Département de Microbiologie Fondamentale, Bâtiment de Biologie, Université de Lausanne, CH-1015 Lausanne, Switzerland
Correspondence
Kathrin Minnig
kathrin.minnig{at}hispeed.ch
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is AJ889011.
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INTRODUCTION |
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In B. subtilis W23, the chromosome region encompassing most of the genes that specify the synthesis of Rbo-P-containing WTA has been cloned, sequenced and analysed (Lazarevic et al., 2002a; Young et al., 1989
). The tar (teichoic acid ribitol) genes are organized in two divergently transcribed operons, tarDF and tarABIJKL. This organization is analogous to that of strain 168 tag (teichoic acid glycerol) genes.
Studies of tarA gene regulation in B. subtilis W23 revealed two growth-phase-dependent increases, which are driven by X and
M, two extracytoplasmic function (ECF)
factors (Minnig et al., 2003
). In strain 168,
M is involved in the response to diverse stresses, all of which disturb the cell envelope:
M is essential for growth and survival at high salt concentration (Horsburgh & Moir, 1999
), and is activated in response to ethanol, heat, acid and superoxide stress (Thackray & Moir, 2003
). Moreover,
M is induced, together with
W, by antibiotics that inhibit cell wall synthesis, e.g. vancomycin (Cao et al., 2002b
), and the bacitracin-resistance gene bcrC was shown to be under control of
M and
X (Cao & Helmann, 2002
). Cao et al. (2002a)
suggested that the signal activating
M is not the antibiotic molecule itself, but structural changes induced by the attack of the cell wall. Thus,
M is considered to be part of a network coordinating antibiosis stress response and cell envelope homeostasis (Mascher et al., 2003
). In strain W23,
M plays an important role not only in wall synthesis and homeostasis, but also in cell division (Minnig et al., 2003
). In both 168 and W23, sigM gene expression is driven by two promoters, recognized by
A and
M, respectively (Horsburgh & Moir, 1999
).
Under phosphate-limiting conditions, B. subtilis WTAs are substituted by phosphate-free teichuronic acids (TUAs) (Ellwood & Tempest, 1972). In B. subtilis 168, this process, referred to as WTATUA switch, is regulated by the PhoPR two-component system, which is activated, together with the general stress response, when the extracellular Pi concentration drops below 100 µM (Hulett, 2002
). During phosphate deprivation, PhoPR activates, among others, the tuaABCDEFGH operon specifying TUA synthesis, while repressing the expression of WTA operons tagAB and tagDEF.
In strain W23, only the biochemical aspects of the replacement of WTA by TUA have been studied so far (Cheah et al., 1982; Wright & Heckels, 1975
). However, the presence of a putative PhoP-binding region, referred to as the Pho-box, in the tarAtarD intergenic region (Fig. 2a
and Lazarevic et al., 2002a
), suggests that the WTATUA switch should also be mediated by the PhoPR system.
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METHODS |
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Transformations and construction of strains.
Competent E. coli DH5 and JM83 cells were prepared by treatment with calcium and manganese (Hanahan et al., 1991
). Transformation of B. subtilis was performed as described by Karamata & Gross (1970)
. Recombinants were selected on solid media supplemented with appropriate antibiotics [for E. coli, ampicillin (Ap) 100 µg ml1; for B. subtilis kanamycin (Km) 5 µg ml1, erythromycin (Em) 0·3 and 100 µg ml1, chloramphenicol (Cm) 5 µg ml1].
Since strain W23 can be transformed with chromosomal DNA, but not with integrative plasmids, the latter were first inserted into 168, and the DNA of the resulting strains was used to transform W23. Construction of the relevant strains is outlined in Table 1. The pDG268 plasmid (Antoniewski et al., 1990
) was used for the construction of strains bearing transcriptional lacZ fusions. To insert these reporter fusions into the amyE locus, strain 168 was transformed by linearized pDG268-derived plasmids. To obtain strain W14900, featuring a lacZ fusion at the end of the tarL gene, circular pW11146 plasmid was used to transform strain L5607, a 168-derived interstrain hybrid containing the tar gene region of W23 (Karamata et al., 1987
). Genomic DNA of the resulting strain was used to transform W23.
Sequencing of the B. subtilis W23 tarO gene.
Plasmid pV019 carrying a segment of strain W23 tuaE gene was integrated into the B. subtilis W23 chromosome by homologous recombination. SalI chromosomal fragments of a chosen Cm-resistant transformant were treated with ligase, and used to transform E. coli TP610 (Hedegaard & Danchin, 1985). The resulting plasmid pV021, recovered by Ap selection, was used as template to determine the nucleotide sequence of the tarO gene (GenBank accession no. AJ889011). Sequencing was performed at Microsynth (Balgach, Switzerland).
-Galactosidase assays.
