Regulatory interactions between the Pho and {sigma}B-dependent general stress regulons of Bacillus subtilis

Zoltán Prágai1 and Colin R. Harwood1

Department of Microbiology and Immunology, The Medical School, University of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne NE2 4HH, UK1

Author for correspondence: Colin R. Harwood. Tel: +44 191 222 7708. Fax: +44 191 222 7736. e-mail: Colin.Harwood{at}ncl.ac.uk


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
When Bacillus subtilis is subjected to phosphate starvation, the Pho and {sigma}B-dependent general stress regulons are activated to elicit, respectively, specific and non-specific responses to this nutrient-limitation stress. A set of isogenic mutants, with a ß-galactosidase reporter gene transcriptionally fused to the inactivated target gene, was used to identify genes of unknown function that are induced or repressed under phosphate limitation. Nine phosphate-starvation-induced (psi) genes were identified: yhaX, yhbH, ykoL and yttP were regulated by the PhoP–PhoR two-component system responsible for controlling the expression of genes in the Pho regulon, while ywmG (renamed csbD), yheK, ykzA, ysnF and yvgO were dependent on the alternative sigma factor {sigma}B, which controls the expression of the general stress genes. Genes yhaX and yhbH are unique members of the Pho regulon, since they are phosphate-starvation induced via PhoP–PhoR from a sporulation-specific {sigma}E promoter or a promoter that requires the product of a {sigma}E-dependent gene. Null mutations in key regulatory genes phoR and sigB showed that the Pho and {sigma}B-dependent general stress regulons of Bacillus subtilis interact to modulate the levels at which each are activated.

Keywords: stress response, psi genes, phosphate starvation, regulatory networks

Abbreviations: APase, alkaline phosphatase; BFA, Bacillus subtilis functional analysis; HPM, high-phosphate medium; LPM, low-phosphate medium; {sigma}B-GS, {sigma}B-dependent general stress


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
During phosphate-starvation stress, Bacillus subtilis responds by activating the Pho and {sigma}B-dependent general stress ({sigma}B-GS) regulons (Hulett, 1996 ; Hecker & Völker, 1998 ; Antelmann et al., 2000 ; Lahooti et al., 2000 ; Prágai et al., 2001 ). Induction or repression of genes of the Pho regulon enables the cell either to transport and use limiting phosphate resources more efficiently or to increase the accessibility of alternative phosphate sources. Genes of the Pho regulon that are induced in response to phosphate starvation include the following: phoA and phoB, encoding alkaline phosphatases (APases) (Bookstein et al., 1990 ; Hulett et al., 1990 ); phoD, encoding a phosphodiesterase/APase, which has a putative role in cell-wall teichoic acid turnover (Eder et al., 1996 ); the pstSACBABB operon, encoding a high-affinity phosphate transporter (Qi et al., 1997 ); glpQ, encoding a glycerophosphoryl diester phosphodiesterase involved in the hydrolysis of deacylated phospholipids (Antelmann et al., 2000 ); the phoPR and resABCDE operons, encoding two-component signal-transduction systems PhoP–PhoR and ResD–ResE (Hulett et al., 1994 ; Hulett, 1996 ; Nakano et al., 2000 ); and two genes, ydhF and ykoL, of unknown function (Antelmann et al., 2000 ; Robichon et al., 2000 ). In addition, the expression of the tagAB and tagDEF operons, involved in the synthesis of a phosphate-containing cell-wall polymer, polyglycerolteichoic acid, is repressed (Müller et al., 1997 ; Liu et al., 1998 ), while the expression of the tuaABCDEFGH operon, responsible for the synthesis of teichuronic acid, a non-phosphate-containing polymer that replaces teichoic acid in the wall, is induced during phosphate starvation (Müller et al., 1997 ; Liu & Hulett, 1998 ; Lahooti & Harwood, 1999 ).

