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.

Two-component signal transduction systems, composed of sensor kinase (SK) and response regulator (RR) proteins, allow bacterial cells to adapt to changes such as environmental flux or the presence of a host. RscS is an SK required for Vibrio fischeri to initiate a symbiotic partnership with the Hawaiian squid Euprymna scolopes, likely due to its role in controlling the symbiosis polysaccharide (syp) genes and thus biofilm formation. To determine which RR(s) functions downstream of RscS, we performed epistasis experiments with a library of 35 RR mutants. We found that several RRs contributed to RscS-mediated biofilm formation in V. fischeri. However, only the syp-encoded symbiosis regulator SypG was required for both biofilm phenotypes and syp transcription induced by RscS. These data support the hypothesis that RscS functions upstream of SypG to induce biofilm formation. In addition, this work also revealed a role for the syp-encoded RR SypE in biofilm formation. To our knowledge, no other study has used a large-scale epistasis approach to elucidate twocomponent signaling pathways. Therefore, this work both contributes to our understanding of regulatory pathways important for symbiotic colonization by V. fischeri and establishes a paradigm for evaluating two-component pathways in the genomics era.

Two-component signal transduction systems are utilized by prokaryotic and eukaryotic cells to sense and respond to environmental stimuli, both to maintain homeostasis and to rapidly adapt to changing conditions. Studies have begun to emerge that utilize a large-scale mutagenesis approach to analyzing these systems in prokaryotic organisms. Due to the recent availability of its genome sequence, such a global approach is now possible for the marine bioluminescent bacterium Vibrio fischeri, which exists either in a free-living state or as a mutualistic symbiont within a host organism such as the Hawaiian squid species Euprymna scolopes. In this work, we identified 40 putative two-component response regulators encoded within the V. fischeri genome. Based on the type of effector domain present, we classified six as NarL type, 13 as OmpR type, and six as NtrC type; the remaining 15 lacked a predicted DNA-binding domain. We subsequently mutated 35 of these genes via a vector integration approach and analyzed the resulting mutants for roles in bioluminescence, motility, and competitive colonization of squid. Through these assays, we identified three novel regulators of V. fischeri luminescence and seven regulators that altered motility. Furthermore, we found 11 regulators with a previously undescribed effect on competitive colonization of the host squid. Interestingly, five of the newly characterized regulators each affected two or more of the phenotypes examined, strongly suggesting interconnectivity among systems. This work represents the first large-scale mutagenesis of a class of genes in V. fischeri using a genomic approach and emphasizes the importance of two-component signal transduction in bacterium-host interactions.The symbiosis between the Hawaiian squid Euprymna scolopes and the bacterium Vibrio fischeri serves as a model for symbiotic bacterium-host interactions. Previous studies have revealed a number of bacterial factors required for host colonization (24, 56), including motility (25, 49, 57) and luminescence (78). Many of these factors were identified by generating and testing specific hypotheses developed from an understanding of the general colonization process. To date, however, large-scale studies of colonization factors have been hampered by a deficit of genetic tools needed for bacterial mutant construction. Recently, however, the genome sequence of V. fischeri has been published (65). In addition, the availability of a useful suicide vector (17) has greatly facilitated mutant construction (73, 90). Thus, it is now possible to approach the investigation of V. fischeri biology from a genomic perspective.

The bacterium Xenorhabdus nematophila engages in phenotypic variation with respect to pathogenicity against insect larvae, yielding both virulent and attenuated subpopulations of cells from an isogenic culture. The global regulatory protein Lrp is necessary for X. nematophila virulence and immunosuppression in insects, as well as colonization of the mutualistic host nematode Steinernema carpocapsae, and mediates expression of numerous genes implicated in each of these phenotypes. Given the central role of Lrp in X. nematophila host associations, as well as its involvement in regulating phenotypic variation pathways in other bacteria, we assessed its function in virulence modulation. We discovered that expression of lrp varies within an isogenic population, in a manner that correlates with modulation of virulence. Unexpectedly, although Lrp is necessary for optimal virulence and immunosuppression, cells expressing high levels of lrp were attenuated in these processes relative to those with low to intermediate lrp expression. Furthermore, fixed expression of lrp at high and low levels resulted in attenuated and normal virulence and immunosuppression, respectively, and eliminated population variability of these phenotypes. These data suggest that fluctuating lrp expression levels are sufficient to drive phenotypic variation in X. nematophila. IMPORTANCEMany bacteria use cell-to-cell phenotypic variation, characterized by distinct phenotypic subpopulations within an isogenic population, to cope with environmental change. Pathogenic bacteria utilize this strategy to vary antigen or virulence factor expression. Our work establishes that the global transcription factor Lrp regulates phenotypic variation in the insect pathogen Xenorhabdus nematophila, leading to attenuation of virulence and immunosuppression in insect hosts. Unexpectedly, we found an inverse correlation between Lrp expression levels and virulence: high levels of expression of Lrp-dependent putative virulence genes are detrimental for virulence but may have an adaptive advantage in other aspects of the life cycle. Investigation of X. nematophila phenotypic variation facilitates dissection of this phenomenon in the context of a naturally occurring symbiosis.

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...