F. Marion Hulett

The PhoP–PhoR two-component system (TCS) controls transcription of the Pho regulon in response to inorganic phosphate limitation in Bacillus subtilis. In addition to its role in phosphate homeostasis, increasing evidence suggests that central metabolic pathways influence Pho regulon activity. Here, we investigated the mechanism underlying phosphate-independent activation of the Pho regulon in a ccpA mutant lacking the global regulator of CCR. Gene expression analyses demonstrated strong glucose-dependent induction of Pho regulon genes in the absence of both CcpA and the cognate histidine kinase PhoR. Using PhosTag gel electrophoresis analysis and a phoasphoablative mutation in phoP (D53A), we detected early PhoP phosphorylation and determined that Pho regulon activation requires a phosphorylatable form of PhoP, even when the cognate histidine kinase phoR gene was deleted. Because PhoR can phosphorylate non-cognate response regulator YycF, we examined whether the essential YycFG signaling system contributes to PhoP activation. Co-immunoprecipitation and in vitro phosphorylation assays confirmed a direct interaction between PhoP and YycG and demonstrated in vitro PhoP phosphorylation by YycG. Taken together, we propose a model in which CcpA limits excessive PhoP accumulation to preserve signaling specificity and prevent aberrant activation through non-cognate kinases. Furthermore, our study reveals a previously unrecognized link between carbon catabolite repression and phosphate starvation signaling and demonstrates another crosstalk between two-component signal transduction systems in the Gram-positive bacterium, B. subtilis.

Induction of the Pho response inBacillus subtilisoccurs when the P_iconcentrations in the growth medium fall below 0.1 mM, a condition which results in slowed cellular growth followed by entry into stationary phase. ThephoPRpromoter region contains three σ^A-responsive promoters; only promoter P_A4is PhoP autoregulated. Expression of thephoPRoperon is postexponential, suggesting the possibility of a repressor role for a transition-state-regulatory protein(s). Expression of aphoPRpromoter-lacZfusion in ascoCloss-of-function mutant strain grown in low-phosphate defined medium was significantly higher than expression in the wild-type strain during exponential growth or stationary phase. Derepression in thescoCstrain from aphoPpromoter fusion containing a mutation in the CcpA binding site (cre1) was further elevated approximately 1.4-fold, indicating that the repressor effects of ScoC and CcpA onphoPexpression were cumulative. DNase I footprinting showed protection of putative binding sites by ScoC, which included the −10 and/or −35 elements of five (P_B1, P_E2, P_A3, P_A4, and P_A6) of the six promoters within thephoPRpromoter region. P_A6was expressedin vivofrom thephoP cre1promoter fusion in both wild-type andscoCstrains. Evidence for ScoC repressionin vivowas shown by primer extension for P_A4and P_A3from the wild-type promoter and for P_A4and P_E2from thephoP cre1promoter. The latter may reflect ScoC repression of sporulation that indirectly affectsphoPRtranscription. ScoC was shown to repress P_A6, P_A4, P_E2, and P_B1in vitro.

The cydABCD operon of Bacillus subtilis encodes products required for the production of cytochrome bd oxidase. Previous work has shown that one regulatory protein, YdiH (Rex), is involved in the repression of this operon. The work reported here confirms the role of Rex in the negative regulation of the cydABCD operon. Two additional regulatory proteins for the cydABCD operon were identified, namely, ResD, a response regulator involved in the regulation of respiration genes, and CcpA, the carbon catabolite regulator protein. ResD, but not ResE, was required for full expression of the cydA promoter in vivo. ResD binding to the cydA promoter between positions -58 and -107, a region which includes ResD consensus binding sequences, was not enhanced by phosphorylation. A ccpA mutant had increased expression from the full-length cydA promoter during stationary growth compared to the wild-type strain. Maximal expression in a ccpA mutant was observed from a 3'-deleted cydA promoter fusion that lacked the Rex binding region, suggesting that the effect of the two repressors, Rex and CcpA, was cumulative. CcpA binds directly to the cydA promoter, protecting the region from positions -4 to -33, which contains sequences similar to the CcpA consensus binding sequence, the cre box. CcpA binding was enhanced upon addition of glucose-6-phosphate, a putative cofactor for CcpA. Mutation of a conserved residue in the cre box reduced CcpA binding 10-fold in vitro and increased cydA expression in vivo. Thus, CcpA and ResD, along with the previously identified cydA regulator Rex (YdiH), affect the expression of the cydABCD operon. Low-level induction of the cydA promoter was observed in vivo in the absence of its regulatory proteins, Rex, CcpA, and ResD. This complex regulation suggests that the cydA promoter is tightly regulated to allow its expression only at the appropriate time and under the appropriate conditions.

