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PubMed2006

Expression of Helicobacter pylori cag12 gene in Lactococcus lactis MG1363 and its oral administration to induce systemic anti-Cag12 immune response in mice.

To develop an oral vaccine against Helicobacter pylori infection, we have expressed the H. pylori cag12 (HP0532) gene, encoding the outer membrane protein Cag12 (31 kDa), in a live delivery vehicle Lactococcus lactis. The cag12 gene was amplified by polymerase chain reaction (PCR) using the genomic DNA of H. pylori K51 isolated from Korean patients. DNA sequence analysis revealed that the cag12 gene of H. pylori K51 has 98.1 and 97.4% identity with individual cag12 genes of the H. pylori 26695 and J99, respectively. The GST-Cag12 fusion protein, produced using the Escherichia coli expression system, was used to raise a rat polyclonal anti-Cag12 antibody. The PCR-amplified cag12 gene of H. pylori K51 was cloned in the E. coli-L. lactis shuttle vector (pMG36e) and transformed into L. lactis. Western blot analysis demonstrated that the Cag12 protein was expressed in the L. lactis transformant, with a maximum level at the log phase without extracellular secretion. The oral administration of the transformant into mice resulted in the generation of the anti-Cag12 antibody in serum in two out of five cases. These results suggest that the recombinant L. lactis, which expresses Cag12, may be applicable as an oral vaccine to induce protective immunity against H. pylori.

Administration, Oral↗

PubMed2005

Modelling the production of nisin by Lactococcus lactis in fed-batch culture.

Nisin production in batch culture and fed-batch cultures (sucrose feeding rates were 6, 7, 8, and 10 g l(-1) h(-1), respectively) by Lactococcus lactis subsp. lactis ATCC 11454 was investigated. Nisin production showed primary metabolite kinetics, and could be improved apparently by altering the feeding strategy. The nisin titer reached its maximum, 4,185 IU ml(-1), by constant addition of sucrose at a feeding rate of 7 g l(-1) h(-1); an increase in 58% over that of the batch culture (2,658 IU ml(-1)). Nisin biosynthesis was affected strongly by the residual sucrose concentration during the feeding. Finally, a mathematical model was developed to simulate the cell growth, sucrose consumption, lactic acid production and nisin production. The model was able to describe the fermentation process in all cases.

Culture Media↗

PubMed2006

Enhancement of recombinant streptokinase production in Lactococcus lactis by suppression of acid tolerance response.

Lactococcus lactis is a potential host for production of recombinant proteins, especially of therapeutic importance. However, in glucose-grown cultures, lowering of pH due to accumulation of lactic acid and the concomitant induction of acid tolerance response (ATR) may affect the recombinant protein produced. In this work, we have analyzed the effect of culture pH and the associated ATR on production of recombinant streptokinase. Streptokinase gene was cloned and expressed as a secretory protein in L. lactis under the control of P170 promoter. It was found to undergo degradation to form inactive products leading to low productivity. The extent of degradation and productivity of streptokinase was greatly influenced by the development of ATR, which was dependent on the pH of the culture and initial phosphate concentration of the medium. It was found that high pH and high initial phosphate concentration leads to suppression of ATR and this results in at least 2.5-fold increase in streptokinase productivity and significant decrease in degradation of streptokinase.

Adaptation, Physiological↗

PubMed2006

Heterologous protein secretion in Lactococcus lactis is enhanced by the Bacillus subtilis chaperone-like protein PrsA.

The Bacillus subtilis lipoprotein PrsA enhances the yield of several homologous and heterologous exported proteins in B. subtilis by being involved in the posttranslocational stage of the secretion process. In this work, we have studied the effect of B. subtilis PrsA on the secretion of Bacillus amyloliquefaciens alpha-amylase (AmyQ), a target protein for PrsA, and Bacillus licheniformis penicillinase (PenP) a nontarget protein for PrsA, in Lactococcus lactis. Two compatible plasmids were constructed and introduced into L. lactis strain NZ9000: one high copy plasmid, expressing the AmyQ gene (amyQ) or the PenP gene (penP), and one low copy plasmid, expressing the PrsA encoding gene (prsA). When amyQ and prsA were simultaneously expressed under the nisin-inducible promoter P( nisA ), Western blotting experiments revealed a 15- to 20-fold increase in the total yield of AmyQ and a sixfold increase in secreted AmyQ activity, compared to a control strain lacking prsA. When expressed under the same induction conditions, PrsA had no effect on the secretion or total yield of PenP. These results show that the secretion yield of some heterologous proteins can be significantly increased in L. lactis when coproduced with the B. subtilis PrsA protein.

