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
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ABSTRACT |
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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; B-GS,
B-dependent general stress
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INTRODUCTION |
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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 PhoPPhoR sensorregulator 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, ResDResE, 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 ResDResE, 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, PhoPP 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 B-GS regulon (Antelmann et al., 2000
). The
B-GS regulon provides a non-specific resistance to stress. Proteins encoded by members of the
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
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
B is controlled by anti-sigma-factor RsbW. In unstressed cells, when the antagonist protein RsbV is phosphorylated, RsbW can bind to, and inactivate,
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,
B is free to activate genes in the
B-GS regulon (Dufour & Haldenwang, 1994
). In the case of the RsbP phosphatase, its PAS domain and RsbQ, an
/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 B-GS regulon by monitoring their expression in phoR-null or sigB-null mutants, respectively. We also investigated the effects of
B on the expression of the Pho regulon and of PhoPPhoR on the
B-GS regulon. The data indicate that the Pho and
B-GS regulons are interacting members of the phosphate stimulon.
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METHODS |
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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 LuriaBertani 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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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 0·11 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 0·20·3, 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 (0·11 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.
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RESULTS |
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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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To confirm that the 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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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
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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Effect of a phoR-null mutation on transcription of ctc, sigB, rsbP and rsbR
Since B modulates the activity of the Pho regulon, we investigated whether PhoPPhoR could influence members of the
B-GS regulon and the expression of sigB itself. Transcriptional fusions were constructed for ctc, a well-characterized
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 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
B-GS regulon (Fig. 2a
).
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Finally, the expression of rsbP, a phosphatase responsible for sensing and signalling energy stress, was studied in PZ133 and PZ133-PR. The expression of A-dependent rsbP was very low and was unaffected by the presence of the phoR-null mutation (data not shown).
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DISCUSSION |
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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 E promoter (Fawcett et al., 2000
), while analysis of the upstream region of yhaX reveals the presence of putative
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 PhoPPhoR from a sporulation-specific
E promoter or a promoter that requires the product of a
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
A promoter.
An interesting observation was the regulatory interactions between the Pho and 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
B on phoPR could account for its influence on the other members of the Pho regulon. The absence of
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 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
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
B-GS regulon, ctc and sigB itself, was enhanced by between two- and fourfold (Fig. 6
). The influence of PhoPPhoR on sigB could account for its influence on the other members of the
B-GS regulon.
We do not, as yet, understand the molecular mechanisms underlying the interactions between the Pho and 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
B-GS, the absence of
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
A and
E). However, analysis of the expression of a constitutive gene, ysxC, showed no evidence for sigma-factor competition (Fig. 3
).
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ACKNOWLEDGEMENTS |
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Received 2 November 2001;
accepted 11 January 2002.