Conjugal Transfer of Genetic Information in Group N Streptococci

Streptococcus lactis strains ML3 and C₂O and S. lactis subsp. diacetylactis strains DRC3, 11007, and WM₄ were found to transfer lactose-fermenting ability to LM0230, an S. lactis C2 lactose-negative (Lac⁻) derivative which is devoid of plasmid deoxyribonucleic acid (DNA). Lactose-positive streptomycin-resistant (Lac⁺ Str^r) recombinants were found when the Lac⁺ Str^s donor was mixed with Lac⁻ Str^r LM0230 in solid-surface matings. Transduction and transformation were ruled out as the mechanism of genetic exchange in strains ML3, DRC3, 11007, and WM₄, nor was reversion responsible for the high number of Lac⁺ Str^r recombinants. Furthermore, chloroform treatment of the donor prevented the appearance of recombinants, indicating that transfer of lactose-fermenting ability required viable cell-to-cell contact. Strain C₂O demonstrated transduction as well as conjugation. Transfer of plasmid DNA during conjugation for all strains was confirmed by demonstrating the presence of plasmid DNA in the transconjugants by using agarose gel electrophoresis. In some instances, a cryptic plasmid was transferred in conjunction with the lactose plasmid by using strains DRC3, 11007, and WM₄. In S. lactis C2 × LM0230 matings, the Str^r marker was transferred from LM0230 to C2, suggesting conjugal transfer of chromosomal DNA. The results confirm conjugation as another mechanism of genetic exchange occurring in dairy starter cultures.     

Development of a lactococcal integration vector by using IS981 and a temperature-sensitive lactococcal replication region.

A vector (pKMP10) capable of Campbell-like integration into the Lactococcus lactis subsp. lactis LM0230 chromosome via homologous recombination with chromosomal IS981 sequences was constructed from the replication region of lactococcal plasmid pSK11L, an internal fragment of IS981, and the erythromycin resistance gene and Escherichia coli replication origin of pVA891. The pSK11L replication region is temperature sensitive for maintenance in L. lactis subsp. lactis LM0230, resulting in loss of unintegrated pKMP10 during growth at greater than 37 degrees C. pKMP10 integrants made up 8 to 75% of LM0230(pKMP10) erythromycin-resistant cells following successive growth at 25 degrees C with selection, 39 degrees C without selection, and 39 degrees C with selection. pKMP10 integrants were also isolated from L. lactis subsp. lactis MG1363(pKMP10) but at a 10-fold-lower frequency (4%). No integrants were isolated form L. lactis subsp. lactis MMS368(pKMP10) (a Rec-deficient strain) or LM0230(pKMP1-E) (the corresponding plasmid lacking the IS981 fragment). Examination of 17 LM0230 integrants by Southern hybridization revealed pKMP10 integration into five different chromosomal sites. Four of the integration sites appeared to be chromosomal IS981 sequences, while one was an uncharacterized chromosomal sequence. The four IS981 integrants seemed to have pKMP10 integrated in a tandem repeat structure of undetermined length. Integrated pKMP10 was more stable (0 to 2% plasmid loss) than unintegrated pKMP10 (100% plasmid loss) when grown for 100 generations at 32 degrees C without selection.     

Development of a lactococcal integration vector by using IS981 and a temperature-sensitive lactococcal replication region.

A vector (pKMP10) capable of Campbell-like integration into the Lactococcus lactis subsp. lactis LM0230 chromosome via homologous recombination with chromosomal IS981 sequences was constructed from the replication region of lactococcal plasmid pSK11L, an internal fragment of IS981, and the erythromycin resistance gene and Escherichia coli replication origin of pVA891. The pSK11L replication region is temperature sensitive for maintenance in L. lactis subsp. lactis LM0230, resulting in loss of unintegrated pKMP10 during growth at greater than 37 degrees C. pKMP10 integrants made up 8 to 75% of LM0230(pKMP10) erythromycin-resistant cells following successive growth at 25 degrees C with selection, 39 degrees C without selection, and 39 degrees C with selection. pKMP10 integrants were also isolated from L. lactis subsp. lactis MG1363(pKMP10) but at a 10-fold-lower frequency (4%). No integrants were isolated form L. lactis subsp. lactis MMS368(pKMP10) (a Rec-deficient strain) or LM0230(pKMP1-E) (the corresponding plasmid lacking the IS981 fragment). Examination of 17 LM0230 integrants by Southern hybridization revealed pKMP10 integration into five different chromosomal sites. Four of the integration sites appeared to be chromosomal IS981 sequences, while one was an uncharacterized chromosomal sequence. The four IS981 integrants seemed to have pKMP10 integrated in a tandem repeat structure of undetermined length. Integrated pKMP10 was more stable (0 to 2% plasmid loss) than unintegrated pKMP10 (100% plasmid loss) when grown for 100 generations at 32 degrees C without selection.     