The PhoPR two-component system activates or represses Pho regulon genes to overcome a phosphate deficiency. The Pho signal transduction network is comprised of three two-component systems, PhoPR, ResDE, and Spo0A. Activated PhoP is required for expression of ResDE from the resA promoter, while ResD is essential for 80% of Pho induction, establishing a positive feedback loop between these two-component systems to amplify the signal received by the Pho system. The role of ResD in the Pho response is via production of terminal oxidases. Reduced quinones inhibit PhoR autophosphorylation in vitro, and it was proposed that the expression of terminal oxidases leads to oxidation of the quinone pool, thereby relieving the inhibition. We show here that the reducing environment generated by dithiothreitol (DTT) in vivo inhibited Pho induction in a PhoR-dependent manner, which is in agreement with our previous in vitro data. A strain containing a PhoR variant, PhoR _C303A , exhibited reduced Pho induction and remained sensitive to inhibition by DTT, suggesting that the mechanisms for Pho reduction via PhoR _C303A and DTT are different. PhoR and PhoR _C303A were similar with regard to cellular concentration, limited proteolysis patterns, rate of autophosphorylation, stability of PhoR∼P, and inhibition of autophosphorylation by DTT. Phosphotransfer between PhoR∼P or PhoR _C303A ∼P and PhoP occurred rapidly; most label from PhoR∼P was transferred to PhoP, but only 10% of the label from PhoR _C303A ∼P was associated with PhoP, while 90% was released as inorganic phosphate. No difference in PhoP∼P or PhoR autophosphatase activity was observed between PhoR and PhoR _C303A that would explain the release of inorganic phosphate. Our data are consistent with a role for PhoR _C303 in PhoR activity via stabilization of the phosphoryl-protein intermediate(s) during phosphotransfer from PhoR∼P to PhoP, which is stabilization that is required for efficient production of PhoP∼P.

The Bacillus subtilis PhoPR two-component system is directly responsible for activation or repression of Pho regulon genes in response to phosphate deprivation. The response regulator, PhoP, and the histidine kinase, PhoR, are encoded in a single operon with a complex promoter region that contains five known transcription start sites, which respond to at least two regulatory proteins. We report here the identification of another direct regulator of phoPR transcription, carbon catabolite protein A, CcpA. This regulator functions in the presence of glucose or other readily metabolized carbon sources. The maximum derepression of phoPR expression in a ccpA mutant compared to a wild-type stain was observed under excess phosphate conditions with glucose either throughout growth in a high-phosphate defined medium or in a low-phosphate defined medium during exponential growth, a growth condition when phoPR transcription is low in a wild-type strain due to the absence of autoinduction. Either HPr or Crh were sufficient to cause CcpA dependent repression of the phoPR promoter in vivo. A ptsH1 (Hpr) crh double mutant completely relieves phoPR repression during phosphate starvation but not during phosphate replete growth. In vivo and in vitro studies showed that CcpA repressed phoPR transcription by binding directly to the cre consensus sequence present in the promoter. Primer extension and in vitro transcription studies revealed that the CcpA regulation of phoPR transcription was due to repression of P _A6 , a previously unidentified promoter positioned immediately upstream of the cre box. Eσ ^A was sufficient for transcription of P _A6 , which was repressed by CcpA in vitro. These studies showed direct repression by CcpA of a newly discovered Eσ ^A -responsive phoPR promoter that required either Hpr or Crh in vivo for direct binding to the putative consensus cre sequence located between P _A6 and the five downstream promoters characterized previously.

