The essential two-component regulatory system encoded by yycF and yycG modulates expression of the ftsAZ operon in Bacillus subtilis
Keisuke Fukuchi1,
Yasuhiro Kasahara1,
Kei Asai1,
Kazuo Kobayashi1,
Shigeki Moriya1 and
Naotake Ogasawara1
Department of Cell Biology, Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5, Takayama, Ikoma, Nara 630-0101, Japan1
Author for correspondence: Naotake Ogasawara. Tel: +81 743 72 5430. Fax: +81 743 72 5439. e-mail: nogasawa{at}bs.aist-nara.ac.jp
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ABSTRACT
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Essential two-component systems are now being identified in bacteria. The Bacillus subtilis yycF gene encoding a response regulator, and its orthologue in Staphylococcus aureus, were reported recently to be essential for cell growth, although genes under their control have yet to be identified. The essential nature of the yycF regulator gene and its cognate kinase gene, yycG, in B. subtilis was also noted during the course of construction of a knockout mutant bank of newly identified genes in the genome sequence project. It was found that yycG could be deleted in the presence of an active form of the YycF protein, thereby suggesting direct interaction between YycG and YycF. Production of mini-cells and reduction in cell length occurred when the YycF regulator was overproduced in B. subtilis. These observations led to the finding that YycF overproduction up-regulated the expression from the P1 promoter of the cell division operon, ftsAZ. In addition, the YycF protein binds to the P1 promoter region in vitro. These results clearly indicate that the essential two-component regulatory system encoded by yycF and yycG genes has the potential to modulate expression of the ftsAZ operon in B. subtilis.
Keywords: YycFG esssential two-component system, Bacillus subtilis, two-component regulatory system, ftsAZ gene
Abbreviations: DAPI, 4',6-diamidino-2-phenylindole;; DIG, digoxigenin
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INTRODUCTION
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The two-component signal-transduction system is a ubiquitous mechanism for sensing and responding to various environmental stimuli in bacteria (Parkinson & Kofoid, 1992
). This system also plays an important role in lower eukaryotes and plants (Loomis et al., 1998
). A typical two-component system consists of two protein components: sensor kinase and response regulator. The sensor kinases monitor environmental signals, and modulate functions of response regulators through phosphotransfer reactions. In many cases, the response regulators modulate gene expression directly as a transcriptional repressor or activator. The two-component system can mediate diverse cellular processes, including adaptation to osmolarity, the redox state, turgor pressure, availability of nutrients, etc. The system enables genetic networks to adapt and survive in specific environmental conditions and is dispensable for growth under usual conditions of culture. However, essential two-component systems are now being identified in bacteria. In Caulobacter crescentus, the CrtA response regulator acts directly at cell cycle-regulated promoters to control DNA replication, DNA methylation and flagellar biogenesis, and is essential for growth (Quon et al., 1996
). An essential sensor kinase, CckA, that is responsible for the CtrA activation has been identified (Jacobs et al., 1999
). Furthermore, the Bacillus subtilis yycF gene and its orthologue in Staphylococcus aureus encode a response regulator and seem to be essential for cell growth, although genes under their control have yet to be identified (Fabret & Hoch, 1998
; Martin et al., 1999
).
We also found the essential nature of the yycFG genes in B. subtilis during construction of a knockout mutant bank of newly identified genes by genome sequencing. The entire genome sequence of B. subtilis revealed the existence of over 4000 ORFs, two-thirds of which had not been characterized in previous genetic and biochemical studies (Kunst et al., 1997
). Systematic analysis of functions of uncharacterized genes is being carried out in two research consortia, one in Japan and the other in Europe (Ogasawara, 2000
). The main approach to assessing gene function is the construction of mutants in target genes and the analysis of the mutant phenotypes. Our group sequenced a 220 kb region containing the replication origin and identified 134 new genes (Ogasawara et al., 1994
; Kasahara et al., 1997
). When we carried out systematic knockout mutagenesis of the 134 genes, several genes were difficult to disrupt; these included two-component sensor kinase and regulator genes, yycF and yycG. Growth of the mutants in which an IPTG-inducible promoter, Pspac, regulates the expression of yycFG became IPTG dependent. We now report that in B. subtilis expression of a cell division gene, ftsZ, is potentially under the direct control of the YycFG system.
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METHODS
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Bacterial strains, plasmids and culture conditions.
