Production of a nisin-like bacteriocin by Lactococcus lactis subsp. lactis A164 isolated from Kimchi

Lactococcus lactis subsp. lactis A164 was isolated from Kimchi (Korean traditional fermented vegetables). The bacteriocin produced by strain A164 was active against closely related lactic acid bacteria and some food-borne pathogens including Staphylococcus aureus, Listeria monocytogenes and Salmonella typhimurium. The antimicrobial spectrum was nearly identical to that of nisin. Bacteriocin activity was not destroyed by exposure to elevated temperatures at low pH values, but the activity was lost at high pH values. This bacteriocin was inactivated by pronase E and alpha, beta-chymotrypsin, but not by trypsin, pepsin, and alpha-amylase. Cultures of L. lactis subsp. lactis A164 maintained at a constant pH of 6.0 exhibited maximum production of the bacteriocin. It was purified to homogeneity by ammonium sulphate precipitation, sequential ion exchange chromatography, and ultrafiltration. Tricine-SDS-PAGE of purified bacteriocin gave the same molecular weight of 3.5 kDa as that of nisin. The gene encoding this bacteriocin was amplified by PCR with nisin gene-specific primers and sequenced. It showed identical sequences to the nisin gene. These results indicate that bacteriocin produced by Lactococcus lactis A164 is a nisin-like bacteriocin.     

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.     

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.     

Characterization of N-deoxyribosyltransferase from Lactococcus lactis subsp. lactis

A nucleoside N-deoxyribosyltransferase-homologous gene was detected by homological search in the genomic DNA of Lactococcus lactis subsp. lactis. The gene yejD is composed of 477 nucleotides encoding 159 amino acids with only 25% identity, which is low in comparison to the amino acid sequences of the N-deoxyribosyltransferases from other lactic acid bacteria, i.e. Lactobacillus leichmannii and Lactobacillus helveticus. The residues responsible for catalytic and substrate-binding sites in known enzymes are conserved at Gln49, Asp73, Asp93 (or Asp95), and Glu101, respectively. The recombinant YejD expressed in Escherichia coli shows a 2-deoxyribosyl transfer activity to and from both bases of purine and pyrimidine, showing that YejD should be categorized as a class II N-deoxyribosyltransferase. Interestingly, the base-exchange activity as well as the heat stability of YejD was enhanced by the presence of monovalent cations such as K(+), NH(4)(+), and Rb(+), indicating that the Lactococcus enzyme is a K(+)-activated Type II enzyme. However, divalent cations including Mg(2+) and Ca(2+) significantly inhibit the activity. Whether or not the yejD gene product actually participates in the nucleoside salvage pathway of Lc. lactis remains unclear, but the lactic acid bacterium possesses the gene coding for the nucleoside N-deoxyribosyltransferase activated by K(+) on its genome.     

Influence of the carbon source on nisin production in Lactococcus lactis subsp. lactis batch fermentations.

Nisin production by Lactococcus lactis subsp. lactis NIZO 22186 was studied in batch fermentation using a complex medium. Nisin production showed primary metabolite kinetics: nisin biosynthesis took place during the active growth phase and completely stopped when cells entered the stationary phase. A stringent correlation could be observed between the expression of the prenisin gene (nisA) and the synthesis of the post-translationally enzymically modified and processed mature nisin peptide. Moreover, it seemed likely that nisin had a growth control function. A physiological link is proposed between sucrose fermentation capacity and nisin production ability. Carbon source regulation appears to be a major control mechanism for nisin production.     

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.     

Characterization of two nisin-producing Lactococcus lactis subsp. lactis strains isolated from a commercial sauerkraut fermentation.

