An antimicrobial peptide produced by Bacillus subtilis 168 was isolated and characterized. It was named sublancin 168, and its behavior during Edman sequence analysis and its NMR spectrum suggested that sublancin is a dehydroalanine-containing lantibiotic. A hybridization probe based on the peptide sequence was used to clone the presublancin gene, which encoded a 56-residue polypeptide consisting of a 19-residue leader segment and a 37-residue mature segment. The mature segment contained one serine, one threonine, and five cysteine residues. Alkylation of mature sublancin showed no free sulfhydryl groups, suggesting that one sulfydryl had formed a ␤-methyllanthionine bridge with a dehydrobutyrine derived by posttranslational modification of threonine; with the other four cysteines forming two disulfide bridges. It is unprecedented for a lantibiotic to contain a disulfide bridge. The sublancin leader was similar to known type AII lantibiotics, containing a double-glycine motif that is typically recognized by dual-function transporters. A protein encoded immediately downstream from the sublancin gene possessed features of a dual-function ABC transporter with a proteolytic domain and an ATP-binding domain. The antimicrobial activity spectrum of sublancin was like other lantibiotics, inhibiting Gram-positive bacteria but not Gram-negative bacteria; and like the lantibiotics nisin and subtilin in its ability to inhibit both bacterial spore outgrowth and vegetative growth. Sublancin is an extraordinarily stable lantibiotic, showing no degradation or inactivation after being stored in aqueous solution at room temperature for 2 years. The fact that sublancin is a natural product of B. subtilis 168, for which a great deal of genetic information is available, including the entire sequence of its genome, suggests that sublancin will be an especially good model for studying the potential of lantibiotics as sources of novel biomaterials.

Nisin is a small gene-encoded antimicrobial protein produced by Lactococcus lactis that contains unusual dehydroalanine and dehydrobutyrine residues. The reactivity of these residues toward nucleophiles was explored by reacting nisin with a variety of mercaptans. The kinetics of reaction with 2-mercaptoethanesulfonate and thioglycolate indicated that the reaction pathway includes a binding step. Reaction of nisin at high pH resulted in the formation of multimeric products, apparently as a result of intramolecular and intermolecular reactions between nucleophilic groups and the dehydro residues. One of the nucleophiles had a PKa of about 9.8. The unique vinyl protons of the dehydro residues that give readily identifiable proton nuclear magnetic resonances were used to observe the addition of nucleophiles to the dehydro moiety. After reaction with nucleophiles, nisin lost its antibiotic activity and no longer showed the dehydro resonances, indicating that the dehydro groups had been modified. The effect of pH on the solubility of nisin was determined; the solubility was quite high at low pH (57 mg/ml at pH 2) and was much lower at high pH (0.25 mg/ml at pH 8 to 12), as measured before significant pH-induced chemical modification had occurred. High-performance liquid chromatography on a C18 column was an effective technique for separating unmodified nisin from its reaction products. The cyanogen bromide cleavage products of nisin were about 90% less active toward inhibition of bacterial spore outgrowth than was native nisin. These results are consistent with earlier observations, which suggested that the dehydro residues of nisin have a role in the mechanism of antibiotic action, in which they act as electrophilic Michael acceptors toward nucleophiles in the cellular target.

Peptides that have antimicrobial activity are synthesized by many prokaryotic and eukaryotic organisms. Antimicrobial peptides commonly contain unusual amino acids that contribute to their properties and functions. Although bacteria synthesize most of these peptides by nonribosomal mechanisms, this review focuses on those that are synthesized by pathways that involve posttranslational modification of ribosomally synthesized precursor peptides. A particularly interesting class of these antimicrobial peptides is the lantibiotics, of which nisin and subtilin are the longest-known examples, although nearly a dozen new lantibiotics have been discovered in recent years. The fact that the lantibiotic structures are derived from gene-encoded peptides means that structural analogs of natural lantibiotics can be constructed by mutagenesis of their structural genes. Recent advances in our understanding of the molecular genetics of lantibiotics has made the construction of novel lantibiotics with enhanced chemical and antimicrobial properties possible. This review describes these advances and proposes future trends of research, as well as potential application of engineered lantibiotics, in the context of the general field of antimicrobial peptides.