The bacterial strains and plasmids used in this study are listed in Tables 1
and 2
. Bacterial strains were grown in LB medium (5 g yeast extract l-1, 10 g tryptone l-1, 5 g NaCl l-1, pH 7·2) at 37 °C. A sporulation medium (DS medium; Schaeffer et al., 1965
) was used for B. subtilis sporulation. When required, antibiotics were added at the following concentrations: ampicillin at 50 µg ml-1, kanamycin at 10 µg ml-1, erythromycin at 0·5 µg ml-1, chloramphenicol at 5µg ml-1. IPTG was added at 1 mM.
Construction of plasmids and B. subtilis strains.
Table 2
summarizes relevant characteristics of the plasmids we constructed. The primers for PCR amplification of B. subtilis chromosomal fragments used to construct the plasmids are listed in Table 3
. Derivatives of pMutinT3 (Vagner et al., 1998
) were used to construct knockout or conditional-null mutants of B. subtilis genes. The BamHISacI fragment of pMutinT3 was cloned into the BamHISacI site of pDL (Yuan & Wong, 1995
) to obtain pDL2, which was used to place the promoterlacZ fusions in the amyE locus of the B. subtilis genome. Plasmids pRB373 (Bruckner, 1992
) and pET15b (Novagen) were used to express the YycF proteins in B. subtilis and Escherichia coli, respectively. Site-directed PCR mutagenesis was done to introduce a point mutation in the yycF gene on pRBYycF. Two primers, FKR021 and FKF022, having one base change which replaces the Asp-54 of the YycF protein with His, were designed, and the 5'- and 3'-areas of yycF were amplified, using primer sets FKF021-FKR021 and FKF022-FKR022, respectively. The two fragments were ligated by PCR with FKF021 and FKR022 primers, and cloned into pRB373 to obtain pRBYycFD54H. Transformation of B. subtilis cells to obtain the strains described in Table 1
was done as described by Moriya et al. (1998)
. Further details of the construction of the plasmids and the B. subtilis strains used in this work are available on request.
Northern blot analysis.
B. subtilis 168 cells were collected by centrifugation at various time points during growth in the sporulation medium. Each cell pellet (cell density 10 OD600 units) was resuspended in 1 ml SET buffer (50 mM Tris/HCl pH 7·6, 50 mM EDTA, 20% sucrose) containing 2 mg lysozyme, and kept on ice for 10 min. Cells were then collected and treated with ISOGEN (Nippon Gene) to prepare RNA fractions. Ten micrograms of total RNA was loaded into each lane of a 1% (w/v) agarose gel. After electrophoresis, RNA was blotted on a positively charged nylon membrane (Hybond-N+, Amersham Life Science). Digoxigenin (DIG)-labelled antisense RNA probes encompassing each of the yycFGHIJ and yyxA genes were prepared in vitro with T7 RNA polymerase using PCR products as templates. Inserts of the pKF001, pKF002, pKF003, pKF004, pKF005 and pKF006 plasmids were amplified with T3T7R, which contains the T7 promoter sequence, and T3F, and used as templates for in vitro RNA synthesis using the DIG RNA Labelling Kit (Boehringer Mannheim). Hybridization and detection of the signals were done following the manufacturers instructions (Boehringer Mannheim).
Fluorescence microscopy.
Cell morphology and nucleoid distribution were examined by fluorescence microscopy after DAPI staining, as described by Hassan et al. (1997)
.
ß-Galactosidase assay.
B. subtilis cells growing in LB medium (OD600 0·4) were collected by centrifugation and the activity of the ß-galactosidase was assayed according to Youngman et al. (1985)
. One unit was defined as 1 nmol 4-methylumbelliferyl ß-D-galactoside hydrolysed in 1 min per mg protein. Protein concentration was determined using the Bio-Rad Protein Assay Kit. All results are the means of three or more assays.
Two-dimensional PAGE and micro-sequencing of proteins.
B. subtilis cells grown in 200 ml LB medium were harvested at OD600 0·4, and washed with 10 mM Tris/HCl (pH 8·0) followed by 10 mM Tris/HCl (pH 8·0) containing 10% sucrose. The cells were then resuspended in 10 mM Tris/HCl (pH 8·0) containing 10% sucrose, 1 mM PMSF and 1 mg lysozyme ml-1, and incubated at 37 °C for 10 min, followed by addition of 9 vols lysis solution [8 M urea, 1 mM DTT, 1 mM PMSF and 1·5% IPG phor (Pharmacia Biotech)]. After removal of insoluble materials by centrifugation at 18000 g for 15 min at 25 °C, protein mixtures were isoelectrophoretically focused at room temperature on 180 mm IPG Drystrip pH 47, using the Multiphor II 2-D system and following the manufacturers instructions (Pharmacia Biotech). The second dimension was run using a precast 1214% gradient gel (Pharmacia Biotech). Proteins separated by two-dimensional PAGE were electroblotted onto a PVDF membrane (Bio-Rad) and the N-terminal sequences were determined using a 470A automated gas-phase sequencer (PE Biosystems).
