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.

Flagellar biogenesis and hence motility of Vibrio fischeri depends upon the presence of magnesium. In the absence of magnesium, cells contain few or no flagella and are poorly motile or nonmotile. To dissect the mechanism by which this regulation occurs, we screened transposon insertion mutants for those that could migrate through soft agar medium lacking added magnesium. We identified mutants with insertions in two distinct genes, VF0989 and VFA0959, which we termed mifA and mifB, respectively, for magnesium-dependent induction of flagellation. Each gene encodes a predicted membrane-associated protein with diguanylate cyclase activity. Consistent with that activity, introduction into V. fischeri of medium-copy plasmids carrying these genes inhibited motility. Furthermore, multicopy expression of mifA induced other phenotypes known to be correlated with diguanylate cyclase activity, including cellulose biosynthesis and biofilm formation. To directly test their function, we introduced the wild-type genes on high-copy plasmids into Escherichia coli. We assayed for the production of cyclic di-GMP using two-dimensional thin-layer chromatography and found that strains carrying these plasmids produced a small but reproducible spot that migrated with an R f value consistent with cyclic di-GMP that was not produced by strains carrying the vector control. Disruptions of mifA or mifB increased flagellin levels, while multicopy expression decreased them. Semiquantitative reverse transcription-PCR experiments revealed no significant difference in the amount of flagellin transcripts produced in either the presence or absence of Mg 2؉ by either vector control or mifA-overexpressing cells, indicating that the impact of magnesium and cyclic-di-GMP primarily acts following transcription. Finally, we present a model for the roles of magnesium and cyclic di-GMP in the control of motility of V. fischeri.The limiting step in understanding signal transduction most often is the identification of the environmental signal that induces a physiological change. This has been true for twocomponent signaling (13, 58) and may also be true for the pathways that control the production of cyclic di-GMP (c-di-GMP) (45). This newly appreciated second messenger is synthesized from two GTP molecules by diguanylate cyclases (DGCs). These enzymes, found in numerous and diverse bacterial genomes, are readily identifiable through their signature GGDEF domains (19,39,40,50,53,56). Furthermore, many bacterial species possess multiple proteins with domains that contain this GGDEF domain (for a recent review, see reference 45). Degradation of c-di-GMP is accomplished by phosphodiesterases containing either EAL or HD-GYP domains (8,15,49,52,57). Together, these activities maintain the steady-state concentration of c-di-GMP (46). c-di-GMP first was discovered as a component of the cellulose biosynthesis enzyme complex from Gluconacetobacter xylinus, where it plays a vital role in promoting cellulose biosynthesis (46). c-di-GMP is now known to control exopolys...

The bacterium Vibrio fischeri requires bacterial motility to initiate colonization of the Hawaiian squid Euprymna scolopes. Once colonized, however, the bacterial population becomes largely unflagellated. To understand environmental influences on V. fischeri motility, we investigated migration of this organism in tryptone-based soft agar media supplemented with different salts. We found that optimal migration required divalent cations and, in particular, Mg 2؉ . At concentrations naturally present in seawater, Mg 2؉ improved migration without altering the growth rate of the cells. Transmission electron microscopy and Western blot experiments suggested that Mg 2؉ addition enhanced flagellation, at least in part through an effect on the steady-state levels of flagellin protein.The symbiosis between the marine bacterium Vibrio fischeri and the Hawaiian squid Euprymna scolopes provides a model for exploring the communication that occurs between a bacterium and its host in a natural setting (30,39,45,55). Juveniles of E. scolopes hatch without V. fischeri cells present inside the symbiotic organ (the light organ), and rapidly acquire these bacteria from the surrounding seawater (31, 57). Colonization begins with aggregation of V. fischeri cells in mucus on the surface of the light organ (37, 38, 40), followed by movement of these bacteria through pores into ducts, apparently toxic passageways that limit nonspecific invaders (11, 40), and ultimately into crypts where the bacteria multiply (46).Only motile cells of V. fischeri initiate symbiotic colonization of E. scolopes. Nonmotile mutants fail to colonize (16,33,59), presumably because they fail to migrate out of the bacterial aggregates formed on the surface of the light organ (40). Apparently, normal initiation also requires optimal motility, because several hypermotile mutants colonize with delayed kinetics (32).Once V. fischeri cells initiate colonization, the majority of symbiotic bacteria within the E. scolopes light organ become nonflagellated (33, 46). However, within an hour of their release from the light organ into seawater, V. fischeri cells regrow their flagella (46). These observations suggest that environmental conditions inside the light organ inhibit flagellation, while those outside favor it (46).Environmental influences on the motility of the enteric bacteria Escherichia coli and Salmonella enterica serovar Typhimurium have been well documented (for reviews, see references 3 and 29). These influences include nutrient availability, temperature, ionic composition, pH, and surface interactions (2,21,23,27,35,48,50). Most known environmental influences act at the level of transcription initiation or, to a lesser extent, message stability. These operate through at least one nucleoid protein (H-NS) and a host of transcription factors, including the cyclic AMP (cAMP) receptor protein, LrhA (CRP), and several two-component response regulators (1,7,14,15,21,22,26,41,(49)(50)(51)54). Control of message stability involves the small RNA-binding protein CsrA ...