Bacillus subtilis↗

PubMed2006

Expression of citrate permease gene of plasmid pCM1 isolated from Lactococcus lactis subsp. lactis biovar diacetylactis NIAI N-7 in Lactobacillus casei L-49-4.

Recombinant vector pJLECit (8,232 bp) was constructed using citrate permease gene contained in the 3,919-bp fragment of plasmid pCM1 (8,280 bp) isolated from Lactococcus lactis subsp. lactis biovar diacetylactis NIAI N-7, repA and ori from pLU1, and pMB1 ori and the erythromycin resistance gene from pJIR418. Lactobacillus casei L-49-4 (plasmid-free mutant of strain L-49) harboring the constructed pJLECit converted citrate into diacetyl/acetoin. Citrate uptake rate of resting cells was the highest at pH 5.5 and 10 mM citrate concentration. Diacetyl formation activity by the cell-free extracts of Lb. casei L-49-4 (pJLECit) grown in de Man-Rogosa-Sharpe (MRS) broth was higher than that of cells grown in MRS broth without citrate. On the other hand, diacetyl reductase activity of cells grown in MRS broth was lower than that of cells grown in MRS broth without citrate.

Acetoin↗

PubMed2006

Transfer of nisin gene cluster from Lactococcus lactis ATCC 11454 into the chromosome of Bacillus subtilis 168.

Nisin is an antimicrobial peptide produced by certain strains of Lactococcus lactis. It is a gene-encoded peptide that contains unusual amino acid residues. These novel residues are introduced by posttranslational modification machinery and confer unique chemical and physical properties that are not attainable by regular amino acid residues. To study the modification mechanisms and to create structural analogs with superior properties, it would be advantageous to insert the nisin genes into a bacterial strain that is amenable to genetic manipulation. In this study, we report the cloning and integration of the complete and intact nisin gene cluster into the Bacillus subtilis 168 chromosome. Furthermore, we demonstrate that the nisin genes are transcriptionally active. These results should greatly facilitate the studies of the genes and proteins involved in nisin expression, as well as provide a standard system for the manipulation and expression of genes involved in other members of the lantibiotic family of antimicrobial peptides.

Bacillus subtilis↗

PubMed2007

Secretory expression of a heterologous nattokinase in Lactococcus lactis.

Nattokinase has been reported as an oral health product for the prevention of atherosclerosis. We developed a novel strategy to express a nattokinase from Bacillus subtilis in a live delivery vehicle, Lactococcus lactis. Promoter P( nisZ) and signal peptide SP(Usp) were used for inducible and secretory expression of nattokinase in L. lactis. Western blotting analysis demonstrated that nattokinase was successfully expressed, and about 94% of the enzyme was secreted to the culture. The recombinant nattokinase showed potent fibrinolytic activity, equivalent to 41.7 urokinase units per milliliter culture. Expression and delivery of such a fibrinolytic enzyme in the food-grade vehicle L. lactis would facilitate the widespread application of nattokinase in the control and prevention of thrombosis diseases.

Bacillus subtilis↗

PubMed1996

Lantibiotic nisin Z fermentative production by Lactococcus lactis IO-1: relationship between production of the lantibiotic and lactate and cell growth.

The influence of several parameters on the fermentative production of nisin Z by Lactococcus lactis IO-1 was studied. Considerable attention has been focused on the relationship between the primary metabolite production of bacteriocin and lactate and cell growth, which has so far not been clarified in detail. Production of nisin Z was optimal at 30 degrees C and in the pH range 5.0-5.5. The addition of Ca2+ to the medium showed a stimulating effect on the production of nisin Z. A maximum activity of 3150 IU/ml was obtained during pH-controlled batch fermentation in the medium supplemented with 0.1 M CaCl2. It was about three times higher than that obtained under the optimal conditions for cell growth and lactic acid production.

Anti-Bacterial Agents↗

PubMed1997

Heterologous gene expression of bovine plasmin in Lactococcus lactis.

Heterologous production of bovine plasmin was studied in the industrially relevant bacterium Lactococcus lactis. Two sets of lactococcal gene expression signals were coupled to the region of the plasmin gene coding for the serine protease domain. When the promoter region of the prtP gene was used, plasmin was detected mainly intracellularly in strain BPL25 by Western blot hybridization. The intracellular presence of plasmin led to physiological stress. Expression of the plasmin gene driven by the promoter and complete signal sequence of the lactococcal usp45 gene resulted in efficient plasmin secretion in strain BPL420. Cell lysis was observed in strains producing plasmin fragments including the catalytic domain, but not in control strains, which only produced a non-catalytic region of plasmin. The plasmin produced was shown to be biologically active.

