The production of the peptide antibiotic (lantibiotic) subtilin in Bacillus subtilis ATCC 6633 is highly regulated. Transcriptional organization and regulation of the subtilin gene cluster encompassing 11 genes was characterized. Two polycistronic mRNAs encoding transcript spaBTC (6.8 kb) and encoding transcript spaIFEG (3.5 kb) as well as the monocistronic spaS (0.3 kb) mRNA were shown by Northern hybridization. Primer extension experiments and β-galactosidase fusions confirmed three independent promoter sites preceding genes spaB, spaS and spaI. β-Galactosidase expression of spaB, spaS and spaI promoter lacZ fusions initiated in mid-exponential growth. Maximal activities were reached at the transition to stationary growth and were collinear with subtilin production. The lacZ activity was dependent on co-expression with the two-component regulatory system spaRK. The presence of subtilin was needed for efficient expression of all three promoter lacZ fusions. This suggests a transcriptional autoregulation according to a quorum-sensing mechanism with subtilin as autoinducer and signal transduction via SpaRK. Additionally, spaR expression was found to be under positive control of the alternative sigma factor H. Deletion of sigma H strongly decreased subtilin production. Full subtilin production could be restored after in-trans complementation of spaR. Deletion of the major B. subtilis transition state regulator AbrB strongly increased subtilin production. These results show that the spaRK two-component regulatory system, and hence subtilin biosynthesis and immunity, is under dual control of two independent regulatory systems: autoinduction via subtilin and transcriptional regulation via sigma factor H.
Here, we report on the regulation of subtilin biosynthesis and immunity, which is closely related to nisin with respect to autoinduction, but is more complex with respect to the regulation of the spaRK two-component regulatory system whose expression is pleiotropically controlled by the major transition state regulator AbrB and sigma factor H.
Transcription units of the spa gene cluster
The transcriptional organization of the subtilin gene cluster and the expression of genes spaB, T, C, S, I, F, E, G, R and K (Fig. 1) was studied in B. subtilis ATCC 6633 at various growth phases. Total RNA was isolated at different time-points from the wild type and as a control from deletion mutants of spaS, spaB and spaI.
With spaB as a probe for Northern hybridization, an intensive band of 6.8 kb was obtained with RNA isolated in the mid-logarithmic growth phase (4–5 h after inoculation; Fig. 2B, lanes 5–6). This signal disappeared after 5 h and was also no longer present in RNA samples taken later then 6 h (Fig. 2B, lanes 7–8). No such signal was found with RNA isolated from the ΔspaB mutant (Fig. 2B, lanes 1–4). The large transcript is in accordance with a polycistronic mRNA compassing the three genes spaB, spaT and spaC (theoretically 6.2 kb).
Northern hybridization using a spaS-specific probe revealed a band of 0.35 kb (Fig. 2C). Here, the most intensive band was detected after 5 h of growth. The transcript was not observed using RNA of a ΔspaS strain (Klein et al., 1992). Therefore, the structural gene of subtilin is located on a small transcript, which confirms previous findings (Banerjee and Hansen, 1988).
To identify the transcript of the immunity genes, probes were directed against spaI and spaE (Fig. 2D). Using wild-type RNA, a transcript of 3.5 kb was obtained in both cases, which is in accordance with a polycistronic RNA transcript comprising all four immunity genes spaI, spaF, spaE and spaG (theoretically 3 kb). As a control, RNA isolated from a ΔspaI deletion mutant was used and revealed no respective signal with the spaI probe. The spaE probe also provided no signal, because of a transcription terminator behind the chloramphenicol cassette, which was used for spaI deletion (Klein and Entian, 1994). Unspecific hybridization of spaI/spaE probes with 23S and 16S B. subtilis rRNA gave signals at 1.5 and 2.9 kb (theoretically 2.96 and 1.54 kb respectively).
To determine the 5′-start sites of the spa gene transcripts, total RNA of B. subtilis ATCC 6633 was isolated after 5 h of growth, where a maximal Northern signal was observed. For spaS primer extension, an oligonucleotide complementary to the coding strand of spaS was used (see Table 2). Assuming that the most intensive band is the primer extension product, transcription of the spaS gene initiates at the T residue 74 bases upstream of the ATG start codon (Fig. 3A). For spaI, the most intensive signal corresponds to a primer extension product starting at an A residue 144 bases upstream the spaI start codon (Fig. 3B). Although no clear consensus sequences were found, comparison of the promoters of spaS and spaI with that of spaB (Chung and Hansen, 1992) revealed some similarities within the –10 and –35 region (Fig. 3C).
