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Serratia liquefaciens MG1 employs a quorum-sensing system, the swr system, to control swarming motility. Swarming of S. liquefaciens MG1 is characterized by differentiation of short motile rods at the periphery of a colony into elongated, polyploid, and hyperflagellated swarm cells that migrate coordinately in rafts atop the agar surface (7, 9). The swr system relies on two proteins, SwrI, which directs the synthesis of the diffusable signal molecules N-butanoyl-L-homoserine lactone (C4-HSL) and N-hexanoyl-L-homoserine lactone in a molar ratio of 10 to 1, and SwrR, which, after binding of the signal molecules, is thought to activate or repress transcription of target genes (8, 9, 12). A global analysis by two-dimensional polyacrylamide gel electrophoresis (PAGE) revealed that at least 28 genes are under control of the swr regulatory system (12).

By transposon mutagenesis, we have recently isolated 19 'luxAB insertion mutants of S. liquefaciens MG44 (swrI) that are responsive to the presence of C4-HSL (16). Only one of these mutants, S. liquefaciens PL10, was unable to form a swarming colony when C4-HSL was provided in the medium. This mutant was demonstrated to bear the insertion in a gene, swrA, which encodes an enzyme that catalyzes the synthesis of the biosurfactant serrawettin W2. This compound, a cyclic lipodepsipentapeptide carrying a 3-hydroxy C₁₀ fatty acid side chain, lowers the surface tension of the surrounding medium, a process that is indispensable for the development of an S. liquefaciens MG1 swarming colony. The swrA gene appears to be the only gene of the swr regulon that is required for swarming motility, since addition of purified serrawettin W2 to swarm plates fully restores swarming of the swrI mutant MG44 in the absence of C4-HSL. Thus, most of the quorum-sensing-regulated genes in S. liquefaciens MG1 are involved in functions not related to swarming motility.

In this report we show that one of the 'luxAB insertion mutants which is responsive to the addition of C4-HSL bears the transposon insertion within the lipB gene, which encodes a component of the Lip exporter. This type I protein secretion system is responsible for the transport of the S. liquefaciens lipase, metalloprotease, and S-layer protein. As a consequence of the involvement of the swr system in the regulation of lipB expression, the levels of extracellular metalloprotease and S-layer protein are significantly lowered in a swrI mutant. However, the amount of secreted lipase is unchanged.

Expression of the lipB gene is quorum sensing regulated. We previously noticed that the S. liquefaciens swrI mutant MG44 exhibits lower proteolytic activity than the wild-type MG1 (8). This suggests that either the expression or the secretion of the S. liquefaciens MG1 protease(s) is under control of the swr system. To address this issue, we tested the 19 previously isolated mutants that bear 'luxAB insertions in quorum-sensing-regulated genes for proteolytic activity on 2% (wt/vol) skim milk plates (14). One mutant, MG3645, was found to be completely protease negative on our test plates, and this mutant was further investigated. When grown in liquid AB medium (6) supplemented with 0.2% glucose and 0.2% Casamino Acids, this mutant showed an approximately fivefold stimulation of bioluminescence in response to addition of 200 nM C4-HSL (Fig. 1). The proteolytic activity of the supernatants of cultures of MG1, MG44, and MG3645 grown in the presence or absence of 200 nM C4-HSL was measured as described previously (4). The extracellular proteolytic activity of the swrI mutant MG44 was about twofold reduced compared with that of the wild type (Fig. 2A). Addition of C4-HSL to the culture medium completely restored protease activity. Supernatants of MG3645 cultures showed virtually no protease activity irrespective of the presence or absence of C4-HSL.

View larger version (16K): [in this window] [in a new window]   FIG. 1.   Induction of bioluminescence in the 'luxAB insertion mutant S. liquefaciens MG3645 in the presence of C4-HSL. Bacterial cultures were grown in AB medium containing 0.2% glucose, 0.2% Casamino Acids, and either no (circles) or 200 nM (squares) C4-HSL. Optical densities at 450 nm (OD450) (open symbols) and bioluminescence (measured in relative light units [RLU]) (closed symbols) were determined along the growth curve. The data presented are representative of those from at least three separate experiments.

View larger version (33K): [in this window] [in a new window]   FIG. 2.   Enzymatic activities of culture supernatants of S. liquefaciens MG1 and different mutant strains. Cultures were grown in LB medium in the presence or absence of 200 nM C4-HSL for 16 h. Sterile filtered supernatants were used for enzymatic measurements of proteolytic (A) and lipolytic (B) activities. Each bar represents the average from at least three independent experiments. Error bars represent the standard errors of the means. OD440, optical density at 440 nm.

Secretion of extracellular metalloprotease is quorum sensing regulated. To further investigate the role of quorum sensing in the regulation of extracellular proteolytic activity, we analyzed supernatants with the aid of zymograms. For that, sodium dodecyl sulfate (SDS)-PAGE of the culture supernatants with 0.2% (wt/vol) azocasein incorporated in the gel matrix (10% polyacrylamide) was performed. The enzymatic activity was visualized by washing the gel two times with 50 mM Tris-HCl (pH 7.5) containing 25% (vol/vol) isopropanol for 15 min, followed by incubation in 50 mM Tris-HCl (pH 7.5) for 1 h at 45°C and destaining with 1 M NaOH. Proteolytic enzyme bands were detected as colorless zones in an orange background. In these zymograms one major band with proteolytic activity was detected with the wild type, and this band was completely missing in the lipB mutant (Fig. 3A). However, these zymograms do not allow quantitative measurements. In fact, the twofold reduction of protease activity of the swrI mutant was not clearly visible in the zymograms.

