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

M. Kleerebezem, R. Bongers, G. Rutten, W.M. de Vos, O.P. Kuipers

Research output: Contribution to journalArticleAcademicpeer-review

31 Citations (Scopus)

Abstract

The production of the type 1 antimicrobial peptide (AMP) subtilin by Bacillus subtilis is regulated in a cell-density-dependent manner [Kleerebezem M, de Vos WM, Kuipers OP. The lantibiotics nisin and subtilin act as extracellular regulators of their own biosynthesis. In: Dunny GM, Winans SC, editors. Cell-cell signaling in bacteria. Washington, D.C., USA: ASM Press; 1999. p. 159-74; Stein T, Borchert S, Kiesau P. Heinzmann S, Moss S, Klein C, Helfrich M, Entian KD. Dual control of subtilin biosynthesis and immunity in Bacillus subtilis. Mol Microbiol 2002;44:403-16; Stein T, Heinzmann S, Kiesau P, Himmel B, Entian KD. The spa-box for transcriptional activation of subtilin biosynthesis and immunity in Bacillus subtilis. Mol Microbiol 2003;47:1627-36]. Three subtilin-responsive promoter elements within the spaBTCSIFEGRK are controlled by the specific cis-acting sequence element called the spa-box, which represents the binding site of the subtilin regulator SpaR [Stein T, Heinzmann S, Kiesau P, Himmel B, Entian KD. The spa-box for transcriptional activation of subtilin biosynthesis and immunity in Bacillus subtilis. Mol Microbiol 2003;47:1627-36]. Here, we describe the functional characterization of the spaB, spaS and spaI promoters by transcriptional fusion with a promoterless P-glucuronidase encoding gusA gene. Within these gusA fusion constructs, transcription initiation start sites of the spaS and spa1 promoters were mapped to be located downstream of the spa-box, which is in contrast to previous reports [Banerjee S, Hansen JN. Structure and expression of a gene encoding the precursor of subtilin, a small protein antibiotic. J Biol Chem 1988;263:9508-14; Stein T, Heinzmann S, Kiesau P, Himmel B, Entian KD. The spa-box for transcriptional activation of subtilin biosynthesis and immunity in Bacillus subtilis. Mol Microbiol 2003;47:1627-36]. Nevertheless, all spa-promoters displayed typical cell-density-dependent activity in a subtilin-producing strain B. subtilis ATCC6633. Moreover, analysis of P-glucuronidase activities in a spaB mutant of B. subtilis ATCC6633 and a derivative of strain 168 that harbors the spaRK genes integrated in the chromosomal amyE locus, confirmed that these promoters are activated by subtilin-triggered, SpaRK-mediated, quorum-sensing control. Quantitative analysis showed that the spaS promoter strength at a given subtilin concentration appeared to be approximately five-fold higher than the spaB promoter, which in turn is approximately two-fold higher than the spa1 promoter. Finally, it is shown that the elementary components involved in subtilin-mediated regulation are the two-component system, SpaRK, and a spa-box containing promoter. (C) 2004, Elsevier Inc. All rights reserved.
Original languageEnglish
Pages (from-to)1415-1424
JournalPeptides
Volume25
Issue number9
DOIs
Publication statusPublished - 2004

Keywords

  • gram-positive bacteria
  • lactic-acid bacteria
  • controlled gene-expression
  • nisin-controlled expression
  • lantibiotic subtilin
  • lactococcus-lactis
  • signal-transduction
  • transcriptional activation
  • dependent regulation
  • immunity

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