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PubMed2001

pH-controlled cell release and biomass distribution of alginate-immobilized Lactococcus lactis subsp. lactis.

AIMS: To investigate the growth and release of Lactococcus lactis subsp. lactis in gel beads and to affect rates of cell release by changing the growth conditions. METHODS AND RESULTS: The rate of release and the distribution of immobilized L. lactis subsp. lactis in alginate beads were studied in continuous fermentations for 48 h. A change in operating pH from 6.5 to 9.25 initially reduced the ratio of the rates of cell release to lactate production by almost a factor of 105. Compared with fermentations at pH 6.5, growth at pH 9.25 also increased the final internal bead biomass concentration by a factor of 5 and increased the final rate of lactate production by 25%. After 48 h, the ratio of the rates of cell release to lactate production was still 10 times lower than in fermentations at pH 6.5. CONCLUSIONS: A change in the operating pH from 6.5 to 9.25 reduced rates of cell release throughout 48 h of fermentation and increased the final rates of lactate production and internal bead biomass concentration. SIGNIFICANCE AND IMPACT OF THE STUDY: These data illustrate that diffusional limitations and corresponding pH gradients can be exploited in affecting the distribution of immobilized growing cells and their concomitant release.

Alginates↗

PubMed1998

Effect of cold shock on protein synthesis and on cryotolerance of cells frozen for long periods in Lactococcus lactis.

Aspects of the cold-shock response in Lactococcus lactis subsp. lactis LL41-1 were investigated. First, it was determined whether new proteins were synthesized in response to cold shock. Cell-free extracts were prepared from a cold-shocked (exposed to 10 degreesC for 5 h) culture (cfe-cs) and from a non-cold-shocked (held at 30 degreesC continuously) culture (cfe-non), and were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis. A protein of approximately 6.3 kDa was present in the cfe-cs and appeared not to be present in the cfe-non. No other changes were evident. Second, the effect of cold shock on cryotolerance of cells that have been frozen at -20 degreesC for up to 1 year was examined. Without the cold-shock treatment prior to freezing the cell viability following freezing for 1 day was 34%, 14 days 32%, 182 days 7%, and 364 days 0.2%. However, with the cold shock treatment it was 83%, 82%, 12%, and 0.8%, respectively. It appears that cold shock significantly improves cryotolerance of the cells for short periods of freezing, but the protective effect was less marked following longer storage periods.

Bacterial Proteins↗

PubMed1990

High efficiency electroporation of Lactococcus lactis subsp. lactis LM0230 with plasmid pGB301.

Electroporation-mediated transformation of Lactococcus lactis with plasmid pGB301, a 9.8 kilobase pair vector (Behnke et al. 1981), has been reported by McIntyre & Harlander (1989a). Improved transformation efficiencies of 10(2)-10(3)/micrograms DNA were achieved by altering the conditions under which the bacteria were grown prior to electroporation (McIntyre & Harlander 1989b). This present investigation sought to improve still further transformation efficiencies in order to provide a reliable high frequency transformation system for Lc. lactis subsp. lactis.

DNA, Bacterial↗

PubMed1996

Inducible gene expression mediated by a repressor-operator system isolated from Lactococcus lactis bacteriophage r1t.

A regulatory region of the temperate Lactococcus lactis bacteriophage r1t chromosome has been cloned and characterized. It encompasses the two divergently oriented genes rro, encoding the phage repressor, and tec. Both genes, of which the transcription start sites have been mapped, are preceded by consensus -35 and -10 promoter sequences. The region contains three 21 bp direct repeats with internal dyad symmetry which probably act as operators. Two of these repeats partially overlap the two promoter sequences. The distant third repeat is located within the tec coding sequence. Gel mobility shift assays demonstrated that Rro specifically binds to this sequence. To study possible transcriptional regulation of the region, a lacZ translational fusion with an open reading frame following tec was constructed. Under conditions that favour the lysogenic life cycle of r1t, beta-galactosidase activity was very low. Expression of the lacZ fusion could be induced 70-fold by the addition of mitomycin C at a concentration which promotes the switch of r1t from the lysogenic to the lytic life cycle. In non-induced cells, promoter activity was repressed by Rro, as a frameshift mutation in rro resulted in constitutive expression of the lacZ gene fusion.