Genes of the Pho regulon are controlled by the interaction of at least three two-component signal-transduction systems (Hulett, 1996 ). The centre of this regulatory network is the PhoP–PhoR sensor–regulator system (Hulett et al., 1994 ). During phosphate starvation, the PhoP response regulator is activated by its cognate sensor-kinase, PhoR. Phosphorylated PhoP (PhoP~P) is required for the activation or repression of genes in the Pho regulon and to enhance the transcription of the phoPR and resABCDE operons. The second signal-transduction system, ResD–ResE, involved in the activation of genes required for aerobic and anaerobic respiration, is required for the full induction of the Pho regulon, while the third response regulator, Spo0A, terminates the phosphate response via AbrB and ResD–ResE, and initiates sporulation if phosphate-starvation conditions persist. Additionally, in the presence of the NhaC Na+/H+ antiporter, Na+ has a repressive effect on the expression of the phoPR operon, while in the absence of this antiporter the phoPR operon is hyper-induced (Prágai et al., 2001 ).

For the activation or repression of Pho-regulon genes, PhoP~P binds to Pho-box sequences, direct repeats of TT(A/T/C)ACA with a 5±2 bp spacer (Eder et al., 1999 ). For efficient PhoP~P binding, four TT(A/T/C)ACA-like sequences with an 11 bp periodicity are required. In the case of genes induced by PhoP~P, the PhoP-binding sites are on the coding strand of the promoter region, while in the case of the tagAD promoter, which is repressed by PhoP~P, these sites are on the non-coding strand (Liu et al., 1998 ).

In addition to the phosphate-starvation-specific Pho regulon, phosphate limitation also induces genes of the {sigma}B-GS regulon (Antelmann et al., 2000 ). The {sigma}B-GS regulon provides a non-specific resistance to stress. Proteins encoded by members of the {sigma}B-GS regulon appear to protect DNA, membranes and proteins from the damage caused by oxidative stress and contribute to survival in extreme environments such as those involving heat, salt and ethanol stress (Gaidenko & Price, 1998 ; Hecker & Völker, 1998 ). The {sigma}B-mediated general stress response is one of the earliest responses to growth arrest and is induced by two independent classes of stress: (1) energy limitation arising from, for example, carbon or phosphate starvation; and (2) environmental stress such as osmotic stress and heat, salt, acid or alkaline shock (Akbar et al., 1997 ; Hecker & Völker, 1998 ). The activity of {sigma}B is controlled by anti-sigma-factor RsbW. In unstressed cells, when the antagonist protein RsbV is phosphorylated, RsbW can bind to, and inactivate, {sigma}B (Benson & Haldenwang, 1993 ). In response to energy stress or environmental stress, RsbP or RsbU PP2C phosphatases, respectively, remove the serine phosphate from RsbV~P (Voelker et al., 1996 ; Yang et al., 1996 ; Vijay et al., 2000 ), allowing it to sequestrate RsbW. As a result, {sigma}B is free to activate genes in the {sigma}B-GS regulon (Dufour & Haldenwang, 1994 ). In the case of the RsbP phosphatase, its PAS domain and RsbQ, an {alpha}/ß-hydrolase, appears to be required to sense energy stress (Vijay et al., 2000 ; Brody et al., 2001 ). In contrast, in the environmental stress pathway, the RsbU phosphatase is activated by elements higher up in the regulatory cascade and which include the RsbS antagonist, the RsbT kinase, the RsbX phosphatase, the RsbR regulator and four RsbR paralogues, namely YkoB, YojH, YqhA and YtvA (Yang et al., 1996 ; Akbar et al., 1997 ; Voelker et al., 1997 ; Akbar et al., 2001 ).

In this paper, we report the results of a systematic search for genes of unknown function that respond to phosphate starvation. The identified genes were assigned to either the Pho or the {sigma}B-GS regulon by monitoring their expression in phoR-null or sigB-null mutants, respectively. We also investigated the effects of {sigma}B on the expression of the Pho regulon and of PhoP–PhoR on the {sigma}B-GS regulon. The data indicate that the Pho and {sigma}B-GS regulons are interacting members of the phosphate stimulon.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains, plasmids and media.
Bacterial strains and plasmids are listed in Table 1. Strains were grown in low-phosphate medium (LPM) or high-phosphate medium (HPM) (Prágai & Harwood, 2000a ). The starting concentration of phosphate was 0·42 mM in LPM and 5·0 mM in HPM. LPA and HPA are LPM and HPM, respectively, solidified with 1·5% agar. When required, the concentrations of antibiotics were as follows: ampicillin, 50 µg ml-1 (for Escherichia coli) and chloramphenicol, 6 µg ml-1; erythromycin 0·3 µg ml-1; kanamycin 10 µg ml-1; lincomycin 25 µg ml-1; neomycin 7 µg ml-1; tetracycline 12·5 µg ml-1 (for B. subtilis). X-Gal was used at 100 µg ml-1 and IPTG at 1 mM.


