1 Section of Molecular Microbiology, BioCentrum-DTU, Building 301, Technical University of Denmark, DK-2800, Kgs. Lyngby, Denmark
2 Lehrstuhl für Mikrobiologie, Technische Universität München, Am Hochanger 4, D-85350 Freising, Germany
3 Danish Institute for Fisheries Research, Department of Seafood Research, Building 221, c/o Technical University of Denmark, DK-2800, Kgs. Lyngby, Denmark
Correspondence
Michael Givskov
immg{at}pop.dtu.dk
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ABSTRACT |
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The GenBank accession numbers for the sequences reported in this paper are AY040208 (16S rDNA of S. proteamaculans), AY040209 (sprIsprR of S. proteamaculans) and AY040210 (slaAlipB of S. proteamaculans).
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INTRODUCTION |
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At the molecular level, the majority of the quorum sensing regulatory R-proteins described to date work as transcriptional activators; however, few R-proteins have been suggested to work as repressors. The most thoroughly described R-protein functioning as a repressor is EsaR of Pantoea stewartii (formerly Erwinia stewartii). It has been suggested that EsaR represses the synthesis of extracellular polysaccharide at low cell density, and that the presence of its cognate signal molecule in micromolar amounts results in derepression (Beck von Bodman & Farrand, 1995; Beck von Bodman et al., 1998
; Minogue et al., 2002
). Other examples of R-proteins that have been proposed to function as repressors include SpnR of Serratia marcescens and YpsP of Yersinia pseudotuberculosis (Atkinson et al., 1999
; Horng et al., 2002
).
The AHL-producing phenotype is widespread among members of the Enterobacteriaceae (e.g. Serratia liquefaciens, Enterobacter agglomerans, Erwinia carotovora, Hafnia alvei and Rahnella aquatilis) (Eberl et al., 1996; Swift et al., 1993
, 1999
). It has been demonstrated that the ability to produce AHLs is a common feature among Enterobacteriaceae isolated from food products such as chilled, vacuum-packed meat and cold-smoked salmon (Gram et al., 1999
; Ravn et al., 2001
). These psychrophilic members of the Enterobacteriaceae are frequently encountered in the microflora of spoiling food products such as milk, cream, fish and minced meat, and are in some products involved in spoilage process (Borch et al., 1996
; Lindberg et al., 1998
).
The activities of hydrolytic enzymes produced by bacteria have been linked to the spoilage of raw food products. Proteolytic and lipolytic activities of psychrophilic Gram-negative bacteria are regarded as a cause of deterioration of milk and other dairy products (Burger et al., 2000; Tan & Miller, 1992
). Also, the soft-rot spoilage of vegetables and fruits is often caused by the pectinolytic activity of pseudomonads or Enterobacteriaceae (mostly Erwinia spp.) (Chatterjee et al., 1994
; Liao, 1989
). Interestingly, the pectinase of Erwinia carotovora is regulated by acylated homoserine lactone (Pirhonen et al., 1993
), suggesting that rot is controlled by a quorum-sensing mechanism. In S. marcescens and S. liquefaciens MG1, a functional lipB operon is required for the secretion of several unrelated and potentially food-quality-relevant proteins such as the lipase LipA, the metalloprotease PrtA and the surface-layer protein (S-layer) SlaA (Akatsuka et al., 1997
; Kawai et al., 1998
; Riedel et al., 2001
). The LipB protein translocation system is a type I secretion apparatus belonging to the superfamily of ATP-binding cassette transporters (ABC) that is dedicated to proteins lacking an N-terminal signal peptide [(for a review see Binet et al. (1997)
]. The structural organization and sequence conservation of the genes encoding the ABC protein translocation systems is highly conserved among the Gram-negative bacteria (Binet et al., 1997
). The lipB operon of S. marcescens consists of three genes, lipBCD, encoding an inner-membrane ATPase (the ABC protein), a membrane fusion protein and an outer-membrane polypeptide, respectively (Akatsuka et al., 1995
, 1997
). S. liquefaciens MG1 has been demonstrated to be under the transcriptional control of N-butyryl-L-homoserine lactone (C4-HSL), which renders the production of extracellular protease activity indirectly under the control of quorum sensing (Riedel et al., 2001
). In the present study, we have analysed quorum sensing regulation of protein expression in S. proteamaculans and have demonstrated that the activities of several exoenzymes are affected by 3-oxo-C6-HSL. This is the first report linking AHL-type signal molecules and chitinolytic activity in Serratia spp. In addition, the activities of food-quality-relevant phenotypes (i.e. expression of lipase and protease) are affected by 3-oxo-C6-HSL, which leads us to conclude that quorum sensing is involved in the control of food-spoilage processes.