Purification of the His6-YycF protein.
E. coli BL21(DE3)pLysS cells (Novagen) containing the pETYycF plasmid were incubated at 30 °C in 10 ml LB medium containing ampicillin (50 µg ml-1). When the culture reached an OD600 of 0·6, IPTG was added to a final concentration of 1 mM. The cells were incubated for another 3 h, then harvested by centrifugation, and the His6-YycF protein was purified according to the manual of the PET system (Novagen). Collected cells were washed with a binding buffer (0·5 M NaCl, 5 mM imidazole, 20 mM Tris/HCl pH 7·9), and resuspended in 25 ml of the same buffer. The cells were broken by sonication on ice, and the lysate was centrifuged at 39000 g for 30 min at 4 °C. The supernatant fraction was loaded on the His-Bind Resin, and the resin was washed with 10 ml of the binding buffer followed by 10 ml of a washing buffer (0·5 M NaCl, 60 mM imidazole, 20 mM Tris/HCl pH 7·9). Protein bound to the resin was eluted with 10 ml each of the binding buffers containing an increasing amount of imidazole (0·1 to 1 M). The fraction containing His6-YycF protein was stored at -80 °C after the addition of glycerol to 10% (v/v).
Gel mobility-shift assay.
DNA fragments covering the -168 to -35 region of the ftsAZ P1 promoter and the -92 to +1 region of the ftsAZ P3 promoter were PCR-amplified, using primer sets FKF201-FKR201 and FKF203-FKR203, respectively. DNA fragments covering the -59 to -35, -77 to -35 and -92 to -35 regions of the P1 promoter were PCR-amplified from plasmids pKF105, pKF106 and pKF107, respectively, using primer set pDLF-FKR201. PCR products were purified using TaKaRa RECOCHIP (Takara) after separation in agarose gel, and end-labelled with DIG using the DIG Labelling Kit (Boehringer Mannheim). The binding reactions (10 µl final volume) were performed by incubating 20 fmol each of the DIG-labelled DNA fragments, 0·5 µg poly(dI-dC) and 024 pmol purified His6-YycF protein in 50 mM PIPES (pH 6·1), 200 mM NaCl, 1 mM EDTA, 4 mM MgCl2, 4 mM DTT, 0·5% Tween 20 and 10% (v/v) glycerol. For binding competition assays, 20 pmol unlabelled fragment was added to the reaction mixture. After incubation at 25 °C for 30 min, 2 µl loading dye (0·1% xylene cyanol in 40%, v/v, glycerol, 22·25 mM Tris, 20 mM boric acid and 0·5 mM EDTA, pH 8·0) was added to stop the reactions and the samples were loaded on 5% polyacrylamide nondenaturing gels (29:1 acrylamide to bisacrylamide in 0·5xTBE buffer). After electrophoresis, DIG-labelled DNA fragments were transferred onto a Hybond-N+ membrane (Amersham Life Science) and detected using the DIG Gel Shift Assay Kit (Boehringer Mannheim).
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RESULTS
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Characterization of an operon containing yycF and yycG
The nucleotide sequence around the yycF and yycG genes suggested that they constitute an operon with additional four genes, yycH, yycI, yycJ and yyxA (Ogasawara et al., 1994
; Fig. 1a
). Northern blot analysis using the yycF- and yyxA-specific RNA probes revealed a common 7·4 kb transcript covering six genes in the early vegetative growth phase (Fig. 1b
, c
). The same transcript was detected with the yycG-, yycH-, yycI- and yycJ-specific probes (data not shown). In addition, the yycF probe detected a 2·4 kb transcript in the early vegetative phase and the yyxA probe a 1·4 kb transcript in the late sporulation phase.

