luxS mutants of Serratia defective in autoinducer-2-dependent ‘quorum sensing’ show strain-dependent impacts on virulence and production of carbapenem and prodigiosin

Sarah J. Coulthurst1, C. Léopold Kurz2 and George P. C. Salmond1

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


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The enzyme LuxS is responsible for the production of autoinducer-2 (AI-2), a molecule that has been implicated in quorum sensing in many bacterial species. This study investigated whether there is a luxS-dependent signalling system in the Gram-negative bacteria Serratia spp. Serratia marcescens is a broad-host-range pathogen and an important cause of nosocomial infections. Production of AI-2 activity was detected in S. marcescens ATCC 274 and Serratia ATCC 39006 and their luxS genes were sequenced. luxS mutants were constructed in these strains and were analysed to determine which phenotypes are regulated by luxS and therefore, potentially, by AI-2. The phenotypes of the luxS mutants included decreased carbapenem antibiotic production in Serratia ATCC 39006 and decreased prodigiosin and secreted haemolysin production in S. marcescens ATCC 274. The luxS mutant of S. marcescens ATCC 274 was also found to exhibit modestly reduced virulence in a Caenorhabditis elegans model. Finally, it was shown that the culture supernatant of a wild-type strain contains a signal, presumably AI-2, capable of complementing the prodigiosin defect of the luxS mutant of another strain, even when substantially diluted. It is concluded that luxS modulates virulence and antibiotic production in Serratia, in a strain-dependent manner, and that, for at least one phenotype, this regulation is via extracellular signalling.


Abbreviations: aHSL, N-acyl-L-homoserine lactone; AI-2, autoinducer-2; CM, conditioned medium; Ecc, Erwinia carotovora subsp. carotovora; QS, quorum sensing

The GenBank accession numbers for the sequences reported in this paper are AJ628150, AJ628151 and AJ628152.


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Quorum sensing (QS) is the process by which bacteria detect their population density and use this information to regulate gene expression accordingly. Bacterial cells produce low-molecular-mass signal molecules which accumulate in their surroundings as the population increases. When the concentration of the molecule exceeds a threshold value, signalling pathways are activated and the bacteria respond by altering gene expression and modulating physiological processes in a concerted manner throughout the population. A wide spectrum of important processes, in diverse bacterial species, is regulated by QS, for example virulence, production of secondary metabolites, symbiosis, sporulation and biofilm formation (Whitehead et al., 2001). The most extensively studied QS systems in Gram-negative bacteria are those utilizing N-acyl-L-homoserine lactone (aHSL) signal molecules, in which LuxI homologues synthesize various aHSL signals and LuxR-type transcriptional regulatory proteins bind their cognate signal and alter gene expression accordingly (reviewed by Whitehead et al., 2001). A second type of QS system has also been described in Gram-negative bacteria. The autoinducer-2 (AI-2) system was discovered in the marine bacterium Vibrio harveyi, where it is one of two converging QS pathways that regulate bioluminescence (the other uses an aHSL signal, AI-1). As reviewed (Schauder et al., 2001; Whitehead et al., 2001), at high cell density, AI-2 binds LuxP, initiating a dephosphorylation cascade which results in the dephosphorylation and inactivation of the response regulator LuxO; inactivation of LuxO, together with positive activation by LuxRVh, allows expression of the bioluminescence operon. The synthesis of AI-2 is dependent on the enzyme LuxS. The structure of AI-2 bound to LuxP has been solved and was found to be a furanosyl borate diester (Chen et al., 2002).

