Histidine biosynthesis genes in Lactococcus lactis subsp. lactis.

The genes of Lactococcus lactis subsp. lactis involved in histidine biosynthesis were cloned and characterized by complementation of Escherichia coli and Bacillus subtilis mutants and DNA sequencing. Complementation of E. coli hisA, hisB, hisC, hisD, hisF, hisG, and hisIE genes and the B. subtilis hisH gene (the E. coli hisC equivalent) allowed localization of the corresponding lactococcal genes. Nucleotide sequence analysis of the 11.5-kb lactococcal region revealed 14 open reading frames (ORFs), 12 of which might form an operon. The putative operon includes eight ORFs which encode proteins homologous to enzymes involved in histidine biosynthesis. The operon also contains (i) an ORF encoding a protein homologous to the histidyl-tRNA synthetases but lacking a motif implicated in synthetase activity, which suggests that it has a role different from tRNA aminoacylation, and (ii) an ORF encoding a protein that is homologous to the 3'-aminoglycoside phosphotransferases but does not confer antibiotic resistance. The remaining ORFs specify products which have no homology with proteins in the EMBL and GenBank data bases.     

Effect of citrate on growth of Lactococcus lactis subsp. lactis in milk

The effect of citrate on the growth of Lactococcus lactis subsp. lactis var. diacetylactis in milk has been investigated. Five strains of Lactococcus lactis subsp. lactis var. diacetylactis were compared to their citrate-negative variants, which lack the plasmid coding for citrate permease. In most cases, acidification kinetics and the final bacterial concentration of pure cultures of parental and variant strains did not differ significantly. Co-cultures of parental and variant strains, however, systematically tended towards the predominance of parental strains. Citrate metabolism is responsible for this change, since the predominance of citrate-positive strains was not observed in the absence of citrate. Continuous culture in milk enabled the difference in growth rates between the parental strain Lactococcus lactis subsp. lactis var. diacetylactis CDI1 and its citrate-negative variant to be quantified by following changes in the populations of the two co-cultured strains. At 26 °C, the growth rate of the parental strain was 7% higher than that of its citrate-negative variant. These results show that citrate metabolism slightly stimulates the growth of lactococci in milk. Received: 18 February 1997 / Received revision: 2 May 1997 / Accepted: 4 May 1997     

Insertion and amplification of foreign genes in the Lactococcus lactis subsp. lactis chromosome

The plasmid pE194 is unable to replicate in Lactococcus lactis subsp. lactis (formerly Streptococcus lactis). When linked to resident bacteriophage sequences, pE194 was able to integrate into the L. lactis subsp. lactis chromosome either by Campbell-like recombination or by double crossing over with deletion. Integration occurred into the DNA of the prophage and prevented its multiplication. When a selective pressure was applied to an integrant in which pE194 was flanked by two direct repeats of prophage fragment, amplification of pE194 and the prophage fragment was observed. The pE194 copy number was assessed at six to nine, and amplification was stable upon growth under nonselective conditions.     

Insertion and amplification of foreign genes in the Lactococcus lactis subsp. lactis chromosome.

The plasmid pE194 is unable to replicate in Lactococcus lactis subsp. lactis (formerly Streptococcus lactis). When linked to resident bacteriophage sequences, pE194 was able to integrate into the L. lactis subsp. lactis chromosome either by Campbell-like recombination or by double crossing over with deletion. Integration occurred into the DNA of the prophage and prevented its multiplication. When a selective pressure was applied to an integrant in which pE194 was flanked by two direct repeats of prophage fragment, amplification of pE194 and the prophage fragment was observed. The pE194 copy number was assessed at six to nine, and amplification was stable upon growth under nonselective conditions.     

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.     

