 in minimal defined medium was also the same as the wild-type S. marcescens 274 (data not shown). SCC6 exhibited a mild decrease in growth rate, but not final cell density, compared to wild-type Serratia 39006 when grown in minimal medium (doubling time increased from 3·1 to 3·8 h). The luxS mutants SCC4 and SCC6 showed no difference in their ability to grow under conditions of oxidative stress, iron starvation or anaerobiosis when compared to the wild-type strains (data not shown).
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The luxS mutant of S. marcescens 274 exhibits reduced haemolysin production
S. marcescens spp. produce a variety of secreted products, including haemolysin and protease (Braun et al., 1993; Hejazi & Falkiner, 1997
). Production of secreted haemolysin by wild-type S. marcescens 274 and the luxS mutant SCC4 was assayed on blood agar plates. SCC4 showed reduced production of haemolytic activity compared to the wild-type (Fig. 3c
). Haemolysin production by SCC4 was restored to wild-type levels by expression of luxS in trans (data not shown). Therefore a functional copy of luxS is required for production of wild-type levels of secreted haemolysin activity. Production of secreted protease and nuclease activity was not affected in SCC4 compared to the wild-type (data not shown).
The luxS mutant of S. marcescens 274 shows altered virulence in a C. elegans model
In order to determine if the virulence of the luxS mutant SCC4 is impaired compared to the wild-type S. marcescens 274 in vivo, the C. elegans model system (Kurz et al., 2003) was used. As shown in Fig. 4
, the rate of killing by SCC4 was altered compared to the wild-type, with a statistically significant increase in the survival of worms grown on the mutant compared to those grown on the wild-type being observed in two independent experiments. Therefore the luxS mutant of S. marcescens 274 exhibits a small but reproducible diminution in virulence.
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DISCUSSION |
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We inactivated the luxS gene in S. marcescens 274 and Serratia 39006, showing that a functional luxS gene is required for AI-2 production by these strains. We then studied the luxS mutants in order to determine which phenotypes are regulated by luxS and therefore potentially by AI-2. A wide range of phenotypes have previously been reported for luxS mutants. These include reduced secreted protease activity and enhanced haemolytic activity in Streptococcus pyogenes, altered carbohydrate metabolism in Streptococcus gordonii, impaired adaptation to iron-limited conditions in Actinobacillus actinomycetemcomitans, altered biofilm formation in Streptococcus mutans, and decreased virulence in V. vulnificus (Fong et al., 2003; Kim et al., 2003
; Lyon et al., 2001
; McNab et al., 2003
; Merritt et al., 2003
). A microarray analysis revealed that expression of
10 % of E. coli genes is altered in the luxS mutant; whereas in Salmonella typhimurium, the only genes found to be differently expressed in a luxS mutant, in an intensive genetic screen, were those of an operon encoding an ABC transporter which apparently functions to import AI-2 into the cell (Sperandio et al., 2001
; Taga et al., 2001
). In general, the growth of luxS mutants is unaffected, especially in rich medium, although there have been several reports of a luxS mutant having some kind of growth defect (Jones & Blaser, 2003
; Lyon et al., 2001
). In Serratia 39006, but not S. marcescens 274, a modest decrease in growth rate (but not final cell density) was observed in minimal medium.
In S. marcescens 274, a considerable decrease in prodigiosin production was observed in the luxS mutant. The physiological role of this red antibiotic pigment is currently unclear; it may be involved in virulence (e.g. it can uncouple vacuolar-type ATPases: Ohkuma et al., 1998), it may act as a proline sink (Hood et al., 1992
) or it may have a different role entirely. The regulation of prodigiosin production is complex, with many environmental inputs (Slater et al., 2003
). Here we have shown that in S. marcescens 274, luxS is involved in regulation of prodigiosin production, whereas in Serratia 39006 it is not. The production of secreted haemolysin activity is also decreased in the luxS mutant of S. marcescens 274. Haemolysin is a well-characterized virulence factor of S. marcescens (Braun et al., 1993
). Its production is dependent on a two-gene operon, shlBA, in which ShlA is the pore-forming protein, and ShlB activates ShlA and transports it out of the cell (Braun et al., 1993
). Altered production of secreted virulence factors has been reported in the luxS mutants of other bacteria, for example reduced production of cysteine protease and haemagglutinin activities in P. gingivalis, decreased toxin production in C. perfringens, and enhanced production of haemolysin activity in Streptococcus pyogenes and V. vulnificus (Burgess et al., 2002
; Kim et al., 2003
; Lyon et al., 2001
; Ohtani et al., 2002
). In contrast with the latter two strains, in S. marcescens 274, haemolysin production is decreased rather than increased in the luxS mutant.
The luxS mutant of Serratia 39006 was found to produce reduced levels of the antibiotic carbapenem. This is in stark contrast to the situation in Photorhabdus luminescens, where carbapenem production is increased in the luxS mutant (Derzelle et al., 2002). However, the regulation of carbapenem production is clearly different in the two genera. In Serratia 39006, in addition to this luxS modulation, carbapenem production is under strict aHSL-mediated QS control (Slater et al., 2003
), whereas in Photorhabdus luminescens, no aHSL system was detected (Derzelle et al., 2002
).