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METHODS |
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PCR amplification of 16S rDNA.
The oligonucleotides used in this study are listed in Table 1. The amplification of the 16S rDNA was performed using the Expand High Fidelity PCR System according to manufacturer's instructions (Boehringer Mannheim). Using approximately 2 µg chromosomal DNA, primers 9F and 1512R and 1·5 mM MgCl2, PCR was performed on a Biometra Thermocycler. The thermal profile consisted of an initial denaturation step of 5 min at 94 °C followed by 30 cycles of 15 s at 94 °C, 30 s at 55 °C and 90 s at 72 °C, followed by a final step of 5 min at 72 °C. The PCR product was purified using a GFX PCR DNA and Gel Band Purification Kit (Amersham Pharmacia Biotech).
DNA manipulations.
Standard techniques for DNA manipulations were used (Sambrook et al., 1989). pUT and pUT-derived vectors were propagated in Escherichia coli CC118
pir. For construction of the pUT-derived luxAB-Smr vector pAC25, the 3·2 kb NotI fragment of pUTtcluxAB (spanning the promoterless luxAB genes) was ligated into the single NotI site of pUTsm. The orientation of the luxAB cassette was investigated by digesting pAC25 with SphI. A 3·2 kb fragment indicated that the luxAB cassette had been inserted in the correct orientation (data not shown). For construction of pAC26, chromosomal DNA of S. proteamaculans AC1 was digested with BglII and ligated into the BamHI site of pLOW1. Kanamycin-resistant Escherichia coli MT102 clones containing pAC26 carried a DNA fragment of approximately 12 kb. For construction of pAC27, chromosomal DNA of S. proteamaculans AC2 was digested with BglII and ligated into the BamHI site of pLOW1. Bioluminescent Escherichia coli MT102 clones containing pAC27 carried a DNA fragment of approximately 6 kb.
Construction of an AHL-negative mutant of S. proteamaculans B5aN.
A bank of random insertion mutants was made using the mini-Tn5 transposon delivery system described by Herrero et al. (1990). In brief, Escherichia coli CC118
pir harbouring pUTkm was used as donor, Escherichia coli HB101 harbouring pRK600 was used as helper and S. proteamaculans B5aN was used as recipient. The three strains were mixed in a ratio of 1 : 1 : 5 (donor : helper : recipient) and incubated on an LB agar plate for 6 h at 30 °C. The mutant bank was plated onto selective LB plates (containing nalidixic acid and kanamycin) and incubated for 12 h. To screen for 3-oxo-C6-HSL-negative mutants, the resulting bank of insertion mutants was replica-plated onto indicator LB plates containing approximately 1 % (v/v) outgrown culture of the monitor bacterium C. violaceum CV026. Putative AHL-negative clones were unable to induce violacein production after 12 h incubation.
Construction of quorum sensing target gene mutants of S. proteamaculans AC1.
A bank of secondary transposon insertion mutants was made using the mini-Tn5 transposon delivery system described by Herrero et al. (1990). Escherichia coli CC118
pir harbouring pAC25 ('luxAB-Smr) was used as donor, Escherichia coli HB101 harbouring pRK600 was used as helper and S. proteamaculans AC1 was used as recipient. The secondary mutant bank was plated onto selective LB plates (containing kanamycin and streptomycin) and incubated for 12 h. To screen for potential quorum-sensing-regulated genes, the resulting bank of double mutants was replica-plated onto selective LB plates and indicator LB plates containing 100 nM of 3-oxo-C6-HSL (Sigma-Aldrich; CAS no. 143537-62-6) and incubated for 12 h. Bioluminescent clones are expected to have the promoterless luxAB cassette inserted as a transcriptional fusion. The activities of bioluminescent transconjugants were compared using a Hamamatsu charge-coupled device camera connected via a Hamamatsu M4314 controller to a Hamamatsu Argus-50 image processor (Hamamatsu Photonics). Ten mutants with elevated bioluminescence expression on LB plates containing 3-oxo-C6-HSL were selected for further analysis.