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 {beta}-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.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains, plasmids and culture conditions.
Bacterial strains and plasmids are described in Table 1. Serratia strains were routinely grown at 30 °C and E. coli strains at 37 °C in Luria broth (LB), containing 10 g tryptone l–1, 5 g yeast extract l–1, 5 g NaCl l–1, and on LB agar. Minimal defined media and AB medium were as described by Greenberg et al. (1979) and Sambrook et al. (1989). Cultures were grown with good aeration (300 r.p.m.) and growth was measured as OD600 using a Unicam He{lambda}ios spectrophotometer and 1 cm path length cuvettes. When required, media were supplemented with antibiotics: kanamycin (Kn) 50 µg ml–1, streptomycin (Sm) 50 µg ml–1, ampicillin (Ap) 100 µg ml–1 and chloramphenicol (Cm) 50 µg ml–1.


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Table 1. Bacterial strains, plasmids and phages used in this study

 
AI-2 bioassay.
The V. harveyi BB170 bioassay was used to detect AI-2 activity (Surette & Bassler, 1998). Samples (10 µl) of cell-free supernatant were added to the wells of a black microtitre plate. A 16 h overnight culture of BB170, grown at 30 °C in AB medium to an OD600 of 1·20, was diluted 1 in 5000 in fresh AB medium and 90 µl added to each sample. The bioassay was incubated for 5 h at 30 °C and light production measured using an Anthos LUCY1 luminometer. Cell-free supernatant samples were prepared by centrifugation at 13 000 r.p.m. for 5 min, followed by passage of the supernatant through a 0·22 µm filter (Millipore), and were stored at –80 °C. Positive control samples were obtained from 5 ml AB overnight cultures of BB152, grown for 16 h to an OD600 of 1·25.

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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Table 2. Oligonucleotide primers used in this study

 
The degenerate primers SC03 and SC04, binding bp 1–20 and 220–245 in E. coli luxS, were used to amplify fragments of luxS from S. marcescens 274 and Ecc ATTn10 genomic DNA. The sequences of these fragments were then used in a single-specific-primer PCR strategy, based on Shyamala & Ames (1989). Briefly, for each strain, genomic DNA was digested with pairs of restriction enzymes which cut in the multiple cloning site (MCS) of pBluescript-II KS+. The resulting fragments were ligated into pBluescript-II KS+ cut with the same restriction enzymes. PCR reactions using combinations of two luxS-specific primers (pointing outwards from the known sequence) and two vector-specific primers (pointing into the MCS) were performed on the ligation mixes. The resulting PCR products were sequenced and the sequence of luxS and flanking regions was assembled and analysed. The luxS-specific primers used were SC10 and SC11 for S. marcescens 274, and SC12 and SC13 for Ecc ATTn10. Vector-specific primers were ST3 and ST7. In Serratia 39006, the luxS gene and surrounding regions were PCR-amplified, using one primer complementary to gshA (SC39) and one to the corE-like gene (SC37), and sequenced.

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 8–10 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.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
S. marcescens 274 and Serratia 39006 produce AI-2 activity and possess a luxS gene
The V. harveyi BB170 bioassay (Bassler et al., 1997) was used to detect whether AI-2 activity was present in the culture supernatants of S. marcescens 274 and Serratia 39006. The sensor strain BB170 responds to the presence of exogenous AI-2 by induction of bioluminescence. As shown in Fig. 1, both S. marcescens 274 and Serratia 39006 produced significant amounts of AI-2 activity. The peak in AI-2 activity was observed in late-exponential phase in S. marcescens 274 and during the transition into stationary phase in Serratia 39006; the activity then decreased into stationary phase in both strains. These profiles of AI-2 production throughout growth are in accordance with published results in other bacteria (Burgess et al., 2002; Surette & Bassler, 1998).



