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.     

Characterization of the Rhizobium (Sinorhizobium) meliloti high- and low-affinity phosphate uptake systems.

Genetic studies have suggested that Rhizobium (Sinorhizobium) meliloti contains two distinct phosphate (Pi) transport systems, encoded by the phoCDET genes and the orfA-pit genes, respectively. Here we present data which show that the ABC-type PhoCDET system has a high affinity for Pi (Km, 0.2 microM) and that Pi uptake by this system is severely inhibited by phosphonates. This high-affinity uptake system was induced under Pi-limiting conditions and was repressed in the presence of excess Pi. Uptake via the OrfA-Pit system was examined in (i) a phoC mutant which showed increased expression of the orfA-pit genes as a result of a promoter-up mutation and (ii) a phoB mutant (PhoB is required for phoCDET expression). Pi uptake in both strains exhibited saturation kinetics (Km, 1 to 2 microM) and was not inhibited by phosphonates. This uptake system was active in wild-type cells grown with excess Pi and appeared to be repressed when the cells were starved for Pi. Thus, our biochemical data show that the OrfA-Pit and PhoCDET uptake systems are differentially expressed depending on the state of the cell with respect to phosphate availability.     

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.     

The regulator gene phoB mediates phosphate stress-controlled synthesis of the membrane lipid diacylglyceryl-N,N,N-trimethylhomoserine in Rhizobium (Sinorhizobium) meliloti

Bacteria react to phosphate starvation by activating genes involved in the transport and assimilation of phosphate as well as other phosphorous compounds. Some soil bacteria have evolved an additional mechanism for saving phosphorous. Under phosphate-limiting conditions, they replace their membrane phospholipids by lipids not containing phosphorus. Here, we show that the membrane lipid pattern of the free-living microsymbiotic bacterium Rhizobium (Sinorhizobium) meliloti is altered at low phosphate concentrations. When phosphate is growth limiting, an increase in sulpholipids, ornithine lipids and the de novo synthesis of diacylglyceryl trimethylhomoserine (DGTS) lipids is observed. Rhizobium meliloti phoCDET mutants, deficient in phosphate uptake, synthesize DGTS constitutively at low or high medium phosphate concentrations, suggesting that reduced transport of phosphorus sources to the cytoplasm causes induction of DGTS biosynthesis. Rhizobium meliloti phoU or phoB mutants are unable to form DGTS at low or high phosphate concentrations. However, the functional complementation of phoU or phoB mutants with the phoB gene demonstrates that, of the two genes, only intact phoB is required for the biosynthesis of the membrane lipid DGTS.     

The chromosomal response regulatory gene chvI of Agrobacterium tumefaciens complements an Escherichia coli phoB mutation and is required for virulence.

In an effort to identify the Agrobacterium tumefaciens phosphate regulatory gene(s), we isolated a clone from an A. tumefaciens cosmid library that restored regulated alkaline phosphatase activity to an Escherichia coli phoB mutant. The gene that complemented phoB was localized by subcloning and deletion analysis, and the DNA sequence was determined. An open reading frame, denoted chvI, was identified that encoded a predicted protein with amino acid similarity to the family of bacterial response regulators and 35% identify to PhoB. Surprisingly, an A. tumefaciens chvI mutant showed normal induction of phosphatase activity and normal virG expression when grown in phosphate-limiting media. However, this mutant was unable to grow in media containing tryptone, peptone, or Casamino Acids and was also more sensitive than the wild type to acidic extracellular pH. This mutant was avirulent on Kalanchoeë diagremontiana and was severely attenuated in vir gene expression. The pH-inducible expression of virG was also abolished. Growth of the chvI mutant was inhibited by K. diagremontiana wound sap, suggesting that avirulence may be due, in part, to the inability of this mutant to survive the plant wound environment.     

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.