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PubMed1991

Engineering of the Lactococcus lactis serine proteinase by construction of hybrid enzymes.

Plasmids containing wild-type and hybrid proteinase genes were constructed from DNA fragments of the prtP genes of Lactococcus lactis strains Wg2 and SK11. These plasmids were introduced into the plasmid-free strain L. lactis MG1363. The serine proteinases produced by these L. lactis strains were isolated, and their cleavage specificity and rate towards alpha s1- and beta-casein was investigated. The catalytic properties of both the SK11 and Wg2 proteinases, which differ in 44 out of 1902 amino acid residues, could be changed dramatically by the reciprocal exchange of specific fragments between the two enzymes. As a result, various L. lactis strains were constructed having new proteolytic properties that differ from those of the parental strains. Furthermore, two segments in the proteinase could be identified that contribute significantly to the cleavage specificity towards casein; within these two segments, several amino acid residues were identified that are important for substrate cleavage rate and specificity. The results also indicate that the lactococcal proteinase has an additional domain involved in substrate binding compared with the related subtilisins. This suggests that the 200 kd L. lactis proteinase may be the representative of a new subclass of subtilisin-like enzymes.

Amino Acid Sequence↗
PubMed1989

Localization of Separate Genetic Loci for Reduced Sensitivity towards Small Isometric-Headed Bacteriophage sk1 and Prolate-Headed Bacteriophage c2 on pGBK17 from Lactococcus lactis subsp. lactis KR2.

The mechanism of reduced sensitivity to the small isometric-headed bacteriophage sk1 encoded on a 19-kilobase (kb) HpaII fragment subcloned from pKR223 of Lactococcus lactis subsp. lactis KR2 was examined. The reduced sensitivity to phage sk1 was due to a modest restriction/modification (R/M) system that was not active against prolate-headed phage c2. The genetic loci for the R/M system against sk1 and the abortive phage infection (Abi) mechanism effective against phage c2 were then localized by restriction mapping, subcloning, and deletion analysis. The restriction gene was localized to a region of a 2.7-kb EcoRV fragment and included an EcoRI site within that fragment. The modification gene was found to be physically separable from the restriction gene and was present on a 1.75-kb BstEII-XbaI fragment. The genetic locus for the Abi phenotype against phage c2 was localized to a region containing a 1.3-kb EcoRI fragment. Attempts to clone the c2 Abi mechanism independent of the sk1 R/M system were unsuccessful, suggesting that expression of the abi genes required sequences upstream of the modification gene. Some pGBK17 (vector pGB301 plus a 19-kb HpaII insert fragment) transformants exhibited the R/M system against phage sk1 but lost the Abi mechanism against phage c2. These transformants contained a 1.2- to 1.3-kb insertion in the Abi region. The data identified genetic loci on a cloned 19-kb HpaII fragment responsible for restriction activity and for modification activity against a small isometric-headed phage and for Abi activity against prolate-headed phage c2. A putative insertion element was also found to inactivate the abi gene(s).

Journal Article↗
PubMed1993

Characterization of Loosely Associated Material from the Cell Surface of Lactococcus lactis subsp. cremoris E8 and Its Phage-Resistant Variant Strain 398.

Loosely associated material (LAM) was isolated by gentle extraction procedures from the cell surface of Lactococcus lactis subsp. cremoris E8 and its phage-resistant variant strain 398. LAM from both strains was chemically characterized, and its role in the adsorption of three small isometric bacteriophages, phi 618, phi 833, and phi 852, to the cell surface of the two strains was investigated. The phage-resistant strain (strain 398) produced LAM which differed significantly from the material produced by the parent strain. The total yield of LAM from strain 398 was two- to threefold higher than that from strain E8, and the material contained fivefold more rhamnose and twofold more galactose. Polyacrylamide gel electrophoretic analysis showed that LAM from strain 398 lacked a 21-kDa protein which was present in LAM from the parent strain. Inhibition studies of phage binding by using isolated LAM from two strains showed that although LAM from strain E8 reduced the titer of phi 618 and phi 852 by 53 and 82% respectively, LAM from strain 398 had no effect on the plaque-forming ability of any of the three phages tested. Treatment of LAM from strain E8 with sodium metaperiodate destroyed its ability to bind with phi 618 and phi 852. Phenotypically, strain 398 differed from its parent strain E8 in that it was more prone to cell lysis and required an osmotically adjusted buffer system for the extraction of LAM.

