Expression of the Pho regulon negatively regulates biofilm formation by Pseudomonas aureofaciens PA147-2

We report the isolation of insertional mutations to the pstC and pstA genes of the phosphate-specific transport (pst) operon that results in loss of biofilm formation by Pseudomonas aureofaciens PA147-2. Consistent with the known roles of the Pst system in Escherichia coli and Pseudomonas aeruginosa, both P. aureofaciens pst mutants were demonstrated to have defects in inorganic phosphate (P(i)) transport and repression of Pho regulon expression. Subsequently, biofilm formation by the wild type was shown to require a threshold concentration of extracellular P(i). The two-component regulatory pair PhoR/PhoB is responsible for upregulation of Pho regulon expression in response to P(i)-limiting environments. By generating phoR mutants that were unable to express the Pho regulon, we were able to restore biofilm formation by P. aureofaciens in P(i)-limiting conditions. This result suggests that gene(s) within the Pho regulon act to regulate biofilm formation negatively in low-P(i) environments, and that phoR mutations uncouple PA147-2 from such regulatory constraints. Furthermore, the inability of pst mutants to repress Pho regulon expression accounts for their inability to form biofilms in non-limiting P(i) environments. Preliminary evidence suggests that the Pst system is also required for antifungal activity by PA147-2. During phenotypic analysis of pst mutants, we also uncovered novelties in relation to P(i) assimilation and Pho regulon control in P. aureofaciens.     

The extended N-terminal region of SphS is required for detection of external phosphate levels in Synechocystis sp. PCC 6803

A novel 47 amino acid extension at the N-terminus of the SphS histidine kinase has been identified in the cyanobacterium Synechocystis sp. PCC 6803. Here, we demonstrate this region is required for activation of the SphS-SphR phosphate-sensing two-component system under phosphate-limiting conditions and mutants lacking this extension do not show constitutive alkaline phosphatase activity when the negative regulator SphU is inactivated. We have also identified a putative membrane-associated domain within this region involved in control of the Pho regulon. In addition, there are two high-affinity ABC-type phosphate uptake systems in this organism. Our results demonstrate that the Pst1 system, but not the Pst2 system, is required for suppression of the Pho regulon under phosphate-sufficient conditions. Deletion of the pst1 operon and disruption of the membrane-spanning domain may both target the same control mechanism since constitutive alkaline phosphatase activity is similar in the double and single mutants.     

The phosphate regulon and bacterial virulence: a regulatory network connecting phosphate homeostasis and pathogenesis

Bacterial pathogens regulate virulence factor gene expression coordinately in response to environmental stimuli, including nutrient starvation. The phosphate (Pho) regulon plays a key role in phosphate homeostasis. It is controlled by the PhoR/PhoB two-component regulatory system. PhoR is an integral membrane signaling histidine kinase that, through an interaction with the ABC-type phosphate-specific transport (Pst) system and a protein called PhoU, somehow senses environmental inorganic phosphate (P(i)) levels. Under conditions of P(i) limitation (or in the absence of a Pst component or PhoU), PhoR activates its partner response regulator PhoB by phosphorylation, which, in turn, up- or down-regulates target genes. Single-cell profiling of PhoB activation has shown recently that Pho regulon gene expression exhibits a stochastic, "all-or-none" behavior. Recent studies have also shown that the Pho regulon plays a role in the virulence of several bacteria. Here, we present a comprehensive overview of the role of the Pho regulon in bacterial virulence. The Pho regulon is clearly not a simple regulatory circuit for controlling phosphate homeostasis; it is part of a complex network important for both bacterial virulence and stress response.     

