RESULTS AND DISCUSSION
Antibiotic production by different B. subtilis strains was investigated by performing antimicrobial activity tests, reversed-phase HPLC separation of culture supernatants, and MALDI-TOF mass spectrometry (Fig. (Fig.1).1). For strain ATCC 6633, a representative strain, subtilin and its isoform [N-alpha-succinyl-Trp1]-subtilin are responsible for the main anti-Micrococcus activity. Consequently, the ΔspaS subtilin deletion strain exhibited no subtilin production. For the previously unidentified active antimicrobial compound in peak II (Fig. (Fig.1B)1B) an m/z value of 3400.7 was determined (Fig. (Fig.1C).1C). Both the m/z value and the elution position are consistent with the properties of authentic subtilosin produced by B. subtilis 168 (3, 19). In addition, peak II (subtilosin) was not observed in the supernatant of the ΔspaS/Δsbo double mutant (Fig. (Fig.1B),1B), clearly demonstrating that the newly identified ATCC 6633 bacteriocin is based on sbo gene expression and demonstrating that it is identical to subtilosin. In fractions eluting around 20 min (Fig. (Fig.1B)1B) the lipopeptides surfactin and mycosubtilin were observed. The production of these compounds was not affected in the gene deletion mutants (data not shown). Surprisingly, all eight B. subtilis wild-type strains investigated, including B. atrophaeus (black-pigmented B. subtilis; formerly B. subtilis DSM 2207 or ATCC 51189) (11), that are listed in Table Table11 have been found to produce subtilosin. This finding was unprecedented because most of the known B. subtilis wild-type strains produce individual antibiotic cocktails. For example, subtilin production has been described only for the ATCC 6633 strain, and distinct lipopeptides are produced only by a few individual strains. The widespread occurrence of subtilosin might reflect an important physiological role. As subtilosin is produced at the end of exponential growth, particularly under stress conditions, a specific function of subtilosin as an antibiotic, killing factor (12) or as a pheromone during anaerobic or biofilm growth of B. subtilis (15) has to be considered.
During cloning of B. subtilis ATCC 6633 DNA we observed restriction sites not present in the genome of strain 168 (15), while proposed sites were absent. We sequenced the sbo genes and flanking regions of all investigated B. subtilis wild-type strains in order to analyze the structural basis of these observations and to evaluate possible evolutionary relationships among the subtilosin producers. The main result of a comparison of the sbo alleles was identification of two distinct B. subtilis classes (Fig. (Fig.2A).2A). Class 1 (168-like) includes strains 60015 (Marburg strain), 168, and 10T (type strain), as well as DSM 1088 and DSM 2109. Class 2 (W23-like) comprises strains ATCC 6633 and DSM 618, as well as DSM 6405, a mutant of the W23 strain. This observation is in good agreement with the recent classification of strain 168 as B. subtilis subsp. subtilis and the recent classification of W23-related strains as B. subtilis subsp. spizizenii based on DNA reassociation studies (21).
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Remarkably, the nucleotide sequences of the sbo genes and flanking regions are identical in strains belonging to the same subspecies, and the sequences differ by three nucleotides in the two subspecies (Fig. (Fig.2A).2A). However, the encoded Sbo prepeptides are identical in all cases. Primer extension analyses of sbo transcripts in representatives of both B. subtilis classes revealed transcriptional start sites that are 47 nucleotides (class 2 strain ATCC 6633) and 45 nucleotides (class 1 strain 168) upstream of the start ATG codon of sbo (Fig. (Fig.3).3). Similar 5′ transcriptional start sites are utilized by both B. subtilis classes. Due to a two-nucleotide insertion into class 2 sequences, the sizes of the transcripts differ by two nucleotides. The −10 and −35 regions derived from the transcriptional start sites resemble promoter regions utilized by sigma factor A (Fig. (Fig.2A).2A). Remarkably, within the −10 region two nucleotide substitutions in both B. subtilis classes were observed, which suggests that there is an effect on sbo expression. However, a region upstream of the −35 region (positions −70 to −110) is perfectly conserved. This region represents a perfect sigma factor H binding site; however, involvement of this region in regulation of subtilosin biosynthesis has not been shown yet.
Downstream of sbo a gene cluster with seven open reading frames (ywiA and ywhRQPOMN) has been identified and sequenced in B. subtilis ATCC 6633, a representative of the B. subtilis subsp. spizizenii strains (accession number {“type”:”entrez-nucleotide”,”attrs”:{“text”:”AJ430547″,”term_id”:”20387045″}}AJ430547). The identified gene cluster exhibits a high level of homology to the sbo-alb gene cluster of B. subtilis 168 involved in the biosynthesis of subtilosin, including the structural gene, as well as genes encoding the posttranslational modification machinery and subtilosin immunity proteins. BLAST alignments (Table (Table2)2) revealed that the first four genes are highly conserved with those of B. subtilis subsp. subtilis (96 to 100% amino acid identity), while the remaining four genes are less conserved (83 to 88% identity). These differences reveal incipient evolutionary divergence of the B. subtilis subspecies. This low level of conservation is unprecedented; for example, thymidylate synthases A (thyA) in B. subtilis subsp. spizizenii ATCC 6633 and W23 and B. subtilis subsp. subtilis (168) exhibit more than 95% amino acid identity (32). Even the average level of amino acid identity for the DNA gyrases (gyrA) in seven Bacillus type strains was 95.1% (7).
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The nucleotide sequences of the closely related species and subspecies can be used for identification of genes and highly conserved regions in the gene products putatively corresponding to functional domains. For example, in strain 168 a new gene with an unknown function, sboX, encoding a bacteriocin-like product, was hypothesized (Fig. (Fig.2A)2A) (34), which resides in an open reading frame overlapping the coding region of sbo. Notably, the expression of sboX would result in a 22-amino-acid truncated peptide in W23-like strains compared to the peptide produced by 168-like strains (Fig. (Fig.2B),2B), which makes it unlikely that SboX is produced by W23-like strains.
YwiA (AlbA) is involved in subtilosin biosynthesis, most likely in the posttranslational modification of presubtilosin (34, 35), although its molecular function is unknown. The amino acid sequences of YwiA in the two B. subtilis subspecies (Fig. (Fig.4)4) were compared, and two highly conserved regions (amino acids 1 to 90 and 109 to 450) separated by a less conserved linker region (amino acids 91 to 105) were identified. The large conserved domain from amino acid 109 to amino acid 450 exhibits homology to proteins belonging to the MoaA-NifB-PqqE family, which carry Fe-S centers in their active sites. Also, this cysteine-rich cofactor binding region is conserved in YwiA (core 1), as is a second CXXC motif near the C terminus (core 2). A pattern search for core 2 of YwiA proteins (Fig. (Fig.4)4) revealed homology to arylsulfatases and metallothioneins. Upstream of this signature sequence an unusual sulfur-rich motif (CMXXXC) with an unknown function has been found in YwiA proteins.
As this study revealed, two distinct B. subtilis subspecies (B. subtilis subsp. subtilis and B. subtilis subsp. spizizenii) are distinguishable only on the basis of their sbo genes. Comparisons between the subtilosin gene clusters of the two subspecies led to identification of highly conserved protein domains and also provided insight into incipient evolutionary divergence.
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