Rapid Genotypic Differentiation of Lactococcus lactis Subspecies and Biovar

Beimfohr, Claudia; Ludwig, Wolfgang; Schleifer, Karl‐Heinz

doi:10.1016/s0723-2020(97)80068-9

Cited by 52 publications

(63 citation statements)

References 13 publications

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“…is based on the detection of the d-alanine-ligase enzyme, antibiotic resistance and specific regions (Dutka-Malen et al, 1995). Species specific PCR is also useful for identifying microorganisms at subspecies level, which cannot be easily achieved with other common techniques (Beimfohr et al, 1997). Another disadvantage is the time required to obtain the sequencing results, which is considerably higher than the time needed to obtain phenotypic test results.…”

Section: Comparison Of Api With Sequencing Of 16s Rdna Genementioning

confidence: 99%

Biochemical and molecular identification of lactic acid bacteria isolated from camel milk in Tunisia

Fguiri

Ziadi

Atigui

et al. 2015

Emir. J. Food Agric

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Section: Comparison Of Api With Sequencing Of 16s Rdna Genementioning

confidence: 99%

Biochemical and molecular identification of lactic acid bacteria isolated from camel milk in Tunisia

Fguiri

Ziadi

Atigui

et al. 2015

Emir. J. Food Agric

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“…To attempt to accurately identify subspecies of L. lactis, various assays have been developed (11-13). These assays employ different molecular techniques such as PCR-restriction fragment length polymorphism (PCR-RFLP) and Southern blot hybridization, targeting genes encoding branched-chain amino acid biosynthesis and glutamate decarboxylase, among others (12,14).In particular, two such methods have been developed for rapid and simple species genotype identification by PCR, based on the 16S rRNA gene and the histidine biosynthesis operon (11,13) …”

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“…In particular, two such methods have been developed for rapid and simple species genotype identification by PCR, based on the 16S rRNA gene and the histidine biosynthesis operon (11,13) …”

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“…cremoris phenotype (cremoris phenotype) (8) and vice versa (9,10). To attempt to accurately identify subspecies of L. lactis, various assays have been developed (11)(12)(13). These assays employ different molecular techniques such as PCR-restriction fragment length polymorphism (PCR-RFLP) and Southern blot hybridization, targeting genes encoding branched-chain amino acid biosynthesis and glutamate decarboxylase, among others (12,14).…”

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

Cavanagh

Casey

Altermann

et al. 2015

Appl Environ Microbiol

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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...

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“…lactis to L. lactis subsp. cremoris and vice versa.As classification according to phenotypic criteria is complicated and requires skill, simple, fast, and reliable molecular methods have been developed (3,5). Classification based on these methods, however, coincides with genotypic identification and does not always correlate with phenotypic characterization.…”

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Rapid PCR-Based Method Which Can Determine Both Phenotype and Genotype of Lactococcus lactis Subspecies

Nomura

Kobayashi

Okamoto

2002

Appl Environ Microbiol

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A highly efficient, rapid, and reliable PCR-based method for distinguishing Lactococcus lactis subspecies (L. lactis subsp. lactis and L. lactis subsp. cremoris) is described. Primers complementary to positions in the glutamate decarboxylase gene have been constructed. PCR analysis with extracted DNA or with cells of different L. lactis strains resulted in specific fragments. The length polymorphism of the PCR fragments allowed a clear distinction of the L. lactis subspecies. The amplified fragment length polymorphism with the primers and the restriction fragment length polymorphism of the amplified products agreed perfectly with the identification based on genotypic and phenotypic analyses, respectively. Isolates from cheese starters were investigated by this method, and amplified fragments of genetic variants were found to be approximately 40 bp shorter than the typical L. lactis subsp. cremoris fragments.Lactococcal strains are essential to milk fermentation, especially in the cheese-making process, providing optimal conditions for curd formation and for the development of texture and flavor. It is important to the dairy industry to identify new strains of Lactococcus lactis for cheese manufacture. Dairy lactococcal strains are subdivided into L. lactis subsp. lactis and L. lactis subsp. cremoris based on a few phenotypic tests: growth in 4% NaCl, pH 9.2, at 40°C; the ability to hydrolyze arginine (7); and sensitivity to lithium chloride (1).Recently, a novel criterion for distinguishing L. lactis subsp. lactis from L. lactis subsp. cremoris has been reported: glutamate decarboxylase (GAD; EC 4.1.1.15) activity, which has been observed in L. lactis subsp. lactis and not in L. lactis subsp. cremoris (8). GAD catalyzes the irreversible decarboxylation of glutamate to ␥-aminobutyric acid (GABA). GAD constitutes a glutamate-dependent acid resistance mechanism with a glutamate-GABA antiporter (12). The gadB gene encoding L. lactis subsp. cremoris GAD was apparently inactivated by a frameshift mutation resulting from an adenine deletion or a thymine insertion and encoded a nonfunctional protein (10).It has been discovered in recent years that L. lactis strains can be divided into two phylogenetic groups based on genotypic analysis (6,11,13), and it has been proposed that the subspecies diagnoses be redefined to reflect these natural relationships (6). The new taxonomic system requires the transfer of several strains across subspecies lines, from L. lactis subsp. lactis to L. lactis subsp. cremoris and vice versa.As classification according to phenotypic criteria is complicated and requires skill, simple, fast, and reliable molecular methods have been developed (3,5). Classification based on these methods, however, coincides with genotypic identification and does not always correlate with phenotypic characterization. The classification of L. lactis subspecies based on phenotypic characteristics is of primary importance in the dairy industry, as phenotypes directly reveal the abilities required in milk fermentation. ...

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Rapid Genotypic Differentiation of Lactococcus lactis Subspecies and Biovar

Cited by 52 publications

References 13 publications

Biochemical and molecular identification of lactic acid bacteria isolated from camel milk in Tunisia

Biochemical and molecular identification of lactic acid bacteria isolated from camel milk in Tunisia

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

Rapid PCR-Based Method Which Can Determine Both Phenotype and Genotype of Lactococcus lactis Subspecies

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