Two Lactococcus lactis subsp. lactis strains, NCK400 and LJH80, isolated from a commercial sauerkraut fermentation were shown to produce nisin. LJH80 was morphologically unstable and gave rise to two stable, nisin-producing (Nip+) derivatives, NCK318-2 and NCK318-3. NCK400 and derivatives of LJH80 exhibited identical morphological and metabolic characteristics, but could be distinguished on the basis of plasmid profiles and genomic hybridization patterns to a DNA probe specific for the iso-ISS1 element, IS946. NCK318-2 and NCK318-3 harbored two and three plasmids, respectively, which hybridized with IS946. Plasmid DNA was not detected in NCK400, and DNA from this strain failed to hybridize with IS946. Despite the absence of detectable plasmid DNA in NCK400, nisin-negative derivatives (NCK402 and NCK403) were isolated after repeated transfer in broth at 37 degrees C. Nisin-negative derivatives concurrently lost the ability to ferment sucrose and became sensitive to nisin. A 4-kbp HindIII fragment containing the structural gene for nisin (spaN), cloned from L. lactis subsp. lactis ATCC 11454, was used to probe genomic DNA of NCK318-2, NCK318-3, NCK400, and NCK402 digested with EcoRI or HindIII. The spaN probe hybridized to an 8.8-kbp EcoRI fragment and a 10-kbp HindIII fragment in the Nip+ sauerkraut isolates, but did not hybridize to the Nip- derivative, NCK402. A different hybridization pattern was observed when the same probe was used against Nip+ L. lactis subsp. lactis ATCC 11454 and ATCC 7962. These phenotypic and genetic data confirmed that unique Nip+ L. lactis subsp. lactis strains were isolated from fermenting sauerkraut.     

Characterization of two nisin-producing Lactococcus lactis subsp. lactis strains isolated from a commercial sauerkraut fermentation.

Two Lactococcus lactis subsp. lactis strains, NCK400 and LJH80, isolated from a commercial sauerkraut fermentation were shown to produce nisin. LJH80 was morphologically unstable and gave rise to two stable, nisin-producing (Nip+) derivatives, NCK318-2 and NCK318-3. NCK400 and derivatives of LJH80 exhibited identical morphological and metabolic characteristics, but could be distinguished on the basis of plasmid profiles and genomic hybridization patterns to a DNA probe specific for the iso-ISS1 element, IS946. NCK318-2 and NCK318-3 harbored two and three plasmids, respectively, which hybridized with IS946. Plasmid DNA was not detected in NCK400, and DNA from this strain failed to hybridize with IS946. Despite the absence of detectable plasmid DNA in NCK400, nisin-negative derivatives (NCK402 and NCK403) were isolated after repeated transfer in broth at 37 degrees C. Nisin-negative derivatives concurrently lost the ability to ferment sucrose and became sensitive to nisin. A 4-kbp HindIII fragment containing the structural gene for nisin (spaN), cloned from L. lactis subsp. lactis ATCC 11454, was used to probe genomic DNA of NCK318-2, NCK318-3, NCK400, and NCK402 digested with EcoRI or HindIII. The spaN probe hybridized to an 8.8-kbp EcoRI fragment and a 10-kbp HindIII fragment in the Nip+ sauerkraut isolates, but did not hybridize to the Nip- derivative, NCK402. A different hybridization pattern was observed when the same probe was used against Nip+ L. lactis subsp. lactis ATCC 11454 and ATCC 7962. These phenotypic and genetic data confirmed that unique Nip+ L. lactis subsp. lactis strains were isolated from fermenting sauerkraut.     

Isolation and characterization of nisin-producing Lactococcus lactis subsp. lactis from bean-sprouts

Bacterial isolates from bean-sprouts were screened for anti- Listeria monocytogenes bacteriocins using a well diffusion method. Thirty-four of 72 isolates inhibited the growth of L.monocytogenes Scott A. One, HPB 1688, which had the biggest inhibition zone against L.monocytogenes Scott A, was selected for subsequent analysis. Both ribotyping and DNAsequencing of 16S ribosomal RNA gene demonstrated that the isolate was Lactococcus lactis subsp. lactis . Polymerase chain reaction and nucleotide sequencing revealed that thegenomic DNA of the bean-sprout isolates contained a nisin Z structural gene. In MRS broth,bean-sprout isolate HPB 1688 survived at 3–4·5°C for at least 20 d, grew at 4°Cand produced anti-listerial compoundsat 5°C. When co-cultured with L. monocytogenes in MRS broth, the isolate inhibited thegrowth of L. monocytogenes at 4°C after 14d and at 10°C after 2 d. When co-inoculatedwith 10²cells g⁻¹ of L.monocytogenes on fresh-cut ready-to-eat Caesar salad, L. lactis subsp. lactis (10⁸cells g⁻¹) was able to reduce the number of L. monocytogenes by 1–1·4 logs after storage for 10 d at 7° and 10°C. A bacteriocin-producing Enterococcusfaecium was also able to reduce the numbers of L. monocytogenes onCaesar salad, butdid not act synergistically when co-inoculated with L. lactis subsp. lactis .     

