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.     

High-resolution amplified fragment length polymorphism typing of Lactococcus lactis strains enables identification of genetic markers for subspecies-related phenotypes

A high-resolution amplified fragment length polymorphism (AFLP) methodology was developed to achieve the delineation of closely related Lactococcus lactis strains. The differentiation depth of 24 enzyme-primer-nucleotide combinations was experimentally evaluated to maximize the number of polymorphisms. The resolution depth was confirmed by performing diversity analysis on 82 L. lactis strains, including both closely and distantly related strains with dairy and nondairy origins. Strains clustered into two main genomic lineages of L. lactis subsp. lactis and L. lactis subsp. cremoris type-strain-like genotypes and a third novel genomic lineage rooted from the L. lactis subsp. lactis genomic lineage. Cluster differentiation was highly correlated with small-subunit rRNA homology and multilocus sequence analysis (MLSA) studies. Additionally, the selected enzyme-primer combination generated L. lactis subsp. cremoris phenotype-specific fragments irrespective of the genotype. These phenotype-specific markers allowed the differentiation of L. lactis subsp. lactis phenotype from L. lactis subsp. cremoris phenotype strains within the same L. lactis subsp. cremoris type-strain-like genomic lineage, illustrating the potential of AFLP for the generation of phenotype-linked genetic markers.     

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.     

Divergence of Genomic Sequences between Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris

Relatedness between Lactococcus lactis subsp. cremoris and L. lactis subsp. lactis was assessed by Southern hybridization analysis, with cloned chromosomal genes as probes. The results indicate that strains of the two subspecies form two distinct groups and that the DNA sequence divergence between L. lactis subsp. lactis and L. lactis subsp. cremoris is estimated to be between 20 and 30%. The previously used phenotypic criteria do not fully discriminate between the groups; therefore, we propose a new classification which is based on DNA homology. In agreement with this revised classification, the L. lactis subsp. lactis and L. lactis subsp. cremoris strains from our collection have distinct phage sensitivities.     

Genetic construction of nisin-producing Lactococcus lactis subsp. cremoris and analysis of a rapid method for conjugation.

Conjugation was used to construct nisin-producing Lactococcus lactis subsp. cremoris strains. Recipients were obtained by electroporation of L. lactis subsp. cremoris strains with the drug resistance plasmid pGK13 or pGB301. A method, direct-plate conjugation, was developed in which donor and recipient cells were concentrated and then combined directly on selective media. This method facilitated transfer of the nisin-sucrose (Nip+ Suc+) phenotype from the donor strain, L. lactis subsp. lactis 11454, to three L. lactis subsp. cremoris recipient strains. Nip+ Suc+ L. lactis subsp. cremoris transconjugants were obtained at frequencies which ranged from 10(-7) to 10(-8) per donor CFU. DNA-DNA hybridization to transconjugant DNAs, performed with an oligonucleotide probe synthesized to detect the nisin precursor gene, showed that this gene was transferred during conjugation but was not associated with detectable plasmid DNA. Further investigation indicated that L. lactis subsp. cremoris Nip+ Suc+ transconjugants retained the recipient strain phenotype with respect to bacteriophage resistance and acid production in milk. Results suggested that it would be feasible to construct nisin-producing L. lactis subsp. cremoris strains for application as mixed and multiple starter systems. Additionally, the direct-plate conjugation method required less time than filter or milk agar matings and may also be useful for investigations of conjugal mechanisms in these organisms.     

Genetic construction of nisin-producing Lactococcus lactis subsp. cremoris and analysis of a rapid method for conjugation

Conjugation was used to construct nisin-producing Lactococcus lactis subsp. cremoris strains. Recipients were obtained by electroporation of L. lactis subsp. cremoris strains with the drug resistance plasmid pGK13 or pGB301. A method, direct-plate conjugation, was developed in which donor and recipient cells were concentrated and then combined directly on selective media. This method facilitated transfer of the nisin-sucrose (Nip+ Suc+) phenotype from the donor strain, L. lactis subsp. lactis 11454, to three L. lactis subsp. cremoris recipient strains. Nip+ Suc+ L. lactis subsp. cremoris transconjugants were obtained at frequencies which ranged from 10(-7) to 10(-8) per donor CFU. DNA-DNA hybridization to transconjugant DNAs, performed with an oligonucleotide probe synthesized to detect the nisin precursor gene, showed that this gene was transferred during conjugation but was not associated with detectable plasmid DNA. Further investigation indicated that L. lactis subsp. cremoris Nip+ Suc+ transconjugants retained the recipient strain phenotype with respect to bacteriophage resistance and acid production in milk. Results suggested that it would be feasible to construct nisin-producing L. lactis subsp. cremoris strains for application as mixed and multiple starter systems. Additionally, the direct-plate conjugation method required less time than filter or milk agar matings and may also be useful for investigations of conjugal mechanisms in these organisms.     