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Fig. 1. Gene organization and transcriptional units of the yycFGHIJ and yyxA region. (a) Arrangement of the yycFGHIJ and yyxA genes is shown schematically. T indicates the location of a possible rho-independent termination signal of transcription. Below the map, transcripts detected by Northern blot analysis are indicated by arrows. (b, c) Northern blot analysis with yycF- (b) and yyxA-specific (c) RNA probes. Each lane contained 10 µg total RNA from the B. subtilis wild-type strain 168 at t-2·5 (lane 1), t-2 (2), t-1·5 (3), t-1 (4), t0 (5), t1 (6), t2 (7), t3 (8), t4 (9) and t5 (10) during growth in sporulation medium. An RNA marker (Boehringer Mannheim) was loaded in lane M and sizes are indicated on the left of the blots. Positions of rRNA are indicated (23S, 2·93 kb 23S rRNA; 16S, 1·55 kb 16S rRNA).
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To characterize functions of yycFGHIJ and yyxA genes, we attempted to knock out each of them by integrating the pMutinT3 plasmid harbouring an internal fragment of each gene into the genome by a Campbell-type recombination (Vagner et al., 1998
). Knockout mutants of yycH, yycI, yycJ and yyxA were easily obtained, and growth defects, including the formation of heat-resistant spores, were not observed in the mutants (data not shown). By contrast, yycF and yycG disruptants could not be obtained, suggesting that yycF and yycG are essential for cell growth. Therefore, we constructed conditional-null mutants by integrating pMutinT3 harbouring the SD sequence and the N-terminal portion of yycF or yycG (Fig. 2a
). If these genes are indeed essential, integration of the plasmids was expected to occur only in the presence of IPTG, where the expression of yycF and yycG would be maintained by an IPTG-inducible promoter, Pspac. As expected, transformants in which the pMutinT3 derivatives were properly integrated into the genome (NIS8033 and NIS8032) were obtained in the presence, but not in the absence, of IPTG.
The growth of the conditional-null mutant of yycFG (NIS8033) was monitored in liquid LB medium, with and without IPTG (Fig. 2b
). When IPTG was removed from the medium, the increase in optical density halted within 2 h and cells began to lose viability. Similar growth defects were also observed in both the sporulation and a defined salt medium (data not shown). Microscopic examination of the mutant cells cultivated for 2 h in the absence of IPTG revealed the presence of ghost cells in which the cytoplasmic contents seemed to be lost (Fig. 2c
). Fabret & Hoch (1998)
reported the same phenomenon for the yycF-ts mutant at the non-permissive temperature. The defect of growth was not clearly observed for the Pspac-yycG mutant (NIS8032) in liquid medium without IPTG, suggesting that a weak residual expression of yycG from Pspac in the absence of IPTG would be sufficient to maintain apparently normal cell growth for several generations (data not shown).
Inactivation of the yycG sensor gene in the presence of a mutated YycF regulator
We then asked if the growth defect in the YycG sensor mutant was due to a deficiency in activation of the YycF regulator. A 1046 bp fragment covering the promoter and the complete coding region of the yycF gene was cloned into a shuttle plasmid, pRB373 (Bruckner, 1992
), and a mutation which replaced the conserved Asp residue (Asp-54, probable phosphorylation site) with His was introduced by site-directed mutagenesis (pRBYycFD54H, Fig. 3a
). We then transformed cells carrying pRBYycFD54H by the pMutinT3-yycG plasmid (pFK002), and inactivated the yycG sensor gene (Fig. 3b
). This finding indicated that the YycFD54H protein was locked in an active form independently of the YycG kinase. Furthermore, this evidence strongly suggested a direct interaction between the YycG kinase and the YycF regulator. The growth defect in the absence of YycG is attributed to a deficiency in activation (phosphorylation) of the YycF regulator.

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Fig. 3. Inactivation of the yycG sensor gene in the presence of a mutated YycF regulator, YycFD54H. (a) Design of primers, FKR022 and FKF022, used to introduce a base change that replaces Asp-54 of the YycF protein with His. The partial nucleotide sequence and the deduced amino acid sequence of yycF, containing the XXXXD motif (X indicates non-polar residues, underlined) conserved in response regulators (Parkinson & Kofoid, 1992 ), are shown. Nucleotide sequences of FKR022 and FKF022 primers are shown below. (b) Wild-type 168 and its derivatives harbouring pRB373, pRBYycF or pRBYycFD54H were transformed with pKF002 containing an internal fragment of yycG. The number of erythromycin-resistant transformants (disruptants of yycG) per ml of competent cells is shown.
