RESUMO
Two new Lactobacillus plantarum strains, KR6-DSM 28780 and M5 isolated from sour turnip and traditional dried fresh cheese, respectively, were evaluated for species identity, antibiotic susceptibility, resistance to gastrointestinal conditions and adaptive response to low pH. Resistance mechanisms involved in the adaptation to acid-induced stress in these two strains were investigated by quantitative PCR of the atpA, cfa1, mleS and hisD genes. In addition to absence of antibiotic resistance, the two L. plantarum strains showed excellent survival rates at pH values as low as 2.4. Adaptive response to low pH was clearly observed in both strains; strain KR6 was superior to M5, as demonstrated by its ability to survive during 3 h incubation at pH 2.0 upon adaptation to moderately acidic conditions. In contrast, acid adaptation did not significantly affect the survival rate during simulated passage through the gastrointestinal tract. In both strains, induction of histidine biosynthesis (hisD) was upregulated during the acid adaptation response. In addition, significant upregulation of the cfa1 gene, involved in modulation of membrane fatty acid composition, was observed during the adaptation phase in strain KR6 but not in strain M5. Cells adapted to moderately acidic conditions also showed a significantly increased viability after the lyophilisation procedure, a cross-protection phenomenon providing additional advantage in probiotic application.
Assuntos
Ácidos/farmacologia , Lactobacillus plantarum/fisiologia , Adaptação Fisiológica/efeitos dos fármacos , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Brassica napus/microbiologia , Queijo/microbiologia , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Lactobacillus plantarum/efeitos dos fármacos , Lactobacillus plantarum/genética , Lactobacillus plantarum/isolamento & purificação , Probióticos/química , Estresse Fisiológico/efeitos dos fármacosRESUMO
The efficiency of gene targeting within different segments of genes in yeast was estimated by transforming yeast cells with double-stranded integrative plasmids, bearing functional gene domains [promoter (P), ORF (O) and terminator (T)] derived from the common genetic markers HIS3, LEU2, TRP1 and URA3. Transformation experiments with circular plasmids carrying a single gene domain demonstrated that the 5' and 3' flanking DNA regions (P and T) of the HIS3 and URA3 genes are preferred as sites for plasmid integration by several fold over the corresponding ORFs. Moreover, when plasmids bearing combinations of two or three regions were linearized to target them to a specific site of integration, three of the ORFs were found to be less preferred as sites for plasmid integration than their corresponding flanking regions. Surprisingly, in up to 50% of the transformants obtained with plasmids that had been linearized within coding sequences, the DNA actually integrated into neighbouring regions. Almost the same frequencies of ORF mis-targeting were obtained with plasmid vectors containing only two functional domains ("PO" or "OT") of the gene URA3, demonstrating that this event is not the consequence of competition between homologous DNA regions distal to the ORF. Therefore, we suggest that coding sequences could be considered to be "cold spots" for plasmid integration in yeast.
Assuntos
Aldose-Cetose Isomerases , DNA Fúngico/metabolismo , Marcadores Genéticos , Fases de Leitura Aberta , Plasmídeos/genética , Recombinação Genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Transformação Genética , Primers do DNA/química , DNA Fúngico/genética , Proteínas Fúngicas/genética , Proteínas de Plantas/genética , Plasmídeos/química , Reação em Cadeia da Polimerase , Estrutura Terciária de ProteínaRESUMO
We studied illegitimate recombination by transforming yeast with a single-stranded (ss) non-replicative plasmid. Plasmid pCW12, containing the ARG4 gene, was used for transformation of yeast strains deleted for the ARG4, either in native (circular) form or after linearization within the vector sequence by the restriction enzyme ScaI. Both circular and linearized ss plasmids were shown to be much more efficient in illegitimate integration than their double-stranded (ds) counterparts and more than two-thirds of the transformants analysed contained multiple tandem integrations of the plasmid. Pulsed-field gel electrophoresis of genomic DNA revealed significant changes in the karyotype of some transformants. Plasmid DNA was frequently detected on more than one chromosome and on mitotically unstable, autonomously replicating elements. Our results show that the introduction of nonhomologous ss DNA into yeast cells can lead to different types of alterations in the yeast genome.