Diversity analysis of dairy and nondairy Lactococcus lactis isolates, using a novel multilocus sequence analysis scheme and (GTG)5-PCR fingerprinting

The diversity of a collection of 102 lactococcus isolates including 91 Lactococcus lactis isolates of dairy and nondairy origin was explored using partial small subunit rRNA gene sequence analysis and limited phenotypic analyses. A subset of 89 strains of L. lactis subsp. cremoris and L. lactis subsp. lactis isolates was further analyzed by (GTG)(5)-PCR fingerprinting and a novel multilocus sequence analysis (MLSA) scheme. Two major genomic lineages within L. lactis were found. The L. lactis subsp. cremoris type-strain-like genotype lineage included both L. lactis subsp. cremoris and L. lactis subsp. lactis isolates. The other major lineage, with a L. lactis subsp. lactis type-strain-like genotype, comprised L. lactis subsp. lactis isolates only. A novel third genomic lineage represented two L. lactis subsp. lactis isolates of nondairy origin. The genomic lineages deviate from the subspecific classification of L. lactis that is based on a few phenotypic traits only. MLSA of six partial genes (atpA, encoding ATP synthase alpha subunit; pheS, encoding phenylalanine tRNA synthetase; rpoA, encoding RNA polymerase alpha chain; bcaT, encoding branched chain amino acid aminotransferase; pepN, encoding aminopeptidase N; and pepX, encoding X-prolyl dipeptidyl peptidase) revealed 363 polymorphic sites (total length, 1,970 bases) among 89 L. lactis subsp. cremoris and L. lactis subsp. lactis isolates with unique sequence types for most isolates. This allowed high-resolution cluster analysis in which dairy isolates form subclusters of limited diversity within the genomic lineages. The pheS DNA sequence analysis yielded two genetic groups dissimilar to the other genotyping analysis-based lineages, indicating a disparate acquisition route for this gene.     

Inactivation of the glutamate decarboxylase gene in Lactococcus lactis subsp. cremoris

Lactococcus lactis subsp. lactis strains show glutamate decarboxylase activity, whereas L. lactis subsp. cremoris strains do not. The gadB gene encoding glutamate decarboxylase was detected in the L. lactis subsp. cremoris genome but was poorly expressed. Sequence analysis showed that the gene is inactivated by the frameshift mutation and encoded in a nonfunctional protein.     

Isolation of Lactococcus lactis subsp. cremoris from nature by colony hybridization with rRNA probes.

Lactococcus lactis subsp. cremoris is widely used in the manufacture of fermented milk products. Despite numerous attempts, efforts to isolate new strains by traditional plating and identification methods have not been successful. Previously, we described oligonucleotide probes for 16S rRNAs which could be used to discriminate L. lactis subsp. cremoris from related strains. These probes were used in colony hybridization experiments to screen large numbers of colonies obtained from enrichment cultures. A total of 170 strains of L. lactis were isolated from six milk samples, two colostrum samples, and one corn sample by using oligonucleotide probe 212RLa specific for the species L. lactis. Fifty-nine of these isolates also hybridized to L. lactis subsp. cremoris-specific probe 68RCa, and 26 of the strains which hybridized to the L. lactis subsp. cremoris-specific probe had the L. lactis subsp. cremoris phenotype.     

16S rRNA和recA、groEL基因分类鉴定乳酸乳球菌乳酸亚种和乳脂亚种的比较

【目的】比较16S rRNA和recA、groEL基因部分序列用于乳酸乳球菌乳酸亚种和乳脂亚种分类鉴定的效果。【方法】对已鉴定的8株分离自传统发酵乳的乳酸乳球菌, 选取recA和groEL基因片段, 通过PCR扩增、测序, 将测序得到的序列比对后构建系统发育树, 并与16S rRNA基因序列分析技术进行比较。【结果】比较分析不同菌株16S rRNA和recA、groEL基因的亲缘关系, recA、groEL基因可以准确地完成乳酸乳球菌乳酸亚种和乳脂亚种的区分和鉴定。【结论】recA和groEL基因序列分析可以实现乳酸乳球菌乳酸亚种和乳脂亚种的区分, 因其具有快速、准确、稳定的特点, 可适合于乳酸乳球菌乳酸亚种和乳脂亚种间的快速分类鉴定。     

