RESUMEN
A slowly milk-coagulating variant (Fmc(-)) of Lactobacillus helveticus CRL 1062, designated S1, was isolated and characterized. Strain S1 possessed all the known essential components required to utilize casein as a nitrogen source, which include functional proteinase and peptidase activities as well as functional amino acid, di- and tripeptide, and oligopeptide transport systems. The amino acid requirements of strain S1 were similar to those of the parental strain. However, on a purine-free, chemically defined medium, the growth rate of the Fmc(-) strain was threefold lower than that of the wild-type strain. L. helveticus S1 was found to be defective in IMP dehydrogenase activity and therefore was deficient in the ability to synthesize XMP and GMP. This conclusion was further supported by the observation that the addition of guanine or xanthine to milk, a substrate poor in purine compounds, restored the Fmc(+) phenotype of L. helveticus S1.
Asunto(s)
IMP Deshidrogenasa/metabolismo , Lactobacillus/metabolismo , Leche/metabolismo , Leche/microbiología , Purinas/biosíntesis , Animales , Caseínas/metabolismo , Endopeptidasas/metabolismo , Guanina/biosíntesis , Lactobacillus/genética , Lactobacillus/crecimiento & desarrollo , Factores de Tiempo , beta-Galactosidasa/metabolismoRESUMEN
The nutritional requirements of Lactobacillus helveticus CRL 1062 were determined with a simplified chemically defined medium (SCDM) and compared with those of L. helveticus CRL 974 (ATCC 15009). Both strains were found to be prototrophic for alanine, glycine, asparagine, glutamine, and cysteine. In addition, CRL 1062 also showed prototrophy for lysine and serine. The microorganisms also required riboflavin, calcium pantothenate, pyridoxal, nicotinic acid, and uracil for growth in liquid SCDM. The growth rate and the synthesis of their cell membrane-bound serine proteinases, but not of their intracellular leucyl-aminopeptidases, were influenced by the peptide content of the medium. The highest proteinase levels were found during cell growth in basal SCDM, while the synthesis of this enzyme was inhibited in SCDM supplemented with Casitone, Casamino Acids, or beta-casein. Low-molecular-mass peptides (<3,000 Da), extracted from Casitone, and the dipeptide leucylproline (final concentration, 5 mM) play important roles in the medium-dependent regulation of proteinase activity. The addition of the dipeptide leucylproline (5 mM) to SCDM reduced proteinase activity by 25%.
Asunto(s)
Endopeptidasas/metabolismo , Lactobacillus/metabolismo , Nitrógeno/metabolismo , Aminopeptidasas/metabolismo , Caseínas/metabolismo , Medios de Cultivo/química , Hidrólisis , Lactobacillus/crecimiento & desarrollo , Serina Endopeptidasas/metabolismoRESUMEN
The technological relevant characteristics of five homofermentative lactobacilli strains, isolated from natural fermented hard cheeses, were studied. Isolates CRL 581 and CRL 654, from Argentinian artesanal hard cheeses, and isolates CRL 1177, CRL 1178, and CRL 1179, from Italian Grana cheeses, were identified as Lactobacillus delbrueckii subsp. lactis and Lactobacillus helveticus, respectively, by physiological and biochemical tests, SDS-PAGE of whole-cell proteins and sequencing of the variable (V1) region of the 16S ribosomal DNA. All strains showed high levels of beta-galactosidase activity. However, proteolytic activity varied widely among isolates. Strains CRL 581, CRL 654, and CRL 1177 hydrolyzed alpha- and beta-caseins and were able to coagulate reconstituted skim milk in less than 16 h at 42 degrees C. According to the substrate specificity, these proteinases have a caseinolytic activity comparable to that of the P(III)-type of lactococcal proteinases. No strains produced inhibitor substances (bacteriocin) and all were insensitive to attack by 14 L. helveticus- and L. delbrueckii subsp. lactis-specific bacteriophages.
Asunto(s)
Productos Lácteos/microbiología , Lactobacillus/aislamiento & purificación , Animales , Argentina , Técnicas de Tipificación Bacteriana , Caseínas/metabolismo , Queso/microbiología , ADN Ribosómico/química , Electroforesis en Gel de Poliacrilamida/métodos , Fermentación , Lactobacillus/clasificación , Lactobacillus/genética , Lactobacillus/metabolismo , Fenotipo , Reacción en Cadena de la Polimerasa/métodos , ARN Ribosómico 16S/genética , Análisis de Secuencia/métodos , Especificidad por SustratoRESUMEN
DNase activity was examined in the extracellular and subcellular fractions of six non-transformable strains belonging to Lactobacillus delbrueckii subsp. lactis (L. lactis) and Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus) and compared with the activity present in Lactobacillus johnsonii NCK 65, a transformable strain of Lactobacillus. In the extracellular fraction of the L. delbrueckii strains, a common protein band of 36 kDa was detected, while a band of 29 kDa was found in the same fraction of L. johnsonii. No nuclease activity was detected in the cytoplasmic fraction of this strain, indicating that the localization of the DNase activity could be a key factor in the uptake of foreign DNA.
Asunto(s)
Proteínas Bacterianas/metabolismo , Desoxirribonucleasas/metabolismo , Lactobacillus/enzimología , Sistema Libre de Células , ADN/metabolismo , Electroforesis en Gel de Poliacrilamida , Espacio Extracelular/enzimología , Lactobacillus/crecimiento & desarrollo , Lactobacillus/metabolismo , Fracciones SubcelularesRESUMEN
The structural gene determinants of lactocin 705, a bacteriocin produced by Lactobacillus casei CRL 705, have been amplified from a plasmid of approximately 35 kb and sequenced. Lactocin 705 is a class IIb bacteriocin, whose activity depends upon the complementation of two peptides (705alpha and 705beta) of 33 amino acid residues each. These peptides are synthesized as precursors with signal sequences of the double-glycine type, which exhibited high identities with the leader peptides of plantaricin S and J from Lactobacillus plantarum, brochocin C from Brochotrix campestris, sakacin P from Lactobacillus sake, and the competence stimulating peptides from Streptococcus gordonii and Streptococcus mitis. However, the two mature bacteriocins 705alpha and 705beta do not show significant similarity to other sequences in the databases.
Asunto(s)
Bacteriocinas/biosíntesis , Bacteriocinas/genética , Genes Bacterianos , Lacticaseibacillus casei/metabolismo , Secuencia de Aminoácidos , Bacteriocinas/química , Bacteriocinas/clasificación , Secuencia de Bases , Lacticaseibacillus casei/genética , Datos de Secuencia Molecular , Péptidos/síntesis química , Péptidos/química , Plásmidos/genética , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADNRESUMEN
A restriction map was constructed of the 37 kb genome of the temperate Lactobacillus delbrueckii subsp. bulgaricus bacteriophage lb539. Restriction analysis and Southern hybridization experiments detected variable levels of homologous regions among the genomes of lb539 and the L. delbrueckii reference phages LL-H (virulent) and mv4 (temperate). The principal homology was observed at the regions encoding the structural proteins. These studies allowed us to construct a partial genetic map of phage lb539 for lysin, the main structural tail protein and the packaging region genes. Furthermore, a short 1.5 kb DNA fragment of the prolate-headed JCL1032 phage genome was observed to be highly homologous with the DNA of the isometric-headed lb539, mv4 and LL-H phages. The described distribution of the homologous regions between the genomes of the phages lb539, LL-H, mv4 and JCL1032 presented here supports the modular evolution theory of the bacteriophages.