Amino Acid Sequence↗

PubMedDate not supplied

Manipulation of activity and orientation of membrane-reconstituted di-tripeptide transport protein DtpT of Lactococcus lactis.

The di-tripeptide transport system (DtpT) of Lactococcus lactis was purified to apparent homogeneity by pre-extraction of crude membrane vesicles with octaethylene glycol monodecyl ether (C10E8), followed by solubilization with n-dodecyl-beta-D-maltoside (DDM) and chromatography on a Ni-NTA resin. The DtpT protein was reconstituted into detergent-destabilized preformed liposomes prepared from E. coli phospholipid/phosphatidylcholine. A variety of detergents were tested for their ability to mediate the membrane reconstitution of DtpT and their effectiveness to yield proteoliposomes with a high transport activity. The highest activities were obtained with TX100, C12E8 and DM, whereas DDM yielded relatively poor activities, in particular when this detergent was used at concentrations beyond the onset of solubilization of the preformed liposomes. Parallel with the low activity, significant losses of lipid were observed when the reconstitution was performed at high DDM concentrations. This explained at least part of the reduced transport activity as the DtpT protein was highly dependent on the final lipid-to-protein ratios in the proteoliposomes. Consistent with the difference in mechanism of DDM- and TX100-mediated membrane protein reconstitution, the orientation of the DtpT protein in the membrane was random with DDM and inside-in when TX100 was used. The methodology to determine the orientation of membrane-reconstituted proteins from the accessibility of cysteines for thiol-specific reagents is critically evaluated.

Bacterial Proteins↗

PubMed1994

Specificity of hydrolysis of bovine kappa-casein by cell envelope-associated proteinases from Lactococcus lactis strains.

The cell envelope-associated proteinases from Lactococcus lactis subsp. cremoris H2 (a PI-type proteinase-producing strain) and SK11 (a PIII-type proteinase-producing strain) both actively hydrolyze the kappa-casein component of bovine milk but with significant differences in the specificity of peptide bond hydrolysis. The peptide bonds Ala-23-Lys-24, Leu-32-Ser-33, Ala-71-Gln-72, Leu-79-Ser-80, Met-95-Ala-96, and Met-106-Ala-107 were cleaved by both proteinase types, although the relative rates of hydrolysis at some of these sites were quite different for the two proteinases. Small histidine-rich peptides were formed as early products of the action of the cell envelope-associated proteinases on kappa-casein, implicating this casein as a possible significant source of histidine, which is essential for starter growth. The major difference between the two proteinase types in their action on kappa-casein was in their ability to cleave bonds near the C-terminal end of the molecule. The bond Asn-160-Thr-161 and, to a lesser extent, the bond Glu-151-Val-152 were very rapidly cleaved by the PIII-type proteinase, whereas hydrolysis of these bonds by the PI-type proteinase was barely detectable (even after 24 h of digestion). Differential hydrolysis of kappa-casein at these sites by the two different proteinase types resulted in the formation of distinctive, high-M(r) products detectable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1996

Heterologous expression and characterization of recombinant Lactococcus lactis neutral endopeptidase (neprilysin).

