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Biosynthesis of the lantibiotic subtilin is regulated by a histidine kinase/response regulator system.

ABSTRACT: 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)

SUBMITTER: Klein C

PROVIDER: S-EPMC202094 | biostudies-other | 1993 Jan

REPOSITORIES: biostudies-other

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Biosynthesis of the lantibiotic subtilin is regulated by a histidine kinase/response regulator system.

Klein C C Kaletta C C Entian K D KD

Applied and environmental microbiology 19930101 1

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 subclon ...[more]

PMID: 8439156

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Project description:Autoinduction via two-component systems is a widespread regulatory mechanism that senses environmental and metabolic changes. Although the lantibiotics nisin and subtilin are closely related and share the same lanthionine ring structure, they autoinduce their biosynthesis in a highly specific manner. Subtilin activates only the two-component system SpaRK of Bacillus subtilis, whereas nisin activates solely the two-component system NisRK of Lactococcus lactis To identify components that determine the specificity of subtilin autoinduction, several variants of the respective lantibiotics were analyzed for their autoinductive capacities. Here, we show that amino acid position 20 is crucial for SpaK activation, as an engineered nisin molecule with phenylalanine at position 20 (nisin N20F) was able to activate SpaK in a specific manner. In combination with the N-terminal tryptophan of subtilin (nisin I1W/N20F), SpaK autoinduction reached almost the level of subtilin-mediated autoinduction. Furthermore, the overall structure of subtilin is also important for its association with the histidine kinase. The destruction of the second lanthionine ring (subtilin C11A, ring B), as well as mutations that interfere with the flexibility of the hinge region located between lanthionine rings C and D (subtilin L21P/Q22P), abolished SpaK autoinduction. Although the C-terminal part of subtilin is needed for efficient SpaK autoinduction, the destruction of lanthionine rings D and E had no measurable impact. Based on these findings, a model for the interaction of subtilin with histidine kinase SpaK was established.IMPORTANCE Although two-component systems are important regulatory systems that sense environmental changes, very little information on the molecular mechanism of sensing or the interaction of the sensor with its respective kinase is available. The strong specificity of linear lantibiotics such as subtilin and nisin for their respective kinases provides an excellent model system to unravel the structural needs of these lantibiotics for activating histidine kinases in a specific manner. More than that, the biosyntheses of lantibiotics are autoinduced via two-component systems. Therefore, an understanding of their interactions with histidine kinases is needed for the biosynthesis of newly engineered peptide antibiotics. Using a Bacillus subtilis-based reporter system, we were able to identify the molecular constraints that are necessary for specific SpaK activation and to provide SpaK specificity to nisin with just two point mutations.

Dataset Information

Biosynthesis of the lantibiotic subtilin is regulated by a histidine kinase/response regulator system.

Publications

Biosynthesis of the lantibiotic subtilin is regulated by a histidine kinase/response regulator system.

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