Autoregulation of subtilin biosynthesis in Bacillus subtilis: the role of the spa-box in subtilin-responsive promoters

Kleerebezem, Michiel; Bongers, Roger S.; Rutten, Ger; Vos, Willem M. de; Kuipers, Oscar P.

doi:10.1016/j.peptides.2003.11.025

Cited by 40 publications

(25 citation statements)

References 32 publications

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“…DRI to DRIII are located upstream of bovA. Similar structures were reported in the subtilin promoter (Kleerebezem et al, 2004). The transcription start site of bovE was a C residue 35 bp upstream of the ATG codon, whereas no obvious 210 or 235 regions or ribosome-binding site were found.…”

Section: Discussionsupporting

confidence: 66%

Characteristics of the bovicin HJ50 gene cluster in Streptococcus bovis HJ50

Liu

Zhong

et al. 2009

Microbiology

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Bovicin HJ50 is a new lantibiotic containing a disulfide bridge produced by Streptococcus bovis HJ50; its encoding gene bovA was reported in our previous publication. To identify other genes involved in bovicin HJ50 production, DNA fragments flanking bovA were cloned and sequenced. The bovicin HJ50 biosynthesis gene locus was encoded by a 9.9 kb region of chromosomal DNA and consisted of at least nine genes in the following order: bovA, -M, -T, -E, -F, ORF1, ORF2, bovK and bovR. A thiol-disulfide oxidoreductase gene named sdb1 was located downstream of bovR. A knockout mutant of this gene retained antimicrobial activity and the molecular mass of bovicin HJ50 in the mutant was the same as that of bovicin HJ50 in S. bovis HJ50, implying that sdb1 is not involved in bovicin HJ50 production. Transcriptional analyses showed that bovA, bovM and bovT constituted an operon, and the transcription start site of the bovA promoter was located at a G residue 45 bp upstream of the translation start codon for bovA, while bovE through bovR were transcribed together and the transcription start site of the bovE promoter was located at a C residue 35 bp upstream of bovE. We also demonstrated successful heterologous expression of bovicin HJ50 in Lactococcus lactis MG1363, which lacks thiol-disulfide oxidoreductase genes; this showed that thiol-disulfide oxidoreductase genes other than sdb1 are not essential for bovicin HJ50 biosynthesis.

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Section: Discussionsupporting

confidence: 66%

Characteristics of the bovicin HJ50 gene cluster in Streptococcus bovis HJ50

Liu

Zhong

et al. 2009

Microbiology

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“…Therefore, a threshold concentration of 1 to 2 mg/ml mersacidin, which will activate transcription of the immunity genes just in time to avoid a growth inhibition, seems plausible. Similar mechanisms have been described for the immunity proteins of other lantibiotics: the promoter of the gene nisF is nisin inducible (6) and the promoter of spaI, which confers immunity against the type A lantibiotic subtilin, exhibited quorum-sensing activity in the producer strain Bacillus subtilis ATCC 6633 (18).…”

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confidence: 66%

The Lantibiotic Mersacidin Is an Autoinducing Peptide

Schmitz

Hoffmann

Szekat

et al. 2006

Appl Environ Microbiol

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The lantibiotic (lanthionine-containing antibiotic) mersacidin is an antimicrobial peptide consisting of 20 amino acids and is produced by Bacillus sp. strain HIL Y-85,54728. The structural gene (mrsA) and the genes for producer self-protection, modification enzymes, transport proteins, and regulator proteins are organized in a 12.3-kb biosynthetic gene cluster on the chromosome of the producer strain. Mersacidin is produced in stationary phase in a synthetic medium (K. Altena, A. Guder, C. Cramer, and G. Bierbaum, Appl. Environ. Microbiol. 66:2565-2571, 2000). To investigate the influence of the alternative sigma factor H on mersacidin biosynthesis, a SigH knockout was constructed. The knockout mutant was asporogenous, and a comparison to the wild-type strain indicated no significant differences concerning mersacidin production and immunity. Characterization of the mrsA promoter showed that the gene is transcribed by the housekeeping sigma factor A. The biosynthesis of some lantibiotic peptides like nisin or subtilin is regulated in a cell-density-dependent manner (M. Kleerebezem, Peptides 25:1405-1414, 2004). When mersacidin was added at a concentration of 2 mg/liter to an exponentially growing culture, an earlier production of antibacterial activity against Micrococcus luteus ATCC 4698 in comparison to that of the control culture was observed, suggesting that mersacidin itself functions as an autoinducer. In real-time PCR experiments, the expression of mrsA was remarkably increased in the induced culture compared to the control. In conclusion, mersacidin is yet another lantibiotic peptide whose biosynthesis can be regulated by an autoinducing mechanism.