Isolation and identification of new nisin-producing Lactococcus lactis subsp. lactis from milk

A method for isolating active nisin-producing strains of mesophilic lactococci was developed. Overall, 55 strains of mesophilic lactic acid bacteria were isolated from fresh cow’s milk obtained from milk farms in various regions throughout Russia; of them, 36 displayed nisin-synthesizing activity. The three most active strains were studied according to morphological, cultural, physiological, and biochemical characteristics and identified as Lactococcus lactis subsp. lactis. The species attribution of the strains studied was confirmed by the similarity of the nucleotide sequences of the 16S rRNA gene. The nucleotide sequences of the 16S rRNA genes were deposited with the GenBank under accession numbers DQ255951–DQ255954. The distinctions between these strains in physiological and biochemical characteristics and the ranges of their bactericide action on the microorganisms capable of developing in agricultural materials and food products were determined. The isolated strains displayed considerably wider ranges of action, which differed from the nisin-producing strain MGU and the commercial nisin preparation (Nisaplin), used as a biological preserving agent.     

Isolation and identification of new nisin-producing Lactococcus lactis subsp. lactis from milk

A method for isolating active nisin-producing strains of mesophilic lactococci was developed. Overall, 55 strains of mesophilic lactic acid bacteria were isolated from fresh cow's milk obtained from milk farms in various regions throughout Russia; of them, 36 displayed nisin-synthesizing activity. The three most active strains were studied according to morphological, cultural, physiological, and biochemical characteristics and identified as Lactococcus lactis subsp. lactis. The species attribution of the strains studied was confirmed by the similarity of the nucleotide sequences of the 16S rRNA gene. The nucleotide sequences of the 16S rRNA genes were deposited with the GenBank under accession numbers DQ255951-DQ255954. The distinctions between these strains in physiological and biochemical characteristics and the ranges of their bactericide action on the microorganisms capable of developing in agricultural materials and food products were determined. The isolated strains displayed considerably wider ranges of action, which differed from the nisin-producing strain MGU and the commercial nisin preparation (Nisaplin), used as a biological preserving agent.     

Improved electroporation efficiency of intact Lactococcus lactis subsp. lactis cells grown in defined media

The impact of growth conditions on electroporation of Lactococcus lactis subsp. lactis LM0230 (previously designated Streptococcus lactis LM0230) was evaluated. Cells grown in M17 broth supplemented with 0.5% glucose (M17-Glu) and two chemically defined synthetic media, FMC and RPMI 1640, all supplemented with 0.24% DL-threonine or 0.5% glycine, were harvested, washed with double-distilled water, diluted, and porated in the presence of 1 microgram of pGB301 DNA with a Transfector 100 (BTX, Inc., San Diego, Calif.) or a Gene Pulser (Bio-Rad Laboratories, Richmond, Calif.). Transformants were recovered at consistently higher efficiencies for cells grown in FMC or RPMI 1640 (10(3) to 10(4) transformants per micrograms of DNA) than for cells grown in M17-Glu (10(1) to 10(2) transformants per micrograms of DNA). Other parameters influencing electroporation of L. lactis cells grown in chemically defined media were growth phase and final concentration of cells, concentration of plasmid DNA, voltage achieved during poration, and expression conditions. A high degree of variability in transformation efficiencies was evident for replicate samples of cells pulsed with either electroporation machine. A trend toward decreased variability was observed for duplicate samples of cells prepared on the same day. In addition, storage studies done with a large batch of cells prepared on the same day indicated that freezing dry cell pellets at -60 degrees C had no deleterious effect on transformation efficiencies over a 30-day period when a new 0.2-cm cuvette was used for porating each sample.     

Improved electroporation efficiency of intact Lactococcus lactis subsp. lactis cells grown in defined media.

