RESUMEN

A 4.5-kb region of chromosomal DNA carrying the locus responsible for the production of plantaricin S, a two-peptide bacteriocin produced by Lactobacillus plantarum LPCO10 (R. Jiménez-Díaz, J. L. Ruiz-Barba, D. P. Cathcart, H. Holo, I. F. Nes, K. H. Sletten, and P. J. Warner, Appl. Environ. Microbiol. 61:4459-4463, 1995), has been cloned, and the nucleotide sequence has been elucidated. Two genes, designated plsA and plsB and encoding peptides alpha and beta, respectively, of plantaricin S, plus an open reading frame (ORF), ORF2, were found to be organized in an operon. Northern blot analysis showed that these genes are cotranscribed, giving a ca. 0.7-kb mRNA, whose transcription start point was determined by primer extension. Nucleotide sequences of plsA and plsB revealed that both genes are translated as bacteriocin precursors which include N-terminal leader sequences of the double-glycine type. The role of ORF2 is unknown at the moment, although it might be expected to encode an immunity protein of the type described for other bacteriocin operons. In addition, several other potential ORFs have been found, including some which may be responsible for the regulation of bacteriocin production. Two of them, ORF8 and ORF14, show strong homology with histidine protein kinase and response regulator genes, respectively, which have been found to be involved in the regulation of the production of other bacteriocins from lactic acid bacteria. A third ORF, ORF5, shows homology with gene agrB from Staphylococcus aureus, which is involved in the mechanism of regulation of the virulence phenotype in this species. Thus, an agr-like regulatory system for the production of plantaricin S is postulated.

Asunto(s)

Bacteriocinas/genética , Lactobacillus/genética , Secuencia de Aminoácidos , Bacteriocinas/biosíntesis , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , Lactobacillus/metabolismo , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Operón , Reacción en Cadena de la Polimerasa , Transcripción Genética

RESUMEN

Plantaricin S, one of the two bacteriocins produced by Lactobacillus plantarum LPCO10, which was isolated from a green-olive fermentation (R. Jiménez-Díaz, R.M. Ríos-Sánchez, M. Desmazeaud, J.L.Ruiz-Barba, and J.-C. Piard, Appl. Environ. Microbiol. 59:1416-1424, 1993), has been purified to homogeneity by ammonium sulfate precipitation, by binding to SP-Sepharose fast-flow, phenyl-Sepharose CL-4B, and C2/C18 reverse-phase chromatographies. The purification resulted in a final yield of 91.6% and a 352,617-fold increase in the specific activity. The bacteriocin activity was associated with two distinct peptides, termed alpha and beta, which were separated by C2/C18 reverse-phase chromatography. Although beta alone appeared to retain a trace of inhibitory activity, the complementary action of both the alpha and beta peptides was required for full bacteriocin activity, as judged by both the agar well diffusion and the microtiter plate assays. From the N-terminal end, 26 and 24 amino acids residues of alpha and beta, respectively, were sequenced. Further attempts at sequencing revealed no additional amino acids residues, suggesting that either modifications in the next amino acid residue blocked the sequencing region or that the C-terminal end had been reached. The amino acid sequences of alpha and beta show no apparent homology to each or to other bacteriocins purified from lactic acid bacteria.

Asunto(s)

Proteínas Bacterianas/aislamiento & purificación , Bacteriocinas/aislamiento & purificación , Lactobacillus/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Bacteriocinas/química , Bacteriocinas/metabolismo , Datos de Secuencia Molecular , Fragmentos de Péptidos/química

RESUMEN

Bacteriocin-producing Lactobacillus plantarum LPCO10 and its non-bacteriocin-producing, bacteriocinimmune derivative, L. plantarum 55-1, were evaluated separately for growth and persistence in natural Spanish-style green olive fermentations. Both strains were genetically marked and selectively enumerated using antibiotic-containing media. Plasmid profile and bacteriocin production (bac) were used as additional markers. When olive brines were inoculated at 10 CFU/ml, the parent strain, LPCO10, proliferated to dominate the epiphytic microflora, sharing high population levels with other spontaneously occurring lactobacilli and persisting throughout the fermentation (12 weeks). In contrast, the derivative strain could not be isolated after 7 weeks. Stability of both plasmid profile and bac (LPCO10 strain) or bac (55-1 strain) phenotype was shown by L. plantarum LPCO10 and L. plantarum 55-1 isolated throughout the fermentation. Bacteriocin activity could be found in the L. plantarum LPCO10-inoculated brines only after ammonium sulfate precipitation and concentration (20 times) of the final brine. Spontaneously occurring lactobacilli and lactic coccus populations, which were isolated from each of the fermenting brines studied during this investigation, were shown to be sensitive to the bacteriocins produced by L. plantarum LPCO10 when tested by the drop diffusion test. The declines in both pH and glucose levels throughout the fermentative process were similar in L. plantarum LPCO10- and in L. plantarum 55-1-inoculated brines and were comparable to the declines in the uninoculated brines. However, the final concentration of lactic acid in L. plantarum LPCO10-inoculated brines was higher than in the L. plantarum 55-1-inoculated brines and uninoculated brines. These results indicated that L. plantarum LPCO10 may be useful as a starter culture to control the lactic acid fermentation of Spanish-style green olives.