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Fig. 1. Production of AI-2 activity throughout growth by wild-type and luxS mutant strains of S. marcescens 274 and Serratia 39006. Production of AI-2 by (a) S. marcescens 274 (wild-type, {bullet}) and SCC4 (luxS, {blacksquare}); and (b) Serratia 39006 (wild-type, {blacktriangleup}) and SCC6 (luxS, {blacklozenge}); was measured during growth in LB. Growth of strains, measured as OD600, is represented by open symbols. At hourly intervals, supernatant samples were harvested and their AI-2 activity was measured using the V. harveyi BB170 bioassay. AI-2 activity induces bioluminescence in the sensor strain, reported as light units (lu). For comparison, positive control samples from V. harveyi BB152 produced 68 lu and 45 lu in bioassays (a) and (b) respectively, and media alone produced <0·04 lu. Each experiment was performed at least in duplicate with essentially the same results.

 
The production of AI-2 activity by S. marcescens 274 and Serratia 39006 suggested the presence of a luxS homologue in these bacteria. For comparison, and because of various phenotypic similarities to Serratia 39006, we also wanted to know whether the phytopathogen Ecc possesses a luxS homologue. The luxS gene was identified in S. marcescens 274 and Ecc ATTn10 by using the high conservation of the gene in different species of bacteria. Degenerate primers designed to conserved regions of the gene were used to PCR-amplify 245 bp fragments from S. marcescens 274 and Ecc ATTn10. These fragments were sequenced and confirmed to be part of a luxS homologue. These known sequences were then used in a PCR-based strategy, single-specific-primer PCR (Shyamala & Ames, 1989), to amplify stretches of DNA containing the rest of the gene and some up- and downstream sequence; 2·85 kb and 1·33 kb of luxS and surrounding regions were sequenced for S. marcescens 274 and Ecc ATTn10 respectively.

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 {gamma}-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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Fig. 2. The luxS loci of S. marcescens 274, Ecc ATTn10 and Serratia 39006. Schematic representation of the organization of the luxS locus in S. marcescens 274, Ecc ATTn10 and Serratia 39006. Distances below genes refer to the length of the gene that was sequenced; for genes that were not sequenced along their entire length, a pair of strokes (//) indicates that the gene extends further in that direction. For comparison, the equivalent region from E. coli K-12 is shown. E. coli K-12 genome sequence data were obtained from NCBI (http://www.ncbi.nlm.nih.gov/).

 
In Serratia 39006, 1·1 kb of luxS and flanking DNA were amplified using PCR primers complementary to conserved regions of gshA and corE. This region contains a luxS gene (luxS39006) with gshA upstream and a corE-like gene downstream, in a similar arrangement to Ecc ATTn10 but in surprising contrast with S. marcescens 274 (Fig. 2). LuxS39006 shows 88 % identity to LuxSSma and 87 % to LuxSEcc.

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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Fig. 3. The luxS mutant of S. marcescens 274 produces reduced levels of prodigiosin and secreted haemolysin; this reduced-prodigiosin phenotype can be complemented by addition of conditioned medium (CM) from a luxS+ (AI-2+) but not an isogenic luxS mutant (AI-2) strain. (a) Production of prodigiosin throughout growth in LB by S. marcescens 274 (wild-type, {bullet}) and SCC4 (luxS, {blacksquare}). Growth is represented by open symbols. Points and error bars represent mean and range respectively of duplicate experiments. Measurement of prodigiosin is described in Methods. (b) Prodigiosin production after 16 h growth in LB by S. marcescens 274 (wild-type, WT); SCC4 (luxS); S. marcescens 274(pSJC20) [vector control]; SCC4(pSJC20); SCC4(pSJC27) [LuxSSma in trans]; SCC4(pSJC16) [LuxSEcc in trans]; SCC4+10 % CM from Serratia 39006 (luxS+, AI-2+); and SCC4+10 % CM from SCC6 (luxS, AI-2). The CM was added at the start of growth and the presence or absence of AI-2 activity had previously been confirmed by V. harveyi BB170 bioassay (see text). Bars show mean±SD (n=3 or 4). (c) The luxS mutant of S. marcescens 274 produces less secreted haemolysin activity than the wild-type. Blood agar was prepared as described in Methods and plates were inoculated with 5 µl of overnight culture, diluted to OD600 0·2, and incubated at 30 °C for 3 days. Production of secreted haemolytic activity by S. marcescens 274 (wild-type, left) and SCC4 (luxS, right) is indicated by zones of clearing around the colonies. The plate shown is representative of three independent experiments.

