Bacillus subtilis is the best-characterized member of the Gram-positive bacteria. Its genome of 4,214,810 base pairs comprises 4,100 protein-coding genes. Of these protein-coding genes, 53% are represented once, while a quarter of the genome corresponds to several gene families that have been greatly expanded by gene duplication, the largest family containing 77 putative ATP-binding transport proteins. In addition, a large proportion of the genetic capacity is devoted to the utilization of a variety of carbon sources, including many plant-derived molecules. The identification of five signal peptidase genes, as well as several genes for components of the secretion apparatus, is important given the capacity of Bacillus strains to secrete large amounts of industrially important enzymes. Many of the genes are involved in the synthesis of secondary metabolites, including antibiotics, that are more typically associated with Streptomyces species. The genome contains at least ten prophages or remnants of prophages, indicating that bacteriophage infection has played an important evolutionary role in horizontal gene transfer, in particular in the propagation of bacterial pathogenesis.

Summary 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.

Bacillus subtilis ATCC 6633 produces the cationic pore-forming lantibiotic subtilin, which preferentially acts on gram-positive microorganisms; self protection of the producer cells is mediated by the four genes spaIFEG. To elucidate the mechanism of subtilin autoimmunity, we transferred different combinations of subtilin immunity genes under the control of an inducible promoter into the genome of subtilin-sensitive host strain B. subtilis MO1099. Recipient cells acquired subtilin tolerance through expression of either spaI or spaFEG, which shows that subtilin immunity is based on two independently acting systems. Cells coordinately expressing all four immunity genes acquired the strongest subtilin protection level. Quantitative in vivo peptide release assays demonstrated that SpaFEG diminished the quantity of cell-associated subtilin, suggesting that SpaFEG transports subtilin molecules from the membrane into the extracellular space. Homology and secondary structure analyses define SpaFEG as a prototype of lantibiotic immunity transporters that fall into the ABC-2 subfamily of multidrug resistance proteins. Membrane localization of the lipoprotein SpaI and specific interaction of SpaI with the cognate lantibiotic subtilin suggest a function of SpaI as a subtilin-intercepting protein. This interpretation was supported by hexahistidine-mediated 0-Å cross-linking between hexahistidinetagged SpaI and subtilin.Bacillus subtilis strain ATCC 6633 produces the cationic peptide antibiotic (lantibiotic) subtilin. Lantibiotics contain unusual thioether amino acids, such as meso-lanthionine and 3-methyl-lanthionine (17), which are incorporated into prepeptides through extensive posttranslational modifications (25,32,41). The subtilin and the closely related ericin gene clusters (35) encompass genes for posttranslational modification (18), transport (18), immunity (20), and regulation (19). Extracellular B. subtilis serine proteases are involved in the final processing step (7, 37). Subtilin biosynthesis and immunity are under the control of the two-component regulatory system SpaK/ SpaR (histidine kinase and response regulator, respectively) and the alternative sigma factor H (36, 38).Lantibiotics act against a wide range of gram-positive bacteria. The antimicrobial action of nisin produced by Lactococcus lactis, a structurally close relative of subtilin, is based on voltage-dependent pore formation that affects the efflux of small molecules and finally the collapse of the proton motive force (for a review see reference 4). The Bacto prenol-bound peptidoglycan precursor lipid II appears to be both a docking molecule assisting membrane targeting (5) and an integral constituent of the lethal pore itself (14). Gram-positive lantibiotic-producing strains need efficient countermeasures to obviate the lethal action of their own products (31). The nisin self protection (immunity) system is composed of ABC transporter homologue NisFEG and lipoprotein NisI (39).In the present study we report on the establishment of subtilin immunit...

A lantibiotic gene cluster was identified inLantibiotics are amphiphilic peptide antibiotics of bacterial origin and are nearly exclusively produced by gram-positive bacteria. They contain unusual constituents like nonproteinogenic didehydroamino acids and lanthionines (49; for reviews, see references 16, 30, 47, and 51). Out of the about 26 known lantibiotics, the nisins (A and Z) of Lactococcus lactis cheese starter organisms (6, 15) are the best-studied members which are also of commercial value (5,14,21,31,33,41). Subtilin was the first lantibiotic isolated from Bacillus subtilis ATCC 6633 (22; for review, see reference 16). A variant of subtilin (subtilin B) was found to have reduced antibiotic activity due to posttranslational succinylation of the amino group of the N-terminal tryptophan residue (7). Sublancin from B. subtilis 168 is quite different and contains a single lanthionine linkage and two disulfide bridges (43). A relative small lantibiotic, mersacidin of Bacillus sp., shows unusual properties with respect to bridging, amphiphilic character, and C-terminal modification (30). Lantibiotics are ribosomally synthesized as precursor peptides consisting of an N-terminal leader and the propeptide sequence. The latter becomes posttranslationally modified by dehydration and thioether formation (49). The biochemistry of these modifications is still unknown but is associated in one group of lantibiotics with proteins LanB and LanC (24,35) and in a second group with LanM (16, 46; for review, see reference 47). A multimeric enzyme complex consisting of LanBTC was demonstrated for subtilin and nisin to be membrane associated and to catalyze modification and transport (32, 50).The B. subtilis strain A1/3 attracted our attention due to a broad spectrum of inhibitory activities against fungi and phytoviruses (28), as well as against diverse bacteria. Notable among these is the causative agent of tomato bacterial canker, Clavibacter michiganensis (20). In this paper we report the discovery of a lantibiotic gene cluster of B. subtilis A1/3, which shows conserved character to subtilin genes but encodes two distinct lantibiotic peptides, ericin S and ericin A. Both ericins were isolated from culture supernatants of B. subtilis A1/3, studied by high-performance liquid chromatography (HPLC), peptidase digestion, and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The complete ericin gene cluster has been sequenced. Mutant studies indicated that both peptides are processed by the LanB homologue EriB. MATERIALS AND METHODSStrains, plasmids, and growth conditions. The original B. subtilis A1/3 (20, 28) contained at least two plasmids. A derivative GB709 of strain A1/3 was cured from plasmid DNA by repeated protoplasting and protoplast regeneration (8) and was used throughout these studies synonymously to A1/3, as it exhibited no detectable phenotypic differences from the parental strain. For antibiotic activity tests the following were used: B. subtilis strains DSM 402 (Spizi...

The deduced amino acid sequence of the gsp gene, located upstream of the 5' end of the gramicidin S operon (grs operon) in BaciUas brevis, showed a high degree of similarity to the sfp gene product, which is located downstream of the srfA operon in B. subtilis. The gsp gene complemented in trans a defect in the sfp gene (s19) and promoted production of the lipopeptide antibiotic surfactin. The functional homology of Gsp and Sfp and the sequence similarity of these two proteins to EntD suggest that the three proteins represent a new class of proteins involved in peptide secretion, in support of a hypothesis published previously (T. H. Grossman, M. Tuckman, S. Ellestad, and M. S. Osburne, J. Bacteriol. 175:6203-6211, 1993).