Journal Article↗
PubMed1993

Transduction of a Plasmid Carrying the Cohesive End Region from Lactococcus lactis Bacteriophage PhiLC3.

Plasmids carrying the cohesive end region from temperate lactococcal bacteriophage PhiLC3 could be packaged in vivo by PhiLC3 and transduced into its host strain, Lactococcus lactis subsp. cremoris NCDO 1201. The transduction frequencies were between 10 and 10 transducing particles per PFU, depending on the size of the phage DNA insert. This transduction system is limited to only certain lactococcal strains. The PhiLC3 cohesive site region (cos) appears to play an important role in plasmid transduction.

Journal Article↗
PubMed2006

Monitoring the cell number of Lactococcus lactis subsp. cremoris FC in human feces by real-time PCR with strain-specific primers designed using the RAPD technique.

Strain-specific PCR primers for Lactococcus lactis subsp. cremoris FC were developed using the randomly amplified polymorphic DNA (RAPD) technique. RAPD was used to generate strain-specific markers. A 1164-bp RAPD marker found to be strain-specific was sequenced, and a primer pair specific for L. lactis subsp. cremoris FC was designed. The specificity of this primer pair was tested with 23 L. lactis subsp. cremoris strains and 20 intestinal bacterial species, and was found to be strain-specific. Subsequently, this primer pair was subjected to the quantification of L. lactis subsp. cremoris FC in the feces of subjects fed fermented milk containing this strain. After administration, L. lactis subsp. cremoris FC was detected in the feces of all 7 subjects, with the maximum number being between 10(5) and 10(9) cells g(-1) of feces. Furthermore, this strain was detected in only one feces sample 2 weeks after administration was stopped. These results suggest that L. lactis subsp. cremoris FC can survive passage through the gastrointestinal tract.

Adult↗
PubMed1990

Differences in short peptide-substrate cleavage by two cell-envelope-located serine proteinases of Lactococcus lactis subsp. cremoris are related to secondary binding specificity.

Various chromophoric peptides have been tested as substates for two genetically related types (PI and PIII) of cell-envelope proteinases of Lactococcus lactis subsp. cremoris. The positively charged peptide methoxy-succinyl-arginyl-prolyl-tyrosyl-p-nitroanilide appeared to be cleaved with the highest catalytic efficiency by both enzymes, although in the case of PIII only at high ionic strength. A cation binding site in the PI-type proteinase that is not present in the related PIII-type appears to be mainly responsible for the difference between these enzymes with respect to the rate of conversion of this chromophoric substrate at relatively low ionic strength. This cation binding site most probably resides in the aspartic acid residue 166, which in PIII is substituted by asparagine. Substitution of the threonine residue 138 by lysine in PIII may also play a role. The binding step in the reaction pathway catalysed by PI at low ionic strength is governed mainly by an ionic interaction involving the cation binding site. In addition, hydrophobic interactions contribute to the binding process. Masking of the cation binding site only increases the Michaelis constant Km; the catalytic constant kcat is not affected. In the absence of the cation binding site (viz. in PIII) the free energy derived from the hydrophobic interactions only is too small to promote binding of the substrate effectively. High activities are measured only if a high ionic strength is introduced. Removal of electrostatic repulsion between the substrate and positively charged residues of the enzyme, among which is lysine 138, may contribute to this activation.(ABSTRACT TRUNCATED AT 250 WORDS)

1-Butanol↗
PubMed1994

Phenotypic Consequences of Altering the Copy Number of abiA, a Gene Responsible for Aborting Bacteriophage Infections in Lactococcus lactis.