The ability to respond to osmotic stress by osmoregulation is common to virtually all living cells. Gramnegative bacteria such as Escherichia coli and Salmonella typhimurium can achieve osmotolerance by import of osmoprotectants such as proline and glycine betaine by an import system encoded in an operon called proU with genes for proteins ProV, ProW, and ProX. In this report, we describe the discovery of a proU-type locus in the gram-positive bacterium Bacillus subtilis. It contains four open reading frames (ProV, ProW, ProX, and ProZ) with homology to the gram-negative ProU proteins, with the B. subtilis ProV, ProW, and ProX proteins having sequence homologies of 35, 29, and 17%, respectively, to the E. coli proteins. The B. subtilis ProZ protein is similar to the ProW protein but is smaller and, accordingly, may fulfill a novel role in osmoprotection. The B. subtilis proU locus was discovered while exploring the chromosomal sequence upstream from the spa operon in B. subtilis LH45, which is a subtilin-producing mutant of B. subtilis 168. B. subtilis LH45 had been previously constructed by transformation of strain 168 with linear DNA from B. subtilis ATCC 6633 (W. Liu and J. N. Hansen, J. Bacteriol. 173:7387-7390, 1991). Hybridization experiments showed that LH45 resulted from recombination in a region of homology in the proV gene, so that the proU locus in LH45 is a chimera between strains 168 and 6633. Despite being a chimera, this proU locus was fully functional in its ability to confer osmotolerance when glycine betaine was available in the medium. Conversely, a mutant (LH45 ⌬proU) in which most of the proU locus had been deleted grew poorly at high osmolarity in the presence of glycine betaine. We conclude that the proU-like locus in B. subtilis LH45 is a gram-positive counterpart of the proU locus in gram-negative bacteria and probably evolved prior to the evolutionary split of prokaryotes into gram-positive and gram-negative forms.Bacteria are capable of active osmoregulatory responses which allow them to adapt to large fluctuations in the osmolarity of their environment (5,12,37,39,46). Study of osmoregulation has important applications to food microbiology (42, 43), plant-microbe interactions (7, 21), and medical microbiology (3,4,8,18,19,23). The ability of cells to provide gene regulatory responses to changes in a physicochemical parameter rather than a specific molecule requires a novel signal transduction mechanism (11). These responses to osmotic stress are often crucial to survival, and the mechanisms by which this is achieved have been studied extensively, particularly in the gram-negative bacteria Escherichia coli and Salmonella typhimurium (2, 6, 13-16, 20, 22, 24). A central response to osmotic stress is the uptake of molecular species that function as osmoprotectants, such as proline and N,N,Ntrimethylglycine (glycine betaine). Although several osmoprotectant uptake systems have been identified, one of the most extensively studied is the osmoregulatory locus known as proU, which is an opero...

Subtilin is a ribosomally synthesized antimicrobial peptide that contains several unusual amino acids as a result of posttranslational modifications. Site-directed mutagenesis was employed to construct a structural variant of subtilin in which the unusual dehydroalanine (Dha) residue at position 5 was changed to alanine. Proton nuclear magnetic resonance spectroscopy, amino acid composition, and N-terminal sequence analysis established that the mutation did not disrupt posttranslational processing of the precursor peptide. This mutant subtilin was devoid of antimicrobial activity as assessed by its lack of inhibitory effects on outgrowth ofBacillus cereus T spores. However, this same mutant subtilin was fully active with respect to its ability to induce lysis of vegetative B. cereus T cells. Because an intact Dha-5 residue is required in the one instance but not in the other, it was concluded that the molecular mechanism by which subtilin inhibits (without lysis) spore outgrowth is not the same as the mechanism by which it inhibits (with lysis) vegetative cells.