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Table 1. Bacterial strains and plasmids

 
DNA manipulation and general methods.
Plasmid and chromosomal DNA extraction, restriction endonuclease digestion, agarose gel electrophoresis, transformation of competent B. subtilis cells, PCR reactions and bioinformatical analyses were carried out as described previously (Prágai et al., 1997 , 2001 ). Enzymes, molecular size markers and deoxynucleotides were purchased from Roche Diagnostics or Amersham Pharmacia Biotech.

Construction of plasmids.
Primers YDCE-FOR and SIGB-REV (Table 2) were used for PCR amplification of a 4067 bp fragment of the ydcEsigB region, and primers RSBU-FOR and SIGB-REV were used to amplify a 1677 bp fragment of the rsbUsigB region. Primers YVFR-FOR and RSBP-REV were used for PCR amplification of a 1673 bp fragment of the yvfRrsbP region, and primers PRS-FOR and CTC-REV were used to amplify a 1205 bp fragment of the prsctc region. The PCR reactions were carried out with Pfu DNA polymerase using chromosomal DNA of B. subtilis 168 as template. After BamHI and EcoRI digestion, the PCR fragments were ligated into a BamHI- and EcoRI-digested pBgaB integrational vector (Mogk et al., 1996 ) and transformed into electrocompetent cells of E. coli XL-1 Blue (Stratagene). Transformants were selected on Luria–Bertani agar medium supplemented with ampicillin. The resulting plasmids, pZP131, pZP132, pZP133 and pZP139 (see Fig. 5), were confirmed by restriction digestion and PCR using the insert-specific primers (Table 2). pZP134 was constructed by releasing a fragment containing the 3'-end of rsbR through to the 5'-end of sigB (see Fig. 5b) from pZP131 with EcoRI digestion.


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Table 2. Primers

 


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Fig. 5. Integrated plasmids used for monitoring the expression of the ctc and rsbR genes, and the sigB and rsbP operons. Schematic representation of inserts cloned into plasmid pBgaB, which carries a bgaB transcriptional reporter gene encoding a heat-stable ß-galactosidase from Bacillus stearothermophilus (Mogk et al., 1996 ). Filled arrows indicate structural genes, promoters are marked with fine arrows, and putative Rho-independent terminators are shown as stem–loop structures. Inserts are shown with thick lines under the chromosomal regions. Only relevant restriction enzyme sites are indicated. (b) ‘PA’ is the {sigma}A promoter and ‘PB’ is the {sigma}B promoter of the sigB operon. pZP139 (a), pZP131, pZP132, pZP134 (b), and pZP133 (c) were integrated into the amyE locus of B. subtilis strains 168 and 168-PR.