Cotransformation of lactococcin-producing, 2.0-mega dalton and erythromycin-resistant pGB 301 plasmids toLactococcus lactis subsp.lactis protoplast

Protoplasts of plasmid-freeLactococcus lactis subsp.lactis LM 0230 and PC₄ strains were cotransformed successfully with the plasmid pools ofL. lactis subsp.lactis 484, a lactosefermenting (Lac⁺), lactococcin-producing (Lap⁺), lactococcin-resistant (Lap^r), sucrosefermenting (Suc⁺) wild strain, its derivatives, and pGB 301 erythromycin resistance plasmid (Ery^r) at the frequencies of 10⁴ transformants/g of DNA. PC₄ protoplasts were transformed at slightly lower frequencies that LM 0230 protoplasts when the same plasmid combinations were used for transformation. Agarose gel electrophoresis of plasmids from three groups of transformants, namely, Lac^–Lap^–Ery^r, Lac⁺Suc⁺Lap⁺Lap^rEry^r, and Lac^–Suc⁺Lap⁺ Lap^rLap^r, confirmed that 2.0 and 65.0 megadalton (MDa) plasmids carried genes for Suc⁺Lap⁺Lap^r and Lac⁺ phenotypes respectively. The protoplasts could be transformed with low-molecular-weight 2.0 MDa Lap plasmid at a relatively higher frequency than those with high-molecular-weight 65.0 MDa Lac plasmid. All the transformants resembled parent culture 484 in terms of lactic acid production (0.810–0.840%), milk curdling time (6 h), and lactococcin activity (7–12 mm, zone of inhibition) againstListeria monocytogenes, Salmonella typhi, andStaphylococcus aureus. The plasmids and their respective phenotypes in PC₄ transformants were genetically more stable than those of LM 0230 protoplasts. The marker plasmid pGB 301 disappeared more frequently from the transformants when present in association with the lowmolecular-weight, high-copy-number 2.0 MDa plasmid, thereby suggesting the incompatibility of these two plasmids.     

Genetic Evidence for Plasmid-Linked Lactose Metabolism in Streptococcus lactis subsp. diacetylactis

It has been previously observed that loss of plasmid pGK4101 occurred concomitantly with loss of lactose-fermenting ability in Streptococcus lactis subsp. diacetylactis 18-16. Transfer of this 41-megadalton plasmid to LM0230, a lactosenegative (Lac⁻) strain of S. lactis, required cell-to-cell contact and resulted in a conversion of LM0230 to the Lac⁺ phenotype. This confirms the linkage of lactose-fermenting ability to the 41-megadalton plasmid in S. lactis subsp. diacetylactis and, in addition, demonstrates transfer by a process resembling conjugation in the group N streptococci.     

Genetic evidence for plasmid-encoded lactococcin production inLactococcus lactis subsp.lactis 484

Lactococcus lactis subsp.lactis 484 produced a proteinaceous antibacterial substance designated as lactococcin capable of inhibiting members of theLactococcus group,Bacillus cereus, Staphylococcus aureus, andSalmonella typhi. Growth of this culture in the presence of 2–30 g/ml of ethidium bromide or acriflavin or novobiocin, and at elevated temperature (39° and 41°C), could not produce any lactococcin-negative (Lap^–) variants. However, protoplast-induced curing with lysozyme was successful in developing Lap^– derivatives. Two types of cured derivatives, namely Lac^– Lap⁺ and Lac^– Lap^–, were obtained. Lap^– variants were also lacking sucrose-fermenting ability (Suc⁺) and lactococcin resistance (Lap^r). The lactose-negative (Lac^–) variants and Lap⁺ were clearly lacking the largest (65 Md) plasmid. However, Lap^– Suc^– Lap^s variants lost a 2 Md plasmid.L. lactis subsp.lactis 484 transferred lactose-fermenting ability as well as Lap⁺ Suc⁺ Lap^r phenotypes simultaneously toL. lactis subsp.lactis LM 2306 and LM 0230 by surface mating at a frequency of 10^–4 and 10^–1 per donor respectively. However, cured Lac^– Lap^– transconjugants could not transfer Lac⁺ Lap⁺ Suc⁺ Lap^r phenotypes to any of these recipient strains. Our results indicate that Lac⁺ and Lap⁺ Suc⁺ Lap^r phenotypes are associated with 65 Md and 2 Md plasmids respectively. Conjugal transfer of 2 Md plasmid is possible only in the presence of a conjugative 65 Md plasmid.     