Primer sequences are given in the direction 5′-3′; changed nucleotides are given in bold face.
spaR in pDG148
spaRK in pDG148
spaI promoter region
spaS probe for Northern hybridization
spaI probe for Northern hybridization
spaE probe for Northern hybridization
primer extension spaS
primer extension spaI
Expression of spa genes
Growth phase dependent expression of lacZ under control of different spa promoters (Fig. 1, Table 1) was investigated after chromosomal integration into the amyE locus of subtilin-producing strain B. subtilis ATCC 6633. As control a promoterless lacZ gene was integrated at the amyE locus (strain 33-Z) without any effects (Fig. 4A).
The lacZ expression under control of spaB-, spaS- or spaI promoters was strongly dependent on the growth phase and coincided with the intensity of the Northern hybridization signals (see Fig. 2). There was no lacZ activity in the early growth phase; expression initiated in the mid-exponential growth and reached maximal β-galactosidase activities at the transition to the stationary growth phase. Interestingly, for spaS and spaB, maximal activities were observed after 6 h, whereas in the case of spaI maximal induction was reached 1–2 h later (Fig. 4A).
To study the influence of spaR on spa gene transcription, lacZ expressions under control of the spa promoters were investigated in B. subtilis MO1099, which originally contains no genes from the subtilin gene cluster. No β-galactosidase activity was observed without IPTG-induced spaR expression. After SpaR induction (with IPTG via Pspac) the lacZ activity strongly increased (Fig. 4B and C). The decrease of activity in the stationary phase is most likely as a result of sporulation of the cells in DSM medium. The expression of spaS and spaI promoter lacZ fusions in B. subtilis MO1099 (Fig. 4C) led to significant higher β-galactosidase activities as compared with expression of the same constructs in B. subtilis ATCC 6633 (Fig. 4A). This is as a result of a higher copy number of plasmid-encoded SpaR in the MO1099 derivatives. Furthermore, the influence of spaK coexpression was investigated as exemplified for the spaI-lacZ construct (strain 99-I-RK) in Fig. 4D. Coexpression of spaR with spaK under the control of the inducible Pspac promoter did not further increase the lacZ activity. This shows that under conditions of strongly induced SpaR, there is no necessity for the SpaK histidine kinase; a phenomenon which has been also described for two-component regulator proteins OmpR in Escherichia coli (Forst et al., 1990) and ComA for B. subtilis (Weinrauch et al., 1990).
Autoinduction of subtilin biosynthesis and immunity
As shown here, subtilin biosynthesis and subtilin immunity require the two-component signal transduction system spaRK. To investigate if subtilin provides the signal for the SpaRK activation, we analysed spa-promoter lacZ fusions in the non-producing strain B. subtilis MO1099, under semi-induced conditions of the Pspac spaRK genes. As shown in Fig. 5A, for the spaS promoter construct (strain 99-S2-RK), no activity was obtained without spaRK induction (no IPTG). At partially induced conditions (0.1 mM IPTG), extracellular addition of subtilin (100 μg l−1) induced β-galactosidase activity twofold. No further increase of activity was obtained with higher subtilin concentrations, which reflects saturation of spaS gene transcription. Similar results were obtained with spaB- and spaI-promoter lacZ fusions. However, spaI and spaB promoter lacZ fusions were further improved even after stronger induction of Pspac spaRK (0.1 and 0.5 mM respectively). Promoters spaB and spaI were obviously more sensitive to subtilin autoinduction than the spaS structural gene.
These data indicate that subtilin acts as a pheromone for the expression of its own structural gene (autoinduction) and for expression of biosynthetic (spaBTC) and subtilin immunity (spaIFEG) genes.
To prove that subtilin autoinduction also occurs in the producer strain B. subtilis ATCC 6633, we determined the amount of SpaC protein in the wild type and in a ΔspaS mutant after induction with sublethal amounts of subtilin (0.3 mg l−1). Lysates of stationary-grown cells were analysed using SDS–PAGE and immunoblotting. Wild-type B. subtilis ATCC 6633 revealed an intensive band for SpaC, whose intensity is similar in both cases, with and without subtilin induction (Fig. 6, lanes 1–2). For the ΔspaS strain, a weak SpaC signal was observed without induction of subtilin (Fig. 6, lane 4), whose intensity increased fourfold in the presence of subtilin (Fig. 6, lane 3). However, the slight SpaC band in the ΔspaS cells indicated that, even without subtilin in the extracellular medium, spa genes were expressed at a low level. As control, the spaB mutant showed no signal for SpaC because a transcription terminator behind the chloramphenicol cassette disrupting the spaB gene (Klein et al., 1992) blocks expression of the following genes spaT and spaC.
