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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 two-component 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 bacterium Xenorhabdus nematophila engages in phenotypic variation with respect to pathogenicity against insect larvae, yielding both virulent and attenuated subpopulations of cells froman isogenic culture. The global regulatory protein Lrp is necessary for X. nematophila virulence and immunosuppression in insects, as well as colonization of themutualistic 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 virulencemodulation.We discovered that expression of lrp varies within an isogenic population, in amanner that correlates withmodulation 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. [ABSTRACT FROM AUTHOR]

The bacterium X enorhabdus nematophila is a mutualist of entomopathogenic S teinernema carpocapsae nematodes and facilitates infection of insect hosts. X . nematophila colonizes the intestine of S . carpocapsae which carries it between insects. In the X . nematophila colonization-defective mutant nilD6:: Tn 5, the transposon is inserted in a region lacking obvious coding potential. We demonstrate that the transposon disrupts expression of a single CRISPR RNA, NilD RNA. A variant NilD RNA also is expressed by X . nematophila strains from S . anatoliense and S . websteri nematodes. Only nilD from the S . carpocapsae strain of X . nematophila rescued the colonization defect of the nilD6:: Tn 5 mutant, and this mutant was defective in colonizing all three nematode host species. NilD expression depends on the presence of the associated Cas6e but not Cas3, components of the Type I- E CRISPR-associated machinery. While cas6e deletion in the complemented strain abolished nematode colonization, its disruption in the wild-type parent did not. Likewise, nilD deletion in the parental strain did not impact colonization of the nematode, revealing that the requirement for NilD is evident only in certain genetic backgrounds. Our data demonstrate that NilD RNA is conditionally necessary for mutualistic host colonization and suggest that it functions to regulate endogenous gene expression. [ABSTRACT FROM AUTHOR]

Microbial symbioses, in which microbes have either positive (mutualistic) or negative (parasitic) impacts on host fitness, are integral to all aspects of biology, from ecology to human health. In many well-studied cases, microbial symbiosis is characterized by a specialized association between a host and a specific microbe that provides it with one or more beneficial functions, such as novel metabolic pathways or defense against pathogens. Even in relatively simple associations, symbiont-derived benefits can be context dependent and influenced by other host-associated or environmental microbes. Furthermore, naturally occurring symbioses are typically complex, in which multiple symbionts exhibit coordinated, competing, or independent influences on host physiology, or in which individual symbionts affect multiple interacting hosts. Here we describe research on the mechanisms and consequences of multipartite symbioses, including consortia in which multiple organisms interact with the host and one another, and on conditional mutualists whose impact on the host depends on additional interacting organisms. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Vibrio 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 mbiosis olysaccharide. 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 σ54 interaction domain. This induction depended on σ54, as a mutation in rpoN abolished syp transcription. Primer extension analysis supported the use of putative σ54 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 σ54-dependent response regulator and uses to promote symbiotic colonization. [ABSTRACT FROM AUTHOR]

Defining appropriate student learning outcomes for general education science courses is a daunting task. We must ask ourselves how to best prepare our students to understand the role of science in their lives and in society at large. In the era of social media and armchair experts, life-changing scientific advancements such as vaccination are being dismissed or actively resisted, emphasizing the critical need to teach science literacy skills. One active classroom method known as problem-based learning promotes self-motivated learning and synthesis skills that, when applied in a science-literacy context, can provide students with the ability to generate informed opinions on new scientific advances throughout their lifetime. This piece describes one such problem-based course, designed to tackle the scientific basis (or lack thereof) of the 5-second rule for eating food dropped on the floor. In this course, first year students experimentally engage this issue, while also applying their developing skill set to sort out scientific controversies such as vaccine safety and genetically modified foods. [ABSTRACT FROM AUTHOR]

Biofilm formation by Vibrio fischeri is a complex process that requires multiple regulators. One such regulator, the NtrC-like response regulator SypG, controls biofilm formation and host colonization by V. fischeri via its impact on transcription of the symbiosis polysaccharide (syp) locus. SypG is predicted to activate syp transcription by binding to the syp enhancer (SE), a conserved sequence located upstream of four syp promoters. In this study, we performed an in-depth analysis of the sequences necessary for SypG to promote syp transcription and biofilm formation. We found that the SE sequence is necessary for SypG-mediated syp transcription, identified individual bases necessary for efficient activation, and determined that SypG is able to bind to syp promoter regions. We also identified SE sequences outside the syp locus and established that SypG recognizes these sequences as well. Finally, deletion of the SE sequence upstream of sypA led to defects in both biofilm formation and host colonization that could be restored by reintroducing the SE sequence into its native location in the chromosome. This work thus fills in critical gaps in knowledge of the Syp regulatory circuit by demonstrating a role for the SE sequence in SypG-dependent control of biofilm formation and host colonization and by identifying new putative regulon members. It may also provide useful insights into other bacteria, such as Vibrio vulnificus and Vibrio parahaemolyticus, that have syp-like loci and conserved SE sequences. [ABSTRACT FROM AUTHOR]

