Cytochrome c2 is a periplasmic redox protein involved in both the aerobic and photosynthetic electron transport chains of Rhodobacter sphaeroides. The process of cytochrome c2 maturation has been analyzed in order to understand the protein sequences involved in attachment of the essential heme moiety to the cytochrome c2 polypeptide and localization of the protein to the periplasm. To accomplish this, five different translational fusions which differ only in the cytochrome c2 fusion junction were constructed between cytochrome c2 and the Escherichia coli periplasmic alkaline phosphatase. All five of the fusion proteins are exported to the periplasmic space. The four fusion proteins that contain the NH2-terminal site of covalent heme attachment to cytochrome c2 are substrates for heme binding, suggesting that the COOH-terminal region of the protein is not required for heme attachment. Three of these hybrids possess heme peroxidase activity, which indicates that they are functional as electron carriers. Biological activity is possessed by one hybrid protein constructed five amino acids before the cytochrome c2 COOH terminus, since synthesis of this protein restores photosynthetic growth to a photosynthetically incompetent cytochrome c2-deficient derivative of R. sphaeroides. Biochemical analysis of these hybrids has confirmed CycA polypeptide sequences sufficient for export of the protein (A. R. Varga and S. Kaplan, J. Bacteriol. 171:5830-5839, 1989), and it has allowed us to identify regions of the protein sufficient for covalent heme attachment, heme peroxidase activity, docking to membrane-bound redox partners, or the capability to function as an electron carrier.
Trimethylamine N-oxide (TMAO) reductases are widespread in bacteria and often function in anaerobic respiration. The regulation and expression of TMAO reductase operons have been well studied in model genera such as Escherichia, Shewanella, and Rhodobacter, although TMAO reductases are present in many other bacteria, including the marine Vibrio species. The genome sequence of Vibrio fischeri revealed three putative TMAO reductase operons, and a previous report identified TMAO reductase activity in symbiotic V. fischeri isolates associated with the light organs of adult Hawaiian bobtail squid, Euprymna scolopes. We examined the roles and regulation of these three operons using mutational analyses and promoter-reporter fusions. We found that the torECA promoter, and to a lesser extent the torYZ and dmsABC promoters, were active during symbiotic colonization of juvenile E. scolopes; however, a V. fischeri strain lacking TMAO reductase activity displays no discernible colonization defect over the first 48 h. Our studies also revealed that torECA has the most active promoter of the putative TMAO reductase operons, and TorECA is the major contributor to TMAO-dependent growth in V. fischeri under the conditions tested. Interestingly, the transcriptional regulation of TMAO reductase operons in V. fischeri appears to differ from that in previously studied organisms, such as Escherichia coli, which may reflect differences in gene arrangement and bacterial habitat. This study lays the foundation for using V. fischeri as a model system for studying TMAO reductases in the Vibrionaceae.
ABSTRACT The symbiosis between Vibrio fischeri and the Hawaiian bobtail squid, Euprymna scolopes , is a tractable and well-studied model of bacteria-animal mutualism. Here, we developed a method to transiently colonize E. scolopes using D-alanine (D-ala) auxotrophy of the symbiont, controlling the persistence of viable infection by supplying or withholding D-ala. We generated alanine racemase ( alr ) mutants of V. fischeri that lack avenues for mutational suppression of auxotrophy or reversion to prototrophy. Surprisingly, an ∆ alr mutant did not require D-ala to grow in a minimal medium, a phenomenon requiring metC , which encodes cystathionine β-lyase. Likewise, overexpression of metC suppressed D-ala auxotrophy in a rich medium. To block potential mechanisms of suppression, we combined the ∆ alr mutation with deletions of metC and/or bsrF , which encodes a broad-spectrum racemase and investigated the suppression rates of four D-ala auxotrophic strains. We then focused on ∆ alr ∆ bsrF mutant MC13, which has a suppression rate of <10 −9 . When D-ala was removed from a growing culture of MC13, cells rounded and lysed within 40 minutes. Transient colonization of E. scolopes was achieved by inoculating squid in seawater containing MC13 and D-ala, and then transferring the squid into water lacking D-ala, which resulted in loss of viable symbionts within hours. Interestingly, the symbionts within crypt 3 persisted longer than those of crypt 1, suggesting a difference in bacterial growth rate in distinct crypt environments. Our study highlights a new approach for inducing transient colonization and provides insight into the biogeography of the E. scolopes light organ. IMPORTANCE The importance of this study is multi-faceted, providing a valuable methodological tool and insight into the biology of the symbiosis between Vibrio fischeri and Euprymna scolopes . First, the study sheds light on the critical role of D-ala for bacterial growth, and the underpinnings of D-ala synthesis. Our observations that metC obviates the need for D-ala supplementation of an alr mutant in minimal medium and that MetC-dependent growth correlates with D-ala in peptidoglycan, corroborate and extend previous findings in Escherichia coli regarding a role of MetC in D-ala production. Second, our isolation of robust D-ala auxotrophs led us to a novel method for studying the squid- Vibrio symbiosis, allowing for transient colonization without the use of antibiotics, and revealed intriguing differences in symbiont growth parameters in distinct light organ crypts. This work and the methodology developed will contribute to our understanding of the persistence and dynamics of V. fischeri within its host.
