Accumulation of ornithine lipids in Vibrio cholerae under phosphate deprivation is dependent on VC0489 (OlsF) and PhoBR system.

Ornithine lipids (OLs) are phosphorus-free lipids found in many bacteria grown under phosphate deprivation, a condition that activates the PhoBR system and leads to phosphate uptake and metabolism. Two OL synthesis pathways have already been described. One depends on OlsB and OlsA acyltransferases to add, respectively, the first and second acyl chains to an ornithine molecule. The other pathway is carried out by OlsF, a bifunctional enzyme responsible for both acylation steps. Although Vibrio cholerae lacks olsBA genes, an olsF homologue (vc0489) was identified in its genome. In this work we demonstrated that V. cholerae produces OLs and expresses vc0489 in response to phosphate depletion, in a PhoBR-dependent manner. In Escherichia coli, under similar condition, vc0489 expression leads to OL accumulation. These results indicate a strong connection between OL synthesis and VC0489 from V. cholerae and, for the first time, a direct regulation of an olsF homologue by the PhoBR system.

Transcriptional and post-transcriptional regulation of pst2 operon expression in Vibrio cholerae O1.

One of the most abundant proteins in V. cholerae O1 cells grown under inorganic phosphate (Pi) limitation is PstS, the periplasmic Pi-binding component of the high-affinity Pi transport system Pst2 (PstSCAB), encoded in pst2 operon (pstS-pstC2-pstA2-pstB2). Besides its role in Pi uptake, Pst2 has been also associated with V. cholerae virulence. However, the mechanisms regulating pst2 expression and the non-stoichiometric production of the Pst2 components under Pi-limitation are unknown. A computational-experimental approach was used to elucidate the regulatory mechanisms behind pst2 expression in V. cholerae O1. Bioinformatics analysis of pst2 operon nucleotide sequence revealed start codons for pstS and pstC genes distinct from those originally annotated, a regulatory region upstream pstS containing potential PhoB-binding sites and a pstS-pstC intergenic region longer than predicted. Analysis of nucleotide sequence between pstS-pstC revealed inverted repeats able to form stem-loop structures followed by a potential RNAse E-cleavage site. Another putative RNase E recognition site was identified within the pstA-pstB intergenic sequence. In silico predictions of pst2 operon expression regulation were subsequently tested using cells grown under Pi limitation by promoter-lacZ fusion, gel electrophoresis mobility shift assay and quantitative RT-PCR. The experimental and in silico results matched very well and led us to propose a pst2 promoter sequence upstream of pstS gene distinct from the previously annotated. Furthermore, V. cholerae O1 pst2 operon transcription is PhoB-dependent and generates a polycistronic mRNA molecule that is rapidly processed into minor transcripts of distinct stabilities. The most stable was the pstS-encoding mRNA, which correlates with PstS higher levels relative to other Pst2 components in Pi-starved cells. The relatively higher stability of pstS and pstB transcripts seems to rely on the secondary structures at their 3' untranslated regions that are known to block 3'-5' exonucleolytic attacks.

Catalases and PhoB/PhoR system independently contribute to oxidative stress resistance in Vibrio cholerae O1.

All cells are subjected to oxidative stress, a condition under which reactive oxygen species (ROS) production exceeds elimination. Bacterial defences against ROS include synthesis of antioxidant enzymes like peroxidases and catalases. Vibrio cholerae can produce two distinct catalases, KatB and KatG, which contribute to ROS homeostasis. In this study, we analysed the mechanism behind katG and katB expression in two V. cholerae O1 pandemic strains, O395 and N16961, of classical and El Tor biotypes, respectively. Both strains express these genes, especially at stationary phase. However, El Tor N16961 produces higher KatB and KatG levels and is much more resistant to peroxide challenge than the classical strain, confirming a direct relationship between catalase activity and oxidative stress resistance. Moreover, we showed that katG and katB expression levels depend on inorganic phosphate (Pi) availability, in contrast to other bacterial species. In N16961, katB and katG expression is reduced under Pi limitation relative to Pi abundance. Total catalase activity in N16961 and its phoB mutant cells was similar, independently of growth conditions, indicating that the PhoB/PhoR system is not required for katB and katG expression. However, N16961 cells from Pi-limited cultures were 50-100-fold more resistant to H2O2 challenge and accumulated less ROS than phoB mutant cells. Together, these findings suggest that, besides KatB and KatG, the PhoB/PhoR system is an important protective factor against ROS in V. cholerae N16961. They also corroborate previous results from our and other groups, suggesting that the PhoB/PhoR system is fundamental for V. cholerae biology.

VCA1008: An Anion-Selective Porin of Vibrio Cholerae.