Animals↗

PubMed1997

The effect of nisin concentration and nutrient depletion on nisin production of Lactococcus lactis.

The kinetics of nisin production was studied in batch cultures using a construct of Lactococcus lactis subsp. lactis C2SmPrt-, containing a transposon (TnNip) that encodes nisin production. The introduction of TnNip into C2SmPrt- significantly lowered the specific growth rate and the maximum A620 reached was reduced from 15.2 to 11.0. The effect of nisin concentration and nutrient depletion on nisin production of the construct, C2SmPrt-(TnNip), was examined. Nisin production was found to be inhibited by high concentrations of nisin, when grown in excess nutrient, even though growth of the culture continued because nutrient limitation was not operating. However, in low nutrient concentrations nisin production was limited by nutrient depletion. The specific growth rate of C2SmPrt-(TnNip) was altered, by using different nutrient concentrations and different sugars, in order to examine the relationship between nisin production and growth. Nisin production was shown to be growth-associated for most of growth, but near the end of growth, when the specific growth rate was 0.05 h-1 or less, the production ceased.

DNA Transposable Elements↗

PubMed1998

Construction of a food-grade multiple-copy integration system for Lactococcus lactis.

A food-grade vector system was developed that allows stable integration of multiple plasmid copies in the chromosome of Lactococcus lactis. The vector consists of the plus origin of replication (Ori+) of the lactococcal plasmid pWV01, the sucrose genes of the lactic acid bacterium Pediococcus pentosaceus PPE1.0 as selectable marker, a multiple-cloning site, and a lactococcal DNA fragment of a well-characterized chromosomal region. The system includes two L. lactis strains, LL108 and LL302, which produce the pWV01 RepA protein essential for replication of the Ori+ vectors. These helper strains allow the construction and isolation of the replicating form of the integration plasmids from a homologous background. Single-crossover integration of the plasmids in L. lactis MG1363 resulted in amplifications to a level of approximately 20 copies/chromosome after selection of the transformants on medium containing sucrose as the only fermentable sugar. The amplifications were stable under selective growth conditions. In glucose-containing medium a limited loss of integrated plasmid copies was detected at a rate of (7.5-15) x 10(-2) copies per generation. One strain, MG124, was isolated that had retained 11 integrated copies after a period of 120 generations of non-selective growth. These results show that the single-cross-over integration system described here represents a simple procedure for the engineering of stable food-grade strains carrying multiple copies of a gene of interest.

Blotting, Southern↗

PubMed1999

Nucleotide sequence and thermostability of pND324, a 3.6-kb plasmid from Lactococcus lactis.

A 3.6-kb plasmid, designated pND324, was isolated from Lactococcus lactis subsp. lactis LL57-1. Sequence analysis revealed the presence of three open reading frames, rep324, orfX1 and orfX2, which are flanked by two non-coding regions, ori324 and cisE. The minimal replication region of pND324 consists of ori324 and rep324, which is closely related to the lactococcal theta-type replicons of the pWV02/pCI305 family. pND324 was stable at both 30 degrees C and 37 degrees C, whereas derivatives that lack cisE were highly unstable at 37 degrees C, indicating that cisE is essential for thermostability. Sequences that are similar to orfX1 are commonly present in the lactococcal theta-type plasmids. The orfX2 product is homologous to TrfA, a 43-kDa protein of the E. coli theta-type plasmid RK2 required for replication and maintenance. Plasmid deletion and stability analyses showed that orfX2 is involved in the thermostability of pND324. Based on the minimal replication region of pND324, an integrative cloning vector, designated pND421, was constructed. In L. lactis LM0230, cells that carried pND421 integrated into its host chromosomal DNA could be recovered readily following incubation at 37 degrees C for 40 generations. The integrated plasmid was totally stable for at least 100 generations without selection at 30 degrees C.

Base Sequence↗

PubMed2001

Redirection of pyruvate catabolism in Lactococcus lactis by selection of mutants with additional growth requirements.

