Lantibiotics, such as nisin and subtilin, are lanthionine-containing peptides that exhibit antimicrobial as well as pheromone-like autoinducing activity. Autoinduction is specific for each lantibiotic, and reporter systems for nisin and subtilin autoinduction are available. In this report, we used the previously reported subtilin autoinduction bioassay in combination with mass spectrometric analyses to identify the novel subtilin-like lantibiotic entianin from Bacillus subtilis subsp. spizizenii DSM 15029T . Linearization of entianin using Raney nickel-catalyzed reductive cleavage enabled, for the first time, the use of tandem mass spectrometry for the fast and efficient determination of an entire lantibiotic primary structure, including posttranslational modifications. The amino acid sequence determined was verified by DNA sequencing of the etnS structural gene, which confirmed that entianin differs from subtilin at 3 amino acid positions. In contrast to B. subtilis ATCC 6633, which produces only small amounts of unsuccinylated subtilin, B. subtilis DSM 15029 T secretes considerable amounts of unsuccinylated entianin. Entianin was very active against several Gram-positive pathogens, such as Staphylococcus aureus and Enterococcus faecalis. The growth-inhibiting activity of succinylated entianin (S-entianin) was much lower than that of unsuccinylated entianin: a 40-fold higher concentration was required for inhibition. For succinylated subtilin (S-subtilin), a concentration 100-fold higher than that of unsuccinylated entianin was required to inhibit the growth of a B. subtilis test strain. This finding was in accordance with a strongly reduced sensing of cellular envelope stress provided by S-entianin relative to that of entianin. Remarkably, S-entianin and S-subtilin showed considerable autoinduction activity, clearly demonstrating that autoinduction and antibiotic activity underlie different molecular mechanisms.

Background:The periplasmic lipoprotein SpaI protects the subtilin-producing Bacillus subtilis against its own lantibiotic by an unknown mechanism. Results: The first structure of a lantibiotic immunity protein, SpaI, reveals a novel fold and its membrane-interacting regions. Conclusion:The membrane interaction is important for SpaI-mediated immunity. Significance: The SpaI structure will help to understand the immunity of B. subtilis against subtilin on a structural level.

Subtilin and the closely related entianin are class I lantibiotics produced by different subspecies of Bacillus subtilis. Both molecules are ribosomally synthesized peptide antibiotics with unusual ring structures. Subtilin-like lantibiotics develop strong antibiotic activities against various Gram-positive organisms with an efficiency similar to that of nisin from Lactococcus lactis. In contrast to nisin, subtilin-like lantibiotics partially undergo an additional posttranslational modification, where the N-terminal tryptophan residue becomes succinylated, resulting in drastically reduced antibiotic activities. A highly sensitive high-performance liquid chromatography (HPLC)-based quantification method enabled us to determine entianin and succinylated entianin (S-entianin) concentrations in the supernatant during growth. We show that entianin synthesis and the degree of succinylation drastically change with culture conditions. In particular, increasing glucose concentrations resulted in higher entianin amounts and lower proportions of S-entianin in Landy-based media. In contrast, no succinylation was observed in medium A with 10% glucose. Interestingly, glucose retarded the expression of entianin biosynthesis genes. Furthermore, deletion of the transition state regulator AbrB resulted in a 6-fold increased entianin production in medium A with 10% glucose. This shows that entianin biosynthesis in B. subtilis is strongly influenced by glucose, in addition to its regulation by the transition state regulator AbrB. Our results suggest that the mechanism underlying the succinylation of subtilin-like lantibiotics is enzymatically catalyzed and occurs in the extracellular space or at the cellular membrane. Lantibiotics are RiPPs (ribosomally synthesized and posttranslationally modified peptide antibiotics) (1) that contain the nonproteinogenic amino acids lanthionine and 3-methyllanthionine (2). They develop strong antimicrobial activities against various Gram-positive organisms, including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) (3, 4). Subtilin and the closely related entianin and ericin are produced by Bacillus subtilis ATCC 6633 (B6633), B. subtilis DSM 15029 (B15029), and B. subtilis A1/3 (BA1/3), respectively. They are representatives of the class I lantibiotics, for which nisin from Lactococcus lactis is the first-described, most prominent member (5). Class I lantibiotics have the distinction of an elongated flexible configuration, which is important for their toxic interaction with lipid II and subsequent pore formation (6, 7). Class I lantibiotics are further characterized by a common posttranslational modification mechanism, which is, in contrast to lantibiotics of classes II to IV, catalyzed by two distinct enzymes, LanB and LanC (1,8,9). Evidence exists that posttranslational modification of the precursor peptide and its transport out of the cell are mediated by a membrane-associated multimeric synthetase complex, LanBTC, which, in the case of subtilin, co...

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