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.     

Bacteriophage receptors of Lactococcus lactis subsp. 'diacetylactis' F7/2 and Lactococcus lactis subsp. cremoris Wg2-1

Bacteriophage P008 revealed irreversible and uniform adsorption to cell walls of L. lactis subsp. 'diacetylactis' F7/2, whereas phage P127 adsorbed reversibly to a limited number of receptor sites on cell walls of L. lactis subsp. cremoris Wg2-1. Neither extraction of lipids, cell wall- and membrane-teichoic acids nor enzymatic degradation of proteins altered the binding efficiencies of both cell wall fractions. However, phage binding was inhibited, when cell walls were subjected to lysozyme, metaperiodate, or acid treatments. This reflects that a carbohydrate component embedded in the peptidoglycan matrix is part of the phage receptors of strains F7/2 and Wg2-1.     

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

Summary The cell wall proteinases of Lactococcus lactis subsp. lactis NCDO 763 and L. lactis subsp. cremoris AC1 hydrolyse -casein with a similar specificity even though some quantitative differences can be observed for a few degradation products analysed by reverse phase HPLC and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The main peptides soluble in 1.1% trifluoroacetic acid and liberated by the two proteinases were identified and have been found to be the same for the two enzymes. They are located in two areas of the -casein sequence (53–93 and the C-terminal part: 129–209) and they include bitter tasting or physiologically active fragments. No narrow specificity was observed for these proteinases. However, glutamine and serine residues are more frequently encountered in position P1 and P1 of the sensitive peptide bond and the close environment (position P2 to P4 and P2 to P4) of the cleaved bond is mainly hydrophobic.     

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.     

Introducing glutathione biosynthetic capability into Lactococcus lactis subsp. cremoris NZ9000 improves the oxidative-stress resistance of the host

This study describes how a metabolic engineering approach can be used to improve bacterial stress resistance. Some Lactococcus lactis strains are capable of taking up glutathione, and the imported glutathione protects this organism against H(2)O(2)-induced oxidative stress. L. lactis subsp. cremoris NZ9000, a model organism of this species that is widely used in the study of metabolic engineering, can neither synthesize nor take up glutathione. The study described here aimed to improve the oxidative-stress resistance of strain NZ9000 by introducing a glutathione biosynthetic capability. We show that the glutathione produced by strain NZ9000 conferred stronger resistance on the host following exposure to H(2)O(2) (150 mM) and a superoxide generator, menadione (30 microM). To explore whether glutathione can complement the existing oxidative-stress defense systems, we constructed a superoxide dismutase deficient mutant of strain NZ9000, designated as NZ4504, which is more sensitive to oxidative stress, and introduced the glutathione biosynthetic capability into this strain. Glutathione produced by strain NZ4504(pNZ3203) significantly shortens the lag phase of the host when grown aerobically, especially in the presence of menadione. In addition, cells of NZ4504(pNZ3203) capable of producing glutathione restored the resistance of the host to H(2)O(2)-induced oxidative stress, back to the wild-type level. We conclude that the resistance of L. lactis subsp. cremoris NZ9000 to oxidative stress can be increased in engineered cells with glutathione producing capability.     

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.     

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.     

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.     

Regulation of the ribonucleotide reductases in Lactococcus lactis subsp. cremoris

Lactococcus lactis has two essential ribonucleotide reductases for DNA biosynthesis and repair which are affected in the presence or absence of oxygen. Expression of glutaredoxin like protein (NrdH), the hydrogen donor for ribonucleotide reductase, was found to be regulated by the FNR like proteins (FlpA and FlpB). Proteomics study demonstrated that expression level of NrdH significantly decreased in the flpA and flpAB deletion mutants. The nrdH gene is located in an nrdHIEF operon and encoding the NrdEF ribonucleotide reductase, which is active under aerobic and anaerobic conditions. Regulation of expression of the nrdHIEF operons was investigated using beta-galactosidase as a reporter gene. The 588 bp fragment containing the nrdH promoter and gene cloned into the pORI vector immediately upstream of a promoterless lacZ gene. Constructed plasmid was transferred into wild type (MG1363), single mutant (flpA orflpB) and double mutant (flpAB). Aerobically, nrdH promoter activity is 15-fold higher than anaerobic expression.     