Sequence analysis upstream from the subtilin structural gene (spaS) puter Group (University of Wisconsin) program package. The best homologies were found with the deduced SpaB protein sequence, which showed extensive homologies to a variety of membrane translocator proteins, such as the HlyB protein, which is involved with export of the hemolysin A protein (hemolysin toxin) in E. coli (6). The homologies between SpaB and HlyB are shown in Fig. 3. Included in the regions of homology are five transmembrane helices and an ATP-binding region. The HlyB protein has previously been shown to have homologies to many different membrane translocators, including human and other mammalian proteins involved with multidrug resistance (3). As would be expected, the SpaB protein also shows homologies to these proteins (data not shown).spaB and spaC have overlapping reading frames. The overlap shown for the SpaB and SpaC ORFs (Fig. 2) is unusual in prokaryotes. The overlapping region contains a sequence that could act as a ribosome binding site for spaC, but it also has characteristics of a frameshift sequence (see the legend to Fig. 2 (Fig. 3). One involves a 30-residue region located about 12 residues from the N terminus of SpaD and a region located about 15 residues from the C terminus of HlyD. The second homology was between a 45-residue region in the

Biosynthesis of lantibiotics such as nisin and subtilin involves post-translational modifications, including dehydration of serines and threonines, formation of thioether cross-linkages, translocation, cleavage of a leader sequence, and release into the medium. We have studied the cellular machinery that performs the modifications by constructing and expressing nisin-subtilin chimeric prepeptides in a strain of Bacillus subtilis 168 that possesses all of the cellular machinery for making subtilin except for the presubtilin gene. The chimeras consisted of a normal subtilin leader region (S L ), fused to nisin-subtilin chimeric structural regions, one of which was S L -Nis 1-11 -Sub 12-32 , in which the N-terminal portion of the structural region was derived from nisin, and the C-terminal portion derived from subtilin. This chimera was accurately and efficiently converted to the corresponding mature lantibiotic, as established by reverse phase high performance liquid chromatography profiles, proton NMR spectroscopy, mass spectral analysis, and biological activity. A succinylated form of the chimera was also produced. Another chimera was in the reverse sense, with subtilin sequence at the N terminus and nisin sequence at the C terminus of the structural region (S L -Sub 1-11 -Nis 12-34 ). It was processed into a heterogeneous mixture of products, none of which had the characteristics of a correctly processed polypeptide, but did contain a minor component that was active, with a specific activity that considerably exceeded nisin itself. These results, together with results published earlier, establish that processing requires specific recognition between the prelantibiotic peptide and the processing machinery, and in order for the processing to occur correctly, there must be an appropriate combination of the N-terminal part of the leader region and the C-terminal part of the structural region of the prepeptide.Nisin (produced by Lactococcus lactis) and subtilin (produced by Bacillus subtilis) are the most thoroughly studied examples of lantibiotics, which are ribosomally synthesized antimicrobial peptides that are characterized by the presence of unusual lanthio and dehydro residues. Their structures are shown in Fig. 1. Their biosynthesis involves several post-translational modifications: dehydration of serines and threonines, formation of thioether cross-linkages between dehydro residues and cysteines, translocation, removal of a leader sequence, and release of the mature antimicrobial peptide into the extracellular medium (reviewed in Refs.

Subtilin is a ribosomally synthesized peptide antibiotic produced by Bacillus subtilis ATCC 6633. B. subtilis 168 was converted to a subtilin producer by competence transformation with chromosomal DNA from B. subtilis ATCC 6633. A chloramphenicol acetyltransferase gene was inserted next to the subtilin structural gene as a selectable marker. The genes that conferred subtilin production were derived from a 40-kb region of the B. subtilis ATCC 6633 chromosome that had flanking homologies to the B. subtilis 168 chromosome. The subtilin produced by the mutant was identical to natural subtilin in its biological activity, chromatographic behavior, amino acid composition, and N-terminal amino acid sequence.