Screening for protease-deficient secondary mutants.
The collection of secondary mutants with insertions in potential quorum sensing target genes was screened for proteolytic activity on LB plates containing 0·5 % (w/v) skim milk powder. One mutant, S. proteamaculans AC2, was unable to make clearing zones on the agar after 24 h incubation.
DNA nucleotide sequencing.
The oligonucleotides used in this study are listed in Table 1. Sequencing was performed at a commercial sequencing facility (K. J. Ross-Petersen AS; http://www.ross.dk/). PCR primers 9F and 1512R were used as sequencing primers for the 16S rDNA. Primers AC14a, AC14b, AC24, AC27, AC30/i-end, AC31/o-end and AC40a were used as sequencing primers for the sprI and sprR genes present on pAC26. The sequencing primer AC50/luxA was used for sequencing of the upstream DNA of the 'luxAB-Smr cassette present on pAC27.
DNA sequence analysis.
BLASTN and BLASTX homology searches for the DNA sequences in the non-redundant sequence databases were performed via the worldwide web at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/BLAST/). Multiple-sequence alignment was performed using the CLUSTAL W algorithm at Network Protein Sequence Analysis (http://pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_clustalw.html).
Southern blot.
Analyses of Southern blots were performed as described previously (Givskov et al., 1995). Chromosomal DNA of S. proteamaculans AC1 was digested with KpnI and PstI, and chromosomal DNA of S. proteamaculans AC2 was digested with BglII and XhoI. A DIG-labelled kanamycin-resistance gene and luxAB were used as probes against S. proteamaculans AC1 and S. proteamaculans AC2, respectively.
SDS-PAGE.
Standard SDS-PAGE was performed as described by Laemmli (1970). Proteins were visualized by silver staining according to Blum et al. (1987)
.
Two-dimensional PAGE analysis of total intracellular proteins.
S. proteamaculans B5aN and S. proteamaculans AC1 were grown in AB minimal medium until an OD450 value of approximately 4 was reached; thereafter the cells were harvested by centrifugation at 5000 g for 20 min, washed in a 0·9 % NaCl solution and resuspended in 100 mM HEPES buffer (pH 7·4). The cells were broken using a French Press and the membrane fraction was removed by centrifugation at 20 000 g for 1 h. To get a clearly focused protein pattern, a phenol extraction on bacterial extracts was performed as described previously (Hanna et al., 2000). In a 2 ml screw-cap tube, 1 ml aliquots of the extracts and 1 ml of phenol were mixed thoroughly and incubated for 10 min at 70 °C. The sample was cooled on ice for 5 min and the phases were separated by centrifugation for 10 min at 5000 g. The top aqueous phase was discarded and 1 ml distilled water was added. After vortexing and incubation for 10 min at 70 °C, the sample was cooled and the phases were separated as before. The aqueous phase was discarded and the proteins were precipitated by adding 1 ml ice-cold acetone. The sample was pelleted by centrifugation for 20 min at 10 000 g, and the pellet washed by adding 1 ml ice-cold acetone. After a final centrifugation step (20 min at 10 000 g), the supernatant was poured off and the pellet was air-dried for 30 min. The protein precipitate from the phenol extraction was solubilized in 1 ml lysis buffer [9·5 M urea, 2 % (w/v) CHAPS, 0·8 % (w/v) Pharmalyte pH 310 (Amersham Pharmacia Biotech), 1 % (w/v) DTT and 5 mM Pefabloc (Merck)]. For the isoelectric focusing, 8 µl of the samples were mixed with 342 µl of rehydration solution [8 M urea, 2 % (w/v) CHAPS, 15 mM DTT and 0·5 % (v/v) IPG-buffer pH 310 (Amersham Pharmacia Biotech)] resulting in a final protein amount of 110 µg per sample. The isoelectric focusing was performed by using immobilized pH gradient (IPG) strips (Amersham Pharmacia Biotech) as described elsewhere (Görg et al., 2000
). The IPG strips (18 cm, pH 310) were rehydrated overnight at 30 V and focused for 3 h at 8000 V at 20 °C under mineral oil. They were then incubated for 10 min in each case in equilibration buffer I [6 M urea, 30 % (w/v) glycerol, 2 % (w/v) SDS and 1 % (w/v) DTT in 0·05 M Tris/HCl buffer, pH 8·8] followed by equilibration buffer II [6 M urea, 30 % (w/v) glycerol, 2 % (w/v) SDS and 4 % (w/v) iodoacetamide in 0·05 M Tris/HCl buffer, pH 8·8]. After the equilibration step, the strips were transferred to 22x22 cm 12·5 % SDS-PAGE gels for the second dimension. Electrophoresis was performed at 150 V and 150 mA at 15 °C for approximately 18 h as described previously (Görg et al., 2000
).
