phoR Genes Research Articles

The PhoR/PhoP two-component system regulates fengycin production in Bacillus subtilis NCD-2 under low-phosphate conditions

Bacillus subtilis strain NCD-2 is an excellent biocontrol agent for plant soil-borne diseases, and the lipopeptide fengycin is one of the active antifungal compounds in strain NCD-2. The regulator PhoP and its sensor kinase PhoR compose a two-component system in B. subtilis. In this study, the phoR- and phoP-knockout mutants were constructed by in-frame deletion and the role of PhoR/PhoP on the production of fengycin was determined. Inactivation of phoR or phoP in B. subtilis decreased its inhibition ability against Botrytis cinerea growth in vitro compared to the strain NCD-2 wild type. The lipopeptides were extracted from strain NCD-2 wild type and its mutant strains by hydrochloric acid precipitate, and the lipopeptides from phoR-null mutant or phoP-null mutant almost lost the inhibition ability against B. cinerea growth compared to the lipopeptides from strain NCD-2 wild type. Fast protein liquid chromatography (FPLC) analysis of the lipopeptides showed that inactivation of phoR or phoP genes reduced the production of fengycin by strain NCD-2. The fengycin production abilities were compared for bacteria under low-phosphate medium (LPM) and high-phosphate medium (HPM), respectively. Results indicated that the regulation of fengycin production by the PhoR/PhoP two-component system occurred in LPM but not in HPM. Reverse transcriptional-PCR confirmed that the fengycin synthetase gene fenC was positively regulated by phoP when cultured in LPM. All of these characteristics could be partially restored by complementation of intact phoR or phoP gene in the mutant. These data indicated that the PhoR/PhoP two-component system greatly regulated fengycin production and antifungal ability in B. subtilis NCD-2 mainly under low-phosphate conditions.

Sequence comparison of phoR, gyrB, groEL, and cheA genes as phylogenetic markers for distinguishing Bacillus amyloliquefaciens and B. subtilis and for identifying Bacillus strain B29.

Given the close genetic relationship between Bacillus amyloliquefaciens and B. subtilis, distinguishing the two solely based on their physiological and biochemical characteristics and 16S rRNA sequences is difficult. Molecular identification was used to discover suitable genes for distinguishing the two bacteria, and to identify the bio-controlling strain B29, due to molecular identification has been paid more and more attention. The similarity of four genes, cheA, gyrB, groEL and phoR, of the two species was compared by the software BLASTN and MAGA, and phylogenetic tree was constructed. The B29 strain was re-identified by using the screened genes. The similarities of the four genes, gyrB, groEL, cheA and phoR, of the two species were 93-95%, 82-84%, 76-78% and 76-77%, respectively. The homologies of the four genes of the strain B29 and the strains of B. amyloliquefaciens strains were more than 95%. We determined how well the phoR and cheA genes could be used to differentiate B. amyloliquefacien and B. subtilis. The previously isolated biological control strain B29, initially classified as B. subtilis, was re-classified as B. amyloliquefaciens. Our data indicate that other than the phoR gene, the cheA gene might be a useful phylogenetic marker for differentiating B. subtilis and B. amyloliquefaciens.

Amelioration of the Fitness Costs of Antibiotic Resistance Due To Reduced Outer Membrane Permeability by Upregulation of Alternative Porins.

The fitness cost of antibiotic resistance is a key parameter in determining the evolutionary success of resistant bacteria. Studies of the effect of antibiotic resistance on bacterial fitness are heavily biased toward target alterations. Here we investigated how the costs in the form of a severely impaired growth rate associated with resistance due to absence of two major outer membrane porins can be genetically compensated. We performed an evolution experiment with 16 lineages of a double mutant of Escherichia coli with the ompCF genes deleted, and reduced fitness and increased resistance to different classes of antibiotics, including the carbapenems ertapenem and meropenem. After serial passage for only 250 generations, the relative growth rate increased from 0.85 to 0.99 (susceptible wild type set to 1.0). Compensation of the costs followed two different adaptive pathways where upregulation of expression of alternative porins bypassed the need for functional OmpCF porins. The first compensatory mechanism involved mutations in the phoR and pstS genes, causing constitutive high-level expression of the PhoE porin. The second mechanism involved mutations in the hfq and chiX genes that disrupted Hfq-dependent small RNA regulation, causing overexpression of the ChiP porin. Although susceptibility was restored in compensated mutants with PhoE overexpression, evolved mutants with high ChiP expression maintained the resistance phenotype. Our findings may explain why porin composition is often altered in resistant clinical isolates and provide new insights into how bypass mechanisms may allow genetic adaptation to a common multidrug resistance mechanism.

