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.     

In vivo action of Lactococcus lactis subsp. lactis isolate (R7) with probiotic potential in the stabilization of cancer cells in the colorectal epithelium

Recent research has contributed to advances in the treatment and prevention of colorectal cancer (CRC); however, the prevalence and mortality rate remain high. Lactococcus lactis subps. lactis is commonly used for industrial purposes, but there are no in vivo studies evaluating its anticarcinogenic potential. This study aimed to explore the protective effects of an isolate of L. lactis subsp. lactis (R7) from ricotta on CRC as well as evaluatingbiochemical and hematological parameters resulting from treatment. Wistar rats received subcutaneous doses of 1,2-dimethylhydrazine (DMH), what is a chemical carcinogen known to induce this carcinogenesis in experimental models, orally a suspension of R7 (10⁸ colony-forming units. mL⁻¹) and a hypercaloric diet. The results demonstrated a positive effect on the stabilization of the cancer cells induced in the colorectal epithelium, demonstrating anti-carcinogenic potential of R7 against CRC. In addition, an immune response was observed and the biochemical parameters showed that R7 reversed the stress caused by DMH and a hypercaloric diet. The results are promising as regards the behavior of R7 in relation to the treatment of CRC.     

Comparison of cell wall proteinases from Lactococcus lactis subsp. cremoris AC1 and Lactococcus lactis subsp. lactis NCDO 763

Summary Cell wall-associated proteinases were isolated from Lactococcus lactis subsp. cremoris AC1 and subsp. lactis NCDO 763 in order to compare their specificities towards different caseins. Two purification strategies were applied. Cells grown in casein-free M17 medium were a suitable starting material for purification, since electrophoretic purity could be achieved after one chromatographic step. Both enzymes has an apparent molecular mass of about 145000 daltons as judged by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Electrophoresis and reversed phase HPLC patterns of hydrolysates of _s1-, _s2-, -, and K-caseins indicated that both proteinases had a similar specificity. The enzyme of L. lactis subsp. lactis split _s1- and _s2-caseins more extensively than that of L. lactis subsp. cremoris.     

PCR amplification of the gene acmA differentiates Lactococcus lactis subsp. lactis and L. lactis subsp. cremoris.

The occurrence of the acmA gene, encoding the lactococcal N-acetylmuramidase in new lactococcal isolates from raw milk cheeses, has been determined. Isolates were genotypically identified to the subspecies level with a PCR technique. On the basis of PCR amplification of the acmA gene, the presence or absence of an additional amplicon of approximately 700 bp correlated with Lactococcus lactis subspecies. L. lactis subsp. lactis exhibits both the expected 1,131-bp product and the additional amplicon, whereas L. lactis subsp. cremoris exhibits a single 1,131-bp fragment.     

6种区分乳酸乳球菌乳酸亚种和乳酸乳球菌乳脂亚种的分子生物学方法比较

【目的】比较并评价6种分子生物学技术对乳酸乳球菌乳酸亚种(Lactococcus lactis subsp.lactis)和乳酸乳球菌乳脂亚种(Lactococcus lactis subsp.cremoris)的区分效果。【方法】采用16S rRNA基因序列分析技术,16S-23S rRNA间区序列多态性分析技术,变性梯度凝胶电泳技术(DGGE),随机扩增多态性分析技术(RAPD),重复基因外回文序列分析技术(rep-PCR)和限制性酶切片段多态性分析技术(RFLP)对4株Lactococcus lactis subsp.lactis和Lactococcus lactis subsp.cremoris参考菌株进行了区分,并对这6种方法的区分效果进行了比较评价。【结果】16S rRNA基因序列分析技术,16S-23S rRNA间区序列多态性分析技术无法区分Lactococcus lactis subsp.lactis和Lactococcus lactis subsp.cremoris,而其余4种技术可以实现区分。【结论】变性梯度凝胶电泳(DGGE),随机扩增多态性分析技术(RAPD)耗时短,操作简单,试验结果准确稳定,更适合Lactococcus lactis subsp.lactis和Lactococcus lactis subsp.cremoris的快速准确区分。     