 
The luxS mutant of S. marcescens 274 exhibits reduced production of prodigiosin
One of the most striking properties of S. marcescens 274 is its production of copious amounts of the red-pigmented antibiotic prodigiosin (see Introduction). Production of prodigiosin by the luxS mutant SCC4 was found to be reduced throughout growth in LB, compared to that of the wild-type S. marcescens 274 (Fig. 3a). To confirm that the pigment defect is a direct consequence of the inactivation of luxS, we attempted to complement the defect by the expression of LuxS in trans. Plasmid pSJC27 contains luxS and flanking regions from S. marcescens 274, together with a separate CmR cassette, cloned into the high-copy vector pBluescript-II KS+. In order to investigate whether the gene is functionally conserved between species, plasmid pSJC16, containing luxSEcc instead of luxSSma, was also constructed. S. marcescens SCC4 was transformed with pSJC27, pSJC16 and the vector control pSJC20. SCC4(pSJC27) and SCC4(pSJC16) were shown to be expressing functional LuxS by using the BB170 bioassay to confirm that SCC4(pSJC27) and SCC4(pSJC16), but not SCC4(pSJC20), produced AI-2 activity. SCC4(pSJC27) and SCC4(pSJC16) induced more than 500 times greater light production than SCC4(pSJC20). As shown in Fig. 3(b), both plasmid pSJC27, containing the native S. marcescens luxS in trans, and plasmid pSJC16, containing the heterologous Ecc ATTn10 luxS in trans, were able to restore the prodigiosin production of SCC4 to wild-type levels (i.e. to the same level as wild-type S. marcescens 274 carrying the vector control pSJC20). Thus the pigment defect of SCC4 is indeed a direct consequence of the inactivation of luxS, and LuxS is functionally conserved between S. marcescens and Ecc. In addition, it is interesting to note that the reduction in pigment production caused by the presence of the vector is more pronounced in the luxS mutant than in the wild-type strain; however, the cause of this increased sensitivity is unclear.

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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Fig. 4. The luxS mutant of S. marcescens 274 is less virulent than the wild-type during infection of C. elegans. Kinetics of killing of C. elegans infected by S. marcescens 274 (wild-type, {bullet}) and SCC4 (luxS, {blacksquare}) are shown. Fifty N2 hermaphrodites were used for each strain and were grown at 20 °C (see Methods). The difference between the two strains is statistically significant (P<0·006). The curves shown are from one representative experiment; a second independent experiment also gave a statistically significant difference between the two strains (P<0·04).

 
The luxS mutant of Serratia 39006 exhibits decreased production of carbapenem antibiotic
We were interested to see if the characteristics of the luxS mutant of Serratia 39006 would be similar to those of SCC4. Unlike S. marcescens 274, Serratia 39006 produces the antibiotic carbapenem. The luxS mutant of Serratia 39006, SCC6, was found to produce decreased levels of carbapenem compared to the wild-type (Fig. 5). The virulence of SCC6 was compared to that of wild-type Serratia 39006 using the C. elegans model, as for S. marcescens 274. No statistically significant difference in survival between worms grown on SCC6 compared to the wild-type was observed in two independent experiments. Serratia 39006 also produces the red pigment prodigiosin. However, unlike the case in S. marcescens 274, the luxS mutant of Serratia 39006 did not show a reduction in pigment production compared to the wild-type. Pigment per cell (100xA534/OD600) after 16 h growth in LB was 4·36±0·34 for the wild-type and 4·40±0·27 for the luxS mutant. SCC6 also showed no discernible difference compared to the wild-type in production of secreted exoenzymes (cellulase and pectate lyase; this strain does not produce secreted protease or haemolysin) or motility. Production of aHSL QS signal molecules was also unaffected in the luxS mutant of Serratia 39006 (data not shown).