Protein spots were visualized by silver staining as described elsewhere (Blum et al., 1987). The gels were scanned with a densitometric ImageScanner (Amersham Pharmacia Biotech) and the raw images were processed using the software IMAGE MASTER 2-D ELITE, version 3.0 (Amersham Pharmacia Biotech). Following spot editing and background subtraction, the protein patterns were matched to each other by visual inspection.
Measurements of bioluminescence.
Bioluminescence was quantified in a Bio-Orbit 1253 luminometer as described previously (Givskov et al., 1998).
Extraction of culture supernatants and analytical TLC.
AHL extractions and TLC were performed as described previously (Ravn et al., 2001). For the detection of AHLs, three different biomonitors were used as described previously, C. violaceum CV026 (McClean et al., 1997
), A. tumefaciens NT1 harbouring pDZLR4 (Cha et al., 1998
) and Escherichia coli MT102 harbouring pSB403 (Winson et al., 1998
).
Enzymic assays.
S. proteamaculans B5aN, S. proteamaculans AC1 and S. proteamaculans AC2 were grown in LB medium until an OD450 value of approximately 10 was reached. Culture supernatants were rescued and filter-sterilized prior to enzymic examinations. Chitinolytic activity was investigated on carboxymethyl-chitin-remazol brilliant violet (CM-chitin-RBV) by incubating 450 µl of a substrate mixture with 150 µl culture supernatant. The substrate mixture contained 1 volume of 0·2 % (w/v) CM-chitin-RBV and 2 volumes of succinate/NaOH buffer [100 mM] (pH 6·0). The mixture was incubated at 37 °C for 6 h. The enzymic reaction was stopped by adding 200 µl of 2 M HCl to the mixture and incubating at 0 °C for 1 h. Prior to spectroscopic measurement, the mixture was centrifuged for 10 min at 15 000 g. The chitinolytic activity was quantified by the determination of the OD550 value. Lipolytic activity was investigated as described previously (Riedel et al., 2001). One millilitre of a reaction mixture containing 100 µl bacterial supernatant and 900 µl substrate mixture was incubated for 1 h at room temperature. The substrate mixture contained 1 volume of 0·3 % (w/v) p-nitrophenyl palmitate in 2-propanol and 9 volumes of 0·2 % (w/v) sodium desoxycholate and 0·1 % (w/v) gummi arabicum in 50 mM sodium phosphate buffer (pH 8·0). The reaction mixture was centrifuged at 15 000 g for 5 min, and the enzymic reaction was terminated by adding 1·0 ml of 1 M Na2CO3 to the supernatant prior to spectroscopic measurement. The lipolytic activity was quantified by determination of the OD410 value. Proteolytic activity was investigated as described previously (Ayora & Gotz, 1994
). An aliquot (150 µl) of filter-sterilized culture supernatant was incubated with 250 µl substrate for 6 h at 37 °C. The substrate contained 1 % (w/v) azoalbumin (pH 7·5). The enzymic reaction was terminated by adding 1·2 ml of 10 % (w/v) trichloroacetic acid. The mixture was incubated for 15 min at room temperature and then centrifuged for 10 min at 15 000 g. Prior to spectroscopic measurement, 600 µl supernatant was rescued and mixed with 750 µl of 1 M NaOH. The proteolytic activity was quantified by the determination of the OD440 value. Specific enzymic activities were determined as the enzymic activity per optical density unit (OD450) of the growth culture.
Zymograms.
