BceA and bceB encode a nucleotide-binding domain (NBD) and membrane-spanning domain (MSD) subunit, respectively, of an ATP-binding cassette (ABC) transporter in Bacillus subtilis. Disruption of these genes resulted in hypersensitivity to bacitracin, a peptide antibiotic that is non-ribosomally synthesized in some strains of Bacillus. Northern hybridization analyses showed that expression of the bceAB operon is induced by bacitracin present in the growth medium. The bceRS genes encoding a two-component regulatory system are located immediately upstream of bceAB. Deletion analyses of the bceAB promoter together with DNase I footprinting experiments revealed that a sensor kinase, BceS, responds to extracellular bacitracin either directly or indirectly and transmits a signal to a cognate response regulator, BceR. The regulator binds directly to the upstream region of the bceAB promoter and upregulates the expression of bceAB genes. The bcrC gene product is additionally involved in bacitracin resistance. The expression of bcrC is dependent on the ECF σ factors, σM and σX, but not on the BceRS two-component system. In view of these results, possible roles of BceA, BceB and BcrC in bacitracin resistance of B. subtilis 168 are discussed.
Bacteria are equipped with devices to cope with external and internal stresses, including toxic compounds produced by other bacteria or themselves. One of the mechanisms of acquiring drug resistance involves the exclusion of compounds by membrane-bound transporter proteins. A number of drug efflux transporters are specific for a given drug or class of drugs, whereas others designated ‘multidrug resistance transporters’ (MDR) display specificity for various compounds with different chemical structures. Transporters are classified into several groups, depending on sequence similarity (Levy, 1992; Paulsen et al., 1996; Putman et al., 2000). The ATP-binding cassette (ABC) transporter family is one of the most abundant of these groups (Fath and Kolter, 1993). The LmrA protein of Lactococcus lactis is the earliest identified MDR efflux transporter of the ABC family in bacteria (van Veen et al., 1996). LmrA pumps out various drugs using a similar mechanism to that proposed for P-glycoprotein of mammalian MDR transporters (van Veen et al., 2000). However, few ABC-type MDR transporters in bacteria have been functionally characterized to date.
Antibiotic expulsion, mediated by a membrane-associated drug efflux transporter, is also a major self-protecting mechanism devised by antibiotic-producing bacteria (Cundliffe, 1989). Drug efflux transporters belonging to the ABC family are frequently involved in these systems (Méndez and Salas, 2001). In bacteria, the biosynthetic operons of peptide antibiotics, such as subtilin, nisin, mersacidin and bacitracin, are associated with genes encoding efflux transporters of the ABC family, which pump out these antibiotics for self-protection (Klein and Entian, 1994; Podlesek et al., 1995; De Ruyter et al., 1996; Altena et al., 2000). Furthermore, genes encoding a two-component signal transduction system, the major mechanism for sensing environmental signals in bacteria (Stock et al., 2000), are commonly located adjacent to the efflux transporter genes and regulate their expression (De Ruyter et al., 1996; Altena et al., 2000; Neumüller et al., 2001; Stein et al., 2002). In the Gram-positive bacterium Bacillus subtilis, 40 operons are predicted to encode efflux transporters of the ABC family (Quentin et al., 1999). Analysis of their genomic location revealed that eight of these operons are located adjacent to genes encoding the two-component regulatory system (Kunst et al., 1997). In view of this finding, we examined whether the ABC transporters and two-component regulatory systems are functionally related. We compared the resistance of the wild-type strain and disruptants of ABC transporter genes to various compounds. Our data show that bceA and bceB (formerly denoted ytsC and ytsD), which encode the nucleotide-binding domain (NBD) and membrane spanning domain (MSD) subunit of the ABC transporter, respectively, are involved in resistance to bacitracin, a peptide antibiotic non-ribosomally synthesized in specific strains of Bacillus (Johnson et al., 1945; Azevedo et al., 1993). We additionally demonstrate that bceRS (formerly known as ytsAB) genes of the two-component regulatory system, located immediately upstream of bceAB, control expression of the bceAB operon by sensing extracellular bacitracin.