A neutral endopeptidase (NEP) from Lactococcus lactis has recently been cloned and shown to contain high sequence homology with the human neutral endopeptidase, endopeptidase 24.11 (I. Mierau et al., J. Bacteriol. 175, 2087-2096, 1993). The gene for the neutral endopeptidase from L. lactis was cloned into the pQE expression vector, resulting in the fusion of a hexahistidine at the N-terminus. The recombinant enzyme was expressed to high levels in Escherichia coli (approximately 10 mg/liter of culture) and purified to homogeneity in a two-step procedure. A number of peptides were studied as substrates for the enzyme. The enzyme cleaves the following peptides at the Gly3-Phe4 bond: enkephalins, dynorphins A-6, A-8, A-9, A-10, A-13, and A-17, and alpha-neo-endorphin. In addition the enzyme hydrolyzes bradykinin, substance P, beta-endorphin, ACTH, and VIP. Although the cleavage patterns observed are similar to that seen with mammalian neutral endopeptidase, the lactococcal enzyme more efficiently cleaves larger peptide substrates. As observed with the mammalian neutral endopeptidase, the lactococcal enzyme exhibits higher kcat/K(m) values for the enkephalins than for their corresponding amides, indicating the functionality of an active-site arginine. Inactivation of the lactococcal endopeptidase by diethyl pyrocarbonate and protection afforded by the substrate dynorphin A-6 indicate the functionality of a positionally conserved active-site histidine. This was confirmed by demonstrating that conversion of this histidine, histidine 587, to glutamine generated inactive enzyme. Similarly, conversion of the putative zinc ligand glutamate 535 to glutamine led to inactive enzyme. These studies indicate a conservation of critical catalytic residues between the two enzymes and suggest that the lactococcal endopeptidase is a better model than thermolysin for the mammalian enzyme.

Amino Acid Sequence↗

PubMed2001

Physiological role of beta-phosphoglucomutase in Lactococcus lactis.

A beta-phosphoglucomutase (beta-PGM) mutant of Lactococcus lactis subsp. lactis ATCC 19435 was constructed using a minimal integration vector and double-crossover recombination. The mutant and the wild-type strain were grown under controlled conditions with different sugars to elucidate the role of beta-PGM in carbohydrate catabolism and anabolism. The mutation did not significantly affect growth, product formation, or cell composition when glucose or lactose was used as the carbon source. With maltose or trehalose as the carbon source the wild-type strain had a maximum specific growth rate of 0.5 h(-1), while the deletion of beta-PGM resulted in a maximum specific growth rate of 0.05 h(-1) on maltose and no growth at all on trehalose. Growth of the mutant strain on maltose resulted in smaller amounts of lactate but more formate, acetate, and ethanol, and approximately 1/10 of the maltose was found as beta-glucose 1-phosphate in the medium. Furthermore, the beta-PGM mutant cells grown on maltose were considerably larger and accumulated polysaccharides which consisted of alpha-1,4-bound glucose units. When the cells were grown at a low dilution rate in a glucose and maltose mixture, the wild-type strain exhibited a higher carbohydrate content than when grown at higher growth rates, but still this content was lower than that in the beta-PGM mutant. In addition, significant differences in the initial metabolism of maltose and trehalose were found, and cell extracts did not digest free trehalose but only trehalose 6-phosphate, which yielded beta-glucose 1-phosphate and glucose 6-phosphate. This demonstrates the presence of a novel enzymatic pathway for trehalose different from that of maltose metabolism in L. lactis.

Culture Media↗

PubMed2002

Effect of wild strains of Lactococcus lactis on the volatile profile and the sensory characteristics of ewes' raw milk cheese.