Overnight B. subtilis cultures with different inoculum sizes were grown in PL medium (Grant, 1979), with aeration by bubbling at 35 °C. The overnight culture with an OD600 of <0·4 was diluted to an OD600 of 0·03, and divided in two tubes containing 40 ml fresh prewarmed PL medium. During 9 h growth at 37 °C with aeration, the OD600 was measured periodically, and 0·5 ml samples were withdrawn, frozen in liquid nitrogen, and stored at 80 °C. After 7 h growth, i.e. 3 or 4 h after phosphate depletion for phoPR+ or phoPR strains, respectively, sodium phosphate buffer (pH 7·0) was added to one of the parallel cultures to obtain a final Pi concentration of 5 mM.
The -galactosidase assay developed for E. coli (Miller, 1972
) was adapted to our conditions (Mauël et al., 1994
).
-Galactosidase activity is expressed in Miller units, i.e. the absorbance x1000 min1 ml1 divided by the OD600 of the culture at the sampling time.
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RESULTS |
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-Galactosidase activity profiles during phosphate limitation were measured for tarD and tarL : : lacZ fusions (Fig. 1d, e
) using strains W4840 (tarD : : lacZ) and W14900 (tarL : : lacZ). The
-galactosidase activity of these fusions decreased continuously as soon as phosphate was exhausted. After phosphate addition,
-galactosidase activity rose again for both strains.
Furthermore, the tarO gene, located in a monocistronic operon downstream of the TUA operon, was sequenced. TarO and its 168 homologue TagO, sharing 97·7 % identity, most likely catalyse the first reaction in the WTA-linkage-unit biosynthesis (Soldo et al., 2002). tarO expression was measured by quantification of the
-galactosidase activity of strain W24078 (tarO : : lacZ) during growth in PL medium (Fig. 1f
).
-Galactosidase activity of the tarO : : lacZ fusion was about 30 % higher after phosphate depletion than in exponential phase. However, tarO does not seem to be regulated by phosphate concentration, as addition of an excess of Pi in stationary phase did not influence its expression.
Thus, three different regulation patterns of tar genes can be deduced from these -galactosidase activity data: (i) tarD and tarL expression are repressed under phosphate starvation; (ii) tarA and tarB expression first decreases, then increases under phosphate starvation; and (iii) tarO does not seem to be regulated by the extracellular phosphate concentration.
PtarA-ext is controlled by the PhoPR two-component system
To dissect the regulatory events that determine the biphasic pattern of tarA expression under phosphate starvation, the activity profiles of its promoters PtarA-ext and PtarA-int were analysed (Fig. 2a). PtarA-ext overlaps the putative PhoP-binding site (Lazarevic et al., 2002a
), and may therefore be repressed under phosphate starvation. The expression data obtained by monitoring
-galactosidase activity of a PtarA-ext : : lacZ fusion (strain W4824) confirmed this hypothesis: PtarA-ext-driven
-galactosidase activity presented a steady level during exponential phase (Fig. 3a
); upon phosphate depletion, its activity decreased continuously. A sharp upregulation of PtarA-ext was observed after addition of an excess of Pi in stationary phase.
-Galactosidase assays of PtarA-ext activity in PL showed that the response to phosphate was abolished in a phoPR mutant, W24069 (Fig. 3a
). The
-galactosidase level of W24069 was higher than in the wild-type, and no repression upon phosphate depletion, or induction after phosphate addition, was detected. Thus, it appears that PtarA-ext is controlled by the PhoPR two-component system, which may indeed act on the putative Pho-box present on the fragment cloned upstream of lacZ.
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Thus, tarA expression under phosphate limitation seems to be controlled by two counteracting phenomena: PtarA-ext is repressed under phosphate starvation by means of the PhoPR two-component system, while PtarA-int is induced under phosphate starvation by means of M.
Regulation of sigM expression and M activity under phosphate limitation
The importance of M for tarA expression under phosphate limitation was confirmed by monitoring
-galactosidase activity of the tarA : : lacZ fusion in a sigM mutant strain (Fig. 4a
). Indeed, after phosphate depletion,
-galactosidase activity of the tarA : : lacZ fusion constantly decreased, the induction present in sigM+ strains being abolished. The impact of
M on tarA expression in PL (Figs 4a and 3b
) suggests that
M itself is activated by phosphate starvation. To test this hypothesis, W14963, a strain carrying a sigMyhdLK : : lacZ fusion, was cultured in PL medium. Indeed, sigM expression, as measured by
-galactosidase activity assays, increased upon phosphate depletion (Fig. 4b
). This increase was immediately stopped by the addition of excess phosphate (5 mM). To get an idea of the activation of the
M factor by an extracellular stimulus, we decided to monitor the activity of a promoter that is dependent solely on
M. Hence, the PM promoter upstream of the sigMyhdLK operon, which was shown to be regulated exclusively by
M (Horsburgh & Moir, 1999
; Thackray & Moir, 2003
), was fused to lacZ in the amyE locus, yielding strain W24076. PM-driven
-galactosidase activity was monitored by cultivation of strain W24076 (PM : : lacZ) in PL medium, and subsequent enzyme assays (Fig. 4c
). The resulting activity profile confirms the sigM expression data, i.e. the PM promoter is induced by phosphate starvation in B. subtilis W23. Thus,
M seems to be activated by a stimulus depending on phosphate depletion in the medium.