Magnesium-dependent induction of Vibrio fischeri flagellar (Mif) biogenesis depends upon two diguanylate cyclases, suggesting an inhibitory role for cyclic di-GMP. Here, we report that cells defective for the sugar phosphotransferase system (PTS) exhibited a magnesium-independent phenotype similar to that of mutants of the Mif pathway. Unlike Mif mutants, PTS mutants also were hyperbioluminescent.The second messenger, cyclic AMP (cAMP), is synthesized by adenylate cyclase (AC). In bacteria of the family Enterobacteriaceae (e.g., Escherichia coli), the activity of AC becomes enhanced by its interaction with the phosphorylated version of EIIA Glc (phospho-EIIA Glc ), the glucose-specific IIA component of the phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS) (6,17,18,20). The glucose-specific PTS is composed of three soluble, cytoplasmic proteins (EI, HPr, and EIIA Glc ) and one integral cytoplasmic membrane protein (EIICB Glc ). These proteins sequentially transfer a phosphoryl group from PEP to glucose, with the concomitant transport of the sugar across the membrane (Fig. 1). The presence of glucose in the environment pulls phosphoryl groups through the PTS, ensuring that the system's components remain essentially unphosphorylated; depletion of that glucose results in the accumulation of phospho-EIIA Glc , which activates AC to synthesize cAMP. This cAMP binds cAMP receptor protein (CRP, also known as CAP), which then regulates the transcription of hundreds of genes, including flhDC, the master regulator of the Enterobacteriaceae flagellar regulon (for reviews, see references 6a, 10, 11, 17, and 30).Cyclic di-GMP (c-di-GMP) is a newly appreciated second messenger, apparently unique to bacteria, that modulates diverse cellular processes (for a recent review, see reference 21). First identified as a positive effector of cellulose synthase in Gluconoacetobacter xylinus (reviewed in reference 22), c-di-GMP regulates transition from the motile, planktonic state to sessile, community-based behaviors, such as biofilm development. It tends to inhibit motility, both flagellar and twitching, while enhancing the biosynthesis of capsular components required by developing biofilms. c-di-GMP is synthesized by diguanylate cyclases (DGC) and degraded by phosphodiesterases (PDE) (21). Together these activities maintain the steadystate concentration of c-di-GMP (22). DGC activity has been associated with the highly conserved GGDEF domain. One PDE activity (to linear di-GMP) has been associated with the highly conserved EAL domain, while a second PDE activity (to two GMPs) has been associated with the HD-GYP domain (21). Finally, a c-di-GMP-binding domain (termed PilZ) was recently reported (2) and shown to inhibit flagellar assembly in E. coli (25).Insights into the control of c-di-GMP production and its targets have come from our investigations of motility in the marine bacterium Vibrio fischeri. This bacterium, found as free-living, motile individuals or as a sessile community in association with the Hawaiia...