Phosphorylation of PhoP Protein Plays Direct Regulatory Role in Lipid Biosynthesis of Mycobacterium tuberculosis

Mycobacterium tuberculosis PhoP is essential for virulence and intracellular growth of the tubercle bacilli. Genetic evidence suggests that PhoP regulates complex lipid biosynthesis, and absence of some of these lipid molecules in a phoP mutant partly accounts for its attenuated growth in macrophages and/or mice. To investigate the mechanism of regulation, here we demonstrate the essentiality of phosphorylation of PhoP in the regulation of complex lipid biosynthesis. We show that phosphorylated PhoP activates transcription of pks2 and msl3, gene(s) encoding polyketide β-ketoacyl synthases through direct DNA binding at the upstream regulatory region(s) of the target genes. Our results identify the genetic determinants recognized by PhoP and show that activation of target genes requires interaction(s) of the phosphorylated regulator at the cognate binding sites. The fact that these sites within the regulatory region of respective genes do not bind in vitro with either unphosphorylated or phosphorylation-deficient PhoP protein is consistent with phosphorylation-dependent assembly of the transcription initiation complex leading to in vivo transcriptional activation. Together, these results reveal so far unknown molecular mechanisms of how PhoP contributes to M. tuberculosis cell wall composition by regulating complex lipid biosynthesis.

Metabolic regulation of Escherichia coli and its phoB and phoR genes knockout mutants under phosphate and nitrogen limitations as well as at acidic condition

BackgroundThe phosphorus compounds serve as major building blocks of many biomolecules, and have important roles in signal transduction. The phosphate is involved in many biochemical reactions by the transfer of phosphoryl groups. All living cells sophisticatedly regulate the phosphate uptake, and survive even under phosphate-limiting condition, and thus phosphate metabolism is closely related to the diverse metabolism including energy and central carbon metabolism. In particular, phosphorylation may play important roles in the metabolic regulation at acidic condition and nitrogen limiting condition, which typically appears at the late growth phase in the batch culture. Moreover, phosphate starvation is a relatively inexpensive means of gene induction in practice, and the phoA promoter has been used for overexpression of heterologous genes. A better understanding of phosphate regulation would allow for optimization of such processes.ResultsThe effect of phosphate (P) concentration on the metabolism in Escherichia coli was investigated in terms of fermentation characteristics and gene transcript levels for the aerobic continuous culture at the dilution rate of 0.2 h-1. The result indicates that the specific glucose consumption rate and the specific acetate production rate significantly increased, while the cell concentration decreased at low P concentration (10% of the M9 medium). The increase in the specific glucose uptake rate may be due to ATP demand caused by limited ATP production under P-limitation. The lower cell concentration was also caused by less ATP production. The less ATP production by H+-ATPase may have caused less cytochrome reaction affecting in quinone pool, and caused up-regulation of ArcA/B, which repressed TCA cycle genes and caused more acetate production. In the case of phoB mutant (and also phoR mutant), the fermentation characteristics were less affected by P-limitation as compared to the wild type where the PhoB regulated genes were down-regulated, while phoR and phoU changed little. The phoR gene knockout caused phoB gene to be down-regulated as well as PhoB regulated genes, while phoU and phoM changed little. The effect of pH together with lower P concentration on the metabolic regulation was also investigated. In accordance with up-regulation of arcA gene expression, the expressions of the TCA cycle genes such as sdhC and mdh were down-regulated at acidic condition. The gene expression of rpoS was up-regulated, and the expression of gadA was up-regulated at pH 6.0. In accordance with this, PhoB regulated genes were up-regulated in the wild type under P-rich and P-limited conditions at pH 6.0 as compared to those at pH 7.0. Moreover, the effect of nitrogen limitation on the metabolic regulation was investigated, where the result indicates that phoB gene was up-regulated, and PhoB regulated genes were also up-regulated under N-limitation, as well as nitrogen-regulated genes.ConclusionThe present result shows the complicated nature of the metabolic regulation for the fermentation characteristics upon phosphate limitation, acidic condition, and nitrogen limitation based on the transcript levels of selected genes. The result implies that the regulations under phosphate limitation, acidic condition, and nitrogen limitation, which occur typically at the late growth phase of the batch culture, are interconnected through RpoS and RpoD together with Pho genes.

