N -Acyl- l -Homoserine Lactone-Mediated Regulation of the Lip Secretion System in Serratia liquefaciens MG1

Abstract

The analysis of Serratia liquefaciens MG1 luxAB insertion mutants that are responsive to N-butanoyl-Lhomoserine lactone revealed that expression of lipB is controlled by the swr quorum-sensing system. LipB is part of the Lip exporter, a type I secretion system, which is responsible for the secretion of extracellular lipase, metalloprotease, and S-layer protein.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 ЈluxAB 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 C 10 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-sensingregulated genes in S. liquefaciens MG1 are involved in functions not related to swarming motility.In this report we show that one of the ЈluxAB 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 Slayer 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 sug...

Quorum-sensing-directed protein expression in Serratia proteamaculans B5a

“…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 , we propose that quorum sensing regulation of the lipB transporter is at least partially responsible for the observed induction of lipolytic and proteolytic activities.…”

“…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 quorumsensing-controlled genes swrA and lipB in S. liquefaciens MG1 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).…”

“…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.…”

“…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)].…”

Metabolic and regulatory engineering of Serratia marcescens: mimicking phage-mediated horizontal acquisition of antibiotic biosynthesis and quorum-sensing capacities

“…In S. marcescens SS-1 (Sma SS-1), prodigiosin, secreted nuclease and sliding motility are under the control of SpnIR (Horng et al, 2002). In the non-pigmented strain S. marcescens MG1 (Sma MG1; formerly S. liquefaciens MG1), swarming motility, production of secreted protease and biofilm formation are under aHSL QS control, via the BHL/HHL synthase, SmaI (Labbate et al, 2004;Riedel et al, 2001).Another type of QS system has also been described in Gramnegative bacteria. As reviewed (Vendeville et al, 2005), autoinducer-2 (AI-2), whose synthesis depends on LuxS, was first described as a QS signalling molecule regulating bioluminescence in Vibrio harveyi, but occurrence of luxS/ AI-2 production now appears to be widespread.…”