 
Construction of strains.
To analyse gene expression in a phoR-null strain, B. subtilis 168-PR (Prágai et al., 2001 ) was transformed with chromosomal DNA from the appropriate B. subtilis functional analysis (BFA) mutants, selecting for Emr, Lmr and Tcr. In these double mutants, phoR was inactivated by the replacement of an internal BalI fragment with a Tcr gene (Hulett et al., 1994 ). For monitoring gene expression in a sigB-deletion strain, ML6 (Igo et al., 1987 ) was transformed with chromosomal DNA from the appropriate BFA mutants, selecting for Emr, Lmr and Cmr. Transcriptional studies of phoA, phoB and phoP were performed in strains ML6K and 168. ML6K was constructed by introducing a Kmr insertion into the Cmr gene of strain ML6 using ScaI-linearized plasmid pCm::Nm (Steinmetz & Richter, 1994 ). The insertion of the Kmr marker was verified by PCR using sigB (SB-FOR and SB-REV) and cat (CAT-FOR and CAT-REV) specific primers (Table 2). The fusions phoAlacZ, phoBlacZ or phoPlacZ were integrated into the amyE locus by transforming strain ML6K with the chromosomal DNA from 168-A, 168-B and 168-P (Prágai et al., 2001 ) and selecting for Kmr Cmr amyE-null transformants. The lacZ fusions were verified by PCR as described previously (Prágai et al., 2001 ). Transcriptional studies of ctc, sigB, rsbP and rsbR were performed in strains 168 and 168-PR. The fusions sigBbgaB, rsbPbgaB, rsbRbgaB or ctcbgaB were integrated into the amyE locus by transforming strains 168 and 168-PR with ScaI-linearized plasmids pZP131, pZP132, pZP133, pZP134 and pZP139, selecting for Nmr amyE-null transformants. The resulting strains were PZ131 and PZ131-PR, PZ132 and PZ132-PR, PZ133 and PZ133-PR, PZ134 and PZ134-PR, and PZ139 and PZ139-PR (Table 1).

Enzyme assays.
Overnight cultures grown in HPM were diluted 200-fold in fresh LPM and HPM medium and grown at 37 °C with shaking at 200 r.p.m. Samples were collected for the determination of optical density at 600 nm, APase activity (40 µl whole culture) and ß-galactosidase activity (cell pellet from 0·1–1 ml of culture). APase and ß-galactosidase samples were stored at -20 °C until required.

For the APase assay, samples and a reagent blank (40 µl LPM) were thawed, 400 µl 1 M Tris/HCl (pH 8·0) containing lysozyme (200 µg ml-1), DNase I (20 µg ml-1), ribonuclease A (20 µg ml-1), chloramphenicol (100 µg ml-1) and 0·0005% SDS were added and incubated for 10 min at 30 °C. To each lysed sample, 300 µl pre-warmed p-nitrophenyl phosphate (1 mg ml-1 in 1 M Tris/HCl, pH 8·0) was added and the mixture incubated at 30 °C for between 5 and 60 min. When the A410 value was ~0·2–0·3, the assay was stopped by the addition of 300 µl 2 M NaOH. The samples were centrifuged for 5 min and the absorbance was measured at 410 nm. Specific APase activity was calculated with the following formula (Nicholson and Setlow, 1990 ): A410x235xV1/ (V2xOD600xT) and expressed as nmol p-nitrophenol min-1 (OD600 unit)-1. In the formula, V1 is the final volume (in ml) of the assay (1·04 ml), V2 is the volume (in ml) of the culture used in the assay (0·04 ml), and T is the reaction time (in min). The value 235 is a constant for the calculation of the p-nitrophenol concentration in nmoles.

The ß-galactosidase assay, described by Miller (1972) , was modified as follows: the frozen pellets were suspended in 800 µl Z buffer (0·06 M Na2HPO4, 0·04 M NaH2PO4, 0·01 M KCl and 0·001 M MgSO4) containing lysozyme (200 µg ml-1), DNase I (20 µg ml-1), ribonuclease A (20 µg ml-1), chloramphenicol (100 µg ml-1) and 0·00025% SDS and incubated for 10 min at 28 °C. Then, 200 µl pre-warmed 2-nitrophenyl ß-D-galactopyranoside (4 mg ml-1 in 0·1 M sodium phosphate buffer, pH 7·5) was added and the mixture incubated at 28 °C for between 5 and 60 min in the case of lacZ and at 55 °C in the case of bgaB (Mogk et al., 1996 ). The assay was stopped by the addition of 0·5 ml 1 M Na2CO3. The samples were centrifuged for 5 min and the absorbance was measured at 420 nm. Specific ß-galactosidase activity was calculated with the following formula: A420xV1/(V2xOD600xTx0·00486) and expressed as nmol 2-nitrophenol min-1 (OD600 unit)-1. In the formula, V1 is the final volume (in ml) of the assay (1·5 ml), V2 is the volume (in ml) of the culture used in the assay (0·1–1 ml), and T is the reaction time (in min). The molar absorption coefficient of 2-nitrophenyl is 4860 M-1 cm-1 at pH 10.