Molecular aspects of phosphate transport in Escherichia coli

Escherichia coli transports inorganic phosphate (Pi) by the low-affinity transport system, Pit. When the level of the external Pi is lower than 20 microM, another transport system, Pst, is induced with a Kt of 0.25 microM. An outer-membrane porin, PhoE, with a Km of about 1 microM is also induced. The outer membrane allows the intake of organic phosphates which are degraded to Pi by phosphatases in the periplasm. The Pi-binding protein will capture the free Pi produced in the periplasm and direct it to the transmembrane channel of the cytoplasmic membrane. The channel consists of two proteins, PstA and PstC, which have six and five transmembrane helices, respectively. On the cytoplasmic side of the membrane the channel is linked to the PstB protein, which carries a nucleotide (probably ATP)-binding site. PstB probably provides the energy required by the channel to free Pi. The Pst system has two functions in E. coli: (i) the transport of Pi, and (ii) the negative regulation of the phosphate regulon (a complex of 20 proteins mostly related to organic phosphate transport). It is remarkable that these two functions are not related, since the repressibility of the regulon depends on the integral structure of Pst (PiBP + PstA + PstC + PstB) and not on the Pi transported. Another gene of the pst operon, phoU, produces a protein involved in the negative regulation of the Pho regulon, but the mechanism of this function has not been explained. Thus the regulatory function of the Pst system remains obscure. Its basal level, present when Pi is abundant, is sufficient to repress the Pho regulon but the negative regulatory function is lost upon Pi starvation.     

The phosphate-starvation response in Vibrio cholerae O1 and phoB mutant under proteomic analysis: disclosing functions involved in adaptation, survival and virulence

A proteomic analysis of a wild-type and of a phoB mutant showed that Vibrio cholerae expresses genes of two major regulons in response to phosphate starvation. The Pho regulon, expressed by the wild-type, allowed the cells to adapt to the new environment. Induction of the general stress regulon was mainly observed in the phoB mutant as a strategy to resist stress and survive. Some functions of the adaptative and survival responses play roles in the pathogenicity of the bacteria. Among the members of the Pho regulon, we found a porin described as an important factor for the intestinal colonisation. Other functions not obviously related to phosphate metabolism, expressed preferentially by the wild-type cells, have also been implicated in virulence. These findings might explain the lack of virulence of the phoB mutant. The Pho regulon picture of V. cholerae, however, will not be complete until minor members and membrane proteins are identified. Among the phosphate-starvation induced genes we have found 13 hypothetical ones and for some of them functions have been assigned. The majority of the genes identified here have not been described before, thus they could be used to expand the proteomic reference map of V. cholerae El Tor.     

Identification of a Streptococcus pneumoniae Gene Locus Encoding Proteins of an ABC Phosphate Transporter and a Two-Component Regulatory System

The Escherichia coli Pst system belongs to the family of ABC transporters. It is part of a phosphate (PHO) regulon which is regulated by extracellular phosphate. Under conditions of phosphate limitation, the response regulator PhoB is phosphorylated by the histidine kinase PhoR and binds to promoters that share a consensus PHO box. Under conditions of phosphate excess, PhoR, Pst, and PhoU downregulate the PHO regulon. Screening of a library of pneumococcal mutants with defects in exported proteins revealed a putative two-component regulatory system, PnpR-PnpS, and a downstream ABC transporter, similar to the Pst system in E. coli including a gene encoding a PhoU protein. Similar to E. coli, mutagenesis of the ATP-binding cassette gene, pstB, resulted in decreased uptake of phosphate. The effects of the loss of the pneumococcal Pst system extended to decreased transformation and lysis. Withdrawal of phosphate led to transformation deficiency in the parent strain R6x but not to penicillin tolerance, suggesting that reduced bacterial death was independent of phosphate. None of these phenotypes was observed in the pneumococcal loss-of-function mutant phoU. By using a lacZ reporter construct, it was demonstrated that expression of the two-component regulatory system PnpR-PnpS was not influenced by different concentrations of phosphate. These results suggest a more complex role of the Pst system in pneumococcal physiology than in that of E. coli.     

