RESUMO
Lactobacillus sakei is a lactic acid bacterium naturally found on meat and often used as starter for the production of dry sausages or other fermented meat products. The gene encoding the green fluorescent protein (GFP) was cloned downstream from the constitutive L-lactate dehydrogenase promoter (pldhL) of L. sakei. The pldhL::gfp fusion was introduced in L. sakei either on a replicative plasmid or by double crossover integration into the chromosome, as a single copy. Both constructions were stable. Expression of GFP did not alter growth and was detectable by epifluorescence microscopy allowing the detection and monitoring of the development of GFP+ specific L. sakei strains both under growth laboratory conditions and in dry sausage samples.
Assuntos
Lactobacillus/metabolismo , Proteínas Luminescentes/metabolismo , Produtos da Carne/microbiologia , Cromossomos Bacterianos , Fermentação , Fluorescência , Microbiologia de Alimentos , Vetores Genéticos , Proteínas de Fluorescência Verde , Indicadores e Reagentes/metabolismo , Ácido Láctico/metabolismo , Lactobacillus/genética , Lactobacillus/crescimento & desenvolvimento , Proteínas Luminescentes/genética , Plasmídeos/genética , Transformação BacterianaRESUMO
16S rRNA targeted oligonucleotide probes were designed by sequence analysis of an rRNA database to discriminate S. carnosus, S. warneri, and S. saprophyticus species. After establishing hybridization conditions by RNA dot blot hybridization with reference species, our probes were shown to be specific. By in situ hybridization only S-S-S.carno-0440-a-A-23 and S-S-S.war-0180-a-A-23 can specifically detect S. carnosus and S. warneri, respectively. The detection of old cells of S. carnosus 833 was more limited by the permeabilisation than by the low rRNA content. One day old cells could be permeabilized with lysostaphin, whereas young cells were permeabilized with lysozyme.
