RESUMEN
A high-resolution amplified fragment length polymorphism (AFLP) methodology was developed to achieve the delineation of closely related Lactococcus lactis strains. The differentiation depth of 24 enzyme-primer-nucleotide combinations was experimentally evaluated to maximize the number of polymorphisms. The resolution depth was confirmed by performing diversity analysis on 82 L. lactis strains, including both closely and distantly related strains with dairy and nondairy origins. Strains clustered into two main genomic lineages of L. lactis subsp. lactis and L. lactis subsp. cremoris type-strain-like genotypes and a third novel genomic lineage rooted from the L. lactis subsp. lactis genomic lineage. Cluster differentiation was highly correlated with small-subunit rRNA homology and multilocus sequence analysis (MLSA) studies. Additionally, the selected enzyme-primer combination generated L. lactis subsp. cremoris phenotype-specific fragments irrespective of the genotype. These phenotype-specific markers allowed the differentiation of L. lactis subsp. lactis phenotype from L. lactis subsp. cremoris phenotype strains within the same L. lactis subsp. cremoris type-strain-like genomic lineage, illustrating the potential of AFLP for the generation of phenotype-linked genetic markers.
Asunto(s)
Análisis del Polimorfismo de Longitud de Fragmentos Amplificados/métodos , Lactococcus lactis/clasificación , Lactococcus lactis/genética , ADN Bacteriano/genética , Marcadores Genéticos , Variación Genética , Genotipo , Tipificación de Secuencias Multilocus , FenotipoRESUMEN
Lactobacillus plantarum is a ubiquitous microorganism that is able to colonize several ecological niches, including vegetables, meat, dairy substrates and the gastro-intestinal tract. An extensive phenotypic and genomic diversity analysis was conducted to elucidate the molecular basis of the high flexibility and versatility of this species. First, 185 isolates from diverse environments were phenotypically characterized by evaluating their fermentation and growth characteristics. Strains clustered largely together within their particular food niche, but human fecal isolates were scattered throughout the food clusters, suggesting that they originate from the food eaten by the individuals. Based on distinct phenotypic profiles, 24 strains were selected and, together with a further 18 strains from an earlier low-resolution study, their genomic diversity was evaluated by comparative genome hybridization against the reference genome of L. plantarum WCFS1. Over 2000 genes were identified that constitute the core genome of the L. plantarum species, including 121 unique L. plantarum-marker genes that have not been found in other lactic acid bacteria. Over 50 genes unique for the reference strain WCFS1 were identified that were absent in the other L. plantarum strains. Strains of the L. plantarum subspecies argentoratensis were found to lack a common set of 24 genes, organized in seven gene clusters/operons, supporting their classification as a separate subspecies. The results provide a detailed view on phenotypic and genomic diversity of L. plantarum and lead to a better comprehension of niche adaptation and functionality of the organism.
Asunto(s)
Biodiversidad , Ambiente , Genoma Bacteriano , Lactobacillus plantarum , Fenotipo , Animales , Análisis por Conglomerados , ADN Bacteriano/genética , Humanos , Lactobacillus plantarum/genética , Lactobacillus plantarum/aislamiento & purificación , Datos de Secuencia Molecular , ARN Ribosómico 16S/genéticaRESUMEN
The diversity in regulatory phenotypes among a collection of 84 Lactococcus lactis strains isolated from dairy and nondairy origin was explored. The specific activities of five enzymes were assessed in cell extracts of all strains grown in two different media, a nutritionally rich broth and a relatively poor chemically defined medium. The five investigated enzymes, branched chain aminotransferase (BcaT), aminopeptidase N (PepN), X-prolyl dipeptidyl peptidase (PepX), alpha-hydroxyisocaproic acid dehydrogenase (HicDH), and esterase, are involved in nitrogen and fatty acid metabolism and catalyze key steps in the production of important dairy flavor compounds. The investigated cultures comprise 75 L. lactis subsp. lactis isolates (including 7 L. lactis subsp. lactis biovar diacetylactis isolates) and 9 L. lactis subsp. cremoris isolates. All L. lactis subsp. cremoris and 22 L. lactis subsp. lactis (including 6 L. lactis subsp. lactis biovar diacetylactis) cultures originated from a dairy environment. All other cultures originated from (fermented) plant materials and were isolated at different geographic locations. Correlation analysis of specific enzyme activities revealed significantly different regulatory phenotypes for dairy and nondairy isolates. The enzyme activities in the two investigated media were in general poorly correlated and revealed a high degree of regulatory diversity within this collection of closely related strains. To the best of our knowledge, these results represent the most extensive diversity analysis of regulatory phenotypes within a single bacterial species to date. The presented findings underline the importance of the availability of screening procedures for, e.g., industrially relevant enzyme activities in models closely mimicking application conditions. Moreover, they corroborate the notion that regulatory changes are important drivers of evolution.