Molecular characterization of the integration of the lactose plasmid from Lactococcus lactis subsp. cremoris SK11 into the chromosome of L. lactis subsp. lactis.

When Lactococcus lactis subsp. lactis LM0230 is transformed by the lactose plasmid (pSK11L) from Lactococcus lactis subsp. cremoris SK11, variants with pSK11L in the integrated state can be derived (J. M. Feirtag, J. P. Petzel, E. Pasalodos, K. A. Baldwin, and L. L. McKay, Appl. Environ. Microbiol. 57:539-548, 1991). In the present study, a 1.65-kb XbaI-XhoI fragment of pSK11L was subcloned for use as a probe in Southern hybridization analyses of the mechanism of integration, which was shown to proceed via a Campbell-like, single-crossover event. Furthermore, the presence of the XbaI-XhoI fragment in a nonreplicating vector facilitated the stable, Rec-dependent integration of the vector into the chromosome of L. lactis subsp. lactis LM0230 and other lactococci. DNA sequence analysis of the fragment revealed an open reading frame of 885 bp with lactococcal expression sequences. The putative gene did not have significant homology with other genes in computer data bases. The XbaI-XhoI fragment is a naturally occurring piece of lactococcal DNA that can be used as a recombinogenic cassette in the construction of integration vectors for the industrially important lactococci.     

Molecular characterization of the integration of the lactose plasmid from Lactococcus lactis subsp. cremoris SK11 into the chromosome of L. lactis subsp. lactis.

When Lactococcus lactis subsp. lactis LM0230 is transformed by the lactose plasmid (pSK11L) from Lactococcus lactis subsp. cremoris SK11, variants with pSK11L in the integrated state can be derived (J. M. Feirtag, J. P. Petzel, E. Pasalodos, K. A. Baldwin, and L. L. McKay, Appl. Environ. Microbiol. 57:539-548, 1991). In the present study, a 1.65-kb XbaI-XhoI fragment of pSK11L was subcloned for use as a probe in Southern hybridization analyses of the mechanism of integration, which was shown to proceed via a Campbell-like, single-crossover event. Furthermore, the presence of the XbaI-XhoI fragment in a nonreplicating vector facilitated the stable, Rec-dependent integration of the vector into the chromosome of L. lactis subsp. lactis LM0230 and other lactococci. DNA sequence analysis of the fragment revealed an open reading frame of 885 bp with lactococcal expression sequences. The putative gene did not have significant homology with other genes in computer data bases. The XbaI-XhoI fragment is a naturally occurring piece of lactococcal DNA that can be used as a recombinogenic cassette in the construction of integration vectors for the industrially important lactococci.     

Replication and temperature-sensitive maintenance functions of lactose plasmid pSK11L from Lactococcus lactis subsp. cremoris.