The production of volatile compounds by wild strains of Lactococcus lactis used as starter cultures and their effect on the sensory characteristics of ewes' raw milk cheese were investigated. Sixteen vats of cheese were manufactured and ripened for 120 d in two experiments, each of them duplicated. In the first experiment, milk was inoculated with different ratios of four wild Lactococcus lactis strains, two producing and two not producing branched-chain volatile compounds, and in the second experiment with different ratios of a commercial starter culture and the two strains producing branched-chain volatile compounds. Cheese pH, proteolysis, and aminopeptidase activity increased when the strains producing branched-chain volatile compounds were inoculated at a higher rate. Fifty volatile compounds were identified in cheeses using a purge and trap system coupled to a gas chromatography-mass spectrometry apparatus. The relative abundances of 30 volatile compounds (8 alcohols, 5 aldehydes, 3 ketones, 12 esters, 1 sulfur compound, and 1 benzenic compound) were influenced by starter culture composition. 2-Methylpropanol, 3-methylbutanol, isobutyl acetate, isoamyl acetate, ethyl butyrate, isobutyl butyrate, and isoamyl butyrate were always more abundant in the cheeses made with a higher level of L. lactis strains producing branched-chain volatile compounds. Flavor intensity was enhanced by a high level of L. lactis strains producing branched-chain volatile compounds in the first experiment, in which four wild L. lactis strains were used as starter culture, but not in the second experiment, in which a combination of two wild L. lactis strains and the commercial starter culture were used. Flavor quality, as judged by trained panelists, was impaired in both experiments by a high level of L. lactis strains producing branched-chain volatile compounds.

Aminopeptidases↗

PubMed2006

Construction and evaluation of food-grade vectors for Lactococcus lactis using aspartate aminotransferase and alpha-galactosidase as selectable markers.

AIMS: We report development of two food-grade cloning vectors for Lactococcus lactis, which utilize either a lactococcal aspartate aminotransferase gene (aspC), or Bifidobacterium longumalpha-galactosidase gene (aglL) as selectable markers. METHODS AND RESULTS: The theta-replicon of lactococcal plasmid, pW563, was combined with aspC and a multiple cloning site. When electroporated into L. lactis JLS400 (AspC-), the resulting vector, pSUW611 (3.9 kbp), restores ability of the mutant to grow in milk thus allowing for selection of the transformants. The vector is stable during 100 generations of nonselective growth (0.2% loss per generation). The second vector, pSUW711 (5.1 kbp), was constructed by exchanging aspC with aglL under the control of usp45 promoter. Lactococcus lactis transformed with pSUW711 produced distinctive colonies within 48-72 h on melibiose-containing plates. Expression of two Lactobacillus helveticus peptidases was attempted using these new vehicles. Introduction of pepN on pSUW611 and pSUW711 into L. lactis led to a sixfold, or 27-fold increase in aminopeptidase activity, respectively. However, no changes in endopeptidase activity were recorded upon transformation with pSUW611 carrying pepO2 under control of three different promoters. Attempts were also made to construct high copy variants of pSUW711. CONCLUSIONS: The aspC and aglL can be employed as food-grade genetic markers for L. lactis. The vectors, pSUW611 and pSUW711, were successfully used to express Lact. helveticus PepN in L. lactis. SIGNIFICANCE AND IMPACT OF THE STUDY: Two novel food-grade vectors were developed which provide simple and convenient selection and maintenance in L. lactis.

Aspartate Aminotransferases↗

PubMed1994

Cloning of promoter-like sequences from Lactobacillus paracasei subsp. paracasei CG11 and their expression in Escherichia coli, Lactococcus lactis, and Lactobacillus reuteri.

Fragments of chromosomal DNA from Lactobacillus paracasei subsp. paracasei CG11 (formerly Lactobacillus casei CG11) capable of functioning as promoters were isolated using the broad host range, promoter-probe vector pGKV210. Five such fragments designated P61, P79, P80, P116, and P144 were completely sequenced and analyzed. Fragment P61 had the highest transcriptional efficiency in Escherichia coli and Lactobacillus reuteri whereas P80 was the most active in Lactococcus lactis. In general, the orders of the transcriptional strengths were almost identical in E. coli and Lactobacillus reuteri but different from that in Lactococcus lactis. Mapping of the 5' end of cat mRNA showed that different regions of fragments P79 and P144 were used as promoters in Lactococcus lactis than in E. coli and Lactobacillus reuteri. Analysis of these DNA sequences revealed that the putative -35 and -10 hexanucleotides resembled those of E. coli, Bacillus subtilis, and lactococci. The spacing between these two hexanucleotides and between the putative -10 hexanucleotide and the transcriptional start point (A residues predominated) ranged from 17 to 18 base pairs and from 5 to 7 base pairs, respectively. Each of the cloned Lactobacillus paracasei CG11 promoter-like fragments contained an AT-rich sequence upstream of the putative -35 region (from 60 to 73%).