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Screening for genes affected by phosphate starvation
Within the framework of the BFA project (Harwood & Wipat, 1996 ), BFA mutants were constructed by integrating pMUTIN (Vagner et al., 1998 ) into genes of unknown function. These mutants were used to identify new phosphate-starvation-induced (psi) and -repressed (psr) genes (Prágai & Harwood, 2000a ), using the spoVGlacZ transcriptional reporter gene of the integrated pMUTIN. BFA mutants were tested on both LPA and HPA supplemented with X-Gal. Since growth was stronger on HPA, mutants were incubated for 24 h on HPA and for 36 h on LPA before ß-galactosidase activities were compared. Mutants carrying psilacZ gene fusions should give blue colonies on LPA and pale blue or white colonies on HPA. Conversely, mutants carrying psrlacZ gene fusions should give blue colonies on HPA and pale blue or white colonies on LPA. Genes which are not affected by the concentration of phosphate should show similar levels of expression in both LPA and HPA. Nine of the 1147 BFA mutants exhibited high ß-galactosidase activity on LPA and little or no ß-galactosidase activity on HPA. These psi genes were ywmG (renamed csbD), yhaX, yhbH, yheK, ykoL, ykzA, ysnF, yttP and yvgO (Kunst et al., 1997 ). No psr genes were detected with this screening system.

Transcriptional activities of psi genes during phosphate starvation
To determine the influence of phosphate on the transcriptional activity of psi genes, ß-galactosidase production by the psi mutants was monitored in LPM and HPM (Fig. 1). The growth kinetics of all the mutants was similar; transition from exponential to stationary phase in LPM was at an OD600 of ~1·1, while in HPM the OD600 was ~3·5. In both LPM and HPM, psi genes showed little or no expression during exponential phase (Fig. 1). In LPM, csbD, ykoL, ykzA, yheK, ysnF, yttP and yvgO were induced at T-1T0 (T-1 is 1 h before transition from the exponential to the stationary phase) (Fig. 1a), which coincided with the point at which APase production was induced (data not shown). The induced specific ß-galactosidase activity of ykoL was markedly higher than that of the other genes. ykzA showed constitutive low-level production of ß-galactosidase in HPM, and in LPM during the exponential phase (Fig. 1). yttP was induced in HPM at T-1T0, but the specific ß-galactosidase activity was at least 10-fold lower in HPM than in LPM (Fig. 1). The other psi genes showed no ß-galactosidase production in HPM (Fig. 1b). The induction of a second group of psi genes, yhaX and yhbH, in LPM was delayed until T2T3 (Fig. 1a). These genes were not expressed in HPM (Fig. 1b).



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Fig. 1. Transcriptional activities of psi genes in LPM (a) and HPM (b). Specific ß-galactosidase activities of yhaX ({blacktriangleup}), yhbH ({bullet}), ykoL ({blacksquare}), yttP ({diamondsuit}), csbD ({triangledown}), yheK ({triangleup}), ykzA ({square}), ysnF ({circ}), yvgO ({lozenge}). (a) The outer abscissa with a higher scale corresponds to the expression of ykoL ({blacksquare}, dashed line). ONP, 2-nitrophenol.

 
Effect of a phoR-null mutation and a sigB-null mutation on the identified psi genes
To determine whether the response of psi genes to phosphate starvation was mediated by the PhoP–PhoR two-component signal-transduction system, phoR-null mutant 168-PR (Prágai et al., 2001 ) was transformed with chromosomal DNA from the BFA psi mutants, selecting for Emr, Lmr and Tcr. When the resulting double mutants (Table 1) were grown in LPM, three of the nine psi genes, yhaX, yhbH and ykoL, were unable to respond to phosphate starvation (Fig. 2a). In addition, the induction of yttP in response to phosphate starvation was reduced fourfold in this background. In contrast, the expression of the remaining five genes, csbD, ykzA, ysnF, yvgO and yheK, was hyper-induced five- to sevenfold in the phoR-null strain (Fig. 2a).



