Evaluation of Lactococcus lactis Isolates from Nondairy Sources with Potential Dairy Applications Reveals Extensive Phenotype-Genotype Disparity and Implications for a Revised Species

Abstract

dLactococcus lactis is predominantly associated with dairy fermentations, but evidence suggests that the domesticated organism originated from a plant niche. L. lactis possesses an unusual taxonomic structure whereby strain phenotypes and genotypes often do not correlate, which in turn has led to confusion in L. lactis classification. A bank of L. lactis strains was isolated from various nondairy niches (grass, vegetables, and bovine rumen) and was further characterized on the basis of key technological traits, including growth in milk and key enzyme activities. Phenotypic analysis revealed all strains from nondairy sources to possess an L. lactis subsp. lactis phenotype (lactis phenotype); however, seven of these strains possessed an L. lactis subsp. cremoris genotype (cremoris genotype), determined by two separate PCR assays. Multilocus sequence typing (MLST) showed that strains with lactis and cremoris genotypes clustered together regardless of habitat, but it highlighted the increased diversity that exists among "wild" strains. Calculation of average nucleotide identity (ANI) and tetranucleotide frequency correlation coefficients (TETRA), using the JSpecies software tool, revealed that L. lactis subsp. cremoris and L. lactis subsp. lactis differ in ANI values by ϳ14%, below the threshold set for species circumscription. Further analysis of strain TIFN3 and strains from nonindustrial backgrounds revealed TETRA values of <0.99 in addition to ANI values of <95%, implicating that these two groups are separate species. These findings suggest the requirement for a revision of L. lactis taxonomy. Lactococcus lactis is a member of the lactic acid bacteria (LAB), a group of organisms used worldwide in the production of fermented dairy products. Three Lactococcus lactis subspecies exist: Lactococcus lactis subsp. cremoris, L. lactis subsp. lactis, and L. lactis subsp. hordniae. Many strains of L. lactis subsp. lactis and L. lactis subsp. cremoris are typically associated with dairy fermentations, but evidence suggests that these organisms originated from a plant niche, and they are now considered to be "domesticated" compared to their so-called "wild" counterparts. Conversely, L. lactis subsp. hordniae is unable to utilize lactose and has not been isolated previously from the dairy environment (1). A citrate-metabolizing biovariety also exists, L. lactis biovar diacetylactis, which is capable of imparting buttery aromas in dairy fermentations (2). Information regarding subspecies classification is important for the application of cultures in dairy fermentations, particularly cheese production, as L. lactis subsp. cremoris strains are often considered more suitable due to their association with cheeses free from off-flavors (3).Before the advent of molecular methods, subspecies classification of L. lactis was based on the possession of a number of phenotypic traits. The ability to grow in the presence of 4% NaCl, at 40°C, and at pH 9.2 and the ability to degrade arginine were assigned as traits for L. lactis subspecies...

Comparative and functional genomics of the Lactococcus lactis taxon; insights into evolution and niche adaptation

“…cremoris . These observations support those made by Cavanagh and colleagues, who recently proposed a re-evaluation of the taxonomic group separating L. lactis into two distinct species L. lactis and L. cremoris based on ANI (average nucleotide identity) and TETRA (tetranucleotide frequency correlation coefficients) [16]. …”

“…lactis isolates in comparison to those from subsp. cremoris isolates, and is in agreement with the described differences in average nucleotide identity and tetranucleotide frequency correlation coefficients between the two subspecies [16]. To investigate the unique allelic types of the two subspecies, a subset of individual highly conserved housekeeping genes, i.e.…”

“…Amino acid transferase activity was determined (for Phe and Met) as previously described by Cavanagh and colleagues [16]. The final absorbance was read at wavelength, 300 nm in triplicate on a DU Series 730 spectrophotometer from Beckman Coulter, blanking the machine between each measurement.…”

“…cremoris MG1363 [13]. To date a number of comparative genome studies have been conducted and have provided novel insights into lipolysis [14], prophage [5, 6], proteolysis [15], taxonomy [16] and niche adaptation functions of these strains [17]. …”

From Genome to Phenotype: An Integrative Approach to Evaluate the Biodiversity of Lactococcus lactis

“…Cavanagh et al [15] determined ANIb values (ANI calculated using the BLAST algorithm) of a set of 19 L. lactis genome sequences. In the same subspecies, ANIb values were comprised between 96.53% and 99.96%.…”