SummarySuccessful colonization of a eukaryotic host by a microbe involves complex microbe-microbe and microbe-host interactions. Previously, we identified in Vibrio fischeri a putative sensor kinase, RscS, required for initiating symbiotic colonization of its squid host Euprymna scolopes. Here, we analysed the role of rscS by isolating an allele, rscS1, with increased activity. Multicopy rscS1 activated transcription of genes within the recently identified symbiosis polysaccharide (syp) cluster. Wild-type cells carrying rscS1 induced aggregation phenotypes in culture, including the formation of pellicles and wrinkled colonies, in a syp-dependent manner. Colonies formed by rscS1-expressing cells produced a matrix not found in control colonies and largely lost in an rscS1-expressing sypN mutant. Finally, multicopy rscS1 provided a colonization advantage over control cells and substantially enhanced the ability of wildtype cells to aggregate on the surface of the symbiotic organ of E. scolopes; this latter phenotype similarly depended upon an intact syp locus. These results suggest that transcription induced by RscSmediated signal transduction plays a key role in colonization at the aggregation stage by modifying the cell surface and increasing the ability of the cells to adhere to one another and/or to squid-secreted mucus.

SummaryVibrio fischeri is the exclusive symbiont residing in the light organ of the squid Euprymna scolopes . To understand the genetic requirements for this association, we searched a library of V. fischeri transposon insertion mutants for those that failed to colonize E. scolopes . We identified four mutants that exhibited severe defects in initiating colonization. Sequence analysis revealed that the strains contained insertions in four different members of a cluster of 21 genes oriented in the same direction. The predicted gene products are similar to proteins involved in capsule, exopolysaccharide or lipopolysaccharide biosynthesis, including six putative glycosyltransferases. We constructed mutations in five additional genes and found that they also were required for symbiosis. Therefore, we have termed this region syp , for symbiosis polysaccharide. Homologous clusters also exist in Vibrio parahaemolyticus and Vibrio vulnificus , and thus these genes may represent a common mechanism for promoting bacteria-host interactions. Using lacZ reporter fusions, we observed that transcription of the syp genes did not occur under standard laboratory conditions, but could be induced by multicopy expression of sypG , which encodes a response regulator with a predicted s s s s 54 interaction domain. This induction depended on s s s s 54, as a mutation in rpoN abolished syp transcription. Primer extension analysis supported the use of putative s s s s 54 binding sites upstream of sypA , sypI and sypM as promoters. Finally, we found that multicopy expression of sypG resulted in robust biofilm formation. This work thus reveals a novel group of genes that V. fischeri controls through a s s s s 54-dependent response regulator and uses to promote symbiotic colonization.

The symbiosis polysaccharide locus, syp, is required for Vibrio fischeri to form a symbiotic association with the squid Euprymna scolopes. It is also required for biofilm formation induced by the unlinked regulator RscS. The syp locus includes 18 genes that can be classified into four groups based on putative function: 4 genes encode putative regulators, 6 encode glycosyltransferases, 2 encode export proteins, and the remaining 6 encode proteins with other functions, including polysaccharide modification. To understand the roles of each of the 14 structural syp genes in colonization and biofilm formation, we generated nonpolar inframe deletions of each gene. All of the deletion mutants exhibited defects in their ability to colonize juvenile squid, although the impact of the loss of SypB or SypI was modest. Consistent with their requirement for colonization, most of the structural genes were also required for RscS-induced biofilm formation. In particular, the production of wrinkled colonies, pellicles, and the matrix on the colony surface was eliminated or severely decreased in all mutants except for the sypB and sypI mutants; in contrast, only a subset of genes appeared to play a role in attachment to glass. Finally, immunoblotting data suggested that the structural Syp proteins are involved in polysaccharide production and/or export. These results provide important insights into the requirements for the syp genes under different environmental conditions and thus lay the groundwork for a more complete understanding of the matrix produced by V. fischeri to enhance cell-cell interactions and promote symbiotic colonization.T he initial interactions between microbes and their hosts are critical to the establishment of both symbiotic and pathogenic associations. The adherence of the microbe to its host and bacterial cell-cell aggregation are two processes that can mediate these initial interactions. The roles of polysaccharides in promoting adherence and cell-cell interactions in pathogenic and symbiotic colonization are well recognized (29, 37). Bacterial lipopolysaccharides (LPS), for example, can mediate the adherence of bacterial cells to various cellular components, such as mannose receptors and mucus (17,34). Capsular polysaccharides (CPS) and/or exopolysaccharides (EPS), present on the bacterial surface and secreted, respectively, can promote adherence to host or abiotic surfaces by facilitating the formation of biofilms and thus increasing colonization efficiency (4).To understand bacterium-host and bacterium-bacterium interactions during the colonization of a host, we have used the symbiosis between the bacterium Vibrio fischeri and its host, the squid Euprymna scolopes, as a model system (30). 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 ...

A panel of cytochrome c maturation (ccm) mutants of Legionella pneumophila displayed a loss of siderophore (legiobactin) expression, as measured by both the chrome azurol S assay and a Legionella-specific bioassay. These data, coupled with the finding that ccm transcripts are expressed by wild-type bacteria grown in deferrated medium, indicate that the Ccm system promotes siderophore expression by L. pneumophila. To determine the basis of this newfound role for Ccm, we constructed and tested a set of mutants specifically lacking individual c-type cytochromes. Whereas ubiquinol-cytochrome c reductase (petC) mutants specifically lacking cytochrome c1 and cycB mutants lacking cytochrome c5 had normal siderophore expression, cyc4 mutants defective for cytochrome c4 completely lacked legiobactin. These data, along with the expression pattern of cyc4 mRNA, indicate that cytochrome c4 in particular promotes siderophore expression. In intracellular infection assays, petC mutants and cycB mutants, but not cyc4 mutants, had a reduced ability to infect both amoebae and macrophage hosts. Like ccm mutants, the cycB mutants were completely unable to grow in amoebae, highlighting a major role for cytochrome c5 in intracellular infection. To our knowledge, these data represent both the first direct documentation of the importance of a c-type cytochrome in expression of a biologically active siderophore and the first insight into the relative importance of c-type cytochromes in intracellular infection events.