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Characterization of the locus responsible for the bacteriocin production in Lactobacillus plantarum C11.

ABSTRACT: Lactobacillus plantarum C11 secretes a small cationic peptide, plantaricin A, that serves as induction signal for bacteriocin production as well as transcription of plnABCD. The plnABCD operon encodes the plantaricin A precursor (PlnA) itself and determinants (PlnBCD) for a signal transducing pathway. By Northern (RNA) and sequencing analyses, four new plantaricin A-induced operons were identified. All were highly activated in concert with plnABCD upon bacteriocin induction. Two of these operons (termed plnEFI and plnJKLR) each encompass a gene pair (plnEF and plnJK, respectively) encoding two small cationic bacteriocin-like peptides with double-glycine-type leaders. The open reading frames (ORFs) encoding the bacteriocin-like peptides are followed by ORFs (plnI and -L, respectively) encoding cationic hydrophobic proteins resembling bacteriocin immunity proteins. On the third operon (termed plnMNOP), a similar bacteriocin-like ORF (plnN) and a putative immunity ORF (either plnM or -P) were identified as well. These findings suggest that two bacteriocins of two-peptide type (mature PlnEF and PlnJK) and a bacteriocin of one-peptide type (mature PlnN) could be responsible for the observed bacteriocin activity. The last operon (termed plnGHSTUV) contains two ORFs (plnGH) apparently encoding an ABC transporter and its accessory protein, respectively, known to be involved in processing and export of peptides with precursor double-glycine-type leaders. Promoter structure was established. A conserved regulatory-like box encompassing two direct repeats was identified in the promoter regions of all five plantaricin A-induced operons. These repeats may serve as regulatory elements for gene expression.

SUBMITTER: Diep DB

PROVIDER: S-EPMC178213 | biostudies-other | 1996 Aug

REPOSITORIES: biostudies-other

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Characterization of the locus responsible for the bacteriocin production in Lactobacillus plantarum C11.

Diep D B DB Håvarstein L S LS Nes I F IF

Journal of bacteriology 19960801 15

Lactobacillus plantarum C11 secretes a small cationic peptide, plantaricin A, that serves as induction signal for bacteriocin production as well as transcription of plnABCD. The plnABCD operon encodes the plantaricin A precursor (PlnA) itself and determinants (PlnBCD) for a signal transducing pathway. By Northern (RNA) and sequencing analyses, four new plantaricin A-induced operons were identified. All were highly activated in concert with plnABCD upon bacteriocin induction. Two of these operons ...[more]

PMID: 8755874

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Project description:BackgroundBacteriocin production in the lactic acid bacterium Lactobacillus plantarum C11 is regulated through a quorum sensing based pathway involving two highly homologous response regulators (59% identity and 76% similarity), PlnC as a transcriptional activator and PlnD as a repressor. Previous in vitro studies have shown that both regulators bind, as homodimers, to the same DNA regulatory repeats to exert their regulatory functions. As the genes for these two proteins are located on the same auto-regulatory operon, hence being co-expressed upon gene activation, it is plausible that their opposite functions must somehow be differentially regulated, either in terms of timing and/or binding kinetics, so that their activities do not impair each other in an uncontrolled manner. To understand the nature behind this potential differentiation, we have studied the binding kinetics of the two regulators on five target promoters (PplnA, PplnM, PplnJ, PplnE and PplnG) from the bacteriocin regulon of L. plantarum C11.ResultsBy using surface plasmon resonance spectroscopy we obtained parameters such as association rates, dissociation rates and dissociation constants, showing that the two regulators indeed differ greatly from each other in terms of cooperative binding and binding strength to the different promoters. For instance, cooperativity is very strong for PlnC binding to the promoter of the regulatory operon (PplnA), but not to the promoter of the transport operon (PplnG), while the opposite is seen for PlnD binding to these two promoters. The estimated affinity constants indicate that PlnC can bind to PplnA to activate transcription of the key regulatory operon plnABCD without much interference from PlnD, and that the repressive function of PlnD might act through a different mechanism than repression of the regulatory operon.ConclusionWe have characterised the DNA binding kinetics of the two regulators PlnC and PlnD from the bacteriocin locus in L. plantarum C11. Our data show that PlnC and PlnD, despite their strong homology to each other, differ greatly from each other in terms of binding affinity and cooperativity to the different promoters of the pln regulon.

Project description:Bacteriocins from lactic acid bacteria (LAB) are of increasing interest in recent years due to their potential as natural preservatives against food and beverage spoilage microorganisms. In a screening study for LAB, we isolated from olives a strain, Lactobacillus plantarum NI326, with activity against the beverage-spoilage bacterium Alicyclobacillus acidoterrestris Genome sequencing of NI326 enabled the identification of a gene cluster (designated plc) encoding a putative circular bacteriocin and proteins involved in its modification, transport, and immunity. This novel bacteriocin, named plantaricyclin A (PlcA), was grouped into the circular bacteriocin subgroup II due to its high degree of similarity with other gassericin A-like bacteriocins. Purification of PlcA from the supernatant of Lb. plantarum NI326 resulted in an active peptide with a molecular mass of 5,570 Da, corresponding to that predicted from the (processed) PlcA amino acid sequence. The plc gene cluster was cloned and expressed in Lactococcus lactis NZ9000, resulting in the production of an active 5,570-Da bacteriocin in the supernatant. PlcA is believed to be produced as a 91-amino-acid precursor with a 33-amino-acid leader peptide, which is predicted to be removed, followed by joining of the N and C termini via a covalent linkage to form the mature 58-amino-acid circular bacteriocin PlcA. We report the characterization of a circular bacteriocin produced by Lb. plantarum The inhibition displayed against A. acidoterrestris highlights its potential use as a preservative in food and beverages.IMPORTANCE In this work, we describe the purification and characterization of an antimicrobial peptide, termed plantaricyclin A (PlcA), produced by a Lactobacillus plantarum strain isolated from olives. This peptide has a circular structure, and all genes involved in its production, circularization, and secretion were identified. PlcA shows antimicrobial activity against different strains, including Alicyclobacillus acidoterrestris, a common spoilage bacterium, which causes substantial economic losses in the beverage industry every year. In this study, we describe a circular antimicrobial peptide, PlcA, for a Lactobacillus plantarum strain.

Dataset Information

Characterization of the locus responsible for the bacteriocin production in Lactobacillus plantarum C11.

Publications

Characterization of the locus responsible for the bacteriocin production in Lactobacillus plantarum C11.

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