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“…Previous studies of the strengths of the spaS, spaB, and spaI promoters have shown that the spaS promoter drives the highest level of transcription activation upon induction with subtilin (22,44). Based on this observation and taking into account the fact that the P spaS spa box contains one mismatch compared to spa boxes found in P spaB and P spaI (22,44), expression vectors containing either the wild-type spaS promoter or a mutant derivative in which the spa box had been changed into a perfect pentanucleotide direct repeat (TTGAT) were constructed, and their transcriptional activation upon subtilin induction was analyzed. To generate a production host, we introduced the subtilin sensor-regulator couple spaRK by integrating plasmid pNZ8900 into the amyE locus of B. subtilis 168, resulting in strain NZ8900 (Table 1).…”

Section: Resultsmentioning

confidence: 99%

“…To construct plasmids harboring the spaS promoter and its mutant derivative that were translationally fused to the gusA reporter gene, both promoter fragments were amplified by PCR using primers PspaS-F and PspaS-R (wild-type spaS promoter) and primers PspaSmut-F and PspaS-R (mutated spaS promoter) and chromosomal DNA of B. subtilis ATCC 6633 as the template. The mutation introduced into the mutated spaS promoter resulted in the presence of a perfect consensus pentanucleotide repeat (TTGAT) in the spa box of this promoter (22,44). After digestion of the resulting 80-bp PCR products with BglII and NcoI, the promoter regions were ligated into the corresponding sites of pNZ8032 (10).…”

Section: Methodsmentioning

confidence: 99%

“…Similar to nisin production in L. lactis, the production of subtilin in B. subtilis is subject to quorum-sensing control that depends on sensing of subtilin by a dedicated sensor histidine kinase (SpaK) and subsequent signal transduction to the corresponding response regulator (SpaR). Upon phosphorylation, SpaR binds to so-called spa boxes in the promoter regions upstream of the spaB, spaI, and spaS genes located in the subtilin biosynthesis gene cluster, thereby triggering promoter activation (22,43,44). Additionally, production of subtilin in B. subtilis has been shown to be dually controlled since spaRK expression is controlled by the transition-state regulator sigma H (43).…”

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confidence: 99%

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Development and Characterization of a Subtilin-Regulated Expression System in Bacillus subtilis : Strict Control of Gene Expression by Addition of Subtilin

Bongers

Veening

Wieringen

et al. 2005

Appl Environ Microbiol

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A system for subtilin-regulated gene expression (SURE) in Bacillus subtilis that is based on the regulatory module involved in cell-density-dependent control of the production of subtilin is described. An integration vector for introduction of the essential sensor-regulator couple spaRK into the amyE locus of the B. subtilis chromosome and a B. subtilis 168-derived production host in which the spaRK genes were functionally introduced were constructed. Furthermore, several expression plasmids harboring the subtilin-inducible wild-type spaS promoter or a mutated derivative of this promoter were constructed, which facilitated both transcriptional and translational promoter-gene fusions. Functional characterization of both spaS promoters and the cognate expression host could be performed by controlled overproduction of the ␤-glucuronidase (GusA) and green fluorescent protein (GFP) reporters. Both spaS promoters exhibited very low levels of basal expression, while extremely high levels of expression were observed upon induction with subtilin. Moreover, the level of expression depended directly on the amount of inducer (subtilin) used. The wild-type spaS promoter appeared to be more strictly controlled by the addition of subtilin, while the highest levels of expression were obtained when the mutated spaS promoter was used. Induction by subtilin led to 110-and 80-fold increases in GusA activity for the spaS promoter and its mutant derivative, respectively. Since the SURE system has attractive functional characteristics, including promoter silence under noninducing conditions and a controlled and high level of expression upon induction, and since it is not subject to catabolite control, we anticipate that it can provide a suitable expression system for various scientific and industrial applications.

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Autoregulation of subtilin biosynthesis in Bacillus subtilis: the role of the spa-box in subtilin-responsive promoters

Cited by 40 publications

References 32 publications

Characteristics of the bovicin HJ50 gene cluster in Streptococcus bovis HJ50

Characteristics of the bovicin HJ50 gene cluster in Streptococcus bovis HJ50

The Lantibiotic Mersacidin Is an Autoinducing Peptide

Development and Characterization of a Subtilin-Regulated Expression System in Bacillus subtilis : Strict Control of Gene Expression by Addition of Subtilin

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