The impact of growth conditions on electroporation of Lactococcus lactis subsp. lactis LM0230 (previously designated Streptococcus lactis LM0230) was evaluated. Cells grown in M17 broth supplemented with 0.5% glucose (M17-Glu) and two chemically defined synthetic media, FMC and RPMI 1640, all supplemented with 0.24% DL-threonine or 0.5% glycine, were harvested, washed with double-distilled water, diluted, and porated in the presence of 1 microgram of pGB301 DNA with a Transfector 100 (BTX, Inc., San Diego, Calif.) or a Gene Pulser (Bio-Rad Laboratories, Richmond, Calif.). Transformants were recovered at consistently higher efficiencies for cells grown in FMC or RPMI 1640 (10(3) to 10(4) transformants per micrograms of DNA) than for cells grown in M17-Glu (10(1) to 10(2) transformants per micrograms of DNA). Other parameters influencing electroporation of L. lactis cells grown in chemically defined media were growth phase and final concentration of cells, concentration of plasmid DNA, voltage achieved during poration, and expression conditions. A high degree of variability in transformation efficiencies was evident for replicate samples of cells pulsed with either electroporation machine. A trend toward decreased variability was observed for duplicate samples of cells prepared on the same day. In addition, storage studies done with a large batch of cells prepared on the same day indicated that freezing dry cell pellets at -60 degrees C had no deleterious effect on transformation efficiencies over a 30-day period when a new 0.2-cm cuvette was used for porating each sample.     

Nutritional factors affecting nisin production by Lactococcus lactis subsp. actis NIZO 22186 in a synthetic medium

L. DE VUYST. 1995 A minimal synthetic medium (SM8) for nisin-producing Lactococcus lactis subsp. lactis strains has been designed; it consists of eight growth-stimulating amino acids (glutamic acid, methionine, valine, leucine, threonine, arginine, isoleucine and histidine), five vitamins (biotin, calcium pantothenate, nicotinic acid, pyridoxine and riboflavin) and the mineral salts dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, magnesium sulphate and trisodium citrate. Nisin biosynthesis is strongly dependent on the presence of a sulphur source, either an inorganic salt (magnesium sulphate or sodium thiosulphate) or the amino acids methionine, cysteine or cystathionine. The amino acids serine, threonine and cysteine highly stimulate nisin production without affecting the final cell yield, indicating their precursor role during nisin biosynthesis.     

sacA and nisA genes are not always linked in Lactococcus lactis subsp. lactis strains

Sixty-seven lactococcal strains arising from dairy habitat were screened for the presence of the sucrose 6-phosphate hydrolase gene by polymerase chain reaction. Of the strains tested, 35.8% were able to ferment sucrose as well as to harbour the sucrose-6-phosphate hydrolase gene, even though they were unable to produce nisin as well as to show the nisin structural gene. After pulsed-field gel electrophoresis and hybridisation all Suc⁺Nis⁻ strains exhibited physical linkage between sacA gene and the left end of lactococcal transposons (Tn5276 or Tn5301) without linkage to nisin genes. However, we were unable to transfer the sacA gene as well as to detect Suc⁻ derivatives from Suc⁺Nis⁻ strains after conjugation and curing experiments.     

Development of a low-cost medium for production of nisin from Lactococcus lactis subsp. lactis

The goal of this project was to develop a lower-cost medium for nisin production, so this bacteriocin could be used in a broader range of industrial fermentation processes. The objectives included: (1) evaluating methods for controlling the inhibitory effect of lactic acid produced during fermentation, and (2) comparing two inexpensive complex media for nisin production. Lactococcus lactis subsp. lactis was cultured in shake flasks on Laurel–Tryptose broth to evaluate a range of buffers for pH control. NaHCO₃ proved to be an effective buffer for increasing nisin production. Subsequent trials then evaluated condensed corn soluble (CCS, a fuel ethanol production byproduct) and cheese whey as inexpensive growth media. CCS was shown to be an efficient, low-cost medium for high nisin titers and yields. These modifications reduced the medium costs for nisin production from $600/kg nisin (based on Laurel–Tryptose broth medium) to $35–40/kg nisin for the corn solubles medium.     

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.