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PubMed2001

Respiration capacity of the fermenting bacterium Lactococcus lactis and its positive effects on growth and survival.

Oxygen is a major determinant of both survival and mortality of aerobic organisms. For the facultative anaerobe Lactococcus lactis, oxygen has negative effects on both growth and survival. We show here that oxygen can be beneficial to L. lactis if heme is present during aerated growth. The growth period is extended and long-term survival is markedly improved compared to results obtained under the usual fermentation conditions. We considered that improved growth and survival could be due to the capacity of L. lactis to undergo respiration. To test this idea, we confirmed that the metabolic behavior of lactococci in the presence of oxygen and hemin is consistent with respiration and is most pronounced late in growth. We then used a genetic approach to show the following. (i) The cydA gene, encoding cytochrome d oxidase, is required for respiration and plays a direct role in oxygen utilization. cydA expression is induced late in growth under respiration conditions. (ii) The hemZ gene, encoding ferrochelatase, which converts protoporphyrin IX to heme, is needed for respiration if the precursor, rather than the final heme product, is present in the medium. Surprisingly, survival improved by respiration is observed in a superoxide dismutase-deficient strain, a result which emphasizes the physiological differences between fermenting and respiring lactococci. These studies confirm respiratory metabolism in L. lactis and suggest that this organism may be better adapted to respiration than to traditional fermentative metabolism.

Anaerobiosis↗
PubMed1993

Functional analysis of the Lactococcus lactis usp45 secretion signal in the secretion of a homologous proteinase and a heterologous alpha-amylase.

The ups45 gene encodes the major extracellular protein from Lactococcus lactis. The deduced sequence of the 27 residue leader peptide revealed the tripartite characteristics of a signal peptide. This leader peptide directed the efficient secretion of the homologous proteinase (PrtP) in L. lactis, indicating that the putative signal peptide of PrtP can be replaced by the 27 residue Usp45 leader peptide. In addition, the 27 residue leader peptide could be used to secrete the Bacillus stearothermophilus alpha-amylase, encoded by the amyS gene. Fusion of the usp45 promoter region and various parts of the leader sequence to an amyS gene devoid of its signal sequence, showed that in Escherichia coli the first 19, 20, and 27 residues of the Usp45 leader are able to direct alpha-amylase secretion. In L. lactis the shorter signal peptides did not result in secretion of alpha-amylase, providing experimental evidence for the hypothesis that gram-positive bacteria require a longer signal peptide for secretion than gram-negative organisms.

Amino Acid Sequence↗
PubMed2006

[Effects of gavage with lactococcus lactis recombinant heme oxygenase-1 gene on inflammation of intestine and bacterial translocation in rats with hemorrhagic shock].

OBJECTIVE: To evaluate the effects of gavage with lactococcus lactis (L. Lactis) recombinant heme oxygenase-1 (HO-1) gene on alleviation of intestinal inflammation and protection of the intestinal mucosa in rats with hemorrhagic shock. METHODS: A model of rats with hemorrhagic shock was reproduced in 30 healthy SD male rats. The rats were randomly divided into the L. Lactis recombinant HO-1 gene group (HO group, n=10), L. Lactis group (LL group, n=10) and phosphate buffer group (PBS group, n=10). These agents were respectively gavaged 24 hours before the experiment. Rats were re-anesthetized 1 hour after fluid resuscitation. The mortality, myeloperoxidase (MPO) activity, bacterial translocation, the pathologic changes, the contents of HO-1, tumor necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) in the intestine were determined and compared. RESULTS: Compared with LL group and PBS group, the mortality, Chiu's grade and the bacterial translocation rate of HO group were significantly decreased (all P<0.05) but the content of HO-1 and the level of IL-10 in HO group were markedly increased (both P<0.05). Compared with HO group and LL group, the MPO activity of PBS group was obviously increased (P<0.05). CONCLUSION: The recombinant L. Lactis has the effect to deliver HO-1, which has protective effect on the intestinal mucosa in lessening the inflammation of the intestine and the incidence of bacterial translocation.

Animals↗
PubMed2006

Combined high pressure and temperature induced lethal and sublethal injury of Lactococcus lactis--application of multivariate statistical analysis.

