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The putative oligosaccharide translocase SypK connects biofilm formation with quorum signaling in Vibrio fischeri.

ABSTRACT: Quorum signaling (QS) describes how bacteria can use small signaling molecules (autoinducers) to coordinate group-level behaviors. In Vibrio fischeri, QS is achieved through a complex regulatory network that ultimately controls bioluminescence, motility, and host colonization. We conducted a genetic screen focused on qrr1, which encodes a small regulatory RNA that is necessary for the core quorum-signaling cascade to transduce autoinducer information into cellular responses. We isolated unique mutants with a transposon inserted into one of two genes within the syp locus, which is involved in biofilm formation. We found that overexpression of sypK, which encodes a putative oligosaccharide translocase, is sufficient to activate qrr1, and, in addition, this effect appears to depend on the kinase activity of the sensor LuxQ. Consistent with the established model for QS in V. fischeri, enhanced expression of qrr1 by the overexpression of sypK resulted in reduced bioluminescence and increased motility. Finally, we found that induction of the syp locus by overexpression of sypG was sufficient to activate qrr1 levels. Together, our results show how conditions that promote biofilm formation impact the quorum-signaling network in V. fischeri, and further highlight the integrated nature of the regulatory circuits involved in complex bacterial behaviors.

SUBMITTER: Miyashiro T

PROVIDER: S-EPMC4263508 | biostudies-literature | 2014 Dec

REPOSITORIES: biostudies-literature

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The putative oligosaccharide translocase SypK connects biofilm formation with quorum signaling in Vibrio fischeri.

Miyashiro Tim T Oehlert Dane D Ray Valerie A VA Visick Karen L KL Ruby Edward G EG

MicrobiologyOpen 20140925 6

Quorum signaling (QS) describes how bacteria can use small signaling molecules (autoinducers) to coordinate group-level behaviors. In Vibrio fischeri, QS is achieved through a complex regulatory network that ultimately controls bioluminescence, motility, and host colonization. We conducted a genetic screen focused on qrr1, which encodes a small regulatory RNA that is necessary for the core quorum-signaling cascade to transduce autoinducer information into cellular responses. We isolated unique m ...[more]

PMID: 25257018

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Project description:Biofilms, complex communities of microorganisms surrounded by a self-produced matrix, facilitate attachment and provide protection to bacteria. A natural model used to study biofilm formation is the symbiosis between Vibrio fischeri and its host, the Hawaiian bobtail squid, Euprymna scolopes Host-relevant biofilm formation is a tightly regulated process and is observed in vitro only with strains that have been genetically manipulated to overexpress or disrupt specific regulators, primarily two-component signaling (TCS) regulators. These regulators control biofilm formation by dictating the production of the symbiosis polysaccharide (Syp-PS), the major component of the biofilm matrix. Control occurs both at and below the level of transcription of the syp genes, which are responsible for Syp-PS production. Here, we probed the roles of the two known negative regulators of biofilm formation, BinK and SypE, by generating double mutants. We also mapped and evaluated a point mutation using natural transformation and linkage analysis. We examined traditional biofilm formation phenotypes and established a new assay for evaluating the start of biofilm formation in the form of microscopic aggregates in shaking liquid cultures, in the absence of the known biofilm-inducing signal calcium. We found that wrinkled colony formation is negatively controlled not only by BinK and SypE but also by SypF. SypF is both required for and inhibitory to biofilm formation. Together, these data reveal that these three regulators are sufficient to prevent wild-type V. fischeri from forming biofilms under these conditions.IMPORTANCE Bacterial biofilms promote attachment to a variety of surfaces and protect the constituent bacteria from environmental stresses, including antimicrobials. Understanding the mechanisms by which biofilms form will promote our ability to resolve them when they occur in the context of an infection. In this study, we found that Vibrio fischeri tightly controls biofilm formation using three negative regulators; the presence of a single one of these regulators was sufficient to prevent full biofilm development, while disruption of all three permitted robust biofilm formation. This work increases our understanding of the functions of specific regulators and demonstrates the substantial negative control that one benign microbe exerts over biofilm formation, potentially to ensure that it occurs only under the appropriate conditions.

Project description:BACKGROUND: In a previous study, we demonstrated that Vibrio scophthalmi, the most abundant Vibrio species among the marine aerobic or facultatively anaerobic bacteria inhabiting the intestinal tract of healthy cultured turbot (Scophthalmus maximus), contains at least two quorum-sensing circuits involving two types of signal molecules (a 3-hydroxy-dodecanoyl-homoserine lactone and the universal autoinducer 2 encoded by luxS). The purpose of this study was to investigate the functions regulated by these quorum sensing circuits in this vibrio by constructing mutants for the genes involved in these circuits. RESULTS: The presence of a homologue to the Vibrio harveyi luxR gene encoding a main transcriptional regulator, whose expression is modulated by quorum-sensing signal molecules in other vibrios, was detected and sequenced. The V. scophthalmi LuxR protein displayed a maximum amino acid identity of 82% with SmcR, the LuxR homologue found in Vibrio vulnificus. luxR and luxS null mutants were constructed and their phenotype analysed. Both mutants displayed reduced biofilm formation in vitro as well as differences in membrane protein expression by mass-spectrometry analysis. Additionally, a recombinant strain of V. scophthalmi carrying the lactonase AiiA from Bacillus cereus, which causes hydrolysis of acyl homoserine lactones, was included in the study. CONCLUSIONS: V. scophthalmi shares two quorum sensing circuits, including the main transcriptional regulator luxR, with some pathogenic vibrios such as V. harveyi and V. anguillarum. However, contrary to these pathogenic vibrios no virulence factors (such as protease production) were found to be quorum sensing regulated in this bacterium. Noteworthy, biofilm formation was altered in luxS and luxR mutants. In these mutants a different expression profile of membrane proteins were observed with respect to the wild type strain suggesting that quorum sensing could play a role in the regulation of the adhesion mechanisms of this bacterium.

Dataset Information

The putative oligosaccharide translocase SypK connects biofilm formation with quorum signaling in Vibrio fischeri.

Publications

The putative oligosaccharide translocase SypK connects biofilm formation with quorum signaling in Vibrio fischeri.

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