The replication region of pSK11L, the lactose plasmid of Lactococcus lactis subsp. cremoris (L. cremoris) SK11, was isolated on a 14.8-kbp PvuII fragment by shotgun cloning into an Escherichia coli vector encoding erythromycin resistance and selection for erythromycin-resistant transformants of L. lactis subsp. lactis (L. lactis) LM0230. Deletion analysis and Tn5 mutagenesis of the resulting plasmid (pKMP1) further localized the replication region to a 2.3-kbp ScaI-SpeI fragment. DNA sequence analysis of this 2.3-kbp fragment revealed a 1,155-bp open reading frame encoding the putative replication protein, Rep. The replication origin was located upstream of rep and consisted of an 11-bp imperfect direct repeat and a 22-bp sequence tandemly repeated three and one-half times. The overall organization of the pSK11L replicon was remarkably similar to that of pCI305, suggesting that pSK11L does not replicate by the rolling-circle mechanism. Like pSK11L, pKMP1 was unstable in L. lactis LM0230. Deletion analysis allowed identification of several regions which appeared to contribute to the maintenance of pKMP1 in L. lactis LM0230. pKMP1 was significantly more stable in L. cremoris EB5 than in L. lactis LM0230 at all of the temperatures compared. This stability was lost by deletion of a 3.1-kbp PvuII-XbaI fragment which had no effect on stability in L. lactis LM0230. Other regions affecting stability in L. cremoris EB5 but not in L. lactis LM0230 were also identified. Stability assays conducted at various temperatures showed that pKMP1 maintenance was temperature sensitive in both L. lactis LM0230 and L. cremoris EB5, although the plasmid was more unstable in L. lactis LM0230. The region responsible for the temperature sensitivity phenotype in L. lactis LM0230 was tentatively localized to a 1.2-kbp ClaI-HindIII fragment which was distinct from the replication region of pSK11L. Our results suggest that the closely related L. lactis and L. cremoris subspecies behave differently regarding maintenance of plasmids.     

A Simple and Rapid Method for Genetic Transformation of Lactic Streptococci by Electroporation

An electroporation procedure for the plasmid-mediated genetic transformation of intact cells of Streptococcus cremoris and Streptococcus lactis was performed. Ten different strains were transformed. The method was simple and rapid and yielded transformant colonies in 14 to 24 h. The method was optimized for S. lactis LM0230, and transformation frequencies of between 1 × 10⁴ and 5 × 10⁵ transformants per μg of purified plasmid (pMU1328) were achieved routinely. The optimized procedure involved lysozyme treatment of cells. Transformation of LM0230 occurred at comparable frequencies with pLS1 (4.4 kilobase pair [kbp]), pMU1328 (7.4 kbp), and pAMβ1 (26.5 kbp). Plasmid DNA isolated from transformants had not undergone detectable deletions or rearrangements. Transformation was possible with plasmid DNA which was religated after restriction endonuclease digestion. Phage DNA-dependent transfection of S. lactis LM0230 and S. lactis C6 was also achieved.     

Stimulation of erythromycin a yield by integration of a chromosomal DNA fragment including the eryC1 gene into the chromosome of Saccharopolyspora erythraea

Summary A 5.2 kbp chromosomal DNA fragment including the eryC1 gene was integrated along with vector DNA into the chromosome of a S. erythraea wild-type strain. The erythromycin A production of transformants in the presence of thiostrepton was two to three times higher in comparison with the non-transformed wild-type strain.     

Genomic organization of lactic acid bacteria

Current knowledge of the genomes of the lactic acid bacteria, Lactococcus lactis and Streptococcus thermophilus, and members of the genera Lactobacillus, Leuconostoc, Pediococcus and Carnobacterium is reviewed. The genomes contain a chromosome within the size range of 1.8 to 3.4 Mbp. Plasmids are common in Lactococcus lactis (most strains carry 4–7 different plasmids), some of the lactobacilli and pediococci, but they are not frequently present in S. thermophilus, Lactobacillus delbrueckii subsp. bulgaricus or the intestinal lactobacilli. Five IS elements have been found in L. lactis and most strains carry multiple copies of at least two of them; some strains also carry a 68-kbp conjugative transposon. IS elements have been found in the genera Lactobacillus and Leuconostoc, but not in S. thermophilus. Prophages are also a normal component of the L. lactis genome and lysogeny is common in the lactobacilli, however it appears to be rare in S. thermophilus. Physical and genetic maps for two L. lactis subsp. lactis strains, two L. lactis subsp. cremoris strains and S. thermophilus A054 have been constructed and each reveals the presence of six rrn operons clustered in less than 40% of the chromosome. The L. lactis subsp. cremoris MG1363 map contains 115 genetic loci and the S. thermophilus map has 35. The maps indicate significant plasticity in the L. lactis subsp. cremoris chromosome in the form of a number of inversions and translocations. The cause(s) of these rearrangements is (are) not known. A number of potentially powerful genetic tools designed to analyse the L. lactis genome have been constructed in recent years. These tools enable gene inactivation, gene replacement and gene recovery experiments to be readily carried out with this organism, and potentially with other lactic acid bacteria and Gram-positive bacteria. Integration vectors based on temperate phage attB sites and the random insertion of IS elements have also been developed for L. lactis and the intestinal lactobacilli. In addition, a L. lactis sex factor that mobilizes the chromosome in a manner reminiscent to that seen with Escherichia coli Hfr strains has been discovered and characterized. With the availability of this new technology, research into the genome of the lactic acid bacteria is poised to undertake a period of extremely rapid information accrual.     

