Regulation of Heterologous Subtilin Production in Bacillus subtilis W168

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 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.

The Epipeptide Biosynthesis Locus epeXEPAB Is Widely Distributed in Firmicutes and Triggers Intrinsic Cell Envelope Stress

The <i>epeXEPAB</i> (formerly <i>yydFGHIJ</i>) locus of <i>Bacillus subtilis</i> encodes a minimalistic biosynthetic pathway for a linear antimicrobial epipeptide, EpeX, which is ribosomally produced and post-translationally processed by the action of the radical-SAM epimerase, EpeE, and a membrane-anchored signal 2 peptide peptidase, EpeP. The ABC transporter EpeAB provides intrinsic immunity against self-produced EpeX, without conferring resistance against extrinsically added EpeX. EpeX specifically targets, and severely perturbs the integrity of the cytoplasmic membrane, which leads to the induction of the Lia-dependent envelope stress response. Here, we provide new insights into the distribution, expression, and regulation of the minimalistic <i>epeXEPAB</i> locus of <i>B. subtilis</i>, as well as the biosynthesis and biological efficiency of the produced epipeptide EpeX*. A comprehensive comparative genomics study demonstrates that the <i>epe</i>-locus is restricted to but widely distributed within the phylum <i>Firmicutes</i>. The gene products of <i>epeXEP</i> are necessary and sufficient for the production of the mature antimicrobial peptide EpeX*. In <i>B. subtilis</i>, the <i>epeXEPAB</i> locus is transcribed from three different promoters, one upstream of <i>epeX</i> (P_<i>epeX</i>) and two within <i>epeP</i> (P_<i>epeA1</i> and P_<i>epeA2</i>). While the latter two are mostly constitutive, P_<i>epeX</i> shows a growth phase-dependent induction at the onset of stationary phase. We demonstrate that this regulation is the result of the antagonistic action of two global regulators: The transition state regulator AbrB keeps the <i>epe</i> locus shut off during exponential growth by direct binding. This tight repression is relieved by the master regulator of sporulation, Spo0A, which counteracts the AbrB-dependent repression of <i>epeXEPAB</i> expression during the transition to stationary phase. The net result of these three ­promoters is an expression pattern that ensures EpeAB-dependent autoimmunity prior to EpeX* production. In the absence of EpeAB, the general envelope stress response proteins LiaIH can compensate for the loss of specific autoimmunity by providing sufficient protection against the membrane-perturbating action of EpeX*. Hence, the transcriptional regulation of <i>epe</i> expression and the resulting intrinsic induction of the two corresponding resistance functions, encoded by <i>epeAB</i> and <i>liaIH</i>, are well balanced to provide a need-based immunity against mature EpeX*.

Synthesis and Succinylation of Subtilin-Like Lantibiotics Are Strongly Influenced by Glucose and Transition State Regulator AbrB

ABSTRACTSubtilin and the closely related entianin are class I lantibiotics produced by different subspecies ofBacillus subtilis. Both molecules are ribosomally synthesized peptide antibiotics with unusual ring structures. Subtilin-like lantibiotics develop strong antibiotic activities against various Gram-positive organisms with an efficiency similar to that of nisin fromLactococcus lactis. In contrast to nisin, subtilin-like lantibiotics partially undergo an additional posttranslational modification, where the N-terminal tryptophan residue becomes succinylated, resulting in drastically reduced antibiotic activities. A highly sensitive high-performance liquid chromatography (HPLC)-based quantification method enabled us to determine entianin and succinylated entianin (S-entianin) concentrations in the supernatant during growth. We show that entianin synthesis and the degree of succinylation drastically change with culture conditions. In particular, increasing glucose concentrations resulted in higher entianin amounts and lower proportions of S-entianin in Landy-based media. In contrast, no succinylation was observed in medium A with 10% glucose. Interestingly, glucose retarded the expression of entianin biosynthesis genes. Furthermore, deletion of the transition state regulator AbrB resulted in a 6-fold increased entianin production in medium A with 10% glucose. This shows that entianin biosynthesis inB. subtilisis strongly influenced by glucose, in addition to its regulation by the transition state regulator AbrB. Our results suggest that the mechanism underlying the succinylation of subtilin-like lantibiotics is enzymatically catalyzed and occurs in the extracellular space or at the cellular membrane.

ScoC Regulates Bacilysin Production at the Transcription Level in Bacillus subtilis

ABSTRACT Bacillus subtilis mutants with high expression of the bacilysin operon ywfBCDEFG were isolated. Comparative genome sequencing analysis revealed that all of these mutants have a mutation in the scoC gene. The disruption of scoC by genetic engineering also resulted in increased expression of ywfBCDEFG. Primer extension and gel mobility shift analyses showed that the ScoC protein binds directly to the promoter region of ywfBCDEFG. Our results indicate that the transition state regulator ScoC, together with CodY and AbrB, negatively regulates bacilysin production in B. subtilis.