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Overproduction of the YycF regulator induces excess cell division
The copy number of pRB373 in B. subtilis was reported to be about 50 (Bruckner, 1992
). Thus, the YycF or YycFD54H protein was expected to be overproduced within cells carrying pRBYycF or pRBYycFD54H, and this was confirmed by two-dimensional PAGE analysis of whole-cell extracts (Fig. 4a
). The molecular mass and pI value of the YycF protein were calculated to be 27·4 kDa and 5·06, respectively. In cells harbouring pRBYycF, the intensity of a spot with an apparent molecular mass of 31 kDa and pI value of 5·15 markedly increased compared with that of the cells harbouring the pRB373 vector. The N-terminal 12 amino acid sequence of the protein in the spot perfectly matched that deduced from the nucleotide sequence of the yycF gene (data not shown). In cells harbouring pRBYycFD54H, there was an additional intense spot with a slightly higher pI value, and the N-terminal amino acid sequence was again identical to that of YycF (data not shown).

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Fig. 4. Effects of YycF and YycFD54H overproduction on cell size. (a) B. subtilis 168 cells harbouring pRB373, pRBYycF or pRBYycFD54H in LB medium were harvested in the vegetative growth phase (OD600 0·4) and crude extracts were analysed by two-dimensional PAGE. Gels were stained using the Pharmacia Biotech Silver Staining Kit. Parts of two-dimensional gels including YycF and YycFD54H spots are shown. (b) Phase-contrast images of 168 cells harbouring pRB373, pRBYycF or pRBYycFD54H. Mini-cells are indicated by arrows. (c) Histograms showing the frequency distribution of cell length of each strain. About 100 cells from the each strain were measured. The mean cell length was 5·6 µm, 4·1 µm and 4·1 µm for cells carrying pRB373, pRBYycF and pRBYycFD54H, respectively (indicated by the vertical arrows in each panel).
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When cells carrying pRBYycF or pRBYycFD54H were examined by phase-contrast microscopy, we noticed production of mini-cells (Fig. 4b
). The proportion of mini-cells was calculated to be 4·8% (49 mini-cells/1020 cells observed) and 2·9% (31/1080) for cells harbouring pRBYycF and pRBYycFD54H, respectively. By contrast, only two mini-cells (0·2%) were found among 1074 cells harbouring pRB373. Furthermore, statistical analysis of cell length revealed that the overproduction of the YycF or YycFD54H protein resulted in shorter cells (Fig. 4c
). Therefore, excess cell division was suggested to occur in cells carrying pRBYycF or pRBYycFD54H.
Overproduction of the YycF regulator stimulates expression from the P1 promoter of the ftsAZ operon
The observation described above suggested that the overproduced YycF regulator promoted the expression of cell-division gene(s). Therefore, we analysed the effect of YycF overproduction on expression of an essential cell division operon, ftsAZ. Three promoter sequences, P1, P2 and P3, have been identified in the B. subtilis ftsZA operon (Fig. 5
; Gonzy-Treboul et al., 1992
). The P1 and P3 promoters were sigma-A dependent and active in the vegetative growth phase, whereas the P2 promoter was sigma-H dependent and important for asymmetric cell division at the onset of sporulation. We constructed cells in which P1, P2, P3 or the whole promoter region was placed in front of the lacZ reporter gene at the amyE locus of the genome (Fig. 5
), then pRB373, pRBYycF and pRBYycFD54H plasmids were introduced into them. The LacZ activities in the vegetative growth phase of the cells thus constructed indicated clearly that overproduced YycF or YycFD54H activates specifically the expression from the P1 promoter about threefold (Fig. 6
). As a consequence, an approximately 1·5-fold increase in the total activity of the three promoters was observed in YycF- or YycFD54H-overproducing cells; this would explain the excess division observed in these cells.

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Fig. 7. Deletion analysis of the regulatory sequence by the YycF regulator. B. subtilis strains containing P1(-138 to +30) (NIS5005), P1(-130 to +30) (NIS5006), P1(-118 to +30) (NIS5007), P1(-108 to +30) (NIS5008), P1(-92 to +30) (NIS5009), P1(-77 to +30) (NIS5010), P1(-59 to +30) (NIS5011) or P1(-45 to +30)lacZ (NIS5012) were transformed with pRB373 or pRBYycF. They were cultured in LB medium at 37 °C to OD600 0·4, and the ß-galactosidase activities were assayed. All results are the means of three assays, and error ranges are indicated by vertical lines.