To analyse the effect of abrB and sigH mutations on subtilin production, a ΔabrB deletion mutant was constructed after transforming competent B. subtilis ATCC 6633 cells with chromosomal DNA of strain TT71 (abrB::neo) and selecting on neomycin resistance and subtilin production. For sigH disruption, the gene was replaced after insertion of a chloramphenicol cassette (see Experimental procedures and Table 1).
As indicated in Fig. 7, subtilin production strongly increased in an ΔabrB deletion mutant, whereas subtilin production reached only 5% of the wild type in a ΔsigH mutant. This clearly showed that subtilin biosynthesis is negatively regulated via AbrB and needs sigma H for its expression.
Whereas the promoters of spaS, spaBTC and spaIFEG contain consensus motifs for sigma A-dependent transcription (Moran et al., 1982; Jarmer et al., 2001), a sigma H consensus motifs was found in the spaRK promoter. To investigate the possible function of sigma H on the expression of spaR, the respective spaR promoter was replaced by the sigma H independent constitutive spo promoter. After transformation with the constitutive spaR gene, subtilin production was completely restored in a sigH mutant of B. subtilis ATCC 6633 (Fig. 7A). Western blot analysis provided a clear SpaR signal in the wild type but not in the sigH mutant (Fig. 7C), which provided evidence that spaRK transcription is sigma H-dependent.
Production of the lantibiotic subtilin in B. subtilis ATCC 6633 starts in the middle of the logarithmic growth phase and reaches its maximum in the stationary phase (Gutowski-Eckel et al., 1994). Using Northern analysis, two polycistronic mRNAs encoding genes spaB, spaT and spaC (6.8 kb), and spaI, spaF, spaE and spaG (3.5 kb), were identified. The subtilin structural gene, spaS, was transcribed as a single transcript of 0.35 kb. Primer extension analyses identified the –10 and –35 regions of spaS and the spaBTC operon which revealed perfect consensus sequences for B. subtilis ATCC 6633 sigma factor A (Moran et al., 1982; Jarmer et al., 2001). The transcription start site of spaS was identified 74 bp upstream of the translational start codon ATG which is different to the start site previously described 223 bp upstream of the start codon (Banerjee and Hansen, 1988). To investigate this discrepancy, two spaS promoter lacZ- fusions were constructed. Expression of β-galactosidase from both constructs, which either contained (33-S3) or lacked (33-S2) the previously described promoter region, was completely functional. This provides strong evidence for the –74 position to be sufficient for regulated spaS transcription.
The polycistronic transcriptional units identified here coincide with the proteins within the multimeric protein complexes for lantibiotic biosynthesis (SpaC, SpaT, SpaB) and immunity (SpaF, SpaE, SpaG), which is useful for the stoichiometric synthesis of the subunits. The structural gene spaS is much more strongly transcribed than the gene cluster encoding the lanthionine–synthetase complex, which ensures that the amount of substrate (presubtilin) strongly exceeds the amount of enzyme (lanthionine–synthetase complex). The expression of immunity genes was delayed as compared with the biosynthetic genes. This ensures that subtilin immunity is established when the amount of subtilin reaches a critical concentration for the producing strain.
To study spa gene expression independent of subtilin production, promoter lacZ fusions were expressed in B. subtilis MO1099, a strain lacking all subtilin genes. Here, gene expression was studied in the presence or absence of spaR, the regulator protein of the two-component regu-latory system spaRK. From the data presented, we conclude that spa gene expression depends on coexpression with spaR. Under conditions in which SpaR expression was semi-induced, we could show that subtilin further induced spaS and spaB transcription. A concentration of less than 100 μg l−1 subtilin was sufficient to induce spaS, spaBTC and SpaIFEG expression. The biosynthetic and immunity genes were significantly more sensitive to subtilin induction as compared with spaS. This is possibly as a result of a much lower expression rate of spaBTC and spaIFEG, which makes these operons more susceptible to further stimulation.