Comparative phenotypic and molecular genetic profiling of wild Lactococcus lactis subsp. lactis strains of the L. lactis subsp. lactis and L. lactis subsp. cremoris genotypes, isolated from starter-free cheeses made of raw milk

Twenty Lactococcus lactis strains with an L. lactis subsp. lactis phenotype isolated from five traditional cheeses made of raw milk with no added starters belonging to the L. lactis subsp. lactis and L. lactis subsp. cremoris genotypes (lactis and cremoris genotypes, respectively; 10 strains each) were subjected to a series of phenotypic and genetic typing methods, with the aims of determining their phylogenetic relationships and suitability as starters. Pulsed-field gel electrophoresis (PFGE) analysis of intact genomes digested with SalI and SmaI proved that all strains were different except for three isolates of the cremoris genotype, which showed identical PFGE profiles. Multilocus sequence typing (MLST) analysis using internal sequences of seven loci (namely, atpA, rpoA, pheS, pepN, bcaT, pepX, and 16S rRNA gene) revealed considerable intergenotype nucleotide polymorphism, although deduced amino acid changes were scarce. Analysis of the MLST data for the present strains and others from other dairy and nondairy sources showed that all of them clustered into the cremoris or lactis genotype group, by using both independent and combined gene sequences. These two groups of strains also showed distinctive carbohydrate fermentation and enzyme activity profiles, with the strains in the cremoris group showing broader profiles. However, the profiles of resistance/susceptibility to 16 antibiotics were very similar, showing no atypical resistance, except for tetracycline resistance in three identical cremoris genotype isolates. The numbers and concentrations of volatile compounds produced in milk by the strains belonging to these two groups were clearly different, with the cremoris genotype strains producing higher concentrations of more branched-chain, derived compounds. Together, the present results support the idea that the lactis and cremoris genotypes of phenotypic Lactococcus lactis subsp. lactis actually represent true subspecies. Some strains of the two subspecies in this study appear to be good starter candidates.     

Rapid PCR-based method which can determine both phenotype and genotype of Lactococcus lactis subspecies

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.     

Cloning, sequencing, and expression of the pyruvate carboxylase gene in Lactococcus lactis subsp. lactis C2

A functional pyc gene was isolated from Lactococcus lactis subsp. lactis C2 and was found to complement a Pyc defect in L. lactis KB4. The deduced lactococcal Pyc protein was highly homologous to Pyc sequences of other bacteria. The pyc gene was also detected in Lactococcus lactis subsp. cremoris and L. lactis subsp. lactis bv. diacetylactis strains.     

Recent genetic transfer between Lactococcus lactis and enterobacteria

The genome sequence of Lactococcus lactis revealed that the ycdB gene was recently exchanged between lactococci and enterobacteria. The present study of ycdB orthologs suggests that L. lactis was probably the gene donor and reveals three instances of gene transfer to enterobacteria. Analysis of ycdB gene transfer between two L. lactis subspecies, L. lactis subsp. lactis and L. lactis subsp. cremoris, indicates that the gene can be mobilized, possibly by conjugation.     

Exopolysaccharide expression in Lactococcus lactis subsp. cremoris Ropy352: evidence for novel gene organization

Lactococcus lactis subsp. cremoris Ropy352 produces two distinct heteropolysaccharides, phenotypically described as ropy and mucoid, when cultured in nonfat milk. One exopolysaccharide precipitated with 50% ethanol as a series of elongated threads and was composed of glucose and galactose in a molar ratio of 3:2. The second exopolysaccharide precipitated with 75% ethanol as a fine flocculant and consisted of galactose, glucose, and mannose with a molar ratio of 67:21:12. A mutant strain, L. lactis subsp. cremoris EK240, lacking the ropy phenotype did not produce the exopolysaccharide that precipitated with 50% ethanol; however, it produced the exopolysaccharide that precipitated with 75% ethanol, indicating that the former exopolysaccharide is essential for the ropy phenotype. Cultures of L. lactis subsp. cremoris Ropy352 in 10% nonfat milk reached a viscosity of 25 Pa-s after 24 h, while those of the nonropy L. lactis subsp. cremoris EK240 mutant did not change. A mutation abolishing ropy exopolysaccharide expression mapped to a region on a plasmid containing two open reading frames, epsM and epsN, encoding novel glycosyltransferases bordered by ISS1 elements oriented in the same direction. Sequencing of this plasmid revealed two other regions involved in exopolysaccharide expression, an operon located between partial IS981 and IS982 elements, and an independent gene, epsU. Two and possibly three of these regions are involved in L. lactis subsp. cremoris Ropy352 exopolysaccharide expression and are arranged in a novel fashion different from that of typical lactococcal exopolysaccharide loci, and this provides genetic evidence for exopolysaccharide gene reorganization and evolution in Lactococcus.     