M has been studied in more detail in strain 168, but, so far, phosphate starvation has not been identified as its activator. Indeed, the PM-driven
-galactosidase activity, assayed in strain L24071, a 168-derived strain carrying the PM : : lacZ fusion, showed no induction of the PM promoter after phosphate depletion (Fig. 4c
). Therefore, the response of
M to phosphate concentration seems to be specific to strain W23.
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DISCUSSION |
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Dissection of the regulatory elements governing tarA expression by lacZ reporter fusions revealed opposite responses to phosphate concentration of its two promoters: PtarA-ext is inhibited under phosphate starvation (Fig. 3a), whereas PtarA-int is induced under the same conditions (Fig. 3b
). The sum of activities of those two promoters results in the biphasic response of tarA expression to phosphate exhaustion (Fig. 1b
).
The decrease in tarA expression following phosphate depletion was shown to be dependent on the PhoPR two-component system, which is at least partially responsible for the WTATUA switch in B. subtilis 168 (Hulett, 2002; Liu et al., 1998
). Repeats of TTHACA-like motifs, with an opposite orientation to the direction of transcription, corresponding to the B. subtilis 168 Pho-box, were identified in the W23 tar regulatory region, overlapping PtarA-ext (Lazarevic et al., 2002a
). Indeed, in a phoPR mutant, this promoter no longer responds to phosphate concentration (Fig. 3a
), indicating that PhoP binding could be responsible for PtarA-ext repression upon phosphate exhaustion.
In rich medium, PtarA-int is recognized by two ECF factors,
X and
M (Minnig et al., 2003
). Therefore, we investigated the influence of those two ECF
factors on PtarA-int-driven
-galactosidase activity in PL medium. Indeed, inactivation of
M abolished the activity increase that took place after phosphate depletion (Fig. 3b
), whereas inactivation of
X did not have any effect (data not shown). Different activation factors have been identified for
M, all of them disturbing the cell envelope (Cao et al., 2002a
; Horsburgh & Moir, 1999
; Thackray & Moir, 2003
). By monitoring sigM expression and PM promoter activity, we were able to show that in B. subtilis W23, but not in strain 168,
M is also induced under phosphate starvation (Fig. 4c
). The underlying mechanism could consist of either (i) the direct perception of low phosphate concentration by its regulators YhdL and YhdK, or (ii) the regulation of
M by another, higher-level regulatory system activated by low phosphate concentration. At least two important regulatory systems have been shown to be directly induced by phosphate starvation in B. subtilis 168: the PhoPR two-component system and the general stress response
factor
B. Consensus sequences of the PhoP-binding site (Hulett, 2002
) and the
B-dependent promoter (Helmann & Moran, 2002
) have been defined. Inspection of the sequence upstream of the B. subtilis W23 sigMyhdLK operon (Minnig et al., 2003
) revealed neither a putative Pho-box nor a
B-controlled promoter. Moreover, no evidence of a
B-mediated control of sigM expression was reported (Horsburgh & Moir, 1999
; Price et al., 2001
; Thackray & Moir, 2003
). Therefore, dependency of sigM expression on
B has not been investigated here.
Even though inspection of the sequence upstream of sigM did not reveal a putative Pho-box, induction of sigM expression under phosphate starvation depends on the PhoPR two-component system. The influence of PhoPR on M thus seems to be indirect. Globally, a phoPR mutant lost its capacity to adapt to phosphate starvation, e.g. the WTA are not replaced by TUA and no growth in phosphate depleted medium can be observed. The WTATUA switch, which brings about a transient impairment of the wall, is probably sensed by the bacterium as stress. In the phoPR mutant, this cell-wall stress does not happen, and
M remains inactive. However, in the wild-type cell,
M would be induced by the cell-wall impairment caused by the WTATUA switch. This hypothesis is consistent with previous studies showing that
M is induced by different kinds of stresses, all of them disturbing the cell envelope, i.e. high salt concentration, heat, ethanol, superoxide stress and cell-wall-targeted antibiotics (Horsburgh & Moir, 1999
; Cao & Helmann, 2002
; Cao et al., 2002a
; Thackray & Moir, 2003
).
As shown in this contribution, M seems to play a role in the adaptation to phosphate-depleted conditions in B. subtilis W23. However, the exact role of
M, and the advantage brought about by the induction of
M and its regulon under phosphate starvation, remain unknown. For example, cultures of sigM mutant strains in PL medium did not reveal any growth defect, which was reported to be characteristic for several 168-derived mutants affected in WTA biosynthesis (Lazarevic et al., 2002b
). Moreover, in B. subtilis 168,
M was not induced under phosphate starvation (Fig. 4c
), and this strain is able to adapt well to phosphate starvation.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 10 March 2005;
revised 20 May 2005;
accepted 25 May 2005.
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