Discrimination of dairy industry isolates of the Lactobacillus casei group.

Lactobacilli are a major part of the microflora of the gut and of many fermented dairy products, and are found in a variety of environments. Lactobacillus casei, Lactobacillus paracasei, Lactobacillus rhamnosus, and Lactobacillus zeae form a closely related taxonomic group within the facultatively heterofermentative lactobacilli. The classification and nomenclature of these bacteria are controversial. In this study, relationships between these species were investigated using type strains and dairy industry isolates examined with DNA-based techniques and conventional carbohydrate use tests. Carbohydrate use patterns gave poor discrimination of some species, but DNA PCR using specific primers targeted to sequences of the 16S rRNA gene discriminated 4 types consistent with the currently recognized species. Pulsed-field agarose gel electrophoresis of chromosomal NotI restriction fragments identified 18 different band patterns from 21 independent Lactobacillus isolates and confirmed the identity of L. casei strains from 2 culture collections (CSCC 5203 and ASCC 290), both representing the type strain of L. casei. Some isolates were reclassified as L. rhamnosus, suggesting that the prevalence of L. rhamnosus as a natural component of the microflora of dairy foods and dairy environments has previously been underestimated. These methods can provide a practical basis for discrimination of the species and identification of individual industrial strains.

Integrated polymerase chain reaction-based procedures for the detection and identification of species and subspecies of the Gram-positive bacterial genus Lactococcus.

AIMS: Five species of the Gram-positive bacterial genus Lactococcus (Lactococcus lactis, L. garvieae, L. plantarum, L. piscium and L. raffinolactis) are currently recognized. The aim of this work was to develop a simple approach for the identification of these species, as well as to differentiate the industrially important dairy subspecies L. lactis subsp. lactis and L. lactis subsp. cremoris. METHODS AND RESULTS: Methods were devised based on specific polymerase chain reaction (PCR) amplifications that exploit differences in the sequences of the 16S ribosomal RNA genes of each species, followed by restriction enzyme cleavage of the PCR products. The techniques developed were used to characterize industrial cheese starter strains of L. lactis and the results were compared with biochemical phenotype and DNA sequence data. CONCLUSIONS: The PCR primers designed can be used simultaneously, providing a simple scheme for screening unknown isolates. Strains of L. lactis show heterogeneity in the 16S ribosomal RNA gene sequence. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides an integrated set of methods for differentiation and identification of lactococcal species associated with agricultural, veterinary, medical and processed food industries.

Stability analysis of the Lactococcus lactis DRC1 lactose plasmid using pulsed-field gel electrophoresis.

Pulsed-field agarose gel electrophoresis of SmaI digests of genomic DNA was used to examine lactose plasmid copy number and stability in Lactococcus lactis. In L. lactis strain DRC1, the plasmid was found to exist as a single-copy plasmid. Transconjugants of strain HID113 carrying this plasmid were unstable. Variants were isolated with improved phenotypic stability resulting from improved maintenance of the lactose plasmid or from integration of part of the plasmid into the lactococcal chromosome.

Rapid genomic fingerprinting of Lactococcus lactis strains by arbitrarily primed polymerase chain reaction with 32P and fluorescent labels.

Arbitrarily primed polymerase chain reaction, with incorporation of either radioactive or fluorescent labels, was used as a rapid and sensitive method for obtaining genomic fingerprints of strains of Lactococcus lactis. Closely related strains produced almost identical fingerprints. Fingerprints of other strains showed only some similarities.

Phage DNA synthesis and host DNA degradation in the life cycle of Lactococcus lactis bacteriophage c6A.

Bacteriophage c6A is a lytic phage that infects strains of Lactococcus lactis. Infection of L. lactis strain C6 resulted in inhibition of culture growth within 10 min, mature intracellular phage particles appeared after 17.5 min, and cell lysis occurred after 25 min. A culture of strain C6 carrying 3H-labelled DNA was infected with c6A, and the fate of the radiolabel was monitored. The results showed that degradation of host cell DNA began within 6 min of infection and that the breakdown products were incorporated into progeny c6A DNA. Quantitative DNA hybridizations indicated that synthesis of phage DNA began within 6 min of infection and continued at an approximately constant rate throughout the latent period.

Species and type phages of lactococcal bacteriophages.

Lactococcal phages are classified according to morphology and DNA homology. Phages are differentiated into 12 phage species, and type phages of each species are proposed. Members and possible members of each species are named. Available data on type phages are tabulated including morphology, DNA characteristics and phage protein bands.

Simultaneous conjugal transfer in Lactococcus to genes involved in bacteriocin production and reduced susceptibility to bacteriophages.

Conjugal matings were performed between Lactococcus lactis DRC1 (a lactose-fermenting (Lac+), bacteriocin-producing (Bac+) strain) and L. lactis HID113 (Lac- and Bac-). Transconjugant derivatives of HID113 were identified on the basis of lactose fermentation, resistance to the DRC1 bacteriocin (dricin) or reduced sensitivity to phage sk1. Regardless of how they were identified, all transconjugants gave fewer and smaller plaques with phages c2 and sk1 than did HID113. All but one of 275 transconjugants tested also produced dricin, suggesting some functional relationship or close genetic linkage between the reduced phage sensitivity and dricin production and resistance. Some transconjugants were also Lac+, but this property was unstable.

Temperate bacteriophages and lysogeny in lactic acid bacteria.

Lysogeny is widespread in the lactic acid bacteria. The majority of lysogens can be induced by UV irradiation or treatment with mitomycin C, but indicator strains which allow lytic growth of the induced phage are often not easy to identify. A few temperate phages have been shown to transduce chromosomal and/or plasmid markers. Information about the molecular biology of the temperate phages from lactic acid bacteria is sparse and needs significant supplementation in order that these potentially valuable phages might be utilized more efficiently as tools for improving existing starter strains in dairy fermentations.

Resistance to In Vitro Restriction of DNA from Lactic Streptococcal Bacteriophage c6A.

DNA isolated from streptococcal bacteriophage c6A was cut only infrequently by many restriction endonucleases. Fragments of c6A DNA cloned in Escherichia coli plasmids were similarly resistant to cleavage. We conclude that the low frequency of cleavage is due to an unusually low number of restriction enzyme recognition sequences in c6A DNA.

Characterization of streptococcal bacteriophage c6A.

Bacteriophage c6A is a lytic phage that infects strains of Streptococcus lactis. Infection of S. lactis C6 under standard conditions yielded 124 +/- 8 p.f.u. per infected cell after a latent period of 25 min at 30 degrees C. The virion of c6A was shown to contain at least 12 polypeptides and a 21.9 kilobase double-stranded, linear DNA genome with complementary 5'-protruding single-stranded termini. The (G + C) content of this DNA was estimated to be 36.7%. A restriction map was constructed which indicates that a number of restriction endonucleases did not digest the DNA and that others cleaved with a much lower frequency than expected.