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The 16S ribosomal RNA gene sequences of Lactococcus lactis ssp. lactis and ssp. cremoris differ by 9-10 bp (depending on strain), within the first 200 bp of the sequence. These differences were used to develop two methods of genetically differentiating lactis and cremoris strains. Primers to conserved sequences in the 16S rRNA gene were used in a PCR reaction to amplify fragments of the 16S rRNA gene. A single base difference at position 180 of the sequence was utilised to develop a ligase chain reaction to differentiate lactis and cremoris sequences. The second method involved digestion of the amplified fragments with restriction endonucleases specific for either the lactis or cremoris sequence. Resolution of the digested fragments on an agarose gel allowed the strains to be identified as genetically lactis or cremoris. This method was used to examine lactococci isolated from raw milk. Of 31 raw milk strains examined, 21 contained the cremoris 16S rRNA sequence, however, all 31 strains exhibited the phenotypic characteristics of the lactis subspecies.

The three enzymes of the D-tagatose 6-phosphate pathway (galactose 6-phosphate isomerase, D-tagatose 6-phosphate kinase, and tagatose 1,6-diphosphate aldolase) were absent in lactose-negative (Lac-) derivatives of Streptococcus lactis Clo, H1, and 133 grown on galactose. The lactose phosphoenolpyruvatedependent phosphotransferase system and phospho-p-galactosidase activities were also absent in Lac-derivatives of strains H1 and 133 and were low (possibly absent) in C1o Lac-. In all three Lac-derivatives, low galactose phosphotransferase system activity was found. On galactose, Lac-derivatives grew more slowly (presumably using the Leloir pathway) than the wild-type strains and accumulated high intracellular concentrations of galactose 6-phosphate (up to 49 mM); no intracellular tagatose 1,6-diphosphate was detected. The data suggest that the Lac phenotype is plasmid linked in the three strains studied, with the evidence being more substantial for strain H1. A Lac' derivative of H1 contained a single plasmid (33 megadaltons) which was absent from the Lacmutant. We suggest that the genes linked to the lactose plasmid in S. lactis are more numerous than previously envisaged, coding for all of the enzymes involved in lactose metabolism from initial transport to the formation of triose phosphates via the D-tagatose 6phosphate pathway.Lactic streptococci (Streptococcus cremoris and S. lactis) transport lactose via a phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) (26, 37). The lactose phosphate formed is hydrolyzed by phospho-f3galactosidase (16), giving D-glucose and D-galactose 6-phosphate (Gal-6P) (37). The latter intermediate is further metabolized to triose phosphates by the three enzymes (see Fig. 2) of the D-tagatose 6-phosphate (Tag-6P) pathway(3).In Staphylococcus aureus, the Tag-6P pathway is required for utilization of galactose as well as lactose (4). Lactic streptococci, however, have the enzymatic potential to metabolize galactose via two initially separate routes, namely, the Tag-6P pathway and the galactose 1phosphate (Gal-1P) pathway (Leloir pathway) (3). Either one or both pathways appear to operate, depending on the strain and the exogenous galactose concentration (36). With most S. lactis and S. cremoris strains, galactose fermentation is heterolactic (36) whereas lactose fermentation is homolactic (33).The instability of lactose metabolism in lactic streptococci was noted by earlier workers (14, 15, 40), and the frequency of lactose-negative (Lac-) variants was increased by curing agents (24,25,27). Subsequent work (for a review, see reference 10) has supported the early suggestion (25) that lactose metabolism is plasmid linked. The lactose-PTS and phospho-,B-galactosidase have been associated with the lactose plasmid in strains of S. lactis and S. cremoris (1,20,21,26,32), and the lactose plasmid influences the metabolism of galactose by S. lactis (21). However, the three enzymes involved in the metabolism of Gal-6P (see Fig. 2) have not been assayed in Lac-variants. A deficie...

Eleven of 150 Streptococcus cremoris strains examined produced the bacteriocin diplococcin. The diplococcin activity spectrum was restricted to S. cremoris and Streptococcus lactis strains, and none of a wide range of other gram-positive or gram-negative strains were inhibited. The diplococcin produced by S. cremoris 346 was purified by ammonium sulfate precipitation and column chromatography. Purified diplococcin was very unstable at room temperature and lost 75% of its activity after heating at 1000C for 1 min. The proteolytic enzymes trypsin, pronase, and a-chymotrypsin completely inactivated diplococcin. The amino acid composition showed a high content of acidic and neutral acids and a correspondingly low content of basic amino acids, including one residue of ornithine per mole. From the amino acid analysis a molecular weight of 5,300 was estimated. Diplococcin was readily distinguished from the S. lactis bacteriocin nisin by its restricted activity spectrum, its biological properties, and by cross-reaction experiments.