ThephoBgene ofBacillus subtilisencodes an alkaline phosphatase (PhoB, formerly alkaline phosphatase III) that is expressed from separate promoters during phosphate deprivation in a PhoP-PhoR-dependent manner and at stage two of sporulation under phosphate-sufficient conditions independent of PhoP-PhoR. Isogenic strains containing either the completephoBpromoter or individualphoBpromoter fusions were used to assess expression from each promoter under both induction conditions. ThephoBpromoter responsible for expression during sporulation,phoB-P_S, was expressed in a wild-type strain during phosphate deprivation, but induction occurred >3 h later than induction of Pho regulon genes and the levels were approximately 50-fold lower than that observed for the PhoPR-dependent promoter,phoB-P_V. Eσ^Ewas necessary and sufficient for P_Sexpression in vitro. P_Sexpression in aphoPRmutant strain was delayed 2 to 3 h compared to the expression in a wild-type strain, suggesting that expression or activation of σ^Eis delayed in aphoPRmutant under phosphate-deficient conditions, an observation consistent with a role for PhoPR in spore development under these conditions. Phosphorylated PhoP (PhoP∼P) repressed P_Sin vitro via direct binding to the promoter, the first example of an Eσ^E-responsive promoter that is repressed by PhoP∼P. Whereas either PhoP or PhoP∼P in the presence of Eσ^Awas sufficient to stimulate transcription from thephoB-P_Vpromoter in vitro, roughly 10- and 17-fold-higher concentrations of PhoP than of PhoP∼P were required for P_Vpromoter activation and maximal promoter activity, respectively. The promoter for a second gene in the Pho regulon,ykoL, was also activated by elevated concentrations of unphosphorylated PhoP in vitro. However, because no Pho regulon gene expression was observed in vivo during P_i-replete growth and PhoP concentrations increased only threefold in vivo duringphoPRautoinduction, a role for unphosphorylated PhoP in Pho regulon activation in vivo is not likely.

The Bacillus subtilis Pho signal transduction network, which regulates the cellular response to phosphate starvation, integrates the activity of three signal transduction systems to regulate the level of the Pho response. This signal transduction network includes a positive feedback loop between the PhoP/PhoR and ResD/ResE two-component systems. Within this network, ResD is responsible for 80% of the Pho response. To date, the role of ResD in the generation of the Pho response has not been understood. Expression of two terminal oxidases requires ResD function, and expression of at least one terminal oxidase is needed for the wild-type Pho response. Previously, our investigators have shown that strains bearing mutations in resD are impaired for growth and acquire secondary mutations which compensate for the loss of the a -type terminal oxidases by allowing production of cytochrome bd . We report here that the expression of cytochrome bd in a Δ resDE background is sufficient to compensate for the loss of ResD for full Pho induction. A ctaA mutant strain, deficient in the production of heme A, has the same Pho induction phenotype as a Δ resDE strain. This demonstrates that the production of a -type terminal oxidases is the basis for the role of ResD in Pho induction. Terminal oxidases affect the redox state of the quinone pool. Reduced quinones inhibit PhoR autophosphorylation in vitro, consistent with a requirement for terminal oxidases for full Pho induction in vivo.

During aerobic respiration, Bacillus subtilis utilizes three terminal oxidases, cytochromes aa ₃ , caa ₃ , and bd . Cytochrome bd is encoded by the cydABCD operon. We report here the first identification of a regulator for the cydABCD operon, YdiH. While working with Δ resDE mutant strains, we identified colonies which contained suppressor mutations ( cmp ) which bypassed the requirement for ResD for all phenotypes not associated with cytochrome aa ₃ or caa ₃ . Mapping identified a class of Tn 10 insertions which were close to the cmp locus (Tn 10 - 2 ) and a second class (Tn 10 - 1 ) which was inserted in cydD , a gene which appears to be essential to the cmp phenotype. Sequencing of the cmp loci from four independent Δ resDE cmp isolates yielded four loss-of-function alleles of ydiH , a gene encoding a protein with homology to AT-rich DNA-binding proteins. Additionally, we determined that cytochrome bd was aberrantly expressed in the Δ resDE cmp background. Together these data led to the hypothesis that YdiH serves as a negative regulator of cydABCD expression, a hypothesis supported by both gel-shift and DNase I footprinting analyses. YdiH protected the cydA promoter region at three 22-bp repeats located in the long 5′ untranslated region (193 bp). Induction of the cydABCD operon in a Δ resDE background showed that expression of the terminal oxidase bd was responsible for the bypass phenotype observed in a Δ resDE cmp strain, indicating that cytochrome bd expression complemented the loss of cytochromes aa ₃ and caa ₃ in the Δ resDE strain.