A putative porin function has been assigned to VCA1008 of Vibrio cholerae. Its coding gene, vca1008, is expressed upon colonization of the small intestine in infant mice and human volunteers, and is essential for infection. In vitro, vca1008 is expressed under inorganic phosphate limitation and, in this condition, VCA1008 is the major outer membrane protein of the bacterium. Here, we provide the first functional characterization of VCA1008 reconstituted into planar lipid bilayers. Our main findings were: 1) VCA1008 forms an ion channel that, at high voltage (~±100 mV), presents a voltage-dependent activity and displays closures typical of trimeric porins, with a conductance of 4.28±0.04 nS (n=164) in 1M KCl; 2) It has a preferred selectivity for anions over cations; 3) Its conductance saturates with increasing inorganic phosphate concentration, suggesting VCA1008 contains binding site(s) for this anion; 4) Its ion selectivity is controlled by both fixed charged residues within the channel and diffusion along the pore; 5) Partitioning of poly (ethylene glycol)s (PEGs) of different molecular mass suggests that VCA1008 channel has a pore exclusion limit of 0.9 nm.

A comparative proteomic analysis of Vibrio cholerae O1 wild-type cells versus a phoB mutant showed that the PhoB/PhoR system is required for full growth and rpoS expression under inorganic phosphate abundance.

PhoB/PhoR is a two-component system originally described as involved in inorganic phosphate (Pi) transport and metabolism under Pi limitation. In order to disclose other roles of this system, a proteomic analysis of Vibrio cholerae 569BSR and its phoB/phoR mutant under high Pi levels was performed. Most of the proteins downregulated by the mutant have roles in energy production and conversion and in amino acid transport and metabolism. In contrast, the phoB/phoR mutant upregulated genes mainly involved in adaptation to atypical conditions, indicating that the absence of a functional PhoB/PhoR caused increased expression of a number of genes from distinct stress response pathways. This might be a strategy to overcome the lack of RpoS, whose expression in the stationary phase cells of V. cholerae seems to be controlled by PhoB/PhoR. Moreover, compared to the wild-type strain the phoB/phoR mutant presented a reduced cell density at stationary phase of culture in Pi abundance, lower resistance to acid shock, but higher tolerance to thermal and osmotic stresses. Together our findings show, for the first time, the requirement of PhoB/PhoR for full growth under high Pi level and for the accumulation of RpoS, indicating that PhoB/PhoR is a fundamental system for the biology of V. cholerae. BIOLOGICAL SIGNIFICANCE: Certain V. cholerae strains are pathogenic to humans, causing cholera, an acute dehydrating diarrhoeal disease endemic in Southern Asia, parts of Africa and Latin America, where it has been responsible for significant mortality and economical damage. Its ability to grow within distinct niches is dependent on gene expression regulation. PhoB/PhoR is a two-component system originally described as involved in inorganic phosphate (Pi) transport and metabolism under Pi limitation. However, Pho regulon genes also play roles in virulence, motility and biofilm formation, among others. In this paper we report that the absence of a functional PhoB/PhoR caused increased expression of a number of genes from distinct stress response pathways, in Pi abundance. Moreover, we showed, for the first time, that the interrelationship between PhoB-RpoS-(p)ppGpp-poly(P) in V. cholerae, is somewhat diverse from the model of inter-regulation between those systems, described in Escherichia coli. The V. cholerae dependence on PhoB/PhoR for the RpoS mediated stress response and cellular growth under Pi abundance, suggests that this system's roles are broader than previously thought.

Molecular analysis of VCA1008: a putative phosphoporin of Vibrio cholerae.

The PhoB/PhoR-dependent response to inorganic phosphate (Pi)-starvation in Vibrio cholerae O1 includes the expression of vc0719 for the response regulator PhoB, vca0033 for an alkaline phosphatase and vca1008 for an outer membrane protein (OMP). Sequences with high identity to these genes have been found in the genome of clinical and environmental strains, suggesting that the Pi-starvation response in V. cholerae is well conserved. VCA1008, an uncharacterized OMP involved in V. cholerae pathogenicity, presents sequence similarity to porins of Gram-negative bacteria such as phosphoporin PhoE from Escherichia coli. A three-dimensional model shows that VCA1008 is a 16-stranded pore-forming beta-barrel protein that shares three of the four conserved lysine residues responsible for PhoE anionic specificity with PhoE. VCA1008 beta-barrel apparently forms trimers that collapse into monomers by heating. Properties such as heat modifiability and resistance to denaturation by sodium dodecyl sulfate at lower temperatures permitted us to suggest that VCA1008 is a classical porin, more precisely, a phosphoporin due to its Pi starvation-induced PhoB-dependent expression, demonstrated by electrophoretic mobility shift assay and promoter fusion-lacZ assays.