Nisin produced by Streptococcus lactis is used as a food preservative and is the most important member of a group of antibiotics containing lanthionine bridges. To understand the genetic basis of these so-called lantibiotics (Schnell et al., Nature 333:276-278, 1988), we characterized the nisin structural gene, nisA, which is located on a plasmid and codes for a 57-amino-acid prepeptide. The prepeptide is processed posttranslationally to the pentacyclic antibiotic. Although nisin and the recently elucidated lantibiotic epidermin from Staphylococcus epidermidis are produced by different organisms, their gene organization is identical. As with epidermin, the nisin propeptide corresponds to the C-terminus of the prepeptide. The N-terminus of the prepeptide is cleaved at a characteristic splice site (Pro--2 Arg--1 Ile-+1). Remarkably, the N-terminus of prenisin shares 70% similarity with preepidermin, although the propeptide sequences are distinctly different. The structural similarities between these two lantibiotics are consistent with the fact that there is a common mechanism of biosynthesis of these lanthionine-containing antibiotics.

333:276-278, 1988). The most important lantibiotics are subtilin and the food preservative nisin, which both have a very similar structure. By using a hybridization probe specific for the structural gene of subtilin, spaS, the DNA region adjacent to spaS was isolated from Bacillus subtilis. Sequence analysis of a 4.9-kb fragment revealed several open reading frames with the same orientation as spaS. Downstream of spaS, no reading frames were present on the isolated XbaI fragment. Upstream of spaS, three reading frames, spaB, spaC, and spaT, were identified which showed strong homology to genes identified near the structural gene of the lantibiotic epidermin. The SpaT protein derived from the spaT sequence was homologous to hemolysin B of Escherichia coli, which indicated its possible function in subtilin transport. Gene deletions within spaB and spaC revealed subtilin-negative mutants, whereas spaT gene disruption mutants still produced subtilin. Remarkably, the spaT mutant colonies revealed a clumpy surface morphology on solid media. After growth on liquid media, spaT mutant cells agglutinated in the mid-logarithmic growth phase, forming longitudinal 3to 10-fold-enlarged cells which aggregated. Aggregate formation preceded subtilin production and cells lost their viability, possibly as a result of intracellular subtilin accumulation. Our results clearly proved that reading frames spaB and spaC are essential for subtilin biosynthesis whereas spaT mutants are probably deficient in subtilin transport.

The pentacyclic hopanoids, a class of eubacterial lipids, are synthesized by squalene-hopene cyclase and side chain-elongating enzymes. With the aid of DNA probes based on the amino-terminal sequence of purified squalene-hopene cyclase from Bacillus acidocaldarius, clones of Escherichia coi that express this enzy'me in the cytoplasmic membrane were isolated. According to the DNA sequence, the cyclase contained 627 amino acids with a molecular mass of 69,473 Da. A high percentage of the amino acids were basic. No significant similarity to existing sequenced proteins was found.Hopanoids are a class of pentacyclic triterpenoids that occur in all crude oils (18) and are also widespread eubacterial lipids (19,24). They have a structure similar to that of sterols (18) and condense phospholipids in mnodel membrane systems (2, 22) and supposedly in cellular membranes. Hopanoids are more effectively produced at higher growth temperatures by Bacillus acidocaldarius (21), Zymomonas mobilis (26), and Rhodospirillum acidophila (12) and at high ethanol levels by Z. mobilis (6). In Mycoplasma mycoides they are able to replace sterols (11). It was recently found that the N2-fixing symbiont Frankia sp. contains high levels of hopanoids (3).In contrast to sterol biosynthesis, the biosynthesis of hopanoids is independent of molecular oxygen as a substrate. Hopanoids are cyclized directly from squalene by squalene-hopene cyclase (28), which may be homologous to sterol cyclases and other triterpenoid cyclases from eucaryotic cells, synthesizing by a similar reaction a similar product with analogous membrane properties. It would therefore be of interest to compare the amino acid sequence of hopene cyclase with those of sterol cyclases, thereby increasing our understanding of the recruitment of these enzymes. If homologies are found, in vitro mutagenesis studies would be of value in understanding the evolution of sterol cyclases. So far no triterpenoid cyclase has been sequenced. As a first step in this direction, the squalene-hopene cyclase gene from B. acidocaldarius was cloned, expressed, and sequenced; this enzyme appears to represent a new gene family. MATERIALS AND METHODSBacterial strains and plasmids. The source of squalenehopene cyclase and genomic DNA was B. acidocaldarius ATCC 27009. Escherichia coli BMH71-18 (14) was used for transformation. Plasmid pUC19 was described previously (31).Culture conditions. B. acidocaldarius was grown on sporulation medium at pH 3 and 60°C (8). E. coli was grown at 37°C in LB medium (GIBCO, Neuisenburg, Germany) or on LB plates, both containing 30 mg of ampicillin per liter, when plasmid-containing cells were selected. * Corresponding author. DNA preparation. B. acidocaldarius grown to the early exponential phase was harvested by centrifugation and washed with 150 mM NaCl-10 mM EDTA (pH 8.0) (SE). The wet cells (1 g) were resuspended in 10 ml of SE. After lysozyme (1 mg/ml) was added, the suspension was incubated at 37°C. After 1 h, the cells were lysed by the addition of 200 ,ul of 20% (wt/v...

A wobbled 14-mer oligonucleotide was derived from the amino acid sequence of the 34-residue propeptide of the lantibiotic Pep5 (Kellner et al. 1989). Using this hybridization probe, the structural gene of Pep5, pepA, was located on the 18.6 kbp plasmid pED503. The nucleotide sequence of pepA codes for a prepeptide with 60 residues and proves that Pep5 is ribosomally synthesized. The N-terminus of the prepeptide has a high alpha-helix probability and a characteristic proteolytic cleavage site precedes the C-terminal 34-residue propeptide. Our present theory is that maturation of Pep5 involves (a) enzymic conversion of Thr, Ser and Cys into dehydrated amino acids and sulfide bridges, (b) membrane translocation and cleavage of the modified prepeptide.

Subtilin is a lanthionine-containing peptide antibiotic (lantibiotic) which is produced by BaciUlus subtilis ATCC 6633. Upstream from the structural gene of subtilin, spaS, three genes (spaB, spaT, and spaC) which are involved in the biosynthesis of subtilin have been identified (C. Klein, C. Kaletta, N. Schnell, and K.-D. Entian, Appl. Environ. Microbiol. 58:132-142, 1992). By using a hybridization probe specific for these genes, the DNA region downstream from spaS was isolated. Further subcloning revealed a 5.2-kb Kpnl-HindIII fragment on which two open reading frames, spaR and spaK, were identified approximately 3 kb downstream from spaS. The spaR gene encodes an open reading frame of 220 amino acids with a predicted molecular mass of 25.6 kDa. SpaR shows 35% similarity to positive regulatory factors OmpR and PhoB. The spaK gene encodes an open reading frame of 387 amino acids with a predicted molecular mass of 44.6 kDa and was highly similar to