Cortina Kaletta’s research while affiliated with Goethe University Frankfurt and other places

Subtilin is a lanthionine-containing peptide antibiotic (lantibiotic) which is produced by Bacillus 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 KpnI-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 histidine kinases previously described (PhoM, PhoR, and NtrB). Hydrophobicity blots suggested two membrane-spanning regions. Thus, spaR and spaK belong to a recently identified family of environmentally responsive regulators. These results indicated a regulatory function of spaR and spaK in subtilin biosynthesis. Indeed, batch culture experiments confirmed the regulation of subtilin biosynthesis starting in the mid-logarithmic growth phase and reaching its maximum in the early stationary growth phase. Gene deletions within spaR and spaK yielded subtilin-negative mutants, which confirms that subtilin biosynthesis is under the control of a two-component regulatory system.(ABSTRACT TRUNCATED AT 250 WORDS)

Lantibiotics are peptide-derived antibiotics with high antimicrobial activity against pathogenic gram-positive bacteria. They are ribosomally synthesized and posttranslationally modified (N. Schnell, K.-D. Entian, U. Schneider, F. Götz, H. Zähner, R. Kellner, and G. Jung, Nature [London] 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 3- to 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 coli that express this enzyme 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.

Using a hybridization probe specific for the structural gene of subtilin, spaS, the DNA regions adjacent to spaS could be isolated. Sequence analysis revealed several open reading frames with the same orientation as spaS. Upstream of spaS three reading frames, spaB, spaC, and spaY were identified (Klein et al, 1992) which showed strong homology to genes identified near the structural gene of the lantibiotic epidermin (Schnell et al, 1992). The SpaY protein derived from the spaY sequence was homologous to hemolysin B of E. coli which indicated its possible function in subtilin transport. Gene deletions within spaB and spaC, revealed subtilin negative mutants whereas spaY gene disruption mutants still produced subtilin (Klein et al., 1992). Remarkably, the spaY - colonies revealed a clumpy surface morphology on solid media and lost their viability in early stationary growth phase, possibly as a result of intracellular subtilin accumulation. Our results clearly proved that reading frames spaB and spaC are essential for subtilin biosynthesis whereas spaY - mutants are probably deficient in subtilin transport.

Binding between analyte and modified probe, resulting in stable hybrid molecules, is most prominently mediated by specific interaction between complementary purine and pyrimidine bases forming A:T and G:C base pairs.

The tetracyclic polypeptide antibiotic cinnamycin (Ro 90-0198) belongs to the duramycin-type lantibiotics and contains the unusual amino acids threo-3-methyl-lanthionine, meso-lanthionine, lysinoalanine and 3-hydroxyaspartic acid. Its structural gene, referred to as cinA, has been identified on isolated chromosomal DNA of the Ro 09-0198-producing strain Streptoverticillium griseoverticillatum via a 39-residue oligonucleotide probe derived from fragment 7-19 of the hypothetical prolantibiotic sequence CRQSCSFGPFTFVCDGNTK. This propeptide part was then found within an open reading frame of 77 amino acids. In contrast to the nisin-type prelantibiotics, this first duramycin-type prelantibiotic has an unusually long leader sequence of 58 amino acids. it also differs in the processing site and the direction of the formation of the threo-3-methyl-lanthionine bridges is from N-terminal cysteine to C-terminal dehydrated threonine residues, whereas the meso-lanthionine and lysinoalanine bridges are formed by addition reactions from C-terminal cysteine or lysine to N-terminal dehyrated serine residues.

Lantibiotica sind Peptidantibiotica, die Lanthionin enthalten. Pep5, eine stark basische Verbindung, wird von Staphylococcus epidermidis 5 durch ribosomale Synthese eines Vorläuferpeptids und posttranslationale Modifizierung produziert. Die antibiotische Wirkung von Pep5 beruht auf der Bildung spannungsabhängiger Poren in Bakterienmembranen. Pep5 ist das bisher größte Lantibioticum (Mr = 3488). Es ist durch einen 2-Oxobutyrylrest N-terminal blockiert und enthält neben 25 Proteinaminosäuren Dehydrobutyrin (2), meso-Lanthionin (2) und 3-Methyllanthionin (1). Automatischer Edman-Abbau, Massenspektrometrie enzymatisch erhaltener Fragmente und chemische Derivatisierung ergaben eine 34-Peptidsequenz mit drei Sulfidringen (Lanthionin = S(CH2 CHNH2 COOH)2).

Lantibiotics are peptide antibiotics which contain lanthionine. Pep 5, a strongly basic compound, is produced from Staphylococcus epidermidis 5 by ribosomal synthesis of a precursor peptide and post‐translational modification. The antibiotic activity of Pep 5 rests on the formation of strain‐dependent pores in bacterial membranes. Pep 5 is the hitherto largest lantibiotic ( M r = 3488). It is N ‐terminally blocked by a 2‐oxobutyryl residue and contains, besides 25 protein amino acids, dehydrobutyrine (2), meso ‐lanthionine (2) and 3‐methyllanthionine (1). Automatic Edman degradation, mass spectrometry of enzymatically obtained fragments and chemical derivatization gave a 34‐peptide sequence with three sulfide rings (lanthionine = S(CH 2 CHNH 2 COOH) 2 ).

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.