Novel gene members in the Pho regulon ofEscherichia coli

The transcriptome profiles of the wild-type and the phoB mutant strains were compared at the time point showing the highest expression levels of the phoB and phoR genes under a P-limiting condition. Among the 18 new putative genes that were found to be under the control of the PhoB transcriptional regulator, five genes that contain the consensus Pho box were identified by sequence analysis. A reporter gene assay was carried out by fusing the upstream regions of these genes to the promoterless enhanced green fluorescent protein gene, followed by expression. It was found that the expressions of the amn (AMP nucleosidase), yibD (metal ion stress response gene) and ytfK (hypothetical protein) genes were activated by PhoB. These results indicate the additional roles of PhoB as a global regulator.

A role for the PhoBR regulatory system homologue in the Vibrio cholerae phosphate-limitation response and intestinal colonization.

To survive and multiply in different environments, Vibrio cholerae has to coordinately regulate the expression of genes involved in adaptive responses. In many pathogens, adaptive responses, including pathogenic responses, are regulated by two-component regulator (TCR) systems. It is likely that members of a TCR family play a role in the regulation of processes involved in intestinal colonization, and therefore pathogenesis, in V. cholerae. We have identified and characterized a TCR system of V. cholerae: this system is a homologue of Escherichia coli PhoBR. The presence of a putative Pho box suggests that the V. cholerae phoBR operon is regulated by inorganic phosphate levels. The phoR and phoB genes are organized the same way as in E. coli. Mutation of the V. cholerae phoB gene affected the expression of the putative Pho regulon, including PhoA, but did not affect the production of cholera toxin. V. cholerae phoB mutants are less able to colonize rabbit intestine than wild-type V. cholerae. The addition of inorganic phosphate at a high concentration to the inoculum only partially restored the ability of the mutants to colonize the intestine, suggesting that the V. cholerae Pho regulon in vivo may not be regulated by inorganic phosphate levels alone.

The cytoplasmic kinase domain of PhoR is sufficient for the low phosphate-inducible expression of pho regulon genes in Bacillus subtilis.

PhoP-PhoR, one of three two-component systems known to be required to regulate the pho regulon in Bacillus subtilis, directly regulates the alkaline phosphatase genes that are used as pho reporters. Biochemical studies showed that B. subtilis PhoR, purified from Escherichia coli, was autophosphorylated in vitro in the presence of ATP. Phosphorylated PhoR showed stability under basic conditions but not acidic conditions, indicating that the phosphorylation probably occurs on a conserved histidine residue. Phospho-PhoR phosphorylated its cognate response regulator, PhoP in vitro. B. subtilis phoR was placed in the Bacillus chromosome under the control of the Pspac promoter, which is IPTG inducible. The wild-type phoR, under either native promoter or Pspac promoter with IPTG induction, resulted in a similar level of alkaline phosphatase production. Under high phosphate conditions, strains containing wild-type phoR, or phoR mutant gene products that lacked either the periplasmic domain, or both N-terminal transmembrane PhoR mutant gene products that lacked either the periplasmic domain, or both N-terminal transmembrane PhoR sequences or various extended N-terminal sequences, showed no significant APase production. Under phosphate starvation conditions, in the presence of IPTG, all strains containing mutated phoR genes showed alkaline phosphatase induction patterns similar to that of the wild-type strain, although the fully induced level was lower in the mutants. The decrease in total alkaline phosphatase production in these mutant strains can be compensated completely or partially by increasing the copy number of the mutant phoR gene. These in vivo results suggest that the C-terminal kinase domain of PhoR is sufficient for the induction of alkaline phosphatase expression under phosphate-limited conditions, and that the regulation for repression of APase under phosphate-replete conditions remains intact.