Recently, we (Ohki et al., 2003) and Cao and Helmann (2002) showed that the bcrC gene (formerly designated ywoA), which encodes a homologue of the possible MSD subunit of the Bacillus licheniformis ABC transporter for bacitracin efflux (bcrC), is involved in bacitracin resistance in B. subtilis 168. Expression of bcrC is dependent on ECF σ factors, σM and σX, but not on the BceRS two-component system. Based on these results, possible roles of BceAB and BcrC in bacitracin resistance of B. subtilis 168 are discussed.
An ABC transporter, BceAB, and a two-component regulatory system, BceRS, are involved in bacitracin resistance in B. subtilis
Among 40 possible ABC efflux transporter operons in B. subtilis, eight (ycbN, natAB, yclIH, yfiLMN, ytsCD, yvcRS, yvfRS and yxdLM) are located next to genes encoding the two-component regulatory system (Kunst et al., 1997). Seven of these transporters (except natAB encoding the Na+ extrusion transporter) and the associated two-component regulatory systems have not yet been functionally characterized (Fabret et al., 1999). To determine the functions of these genes, we compared the resistance of the wild-type strain and pMutin disruptants of ABC transporter genes to various drugs. Mutants of ytsC and ytsD encoding NBD and MSD subunits, respectively, displayed hypersensitivity to bacitracin, as shown in Table 1. The bcrC gene product is additionally involved in bacitracin resistance in B. subtilis 168 (Cao and Helmann, 2002; Ohki et al., 2003). Disruptants of ytsC or ytsD were more sensitive to bacitracin, compared with a bcrC mutant.
Table 1. . Bacitracin sensitivity of various mutants.
The ytsC and ytsD genes constitute an operon. Therefore, it is necessary to confirm that the bacitracin-sensitive phenotype of YTSCd results from inactivation of the ytsC gene, and not the polar effect of the pMutin insertion on downstream ytsD expression. In pMutin disruptants, downstream genes are placed under the control of the spac promoter, which is induced by the addition of IPTG (Vagner et al., 1998). Northern hybridization analyses using total RNA isolated from YTSCd grown in the presence or absence of IPTG demonstrated that a transcript detected with a probe specific for ytsD increased 25-fold with IPTG (Supplementary material, Fig. S1A). However, bacitracin resistance was not increased upon addition of IPTG, indicating that YtsC is required for bacitracin resistance (data not shown). Moreover, disruption of these genes had no effect on resistance to the other drugs examined, including vancomycin, tunicamycin, surfactin, iturin A and several cationic dyes (data not shown).
The ytsA and ytsB genes, located immediately upstream of ytsCD, encode a response regulator and a histidine sensor kinase, respectively, of the two-component regulatory system. Disruption of either ytsA or ytsB, which constitute an operon, caused a similar decrease in bacitracin resistance to that observed in ytsC and ytsD mutants (Table 1). In YTSAd, the addition of IPTG resulted in a more than 10-fold increase in the amount of 1.7 kb transcript detected with a probe specific for ytsB (Supplementary material, Fig. S1B), but had no effect on bacitracin resistance (data not shown).
The results collectively suggest that ytsC and ytsD constitute subunits of a bacitracin efflux transporter, whereas ytsA and ytsB are involved in the regulation of bacitracin resistance. Accordingly, we designated ytsC and ytsD as bceA and bceB (bacitracin efflux), and ytsA and ytsB as bceR and bceS respectively.
Identification of the bceRS and bceAB transcriptional units and induction of bceAB expression by external bacitracin
It is predicted that bceRS and bceAB genes are transcribed in the same direction, and the open reading frames (ORFs) of bceR and bceS, as well as of bceA and bceB, overlap by two and three codons, respectively, as shown schematically in Fig. 1E. Northern blot experiments using total RNA isolated from wild-type cells revealed a 1.7 kb band with probes specific for bceR or bceS and a 2.7 kb band with probes specific for bceA or bceB (Fig. 1). Levels of both transcripts were low during all the growth phases examined.
The addition of 40 U ml−1 bacitracin to LB inhibited the growth of wild-type cells by about 50%. An approximately twofold increase in bacitracin resistance was observed with wild-type cells grown in LB medium containing 10 U ml−1 bacitracin without apparent growth inhibition (Ohki et al., 2003). As shown in Fig. 1C and D, bceAB mRNA levels increased by more than 50-fold upon addition of 10 U ml−1 bacitracin to the growth medium. This finding suggests that the increase in bacitracin resistance is induced as a result of increased bceAB expression. Conversely, bceRS mRNA levels were decreased under the same growth conditions (Fig. 1A and B).