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Fig. 6. Effect of YycF and YycFD54H overproduction on ftsAZ promoter acitivity. B. subtilis strains containing a P1 (NIS5001), P2 (NIS5002), P3 (NIS5003) or P123lacZ fusion (NIS5004) were transformed with pRB373, pRBYycF or pRBYycFD54H, cultured in LB medium at 37 °C to OD600 0·4 and ß-galactosidase activities assayed. All results are the means of three assays, and the error ranges are indicated by vertical lines.
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Deletion analysis of a regulatory region of the ftsZA P1 promoter
To further characterize regulation of expression from the P1 promoter by the YycF regulator, we gradually shortened the upstream sequence of the P1 promoter in the P1lacZ assay (Fig. 7
; see also Fig. 5
). The P1 fragment used in the previous assay contained a 118 bp sequence upstream of the start site of the transcription, and the same level of activation by YycF was observed in cells containing a 16 bp shorter sequence (-92). By contrast, deletion of a further 15 bp abolished the activation (-77), although a basal level of expression from the P1 promoter was maintained. These results indicated that a region between -92 and -77 was essential for activation of the P1 promoter by the YycF protein.
The YycF regulator binds to the P1 promoter sequence in vitro
Finally, we examined the direct interaction between the YycF protein and the P1 promoter sequence in gel mobility-shift assays. The YycF protein was expressed in E. coli as a fused protein with a histidine tag (His6) and purified to near homogeneity as described in Methods (Fig. 8a
). A 134 bp fragment covering the P1 promoter and a 92 bp fragment covering the P3 promoter were PCR-amplified (see Fig. 5
), end-labelled with DIG, and used as probes for gel mobility-shift assays (Fig. 8b
). When incubated with 20 fmol each of the DIG-labelled DNA fragments, 18 pmol His6-YycF caused a complete shift of the P1 but not the P3 fragment (lane 5). Specificity of the interaction of His6-YycF with the P1 sequence was further tested in competition experiments. When 20 pmol unlabelled P1 fragment was added to the reaction mixture, a clear competition was observed (lane 7). These results demonstrated that the YycF protein bound specifically to the ftsAZ P1 promoter sequence. The in vivo deletion analysis of the regulatory sequence for the P1 activation by YycF suggested that the -92 to -77 region would be the binding site of YycF. When a fragment covering -92 to -35 of the P1 sequence was used as probe, the same mobility shift was observed as with the 134 bp P1 fragment (Fig. 8c
, lanes 912). As expected, deletion of the -92 to -78 region from the probe markedly reduced the binding affinity to YycF (lane 58), and further deletion to -59 completely abolished the YycF binding (lanes 14). Thus the DNA-binding property of the YycF protein in vitro was in agreement with the result of the deletion analysis of the in vivo regulatory sequence.

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Fig. 8. Direct binding of the YycF regulator to the ftsZA P1 promoter region. (a) SDS-PAGE of protein samples from each purification step of His6-YycF. The proteins were separated in a 15% acrylamide gel and stained with Coomassie brilliant blue. Lanes: 1, standard proteins (Bio-Rad), with the molecular masses given on the left; 2, crude extract; 3, flowthrough from the nickel resin; 4, wash by 1 mM imidazole; 5, wash by 30 mM imidazole; 6, elution by 100 mM imidazole; 7, elution by 200 mM imidazole; 8, elution by 300 mM imidazole; 9, elution by 400 mM imidazole; 10, elution by 1 M imidazole. The protein fraction eluted by 100 mM idazole (lane 6) was used for the gel mobility-shift assay. (b) Gel mobility-shift assay for the binding of His6-YycF to the P1 and P3 promoter regions. A 134 bp fragment covering the P1 promoter and a 92 bp fragment covering the P3 promoter (see Fig. 5 ) were end-labelled with DIG. Twenty femtomoles each of the DIG-labelled DNA fragments were incubated with 0 pmol (lane 1), 3 pmol (lane 2), 6 pmol (lane 3), 12 pmol (lane 4), 18 pmol (lane 5) or 24 pmol (lanes 6 and 7) of purified His6-YycF. Non-labelled P1 fragment (20 pmol) was added to the reaction mixture in lane 7. The samples were run on 5% polyacrylamide nondenaturing gel and the DIG-labelled fragments were detected using the DIG Gel Shift Assay Kit (Boehringer Mannheim). (c) Deletion analysis of the YycF-binding sequence. DNA fragments covering the -59 to -35 (lanes 14), -77 to -35 (lanes 59) or -92 to -35 (lanes 912) region of the P1 sequence were labelled with DIG and incubated with 0 pmol (lanes 1, 5, 9), 12 pmol (lane 2, 6, 10), 18 pmol (lanes 3, 7, 11) or 24 pmol (lanes 4, 8, 12) of purified His6-YycF. Then the DIG-labelled fragments were separated and detected as described in (b).