Exploring the rearrangement of sensory intelligence in proteobacteria: insight of <Emphasis Type="Italic">Pho regulon</Emphasis>

Pho regulon is a highly evolved and conserved mechanism across the microbes to fulfil their phosphate need. In this study, 52 proteobacteria genomes were analyzed for the presence of phosphorus acquisition genes, their pattern of arrangement and copy numbers. The diverse genetic architecture of the Pho regulon genes indicates the evolutionary challenge of nutrient limitation, particularly phosphorus, faced by bacteria in their environment. The incongruence between the Pho regulon proteins phylogeny and species phylogeny along with the presence of additional copies of pstS and pstB genes, having cross similarity with other genera, suggest the possibility of horizontal gene transfer event. The substitution rate analysis and multiple sequence alignment of the Pho regulon proteins were analyzed to gain additional insight into the evolution of the Pho regulon system. This comprehensive study confirms that genes perform the regulatory function (phoBR) were vertically inherited, whereas interestingly, genes whose product involved in direct interaction with the environment (pstS) acquired by horizontal gene transfer. The substantial amino acid substitutions in PstS most likely contribute to the successful adaptation of bacteria in different ecological condition dealing with different phosphorus availability. The findings decipher the intelligence of the bacteria which enable them to carry out the targeted alteration of genes to cope up with the environmental condition.     

Genome-Wide Analysis of the Pho Regulon in a pstCA Mutant of Citrobacter rodentium

The phosphate-specific transport operon, pstSCAB-phoU, of Gram-negative bacteria is an essential part of the Pho regulon. Its key roles are to encode a high-affinity inorganic phosphate transport system and to prevent activation of PhoB in phosphate-rich environments. In general, mutations in pstSCAB-phoU lead to the constitutive expression of the Pho regulon. Previously, we constructed a pstCA deletion mutant of Citrobacter rodentium and found it to be attenuated for virulence in mice, its natural host. This attenuation was dependent on PhoB or PhoB-regulated gene(s) because a phoB mutation restored virulence for mice to the pstCA mutant. To investigate how downstream genes may contribute to the virulence of C. rodentium, we used microarray analysis to investigate global gene expression of C. rodentium strain ICC169 and its isogenic pstCA mutant when grown in phosphate-rich medium. Overall 323 genes of the pstCA mutant were differentially expressed by at least 1.5-fold compared to the wild-type C. rodentium. Of these 145 were up-regulated and 178 were down-regulated. Differentially expressed genes included some involved in phosphate homoeostasis, cellular metabolism and protein metabolism. A large number of genes involved in stress responses and of unknown function were also differentially expressed, as were some virulence-associated genes. Up-regulated virulence-associated genes in the pstCA mutant included that for DegP, a serine protease, which appeared to be directly regulated by PhoB. Down-regulated genes included those for the production of the urease, flagella, NleG8 (a type III-secreted protein) and the tad focus (which encodes type IVb pili in Yersinia enterocolitica). Infection studies using C57/BL6 mice showed that DegP and NleG8 play a role in bacterial virulence. Overall, our study provides evidence that Pho is a global regulator of gene expression in C. rodentium and indicates the presence of at least two previously unrecognized virulence determinants of C. rodentium, namely, DegP and NleG8.     

Phosphate-dependent modulation of c-di-GMP levels regulates Pseudomonas fluorescens Pf0-1 biofilm formation by controlling secretion of the adhesin LapA

Biofilm formation is commonly described as a developmental process regulated by environmental cues. In the current study we present a mechanistic model to explain regulation of Pseudomonas fluorescens biofilm formation by the environmentally relevant signal inorganic phosphate (P(i)). We show that activation of the Pho regulon, the major pathway for adaptation to phosphate limitation, results in conditional expression of a c-di-GMP phosphodiesterase referred to as RapA. Genetic analysis indicated that RapA is an inhibitor of biofilm formation and required for loss of biofilm formation in response to limiting P(i). Our results suggest that RapA lowers the level of c-di-GMP, which in turn inhibits the secretion of LapA, a large adhesion required for biofilm formation by P. fluorescens. The ability of c-di-GMP to modulate protein secretion is a novel finding and further extends the biological influence of c-di-GMP beyond that of regulating exopolysaccharide synthesis and motility. Interestingly, Pho regulon expression does not impinge on the rate of attachment to a surface but rather inhibits the transition of cells to a more stable interaction with the surface. We hypothesize that Pho regulon expression confers a surface-sensing mode on P. fluorescens and suggest this strategy may be broadly applicable to other bacteria.