Genotypic and phenotypic characterization of Lactobacillus casei isolated from different environments

Lactobacillus casei is an ecologically versatile and industrially important lactic acid bacterium (LAB). To gain comprehensive knowledge of their evolutionary adaptation to different ecological niches, we assembled 52 Lb. casei strains from various sources and studied these strains as follows: (i) Determination of the evolutionary relatedness of 40 Lb. casei strains by multilocus sequence typing (MLST) and pulse-field gel electrophoresis (PFGE). MLST revealed 36 sequence types. PFGE using SfiI separated all 40 strains, suggesting that point mutations have accumulated at a slower rate than indels and genome rearrangements in Lb. casei. MLST phylogenetic analysis grouped Lb. casei strains into a cheese cluster, a silage cluster, and a cluster with strains of different origins. Frequent intraspecies recombination was observed in Lb. casei. (ii) Characterization of Lb. casei ATCC 334 genome and determination of the genome inventory differences of 22 Lb. casei strains by comparative genome hybridization (CGH). A high number of genes involved in carbohydrate metabolism and transcriptional regulation was identified in ATCC 334. Comparative genomics and CGH analysis revealed genomic islands and hypervariable regions, including a lifestyle adaptation island. A distinct subpopulation of cheese isolates that have undergone significant gene decay was identified. (iii) Screening of 22 Lb. casei strains for their ability to dominate the non-starter LAB (NSLAB) microbiota, produce volatile flavor compounds, and hydrolyze a model bitter peptide beta-CN(fl93-209) in Cheddar cheese extract prepared from 4 month old Cheddar cheese and supplemented with citrate, under Cheddar cheese ripening condition (pH5.1, 3.1% NaCl, and 8°C). None of the Lb. casei strains examined could degrade the bitter peptide. Six strains exhibited growth parameters in the model system likely to be associated with the ability to dominate the NSLAB microbiota. Significant strain-specific differences in the concentrations of 2,3-butanedione, phenylethanal, and phenylethanol accumulation were observed. In summary, Lb. casei strains analyzed demonstrated both diversity and specificity to different ecological niches. Results from comparative genomics and CGH analysis provided valuable insights into their niche-associated evolution. Studies on growth and production of volatile compounds provided a starting point for rational selection of Lb. casei adjunct cultures to control Cheddar cheese flavor development.