Asunto(s)
Proteínas Bacterianas/metabolismo , Enzimas/metabolismo , Regulación Bacteriana de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Variación Genética , Lactococcus lactis/clasificación , Lactococcus lactis/fisiología , Medios de Cultivo/química , Productos Lácteos/microbiología , Lactococcus lactis/genética , Lactococcus lactis/aislamiento & purificación , Plantas Comestibles/microbiologíaRESUMEN
Application of phytohemagglutinin (PHA) in weaning feed has been suggested to stimulate intestinal epithelium maturation. In this study, PHA strongly affected the fecal bacterial population structure of rats. Escherichia coli overgrowth was not prevented by probiotic mannose-adhering Lactobacillus plantarum 299v. Therefore, use of PHA in weaning feed deserves careful evaluation.
Asunto(s)
Escherichia coli/fisiología , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/microbiología , Lactobacillus plantarum/fisiología , Fitohemaglutininas/farmacología , Probióticos , Alimentación Animal/microbiología , Animales , Antibiosis , Adhesión Bacteriana , Recuento de Colonia Microbiana , Dermatoglifia del ADN , ADN Bacteriano/genética , ADN Espaciador Ribosómico/genética , Diarrea/microbiología , Escherichia coli/genética , Escherichia coli/aislamiento & purificación , Heces/microbiología , Lactobacillus plantarum/genética , Lactobacillus plantarum/aislamiento & purificación , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Ratas , Ratas Wistar , DesteteRESUMEN
The diversity of a collection of 102 lactococcus isolates including 91 Lactococcus lactis isolates of dairy and nondairy origin was explored using partial small subunit rRNA gene sequence analysis and limited phenotypic analyses. A subset of 89 strains of L. lactis subsp. cremoris and L. lactis subsp. lactis isolates was further analyzed by (GTG)(5)-PCR fingerprinting and a novel multilocus sequence analysis (MLSA) scheme. Two major genomic lineages within L. lactis were found. The L. lactis subsp. cremoris type-strain-like genotype lineage included both L. lactis subsp. cremoris and L. lactis subsp. lactis isolates. The other major lineage, with a L. lactis subsp. lactis type-strain-like genotype, comprised L. lactis subsp. lactis isolates only. A novel third genomic lineage represented two L. lactis subsp. lactis isolates of nondairy origin. The genomic lineages deviate from the subspecific classification of L. lactis that is based on a few phenotypic traits only. MLSA of six partial genes (atpA, encoding ATP synthase alpha subunit; pheS, encoding phenylalanine tRNA synthetase; rpoA, encoding RNA polymerase alpha chain; bcaT, encoding branched chain amino acid aminotransferase; pepN, encoding aminopeptidase N; and pepX, encoding X-prolyl dipeptidyl peptidase) revealed 363 polymorphic sites (total length, 1,970 bases) among 89 L. lactis subsp. cremoris and L. lactis subsp. lactis isolates with unique sequence types for most isolates. This allowed high-resolution cluster analysis in which dairy isolates form subclusters of limited diversity within the genomic lineages. The pheS DNA sequence analysis yielded two genetic groups dissimilar to the other genotyping analysis-based lineages, indicating a disparate acquisition route for this gene.