This shows that subtilin acts as an autoinducer for its own biosynthesis. This is similar as previously described for nisin biosynthesis and immunity, in which nisin was identified as the signal inducing its own biosynthesis and immunity (Kuipers et al., 1995; de Ruyter et al., 1996). Although the genetic components of subtilin and nisin biosynthesis and immunity are very specific for the respective lantibiotic, this result shows that the genetic organization and the regulation of the two lantibiotics is closely related.
Most probably the histidine kinase SpaK represents the subtilin sensor, which transfers the signal to the regulatory (DNA binding) SpaR protein. SpaK contains two transmembrane domains and characteristic sequence motifs, which strongly suggest a membrane localization (Klein et al., 1993). After sensing extracellular subtilin, SpaK is probably autophosphorylated at a conserved histidine residue. Subsequently, the response regulator SpaR is activated by transfer of the phosphoryl group to a conserved asparagine residue, by which SpaR is able to bind to promoter elements and induce gene transcription. A repeated pentanucleotide motif WTGAT (W = A/T) spaced by six basepairs was recently identified as the DNA sequence recognized by SpaR, which is present in front of each transcriptional unit (T. Stein, S. Heinzmann, P. Kiesau and K.-D. Entian, manuscript in preparation).
Interestingly, we found subtilin production under positive control of B. subtilis sigma factor SigH. This alter-native sigma factor represents a typical regulator of late-growth activities (Carter and Moran, 1986; Dubnau et al., 1988) affecting a broad range of genes involved in sporulation, competence development, motility and production of degradative enzymes. Additionally, sigma factor H is required for transcription of stress-inducible genes (Solomon et al., 1995). Deletion of sigH in B. subtilis ATCC 6633 diminished subtilin production by 95%. Transcription of sigH is under negative control of the general transition state regulator AbrB (Weir et al., 1991; Strauch, 1995), whose synthesis is repressed by the key regulator of late-growth processes, Spo0A (Strauch et al., 1990). Consequently, a abrB deletion mutant revealed a fourfold increased subtilin production and production started already in the logarithmic growth phase.
As the target for AbrB/sigma H-dependent regulation, spaRK was identified. This was indirectly shown by restored subtilin production in a ΔsigH mutant after constitutive expression of SpaR and directly proven by the absence of the SpaR protein in a ΔsigH mutant. However, an indirect effect of the global B. subtilis regulators AbrB/sigma H on subtilin production (via SpaR) cannot be excluded.
Subtilin autoinduction shows characteristic properties of quorum-sensing systems, which have been described in several bacteria for important physiological processes, for example, competence (Tortosa et al., 2001), sporulation, motility, biofilm formation, bioluminescence and virulence (Kleerebezem and Quadri, 2001). In Bacillus subtilis, quorum sensing by autoinducing peptides was described for phosphatase regulation (phr) (Lazazzera, 2001; Perego and Brannigan, 2001) controlled by sigma factor H (McQuade et al., 2001) and also for ComX, a natural competence regulating pheromone (Solomon et al., 1995; Tortosa et al., 2001). For nisin of L. lactis, autoinduction of the two-component system nisR and nisK was also discussed in the light of a quorum-sensing mechanism (Kuipers et al., 1995; de Ruyter et al., 1996), although the strong definition of quorum sensing allows bacteria to sense their population density and to respond through appropriate changes in gene expression (Kleerebezem et al., 1997). The AbrB-dependent regulation for subtilin shows the interaction of growth phase-dependent regulation and autoinduction. Quorum sensing with subtilin would also enable producing cells to communicate with neighbouring cells, for example to signal/ sense non-ideal conditions, and may provide an example for a B. subtilis peptide antibiotic used as a pheromone for quorum sensing.
In summary, the expression of subtilin biosynthesis and immunity genes is regulated by two different mechanisms (Fig. 8). During exponential growth phase, the transition state regulator protein AbrB acts as a repressor of subtilin biosynthesis and only a basal level of biosynthetic proteins SpaBTC are synthesized (see SpaC content in ΔspaS strain). Consequently, low amounts of subtilin appeared in the culture, which were not sufficient to induce the regulatory cascade. At the end of exponential growth, AbrB synthesis is repressed (Perego et al., 1988; Strauch et al., 1989; Strauch et al., 1990), followed by derepression of the alternative sigma factor H (Weir et al., 1991; Strauch, 1995). Sigma factor induces spaRK transcription and the SpaK histidine kinase is expressed. From a critical concentration, subtilin acts via SpaRK as inducer of spaS and spaBTC transcription, resulting in drastically increased subtilin production. Additionally, the immunity system spaIFEG is induced to protect the producer from the lethal activity of its own lantibiotic. The dual control tunes subtilin production and the physiological state of the cell.