Regulation of the phosphate regulon of Escherichia colt Properties of phoR deletion mutants and subcellular localization of PhoR protein

The phoR gene is a bifunctional regulatory gene for the phosphate regulon of Escherichia coli. It acts as a negative regulator in the presence of excess phosphate and as a positive regulator with limited phosphate, through modification of PhoB protein. We constructed several phoR genes, with various deletions in the 5' regions, which were regulated by the trp-lac hybrid promoter. The PhoR1084 and PhoR1159 proteins that lack the 83 and 158 N-terminal amino acids, respectively, retained the positive function for the expression of phoA that codes for alkaline phosphatase, but lacked the negative function. The PhoR1263 protein that lacks the 262 N-terminal amino acids was deficient in both functions. An antiserum against PhoR1084 protein was prepared. Western blot analysis of the subcellular fractions obtained by differential centrifugation indicated that the intact PhoR and PhoR1084 proteins are located in the inner membrane and cytoplasmic fractions, respectively. The results suggest that PhoR protein is anchored to the cytoplasmic membrane by the amino-terminal region.

Phosphate regulon in members of the family Enterobacteriaceae: comparison of the phoB-phoR operons of Escherichia coli, Shigella dysenteriae, and Klebsiella pneumoniae

The structure and function of the phoB and phoR genes of Shigella dysenteriae strains and Klebsiella pneumoniae, which are involved in regulation of the phosphate regulon, were analyzed. Complementation tests among the genes of Escherichia coli, S. dysenteriae strains, and K. pneumoniae for production of alkaline phosphatase indicate that S. dysenteriae serotype 2 and serotype 3 strains and K. pneumoniae are phoA+ phoB+ phoR+ but S. dysenteriae Sh and serotype 1 strains are phoA phoB+ phoR. Nucleotide sequences of phoB and phoR of S. dysenteriae Sh and K. pneumoniae are highly homologous to those of E. coli, except for a single base insertion found in phoR of S. dysenteriae Sh.

Regulation of the phosphate regulon of Escherichia coli: analysis of mutant phoB and phoR genes causing different phenotypes.

The phoB gene product of Escherichia coli is the transcriptional activator for the genes in the phosphate regulon as well as for phoB itself, all of which are induced by phosphate starvation. The phoR gene product modulates PhoB function in response to the phosphate concentrations in the medium. We quantitatively compared the levels of expression of the phoA, phoB, phoE, and pstS genes in several phoB mutants with different phenotypes by constructing operon fusions of these genes with the gene for chloramphenicol acetyltransferase. Although all the phoB mutants examined had little activator function for phoA, three among the four mutants showed various levels of the activator function for phoB, pstS, and phoE. To study the functional motifs of the PhoB and PhoR proteins, we cloned and sequenced the four classical phoB and six phoR mutant genes. All of the phoB mutations and one of the phoR mutations were missense mutations, and most of the altered amino acids were in the highly conserved amino acids among the regulatory proteins homologous to PhoB or PhoR protein, such as the OmpR, SfrA, and VirG proteins or the EnvZ, CpxA, and VirA proteins. The other five phoR mutations were nonsense mutations.

The phoBR operon in Escherichia coli K-12.

