RESUMEN
AIMS: The organization of ribosomal RNA (rrn) operons in Lactobacillus sanfranciscensis was studied in order to establish an easy-to-perform method for identification of L. sanfranciscensis strains, based on the length and sequence polymorphism of the 16S-23S rDNA intergenic spacer region (ISR). METHODS AND RESULTS: PCR amplification of the 16S-23S rDNA ISRs of L. sanfranciscensis gave three products distinguishing this micro-organism from the remaining Lactobacillus species. Sequence analysis revealed that two of the rrn operons were organized as in previously reported lactobacilli: large spacer (L-ISR), containing tRNA(Ile) and tRNA(Ala) genes; small spacer (S-ISR) without tRNA genes. The third described spacer (medium, M-ISR), original for L. sanfranciscensis, harboured a tRNA-like structure. An oligonucleotide sequence targeting the variable region between tDNA(Ile) and tDNA(Ala) of L. sanfranciscensis L-ISR was approved to be suitable in species-specific identification procedure. Analysis by pulse-field gel electrophoresis of the chromosomal digest with the enzyme I-CeuI showed the presence of seven rrn clusters. Lactobacillus sanfranciscensis genome size was estimated at c. 1.3 Mb. CONCLUSIONS: Direct amplification of 16S-23S ISRs or PCR with specific primer derived from L-ISR showed to be useful for specific typing of L. sanfranciscensis. This was due to the specific rrn operon organization of L. sanfranciscensis strains. SIGNIFICANCE AND IMPACT OF THE STUDY: In this paper, we have reported a rapid procedure for L. sanfranciscensis identification based on specific structures found in its rrn operon.
Asunto(s)
ADN Bacteriano/genética , ADN Espaciador Ribosómico/genética , Lactobacillus/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , Secuencia de Bases/genética , Cartilla de ADN/genética , ADN Intergénico , Microbiología de Alimentos , Genes Bacterianos/genética , Lactobacillus/genética , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Operón/genética , Polimorfismo Genético/genética , ARN Bacteriano/genética , ARN de Transferencia/genética , Alineación de Secuencia , Análisis de Secuencia de ADN/métodos , Especificidad de la Especie , Transcripción Genética/genéticaRESUMEN
AIMS: The restriction fragment length polymorphism (RFLP) method was used to differentiate Lactobacillus species having closely related identities in the 16S-23S rDNA intergenic spacer region (ISR). Species-specific primers for Lact. farciminis and Lact. alimentarius were designed and allowed rapid identification of these species. METHODS AND RESULTS: The 16S-23S rDNA spacer region was amplified by primers tAla and 23S/p10, then digested by HinfI and TaqI enzymes and analysed by electrophoresis. Digestion by HinfI was not sufficient to differentiate Lact. sakei, Lact. curvatus, Lact. farciminis, Lact. alimentarius, Lact. plantarum and Lact. paraplantarum. In contrast, digestion carried out by TaqI revealed five different patterns allowing these species to be distinguished, except for Lact. plantarum from Lact. paraplantarum. The 16S-23S rDNA spacer region of Lact. farciminis and Lact. alimentarius were amplified and then cloned into vector pCR(R)2.1 and sequenced. The DNA sequences obtained were analysed and species-specific primers were designed from these sequences. The specificity of these primers was positively demonstrated as no response was obtained for 14 other species tested. RESULTS AND CONCLUSIONS: The species-specific primers for Lact. farciminis and Lact. alimentarius were shown to be useful for identifying these species among other lactobacilli. The RFLP profile obtained upon digestion with HinfI and TaqI enzymes can be used to discriminate Lact. farciminis, Lact. alimentarius, Lact. sakei, Lact. curvatus and Lact. plantarum. SIGNIFICANCE AND IMPACT OF THE STUDY: In this paper, we have established the first species-specific primer for PCR identification of Lact. farciminis and Lact. alimentarius. Both species-specific primer and RFLP, could be used as tools for rapid identification of lactobacilli up to species level.
Asunto(s)
Técnicas de Tipificación Bacteriana/métodos , Lactobacillus/clasificación , Secuencia de Bases , Cartilla de ADN , ADN Bacteriano/genética , ADN Espaciador Ribosómico/genética , Electroforesis en Gel de Agar , Lactobacillus/genética , Datos de Secuencia Molecular , Polimorfismo de Longitud del Fragmento de Restricción , ARN Bacteriano/genética , ARN Ribosómico 16S/genética , ARN Ribosómico 23S/genética , Alineación de Secuencia , Especificidad de la EspecieRESUMEN
A strain of Enterococcus faecium isolated from Bulgarian yellow cheese "kashkaval" produced a bacteriocin-like substance named enterococcin A 2000. The antibacterial substance had a low molar mass (< 2 kDa), was relatively stable toward heat but was sensitive to selected proteolytic enzymes. It was active against Gram-positive bacteria including enterococci, such as Listeria, Bacillus and Streptococcus, and also against Gram-negative E. coli. Production of enterococcin A 2000 has a maximum near the end of the exponential phase of producer growth. The peptide was purified by ammonium sulfate precipitation, butanol extraction, followed by cation-exchange chromatography and reversed-phase chromatography. A partial sequence of purified enterococcin A 2000 indicated that this substance does not belong to the class IIa of bacteriocins presenting the consensus anti-Listeria motif YGNGV.
Asunto(s)
Antibacterianos/aislamiento & purificación , Bacteriocinas/aislamiento & purificación , Enterococcus faecium/química , Secuencia de Aminoácidos , Aminoácidos/análisis , Antibacterianos/química , Antibacterianos/farmacología , Bacteriocinas/química , Bacteriocinas/genética , Bacteriocinas/farmacología , Secuencia de Bases , Queso/microbiología , ADN Bacteriano/genética , Enterococcus faecium/genética , Escherichia coli/efectos de los fármacos , Listeria/efectos de los fármacos , Peso Molecular , Reacción en Cadena de la Polimerasa , ARN Bacteriano/genética , ARN Ribosómico 16S/genética , ARN Ribosómico 23S/genéticaRESUMEN
The production of a novel broad-spectrum antimicrobial peptide enterococcin A 2000, active against Gram-positive and Gram-negative microorganisms including Listeria subsp. and Escherichia coli, by Enterococcus faecium strain A 2000 isolated from the surface of traditional Bulgarian yellow cheese "kash-kaval" is considerably influenced by complex nitrogen sources in the production medium. Medium components, especially peptone and yeast extract, and their concentration contributed to the increase in bacteriocin production during the stationary phase (16-46 h) of cultivation even in the absence of one of the components present in the basal cultivation MRS medium.