Identification of four phage resistance plasmids from Lactococcus lactis subsp. cremoris HO2

The bacteriophage-host sensitivity patterns of 16 strains of Lactococcus lactis originally isolated from a mixed strain Cheddar cheese starter culture were determined. Using phages obtained from cheese factory whey, four of the strains were found to be highly phage resistant. One of these isolates, Lactococcus lactis subsp. cremoris HO2, was studied in detail to determine the mechanisms responsible for the phage insensitivity phenotypes. Conjugal transfer of plasmid DNA from strain HO2 allowed a function to be assigned to four of its six plasmids. A 46-kb molecule, designated pCI646, was found to harbor the lactose utilization genes, while this and plasmids of 58 kb (pCI658), 42 kb (pCI642), and 4.5 kb (pCI605) were shown to be responsible for the phage resistance phenotypes observed against the small isometric-headed phage phi712 (936 phage species) and the prolate-headed phage phic2 (c2 species). pCI658 was found to mediate an adsorption-blocking mechanism and was also responsible for the fluffy pellet phenotype of cells containing the molecule. pCI642 and pCI605 were both shown to be required for the operation of a restriction-modification system.     

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.     

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.     

Complete genome sequence of Lactococcus lactis subsp. cremoris A76

We report the complete genome sequence of Lactococcus lactis subsp. cremoris A76, a dairy strain isolated from a cheese production outfit. Genome analysis detected two contiguous islands fitting to the L. lactis subsp. lactis rather than to the L. lactis subsp. cremoris lineage. This indicates the existence of genetic exchange between the diverse subspecies, presumably related to the technological process.     

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.     

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.     

Monoclonal Antibodies to the Cell-Wall-Associated Proteinase of Lactococcus lactis subsp. cremoris Wg2

Twelve monoclonal antibodies directed to the cell-wall-associated proteinase of Lactococcus lactis subsp. cremoris Wg2 were isolated after immunization of BALB/c mice with a partially purified preparation of the proteinase. The monoclonal antibodies reacted with the 126-kilodalton proteinase band in a Western immunoblot. All but one of the monoclonal antibodies reacted with protein bands with a molecular weight below 126,000, possibly degradation products of the proteinase. The monoclonal antibodies could be divided into six groups according to their different reactions with the proteinase degradation products in the Western blot. Different groups of monoclonal antibodies reacted with different components of the L. lactis subsp. cremoris Wg2 proteinase. Crossed immunoelectrophoresis showed that monoclonal antibody groups I, II, and III react with proteinase component A and that groups IV, V, and VI react with proteinase component B. The isolated monoclonal antibodies cross-reacted with the proteinases of other L. lactis subspecies. Monoclonal antibodies of group IV cross-reacted with proteinase component C of other L. lactis subsp. cremoris strains. The molecular weight of the proteinase attached to the cells of L. lactis subsp. cremoris Wg2 was 200,000, which is different from the previously reported values. This could be analyzed by immunodetection of the proteinase on a Western blot. This value corresponds to the molecular weight calculated from the amino acid sequence of the cloned L. lactis subsp. cremoris Wg2 proteinase gene.     

