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Comparative phenotypic and molecular genetic profiling of wild Lactococcus lactis subsp. lactis strains of the L. lactis subsp. lactis and L. lactis subsp. cremoris genotypes, isolated from starter-free cheeses made of raw milk.

ABSTRACT: Twenty Lactococcus lactis strains with an L. lactis subsp. lactis phenotype isolated from five traditional cheeses made of raw milk with no added starters belonging to the L. lactis subsp. lactis and L. lactis subsp. cremoris genotypes (lactis and cremoris genotypes, respectively; 10 strains each) were subjected to a series of phenotypic and genetic typing methods, with the aims of determining their phylogenetic relationships and suitability as starters. Pulsed-field gel electrophoresis (PFGE) analysis of intact genomes digested with SalI and SmaI proved that all strains were different except for three isolates of the cremoris genotype, which showed identical PFGE profiles. Multilocus sequence typing (MLST) analysis using internal sequences of seven loci (namely, atpA, rpoA, pheS, pepN, bcaT, pepX, and 16S rRNA gene) revealed considerable intergenotype nucleotide polymorphism, although deduced amino acid changes were scarce. Analysis of the MLST data for the present strains and others from other dairy and nondairy sources showed that all of them clustered into the cremoris or lactis genotype group, by using both independent and combined gene sequences. These two groups of strains also showed distinctive carbohydrate fermentation and enzyme activity profiles, with the strains in the cremoris group showing broader profiles. However, the profiles of resistance/susceptibility to 16 antibiotics were very similar, showing no atypical resistance, except for tetracycline resistance in three identical cremoris genotype isolates. The numbers and concentrations of volatile compounds produced in milk by the strains belonging to these two groups were clearly different, with the cremoris genotype strains producing higher concentrations of more branched-chain, derived compounds. Together, the present results support the idea that the lactis and cremoris genotypes of phenotypic Lactococcus lactis subsp. lactis actually represent true subspecies. Some strains of the two subspecies in this study appear to be good starter candidates.

SUBMITTER: Fernandez E

PROVIDER: S-EPMC3147482 | biostudies-literature | 2011 Aug

REPOSITORIES: biostudies-literature

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Comparative phenotypic and molecular genetic profiling of wild Lactococcus lactis subsp. lactis strains of the L. lactis subsp. lactis and L. lactis subsp. cremoris genotypes, isolated from starter-free cheeses made of raw milk.

Fernández Elena E Alegría Angel A Delgado Susana S Martín M Cruz MC Mayo Baltasar B

Applied and environmental microbiology 20110610 15

Twenty Lactococcus lactis strains with an L. lactis subsp. lactis phenotype isolated from five traditional cheeses made of raw milk with no added starters belonging to the L. lactis subsp. lactis and L. lactis subsp. cremoris genotypes (lactis and cremoris genotypes, respectively; 10 strains each) were subjected to a series of phenotypic and genetic typing methods, with the aims of determining their phylogenetic relationships and suitability as starters. Pulsed-field gel electrophoresis (PFGE) a ...[more]

PMID: 21666023

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Project description:Lactococcus lactis is one of the most commonly used lactic acid bacteria in the dairy industry. Activation of competence for natural DNA transformation in this species would greatly improve the selection of novel strains with desired genetic traits. Here, we investigated the activation of natural transformation in L. lactis subsp. cremoris KW2, a strain of plant origin whose genome encodes the master competence regulator ComX and the complete set of proteins usually required for natural transformation. In the absence of knowledge about competence regulation in this species, we constitutively overproduced ComX in a reporter strain of late competence phase activation and showed, by transcriptomic analyses, a ComX-dependent induction of all key competence genes. We further demonstrated that natural DNA transformation is functional in this strain and requires the competence DNA uptake machinery. Since constitutive ComX overproduction is unstable, we alternatively expressed comX under the control of an endogenous xylose-inducible promoter. This regulated system was used to successfully inactivate the adaptor protein MecA and subunits of the Clp proteolytic complex, which were previously shown to be involved in ComX degradation in streptococci. In the presence of a small amount of ComX, the deletion of mecA, clpC, or clpP genes markedly increased the activation of the late competence phase and transformability. Altogether, our results report the functionality of natural DNA transformation in L. lactis and pave the way for the identification of signaling mechanisms that trigger the competence state in this species.IMPORTANCELactococcus lactis is a lactic acid bacterium of major importance, which is used as a starter species for milk fermentation, a host for heterologous protein production, and a delivery platform for therapeutic molecules. Here, we report the functionality of natural transformation in L. lactis subsp. cremoris KW2 by the overproduction of the master competence regulator ComX. The developed procedure enables a flexible approach to modify the chromosome with single point mutation, sequence insertion, or sequence replacement. These results represent an important step for the genetic engineering of L. lactis that will facilitate the design of strains optimized for industrial applications. This will also help to discover natural regulatory mechanisms controlling competence in the genus Lactococcus.

Dataset Information

Comparative phenotypic and molecular genetic profiling of wild Lactococcus lactis subsp. lactis strains of the L. lactis subsp. lactis and L. lactis subsp. cremoris genotypes, isolated from starter-free cheeses made of raw milk.

Publications

Comparative phenotypic and molecular genetic profiling of wild Lactococcus lactis subsp. lactis strains of the L. lactis subsp. lactis and L. lactis subsp. cremoris genotypes, isolated from starter-free cheeses made of raw milk.

Similar Datasets

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