To survive and multiply in different environments, Vibrio cholerae has to coordinately regulate the expression of genes involved in adaptive responses. In many pathogens, adaptive responses, including pathogenic responses, are regulated by two-component regulator (TCR) systems. It is likely that members of a TCR family play a role in the regulation of processes involved in intestinal colonization, and therefore pathogenesis, in V. cholerae. We have identified and characterized a TCR system of V. cholerae : this system is a homologue of Escherichia coli PhoBR. The presence of a putative Pho box suggests that the V. cholerae phoBR operon is regulated by inorganic phosphate levels. The phoR and phoB genes are organized the same way as in E. coli. Mutation of the V. cholerae phoB gene affected the expression of the putative Pho regulon, including PhoA, but did not affect the production of cholera toxin. V. cholerae phoB mutants are less able to colonize rabbit intestine than wild-type V. cholerae. The addition of inorganic phosphate at a high concentration to the inoculum only partially restored the ability of the mutants to colonize the intestine, suggesting that the V. cholerae Pho regulon in vivo may not be regulated by inorganic phosphate levels alone.

Gluconacetobacter diazotrophicus is a plant-growth-promoting bacterium that colonizes sugarcane. In order to investigate molecular aspects of the G. diazotrophicus-sugarcane interaction, we performed a quantitative mass spectrometry-based proteomic analysis by (15)N metabolic labeling of bacteria, root samples, and co-cultures. Overall, more than 400 proteins were analyzed and 78 were differentially expressed between the plant-bacterium interaction model and control cultures. A comparative analysis of the G. diazotrophicus in interaction with two distinct genotypes of sugarcane, SP70-1143 and Chunee, revealed proteins with fundamental roles in cellular recognition. G. diazotrophicus presented proteins involved in adaptation to atypical conditions and signaling systems during the interaction with both genotypes. However, SP70-1143 and Chunee, sugarcane genotypes with high and low contribution of biological nitrogen fixation, showed divergent responses in contact with G. diazotrophicus. The SP70-1143 genotype overexpressed proteins from signaling cascades and one from a lipid metabolism pathway, whereas Chunee differentially synthesized proteins involved in chromatin remodeling and protein degradation pathways. In addition, we have identified 30 bacterial proteins in the roots of the plant samples; from those, nine were specifically induced by plant signals. This is the first quantitative proteomic analysis of a bacterium-plant interaction, which generated insights into early signaling of the G. diazotrophicus-sugarcane interaction.

A proteomic analysis of a wild-type and of a phoB mutant showed that Vibrio cholerae expresses genes of two major regulons in response to phosphate starvation. The Pho regulon, expressed by the wild-type, allowed the cells to adapt to the new environment. Induction of the general stress regulon was mainly observed in the phoB mutant as a strategy to resist stress and survive. Some functions of the adaptative and survival responses play roles in the pathogenicity of the bacteria. Among the members of the Pho regulon, we found a porin described as an important factor for the intestinal colonisation. Other functions not obviously related to phosphate metabolism, expressed preferentially by the wild-type cells, have also been implicated in virulence. These findings might explain the lack of virulence of the phoB mutant. The Pho regulon picture of V. cholerae, however, will not be complete until minor members and membrane proteins are identified. Among the phosphate-starvation induced genes we have found 13 hypothetical ones and for some of them functions have been assigned. The majority of the genes identified here have not been described before, thus they could be used to expand the proteomic reference map of V. cholerae El Tor.

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A proteome reference map has been constructed for Vibrio cholerae El Tor, in the pI range of 4.0 to 7.0. The map is based on two-dimensional gels (2-D) and the identification, by peptide mass fingerprint, of proteins in 94 spots, corresponding to 80 abundant proteins. Two strains are compared, strain N16961 and a Latin American El Tor strain C3294. The consensus map contains 340 spots consistently seen with both strains grown in Luria-Bertani broth (LB) or minimal M9 medium. The results were obtained from nine gels run with 18 cm immobilized pH gradient strips and precast gels. The 2-D gels were anchored to real N16961 proteins identified by mass spectrometry. Various energy metabolism components and periplasmic ATP-binding cassette (ABC) transporter proteins were identified among the abundant proteins. Two isoforms of OmpU were found. Five operons are proposed and seven hypothetical proteins were experimentally confirmed. Comparisons are made with protein 2-D gels for a classical strain and to microarray analysis available for the N16961 El Tor strain. New results were obtained from the proteome analysis, indicating an abundance of periplasmic ABC transporter proteins not found in microarray studies.