The ldh phylogeny for environmental isolates of Lactococcus lactis is consistent with rRNA genotypes but not with phenotypes.

Lactate dehydrogenase (ldh) gene sequences, levels of 16S rRNA group-specific probe binding, and phenotypic characteristics were compared for 45 environmental isolates and four commercial starter strains of Lactococcus lactis to identify evolutionary groups best suited to cheddar cheese manufacture, ldh sequences from the environmental isolates showed high similarity to those from two groups of L. lactis used for industrial fermentations, L. lactis subsp. cremoris and subsp. lactis. Within each phylogenetically defined subspecies, ldh sequence similarities were greater than 99.1%. Strains with phenotypic traits formerly diagnostic for both subspecies were found in each ldh similarity group, but only strains belonging to L. lactis subsp. cremoris by both the newer, genetic and the older, superseded phenotypic criteria were judged potentially suitable for the commercial production of cheddar cheese. Identical evolutionary relationships were inferred from ldh sequences and from binding of subspecies-specific, 16S rRNA-directed oligonucleotide probes. However, groups defined according to these chromosomal traits bore no relationship to patterns of arginine deamination, carbon substrate utilization, or bacteriophage sensitivity, which may be encoded by cryptic genes or sexually transmissible genetic elements. Fourteen new L. lactis subsp. cremoris isolates were identified as suitable candidates for cheddar cheese manufacture, and 10 of these were completely resistant to three different batteries of commercial bacteriophages known to reduce starter activity.     

Cooperation between Lactococcus lactis and nonstarter lactobacilli in the formation of cheese aroma from amino acids

In Gouda and Cheddar type cheeses the amino acid conversion to aroma compounds, which is a major process for aroma formation, is essentially due to lactic acid bacteria (LAB). In order to evaluate the respective role of starter and nonstarter LAB and their interactions in cheese flavor formation, we compared the catabolism of phenylalanine, leucine, and methionine by single strains and strain mixtures of Lactococcus lactis subsp. cremoris NCDO763 and three mesophilic lactobacilli. Amino acid catabolism was studied in vitro at pH 5.5, by using radiolabeled amino acids as tracers. In the presence of alpha-ketoglutarate, which is essential for amino acid transamination, the lactobacillus strains degraded less amino acids than L. lactis subsp. cremoris NCDO763, and produced mainly nonaromatic metabolites. L. lactis subsp. cremoris NCDO763 produced mainly the carboxylic acids, which are important compounds for cheese aroma. However, in the reaction mixture containing glutamate, only two lactobacillus strains degraded amino acids significantly. This was due to their glutamate dehydrogenase (GDH) activity, which produced alpha-ketoglutarate from glutamate. The combination of each of the GDH-positive lactobacilli with L. lactis subsp. cremoris NCDO763 had a beneficial effect on the aroma formation. Lactobacilli initiated the conversion of amino acids by transforming them mainly to keto and hydroxy acids, which subsequently were converted to carboxylic acids by the Lactococcus strain. Therefore, we think that such cooperation between starter L. lactis and GDH-positive lactobacilli can stimulate flavor development in cheese.     

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.     

Tolerance to high osmolality of Lactococcus lactis subsp. lactis and cremoris is related to the activity of a betaine transport system

Lactococcus lactis strains from the subsp. cremoris are described as more sensitive to osmotic stress than subsp. lactis strains. We examined the relation between osmotic tolerance and the activity of the betaine transporter BusA among 34 strains of L. lactis. The cremoris strains that showed reduced growth at high osmolality failed to accumulate betaine. The nature of the defect was found to vary among cremoris strains: lack of the busA encoding region, absence of synthesis or synthesis of an inactive form of BusA. The results suggest that the selection of strains well fitted to the dairy production lead to the loss of an otherwise efficient adaptation mechanism.