Chitinases were analysed by SDS-PAGE (15 % polyacrylamide). The substrate CM-chitin-RBV was incorporated into the gel matrix to a final concentration of 0·05 % (w/v). After electrophoresis, the proteins were renatured by washing the gel twice in a mixture containing 50 mM Tris/HCl (pH 6·0) and 25 % (v/v) 2-propanol for 15 min at room temperature. After renaturation, the zymogram was incubated for 24 h at 30 °C in 50 mM Tris/HCl (pH 6·0). Enzymic activity was detected as clear zones on a violet background. To improve contrast, the colours of the picture were inverted. Exolipase (or esterase) was analysed by SDS-PAGE (12 % polyacrylamide). After electrophoresis, the proteins were renaturated by washing the gel twice in a mixture containing 50 mM Tris/HCl (pH 7·5) and 25 % (v/v) 2-propanol for 15 min at room temperature and once in 50 mM Tris/HCl (pH 7·5). The renaturated gels were overlaid with a fluorescent substrate, 0·01 M methylumbelliferyl butyrate dissolved in N,N-dimethylformamide (Sigma), and incubated for 30 min at 30 °C. The enzymes could be detected with UV light (360 nm) as blue fluorescent bands. Exoproteases were analysed by SDS-PAGE of culture supernatants with 0·2 % azoalbumin incorporated in the gel matrix (12 % polyacrylamide). After electrophoresis, the proteins were renaturated by washing the gel twice in a mixture containing 50 mM Tris/HCl (pH 7·5) and 25 % (v/v) 2-propanol for 15 min at room temperature. After renaturation, the zymogram was incubated for 4 h in 50 mM Tris/HCl (pH 7·5). Prior to detection of the proteins, the gel was washed in 1 M NaOH for 5 min. Protease activity could be detected as colourless zones in an orange background.
Food spoilage.
Samples of pasteurized and homogenized cows milk containing 3·4 % protein, 1·5 % lipid and 4·8 % carbohydrates were inoculated with approximately 1x106 c.f.u. ml-1 of S. proteamaculans B5aN, S. proteamaculans AC1 or the S. proteamaculans AC2. Samples inoculated with S. proteamaculans AC1 or S. proteamaculans AC2 were grown in the absence and presence of 200 nM of 3-oxo-C6-HSL. The samples were incubated at room temperature for 18 h.
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RESULTS AND DISCUSSION |
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Construction of an AHL-deficient mutant
Using TLC on extracted, spent media, we have previously shown that the major AHL signal molecule produced by S. proteamaculans B5a is 3-oxo-C6-HSL (Gram et al., 1999). Interestingly, the close relatives S. marcescens and S. liquefaciens MG1 produce C4-HSL as their main products (Eberl et al., 1996
). S. liquefaciens MG1 produces C4-HSL and N-hexanoyl-L-homoserine lactone in a ratio of 10 : 1 (Eberl et al., 1996
).
An AHL-deficient S. proteamaculans mutant (denoted AC1) was constructed by random transposon mutagenesis as described in Methods. A Southern blot analysis confirmed that a single copy of the transposable element had been integrated into the chromosome of this mutant (data not shown). DNA sequence analysis on the flanking regions of the kanamycin insertion in S. proteamaculans AC1 situated the insertion to 220 bases downstream from the translation start in a gene belonging to the family of luxI homologous AHL synthases (Fig. 1a). Extracted, spent medium from the sprI mutant (AC1) was subjected to a TLC-based analysis and developed using CviR-, LuxR- and TraR-based AHL monitor systems. This combination of monitors covers the entire range of known AHL signal molecules (Andersen et al., 2001
; Ravn et al., 2001
). However, no responses in any of these monitors were detected (data not shown). Furthermore, cloning of chromosomal DNA from the wild-type S. proteamaculans B5aN digested with three different restriction enzymes and subsequent TLC analysis on extracts from AHL-producing Escherichia coli clones did not indicate the presence of additional AHL-synthase-encoding genes (data not shown). Taken together, these results strongly suggest that sprI encodes the sole AHL-synthesizing enzyme in S. proteamaculans.