Bacterial strains, growth conditions and construction of plasmids
Bacillus subtilis cells were grown in Difco sporulation medium (DSM, Difco; Harwood and Cutting, 1990) and TY medium (0.8% tryptone, 0.5% yeast extract, 0.5% NaCl). Escherichia coli strains were grown on Luria–Bertani (LB) medium (Gibco). The concentrations of antibiotics used were: ampicillin (40 μg ml−1) and chloramphenicol (20 μg ml−1) for E. coli, and chloramphenicol (5 μg ml−1), spectinomycin (25 μg ml−1), neomycin (7 μg ml−1), phleomycin (5 μg ml−1) and kanamycin (10 μg ml−1) for B. subtilis. The B. subtilis strains used in this study are listed in Table 1; a schematic representation of the different strains is given in Fig. 1. EcoRI–BamHI (BamHI–HindIII in the case of spaI) fragments of putative spa gene promoters were amplified by polymerase chain reaction (PCR) (primer sequences are detailed in Table 2) and cloned into pDG268 (Antoniewski et al., 1990), which contains a promoterless lacZ gene, an upstream ribosomal binding-site for B. subtilis and a chloramphenicol resistance cassette for chromosomal integration into B. subtilis amyE locus (Table 2). Promoter regions consisted of promoter regions of spaB comprising 251 bp, the promoter region of spaS with two different lengths comprising 110 and 202 bp, the spaI promoter (486 bp), and the putative spaR promoter (497 bp). Linearized plasmids were used to transform competent cells of B. subtilis ATCC 6633 and MO1099; resulting strains are summarized in Table 1. Correct integration of the spa promoter lacZ fusions were analysed by PCR. Recombinant plasmids were amplified in E. coli RR1 cells. For expression of spaR and spaRK, genes were PCR-amplified and cloned into pDG148 (Stragier et al., 1988) under control of an IPTG-inducible promoter (Pspac).
Molecular biology techniques
Established protocols were followed for molecular biology techniques (Sambrook et al., 1989). DNA was cleaved according to the conditions recommended by the commercial supplier of the restriction enzymes (Boehringer GmbH). Restriction endonuclease-digested DNA was eluted from 0.7% agarose gels by the freeze-squeeze method.
Plasmid isolation and PCR
The alkaline extraction procedure (Birnboim and Doly, 1979) was followed for E. coli plasmid isolation. PCR was carried out following standard procedures (Sambrook et al., 1989) in a Hybaid combi-thermal-reactor R2, Taq DNA polymerase (Boehringer Mannheim) was used.
Total RNA of B. subtilis ATCC 6633 was isolated by the acid/phenol method (Volker et al., 1994). For primer extension analysis, RNA was isolated from cells at the end of exponential growth. To exclude contamination by DNA, each sample was treated by DNase I (2 U per μg RNA, Pharmacia, Biotech) in assay buffer (40 mM Tris-HCl pH 7.5, 60 mM MgCl2) for 10 min at 37°C. After additional phenol/ chloroform extraction and ethanol precipitation, the RNA was dissolved in 20 μl of water. RNA concentrations were measured photometrically at 260 nm or estimated by comparison with 16 S and 23 S RNA band intensities.
A total of 30 μg of RNA was fractionated on a 1.5% agarose gel containing formaldehyde (final concentration 1.2%) and stained with ethidium bromide. After washing with water (15 min), and five times with 10× standard saline citrate (SSC: 1.5 M NaCl, 150 mM sodium citrate, pH 7) RNA was transferred to Biodyne B transfer membrane (Hybond-N). RNA was cross-linked to the dried membrane by UV light (312 nm, 7 min) after briefly rinsing with 2× SSC buffer. The spaB probe (1066 bp) was obtained after EcoRI clearage of plas-mid pCE42 (Klein et al., 1992). For spaS, spaI, and spaE probes, PCR-amplified DNA was used (550, 300 and 300 bp respectively; for primers see Table 2). DNA probes were labelled with [γ-32P]-dCTP using the Ready-to-go-kit (Pharmacia Biotech). For hybridization, membranes were incubated for 3 h in a solution containing 5× SSC, 1× blocking agent (Boehringer), 0.02% (w/v) sodium dodecyl sulphate (SDS) and 0.1% (w/v) N-laurylsarcosine at 42°C. Hybridization was performed with a denatured DNA probe in the same buffer overnight. Hybridized blots were washed in 5× SSC buffer containing 0.01% (w/v) SDS. Radioactivity was detected using autoradiography.