The abiA gene (formerly hsp) encodes an abortive phage infection mechanism which inhibits phage DNA replication. To analyze the effects of varying the abiA gene dosage on bacteriophage resistance in Lactococcus lactis, various genetic constructions were made. An IS946-based integration vector, pTRK75, was used to integrate a single copy of abiA into the chromosomes of two lactococcal strains, MG1363 and NCK203. In both strains, a single copy of abiA did not confer any significant phage resistance on the host except for one of the MG1363 integrants, NCK625, which exhibited a slightly higher level of resistance to phages sk1 and p2. Hybridization of the total cellular RNA from NCK625 to an abiA-specific probe indicated that the integration took place downstream of a promoter causing stronger expression of abiA in this integrant. Three abiA-containing plasmids of various copy numbers were introduced into both strains, and the recombinants were evaluated for resistance to phages c2, p2, sk1, and phi31. Plasmid pTRK18 has a copy number of approximately six (cn = 6) and caused a decreased plaque size for all phages evaluated. Integration of pTRK75 into a native plasmid of NCK203 generated pTRK362 (cn = 13), which caused a reduced efficiency of plaquing (EOP = 10) and reduced plaque size. A high-copy-number abiA plasmid (pTRK363), based on the pAMbeta1 origin of replication, was also constructed (cn = 100). Plasmid pTRK363 caused a significant reduction in EOP (10 to 10) and plaque size for all phages tested, although in some cases, this plasmid caused the evolution of AbiA-resistant phage derivatives. Altering the gene dosage or expression level of abiA significantly affects the phage resistance levels.

Journal Article↗
PubMed1997

Purification and Characterization of Cystathionine (gamma)-Lyase from Lactococcus lactis subsp. cremoris SK11: Possible Role in Flavor Compound Formation during Cheese Maturation.

A cystathionine (gamma)-lyase (EC 4.4.1.1) ((gamma)-CTL) was purified to homogeneity from a crude cell extract of Lactococcus lactis subsp. cremoris SK11 by a procedure including anion-exchange chromatography, hydrophobic interaction chromatography, and gel filtration chromatography. The activity of SK11 (gamma)-CTL is pyridoxal-5(prm1)-phosphate dependent, and the enzyme catalyzes the (alpha),(gamma)-elimination reaction of L-cystathionine to produce L-cysteine, (alpha)-ketobutyrate, and ammonia. The native enzyme has a molecular mass of approximately 120 to 200 kDa and apparently consists of at least six identical subunits of 20 kDa. In this respect, the SK11 enzyme clearly differs from other bacterial cystathionine lyases, which are all tetrameric proteins with identical subunits of approximately 40 kDa. In addition, the specific activity of purified SK11 (gamma)-CTL toward L-cystathionine is relatively low compared with those reported for other bacterial cystathionine lyases. The SK11 enzyme shows a broad substrate specificity. In the case of L-methionine, the action of SK11 (gamma)-CTL results in the formation of methanethiol, a volatile sulfur compound known to be required in flavor development in cheddar cheese. The (alpha),(beta)-elimination reaction of L-cysteine is also efficiently catalyzed by the enzyme, resulting in the formation of hydrogen sulfide. Although the conditions are far from optimal, cystathionine (gamma)-lyase is still active under cheddar cheese-ripening conditions, namely, pH 5.0 to 5.4 and 5% (wt/vol) NaCl. The possible role of the enzyme in cheese flavor development is discussed.

Journal Article↗
PubMed1990

Stability of Integrated Plasmids in the Chromosome of Lactococcus lactis.

Derivatives of plasmids pBR322, pUB110, pSC101, and pTB19, all containing an identical fragment of lactococcal chromosomal DNA, were integrated via a Campbell-like mechanism into the same chromosomal site of Lactococcus lactis MG1363, and the transformants were analyzed for the stability of the integrated plasmids. In all cases the erythromycin resistance gene of pE194 was used as a selectable marker. Transformants obtained by integration of the pBR322 derivatives contained a head-to-tail arrangement of several plasmid copies, which most likely was caused by integration of plasmid multimers. Single-copy integrations were obtained with the pSC101 and pTB19 derivatives. In all of these transformants no loss of the erythromycin gene was detected during growth for 100 generations in the absence of the antibiotic. In contrast, transformants containing integrated amplified plasmid copies of pUB110 derivatives were unstable under these conditions. Since pUB110 appeared to have replicative activity in L. lactis, we suggest that this activity destabilized the amplified structures in L. lactis.