The phoB and phoR genes encode a transcription activator and a sensory protein of the phosphate regulon, respectively. It is shown here that they were transcribed as an operon in which the phoB gene was promoter proximal. Although an operon structure was suggested previously (K. Makino, H. Shinagawa, M. Amemura, and A. Nakata, J. Mol. Biol. 190:37-44 and 192:549-556, 1986), previous results showed only that phoR gene expression during phosphate limitation is dependent on the upstream phoB promoter. The phoR gene could still have had its own promoter for expression in the presence of phosphate. Two polar transposon-induced mutations are described which simultaneously abolished phoB and phoR gene function in cis; one mutation mapped in the phoB gene, and the other mapped upstream of the phoB gene. These results demonstrate an operon structure, in which phoR gene function required expression from the phoB promoter. Unexpectedly, an antisense pho omega Mu d1(lacZ) insertion within the promoter-proximal end of the phoB gene expressed the lacZ reporter gene, thus allowing for the possibility that the phoBR operon is regulated by an antisense RNA.

Nucleotide sequence of the phoR gene, a regulatory gene for the phosphate regulon of Escherichia coli

Genes in the phosphate regulon of Escherichia coli are positively regulated by the products of the phoB and phoR genes with limited phosphate, and negatively regulated by the product of the phoR gene with excess phosphate. We present here the complete nucleotide sequence of the phoR gene. Together with the DNA sequence of the upstream phoB gene that we determined previously, this region shows the following features. (1) The flanking regions of the operon are abundant in A · T base-pairs. (2) A possible stem-and-loop structure of the transcript followed by several U residues characteristic of rho-independent transcriptional terminators was distal to the phor coding region. The operon is probably composed of only two cistrons. (3) The nucleotide sequence of phor indicates that its protein consists of 431 amino acid residues and has a molecular weight of 49,666. (4) The amino acid sequence of the PhoR protein has significant homology with that of the EnvZ protein, which is a regulator for the omp regulon. Therefore, the sequences of the PhoB and PhoR proteins have considerable homologies with those of the OmpR and EnvZ proteins, respectively, indicating that the two operons share a common ancestor. (5) The PhoR protein contains an extensive hydrophobic region in the amino-terminal portion. Thus the protein may be a membrane protein and function as a component of a signal transducer.

Regulation of the lkyB gene expression in Escherichia coli K-12 strains carrying an lkyB-lacZ gene fusion

Phage MudII301 was used to isolate new periplasmic-leaky mutants of Escherichia coli K12 carrying an lkyB-lacZ gene fusion. The properties of strain JC2299 carrying the lkyB-2299 insertion mutation were identical to those of strain JC207 carrying the previously described lkyB-207 mutation. The LkyB-beta-galactosidase hybrid protein was partially extracellular and membrane bound. It was shown that both a nonsense (envZ-22) and a polar (ompR::Tn10) mutation in the ompB operon led to an increase of beta-galactosidase activity in the lkyB-lacZ fusion strain. On the other hand, mutations in the phoB, phoR, phoS, phoT, malT or envY genes had no effect on lkyB gene expression.

Alkaline phosphatase secretion-negative mutant of Bacillus licheniformis 749/C.

An alkaline phosphatase secretion-blocked mutant of Bacillus licheniformis 749/C was isolated. This mutant had defects in the phoP and phoR regions of the chromosome. The selection procedure was based on the rationale that N-methyl-N'-nitro-N-nitrosoguanidine can induce mutations of closely linked multiple genes. The malate gene and the phoP and phoR genes are located at the 260-min position in the Bacillus subtilis chromosome; hence, the malate gene could be used as a marker for the mutation of the phoP and phoR regions of the chromosome. In a two-step selection procedure, strains defective in malate utilization were first selected with the cephalosporin C procedure. Second, these malate-defective strains were further screened in a dye medium to select strains with defects in alkaline phosphatase secretion. One stable mutant (B. licheniformis 749/cNM 105) had a total secretion block for alkaline phosphatase and had the following additional characteristics: (i) the amount of alkaline phosphatase synthesized was comparable to that in the wild type; (ii) the alkaline phosphatase was membrane bound; (iii) the mutant strain alkaline phosphatase, in contrast to that of the wild type, could not be extracted with MgCl2, although the amounts of protein extracted from each strain were comparable; (iv) the sodium dodecyl sulfate-polyacrylamide gel pattern of MgCl2-extracted proteins from the mutant strain was different from that of the wild-type proteins; (v) the mutant, unlike the wild type, could not use malate as a sole source of carbon; and (vi) the outside surface of the wall of the mutant cells contained an additional electron-dense layer that was not present on the wild-type cell wall surface.