Overexpression of BceR in a mutant of the sensor kinase gene, bceS (BCESd), results in increased expression of the bceAB operon and restores bacitracin resistance
Overproduction of response regulators in the absence of cognate sensor kinases may result in the constitutive expression of regulator target genes (Kobayashi et al., 2001; Ogura et al., 2001). We transformed wild-type and bceS-disrupted (BCESd) strains with a multicopy plasmid, pDG148bceR, expressing bceR from the IPTG-inducible spac promoter (Kobayashi et al., 2001). Northern hybridization analyses showed that, in the absence of IPTG, the cellular level of bceAB mRNA was low in both wild-type and BCESd strains harbouring pDG148bceR. Overexpression of bceR after IPTG addition led to a 50-fold increase in bceAB mRNA in both strains (Supplementary material, Fig. S2). In contrast, no changes were observed in the bceRS mRNA level in wild-type cells harbouring pDG148bceR after IPTG addition. This finding strongly suggests that the BceRS two-component system regulates BceAB transporter expression. Consistently, a bacitracin-hypersensitive phenotype of BCESd was suppressed upon overexpression of BceR in cells harbouring pDG148bceR(Table 2).
Table 2. . Bacitracin resistance of bceS-disrupted strains harbouring a plasmid containing bceR.
. Relative resistance was determined by dividing the IC50 for mutant strains by that for wild-type strain 168. The IC50 value for strain 168 is 39.9 U ml−1.
The cis-acting sequence is required for bceAB expression
To analyse further the regulation of bceAB expression by the BceRS two-component system, we determined the 5′ end of the bceAB transcript by primer extension analysis. Using total RNA from B. subtilis 168 cells grown in the presence of 10 U ml−1 bacitracin and 168 cells harbouring pDG148bceR grown in the presence of IPTG, we identified the transcription start site at 26 bp upstream of the initiation codon for bceA(Fig. 2). Consistent with bceAB transcript expression from early log phase growth (Fig. 1C and D), a −10 consensus sequence for σA, TATAAT, was detected 6 bp upstream of the transcription start site. On the other hand, no clear −35 sequence was observed at the appropriate position from the −10 sequence, which is typical of promoters regulated by transcriptional activators (see Fig. 4C).
Next, to determine the cis-acting region necessary for bceAB transcription, we constructed a series of transcriptional fusion strains with progressively shorter bceAB promoter sequences fused to the lacZ reporter gene at the amyE locus on the B. subtilis chromosome (Fig. 3). Strains were grown with or without 10 U ml−1 bacitracin to early log phase. The β-galactosidase (LacZ) activities of cells grown in the absence of bacitracin were at background levels for all the constructs. However, in cells containing constructs that included at least 81 bp of the upstream region of the bceAB transcription start site, LacZ activity was strongly induced upon addition of bacitracin to the culture medium. Interestingly, deletion of a further 18 bp from the 81 bp sequence completely abolished induction. We also monitored the effect of BceR overexpression in these strains, after the transfection of the pDG148bceR plasmid. Measurement of LacZ activities of cells harbouring pDG148bceR at exponential growth phase in the presence of IPTG revealed that BceR overproduction also induces bceAB promoter activity if the 81 bp sequence is retained. These results indicate that a sequence between −81 and −63 bp upstream of the bceAB transcription initiation site is essential for the induction of bceAB transcription by bacitracin. Moreover, transcription is dependent on the binding of the BceR regulator to this site.
The BceR regulator binds directly upstream of the bceAB promoter
To confirm that BceR indeed binds to the cis-acting sequence required for bceAB expression, the protein was fused to glutathione S-transferase (GST) at the N-terminus, expressed in Escherichia coli and purified to homogeneity using glutathione Sepharose (data not shown). Gel shift experiments showed that the purified protein specifically binds to a DNA fragment containing a sequence from −373 bp to +78 bp relative to the bceAB transcription start site (data not shown). We performed DNase I footprinting experiments to map the BceR binding site precisely. A 279 bp fragment from −200 to +78 bp relative to the bceA transcription start site was labelled radioactively at either end and incubated with increasing amounts of purified GST-BceR protein, followed by partial digestion with DNase I. The results indicate that the cis-regulatory region for bceAB induction identified by promoter deletion experiments is protected by GST-BceR in both coding and non-coding strands (Fig. 4A and B). Regulatory elements for the induction of bceAB transcription are summarized in Fig. 4C. Notably, an inverted repeat sequence, AAGCgTGTGACgaaaatGTCACAtGCTT, overlapping the region protected by DNase I, may be a recognition sequence for BceR.