In many bacteria, the twin arginine translocation (Tat) system transports folded proteins across the cytoplasmic membrane, and these proteins can play a role in symbiotic or pathogenic infections. A role for the Vibrio fischeri Tat system was identified during symbiotic colonization of its host Euprymna scolopes, demonstrating a function for the Tat system in host colonization by a member of the Vibrionaceae. Using bioinformatics, mutant analyses, and green fluorescent protein fusions, a set of Tat-targeted proteins in V. fischeri was identified.
Summary Although it is accepted that bacteria‐colonizing host tissues are commonly faced with iron‐limiting conditions and that pathogenic bacteria often utilize iron from host‐derived haem‐based compounds, the mechanisms of iron acquisition by beneficial symbiotic bacteria are less clear. The bacterium Vibrio fischeri mutualistically colonizes the light organ of the squid Euprymna scolopes . Genome sequence analysis of V. fischeri revealed a putative haem‐uptake gene cluster, and through mutant analysis we confirmed this cluster is important for haemin use by V. fischeri in culture. LacZ reporter assays demonstrated Fur‐dependent transcriptional regulation of cluster promoter activity in culture. GFP‐based reporter assays revealed that gene cluster promoter activity is induced in symbiotic V. fischeri as early as 14 h post inoculation, although colonization assays with the haem uptake mutant suggested an inability to uptake haem does not begin to limit colonization until later stages of the symbiosis. Our data indicate that the squid light organ is a low iron environment and that haem‐based sources of iron are used by symbiotic V. fischeri cells. These findings provide important additional information on the availability of iron during symbiotic colonization of E. scolopes by V. fischeri , as well as the role of haem uptake in non‐pathogenic host‐microbe interactions.
is recognized for its ability to colonize diverse habitats and cause disease in a variety of hosts, including plants, invertebrates, and mammals. Understanding how this bacterium is able to occupy wide-ranging niches is important for deciphering its ecology. We used transposon sequencing [Tn-Seq, also known as insertion sequencing (INSeq)] to identify genes in
Vibrio fischeri uses the AinS/AinR pheromone-signaling system to control bioluminescence and other symbiotic colonization factors. The Ain system is thought to initiate cell-cell signaling at moderate cell densities and to prime the LuxI/LuxR signaling system. Here we compared and analyzed the ain locus from two V. fischeri strains and a Vibrio salmonicida strain to explore ain regulation. The ainS and ainR genes were predicted to constitute an operon, which we corroborated using RT-PCR. Comparisons between strains revealed a stark area of conservation across the ainS-ainR junction, including a large inverted repeat in ainR. We found that this inverted repeat in cis can affect accumulation of the AinS-generated pheromone N-octanoyl homoserine lactone, which may account for the previously unexplained low-signal phenotype of a ∆ainR mutant, although the mechanism behind this regulation remains elusive. We also extended the previous observation of a possible "lux box" LuxR binding site upstream of ainS by showing the conservation of this site as well as a second putative lux box. Using a plasmid-based reporter we found that LuxR can mediate repression of ainS, providing a negative feedback mechanism in the Ain/Lux signaling cascade. Our results provide new insights into the regulation, expression, and evolution of ainSR.
Biocontrol involves harnessing disease-suppressive microor? ganisms to improve plant health. Disease suppression by biocontrol agents is the sustained manifestation of interactions among the plant, the pathogen, the biocontrol agent, the microbial community on and around the plant, and the physi? cal environment. Even in model laboratory systems, the study of biocontrol involves interactions among a minimum of three organisms. Therefore, despite its potential in agricultural ap? plications, biocontrol is one of the most poorly understood areas of plant-microbe interactions. The complexity of these systems has influenced the accep? tance of biocontrol as a means of controlling plant diseases in two ways. First, practical results with biocontrol have been variable. Thus, despite some stunning successes with biocon? trol agents in agriculture, there remains a general skepticism born of past failures (Cook and Baker, 1983; Weiler, 1988). Second, progress in understanding an entire system has been slow. Recently, however, substantial progress has been made in a number of biocontrol systems through the application of genetic and mathematical approaches that accommodate the complexity. Biocontrol of soilborne diseases is particularly complex be? cause these diseases occur in the dynamic environment at the interface of root and soil known as the rhizosphere, which is defined as the region surrounding a root that is affected by it. The rhizosphere is typified by rapid change, intense microbial activity, and high populations of bacteria compared with nonrhizosphere soil. Plants release metabolically active cells from their roots and deposit as much as 20% of the carbon allocated to roots in the rhizosphere, suggesting a highly evolved relationship between the plant and rhizosphere microorgan? isms. The rhizosphere is subject to dramatic changes on a short temporal scale?rain events and daytime drought can result in fluctuations in salt concentration, pH, osmotic poten? tial, water potential, and soil particle structure. Over longer temporal scales, the rhizosphere can change due to root growth, interactions with other soil biota, and weathering processes. It is the dynamic nature of the rhizosphere that makes it an interesting setting for the interactions that lead to disease and biocontrol of disease (Rovira, 1965,1969,1991; Hawes, 1991; Waisel et al., 1991).