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Fig. 2. Transcriptional activities of psi genes in phoR-null (a) and sigB-null (b) mutants. The mutants were grown in LPM. The specific ß-galactosidase activities of yhaX ({blacktriangleup}), yhbH ({bullet}), ykoL ({blacksquare}), yttP ({diamondsuit}), csbD ({triangledown}), yheK ({triangleup}), ykzA ({square}), ysnF ({circ}) and yvgO ({lozenge}) are shown. The outer abscissa with a higher scale corresponds to the expression of psi genes (dashed lines). The inner abscissa corresponds to the expression of psi genes (continuous lines).

 
To determine whether the response of any of the psi genes to phosphate starvation was mediated by {sigma}B, sigB-null mutant ML6 (Igo et al., 1987 ) was transformed with chromosomal DNA from the psi BFA mutants. When the double mutants were grown in LPM, five of the nine psi genes, csbD, ykzA, ysnF, yvgO and yheK, were unable to respond to the growth arrest associated with phosphate starvation (Fig. 2b). These were the same genes that were hyper-induced in the absence of PhoR. Three of the four phoR-dependent genes (yhaX, yhbH and yttP) were hyper-induced two- to fourfold in the sigB-null strain, while the remaining gene, ykoL, was unaffected by this mutation (Fig. 2b).

To confirm that the {sigma}B- and PhoR-dependent responses to phosphate starvation were specific, we determined the influence of these regulatory proteins on the expression of a gene, ysxC, that is expressed in both exponential and stationary phase (Prágai & Harwood, 2000b ). The data (Fig. 3) show that the pattern of expression of ysxC is similar in LPM and HPM, and that the absence of PhoR and SigB did not lead to the hyper-induction of this gene.



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Fig. 3. Transcriptional activities of ysxC in LPM ({blacksquare}), in HPM ({diamondsuit}) and in phoR-null ({blacktriangleup}) and sigB-null ({bullet}) mutants grown in LPM.

 
Effect of a sigB-null mutation on transcription of phoA, phoB and phoPR
Our results (Fig. 2b) indicate that {sigma}B influences the expression of three of the four phoR-dependent psi genes. We therefore investigated whether {sigma}B influenced the expression of known members of the Pho regulon and, if so, whether this effect was mediated via PhoP–PhoR.

The genes encoding APaseA and APaseB, responsible for 98% of the APase activity synthesized in response to phosphate starvation, are well-characterized members of the Pho regulon (Hulett et al., 1994 ). To study the effect of {sigma}B on the transcription of these APase genes, phoAlacZ or phoBlacZ transcriptional fusions from strains 168-A or 168-B (Prágai et al., 2001 ), respectively, were integrated at the amyE locus of strain ML6K, resulting in strains ML6K-A and ML6K-B. ML6K was ML6 in which the Cmr gene used to inactivate sigB was itself disrupted by the insertion of a Kmr gene. When strains ML6K-A, ML6K-B, 168-A and 168-B were grown in LPM (Fig. 4a), phoA and phoB expression (Fig. 4a) and APase synthesis (data not shown) were induced as the cells entered stationary phase. At all subsequent time points, the transcriptional activity of phoA and phoB and APase production was approximately twofold higher in the sigB-null mutant, confirming the results obtained with three of the phoR-dependent psi mutants.



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Fig. 4. Influence of a sigB-null mutation on APase production and the transcription of phoA, phoB and the phoPR operon. (a) Specific ß-galactosidase activities for strains 168-A (phoAlacZ) ({diamondsuit}), 168-B (phoBlacZ) ({blacksquare}), ML6K-A (phoAlacZ sigB) ({lozenge}) and ML6K-B (phoBlacZ sigB) ({square}) grown in LPM medium. (b) APase production (open symbols) and specific ß-galactosidase activities (closed symbols) for strains 168-P (phoPlacZ) ({diamondsuit},{lozenge}), ML6K-P (phoPlacZ, sigB) ({blacksquare},{square}) grown in LPM. PNP, p-nitrophenol.

 
Since all of the tested Pho-regulon genes were hyper-induced in the sigB-null background, we analysed the influence of {sigma}B on the transcription of the phoPR operon. A phoPlacZ transcriptional fusion, derived from strain 168-P (Prágai et al., 2001 ), was integrated at the amyE locus of strain ML6K, resulting in strain ML6K-P. When ML6K-P and 168-P were grown in LPM (Fig. 4b), both strains showed a constitutive, low level of phoPR expression during exponential growth phase that was induced as the cells became phosphate-starved (T0). In the absence of {sigma}B (ML6K-P), the expression of phoPR was about threefold higher than in its presence (168-P).