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The sprI open reading frame (ORF) encodes a putative protein of 210 aa with a predicted molecular mass of 24·1 kDa (GenBank accession no. AAK76733). SprI showed its highest degree of relative sequence similarity (79 %) to EsaI of Pantoea stewartii (GenBank accession no. P54656). The degrees of relative sequence similarity to other LuxI homologous proteins were 77 % to SpnI of S. marcescens (GenBank accession no. AAN52498), 61 % to SwrI of S. liquefaciens MG1 (GenBank accession no. P52989) and 42 % to LuxI of V. fischeri (GenBank accession no. CAA68562). Adjacent of the sprI gene, a reading frame showing similarity to the family of luxR homologous genes was located (Fig. 1). The two genes are transcribed convergently and their reading frames contain 23 overlapping bases. Interestingly, a similar genetic arrangement has been reported in various members of the Enterobacteriaceae, including Pantoea stewartii and S. marcescens (Beck von Bodman & Farrand, 1995
; Horng et al., 2002
). By contrast, the luxI and luxR genes of V. fischeri are transcribed divergently (Engebrecht & Silverman, 1984
). The sprR ORF encodes a putative protein of 249 aa with a predicted molecular mass of 28·3 kDa (GenBank accession no. AAK76734), and it showed the highest degree of relative sequence similarity (86 %) to SpnR of S. marcescens (GenBank accession no. AAN52499). The degrees of relative sequence similarity of SprR to other LuxR homologous proteins were 84 % to EsaR of Pantoea stewartii (GenBank accession no. P54293), 66 % to SwrR of S. liquefaciens MG1 (L. Eberl, M. K. Winson, C. Sternberg, G. S. Stewart, G. Christiansen, S. R. Chhabra, B. Bycroft, P. Williams, S. Molin & M. Givskov, unpublished data) and 45 % to LuxR of V. fischeri (GenBank accession no. S06314). A helixturnhelix motif could be identified in the C-terminal part of SprR by similarity to other R-proteins. Surprisingly, the putative I- and R-proteins of the two phylogenetically closely related Serratia species S. liquefaciens MG1 and S. proteamaculans B5aN exhibit an unexpectedly low degree of relative sequence similarity. Though speculative, the high degree of relative sequence similarity to SpnR and EsaR of S. marcescens and Pantoea stewartii, respectively, suggests that SprR might function as a repressor analogous to these R-proteins. The regulatory properties of SprR are currently under investigation.
The quorum sensing regulon
To obtain an estimate of the number of proteins that are responsive to 3-oxo-C6-HSL in S. proteamaculans B5aN, the protein patterns of S. proteamaculans AC1 (sprI mutant) grown in the absence and presence of 3-oxo-C6-HSL were compared by means of two-dimensional PAGE (Fig. 2). The cells were grown in minimal media and harvested in the early-stationary phase. Two-dimensional PAGE analysis of the intracellular proteins from the sprI mutant (AC1) demonstrated a complex 3-oxo-C6-HSL-directed protein expression pattern. Out of a total of approximately 400 separated proteins, a subset of 23 proteins was found to be repressed in the presence of 3-oxo-C6-HSL and induced in the absence of 3-oxo-C6-HSL (Fig. 2b, c
). Yet another subset of 16 proteins was found to be repressed in the absence of 3-oxo-C6-HSL and induced in the presence of 100 nM of 3-oxo-C6-HSL (Fig. 2b, c
). The wild-type (B5aN) displayed a protein expression profile that was similar to the protein expression profile of the sprI mutant grown in the presence of exogenously added 3-oxo-C6-HSL (Fig. 2a, c
). This analysis, therefore, suggested the presence of a quorum sensing system in control of a minimum of 39 proteins. Quorum sensing thus governs control over approximately 10 % of the detectable proteins. However, since the present analysis was performed on early-stationary-phase cells, it is an underestimate of the total number of proteins produced during the growth cycle and cannot represent the entire proteome. It has been estimated that 34 % of the genes in Pseudomonas aeruginosa are under quorum sensing control (Whiteley et al., 1999
). Global protein expression analysis of the quorum sensing regulon in S. liquefaciens MG1 demonstrated that at least 28 proteins are subjected to control by a C4-HSL-dependent regulatory quorum sensing system (Givskov et al., 1998
). Thus the observation that a minimum of 39 proteins are controlled by a 3-oxo-C6-HSL-responsive quorum sensing system in S. proteamaculans B5aN is in good agreement with similar studies. The pleiotropic 3-oxo-C6-HSL-induced changes in the profile of intracellular proteins indicate that the putative R-protein might work as a repressor on one subset of target genes and yet as an activator on a second subset of the target genes. Alternatively, the putative SprR protein might work in conjunction with another regulatory component to modulate gene expression at the transcriptional level. Similar complex signal-molecule-induced expression profiles have been observed in Yersinia enterocolitica (Throup et al., 1995
) and S. liquefaciens MG1 (Givskov et al., 1998
). Multiple luxR homologous genes have been identified in S. marcescens, Erwinia carotovora and Pseudomonas aeruginosa (Latifi et al., 1995
; Salmond et al., 1995
; Thomson et al., 2000
). Thus, we cannot exclude the possibility that an additional luxR homologous gene is present in S. proteamaculans.