ThephoPRoperon encodes a response regulator, PhoP, and a histidine kinase, PhoR, which activate or repress genes of theBacillus subtilisPho regulon in response to an extracellular phosphate deficiency. Induction ofphoPRupon phosphate starvation required activity of both PhoP and PhoR, suggesting autoregulation of the operon, a suggestion that is supported here by PhoP footprinting on thephoPRpromoter. Primer extension analyses, using RNA from JH642 or isogenicsigEorsigBmutants isolated at different stages of growth and/or under different growth conditions, suggested that expression of thephoPRoperon represents the sum of five promoters, each responding to a specific growth phase and environmental controls. The temporal expression of thephoPRpromoters was investigated using in vitro transcription assays with RNA polymerase holoenzyme isolated at different stages of Pho induction, from JH642 or isogenicsigEorsigBmutants. In vitro transcription studies using reconstituted Eσ^A, Eσ^B, and Eσ^Eholoenzymes identified P_A4and P_A3as Eσ^Apromoters and P_E2as an Eσ^Epromoter. Phosphorylated PhoP (PhoP∼P) enhanced transcription from each of these promoters. Eσ^Bwas sufficient for in vitro transcription of the P_B1promoter. P₅was active only in asigBmutant strain. These studies are the first to report a role for PhoP∼P in activation of promoters that also have activity in the absence of Pho regulon induction and an activation role for PhoP∼P at an Eσ^Epromoter. Information concerning P_B1and P₅creates a basis for further exploration of the regulatory coordination or overlap of the PhoPR and SigB regulons during phosphate starvation.

Bacillus subtilis PhoP is a member of the OmpR family of response regulators that activates or represses genes of the Pho regulon upon phosphorylation by PhoR in response to phosphate deficiency. Because PhoP binds DNA and is a dimer in solution independent of its phosphorylation state, phosphorylation of PhoP may optimize DNA binding or the interaction with RNA polymerase. We describe alanine scanning mutagenesis of the PhoP α loop and α helix 3 region of PhoPC (Val190 to E214) and functional analysis of the mutated proteins. Eight residues important for DNA binding were clustered between Val202 and Arg210. Using in vivo and in vitro functional analyses, we identified three classes of mutated proteins. Class I proteins (PhoP _I206A , PhoP _R210A , PhoP _L209A , and PhoP _H208A ) were phosphorylation proficient and could dimerize but could not bind DNA or activate transcription in vivo or in vitro. Class II proteins (PhoP _H205A and PhoP _V204A ) were phosphorylation proficient and could dimerize but could not bind DNA prior to phosphorylation. Members of this class had higher transcription activation in vitro than in vivo. The class III mutants, PhoP _V202A and PhoP _D203A , had a reduced rate of phosphotransfer and could dimerize but could not bind DNA or activate transcription in vivo or in vitro. Seven alanine substitutions in PhoP (PhoP _V190A , PhoP _W191A , PhoP _Y193A , PhoP _F195A , PhoP _G197A, PhoP _T199A , and PhoP _R200A ) that specifically affected transcription activation were broadly distributed throughout the transactivation loop extending from Val190 to as far toward the C terminus as Arg200. PhoP _W191A and PhoP _R200A could not activate transcription, while the other five mutant proteins showed decreased transcription activation in vivo or in vitro or both. The mutagenesis studies may indicate that PhoP has a long transactivation loop and a short α helix 3, more similar to OmpR than to PhoB of Escherichia coli .