Amino Acid Sequence↗

PubMed1993

Partial characterization of an rpoD-like gene of Lactococcus lactis subsp. lactis ML3 with a polymerase chain reaction-based approach.

With degenerated oligonucleotide primers for conserved regions of bacterial sigma factor proteins, a 117-bp internal DNA fragment of an rpoD-like gene of Lactococcus lactis subsp. lactis ML3 was amplified by the polymerase chain reaction (PCR). The DNA sequence of this PCR product was determined by cycle sequencing, and the deduced amino acid sequence of this internal fragment showed an extensive homology with the known sigma factor sequences from six other microorganisms and present a 13-amino acid region corresponding to the typical "RpoD box" of primary sigma factors. This PCR product was used as a probe to specifically detect sigma homologs in Pediococcus acidilactici, Leuconostoc lactis, Lactobacillus helveticus, Lactobacillus acidophilus, Enterococcus faecalis, Streptococcus thermophilus, and Lactococcus lactis subsp. cremoris. These data are consistent with the existence of a high similarity between the primary sigma factors from diverse Gram-positive microorganisms.

Amino Acid Sequence↗

PubMed2001

[Cloning and expression of nisZ gene in Lactococcus lactis].

The gene encoding the precursor of nisin was amplified by PCR using the lambda HJ-3 DNA as the template, which contained the entire nisin biosynthesis gene cluster from Lactococcus lactis AL2 with high yield of nisin, and was cloned into pMG36e. The recombinant plasmid pHJ201 was introduced into Lactococcus lactis NZ9800 by electroporation. pHJ201 is very stable in L. lactis NZ9800. Antimicrobial activity test and Tricine-SDS-PAGE analysis revealed that L. lactis NZ9800 harbouring pHJ201 restored ability of nisin production, but the production level was markedly lower than L. lactis AL2. The result of DNA sequence analysis indicated that Nisin Z is produced by L. lactis AL2.

Amino Acid Sequence↗

PubMed2005

Oral vaccination of mice against rodent malaria with recombinant Lactococcus lactis expressing MSP-1(19).

AIM: To construct the recombinant Lactococcus lactis as oral delivery vaccination against malaria. METHODS: The C-terminal 19-ku fragments of MSP1 (MSP-1(19)) of Plasmodium yoelii 265-BY was expressed in L. lactis and the recombinant L. lactis was administered orally to BALB/c and C57BL/6 mice. After seven interval vaccinations within 4 wk, the mice were challenged with P. yoelii 265-BY parasites of erythrocytic stage. The protective efficacy of recombinant L. lactis was evaluated. RESULTS: The peak parasitemias in average for the experiment groups of BALB/c and C57BL/6 mice were 0.8+/-0.4% and 20.8+/-26.5%, respectively, and those of their control groups were 12.0+/-0.8% and 60.8+/-9.6%, respectively. None of the BALB/c mice in both experimental group and control group died during the experiment. However, all the C57BL/6 mice in the control group died within 23 d and all the vaccinated mice survived well. CONCLUSION: The results imply the potential of recombinant L. lactis as oral delivery vaccination against malaria.

Administration, Oral↗

PubMed1994

Starvation-Induced Stress Resistance in Lactococcus lactis subsp. lactis IL1403.

Carbohydrate-starved cultures of Lactococcus lactis subsp. lactis IL1403 showed enhanced resistance to heat, ethanol, acid, osmotic, and oxidative stresses. This cross-protection seems to be established progressively during the transitional growth phase, with maximum resistance occurring when cells enter the stationary phase. Chloramphenicol or rifamycin treatment does not abolish the development of a tolerant cell state but, on the contrary, seems to provoke this response in L. lactis subsp. lactis.