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Transcription of the lipB promoter was investigated throughout the growth cycle (LB medium) by measuring the specific activity of bioluminescence activity from the chromosomal lipB : : luxAB fusion in the AC2 double mutant (Fig. 3). Induction of lipB transcription was observed at an OD450 value of approximately 3·5, which is in the late-exponential phase of growth. The addition of 100 nM of 3-oxo-C6-HSL resulted in a steeper induction profile where induction occurred at a significantly lower cell density (OD450 value of approximately 2·5), leading to an approximately 50 % increase in specific activity (Fig. 3
). The elevated, 3-oxo-C6-HSL-induced, transcription from the lipB promoter suggests that the lipB transporter is a target for the quorum sensing regulatory system in S. proteamaculans B5aN. By definition, the expression of quorum-sensing-regulated genes is affected when the cell population reaches a certain size. This reflects the requirement for signal molecule accumulation. However, exogenous addition of 3-oxo-C6-HSL to the sprI lipB double mutant (AC2) in the early-exponential phase did not lead to immediate induction of lipB, indicating that expression of lipB requires additional regulatory elements. The delayed mode of induction is in contrast to the direct induction of the luxI promoter from V. fischeri observed in LuxR-based AHL monitor systems (Andersen et al., 2001
), but is in accordance with observations made with the quorum-sensing-controlled genes swrA and lipB in S. liquefaciens MG1 (Lindum et al., 1998
; Riedel et al., 2001
) and the induction of carA in S. marcescens (Thomson et al., 2000
). Interestingly, both immediate and delayed responses to AHL signal molecules have been reported in Pseudomonas aeruginosa (Whiteley et al., 1999
).
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The chitinase zymogram (Fig. 4a) presented one band with chitinolytic activity with an approximate molecular mass of 48 kDa. Production and secretion of several exoenzymes have been reported in S. marcescens, and at least three different chitinase-encoding genes have been identified: chiA encodes a 57 kDa enzyme, chiB encodes a 52 kDa enzyme and chiC encodes a 48 kDa enzyme (Watanabe et al., 1997
). We therefore presume that the chitinase from S. proteamaculans corresponds to ChiC of S. marcescens. The presence of a 48 kDa chitinolytic band in spent media from the lipB mutant indicates that the chitinase is secreted via a LipB-independent transporting system and that the chitinase is not subjected to proteolytic processing by LipB-dependent proteases. Gal et al. (1998)
have reported the proteolytic processing of a 52 kDa chitinase into an active 35 kDa enzyme by S. marcescens. The quantitative enzymic assays for chitinolytic activity (Fig. 4d
) demonstrated that the chitinolytic activity from the sprI mutant (AC1) and sprI lipB double mutant (AC2) were similar, indicating that the chitinase is not secreted via the LipB transporter. Therefore, the observation that chitinolytic activities were twofold-inducible upon 3-oxo-C6-HSL addition suggested that the quorum sensing system controls transcription of the chitinase gene directly. We cannot, however, exclude the existence of a specific chitinase transporter which might be under the control of quorum sensing and thereby subjecting chitinase production to indirect quorum sensing control. The biological significance of chitinases includes biocontrol of growth and development of certain insects, nematodes and fungi (Herrera-Estrella & Chet, 1999
). Serratia spp. are considered potential insect pathogens (Bucher, 1960
); moreover, antifungal properties have been attributed to members of this genus (Kalbe et al., 1996
). It is therefore likely that quorum sensing control of Serratia chitinases plays a key role in the expression of biocontrol phenotypes.