We investigated whether either of the Serratia luxS mutants exhibits altered virulence in an in vivo model. The nematode C. elegans is a very useful model system for the identification of bacterial virulence factors (Ewbank, 2002; Kurz et al., 2003
). Using this model, we showed that the S. marcescens 274 luxS mutant exhibits a subtle but reproducible virulence defect. Attenuation of virulence has been seen in various luxS mutants, for example in N. meningitidis, V. vulnificus and Streptococcus pneumoniae; although in other bacteria, virulence is not significantly altered in the luxS mutant, at least in the chosen model (Burgess et al., 2002
; Kim et al., 2003
; Schneider et al., 2002
; Stroeher et al., 2003
; Winzer et al., 2002b
). The reduced production of haemolysin and/or prodigiosin by the luxS mutant of S. marcescens 274 may contribute to its altered virulence.
An important issue generally is whether the phenotypes observed for luxS mutants are due to a genuine signalling defect or simply a metabolic one (see Introduction). It should also be remembered that AI-2 could be a signal reporting on environmental conditions or the presence (or metabolic status) of other cells, rather than on cell density per se. In this study, we have shown that luxS regulation of prodigiosin production in S. marcescens 274 is indeed via an extracellular signal. This luxS-dependent signal is non-species-specific, as shown by the fact that complementation of a S. marcescens 274 luxS mutant is seen with CM from Serratia 39006 (Fig. 3b) and also with CM from the phytopathogen Ecc ATTn10 (wild-type, but not an isogenic luxS mutant; unpublished results). The simplest explanation is that the signal is AI-2, although it could also be another molecule the presence of which is indirectly dependent on LuxS activity, for example the luxS-dependent extracellular factor AI-3, distinct from AI-2, recently reported to be controlling motility and type III secretion in E. coli (Sperandio et al., 2003
).
Other studies where CM from a luxS+ source was used to complement luxS mutant phenotypes have also been reported. For example, the toxin production defect in luxS mutants of C. perfringens and altered protease and haemolysin production in luxS mutants of V. vulnificus have been complemented by luxS+ but not luxS CM (Kim et al., 2003; Ohtani et al., 2002
). Conclusive proof that AI-2 is the active signal in these cases awaits complementation with pure, synthetic or purified, AI-2. This is not experimentally trivial, however, since it is not clear that the structure of AI-2 is the same in every species or environment (Chen et al., 2002
). Although we have shown that pigment production in S. marcescens 274 is regulated by extracellular signalling, it remains possible that one or more of the other phenotypes we observed is at least partly due to a metabolic defect.
An important point to have emerged in this study is the strain-dependence of luxS regulation in Serratia spp. For example, production of prodigiosin is affected in the luxS mutant of S. marcescens 274 but not Serratia 39006, and only the S. marcescens 274 luxS mutant has a statistically significant difference in virulence. A luxS mutant was also generated in a non-pigmented isolate of S. marcescens, S. marcescens strain 12. However in this strain, the only phenotype observed in the luxS mutant, apart from loss of AI-2 production, was an increase in mucoidy on the C. elegans growth medium. The mutant was unaffected in all other phenotypes examined, including virulence, haemolysin production and motility (data not shown). Our results suggest that luxS has a more significant regulatory role in S. marcescens 274 than in Serratia 39006 or S. marcescens strain 12. In different bacterial species, a wide variety of phenotypes have been reported to be luxS dependent; however, not all bacteria with LuxS necessarily use AI-2 for signalling (McNab & Lamont, 2003; Winzer et al., 2002a
). Our observations highlight the importance of strain-dependent effects in luxS/AI-2 signalling, but also in the study of QS generally.
In conclusion, in this work we have shown for the first time that luxS regulates production of antibiotic secondary metabolites in Serratia spp., that luxS-mediated regulation is strain-dependent and that, in at least one strain of the opportunistic pathogen S. marcescens, luxS is involved in modulating virulence. We have also shown that for at least one phenotype, prodigiosin production by S. marcescens 274, this regulation is via extracellular signalling.
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ACKNOWLEDGEMENTS |
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Winzer, K., Hardie, K. R., Burgess, N. & 8 other authors (2002a). LuxS: its role in central metabolism and the in vitro synthesis of 4-hydroxy-5-methyl-3(2H)-furanone. Microbiology 148, 909922.
Winzer, K., Sun, Y. H., Green, A., Delory, M., Blackley, D., Hardie, K. R., Baldwin, T. J. & Tang, C. M. (2002b). Role of Neisseria meningitidis luxS in cell-to-cell signaling and bacteremic infection. Infect Immun 70, 22452248.
Received 28 November 2003;
revised 2 February 2004;
accepted 9 February 2004.
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