Expression of bcrC is not regulated by the BceRS two-component regulatory system, and bceA–bcrC, bceB–bcrC and bcrC–bceR double mutants are more sensitive to bacitracin than individual single mutants
Disruption of bcrC, which encodes a homologue of the presumed MSD subunit of the Bacillus licheniformis bacitracin transporter, BcrC, results in hypersensitivity to bacitracin. Expression of bcrC is dependent on σM and σX (Cao and Helmann, 2002; Ohki et al., 2003). If BcrC, BceA and BceB constitute the bacitracin efflux transporter, expression may additionally be modulated by the BceRS two-component regulatory system. However, Northern hybridization analyses revealed that bcrC mRNA ex-pression in wild-type and BCESd strains harbouring pDG148bceR are not affected by IPTG (Fig. 5), indicating that the BceRS two-component regulatory system is not involved in this process.
Next, we examined whether the combined absence of BcrC, BceA and BceB has an additive effect on bacitracin resistance by generating double mutants of BCEAd/bcrC::tet and BCEBd/bcrC::tet. As shown in Table 3, bacitracin resistance was further decreased in double mutants, compared with single mutants. Moreover, bacitracin resistance in a double mutant, bcrC::tet/bceR::cat, decreased to one-tenth that in bceR::cat (data not shown). These results suggest that BcrC and BceAB are involved in different mechanisms of bacitracin resistance in B. subtilis.
Table 3. . Bacitracin sensitivity of bceA/bcrC and bceB/bcrC double mutants.
. Relative resistance was determined by dividing the IC50 for mutant strains by that for bcrC::tet. IC50 value for strain bcrC::tet is 0.68 U ml−1.
Bacitracin is non-ribosomally synthesized in some strains of Bacillus (Johnson et al., 1945; Azevedo et al., 1993) and has potent activity against Gram-positive bacteria (Toscano and Storm, 1982). In this study, we identified four B. subtilis genes involved in bacitracin resistance, specifically bceA, bceB, bceR and bceS. The amino acid sequences of these proteins suggest that BceA and BceB are NBD and MSD subunits, respectively, of an ABC transporter, whereas BceR is a response regulator, and BceS is a sensor histidine kinase of a two-component regulatory system. This study is the first report showing that, in antibiotic non-producing bacteria, expression of the ABC transporter involved in antibiotic resistance is regulated by a two-component transduction system that senses the presence of antibiotic in the extracellular medium.
Bacitracin-producing B. licheniformis contains an ABC transporter that is assumed to mediate bacitracin efflux for self-protection (Podlesek et al., 1995). Furthermore, expression of these proteins is regulated by a two-component system consisting of BacS and BacR (Neumüller et al., 2001). In contrast to BceR of B. subtilis, BacR negatively regulates the expression of bcrAB in B. licheniformis. Additionally, B. subtilis BceRS and BceAB are not closely related in structure to the bacitracin-sensing and -extruding proteins of B. licheniformis. The target site of bacitracin is 55C-isoprenyl pyrophosphate, a carrier of a peptidoglycan unit or a disaccharide with pentapeptide in the membrane. The B. subtilis efflux transporter might use a mechanism similar to the hydrophobic vacuum cleaner model in which the efflux transporter removes bacitracin directly from the lipid bilayer to the extracellular medium (Bolhuins et al., 1996). However, the exact mechanism by which the BceAB efflux transporter removes bacitracin from the target site in the membrane remains to be elucidated.
We demonstrate that the sensor kinase, BceS, responds to extracellular bacitracin and activates the response regulator, BceR, which binds specifically upstream of the bceAB operon to induce transcription of bceAB. An unphosphorylated protein was used in DNase I footprinting experiments. Protection was apparent at high concentrations of protein (more than 500-fold excess with respect to DNA), suggesting that BceR phosphorylation increases affinity for DNA and is required for responses in wild-type cells. However, there is currently no direct evidence on the role of BceR phosphorylation. Recently, Joseph et al. (2002) reported that expression of the B. subtilis ytsCD (bceAB) transporter genes was activated by overexpression of ytsA (bceR), although the biological roles of these genes were not explored in detail.