Journal Article↗
PubMed1996

Cloning and analysis of the restriction-modification system LlaBI, a bacteriophage resistance system from Lactococcus lactis subsp. cremoris W56.

The genes coding for the type II restriction-modification (R/M) system LlaBI, which recognized the sequence 5'-C decreases TRYAG-3', have been cloned from a plasmid in Lactococcus lactis subsp. cremoris W56 and sequenced. The DNA sequence predicts an endonuclease of 299 amino acids (33 kDa) and a methylase of 580 amino acids (65 kDa). A 4.0-kb HindIII fragment in pSA3 was able to restrict bacteriophages, showing that the cloned R/M system can function as a phage defense mechanism in L. lactis.

Amino Acid Sequence↗
PubMed1998

Cloning, expression, and chromosomal stabilization of the Propionibacterium shermanii proline iminopeptidase gene (pip) for food-grade application in Lactococcus lactis.

Proline iminopeptidase produced by Propionibacterium shermanii plays an essential role in the flavor development of Swiss-type cheeses. The enzyme (Pip) was purified and characterized, and the gene (pip) was cloned and expressed in Escherichia coli and Lactococcus lactis, the latter species being an extensively studied, primary cheese starter culture that is less fastidious in its growth condition requirements than P. shermanii. The levels of expression of the pip gene could be enhanced with a factor 3 to 5 by using a strong constitutive promoter in L. lactis or the inducible tac promoter in E. coli. Stable replication of the rolling-circle replicating (rcr) plasmid, used to express pip in L. lactis, could only be obtained by providing the repA gene in trans. Upon the integration of pip, clear gene dosage effects were observed and stable multicopy integrants could be maintained upon growth under the selective pressure of sucrose. The multicopy integrants demonstrated a high degree of stability in the presence of glucose. This study examines the possibilities to overexpress genes that play an important role in food fermentation processes and shows a variety of options to obtain stable food-grade expression of such genes in L. lactis.

Aminopeptidases↗
PubMed1990

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.

Journal Article↗
PubMed1994

Evolution of a Lytic Bacteriophage via DNA Acquisition from the Lactococcus lactis Chromosome.

We discovered a phage-host interaction in which the lytic phage ul36, in response to pressure exerted by an abortive phage resistance mechanism, acquired a large DNA fragment from the chromosome of Lactococcus lactis NCK203 to form a new phage, ul37. Phage ul37 was characterized at morphological, phenotypic, and genotypic levels and was found to be a member of the P335 species. Although it exhibits a high level of DNA homology with ul36, phage ul37 is resistant to the abortive mechanism and has a longer tail, a different base plate, and apparently a different origin of replication. The chromosomal DNA implicated in the formation of new phage ul37 was disrupted by site-specific integration in NCK203. This strategy prevented the appearance of ul37 during subsequent infections with ul36.

Journal Article↗
PubMed2002

Characterization of anti-Listeria monocytogenes bacteriocins from Enterococcus faecalis, Enterococcus faecium, and Lactococcus lactis strains isolated from Raïb, a Moroccan traditional fermented milk.

Seventy-four samples of raïb, a Moroccan traditional fermented milk, were screened for their anti-Listeria monocytogenes activity. Nine lactic acid bacteria with antilisterial activity were isolated and identified as Lactococcus lactis [4], Enterococcus faecium [4], and E. faecalis [1]. Antibacterial spectra, determined against 45 target strains, led to the selection of four antibacterial-producing strains, which were further characterized. Their anti-microbial agents, inactivated by one or more proteases, were designed as bacteriocins. Lactococcin R9/2 and R10/1 showed the broadest range of inhibitory action. Anti-bacterial spectra and physico-chemical properties suggest that these bacteriocins were similar to nisin. Enterocin R69 had a specificity of action against Listeria spp., whereas Enterocin R18 had a broad spectrum of activity. Lc. lactis R9/2 and E. faecalis R18 were able to coagulate sterilised UHT milk at 30 degrees C in 24 h and induced a 2 log reduction in L. monocytogenes ATCC 15313 population.