View larger version (79K): [in this window] [in a new window]   FIG. 3.   Analysis of extracellular proteins produced by S. liquefaciens MG1 and different mutant strains. Cultures were grown in LB medium in the presence or absence of 200 nM C4-HSL for 16 h as indicated. The protein molecular mass standard (lanes S) consists of proteins of 196, 118, 90, 70, 55, 38, and 33 kDa. Enzymatic activities of sterile filtered supernatants were visualized with the aid of zymograms as described in the text. The SlaA protein was visualized by immunoblotting with antisera against SlaA. wt, wild type.

Role of the swr system in the secretion of extracellular lipase and S-layer protein. Since the Lip exporter of S. marcescens is responsible not only for the secretion of metalloprotease but also for the secretion of lipase and S-layer protein (15), we next investigated whether the production of the latter proteins is also controlled by the swr system in S. liquefaciens MG1.

Processing of the phospholipase by the metalloprotease. The zymograms used for the detection of the lipase showed an additional band with a molecular mass of about 30 kDa. Interestingly, this band was found to be shifted to a higher molecular mass (38 kDa) in the lipB mutant. Since S. liquefaciens MG1 produces an extracellular phospholipase (PhlA) with a molecular mass of about 38 kDa which is known to show some activity with Tween 20 as a substrate (11), we speculated that this band could represent this enzyme. In fact, when polyacrylamide gels were overlaid with egg yolk agar, a substrate that is specific for the phospholipase, the same pattern of bands was detected (Fig. 3C). In S. liquefaciens MG1 expression and secretion of the phospholipase are coupled to synthesis and export of flagella via the flhDC master regulator (10). In agreement with phlA being part of the flagellar regulon, we observed that a mutation in neither swrI nor lipB affected the amount of extracellular phospholipase. However, the phospholipase was apparently processed in the wild type and the swrI mutant but not in the lipB mutant. Since the lipB mutant does not produce metalloprotease, we tested whether PhlA is proteolytically processed by this enzyme. When supernatants of the lipB mutant were incubated at 30°C for 1 h with purified metalloprotease, the phospholipase was indeed converted to its processed form (data not shown). The swrI mutant apparently produces sufficient amounts of metalloprotease to process all extracellular phospholipase (Fig. 3C). Given that both forms of the enzyme exhibit phospholipolytic activity in our assay, the biological relevance of the PhlA processing remains unclear.

Expression of slaA is independent of the swr regulatory system. While our results show that expression of the Lip exporter is under control of the swr system, these data do not exclude the possibility that expression of metalloprotease or S-layer protein itself is swr regulated. Moreover, since slaA is located upstream of the lip operon and is transcribed in the same direction, it is possible that the lip operon is, at least in part, cotranscribed with slaA. To address this issue, we constructed transcriptional fusions of the slaA and lipB promoter regions to the promoterless luxAB genes. The two promoter regions were PCR amplified using the primer pair LipB1 (5'-CGCGAAGGATCCACCCGGTCG-3') and LipB2 (5'-GTGTGGGATCCGTTGTATTCAGC-3') (BamHI restriction sites are underlined) for P_lipB and the primer pair SlaA1 (5'-GCTGGATCCGATAAGCTTTACGTC-3') and SlaA2 (5'-AAAGGATCCCGGCAACTTCCTTCTGG-3') for P_slaA. Following restriction with BamHI, the two DNA fragments (685 bp for P_lipB and 733 bp for P_slaA) were inserted into the promoter probe vector pGA-L9 cut with the same enzyme. Plasmids which contain the inserts in the orientation placing the promoters upstream of the promoterless luxAB genes of the vector were chosen, and these constructs were designated pTO1 (P_lipB::luxAB) and pTO21 (P_slaA::luxAB). The plasmids were then transferred to the S. liquefaciens wild-type strain MG1 and the swrI mutant MG44. Measurements of bioluminescence revealed that transcription of slaA is entirely independent of the swr system (Table 1). In contrast, transcription of lipB was found to be about threefold reduced in the swrI mutant compared with the wild type, and addition of 200 nM C4-HSL to the growth medium completely restored bioluminescence levels. These data clearly show that expression of lipB but not of slaA is subject to control by the swr system.

Conclusions. The genetic and physiological characterization of a 'luxAB insertion mutant of S. liquefaciens MG44, which responds to the presence of C4-HSL with increased bioluminescence, revealed that the strain bears the transposon in the lipB gene. This gene encodes an ATP-binding cassette transporter protein that is part of the Lip exporter, a type I secretion system, which is required for the secretion of lipase, metalloprotease, and S-layer protein in both S. marcescens (15) and S. liquefaciens MG1 (this study). Our results are reminiscent of the situation found with Pseudomonas aeruginosa, in which expression of the xcp secretion system was shown to be regulated by the two quorum-sensing systems (the las and rhl systems) that operate in this opportunistic pathogen (5). Various virulence factors, including exotoxin A, lipase, the LasA and LasB proteases, and phospholipase C, are secreted with the aid of amino-terminal signal sequences of precursor proteins across the inner membrane and then from the periplasmic space via the Xcp translocation machinery across the outer membrane (21). This two-step secretion mechanism is known as the general or type II secretory pathway (18).