title: Fine-tuning control of phoBR expression in Vibrio cholerae by binding of phoB to multiple pho boxes.
abstract: 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 PhoB~box2 (PhoB bound to box 2) over the transcriptional activity induced by PhoB~box1. The similarity between lacZ expression levels from promoter fragments containing the three boxes or box 1 alone showed that PhoB~box3 eliminated the repressive effect imposed by PhoB~box2 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 ΔphoBR 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.
authors: Unidade Multidisciplinar de Genômica, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
authors: Michelle Menezes Passos
authors: MM
authors: Diniz
authors: Carolina Lage
authors: CL
authors: Goulart
authors: Livia Carvalho
authors: LC
authors: Barbosa
authors: Júlia
authors: J
authors: Farache
authors: Letícia Miranda Santos
authors: LM
authors: Lery
authors: Ana Beatriz Furlanetto
authors: AB
authors: Pacheco
authors: Paulo Mascarello
authors: PM
authors: Bisch
authors: Wanda Maria de Almeida
authors: WM
authors: von Krüger
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D009876:Operon; D010710:Phosphates / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D014734:Vibrio cholerae / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D010710:Phosphates; C061278:PhoB protein, Bacteria; C061279:PhoR protein, Bacteria