It was the aim of this work to determine the combined effects of pressure, temperature, and co-solutes on Lactococcus lactis, and to detect correlations between culture-dependent and culture-independent methods for assessment of cellular viability and sublethal injury. Therefore, the pressure induced inactivation of L. lactis MG 1363 was investigated in buffer and in buffer with 1.5 M sucrose or 4 M NaCl at a pressure range of 0.1 to 500 MPa and a temperature range of 5 to 50 degrees C. The inactivation was characterised by viable cell counts, stress resistant cell counts, membrane integrity, metabolic activity, and the activity of the multi-drug-resistance transport enzyme LmrP. L. lactis was most resistant to pressure application at 20-30 degrees C. Sucrose protected towards inactivation at any temperature, NaCl provided protection at high temperatures only. By using Principal Component Analysis, correlations were detected between viable cell counts and metabolic activity as well as stress resistant cell counts and LmrP activity. In conclusion, the pressure-inactivation of L. lactis is strongly temperature dependent, baroprotection by sucrose occurs at any temperature but the baroprotective effects of NaCl is temperature dependent. Further on, a combination of two experimental methods fully describe lethal and sublethal injury of pressure treated cells. These simplification of data acquisition and model development facilitates the establishment of pressure processes in food technology.

Bacterial Proteins↗
PubMed1999

Rapid fluorescence assessment of the viability of stressed Lactococcus lactis.

The aim of this study was to establish the use of the fluorescent probes carboxyfluorescein (cF) and propidium iodide (PI) for rapid assessment of viability, using Lactococcus lactis subsp. lactis ML3 exposed to different stress treatments. The cF labeling indicated the reproductive capacity of mixtures of nontreated cells and cells killed at 70 degrees C very well. However, after treatment up to 60 degrees C the fraction of cF-labeled cells remained high, whereas the survival decreased for cells treated at above 50 degrees C and was completely lost for those treated at 60 degrees C. In an extended series of experiments, cell suspensions were exposed to heating, freezing, low pH, or bile salts, after which the colony counts, acidification capacity, glycolytic activity, PI exclusion, cF labeling, and cF efflux were measured and compared. The acidification capacity corresponded with the number of CFU. The glycolytic activity, which is an indicator of vitality, was more sensitive to the stress conditions than the reproduction, acidification, and fluorescence parameters. The cF labeling depended on membrane integrity, as was confirmed by PI exclusion. The fraction of cF-labeled cells was not a general indicator of reproduction or acidification, nor was PI exclusion or cF labeling capacity (the internal cF concentration). When the cells were labeled by cF, a subsequent lactose-energized efflux assay was needed for decisive viability assessment. This novel assay proved to be a good and rapid indicator of the reproduction and acidification capacities of stressed L. lactis and has potential for physiological research and dairy applications related to lactic acid bacteria.

Bile Acids and Salts↗
PubMed1995

Genetic aspects of aromatic amino acid biosynthesis in Lactococcus lactis.

Polymerase chain reaction (PCR) primers designed from a multiple alignment of predicted amino acid sequences from bacterial aroA genes were used to amplify a fragment of Lactococcus lactis DNA. An 8 kb fragment was then cloned from a lambda library and the DNA sequence of a 4.4 kb region determined. This region was found to contain the genes tyrA, aroA, aroK, and pheA, which are involved in aromatic amino acid biosynthesis and folate metabolism. TyrA has been shown to be secreted and AroK also has a signal sequence, suggesting that these proteins have a secondary function, possibly in the transport of amino acids. The aroA gene from L. lactis has been shown to complement an E. coli mutant strain deficient in this gene. The arrangement of genes involved in aromatic amino acid biosynthesis in L. lactis appears to differ from that in other organisms.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed1992

Protein export elements from Lactococcus lactis.