Animals↗
PubMed1997

Crystallization and preliminary X-ray diffraction analysis of the heterotetrameric dihydroorotate dehydrogenase B of Lactococcus lactis, a flavoprotein enzyme system consisting of two PyrDB subunits and two iron-sulfur cluster containing PyrK subunits.

Dihydroorotate dehydrogenases are flavin-containing enzymes which catalyze the conversion of (S)-dihydroorotate to orotate. Dihydroorotate dehydrogenase B (DHODB) from Lactococcus lactis is a heterotetramer containing two subunits of the protein encoded by the pyrDb gene (PyrDB) and two subunits of the protein encoded by the pyrK gene (PyrK). In addition, DHODB contains two molecules of flavin mononucleotide, two molecules of flavin adenine dinucleotide and two [2Fe-2S] iron-sulfur clusters as tightly bound cofactors. Yellow crystals of this enzyme have been grown using the hanging-drop vapour-diffusion technique from solutions of 2.5 M ammonium sulfate and 0.1 M sodium acetate, pH 4.6. The crystals have been shown to contain both the PyrDB and the PyrK subunits and fluorescence measurements indicate that the two different subunits interact very closely with each other in the active-site region. Native data sets have been collected to 2.6 A with a conventional X-ray source and to 2.2 A using synchrotron radiation. The crystals are rhombohedral, space group R32, with correspondin8 hexagonal unit-cell dimensions a = b = 202.3 and c = 81.0 A. The asymmetric unit in the crystal contains one PyrDB subunit and one PyrK subunit, which suggests that the two halves of the heterotetramer are related by a crystallographic twofold axis.

Journal Article↗
PubMed1995

Expression of a Lactococcus lactis Phage Resistance Mechanism by Streptococcus thermophilus.

The 7.8-kb lactococcal plasmid pSRQ700 encodes the LlaII restriction/modification system which recognizes and cleaves the sequence 3(prm1)-GATC-5(prm1). When the plasmid pSRQ700 is introduced into a phage-sensitive Lactococcus lactis strain, strong phage resistance is conferred by the LlaII system. In this report, we show that pSRQ700 cannot replicate in Streptococcus thermophilus. However, if cloned into the vector pNZ123, the native LlaII system is expressed and strong phage resistance is conferred to various industrial S. thermophilus strains. Resistance against phages isolated from yogurt and mozzarella wheys was observed. To our knowledge, this is the first report of increased phage resistance in S. thermophilus.

Journal Article↗
PubMed1999

Specificity of lactococcus lactis subsp. cremoris SK11 proteinase, lactocepin III, in low-water-activity, high-salt-concentration humectant systems and its stability compared with that of lactocepin I

Marked changes in the specificity of hydrolysis of alphas1-, beta-, and kappa-caseins by lactocepin III from Lactococcus lactis subsp. cremoris SK11 were found in humectant systems giving the equivalent water activity (aw) and salt concentration of cheddar cheese. Correlations were noted between certain peptides produced by the activity of lactocepin III in the humectant systems and peptides found in cheddar cheese. The stability of lactocepin III was compared with that of lactocepin I from L. lactis subsp. cremoris HP in the humectant systems at different pHs. Significant differences between the stability of each of the lactocepin types were evident. The relationship between stability and humectant type, aw, pH, and NaCl concentration was complex. Nevertheless, in those systems where aw, pH, and NaCl concentration were equivalent to those in cheddar cheese, lactocepin I was generally more stable than lactocepin III. It was concluded that differences in the specificity and/or stability of various lactocepin types are likely to persist in cheese itself and therefore potentially contribute to differences in the peptide composition of ripened cheese.

Journal Article↗
PubMed2001

4-Hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF) production in simple media by lactic acid bacterium, Lactococcus lactis subsp. cremoris IFO 3427.

4-Hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF) was detected in simple media prepared with heated sugar and amino acid solution as a result of the Maillard reaction. However, an increase in the amount of HDMF was observed in the media fermented by Lactococcus lactis subsp. cremoris IFO 3427. 4-Hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone (HEMF) was not detected in all the simple media examined in this study.

Journal Article↗