Effect of a phoR-null mutation on transcription of ctc, sigB, rsbP and rsbR
Since {sigma}B modulates the activity of the Pho regulon, we investigated whether PhoP–PhoR could influence members of the {sigma}B-GS regulon and the expression of sigB itself. Transcriptional fusions were constructed for ctc, a well-characterized {sigma}B-dependent gene, sigB, rsbP and rsbR.

Plasmids pZP131, pZP132, pZP133, pZP134 and pZP139 (Fig. 5, Table 1) were integrated independently into the chromosomes of strains 168 and 168-PR to generate isogenic pairs PZ131/PZ131-PR, PZ132/PZ132-PR, PZ133/PZ133-PR, PZ134/PZ134-PR and PZ139/PZ139-PR, respectively (Table 1).

PZ139 and PZ139-PR, encoding the ctcbgaB reporter fusion, were used to confirm the influence of phoR on the activity of the {sigma}B-GS regulon. The data show that ctc is induced at T0 in response to phosphate starvation (Fig. 6). In the phoR-null mutant (PZ139-PR), the induction level was approximately fourfold higher than that of the wild-type, in agreement with the data from the newly identified members of the {sigma}B-GS regulon (Fig. 2a).



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Fig. 6. Influence of a phoR-null mutation on the transcription of ctc, sigB and rsbR. Specific ß-galactosidase activities are shown for strains PZ131 (sigBbgaB) ({diamondsuit}), PZ131-PR (sigBbgaB phoR) ({lozenge}), PZ132 (sigBbgaB) ({blacksquare}), PZ132-PR (sigBbgaB phoR) ({square}), PZ134 (rsbRbgaB) ({bullet}), PZ134-PR (rsbRbgaB phoR) ({circ}), PZ139 (ctcbgaB) ({blacktriangleup}) and PZ139-PR (ctcbgaB phoR) ({triangleup}) grown in LPM medium. The outer abscissa corresponds to the expression of the ctc gene (dashed lines).

 
PZ131 and PZ131-PR encode a sigBbgaB reporter fusion located downstream of both the {sigma}A promoter and the {sigma}B promoter. sigB was induced at T0 in response to phosphate starvation, and at T5 the induction level in the phoR-null mutant was more than twice that of the wild-type, and the initial rate of induction was approximately 10-fold higher (Fig. 6). To determine whether the hyper-induction of sigB was due to {sigma}A- or {sigma}B-directed transcription, ß-galactosidase activities were determined in fusion strains in which the activities of these promoters could be determined independently. PZ134 and PZ134-PR encode an rbsRbgaB reporter fusion downstream of the {sigma}A promoter. When grown in LPM, both mutants showed an identical, very low, constitutive level of expression, confirming that the {sigma}A promoter at the 5'-end of the rsbRrsbX operon is not involved in the induction of sigB in response to phosphate starvation. PZ132 and PZ132-PR, encoding a sigBbgaB reporter fusion downstream of the internal {sigma}B promoter, exhibited a similar response to that of PZ131/PZ131-PR, except that the levels of induction were enhanced about twofold (Fig. 6). These data confirm that the {sigma}B promoter upstream of sigB is responsible for the induction of this gene in response to phosphate starvation, and that this response was enhanced in the phoR-null background. The enhanced transcription of sigB in PZ132/PZ132-PR as compared with PZ131/PZ131-PR presumably reflects the fact that the former has only single copies of the rsbR, rsbS, rsbT and rsbU genes.

Finally, the expression of rsbP, a phosphatase responsible for sensing and signalling energy stress, was studied in PZ133 and PZ133-PR. The expression of {sigma}A-dependent rsbP was very low and was unaffected by the presence of the phoR-null mutation (data not shown).


