The PhoB regulatory system modulates biofilm formation and stress response in El Tor biotype Vibrio cholerae

The PhoBR regulatory system is required for the induction of multiple genes under conditions of phosphate limitation. Here, we examine the role of PhoB in biofilm formation and environmental stress response in Vibrio cholerae of the El Tor biotype. Deletion of phoB or hapR enhanced biofilm formation in a phosphate-limited medium. Planktonic and redispersed biofilm cells of the Δ phoB mutant did not differ from wild type for the expression of HapR, suggesting that PhoB negatively affects biofilm formation through an HapR-independent pathway. The Δ phoB mutant exhibited elevated expression of exopolysaccharide genes vpsA and vpsL compared with the wild type. Deletion of hapR enhanced the expression of the positive regulator vpsT , but had no effect on the expression of vpsR . In contrast, deletion of phoB enhanced the expression of the positive regulator vpsR , but had no effect on the expression of hapR and vpsT . The Δ phoB mutant was more sensitive to hydrogen peroxide compared with the wild type and with an isogenic Δ rpoS mutant. Conversely, the Δ phoB mutant was more resistant to acidic conditions and high osmolarity compared with the wild type and with an isogenic Δ rpoS mutant. Taken together, our data suggest that phosphate limitation induces V. cholerae to adopt a free-swimming life style in which PhoB modulates environmental stress response in a manner that differs from the general stress response regulator RpoS.     

Transcriptome analysis of phosphate starvation response in Escherichia coli

Escherichia coli has a PhoR-PhoB two-component regulatory system to detect and respond to the changes of environmental phosphate concentration. For the E. coli W3110 strain growing under phosphate-limiting condition, the changes of global gene expression levels were investigated by using DNA microarray analysis. The expression levels of some genes that are involved in phosphate metabolism were increased as phosphate became limited, whereas those of the genes involved in ribosomal protein or amino acid metabolism were decreased, owing to the stationary phase response. The upregulated genes could be divided into temporarily and permanently inducible genes by phosphate starvation. At the peak point showing the highest expression levels of the phoB and phoR genes under phosphate-limiting condition, the phoB- and/or phoR-dependent regulatory mechanisms were investigated in detail by comparing the gene expression levels among the wild-type and phoB and/or phoR mutant strains. Overall, the phoB mutation was epistatic over the phoR mutation. It was found that PhoBR and PhoB were responsible for the upregulation of the phosphonate or glycerol phosphate metabolism and high-affinity phosphate transport system, respectively. These results show the complex regulation by the PhoR-PhoB two-component regulatory system in E. coli.     

Regulation of the Phosphate Stress Response in Rhizobium meliloti by PhoB

Alkaline phosphatase activity and phosphate transport rates in Rhizobium meliloti increased significantly when medium phosphate levels decreased to approximately 10 (mu)M. Both responses were abolished in a Tn5:: phoB mutant, but the mutant could be complemented by a plasmid that contained cloned R. meliloti phoB. The PhoB(sup-) mutant had a normal symbiosis phenotype under growth conditions that supplied either limiting or nonlimiting levels of phosphate to the host plant Medicago sativa, suggesting that induction of genes by PhoB was not required for normal symbiotic function.     

Use of a phoB'-'lacZ fusion gene to determine the N-terminal amino acid sequence of the phoB protein and to prepare antiserum against the protein

phoB is a positive regulatory gene of the phosphate regulon of Escherichia coli. A plasmid carrying a phoB'-'lacZ fusion gene was constructed by in vitro recombination. A PhoB-LacZ hybrid protein was purified from the cells carrying the plasmid by monitoring beta-galactosidase activity. The amino-terminal amino acid sequence of the PhoB protein was determined by utilizing the hybrid protein. Antiserum against the PhoB protein was prepared by immunizing rabbits with the hybrid protein. The serum thus prepared showed specificity for both the PhoB protein and beta-galactosidase.     

Regulation of the pho regulon in Escherichia coli K-12. Genetic and physiological regulation of the positive regulatory gene phoB

phoB is a positive regulatory gene for phoA, which codes for alkaline phosphatase, as well as for other genes belonging to the phosphate (pho) regulon whose expression is inducible by phosphate limitation in Escherichia coli. A hybrid plasmid that contains a phoB-lacZ fused gene was constructed in vitro. This plasmid enabled us to study phoB gene expression by measuring the beta-galactosidase level in the cells. The plasmid was introduced into various regulatory mutants related to the phosphate regulon, and phoB gene expression in these strains was studied under limited and excess phosphate conditions. It was found that the regulation of phoB expression was very similar to that of phoA expression. Expression of both genes was induced by phosphate starvation. Both genes were constitutively expressed in phoR, phoS, phoT and phoU mutants and were not expressed in a phoR-phoM double mutant. The implications of these findings for the regulatory mechanism of the pho regulon are discussed.     

From cell membrane to nucleotides: the phosphate regulon in Escherichia coli

Most of the essential cellular components, like nucleic acids, lipids and sugars, are phosphorylated. The phosphate equilibrium in Escherichia coli is regulated by the phosphate (Pi) input from the surrounding medium. Some 90 proteins are synthesized at an increased rate during Pi starvation and the global control of the cellular metabolism requires cross-talk with other regulatory mechanisms. Since the Pi concentration is normally low in E. coli's natural habitat, these cells have devised a mechanism for synthesis of about 15 proteins to accomplish two specific functions: transport of Pi and its intracellular regulation. The synthesis of these proteins is controlled by two genes (the phoB-phoR operon), involving both negative and positive functions. PhoR protein is a histidine protein kinase, induced in Pi starvation and is a transmembrane protein. It phosphorylates the regulator protein PhoB which is also Pi starvation-induced. The PhoB phosphorylated form binds specifically to a DNA sequence of 18 nucleotides (the pho Box), which is part of the promoters of the Pho genes. The genes controlled by phoB constitute the Pho regulon. The repression of phoA (the gene encoding alkaline phosphatase) by high Pi concentrations in the medium requires the presence of an intact Pst operon (pstS, pstC, pstA, pstB and phoU) and phoR. The products of pstA and pstC are membrane bound, whereas the product of pstS is periplasmic and PstB and PhoU proteins are cytoplasmic. The function of the PhoU protein may be regulated by cofactor nucleotides and may be involved in signaling the activation of the regulon via PhoR.