Improvement of plasmid encoded lactococcal bacteriophage resistance by mutator strain Epicurian coli XL1-Red

A random mutation strategy using mutator strain, Epicurian coli XL1-Red, was applied to a plasmid, pND018, constructed by inserting a Lactococcus lacis bacteriophage resistance gene (abiI) into a L. lactis/E. coli shuttle vector (pDL278), to introduce random mutations throughout the plasmid. Following transformation of the mutated plasmid library to a plasmid free and phage sensitive strain of L. lactis (LM0230), mutated plasmids were screened by cross-streaking and efficiency of plaquing (EOP) assays. Two strains with enhanced resistance were obtained, as well as several phage sensitive strains. Repeated transformation of the mutated plasmids to LM0230 confirmed that the observed phenotypes were caused by mutations located on the plasmids. The EOP values and plaque morphology of two enhanced phage resistance mutants were characterized at 30°C and 37°C. These results indicate that this simple procedure can be applied to generate modified plasmids with improved phage resistance, which may be of commercial value.     

Extrachromosomal inheritance inSchizosaccharomyces pombe

Summary Spontaneous chloramphenicol (cap ^r)- and erythromycin (ery ^r)-resistant mutants were isolated from strain ade7–50 h ^- and the antimycin-resistant mutant ana ^r-8 ade 7–50 h^- of Schizosaccharomyces pombe (Sch. p.). By mitotic segregation analysis all 154 cap ^r- and 120 ery ^r-mutants derived from ade 7–50 h ^- proved to be recessive chromosomal, whereas all 108 cap ^r- and 200 ery ^r-mutants originating from ana ^r-8 were extrachromosomally inherited. The rate of spontaneous cap ^r- and ery ^r-mutants was about hundredfold in ana ^r-8 compared to ade 7–50 h ^-. Growth of cap ^r-and ery ^r-mutants was not inhibited by chloramphenicol or erythromycin, respectively, in glucose-medium and only slightly in glycerol-medium at concentrations which completely inhibited ana ^r-8. By mitotic segregation-, tetrad-, and mitotic haploidization-analysis the extrachromosomal inheritance of mutants derived from ana ^r-8 was established. Segregational patterns of cap ^r- and ery ^r-determinats during mitosis, meiosis, and mitotic haplidization of diploids are discussed.     

Loss of IrpT Function in Lactococcus lactis subsp. lactis N8 Results in Increased Nisin Resistance

The antibiotic nisin, produced by Lactococcus lactis subsp. lactis N8, offers an extensive commercial prospect as natural food preservatives. The nisin immunity of the L. lactis strains is regulated by a variety of mechanisms. In this study, we isolated a L. lactis L31 strain with increased nisin resistance from a mini-Mu transposon mutant pool of strain N8. The single Mu insertion in strain L31 was in the irpT gene with unknown function. By comparing the proteomic profiles of L. lactis L31 and its parental strain, we found that changes occurred in the synthesis of a protein involved in cell wall biosynthesis (RmlD). Strain L31 had 13.7% higher content of rhamnose in the cell wall than the N8 strain. Overexpression of RmlD involved in the synthesis of dTDP-l-rhamnose in the nisin-sensitive MG1363 strain increased nisin resistance of the strain. The results indicate that these cellular proteins effected nisin resistance in L. lactis N8.     