Primer extension analysis
Primer extension experiments were performed according to (Sambrook et al., 1989). A total of 5 μg of B. subtilis RNA was incubated with 10 pmol oligonucleotide primer (Table 2) and 20 U RNase inhibitor (Boehringer Mannheim) for 5 min at 96°C. After annealing (cool down to 42°C in 60 min), 5 mM DTT, 2 μl of 10-fold reaction buffer (500 mM Tris-HCl, pH 8.3, 60 mM MgCl2, 400 mM KCl, and 500 μg ml−1 BSA) were added. To 20 μl of this reaction mixture, 20 U RNase inhibitor, 10 μCi [α-32P]-dCTP, 7.5 μM dNTP-mix without dCTP and 14.5 U AMVRT (reverse transcriptase from Avian Myeloblastosis virus; AGS GmbH) were added. After 5 min of pre-incubation at 42°C, the dNTP mix was added to a final concentration of 23 mM, and the incubation was extended to 25 min at 42°C. By adding 5 μl of stop-mix (95% formamid, 20 mM EDTA, 0.05% bromophenol blue, 0.05 xylen cyanol FF) and a denaturating step (5 min, 96°C), the reaction was stopped. Primer extension reaction and sequencing reactions with the corresponding primer were run on an 8% polyacrylamide gel, radioactivity was detected by autoradiography by exposure of dried gels to Kodak X-Omat-RP films at –80°C.
Histochemical screening for β-galactosidase activity by selecting for blue colonies was performed by including isopropyl-β-D-thiogalactoside (IPTG) at a final concentration of 50 μg ml−1 in DSM plates. For quantitative β-galactosidase activities, standard methods were used (Zuber and Losick, 1983; Miller, 1992). A total of 200 ml of TY/DSM medium was inoculated (1:100) with an overnight culture of B. subtilis cells carrying the reporter gene. Then,1 ml samples were taken at different times of the growth curve, sedimented and washed with 1 ml of 50 mM Tris-HCl (pH = 8.0). Cells were solved in 1 ml Z-buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4, 50 mM β-mercaptoethanol, pH 7.0). After appropriate dilution in 1 ml of the same buffer, the cells were permeabilized by adding 20 μl of toluene. After removal of toluene (37°C for 45 min), the β-galactosidase reaction was started with 200 μl of o-nitrophenyl-β-D-galactoside (ONPG, 4 mg ml−1 in Z-buffer) at 28°C and stopped with 500 μl of 1 M Na2CO3.
SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and Western blot analysis
SDS–PAGE and Western blot analyses were performed as described previously (Kiesau et al., 1997). Selectivity of the SpaC-directed immunoserum was significantly improved after immunoaffinity purification (Harlow and Lane, 1999) using purified SpaC–GST fusion protein heterologously expressed in E. coli. For SpaR-directed antisera, SpaR was isolated from E. coli cells expressing spaR under control of the T7 promoter. Escherichia coli cell extract (incubation in 100 mM NaPO4, pH 7.0, 0.5%β-mercaptoethanol, 4% SDS, 8 M urea and 0.02% bromophenol blue for 10 min at 95°C) was separated by preparative SDS–PAGE; SpaR was isolated by electroelution and used for rabbit immunization (Eurogentec).
Analysis of subtilin production by reversed phase (RP)-HPLC
Stationary B. subtilis cultures were centrifuged at 10 000 g for 10 min. RP-HPLC analyses of the supernatant were performed by means of a Beckman Gold HPLC System, a semi-preparative reversed phase Lichrospher column (particle size: 10 μm, width and length: 4 × 200 mm, Merck) and an analytical ODS Hypersil column (particle size: 5 μm, width and length: 4 × 300 mm, Maisch). Samples were applied using solvent A (aqueous solution of 20% acetonitrile and 0.1% trifluoroacetic v/v). Elution was performed using linear gradients to solvent B (0.1% trifluoroacetic acid in acetonitrile v/v). Eluting peptides were detected measuring the absorption at 214 nm.