Secretion of TEM beta-lactamase with signal sequences isolated from the chromosome of Lactococcus lactis subsp. lactis.

With TEM beta-lactamase as a reporter gene, a set of expression-secretion-promoting fragments were isolated from the chromosome of Lactococcus lactis subsp. lactis. The fact that only translocated beta-lactamase renders cells resistant to ampicillin allowed direct ampicillin selection with an Escherichia coli vector (pKTH33). The clones showing the greatest ampicillin resistance were subcloned onto a replicon capable of replication in lactic acid bacteria (pVS2), and the nucleotide sequences of the relevant fragments were determined. The structure of the secretion-promoting fragments in general resembled that of gram-positive true signal sequences, with a strongly positively charged N terminus, a long hydrophobic core, and a putative signal peptidase recognition site. The promoterlike sequences preceding the signal sequences matched well with those of previously published lactococcal promoters. In addition to E. coli, the functioning of these expression-secretion cassettes was studied in three gram-positive hosts: Bacillus subtilis, L. lactis, and Lactobacillus plantarum. Efficient expression and secretion of TEM beta-lactamase into the culture medium of each gram-positive host was obtained. Furthermore, when a strain of L. lactis subsp. lactis showing increased sensitivity to lysozyme was compared with a standard laboratory strain, threefold-higher secreted enzyme activities were detected.     

Secretion of TEM beta-lactamase with signal sequences isolated from the chromosome of Lactococcus lactis subsp. lactis

With TEM beta-lactamase as a reporter gene, a set of expression-secretion-promoting fragments were isolated from the chromosome of Lactococcus lactis subsp. lactis. The fact that only translocated beta-lactamase renders cells resistant to ampicillin allowed direct ampicillin selection with an Escherichia coli vector (pKTH33). The clones showing the greatest ampicillin resistance were subcloned onto a replicon capable of replication in lactic acid bacteria (pVS2), and the nucleotide sequences of the relevant fragments were determined. The structure of the secretion-promoting fragments in general resembled that of gram-positive true signal sequences, with a strongly positively charged N terminus, a long hydrophobic core, and a putative signal peptidase recognition site. The promoterlike sequences preceding the signal sequences matched well with those of previously published lactococcal promoters. In addition to E. coli, the functioning of these expression-secretion cassettes was studied in three gram-positive hosts: Bacillus subtilis, L. lactis, and Lactobacillus plantarum. Efficient expression and secretion of TEM beta-lactamase into the culture medium of each gram-positive host was obtained. Furthermore, when a strain of L. lactis subsp. lactis showing increased sensitivity to lysozyme was compared with a standard laboratory strain, threefold-higher secreted enzyme activities were detected.     

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.     

Isolation and characterization of Lactococcus lactis subsp. lactis promoters.

DNA fragments with promoter activity were isolated from the chromosome of Lactococcus lactis subsp. lactis. For the isolation, a promoter probe vector based on the cat gene was constructed, which allowed direct selection with chloramphenicol in Bacillus subtilis and L. lactis. Four of the putative promoters (P1, P2, P10, and P21) were analyzed further by sequencing, mapping of the 5' end of the mRNA, Northern (RNA blot) hybridization, and chloramphenicol acetyltransferase activity measurements. From these fragments, -10 and -35 regions resembling the consensus Escherichia coli sigma 70 and B. subtilis sigma 43 promoters were identified. Another set of promoters, together with a signal sequence, were also isolated from the same organism. These fragments promoted secretion of TEM beta-lactamase from L. lactis. When the two sets of promoters were compared, it was found that the ones isolated with the cat vector were more efficient (produced more mRNA). By changing the promoter part of the promoter-signal sequence fragment giving the best TEM beta-lactamase secretion into a more efficient one (P2), a 10-fold increase in enzyme production was obtained.