Conjugal Transfer in Lactic Streptococci of Plasmid-Encoded Insensitivity to Prolate- and Small Isometric-Headed Bacteriophages

Eight of 40 strains of Streptococcus lactis and S. lactis subsp. diacetylactis were able to conjugally transfer a degree of phage insensitivity to Streptococcus lactis LM0230. Transconjugants from one donor strain, S. lactis subsp. diacetylactis 4942, contained a 106-kilobase (kb) cointegrate plasmid, pAJ1106. The plasmid was conjugative (Tra⁺) and conferred phage insensitivity (Hsp) and lactose-fermenting ability (Lac) in S. lactis and Streptococcus cremoris transconjugants. The phage resistance mechanism was effective against prolate- and small isometric-headed phages at 30°C. In S. lactis transconjugants, the phage resistance mechanism was considerably weakened at elevated temperatures. A series of deletion plasmids was isolated from transconjugants in S. cremoris 4854. Deletion plasmids were pAJ2074 (74 kb), Lac⁺, Hsp⁺, Tra⁺; pAJ3060 (60 kb), Lac⁺, Hsp⁺; and pAJ4013 (13 kb), Lac⁺. These plasmids should facilitate mapping Hsp and tra genes, with the aim of constructing phage-insensitive strains useful to the dairy industry.     

Thermosensitive plasmid replication, temperature-sensitive host growth, and chromosomal plasmid integration conferred by Lactococcus lactis subsp. cremoris lactose plasmids in Lactococcus lactis subsp. lactis.

Evidence is presented that lactose-fermenting ability (Lac+) in Lactococcus lactis subsp. cremoris AM1, SK11, and ML1 is associated with plasmid DNA, even though these strains are difficult to cure of Lac plasmids. When the Lac plasmids from these strains were introduced into L. lactis subsp. lactis LM0230, they appeared to replicate in a thermosensitive manner; inheritance of the plasmid was less efficient at 32 to 40 degrees C than at 22 degrees C. The stability of the L. lactis subsp. cremoris Lac plasmids in lactococci appeared to be a combination of both host and plasmid functions. Stabilized variants were isolated by growing the cultures at 32 to 40 degrees C; these variants contained the Lac plasmids integrated into the L. lactis subsp. lactis LM0230 chromosome. In addition, the presence of the L. lactis subsp. cremoris Lac plasmids in L. lactis subsp. lactis resulted in a temperature-sensitive growth response; growth of L. lactis subsp. lactis transformants was significantly inhibited at 38 to 40 degrees C, thereby resembling some L. lactis subsp. cremoris strains with respect to temperature sensitivity of growth.     

Peptide analyses of a protein component, 50-8, of 50s ribosomal subunit from erythromycin resistant mutants of Escherichia coli and Escherichia freudii

Summary Chromatographic analyses on a Dowex 50x8 column of tryptic digests of the mutationally altered 50-8 protein component from several erythromycin resistant (ery ^r) mutants of Escherichia coli and Escherichia freundii have been performed. It was found that (1) the difference in the elution profile of the altered components detected with carboxymethyl cellulose column chromatography reflects the difference in their amino acid sequence, (2) the structural change(s) of the 50-8 protein from three E. coli ery ^r mutants examined seems to exist only in the same single peptide fragment and (3) the primary structure of the 50-8(R) protein of E. freundii (ery ^s: wild type) differs from that of E. coli Q13 (ery ^s) and the structural change in 50-8(R) component of E. freundii caused by the ery ^r mutation was found to take place in different peptide fragments from that in which the mutational change of the E. coli 50-8 component occurred.     

Thermosensitive plasmid replication, temperature-sensitive host growth, and chromosomal plasmid integration conferred by Lactococcus lactis subsp. cremoris lactose plasmids in Lactococcus lactis subsp. lactis

Evidence is presented that lactose-fermenting ability (Lac+) in Lactococcus lactis subsp. cremoris AM1, SK11, and ML1 is associated with plasmid DNA, even though these strains are difficult to cure of Lac plasmids. When the Lac plasmids from these strains were introduced into L. lactis subsp. lactis LM0230, they appeared to replicate in a thermosensitive manner; inheritance of the plasmid was less efficient at 32 to 40 degrees C than at 22 degrees C. The stability of the L. lactis subsp. cremoris Lac plasmids in lactococci appeared to be a combination of both host and plasmid functions. Stabilized variants were isolated by growing the cultures at 32 to 40 degrees C; these variants contained the Lac plasmids integrated into the L. lactis subsp. lactis LM0230 chromosome. In addition, the presence of the L. lactis subsp. cremoris Lac plasmids in L. lactis subsp. lactis resulted in a temperature-sensitive growth response; growth of L. lactis subsp. lactis transformants was significantly inhibited at 38 to 40 degrees C, thereby resembling some L. lactis subsp. cremoris strains with respect to temperature sensitivity of growth.