Broad-host-range plasmids carrying alpha-amylase or beta-lactamase reporter genes lacking a signal sequence were used to select export elements from Lactococcus lactis chromosomal DNA that could function as signal sequences. Fragments containing such elements were identified by their ability to direct the export of the reporter proteins in Escherichia coli. Several of the selected export elements were also active in Bacillus subtilis and L. lactis, although the efficiencies depended strongly on the host organism and reporter gene used. The export elements AL9 and BL1 were highly efficient in L. lactis in the expression and secretion of at least two heterologous proteins (Bacillus licheniformis alpha-amylase and E. coli TEM-beta-lactamase). AL9 even permitted growth of this organism on starch as the sole carbon source. Nucleotide sequence analysis of five selected fragments indicated that these encode oligopeptides with the major characteristics of typical signal peptides. The putative expression signals had a limited similarity to previously described expression signals for E. coli, B. subtilis and L. lactis. Differences in both expression and export efficiency are likely to underlie the host-specific effects.

Amino Acid Sequence↗
PubMed2004

Proteome analyses of heme-dependent respiration in Lactococcus lactis: involvement of the proteolytic system.

Sugar fermentation was long considered the sole means of energy metabolism available to lactic acid bacteria. We recently showed that metabolism of Lactococcus lactis shifts progressively from fermentation to respiration during growth when oxygen and heme are available. To provide insights into this phenomenon, we compared the proteomic profiles of L. lactis under fermentative and respiratory growth conditions in rich medium. We identified 21 proteins whose levels differed significantly between these conditions. Two major groups of proteins were distinguished, one involved in carbon metabolism and the second in nitrogen metabolism. Unexpectedly, enzymes of the proteolytic system (PepO1 and PepC) which are repressed in rich medium in fermentation growth were induced under respiratory conditions despite the availability of free amino acids. A triple mutant (dtpT dtpP oppA) deficient in oligopeptide transport displayed normal respiration, showing that increased proteolytic activity is not an absolute requirement for respiratory metabolism. Transcriptional analysis confirmed that pepO1 is induced under respiration-permissive conditions. This induction was independent of CodY, the major regulator of proteolytic functions in L. lactis. We also observed that pepO1 induction is redox sensitive. In a codY mutant, pepO1 expression was increased twofold in aeration and eightfold in respiration-permissive conditions compared to static conditions. These observations suggest that new regulators activate proteolysis in L. lactis, which help to maintain the energetic needs of L. lactis during respiration.

Bacterial Proteins↗
PubMed1992

Batch cultures of recombinant Lactococcus lactis subsp. lactis in a stirred fermentor. I. Effect of plasmid content on bacterial growth and on genetic stability in pure cultures.

The effect of plasmid introduction into Lactococcus lactis subsp. lactis IL2661 on the growth of this strain and on plasmid stability was studied in pure batch cultures. The plasmids used (coding for erythromycin or chloramphenicol resistance) were the following: pIL205 (42 kb), pIL252 (4.6 kb, 6-9 copies), pIL253 (4.8 kb, 45-85 copies) and pE194 (inserted in the chromosome). Growth and acidification of L. lactis subsp. lactis IL2661 were similar to those of the derived recombinant lactococci. The maximal population at the end of the fermentation (9 h) was about 1.1 +/- 0.3 x 10(10) cfu/ml, and maximal growth rate 0.92 +/- 0.07 h-1. Growth yield and lactic acid concentrations were 3.9 +/- 0.8 x 10(11) cfu/g lactose consumed and 25.6 +/- 2.3 g/l, respectively. Different levels of plasmid stability were detected. Plasmid pE194, and plasmids pIL252 and pIL253 in the absence of pIL205, were stable after 10 h of culture. A slight loss (1-2%) of pIL205 was observed in all strains. In the presence of pIL205, plasmids pIL252 and pIL253 were maintained in only 56-95% of the cells. This result suggested an incompatibility between pIL205 and pIL252 or pIL253.

Biotechnology↗
PubMed1997

Differential induction of the chaperonin GroEL and the Co-chaperonin GroES by heat, acid, and UV-irradiation in Lactococcus lactis subsp. lactis.

Microsequencing of a polypeptide with MW of 14.5 and pI of 5.0 induced by heat treatment at 42 degrees C and 50 degrees C in Lactococcus lactis subsp. lactis revealed that it corresponds to the co-chaperonin GroES. Quantitative analysis of analytical 2-D gels showed a relative induction of 12- and 11-fold after 30 min of heat adaptation at 42 degrees C and 50 degrees C, respectively. GroES is also induced by an acid shift from pH 7 to pH 5.5 and by UV254 nm-irradiation, with relative induction factors of 3.8 and 2.3, respectively. To our knowledge this is the first report showing induction of GroES by mild acid treatment. Contrasting to the relative induction of the groEL gene product, the second protein encoded by the groESL operon, GroES shows significantly higher induction under all stress situations.