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The protease zymogram (Fig. 4c) presented two bands with proteolytic activity with approximate masses of 52 and 48 kDa, respectively. Whether the two proteolytic bands represent two different enzymes or processed versions of the same one remains to be elucidated. The protease from S. proteamaculans B5aN has approximately the same mass as the 52 kDa metalloprotease PrtA from S. liquefaciens MG1 and S. marcescens (Nakahama et al., 1986
; Riedel et al., 2001
). The quantitative enzymic assay for proteolytic activity (Fig. 4f
) on spent culture media from the wild-type (B5aN) and the sprI mutant (AC1) demonstrated a twofold induction upon AHL addition; this is in accordance with the observations made in S. liquefaciens MG1 (Riedel et al., 2001
). The sprI lipB double mutant was devoid of proteolytic and lipolytic activity in our quantitative enzymic assays, indicating that both lipase and proteases are secreted through the lipB transporter. These findings support the observations made in S. marcescens and S. liquefaciens MG1 (Akatsuka et al., 1995
; Riedel et al., 2001
). Hence, since the activities of the lipB promoters and the proteases are inducible by AHLs in both S. proteamaculans B5aN and S. liquefaciens MG1 (Riedel et al., 2001
), we propose that quorum sensing regulation of the lipB transporter is at least partially responsible for the observed induction of lipolytic and proteolytic activities.
SDS-PAGE analysis of the exoprotein profiles (Fig. 5) showed that seven exoproteins are present in spent culture media from the wild-type (B5aN) and the sprI mutant (AC1), but are undetectable in spent culture media from the sprI lipB double mutant (AC2), suggesting that the secretion of these proteins is dependent on the lipB transporter. We cannot exclude the possibility that the observed exoprotein profiles from the wild-type (B5aN) and sprI mutant (AC1) are a result of proteolytic degradation; likewise, the numerous exoprotein bands that are absent in the wild-type (B5aN) and sprI mutant (AC1) background, but present in the sprI lipB double mutant (AC2) background, could be caused by the lack of proteolytic activity by the lipB-dependent proteases. Therefore, the observed LipB-dependent exoproteins might represent fewer than seven distinct proteins.
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S. proteamaculans B5a was isolated from cold-smoked salmon, yet since the spoilage process of cold-smoked salmon is complex and involves interactions between lactic acid bacteria and Gram-negative bacteria (Jørgensen et al., 2000), we have investigated whether quorum sensing contributes to the spoilage process of food products where spoilage may be caused by the growth of psychrophilic members of the Enterobacteriaceae alone. We chose liquid milk in which Enterobacteriaceae spoilage can manifest itself as clotting due to proteolytic degradation of the casein micelles. Here, we present data demonstrating that quorum sensing regulation of protease and/or lipase contributes to the spoilage process of milk (Fig. 6
). Samples of commercially purchased cows milk were inoculated with approximately 1x106 c.f.u. ml-1 of the wild-type S. proteamaculans B5aN, the communication-deficient sprI mutant S. proteamaculans AC1 or the exolipase- and exoprotease-deficient sprI lipB double mutant S. proteamaculans AC2. Samples inoculated with S. proteamaculans AC1 or S. proteamaculans AC2 were grown in the absence and presence of 200 nM of 3-oxo-C6-HSL. After 18 h incubation at room temperature, samples inoculated with the wild-type and sprI mutant strain complemented with 200 nM of 3-oxo-C6-HSL were spoiled. However, samples inoculated with the sprI mutant in the absence of 3-oxo-C6-HSL, the sprI lipB double mutant inoculated without 3-oxo-C6-HSL and the sprI lipB double mutant in the presence of 200 nM of 3-oxo-C6-HSL appeared unspoiled. At the end point, all samples contained approximately 1x109 c.f.u. ml-1, and all samples showed a pH value of 6·2. These results demonstrate that the quorum sensing system controls phenotypes involved in the spoilage of food. Moreover, since the sprI lipB double mutant could not be complemented by exogenous 3-oxo-C6-HSL, it is most likely that quorum sensing controls spoilage through transcriptional control of the secretion apparatus encoded by lipB. We, therefore, suggest that spoilage is caused by the activity of these enzymes. Notably, proteolytic enzymes have been reported to cause clotting of milk (Payens, 1982
). The fact that all samples contained the same concentration of bacteria emphasizes that the amount of spoilage metabolite (or spoilage-generating metabolites) produced per cell (or consortium of cells) is a critical parameter when evaluating the spoilage potential and activity of a strain.
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ACKNOWLEDGEMENTS |
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Received 12 March 2002;
revised 27 September 2002;
accepted 21 October 2002.
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