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The in vitro binding affinity seemed to be relatively low, as about 1000-fold excess molecules of the His6-YycF protein were needed for a complete shift, suggesting that our His6-YycF preparation was in a non-phosphorylated form. We purified YycG kinase, using the His-tag system in E. coli, and attempted to phosphorylate the YycF regulator in vitro, so far without success. Acetyl phosphate also failed to activate the purified YycF protein.
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DISCUSSION
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The B. subtilis yycF gene encoding a response regulator and its orthologue in S. aureus was found by other researchers to be essential for cell growth (Fabret & Hoch, 1998
; Martin et al., 1999
). We obtained supportive evidence for these findings during construction of a knockout mutant bank of genes newly identified in the genome sequence project. Furthermore we found that the yycG gene could be deleted in the presence of an active form of the YycF protein, YycFD54H, thereby suggesting direct interactions between YycG and YycF. The molecular mechanism involved in locking YycF in the activated form by replacement of Asp-54 with His has yet to be clarified.
The YycFG two-component regulatory system is expected to regulate an essential gene function in B. subtilis and S. aureus. Based on the observation that overproduction of the YycF regulator caused excess cell division, we demonstrated that the overproduced YycF protein stimulated the P1 promoter activity of the cell-division operon ftsAZ. Furthermore, we showed that the region between -92 and -77 was essential for the activation of the P1 promoter in vivo, and indeed the YycF protein was shown to bind specifically to the -92 to -59 region of the P1 sequence in vitro. These results clearly indicate that the essential two-component regulatory system encoded by yycF and yycG has the potential to modulate expression of the ftsAZ operon in B. subtilis. However, characteristic features in the sequence between -92 and -59 of the P1 promoter remain to be identified.
In E. coli, the expression of ftsZ is controlled by at least six promoters. In addition, at least two transcriptional activators SdiA, which is a part of a quorum-sensing pathway, and RcsB, which is involved in the regulatory pathway of capsular polysaccharide biosynthesis have been found to modulate ftsZ expression (reviewed by Vicente et al., 1999
). In B. subtilis, three promoter sequences for ftsZ expression were identified, and our results show that YycF is the transcriptional factor with the potential to regulate ftsZ expression. Gonzy-Treboul et al. (1992)
reported that the P1 promoter could be deleted without interference with cell growth. We confirmed that the P1 promoter could be deleted, but the resultant cells became elongated, indicating that the P2 and P3 promoters alone are not sufficient to maintain normal cell division (data not shown). However, deletion of a region necessary for YycF regulation did not clearly affect P1 promoter activity in wild-type cells (Fig. 7
). Therefore, the precise role of the YycFG system under normal expression in the control of cell division awaits further characterization.
The essential nature of the YycFG two-component system cannot be explained solely by its potential to modulate ftsAZ expression; it must regulate hitherto unidentified essential gene(s). Alternatively, it may regulate several genes important for cell growth, and simultaneous inhibition of their expression may be lethal. We and others (Fabret & Hoch, 1998
) observed ghost cells that had apparently lost cellular contents by depletion of the B. subtilis YycF protein or by thermal inactivation of the ts-mutant protein. The temperature-sensitive yycF mutant of S. aureus became hypersensitive to macrolide antibiotics at the permissive temperature, probably due to defects in the permeability barrier (Martin et al., 1999
). Based on these observations, we speculate that the YycFG system may perhaps be involved in global regulation of cell envelope synthesis. Further characterization of genes under the control of the YycFG system is essential to determine precise roles in the control of cell growth and to identify the effector molecule that activates the YycFG system. We are currently characterizing the YycF-binding motif in the ftsZA P1 promoter sequence; once the motif is known, we will survey candidate genes that may belong to the yycFG regulon on the complete genome sequence of B. subtilis.
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ACKNOWLEDGEMENTS
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We are grateful to Dr Tatsuo Nakayama for determining the N-terminal sequences of YycF and YycFD54H proteins. We also thank colleagues in our laboratory for valuable discussions and M. Ohara for language assistance. This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas and a Grant-in-Aid for Scientific Research (C) from the Ministry of Education, Science, Sports and Culture of Japan, and grant JSPS-RFTF96L00105 from the Japan Society for the Promotion of Science.
