Functional characterization of a composite bacteriocin locus from malt isolate Lactobacillus sakei 5 - PubMed (original) (raw)

Functional characterization of a composite bacteriocin locus from malt isolate Lactobacillus sakei 5

Anne Vaughan et al. Appl Environ Microbiol. 2003 Dec.

Abstract

Lactobacillus sakei 5, isolated from malted barley, produces three bacteriocins. Genetic and functional analysis of the purified bacteriocins showed that this strain produces a plasmid-encoded bacteriocin that is identical to sakacin P, as well as two novel, chromosomally encoded bacteriocins, which were designated sakacin T and sakacin X. The structural genes specifying sakacin T and sakacin X are part of the sakacin TX locus, which consists of two adjacent but divergently oriented gene clusters. The first gene cluster includes stxP, stxR, stxK, and stxT, which, based on functional and comparative sequence analysis, are believed to encode an inducing peptide and proteins involved in regulation and secretion of these bacteriocins. The second gene cluster includes the structural and immunity genes for sakacin T, a class IIb two-peptide bacteriocin composed of SakTalpha and SakTbeta, and sakacin X, a class IIa bacteriocin. Interestingly, a so-called transport accessory protein was absent from the locus, and based on our results it appears that a dedicated accessory protein is not required for processing and transport of sakacin T and sakacin X.

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Figures

FIG. 1.

Schematic representation of the sakacin TX locus sequenced to date, obtained by using a combination of ligation-anchored PCR and the primer walking strategy. A total of 22 potential ORFs are shown. The arrows indicate ORFs and the proposed directions of transcription. ORFs with deduced functions in production and secretion of active sakacin T and sakacin X are indicated by stx or sak. Sections of the stx locus assumed to be dedicated to production, immunity, regulation, and transport are indicated.

FIG. 2.

(A) PCR-derived inserts in pLPV111 derivatives with bacteriocin structural and immunity genes. The plasmid designations are indicated on the left, and the phenotypes when the plasmids are introduced (in conjunction with pSAK20) into L. sakei Lb790 are indicated on the right. (B) Inserts in pSAK20, containing the genes necessary for processing and transport of the bacteriocins used, and pSAK20 derivatives used in this study. The phenotypes when these constructs were introduced into L. sakei Lb790 or L. sakei 23K harboring the pLPV111 derivatives with bacteriocin structural and immunity genes are indicated.

FIG. 3.

Bacteriocin activity and complementation of the SakTα and SakTβ peptides. L. sakei Lb790(pSAK20) containing pLPT8 (SakTα) and L. sakei Lb790(pSAK20) containing pLPT6 (SakTβ) were grown and overlaid with the sensitive indicator strain L. sakei LMG2313. Cultures were grown adjacent to each other and as a mixed culture (SakTα + SakTβ↓).

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Functional characterization of a composite bacteriocin locus from malt isolate Lactobacillus sakei 5 - PubMed (original) (raw)