Journal Article↗

PubMed2003

Glutathione protects Lactococcus lactis against oxidative stress.

Glutathione was found in several dairy Lactococcus lactis strains grown in M17 medium. None of these strains was able to synthesize glutathione. In chemically defined medium, L. lactis subsp. cremoris strain SK11 was able to accumulate up to approximately 60 mM glutathione when this compound was added to the medium. Stationary-phase cells of strain SK11 grown in chemically defined medium supplemented with glutathione showed significantly increased resistance (up to fivefold increased resistance) to treatment with H2O2 compared to the resistance of cells without intracellular glutathione. The resistance to H2O2 treatment was found to be dependent on the accumulation of glutathione in 16 strains of L. lactis tested. We propose that by taking up glutathione, L. lactis might activate a glutathione-glutathione peroxidase-glutathione reductase system in stationary-phase cells, which catalyzes the reduction of H2O2. Glutathione reductase, which reduces oxidized glutathione, was detectable in most strains of L. lactis, but the activities of different strains were very variable. In general, the glutathione reductase activities of L. lactis subsp. lactis are higher than those of L. lactis subsp. cremoris, and the activities were much higher when strains were grown aerobically. In addition, glutathione peroxidase is detectable in strain SK11, and the level was fivefold greater when the organism was grown aerobically than when the organism was grown anaerobically. Therefore, the presence of glutathione in L. lactis could result in greater stability under storage conditions and quicker growth upon inoculation, two important attributes of successful starter cultures.

Culture Media↗

PubMed1993

The primary structure of phosphofructokinase from Lactococcus lactis.

The primary amino acid sequence of phosphofructokinase (EC2.7.1.11) from Lactococcus lactis, obtained by Edman analysis of peptides obtained from proteolytic digestions, is MKRIAVLTSGGDAPGMNAAIRAVVRKAISEGIEVYGINHGYAGMVAGDIF PLTSASVGDKIGRGGTFLYSARYPEFAQVEGQLAGIEQLKKFGIEGVVVI GGDGSYHGAMRLTEHGFPAVGLPGTIDNDIVGTDFTIGFDTAVSTVVDAL DKIRDTSSSHNRTFVVEVMGRNAGDIALNAGIAAGADDISIPELEFKFEN VVNNINKGYEKGKNHHIIIVAEGVMTGEEFATKLKEAGYKGDLRVSVLGH IQRGGSPTARDRVLASRMGARAVELLRDGIGGVAVGIRNEELVESPILGT AEEGALFSLTTEGGIKVNNPHKAGLELYRLNSALNNLNL.

Amino Acid Sequence↗

PubMed1991

A host factor absent from Lactococcus lactis subspecies lactis MG1363 is required for conjugative transposition.

In matings between Lactococcus lactis strains, the conjugative transposons Tn916 and Tn919 are found in the chromosome of the transconjugants in the same place as in the chromosome of the donor, indicating that no transposition has occurred. In agreement with this, the frequency of L. lactis transconjugants from intraspecies matings is the same whether the donor contains the wild-type form of the transposon or the mutant Tn916-int1, which has an insertion in the transposon's integrase gene. However, in intergeneric crosses with Bacillus subtilis or Enterococcus faecalis donors, Tn916 and Tn919 transpose to different locations on the chromosome of the L. lactis transconjugants. Moreover, Tn916 and Tn919 could not be transferred by conjugation from L. lactis and B. subtilis, E. faecalis or Streptococcus pyogenes. This suggests that excision of these elements does not occur in L. lactis. When cloned into E. coli with adjacent chromosomal DNA from L. lactis, the conjugative transposons were able to excise, transpose and promote conjugation. Therefore, the inability of these elements to excise in L. lactis is not caused by a permanent structural alteration in the transposon. We conclude that L. lactis lacks a factor required for excision of conjugative transposons.

Bacillus subtilis↗