The control of Vibrio cholerae phoBR expression by PhoB involves its binding to Pho boxes at ؊35 (box 1), ؊60 (box 2), and ؊80 (box 3) from the putative phoB translation start site. These loci were located in the sense (box 1) and antisense (boxes 2 and 3) strands of the phoBR regulatory region, and PhoB binds to these individual boxes with distinct affinities. Fusions of sequences containing different combinations of these boxes upstream of the lacZ reporter in a plasmid demonstrated that only those carrying boxes 1, 2, and 3, or 1 alone, activated transcription under inorganic phosphate (P i ) limitation. When a fragment, including only boxes 1 and 2, was fused to lacZ, expression was no longer induced by low P i , suggesting a repressive role for PhoBϳbox2 (PhoB bound to box 2) over the transcriptional activity induced by PhoBϳbox1. The similarity between lacZ expression levels from promoter fragments containing the three boxes or box 1 alone showed that PhoBϳbox3 eliminated the repressive effect imposed by PhoBϳbox2 on phoBR transcription. Complementation assays with a phoBR-containing plasmid demonstrated that the 234-bp promoter fragment carrying the three boxes is absolutely required for operon expression in Vibrio cholerae ⌬phoBR cells. This was observed under P i abundance, when phoBR was expressed at a basal level and, also in low P i conditions, when Pho regulon genes were fully expressed. Thus, under P i limitation, PhoB exerts dual regulatory functions by binding sequentially distinct Pho boxes to enable the fine-tuning and precise control of phoBR expression in V. cholerae cells.
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.
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.
Crithidia deanei is a trypanosomatid protozoan that harbours a symbiotic bacterium. The partners maintain a mutualistic relationship, thus constituting an excellent model for studying metabolic exchanges between the host and the symbiont, the origin of organelles and cellular evolution. According to molecular analysis, symbionts of different trypanosomatid species share high identity and descend from a common ancestor, a b-proteobacterium of the genus Bordetella. The endosymbiont is surrounded by two membranes, like Gram-negative bacteria, but its envelope presents special features, since phosphatidylcholine is a major membrane component and the peptidoglycan layer is highly reduced, as described in other obligate intracellular bacteria. Like the process that generated mitochondria and plastids, the endosymbiosis in trypanosomatids depends on pathways that facilitate the intensive metabolic exchanges between the bacterium and the host protozoan. A search of the annotated symbiont genome database identified one sequence with identity to porin-encoding genes of the genus Bordetella. Considering that the symbiont outer membrane has a great accessibility to cytoplasm host factors, it was important to characterize this single porin-like protein using biochemical, molecular, computational and ultrastructural approaches. Antiserum against the recombinant porin-like molecule revealed that it is mainly located in the symbiont envelope. Secondary structure analysis and comparative modelling predicted the protein 3D structure as an 18-domain b-barrel, which is consistent with porin channels. Electrophysiological measurements showed that the porin displays a Abbreviations: ASB-14, amidosulfobetaine-14; CD, circular dichroism; IM, inner membrane; OM, outer membrane; RMSD, root mean square deviation; VDAC, voltage-dependent anion-selective channel.The GenBank/EMBL/DDBJ accession number for the porin sequence of the Crithidia deanei endosymbiont is HM480845.Three supplementary figures, showing a sequence alignment of the endosymbiont protein and Delftia acidovorans Omp32, a 3D structure model of the endosymbiont C. deanei porin, and nucleotide and amino acid sequences of the C. deanei endosymbiont porin, are available with the online version of this paper. Microbiology
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