Notably, no self-induction of bceRS expression was observed upon addition of bacitracin in growth medium or overexpression of BceR. However, a decrease in the level of bceRS mRNA was observed after incubation with bacitracin. The mechanism of this effect of bacitracin requires further clarification.
Our group (Ohki et al., 2003) and that of Cao and Helmann (2002) reported that disruption of the bcrC gene in B. subtilis 168 results in hypersensitivity to bacitracin and that transcription of bcrC is dependent on the ECF σ factors, σM and σX. Here, we show that bcrC expression is independent of the BceRS two-component system. Cao and Helmann (2002) observed the σM-dependent induction of bcrC expression and increased resistance to bacitracin between 2 and 30 min after addition. Significantly, no changes in bcrC mRNA levels were observed after longer incubation periods (more than 2 h) with bacitracin, although increased resistance to bacitracin was maintained (Ohki et al., 2003). These results indicate that the σM-dependent response of bcrC expression is transient. In contrast, the data presented here imply that induction of BceAB expression through the BceRS signal transduction system is consistent. Thus, increased resistance observed in our previous study may have resulted mainly from increased expression of bceAB genes.
The BcrC protein displays 28% identity to BcrC of B. licheniformis and contains seven transmembrane helices. It is proposed that the bcrC gene of B. licheniformis encodes an MSD subunit of the bacitracin efflux transporter, despite the low amino acid sequence homology to known MSD subunits of ABC transporters. This assumption is largely based on the fact that bcrC constitutes an operon with bcrA and bcrB genes encoding typical NBD and MSD subunits, respectively, of an ABC transporter (Podlesek et al., 1995; Neumüller et al., 2001). However, the distinct regulatory mechanisms for bceAB and bcrC expression in B. subtilis suggest that these proteins contribute independently to bacitracin resistance. In support of this hypothesis, bceA/bcrC and bceB/bcrC double mutants are more sensitive to bacitracin than individual single mutants. In this respect, it is interesting that BcrC belongs to the PAP2 superfamily of phosphatases in the Pfam database (Bateman et al., 2002) and is classified as a membrane-associated phospholipid phosphatase in the COG database (http:www.ncbi.nlm.nih.govcgi-binCOG). Further studies are required to determine the precise role of BcrC in bacitracin resistance.
Tsuda et al. (2002) reported recently that disruption of the homologous genes in Streptococcus mutans resulted in a bacitracin-sensitive phenotype. Disruption of mbrA and mbrB genes, which encode the NBD and MSD subunits, respectively, of the ABC transporter, resulted in an approximately 100-fold increase in bacitracin sensitivity. The mbrC and mbrD genes located immediately downstream of mbrAB encode a response regulator and a sensor kinase, respectively, of a two-component regulatory system. Disruption of either mbrC or mbrD additionally causes a decrease in bacitracin resistance similar to that observed in mbrA and mbrB mutants.
Various peptide antibiotics are synthesized in different bacteria. These organisms need to evolve defence systems against antibiotics produced by themselves or others. Clusters of the two-component system and ABC transporter genes that are similar to the B. subtilis bceRS and bceAB, respectively, are widespread in the Bacillus/Clostridium group (Joseph et al., 2002). Many of these genes may be involved in defence against peptide antibiotics. Identification of the substrates sensed and excluded by these molecules is important for our understanding of the evolution of bacterial communities.
The PCR primers used in this study are listed in Supplementary material (Table S1).
Construction of mutant strains and plasmids
The pMutin insertion mutants, BCESd, BCEAd, BCEBd and BCRCd, were constructed by members of the Japan and European Union consortia of B. subtilis functional genomics (Kobayashi et al., 2003). The bceR::pMutin2 (BCERd) and bceR::cat mutants were constructed in this study. To generate double mutants of bceR and bcrC (BCRCd/bceR::cat), the bceR gene was replaced with a chloramphenicol-resistant gene (cat). Next, BCRCd was transformed with chromosomal DNA prepared from bceR::cat, and transformants were selected for chloramphenichol and erythromycin resistance. To obtain double mutants BCEAd/bcrC::tet and BCEBd/bcrC::tet, an erythromycin-resistant gene of BCRCd was disrupted by the insertion of a tetracycline-resistant gene (bcrC::tet) and transformed with chromosomal DNA prepared from BCEAd and BCEBd with selection for erythromycin and tetracycline resistance. pDG148bceR (ytsA) was used to overexpress BceR in B. subtilis cells, as described previously (Kobayashi et al., 2001).
