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Genotypic and phenotypic analysis of dairy Lactococcus lactis biodiversity in milk: volatile organic compounds as discriminating markers.

ABSTRACT: The diversity of nine dairy strains of Lactococcus lactis subsp. lactis in fermented milk was investigated by both genotypic and phenotypic analyses. Pulsed-field gel electrophoresis and multilocus sequence typing were used to establish an integrated genotypic classification. This classification was coherent with discrimination of the L. lactis subsp. lactis bv. diacetylactis lineage and reflected clonal complex phylogeny and the uniqueness of the genomes of these strains. To assess phenotypic diversity, 82 variables were selected as important dairy features; they included physiological descriptors and the production of metabolites and volatile organic compounds (VOCs). Principal-component analysis (PCA) demonstrated the phenotypic uniqueness of each of these genetically closely related strains, allowing strain discrimination. A method of variable selection was developed to reduce the time-consuming experimentation. We therefore identified 20 variables, all associated with VOCs, as phenotypic markers allowing discrimination between strain groups. These markers are representative of the three metabolic pathways involved in flavor: lipolysis, proteolysis, and glycolysis. Despite great phenotypic diversity, the strains could be divided into four robust phenotypic clusters based on their metabolic orientations. Inclusion of genotypic diversity in addition to phenotypic characters in the classification led to five clusters rather than four being defined. However, genotypic characters make a smaller contribution than phenotypic variables (no genetic distances selected among the most contributory variables). This work proposes an original method for the phenotypic differentiation of closely related strains in milk and may be the first step toward a predictive classification for the manufacture of starters.

SUBMITTER: Dhaisne A

PROVIDER: S-EPMC3719511 | biostudies-literature | 2013 Aug

REPOSITORIES: biostudies-literature

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Genotypic and phenotypic analysis of dairy Lactococcus lactis biodiversity in milk: volatile organic compounds as discriminating markers.

Dhaisne Amandine A Guellerin Maeva M Laroute Valérie V Laguerre Sandrine S Cocaign-Bousquet Muriel M Le Bourgeois Pascal P Loubiere Pascal P

Applied and environmental microbiology 20130524 15

The diversity of nine dairy strains of Lactococcus lactis subsp. lactis in fermented milk was investigated by both genotypic and phenotypic analyses. Pulsed-field gel electrophoresis and multilocus sequence typing were used to establish an integrated genotypic classification. This classification was coherent with discrimination of the L. lactis subsp. lactis bv. diacetylactis lineage and reflected clonal complex phylogeny and the uniqueness of the genomes of these strains. To assess phenotypic d ...[more]

PMID: 23709512

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Project description: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.

Project description:BACKGROUND:Important industrial traits have been linked to plasmids in Lactococcus lactis. RESULTS:The dairy isolate L. lactis subsp. lactis biovar diacetylactis FM03P was sequenced revealing the biggest plasmidome of all completely sequenced and published L. lactis strains up till now. The 12 plasmids that were identified are: pLd1 (8277 bp), pLd2 (15,218 bp), pLd3 (4242 bp), pLd4 (12,005 bp), pLd5 (7521 bp), pLd6 (3363 bp), pLd7 (30,274 bp), pLd8 (47,015 bp), pLd9 (15,313 bp), pLd10 (39,563 bp), pLd11 (9833 bp) and pLd12 (3321 bp). Structural analysis of the repB promoters and the RepB proteins showed that eleven of the plasmids replicate via the theta-type mechanism, while only plasmid pLd3 replicates via a rolling-circle replication mechanism. Plasmids pLd2, pLd7 and pLd10 contain a highly similar operon involved in mobilisation of the plasmids. Examination of the twelve plasmids of L. lactis FM03P showed that 10 of the plasmids carry putative genes known to be important for growth and survival in the dairy environment. These genes encode technological functions such as lactose utilisation (lacR-lacABCDFEGX), citrate uptake (citQRP), peptide degradation (pepO and pepE) and oligopeptide uptake (oppDFBCA), uptake of magnesium and manganese (2 mntH, corA), exopolysaccharides production (eps operon), bacteriophage resistance (1 hsdM, 1 hsdR and 7 different hsdS genes of a type I restriction-modification system, an operon of three genes encoding a putative type II restriction-modification system and an abortive infection gene) and stress resistance (2 uspA, cspC and cadCA). Acquisition of these plasmids most likely facilitated the adaptation of the recipient strain to the dairy environment. Some plasmids were already lost during a single propagation step signifying their instability in the absence of a selective pressure. CONCLUSIONS:Lactococcus lactis FM03P carries 12 plasmids important for its adaptation to the dairy environment. Some of the plasmids were easily lost demonstrating that propagation outside the dairy environment should be minimised when studying dairy isolates of L. lactis.

Dataset Information

Genotypic and phenotypic analysis of dairy Lactococcus lactis biodiversity in milk: volatile organic compounds as discriminating markers.

Publications

Genotypic and phenotypic analysis of dairy Lactococcus lactis biodiversity in milk: volatile organic compounds as discriminating markers.

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