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

Abstract

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. ...

Glutamate Decarboxylase-Mediated Nisin Resistance in Listeria monocytogenes

“…lactis strains are GAD ϩ , L. lactis subsp. cremoris strains, as a consequence of inactivation of their gadB gene by a frameshift mutation, do not possess GAD activity (20). Thus, to further investigate the link between the mutation, natural or otherwise, of GAD and nisin sensitivity, a selection of L. lactis subsp.…”

Identification and Probiotic Characteristics of Lactococcus Strains from Plant Materials

“…A 600 bp PCR fragment derived from the gadB gene should be obtained from the chromosomal DNA of the strains tested if the strain belongs to L. lactis subsp. lactis 20 . Fig.1 showed that DNA from the tested strains generated fragments corresponding to the 600 bp fragment of L. lactis subsp.…”

“…A 600 bp PCR fragment should be obtained from the chromosomal DNA of the strains tested if the strain belongs to L. lactis subsp. lactis 20 .…”

“…Raffinose fermentation was determined by growth in raffinose-containing M17 broth. PCR (polymerase chain reaction) -based method was also conducted for specific L. lactis identification 20 . Briefly, bacterial cultures (10 7 cfu/mL) were used as templates for PCR analyses.…”

Gene-Specific PCR Amplification of Technologically Important Lactococcal Genes

“…(13,14). Reliable PCR methods for confirmation of the species L. lactis and its two subspecies lactis and cremoris as well as for detection of DNA sequences related to the presence of each of the above genes have been developed (10,12,13).…”