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.

The marine bacterium Vibrio fischeri uses a biofilm to promote colonization of its eukaryotic host Euprymna scolopes. This biofilm depends on the symbiosis polysaccharide (syp) locus, which is transcriptionally regulated by the RscS-SypG two-component regulatory system. An additional response regulator (RR), SypE, exerts both positive and negative control over biofilm formation. SypE is a novel RR protein, with its three putative domains arranged in a unique configuration: a central phosphorylation receiver (REC) domain flanked by two effector domains with putative enzymatic activities (serine kinase and serine phosphatase). To determine how SypE regulates biofilm formation and host colonization, we generated a library of SypE domain mutants. Our results indicate that the N-terminus inhibits biofilm formation, while the C-terminus plays a positive role. The phosphorylation state of SypE appears to regulate these opposing activities, as disruption of the putative site of phosphorylation results in a protein that constitutively inhibits biofilm formation. Furthermore, SypE restricts host colonization: (1) sypE mutants with constitutive inhibitory activity fail to efficiently initiate host colonization, and (2) loss of sypE partially alleviates the colonization defect of an rscS mutant. We conclude that SypE must be inactivated to promote symbiotic colonization by V. fischeri.

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.

Microbial growth curves are used to study differential effects of media, genetics, and stress on microbial population growth. Consequently, many modeling frameworks exist to capture microbial population growth measurements. However, current models are designed to quantify growth under conditions for which growth has a specific functional form. Extensions to these models are required to quantify the effects of perturbations, which often exhibit nonstandard growth curves. Rather than assume specific functional forms for experimental perturbations, we developed a general and robust model of microbial population growth curves using Gaussian process (GP) regression. GP regression modeling of high-resolution time-series growth data enables accurate quantification of population growth and allows explicit control of effects from other covariates such as genetic background. This framework substantially outperforms commonly used microbial population growth models, particularly when modeling growth data from environmentally stressed populations. We apply the GP growth model and develop statistical tests to quantify the differential effects of environmental perturbations on microbial growth across a large compendium of genotypes in archaea and yeast. This method accurately identifies known transcriptional regulators and implicates novel regulators of growth under standard and stress conditions in the model archaeal organism Halobacterium salinarum. For yeast, our method correctly identifies known phenotypes for a diversity of genetic backgrounds under cyclohexamide stress and also detects previously unidentified oxidative stress sensitivity across a subset of strains. Together, these results demonstrate that the GP models are interpretable, recapitulating biological knowledge of growth response while providing new insights into the relevant parameters affecting microbial population growth.

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...