Location and characterization of aminopeptidase N in Lactococcus lactis subsp. cremoris HP

Summary One single, cytosolic aminopeptidase (AP N, EC 3.4.11.2) is found to be responsible for both leucyl-(leucylAP) and lysylaminopeptidese (lysylAP) activity detectible with whole cells of Lactococcus lactis subsp. cremoris strain HP. The existence of a cell-envelope-located form of this enzyme could be excluded. No restriction on the activity of the enzyme is imposed by the cell membrane if leucine-p-nitroanilide is used as the substrate; with lysine-p-nitroanilide the activity is highly cryptic. The enzyme has been purified and characterized. It is a metalloaminopeptidase with a molecular mass of 95 kDa. Co²⁺ appears to be the most potent ion to (re)activate the enzyme; Zn²⁺ and Mn²⁺ are less effective. The AP N releases the positively charged amino acids and several uncharged (including proline) from the N-terminus. Ammonium salts affect the preference of the enzyme with respect to the N-terminal residue. A preferential interaction of the ammonium ion with an essential cation binding site seems to be responsible for the inhibition of lysylAP activity.Trainee from the Laboratory School Friesland, Leeuwarden, The Netherlands Offprint requests to: F. A. Exterkate     

Phenotypic variation in Lactococcus lactis subsp. lactis isolates derived from intestinal tracts of marine and freshwater fish

Aims:  We compared phenotypic characteristics of Lactococcus lactis subsp. lactis derived from different sources including the intestinal tract of marine fish and freshwater fish, and cheese starter culture.
Methods and Results:  In the phylogenetic analysis based on partial 16S rRNA gene nucleotide sequences (1371 bp), freshwater fish-, marine fish- and cheese starter culture-derived strains were identical to that of L. lactis subsp. lactis previously reported. Fermentation profiles determined using the API 50 CH system were similar except for fermentation of several sugars including l -arabinose, mannitol, amygdalin, saccharose, trehalose, inulin and gluconate. The strains did have distinct levels of halotolerance: marine fish-derived strains > cheese starter-derived strain > freshwater fish-derived isolate.
Conclusions:  Lactococcus lactis subsp. lactis showed extensive diversity in phenotypic adaptation to various environments. The phenotypic properties of these strains suggested that L. lactis subsp. lactis strains from fish intestine have additional functions compared with the cheese starter-derived strain that has previously described.
Significance and Impact of the Study:  The unique phenotypic traits of the fish intestinal tract-derived L. lactis subsp. lactis might make them useful as a probiotics in aquaculture, and contribute to the development of functional foods and novel food additives, since the strains derived from fish intestines might have additional functions such as antibacterial activity.     

pAMbeta1-Associated Mobilization of Proteinase Plasmids from Lactococcus lactis subsp. lactis UC317 and L. lactis subsp. cremoris UC205

A combination of plasmid curing and DNA-DNA hybridization data facilitated the identification of proteinase plasmids of 75 (pCI301) and 35 kilobases (pCI203) in the multi-plasmid-containing strains Lactococcus lactis subsp. lactis UC317 and L. lactis subsp. cremoris UC205, respectively. Both plasmids were transferred by conjugation to a plasmid-free background only after introduction of the conjugative streptococcal plasmid, pAMbeta1. All Prt transconjugants from matings involving either donor contained enlarged recombinant Prt plasmids. UC317-derived transconjugants were separable into different classes based on the presence of differently sized cointegrate plasmids and on segregation of the pCI301-derived Lac and Prt markers. All UC205-derived transconjugants harbored a single enlarged plasmid that was a cointegrate between pCI203 and pAMbeta1. The identification of prt genes on pCI301 and pCI203 derivatives was achieved by a combination of restriction enzyme and hybridization analyses.     