Xenorhabdus nematophila bacteria are mutualistic symbionts of Steinernema carpocapsae nematodes and pathogens of insects. The X. nematophila global regulator Lrp controls the expression of many genes involved in both mutualism and pathogenic activities, suggesting a role in the transition between the two host organisms. We previously reported that natural populations of X. nematophila exhibit various levels of Lrp expression and that cells expressing relatively low levels of Lrp are optimized for virulence in the insect Manduca sexta. The adaptive advantage of the high-Lrp-expressing state was not established. Here we used strains engineered to express constitutively high or low levels of Lrp to test the model in which high-Lrp-expressing cells are adapted for mutualistic activities with the nematode host. We demonstrate that high-Lrp cells form more robust biofilms in laboratory media than do low-Lrp cells, which may reflect adherence to host tissues. Also, our data showed that nematodes cultivated with high-Lrp strains are more frequently colonized than are those associated with low-Lrp strains. Taken together, these data support the idea that high-Lrp cells have an advantage in tissue adherence and colonization initiation. Furthermore, our data show that high-Lrp-expressing strains better support nematode reproduction than do their low-Lrp counterparts under both in vitro and in vivo conditions. Our data indicate that heterogeneity of Lrp expression in X. nematophila populations provides diverse cell populations adapted to both pathogenic (low-Lrp) and mutualistic (high-Lrp) states. IMPORTANCE Host-associated bacteria experience fluctuating conditions during both residence within an individual host and transmission between hosts. For bacteria that engage in evolutionarily stable, long-term relationships with particular hosts, these fluctuations provide selective pressure for the emergence of adaptive regulatory mechanisms. Here we present evidence that the bacterium Xenorhabdus nematophila uses various levels of the transcription factor Lrp to optimize its association with its two animal hosts, nematodes and insects, with which it behaves as a mutualist and a pathogen, respectively. Building on our previous finding that relatively low cellular levels of Lrp are optimal for pathogenesis, we demonstrate that, conversely, high levels of Lrp promote mutualistic activities with the Steinernema carpocapsae nematode host. These data suggest that X. nematophila has evolved to utilize phenotypic variation between high- and low-Lrp-expression states to optimize its alternating behaviors as a mutualist and a pathogen. [ABSTRACT FROM AUTHOR]

Xenorhabdus nematophila bacteria are mutualistic symbionts of Steinernema carpocapsae nematodes and pathogens of insects. The X. nematophila global regulator Lrp controls the expression of many genes involved in both mutualism and pathogenic activities, suggesting a role in the transition between the two host organisms. We previously reported that natural populations of X. nematophila exhibit various levels of Lrp expression and that cells expressing relatively low levels of Lrp are optimized for virulence in the insect Manduca sexta The adaptive advantage of the high-Lrp-expressing state was not established. Here we used strains engineered to express constitutively high or low levels of Lrp to test the model in which high-Lrp-expressing cells are adapted for mutualistic activities with the nematode host. We demonstrate that high-Lrp cells form more robust biofilms in laboratory media than do low-Lrp cells, which may reflect adherence to host tissues. Also, our data showed that nematodes cultivated with high-Lrp strains are more frequently colonized than are those associated with low-Lrp strains. Taken together, these data support the idea that high-Lrp cells have an advantage in tissue adherence and colonization initiation. Furthermore, our data show that high-Lrp-expressing strains better support nematode reproduction than do their low-Lrp counterparts under both in vitro and in vivo conditions. Our data indicate that heterogeneity of Lrp expression in X. nematophila populations provides diverse cell populations adapted to both pathogenic (low-Lrp) and mutualistic (high-Lrp) states. IMPORTANCE Host-associated bacteria experience fluctuating conditions during both residence within an individual host and transmission between hosts. For bacteria that engage in evolutionarily stable, long-term relationships with particular hosts, these fluctuations provide selective pressure for the emergence of adaptive regulatory mechanisms. Here we present evidence that the bacterium Xenorhabdus nematophila uses various levels of the transcription factor Lrp to optimize its association with its two animal hosts, nematodes and insects, with which it behaves as a mutualist and a pathogen, respectively. Building on our previous finding that relatively low cellular levels of Lrp are optimal for pathogenesis, we demonstrate that, conversely, high levels of Lrp promote mutualistic activities with the Steinernema carpocapsae nematode host. These data suggest that X. nematophila has evolved to utilize phenotypic variation between high- and low-Lrp-expression states to optimize its alternating behaviors as a mutualist and a pathogen., (Copyright © 2017 American Society for Microbiology.)

Biofilm formation by Vibrio fischeri is a complex process that requires multiple regulators. One such regulator, the NtrC-like response regulator SypG, controls biofilm formation and host colonization by V. fischeri via its impact on transcription of the symbiosis polysaccharide (syp) locus. SypG is predicted to activate syp transcription by binding to the syp enhancer (SE), a conserved sequence located upstream of four syp promoters. In this study, we performed an in-depth analysis of the sequences necessary for SypG to promote syp transcription and biofilm formation. We found that the SE sequence is necessary for SypG-mediated syp transcription, identified individual bases necessary for efficient activation, and determined that SypG is able to bind to syp promoter regions. We also identified SE sequences outside the syp locus and established that SypG recognizes these sequences as well. Finally, deletion of the SE sequence upstream of sypA led to defects in both biofilm formation and host colonization that could be restored by reintroducing the SE sequence into its native location in the chromosome. This work thus fills in critical gaps in knowledge of the Syp regulatory circuit by demonstrating a role for the SE sequence in SypG-dependent control of biofilm formation and host colonization and by identifying new putative regulon members. It may also provide useful insights into other bacteria, such as Vibrio vulnificus and Vibrio parahaemolyticus, that have syp-like loci and conserved SE sequences.

Vibrio 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 sigma54 interaction domain. This induction depended on sigma54, as a mutation in rpoN abolished syp transcription. Primer extension analysis supported the use of putative sigma54 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 sigma54-dependent response regulator and uses to promote symbiotic colonization.