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Fig. 5. Production of carbapenem by Serratia 39006 and its luxS mutant SCC6. A 5 ml agar overlay containing 10 µl ESS overnight culture was inoculated with 3 µl spots of overnight culture, diluted to OD600 0·2, of the test strains and incubated for 24 h at 30 °C. Carbapenem production is indicated by zones of antibiosis around the colonies of Serratia 39006 (wild-type, right) and SCC6 (luxS, left). The plate shown is representative of three independent experiments.

 
Complementation of the prodigiosin production defect of SCC4 by conditioned medium from a luxS+ strain, but not an isogenic luxS mutant
Finally, we wanted to address the issue of whether the observed phenotypic differences of the luxS mutants are caused by the absence of a signalling molecule or simply the lack of the metabolic functions of the LuxS enzyme. The prodigiosin production defect of SCC4 was chosen for this experiment since it is both quantifiable and experimentally convenient. Conditioned medium (CM) was harvested from wild-type Serratia 39006 and its luxS mutant SCC6 after 10 h growth in LB and was shown to contain substantial, and no, AI-2 activity respectively, using the BB170 bioassay (light units in the presence of wild-type CM >500x light units with SCC6 CM). Addition of 10 % CM from Serratia 39006 (luxS+/AI-2+) to SCC4 resulted in restoration of pigment production to wild-type levels, whereas addition of 10 % CM from SCC6 (luxS/AI-2) caused no significant change (Fig. 3b). When the experiment was repeated with 1 % CM, substantial, but incomplete, complementation of the pigment defect of SCC4 was observed (the increase in pigment production on addition of luxS+ CM, relative to luxS CM, was 1·68-fold on addition of 1 % CM, compared to 2·40-fold on addition of 10 % CM). Therefore the supernatant of luxS+ but not luxS Serratia 39006 does indeed contain a signal, presumably AI-2, that is able to complement the pigment defect of a luxS strain of S. marcescens.


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In this work, we aimed to determine whether several strains of Serratia produce luxS-dependent AI-2 activity and to investigate whether any other phenotypes are dependent on luxS in these strains. Initially, we showed that both S. marcescens 274 and Serratia 39006 are able to produce extracellular AI-2 activity, and sequenced the luxS gene and surrounding regions in these strains and the phytopathogen Ecc ATTn10. As observed in other enteric bacteria (e.g. E. coli and Salmonella), the gene upstream of luxS in all cases is gshA, whereas the gene downstream of luxS is variable (Schneider et al., 2002; and genome databases). Our results show that the identity of the downstream gene varies even between different strains of Serratia, being a corB/yfjD-like gene in S. marcescens 274 but a corE-like gene in Serratia 39006. The reason for this variability downstream of luxS is unclear, but it is also seen in Gram-positive bacteria, where the genes upstream of luxS are conserved between Bacillus anthracis and Bacillus subtilis, but the genes downstream are different between the two organisms (Jones & Blaser, 2003).

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.


   ACKNOWLEDGEMENTS
 
We would like to thank Bonnie Bassler for the kind gift of V. harveyi strains BB170 and BB152. We would also like to thank Graham Plastow and Martin Welch for valuable discussions and Ian Foulds for excellent technical support. Work by C. L. K. was conducted as part of his PhD thesis in the laboratory of Jonathan Ewbank, who we thank for support and critical reading of the manuscript. S. J. C. is funded by a BBSRC CASE studentship supported by Sygen International PLC; C. L. K. received an MENRT pre-doctoral fellowship; and G. P. C. S. is supported by BBSRC, NERC and the SGM.


   REFERENCES
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METHODS
RESULTS
DISCUSSION
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Received 28 November 2003; revised 2 February 2004; accepted 9 February 2004.



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