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REFERENCES
|
---|
Bruckner, R. (1992). A series of shuttle vectors for Bacillus subtilis and Escherichia coli.Gene 122, 187-192.[Medline]
Fabret, C. & Hoch, J. A. (1998). A two-component signal transduction system essential for growth of Bacillus subtilis: implications for anti-infective therapy. J Bacteriol 180, 6375-6383.[Abstract/Free Full Text]
Gonzy-Treboul, G., Karmazyn-Campelli, C. & Stragier, P. (1992). Developmental regulation of transcription of the Bacillus subtilis ftsAZ operon. J Mol Biol 224, 967-979.[Medline]
Hassan, A. K., Moriya, S., Ogura, M., Tanaka, T., Kawamura, F. & Ogasawara, N. (1997). Suppression of initiation defects of chromosome replication in Bacillus subtilis dnaA and oriC-deleted mutants by integration of a plasmid replicon into the chromosomes. J Bacteriol 179, 2494-2502.[Abstract]
Jacobs, C., Domian, I. J., Maddock, J. R. & Shapiro, L. (1999). Cell cycle-dependent polar localization of an essential bacterial histidine kinase that controls DNA replication and cell division. Cell 97, 111-120.[Medline]
Kasahara, Y., Nakai, S. & Ogasawara, N. (1997). Sequence analysis of the 36-kb region between gntZ and trnY genes of Bacillus subtilis genome. DNA Res 4, 155-159.[Medline]
Kunst, F., Ogasawara, N., Moszer, I. & 148 other authors (1997). The complete genome sequence of the gram-positive bacterium Bacillus subtilis. Nature 390, 249256.[Medline]
Loomis, W. F., Kuspa, A. & Shaulsky, G. (1998). Two-component signal transduction systems in eukaryotic microorganisms. Curr Opin Microbiol 1, 643-648.[Medline]
Martin, P. K., Li, T., Sun, D., Biek, D. P. & Schmid, M. B. (1999). Role in cell permeability of an essential two-component system in Staphylococcus aureus. J Bacteriol 181, 3666-3673.[Abstract/Free Full Text]
Moriya, S., Tsujikawa, E., Hassan, A. K., Asai, K., Kodama, T. & Ogasawara, N. (1998). A Bacillus subtilis gene-encoding protein homologous to eukaryotic SMC motor protein is necessary for chromosome partition. Mol Microbiol 29, 179-187.[Medline]
Ogasawara, N. (2000). Systematic function analysis of Bacillus subtilis genes. Res Microbiol 151, 129134.[Medline]
Ogasawara, N., Nakai, S. & Yoshikawa, H. (1994). Systematic sequencing of the 180 kilobase region of the Bacillus subtilis chromosome containing the replication origin. DNA Res 1, 1-14.[Medline]
Parkinson, J. S. & Kofoid, E. C. (1992). Communication modules in bacterial signaling proteins. Annu Rev Genet 26, 71-112.[Medline]
Quon, K. C., Marczynski, G. T. & Shapiro, L. (1996). Cell cycle control by an essential bacterial two-component signal transduction protein. Cell 84, 83-93.[Medline]
Schaeffer, P., Millet, J. & Aubert, J.-P. (1965). Catabolite repression of bacterial sporulation. Proc Natl Acad Sci USA 54, 704-711.[Medline]
Vagner, V., Dervyn, E. & Ehrlich, S. D. (1998). A vector for systematic gene inactivation in Bacillus subtilis. Microbiology 144, 3097-3104.[Abstract]
Vicente, M., Chater, K. F. & De Lorenzo, V. (1999). Bacterial transcription factors involved in global regulation. Mol Microbiol 33, 8-17.[Medline]
Youngman, P., Perkins, J. & Sandman, K. (1985). Use of Tn917-mediated transcriptional gene fusions to lacZ and cat-86 for the identification and study of regulated genes in the Bacullus subtilis chromosome. In Molecular Biology of Microbial Differentiation, pp. 47-54. Edited by J. A. Hoch & P. Setlow. Washington, DC: American Society for Microbiology.
Yuan, G. & Wong, S. L. (1995). Regulation of groE expression in Bacillus subtilis: the involvement of the
A-like promoter and the roles of the inverted repeat sequence (CIRCE). J Bacteriol 177, 5427-5433.[Abstract]
Received 28 March 2000;
accepted 17 April 2000.