Purification and characterization of x-prolyl-dipeptidyl aminopeptidase from Lactococcus lactis subsp. cremoris NRRL 634

Summary An X-prolyl-dipeptidylaminopep tidase (Pep-XP) was purified from the crude intracellular extract of Lactococcus lactis subsp. cremoris NRRL 634 by ion exchange and gel filtration chromatographies. The enzyme was purified 80-fold with a recovery of 6%, and appeared as a single band with a molecular weight of about 80 kDa on polyacrylamide gel electrophoresis with sodium dodecyl sulphate (SDS-PAGE). The peptidase showed its maximal activity on arginyl-proline-p-nitroanilide at pH 7.0 and at a temperature of 45 °C, although there was a good activity of Pep-XP in the pH range of 5.5–7.0 and temperatures between 40 and 50 °C. The Michaelis constant (K _m) and the maximum reaction velocity (V _max) values were 0.92 mM and 7.9 U/mg protein min, respectively. The activity of Pep-XP was completely inhibited by phenylmethanesulphonyl fluoride, an inhibitor of serine peptidases, and metal chelators had little effect on enzyme activity. The purified enzyme hydrolyzed synthetic substrates whose structure is X-Pro-Y like Lys-Pro-pNA, but did not hydrolyse Pro-pNA or azocasein, showing that the enzyme did not have aminopeptidase or endopeptidase activities.     

Purification and characterization of an endopeptidase from Lactococcus lactis subsp. cremoris Wg2.

An endopeptidase has been purified to homogeneity from a crude cell extract of Lactococcus lactis subsp. cremoris Wg2 by a procedure that includes diethyl-aminoethane-Sephacel chromatography, phenyl-Sepharose chromatography, hydroxylapatite chromatography, and fast protein liquid chromatography over an anion-exchange column and a hydrophobic-interaction column. Gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated a molecular mass of the purified enzyme of 70,000 Da. The endopeptidase can degrade several oligopeptides into various tetra-, tri-, and dipeptides. The endopeptidase has no aminopeptidase, carboxypeptidase, dipeptidase, or tripeptidase activity. It is optimally active at pH 6.0 to 6.5 and in the temperature range of 30 to 38 degrees C. The enzyme is inactivated by the chemical agents 1,10-phenanthroline, ethylenedinitrilotetraacetate, beta-mercaptoethanol, and phenylmethylsulfonyl fluoride and is inhibited by Cu2+ and Zn2+. The ethylenedinitrilotetraacetate- or 1,10-phenanthroline-treated enzyme can be reactivated by Co2+. Immunoblotting with specific antibodies raised against the purified endopeptidase indicated that the enzyme is also present in other Lactococcus spp., as well as in Lactobacillus spp. and Streptococcus salivarius subsp. thermophilus.     

Physical and genetic map of the Lactococcus lactis subsp. cremoris MG1363 chromosome: comparison with that of Lactococcus lactis subsp. lactis IL 1403 reveals a large genome inversion.

A physical and genetic map of the chromosome of the Lactococcus lactis subsp. cremoris reference strain MG1363 was established. The physical map was constructed for NotI, ApaI, and SmaI enzymes by using a strategy that combines creation of new rare restriction sites by the random-integration vector pRL1 and ordering of restriction fragments by indirect end-labeling experiments. The MG1363 chromosome appeared to be circular and 2,560 kb long. Seventy-seven chromosomal markers were located on the physical map by hybridization experiments. Integration via homologous recombination of pRC1-derived plasmids allowed a more precise location of some lactococcal genes and determination of their orientation on the chromosome. The MG1363 chromosome contains six rRNA operons; five are clustered within 15% of the chromosome and transcribed in the same direction. Comparison of the L. lactis subsp. cremoris MG1363 physical map with those of the two L. lactis subsp. lactis strains IL1403 and DL11 revealed a high degree of restriction polymorphism. At the genetic organization level, despite an overall conservation of gene organization, strain MG1363 presents a large inversion of half of the genome in the region containing the rRNA operons.     

