The Putative Hybrid Sensor Kinase SypF Coordinates Biofilm Formation inVibrio fischeriby Acting Upstream of Two Response Regulators, SypG and VpsR

Abstract

Colonization of the Hawaiian squid Euprymna scolopes by the marine bacterium Vibrio fischeri requires the symbiosis polysaccharide (syp) gene cluster, which contributes to symbiotic initiation by promoting biofilm formation on the surface of the symbiotic organ. We previously described roles for the syp-encoded response regulator SypG and an unlinked gene encoding the sensor kinase RscS in controlling syp transcription and inducing syp-dependent cell-cell aggregation phenotypes. Here, we report the involvement of an additional syp-encoded regulator, the putative sensor kinase SypF, in promoting biofilm formation. Through the isolation of an increased activity allele, sypF1, we determined that SypF can function to induce syp transcription as well as a variety of biofilm phenotypes, including wrinkled colony formation, adherence to glass, and pellicle formation. SypF1-mediated transcription of the syp cluster was entirely dependent on SypG. However, the biofilm phenotypes were reduced, not eliminated, in the sypG mutant. These phenotypes were also reduced in a mutant deleted for sypE, another syp-encoded response regulator. However, SypF1 still induced phenotypes in a sypG sypE double mutant, suggesting that SypF1 might activate another regulator(s). Our subsequent work revealed that the residual SypF1-induced biofilm formation depended on VpsR, a putative response regulator, and cellulose biosynthesis. These data support a model in which a network of regulators and at least two polysaccharide loci contribute to biofilm formation in V. fischeri.In nature, the preferred lifestyle of many bacterial cells is growth within a biofilm, a community of microbes attached to a surface and/or to each other and embedded in a matrix consisting primarily of secreted polysaccharides. Biofilm cells exhibit different properties than individual, planktonic cells, such as increased resistance to antimicrobial agents, altered gene expression, and reduced metabolic rates (reviewed in references 11, 13, 14a, and 40). Because of these properties, and the fact that many human infections occur in the context of a biofilm, a critical area of research is in understanding how biofilms form, persist, and disperse.To date, a number of organisms have been intensively studied for their biofilm-forming properties, including Pseudomonas aeruginosa (17,25,36,46), Staphylococcus spp. (1,3,30,48), and Vibrio cholerae (23,42,45,50,51). In these model systems, it has become apparent that numerous traits, such as motility and polysaccharide production, contribute to optimal biofilm formation. It is further evident that the regulatory control over these processes can be quite complex. For example, V. cholerae, which forms biofilms in the natural environment and during intestinal colonization of individuals with cholera (14, 41), uses multiple regulators to control biofilm formation. One of these is VpsR, a putative two-component response regulator protein that alters the transcriptome to produce a biofilm-competent state under specific environmental cond...

The syp Enhancer Sequence Plays a Key Role in Transcriptional Activation by the 54-Dependent Response Regulator SypG and in Biofilm Formation and Host Colonization by Vibrio fischeri

“…Evidence gathered to date suggest that SypG, a putative 54 -dependent transcriptional activator, serves as the direct transcriptional activator of the syp locus ( Fig. 1) (10,18,40). However, whether SypG activates all four syp promoters (sypA, sypI, sypM, and sypP) and whether its activity depends on the conserved SE sequences located upstream of these promoters remain unknown.…”

The Cyclic-di-GMP Phosphodiesterase BinA Negatively Regulates Cellulose-Containing Biofilms inVibrio fischeri

“…V. fischeri's ability to form biofilms in culture, however, is greatly enhanced when the syp biosynthetic gene cluster ( Fig. 1) is induced by overexpression of the response regulator SypG, the sensor kinase RscS, or the sensor kinase SypF (12,59,60).…”

“…The marine bacterium Vibrio fischeri is known to produce at least two different exopolysaccharides that play roles in biofilm formation, the symbiosis polysaccharide (Syp) and cellulose (12,59,60). The Syp polysaccharide is critical for the formation of a biofilm-like aggregate at the initiation of symbiosis with the Hawaiian bobtail squid Euprymna scolopes (59).…”

Roles of the Structural Symbiosis Polysaccharide (syp) Genes in Host Colonization, Biofilm Formation, and Polysaccharide Biosynthesis in Vibrio fischeri

“…We previously obtained evidence that one or more polysaccharides are important for the ability of V. fischeri to colonize its host (5,36,49,50). In particular, V. fischeri depends upon the 18-gene symbiosis polysaccharide (syp) locus for efficient colonization: the insertional mutation of several syp genes reduced colonization by about 1,000-fold (50).…”

“…(ii) SypE is a second response regulator, which appears to play both positive and negative roles in biofilm formation (16,27). (iii) SypF, a predicted sensor kinase, also influences biofilm formation (5)…”