The bacteriophage kh receptor of Lactococcus lactis subsp. cremoris KH is the rhamnose of the extracellular wall polysaccharide

A receptor for bacteriophages of lactic acid bacteria, including Lactococcus lactis subsp. cremoris KH, was found on the cell wall and not on the cell membrane, as determined by a phage-binding assay of sodium dodecyl sulfate- and mutanolysin-treated cell walls. The cell wall carbohydrates of L. lactis subsp. cremoris KH were analyzed by gas chromatography and mass spectrometry and found to contain rhamnose, galactose, glucose and N-acetylglucosamine. Similar analysis of mutants that were reduced in the ability to bind phages kh, 643, c2, ml3, and 1 indicated that galactose was essential for binding all phages. In addition, rhamnose was required for binding phages kh and ml3. Inhibition studies of phage binding by using two different lectins with a specificity for galactose indicated that phage kh may not bind directly to galactose. Rather, galactose may be an essential structural component located in the vicinity of the receptor. Incubation of any of the five phages with rhamnose or of phage kh with purified cell walls inactivated the phages. Inactivation required divalent cations and was irreversible. Inactivation of phages was stereospecific for rhamnose, as neither L-(+)- nor D-(-)-fucose (the stereoisomers of rhamnose) inhibited the phage. Furthermore, phage infection of a culture was completely inhibited by the addition of rhamnose to the medium. Therefore, the receptor for phage kh appears to be a rhamnose component of the extracellular wall polysaccharide.     

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.     

The bacteriophage kh receptor of Lactococcus lactis subsp. cremoris KH is the rhamnose of the extracellular wall polysaccharide.

A receptor for bacteriophages of lactic acid bacteria, including Lactococcus lactis subsp. cremoris KH, was found on the cell wall and not on the cell membrane, as determined by a phage-binding assay of sodium dodecyl sulfate- and mutanolysin-treated cell walls. The cell wall carbohydrates of L. lactis subsp. cremoris KH were analyzed by gas chromatography and mass spectrometry and found to contain rhamnose, galactose, glucose and N-acetylglucosamine. Similar analysis of mutants that were reduced in the ability to bind phages kh, 643, c2, ml3, and 1 indicated that galactose was essential for binding all phages. In addition, rhamnose was required for binding phages kh and ml3. Inhibition studies of phage binding by using two different lectins with a specificity for galactose indicated that phage kh may not bind directly to galactose. Rather, galactose may be an essential structural component located in the vicinity of the receptor. Incubation of any of the five phages with rhamnose or of phage kh with purified cell walls inactivated the phages. Inactivation required divalent cations and was irreversible. Inactivation of phages was stereospecific for rhamnose, as neither L-(+)- nor D-(-)-fucose (the stereoisomers of rhamnose) inhibited the phage. Furthermore, phage infection of a culture was completely inhibited by the addition of rhamnose to the medium. Therefore, the receptor for phage kh appears to be a rhamnose component of the extracellular wall polysaccharide.     

Mechanism of Proteinase Release from Lactococcus lactis subsp. cremoris Wg2

The procedure generally used for the isolation of extracellular, cell-associated proteinases of Lactococcus lactis species is based on the release of the proteinases by repeated incubation and washing of the cells in a Ca²⁺-free buffer. For L. lactis subsp. cremoris Wg2, as many as five incubations for 30 min at 29°C are needed in order to liberate 95% of the proteinase. Proteinase release was not affected by chloramphenicol, which indicates that release is not the result of protein synthesis during the incubations. Ca²⁺ inhibited, while ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) stimulated, proteinase release from the cells. The pH optimum for proteinase release ranged between 6.5 and 7.5, which was higher than the optimum pH of the proteinase measured for casein hydrolysis (i.e., 6.4). Treatment of cells with the serine proteinase inhibitor phenylmethylsulfonyl fluoride prior to the incubations in Ca²⁺-free buffer reduced the release of the proteinase by 70 to 80%. The residual proteinase remained cell associated but could be removed by the addition of active L. lactis subsp. cremoris Wg2 proteinase. This suggests that proteinase release from cells of L. lactis subsp. cremoris Wg2 is the result of autoproteolytic activity. From a comparison of the N-terminal amino acid sequence of the released proteinase with the complete amino acid sequence determined from the nucleotide sequence of the proteinase gene, a protein of 180 kilodaltons would be expected. However, a proteinase with a molecular weight of 165,000 was found, which indicated that further hydrolysis had occurred at the C terminus.