Dataset Information

Regulation of the Bacillus subtilis acetate kinase gene by CcpA.

ABSTRACT: The Bacillus subtilis gene encoding acetate kinase was identified on the basis of sequence similarity to the Escherichia coli ackA gene and to a second E. coli gene closely related to ackA. Insertional inactivation of this region of the B. subtilis chromosome resulted in the disappearance of acetate kinase enzyme activity in cell extracts. The ackA gene was mapped to a site close to the ccpA gene, at 263 degrees. The transcriptional start site for B. subtilis ackA was located 90 bp upstream from the start of the coding region, and expression was increased by growth in the presence of excess glucose. Growth of the AckA- mutant was inhibited by glucose, suggesting that acetate kinase is important for excretion of excess carbohydrate. The stimulation of ackA expression by glucose was blocked in a CcpA- mutant, indicating that CcpA, which is required for glucose repression of certain carbon source utilization genes, including amyE, may also be involved in activation of carbon excretion pathways. Two sequences resembling the amyO operator site were identified upstream of the ackA promoter; removal of this region resulted in loss of glucose activation of ackA expression.

SUBMITTER: Grundy FJ

PROVIDER: S-EPMC206879 | biostudies-other | 1993 Nov

REPOSITORIES: biostudies-other

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Regulation of the Bacillus subtilis acetate kinase gene by CcpA.

Grundy F J FJ Waters D A DA Allen S H SH Henkin T M TM

Journal of bacteriology 19931101 22

The Bacillus subtilis gene encoding acetate kinase was identified on the basis of sequence similarity to the Escherichia coli ackA gene and to a second E. coli gene closely related to ackA. Insertional inactivation of this region of the B. subtilis chromosome resulted in the disappearance of acetate kinase enzyme activity in cell extracts. The ackA gene was mapped to a site close to the ccpA gene, at 263 degrees. The transcriptional start site for B. subtilis ackA was located 90 bp upstream from ...[more]

PMID: 8226682

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Project description:BackgroundSubtilin is a peptide antibiotic (lantibiotic) natively produced by Bacillus subtilis ATCC6633. It is encoded in a gene cluster spaBTCSIFEGRK (spa-locus) consisting of four transcriptional units: spaS (subtilin pre-peptide), spaBTC (modification and export), spaIFEG (immunity) and spaRK (regulation). Despite the pioneer understanding on subtilin biosynthesis, a robust platform to facilitate subtilin research and improve subtilin production is still a poorly explored spot.ResultsIn this work, the intact spa-locus was successfully integrated into the chromosome of Bacillus subtilis W168, which is the by far best-characterized Gram-positive model organism with powerful genetics and many advantages in industrial use. Through systematic analysis of spa-promoter activities in B. subtilis W168 wild type and mutant strains, our work demonstrates that subtilin is basally expressed in B. subtilis W168, and the transition state regulator AbrB strongly represses subtilin biosynthesis in a growth phase-dependent manner. The deletion of AbrB remarkably enhanced subtilin gene expression, resulting in comparable yield of bioactive subtilin production as for B. subtilis ATCC6633. However, while in B. subtilis ATCC6633 AbrB regulates subtilin gene expression via SigH, which in turn activates spaRK, AbrB of B. subtilis W168 controls subtilin gene expression in SigH-independent manner, except for the regulation of spaBTC. Furthermore, the work shows that subtilin biosynthesis in B. subtilis W168 is regulated by the two-component regulatory system SpaRK and strictly relies on subtilin itself as inducer to fulfill the autoregulatory circuit. In addition, by incorporating the subtilin-producing system (spa-locus) and subtilin-reporting system (PpsdA-lux) together, we developed "online" reporter strains to efficiently monitor the dynamics of subtilin biosynthesis.ConclusionsWithin this study, the model organism B. subtilis W168 was successfully established as a novel platform for subtilin biosynthesis and the underlying regulatory mechanism was comprehensively characterized. This work will not only facilitate genetic (engineering) studies on subtilin, but also pave the way for its industrial production. More broadly, this work will shed new light on the heterologous production of other lantibiotics.

Dataset Information

Regulation of the Bacillus subtilis acetate kinase gene by CcpA.

Publications

Regulation of the Bacillus subtilis acetate kinase gene by CcpA.

Similar Datasets

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