   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In this paper, we have identified nine B. subtilis genes of unknown function that are induced in response to phosphate starvation. These genes are therefore members of the phosphate-stress stimulon. We used a combination of reporter-gene technology and phoR- and sigB-null mutants to assign these genes into specific regulons; yhaX, yhbH, ykoL and yttP are members of the Pho regulon, while yheK, ykzA, ysnF, yvgO and ywmG (now csbD) are members of the {sigma}B-GS regulon. No phosphate-starvation-repressed genes were detected.

All but two of the genes were induced during the transition from exponential phase to stationary phase (T-1T0). The exceptions were yhaX and yhbH, both of which are PhoR-dependent genes. yhbH has a predicted {sigma}E promoter (Fawcett et al., 2000 ), while analysis of the upstream region of yhaX reveals the presence of putative {sigma}E consensus sequence (5'-1055945TTCTAAA-14 bp-CATATCCT1055973-3'; coordinates according to Kunst et al., 1997 ). When we transformed their respective mutants (BFA1631 and BFA1743) with a sigE-null mutation, both lost their ability to be induced in response to phosphate starvation (data not shown). These genes are therefore unique in being phosphate-starvation-induced via PhoP–PhoR from a sporulation-specific {sigma}E promoter or a promoter that requires the product of a {sigma}E-dependent gene. One other member of the Pho regulon, namely phoB, is known to be transcribed during sporulation, but in this case from a phosphate-starvation-independent, sporulation-specific promoter (Chesnut et al., 1991 ; Birkey et al., 1994 ); phoB is also transcribed by a separate, phosphate-starvation-inducible {sigma}A promoter.

An interesting observation was the regulatory interactions between the Pho and {sigma}B-GS regulons. In a sigB-null mutant, the response of three of the four newly described members of the Pho regulon, namely yhaX, yhbH and yttP, to phosphate starvation was enhanced about two- to fourfold (Fig. 2b). Similar results were obtained for three well-established members of the Pho regulon, i.e. phoA, phoB and phoPR (Fig. 4). The influence of {sigma}B on phoPR could account for its influence on the other members of the Pho regulon. The absence of {sigma}B had no effect on the expression of the remaining Pho-regulon gene ykoL (Fig. 2b), showing that a sigB mutation does not influence the expression of all members of the Pho regulon.

The situation with members of the {sigma}B-GS regulon was comparable to that of the Pho regulon. In a phoR-null mutant, the response of all five identified members of the {sigma}B-GS regulon, namely csbD, ykzA, ysnF, yvgO and yheK, to phosphate starvation was enhanced about five-to sevenfold (Fig. 2a). Similarly, the expression of well-established members of the {sigma}B-GS regulon, ctc and sigB itself, was enhanced by between two- and fourfold (Fig. 6). The influence of PhoP–PhoR on sigB could account for its influence on the other members of the {sigma}B-GS regulon.

We do not, as yet, understand the molecular mechanisms underlying the interactions between the Pho and {sigma}B-GS regulons. These regulons provide, respectively, specific and general responses to phosphate-starvation stress and contribute to the survival under phosphate limitation. The absence of one of these regulons is likely to enhance the strength of the signal sensed by the other regulon, resulting in its elevated expression. In the case of a defective {sigma}B-GS, the absence of {sigma} B may also reduce the sigma-factor competition for core RNA polymerase, leading to a general increase in the transcription of genes controlled by other sigma factors (such as {sigma}A and {sigma}E). However, analysis of the expression of a constitutive gene, ysxC, showed no evidence for sigma-factor competition (Fig. 3).


   ACKNOWLEDGEMENTS
 
We thank H. Antelmann for the gift of strain ML6, W. Schumann for pBgaB, and the Bacillus Genetic Stock Center for pCm::Nm. We are grateful to all participants of the BFA consortium for the construction of BFA mutants. BFA214 was constructed in the laboratory of P. Stragier; BFA1036 was constructed in the laboratory of S. Aymerich; BFA1631 and BFA1743 were constructed in the laboratory of S. Bron; and BFA1818 and BFA1843 were constructed in the laboratory of K. Devine. This work was funded by the European Commission (BIO4-CT95-0278 and QLG2-CT-1999-01455) and the UK Biotechnology and Biological Sciences Research Council (13/PRES/12179).


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 2 November 2001; accepted 11 January 2002.