Mutants affected in alkaline phosphatase, expression: evidence for multiple positive regulators of the phosphate regulon in Escherichia coli.

The expression of alkaline phosphatase (the product of the phoA gene) in Escherichia coli is believed to be subject to both positive control by the phoB gene product and negative control by the phoR gene product. We have isolated a large number of PhoA- mutants in the phoR- genetic background. Among mutants altered in the positive control of alkaline phosphatase, some were phoB mutants; others had a mutation in a new gene, designated phoM. We believe that the phoM gene codes for a positive regulator that acts together with the phoB gene product in phoA gene expressions.--The phoM phenotype was found to be masked in phoR+ strains. This and other evidence support a positive regulatory role for the phoR gene product as well.--Our experiments demonstrate that phoA is under positive control by three different positive regulators: the product of the phoB, phoM and phoR genes. The phoB gene product is always needed together with either the phoR or phoM gene product. In addition, the phoR gene product acts as a negative regulator.--We describe a model for phoA gene expression consistent with this new evidence.

Analysis of the regulation of Escherichia coli alkaline phosphatase synthesis using deletions and phi80 transducing phages.

Abstract φ80 transducing phages for the proC † , phoA and phoB genes of Escherichia coli have been obtained. Two mutants have been isolated, in which the brnQ, phoA, proC, phoB (and possibly phoR ) genes have been deleted. Derivatives of a phoA, phoB deletion strain which are lysogenic for a φ80 phoA transducing phage make only very low levels of alkaline phosphatase activity. These results are in agreement with a positive control mechanism for the regulation of alkaline phosphatase synthesis.

Isolation and characterization of recessive, constitutive mutations for repressible acid phosphatase synthesis in Saccharomyces cerevisiae.

Two new classes of mutants containing recessive constitutive mutations, phoT and phoU, that affect the repressible acid phosphatase (EC 3.1.3.2) in Saccharomyces cerevisiae were isolated along with many previously known phoR mutants. These loci segregated independently from each other, from the phoS gene, and from another regulatory gene, phoD, that exerts positive control for acid phosphatase synthesis. The phoR and phoU mutations showed the same genetic behavior in the double mutants, which also contained the phoS or phoD mutation. In contrast, the phoT mutation could not suppress the phoS mutation, which caused a loss of enzyme activity. Many mutant alleles of phoR and phoU were found to be temperature sensitive (ts), whereas those of phoT were not. These ts mutants were constitutive at 35 C but severely repressible at 25 C. These facts strongly suggest that both the phoR and phoU genes are cooperatively concerned with the production of the repressor, whereas the phoT gene might be involved in another mechanism distinct from that in which phoR and phoU are involved. No single mutation of phoR, phoT, or phoU result in an enzyme level comparable to that of fully derepressed enzyme activities, and the temperature sensitivity of the ts phoR and ts phoU mutations in such combinations almost disappeared. In addition to these observations, since the ts phoR phoS and ts phoU phoS double mutants showed some enzyme synthesis at 25 C under derepressing conditions, a defect in the ts mutant repressors was strongly suggested, even at 25 C.

Genetic mapping of the phoR regulator gene of alkaline phosphatase in Escherichia coli.

SUMMARY: Genetic mapping of the structural phoA and the linked regulatory phoR genes for alkaline phosphatase synthesis in Escherichia coli was carried out by conjugation. Distal markers were selected and the segregation of proximal markers was determined. The gene order lac-phoA-phoR-tsx is proposed.