To construct strains with transcriptional fusion of the gradually deleted bceAB promoter and the lacZ reporter gene at the amyE locus on the B. subtilis chromosome, pDL2 (Fukuchi et al., 2000) was used. The eight polymerase chain reaction (PCR) products obtained with a reverse primer, GYR11, and forward primers, GYF03, GYF04, GYF05, GYF06, GYF07, GYF08, GYF09 and GYF10, were digested at the EcoRI and BamHI sites incorporated within the primers and cloned between the corresponding sites of pDL2 to generate pGY001, pGY002, pGY003, pGY004, pGY005, pGY006, pGY007 and pGY008 respectively. The pDL2 derivatives were linearized by PstI digestion and used to transform B. subtilis 168 cells by double cross-over with selection for chloramphenicol resistance to obtain BSGY001, BSGY002, BSGY003, BSGY004, BSGY005, BSGY006, BSGY007 and BSGY008 respectively.
To express the BceR regulator (from the initiation to the termination codon) in E. coli as a fusion with the GST protein at the N-terminus, the entire coding sequence of bceR was amplified by PCR using GYF12 and GYR13 primers. The amplified fragment was digested at the EcoRI and SalI sites within the primers and inserted between the corresponding sites of the pGEX-4T-1 plasmid (Amersham Pharmacia Biotech) to obtain pGEX-bceR.
Bacterial strains were cultured in LB medium (10 g l−1 polypeptone, 5 g l−1 yeast extract, 5 g l−1 NaCl, pH 7.2). Where necessary, IPTG (0.5 mM) and antibiotics were added to medium, specifically, ampicillin (100 µg ml−1), chloramphenicol (20 µg ml−1 for E. coli, 5 µg ml−1 for B. subtilis), kanamycin (20 µg ml−1 for E. coli, 10 µg ml−1 for B. subtilis) and erythromycin (100 µg ml−1 for E. coli, 1 µg ml−1 for B. subtilis). Transformation of B. subtilis cells was performed as described previously (Moriya et al., 1998).
Northern hybridization analysis
Total RNA was isolated from cells grown in LB medium to early (OD530, 0.5), middle (OD530, 1.2) or late (OD530, 2.0) log phase. Cells were resuspended in SET buffer (20% sucrose, 50 mM EDTA, 50 mM Tris-HCl, pH 7.6) containing 2 mg ml−1 lysozyme and incubated on ice for 10 min. RNA was extracted with hot phenol according to the procedure of Aiba et al. (1981). Total RNA (5 µg) was electrophoresed on a 1.5% formaldehyde–agarose gel and blotted onto a nylon membrane. DNA fragments amplified by PCR using the following primers were used as probes: bceS, BceSF, BceSR; bceR, BceRF, BceRR; bceA, BceAF, BceAR; bceB, BceBF, BceBR; bcrC, BcrCF, BcrCR. Probes were labelled with [α-32P]-dCTP using the Multiprime labelling system (Amersham Pharmacia Biotech). Hybridization was performed at 42°C as described previously (Ohki et al., 2003).
Assay of the bacitracin-resistant phenotype
Bacitracin (Sigma; 70 U mg−1) resistance was assayed in LB medium, as described in an earlier report (Ohki et al., 2003). To calculate the relative resistance for various mutants, the concentration of bacitracin leading to 50% inhibition of growth (IC50) was expressed as a ratio, relative to the IC50 value in the control strain.