Asunto(s)
Variación Genética , Lactococcus lactis/genética , Reacción en Cadena de la Polimerasa/métodos , Análisis por Conglomerados , ADN Bacteriano/química , ADN Bacteriano/genética , Productos Lácteos/microbiología , Genotipo , Lactococcus lactis/clasificación , Datos de Secuencia Molecular , Fenotipo , Filogenia , ARN Ribosómico/genética , Análisis de Secuencia de ADNRESUMEN
The effectiveness of high-temperature, short holding time (HTST) pasteurization and homogenization with respect to inactivation of Mycobacterium avium subsp. paratuberculosis was evaluated quantitatively. This allowed a detailed determination of inactivation kinetics. High concentrations of feces from cows with clinical symptoms of Johne's disease were used to contaminate raw milk in order to realistically mimic possible incidents most closely. Final M. avium subsp. paratuberculosis concentrations varying from 10(2) to 3.5 x 10(5) cells per ml raw milk were used. Heat treatments including industrial HTST were simulated on a pilot scale with 22 different time-temperature combinations, including 60 to 90 degrees C at holding (mean residence) times of 6 to 15 s. Following 72 degrees C and a holding time of 6 s, 70 degrees C for 10 and 15 s, or under more stringent conditions, no viable M. avium subsp. paratuberculosis cells were recovered, resulting in >4.2- to >7.1-fold reductions, depending on the original inoculum concentrations. Inactivation kinetic modeling of 69 quantitative data points yielded an E(a) of 305,635 J/mol and an lnk(0) of 107.2, corresponding to a D value of 1.2 s at 72 degrees C and a Z value of 7.7 degrees C. Homogenization did not significantly affect the inactivation. The conclusion can be drawn that HTST pasteurization conditions equal to 15 s at > or =72 degrees C result in a more-than-sevenfold reduction of M. avium subsp. paratuberculosis.
Asunto(s)
Heces/microbiología , Microbiología de Alimentos , Leche/microbiología , Mycobacterium avium subsp. paratuberculosis/aislamiento & purificación , Animales , Bovinos , Desinfección/instrumentación , Desinfección/métodos , Manipulación de Alimentos/instrumentación , Manipulación de Alimentos/métodos , Calor , Humanos , Técnicas In Vitro , Cinética , Modelos Biológicos , Mycobacterium avium subsp. paratuberculosis/crecimiento & desarrollo , Mycobacterium avium subsp. paratuberculosis/patogenicidadRESUMEN
A group of 85 isolates of haloalkaliphilic obligately chemolithoautotrophic sulphur-oxidizing bacteria belonging to the genus Thioalkalivibrio were recently obtained from soda lakes in Mongolia, Kenya, California, Egypt and Siberia. They have been analyzed by repetitive extragenic palindromic (rep)-PCR genomic fingerprinting technique with BOX- and (GTG)5-primer set. Cluster analysis was performed using combined fingerprint profiles and a dendrogram similarity value (r) of 0.8 was used to define the same genotype. Fifty-six genotypes were found among the isolates, revealing a high genetic diversity. The strains can be divided into two major clusters, including isolates from the Asiatic (Siberia and Mongolia) and the African (Kenya and Egypt) continents, respectively. The majority (85.9%) of the genotypes were found in only one area, suggesting an endemic character of the Thioalkalivibrio strains. Furthermore, a correlation between fingerprint clustering, geographic origin and the characteristics of the lake of origin was found.
Asunto(s)
Ectothiorhodospiraceae/crecimiento & desarrollo , Ectothiorhodospiraceae/genética , Sedimentos Geológicos/microbiología , Microbiología del Agua , África , Asia , California , Análisis por Conglomerados , Dermatoglifia del ADN , ADN Bacteriano/química , ADN Bacteriano/genética , Electroforesis en Gel de Agar , Variación Genética , Procesamiento de Imagen Asistido por Computador , Reacción en Cadena de la PolimerasaRESUMEN
Lactococcus lactis is the primary model organism for lactic acid bacteria (LAB) and is widely used in the production of fermented dairy products. In recent years there has been increasing interest in strains isolated from non-dairy environments, as these exhibit a high metabolic diversity and have unique flavour-forming activities. Recent progress has been made in understanding the natural diversity and adaptive responses of L. lactis from dairy and non-dairy origins. Genome sequencing and comparative genomics have also had an impact on understanding natural diversity within the species, and have provided new opportunities for industrial strain development.