Based on requirements for acetate or lipoic acid for aerobic (but not anaerobic) growth, Lactococcus lactis subsp. lactis mutants with impaired pyruvate catabolism were isolated following classical mutagenesis. Strains with defects in one or two of the enzymes, pyruvate formate-lyase (PFL), lactate dehydrogenase (LDH) and the pyruvate dehydrogenase complex (PDHC) were obtained. Growth and product formation of these strains were characterized. A PFL-defective strain (requiring acetate for anaerobic growth) displayed a two-fold increase in specific lactate production compared with the corresponding wild-type strain when grown anaerobically. LDH defective strains directed 91-96% of the pyruvate towards alpha-acetolactate, acetoin and diacetyl production when grown aerobically in the presence of acetate and absence of lipoic acid (a similar characteristic was observed in an LDH and PDHC defective strain in the presence of both acetate and lipoic acid) and more than 65% towards formate, acetate and ethanol production under anaerobic conditions. Another strain with defective PFL and LDH was strictly aerobic. However, a variant with strongly enhanced diacetyl reductase activities (NADH/NAD+ dependent diacetyl reductase, acetoin reductase and butanediol dehydrogenase activities) was selected from this strain under anaerobic conditions by supplementing the medium with acetoin. This strain is strictly aerobic, unless supplied with acetoin.

Acetyltransferases↗

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↗

PubMed2003

Identification and characteristics of nisin Z-producing Lactococcus lactis subsp. lactis isolated from Kimchi.

We isolated bacteriocin-producing Lactococcus lactis subsp. lactis from Kimchi. The bacteriocin inhibited strains of Clostridium perfringens, C. difficile, Listeria monocytogenes, vancomycin-resistant Enterococcus, and one out of four methicillin-resistant Staphylococcus aureus strains, as well as some closely related lactic acid bacteria. In tricine-SDS-PAGE, the bacteriocin migrated with an apparent molecular weight of about 4 kDa to the same location as nisin A and crude nisin Z. The gene encoding this bacteriocin was found to be identical to that of nisin Z with direct PCR sequence methods. The inhibitory activity was stable against heat and pH, but it was lost at 100 degrees C for 1 h and at 121 degrees C for 15 min. The bacteriocin was inactivated by proteolytic enzymes, but was not affected by lysozyme, lipase, catalase, or beta-glucosidase. There were some differences in characteristics from those of nisins described previously.

Bacteria↗

PubMed2003

Growth kinetics of Lactococcus lactis ssp diacetylactis harboring different plasmid content.

The effect of plasmid content on growth of Lactococcus lactis ssp. diacetylactis harboring different plasmids and on plasmid stability was studied. Strain DRC-2C is a plasmid Lac(+)- and Prt(+)-free strain. Strain DRC-2 utilizes lactose as carbohydrate and has proteinase activity. The plasmid-free strain DRC-2C exhibited none of these features. Plasmid-encoded properties were clearly identified. Results showed that plasmid content decreased bacterial growth in terms of the specific growth rate determined. Slightly lower specific growth rate and lactic acid production were observed in the strain of higher plasmid content owing to the plasmid presence, causing metabolic burden to the host cell. The plasmid profile results showed that the number of bands in the two strains before and after fermentation were the same. This indicated that the plasmids were stably maintained and unchanged during the fermentation.

Culture Media↗

PubMed2005

Functional expression of bacterial Zymobacter palmae pyruvate decarboxylase gene in Lactococcus lactis.

A pyruvate decarboxylase (PDC) gene from bacterial Zymobacter palmae (Zymopdc) was cloned, characterized, and introduced into Lactococcus lactis via a shuttle vector pAK80 as part of a research strategy to develop an efficient ethanol-producing lactic acid bacteria (LAB). The expression levels of Zymopdc gene in the host, as measured by a colorimetric assay based on PDC catalyzed formation of (R)-phenylacetylcarbinol ((R)-PAC), appeared to be dependent on the strength of corresponding Gram-positive promoters. A constitutive, highly expressed promoter conferred the greatest PDC activity, and an acid-inducible promoter demonstrated acid-inducible expression. The metabolic production of ethanol and other products was examined in flask fermentations. More than eightfold increases in acetaldehyde concentrations were detected in two recombinant strains. However, no detectable differences for ethanol fermentation in these engineered strains were observed compared with that of the strain carrying lacZ reporter.

Base Sequence↗

PubMed2005

Production and characterization of nisin-like peptide produced by a strain of Lactococcus lactis isolated from fermented milk.

An isolate of Lactococcus lactis from fermented milk was found to produce a bacteriocin peptide. The isolate could grow in a medium with an initial pH of 11.0, in which it produced the bacteriocin extracellularly at the highest level. The level of the bacteriocin in the medium increased in parallel to the bacterial growth and reached its peak during the late exponential phase; thereafter it plateaued. The bacteriocin had a broad antibacterial spectrum similar to that of nisin and inhibited several related species of lactic acid bacteria and other gram-positive bacteria. The inhibitory activity of the bacteriocin was found to be stable over a wide range of pH and temperature. The molecular weight of the peptide was judged to be 2.5 kDa by SDS-polyacrylamide gel electrophoresis.

Anti-Bacterial Agents↗