Primer extension analysis
Total RNA was extracted from B. subtilis 168 cells grown in LB medium (OD600, 0.5) with and without bacitracin, and from 168 cells harbouring pDG148bceR grown in LB medium with IPTG (OD600, 0.5), using the method of Igo and Losick (1986). The GYR02 primer was labelled with [γ-32P]-ATP (Amersham Pharmacia Biotech) using T4 polynucleotide kinase (Takara Shuzo). RNA (10 µg) was incubated with the end-labelled primer (0.5 pmol) for 60 min at 60°C. Reverse transcription was performed for 60 min at 42°C using a Superscript reverse transcriptase, according to the manufacturer's instructions (Gibco BRL). The cDNA products were electrophoresed through an 8% polyacrylamide–urea gel. Radioactive bands were detected with an imaging plate and a BAS2500 scanner (Fuji Photo Film). DNA ladders for use as size markers were created with the same end-labelled primer and a cycle sequencing kit (Takara Shuzo). Primers GYF01 and GYR02 were used to amplify template DNA for the sequencing reaction.
Bacillus subtilis cells in exponential phase of growth were collected by centrifugation, and β-galactosidase activity was assayed according to the method of Youngman et al. (1985). One unit was defined as 1 pmol of 4-methylumbelliferyl β-D-galactoside hydrolysed in 1 min per mg of protein. Protein concentration was determined using the Bio-Rad protein assay kit. All results are presented as mean values of three assays.
Purification of GST-BceR
Escherichia coli BL21(DE3)pLysS cells (Novagen) harbouring pGEX-bceR were incubated at 30°C in 100 ml of LB medium containing ampicillin. At an OD600 of 0.7, IPTG was added to a final concentration of 1 mM. Cells were incubated for another 3 h and harvested by centrifugation. Collected cells were washed with phosphate-buffered saline (PBS; 140 mM NaCl, 2.7 mM KCl, 10.1 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.3) and resuspended in 20 ml of the same buffer. Cells were sonicated, and the lysate was centrifuged at 8000 r.p.m. for 30 min at 4°C. The supernatant fraction was mixed with glutathione Sepharose beads (Amersham Pharmacia Biotech). Beads were washed three times with 10 ml of PBS. GST-BceR was eluted with 1 ml of 10 mM gluthathione in 50 mM Tris-HCl (pH 8.0).
DNase I footprinting assays
A 279 bp fragment encompassing −200 to +78 bp relative to the bceA transcription start site was amplified by PCR using GYF14 and GYR11 primers, and subsequently used as a probe for DNase I footprinting assays. To prepare one-strand labelled probes, one of the primers was initially end-labelled with [γ-32P]-ATP. A constant amount of probe (300 000 c.p.m., 9.25 fmol) and 5 µg of poly-(dI–dC) as competitor DNA were incubated with 5, 10 or 15 pmol of GST-BceR (100, 200 and 300 nM respectively) in 50 µl of binding buffer (50 mM PIPES, 200 mM NaCl, 4 mM MgCl2, 4 mM dithiothreitol, 0.5% Tween 20 and 10% glycerol) at 25°C for 30 min. DNase I (0.14 U; Takara Shuzo) was added to the reaction mixture. After incubation for 1 min at 20°C, the reaction was stopped by adding 100 µl of 20 mM EDTA. The mixture was subjected to phenol–chloroform–isoamyl alcohol (24:24:1) extraction and ethanol precipitation. The pellet was dissolved in loading buffer (0.01% bromophenol blue, 0.01% xylene cyanol and 1 mM EDTA in 90% formamide) and separated on a denaturing 6% polyacrylamide gel. DNA ladders for use as size markers were created with the appropriate end-labelled primer and a cycle sequencing kit (Takara Shuzo). After electrophoresis, radioactive bands were detected with an imaging plate and a BAS2500 scanner.
We are grateful to members of the Japan and EU consortia of B. subtilis functional genomics for providing the pMutin mutants used in this study. We also thank S. Dusko Ehrlich for valuable discussions. This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas, Genome Biology, from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
Fig. S1. Northern hybridization analysis of bceB and bceS transcripts from the spac promoter in BCEAd and BCERd mutants respectively. Overnight cultures of BCEAd (A) and BCERd (B) mutants grown in LB medium were inoculated into LB medium (OD530, 0.05) with or without 0.5 mM IPTG. Samples were withdrawn during early (OD530, 0. 5; lanes 1 and 3) and middle (OD530, 1.2; lanes 2 and 4) log phase growth. Total RNA (5 µg) was loaded per lane. 32P-labelled bceB (A) and bceS (B) were used as indicated.
Fig. S2. Northern hybridization analysis of RNA prepared from wild-type and bceS-disrupted mutant strains harbouring a plasmid containing bceR. A 32P-labelled bceA probe was used.