Analysis of genes involved in biosynthesis of the lantibiotic subtilin.
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.
Klein
C
C
Institute for Microbiology, Johann Wolfgang Goethe-Universität, Frankfurt am Main, Germany.
Kaletta
C
C
Schnell
N
N
Entian
K D
KD
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000096:biosynthesis*; D001412:Bacillus subtilis / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins; D001430:Bacteriocins; D001483:Base Sequence; D015139:Blotting, Southern; D004269:DNA, Bacterial; D008565:Membrane Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D016366:Open Reading Frames; D010455:Peptides; D010456:Peptides, Cyclic / Q000096:biosynthesis / Q000235:genetics; D010641:Phenotype; D015183:Restriction Mapping; D016415:Sequence Alignment
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D001430:Bacteriocins; D004269:DNA, Bacterial; D008565:Membrane Proteins; D010455:Peptides; D010456:Peptides, Cyclic; C104626:SpaB protein, Bacillus subtilis; C104625:SpaC protein, bacteria; C104627:SpaT protein, Bacillus subtilis; C002836:subtilin