Mutation of the oxaloacetate decarboxylase gene of Lactococcus lactis subsp. lactis impairs the growth during citrate metabolism

Aims:  Citrate metabolism generates metabolic energy through the generation of a membrane potential and a pH gradient. The purpose of this work was to study the influence of oxaloacetate decarboxylase in citrate metabolism and intracellular pH maintenance in relation to acidic conditions.
Methods and Results:  A Lactococcus lactis oxaloacetate decarboxylase mutant [ILCitM (pFL3)] was constructed by double homologous recombination. During culture with citrate, and whatever the initial pH, the growth rate of the mutant was lower. In addition, the production of diacetyl and acetoin was altered in the mutant strain. However, our results indicated no relationship with a change in the maintenance of intracellular pH. Experiments performed on resting cells clearly showed that oxaloacetate accumulated temporarily in the supernatant of the mutant. This accumulation could be involved in the perturbations observed during citrate metabolism, as the addition of oxaloacetate in M17 medium inhibited the growth of L. lactis .
Conclusions:  The mutation of oxaloacetate decarboxylase perturbed citrate metabolism and reduced the benefits of its utilization during growth under acidic conditions.
Significance and impact of the study:  This study allows a better understanding of citrate metabolism and the role of oxaloacetate decarboxylase in the tolerance of lactic acid bacteria to acidic conditions.     

Cloning and sequence analysis of a plasmid replicon from Lactococcus lactis subsp. cremoris FG2

A replication region from one of the Lactococcus lactis subsp. cremoris FG2 plasmids was isolated by cloning of a 4.8-kb XbaI fragment into a replication probe vector and transformation into L. lactis LM0230. A 1.8-kb region within this fragment was sequenced and confirmed by PCR subcloning to encode a functional replicon in LM0230. The replicon consists of an open reading frame encoding a putative replication protein (Rep) of 386 amino acids and a non-coding region (ori) which features several structural motifs typical of other known replication origins, including a 22-bp iteron sequence tandemly repeated three and a half times, a 10-bp direct repeat and two sets of inverted repeats. The ori region could drive replication of its plasmid when supplied with the replication region in-trans. The lack of detectable single-stranded DNA during replication and the existence of extensive homology with other known lactococcal theta replicons strongly suggest that this region encodes a theta-replicating mechanism.     

Purification and characterization of a cell wall peptidase from Lactococcus lactis subsp. cremoris IMN-C12.

A peptidase from the cell wall fraction of Lactococcus lactis subsp. cremoris IMN-C12 has been purified to homogeneity by hydrophobic interaction chromatography, two steps of anion-exchange chromatography, and gel filtration. The molecular mass of the purified enzyme was estimated to be 72 kDa by gel filtration and 23 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme has a pI of 4.0, and it has the following N-terminal sequence from the 2nd to the 17th amino acid residues: -Arg-Leu-Arg-Arg-Leu-?-Val-Pro-Gly-Glu-Ileu-Val-Glu-Glu-Leu-Leu. The peptidase is most active at pH 5.8 and at 33 degrees C with trileucine as the substrate. Reducing agents such as dithiothreitol, beta-mercaptoethanol, and cysteine strongly stimulated enzyme activity, while p-chloromercuribenzoate had an inhibitory effect. Also, metal chelators lowered the peptidase activity, which could not be restored with Ca2+ and Mg2+. The divalent cations Cu2+, Zn2+, Fe2+, and Hg2+ completely inhibited peptidase activity. The peptidase is capable of hydrolyzing tripeptides and some dipeptides, with a preference for peptides containing leucine and with the highest activity towards the tripeptides Leu-Leu-Leu, Leu-Trp-Leu, and Ala-Leu-Leu, which were hydrolyzed with Kms of 0.37, 0.18, and 0.61 mM, respectively.