Amino Acid Sequence↗
PubMed1992

Effect of the unsaturation of phospholipid acyl chains on leucine transport of Lactococcus lactis and membrane permeability.

The effect of the degree of unsaturation of the phospholipid acyl chains on the branched-chain amino acid transport system of Lactococcus lactis was investigated by the use of a membrane fusion technique. Transport activity was analyzed in hybrid membranes composed of equimolar mixtures of synthetic unsaturated phosphatidylethanolamine (PE) and phosphatidylcholine (PC) in which the number of cis double bonds in the 18-carbon acyl chains was varied. The accumulation level and initial rate of both counterflow and protonmotive-force driven transport of leucine decreased with increasing number of double bonds. The reduction in transport activity with increasing number of double bonds correlated with an increase in the passive permeability of the membranes to leucine. The membrane fluidity was hardly affected by the double bond content. It is concluded that the degree of lipid acyl chain unsaturation is a minor determinant of the activity of the branched chain amino acid transport system, but effects strongly the passive permeability of the membrane.

Biological Transport↗
PubMed1991

Transposon-encoded sucrose metabolism in Lactococcus lactis. Purification of sucrose-6-phosphate hydrolase and genetic linkage to N5-(L-1-carboxyethyl)-L-ornithine synthase in strain K1.

Sucrose-6-phosphate hydrolase from Lactococcus lactis subsp. lactis K1-23 (formerly Streptococcus lactis K1-23) has been purified 600-fold to electrophoretic homogeneity. Purification of the enzyme was achieved by DEAE-Sephacel, phosphocellulose P-11, and gel exclusion (Ultrogel AcA 54) chromatography. The purified enzyme (specific activity 31 units/mg) catalyzed the hydrolysis of both 6-O-phosphoryl-alpha-D-glucopyranosyl-1,2-beta-D-fructofuranoside (sucrose 6-phosphate) and sucrose (Km = 0.1 and 100 mM, respectively). Ultracentrifugal analysis of sucrose-6-phosphate hydrolase indicated an Mr = 52,200. The purified enzyme migrated as a single protein during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr = 52,000). However, four distinct polypeptides were detected by analytical electrofocusing, and all four species hydrolyzed sucrose and sucrose 6-phosphate. The amino acid composition of sucrose-6-phosphate hydrolase, and the sequence of the first 12 amino acids from the NH2 terminus, have been determined. Hybridization studies with oligonucleotide probes show that the genes for sucrose-6-phosphate hydrolase (scrB), Enzyme IIScr of the phosphoenolypyruvate-dependent sucrose:phosphotransferase system (scrA), and N5-(carboxyethyl)ornithine synthase (ceo) are encoded by the same approximately 20-kilobase EcoRI fragment. This fragment is part of a large transposon Tn5306 that also encodes the nisin precursor gene, spaN, and IS904. In L. lactis ATCC 11454, spaN, IS904, scrA, and scrB (but not ceo) are encoded on a related transposon, Tn5307.

Amino Acid Oxidoreductases↗
PubMed2006

The riboflavin transporter RibU in Lactococcus lactis: molecular characterization of gene expression and the transport mechanism.

This study describes the characterization of the riboflavin transport protein RibU in the lactic acid bacterium Lactococcus lactis subsp. cremoris NZ9000. RibU is predicted to contain five membrane-spanning segments and is a member of a novel transport protein family, not described in the Transport Classification Database. Transcriptional analysis revealed that ribU transcription is downregulated in response to riboflavin and flavin mononucleotide (FMN), presumably by means of the structurally conserved RFN (riboflavin) element located between the transcription start site and the start codon. An L. lactis strain carrying a mutated ribU gene exhibits altered transcriptional control of the riboflavin biosynthesis operon ribGBAH in response to riboflavin and FMN and does not consume riboflavin from its growth medium. Furthermore, it was shown that radiolabeled riboflavin is not taken up by the ribU mutant strain, in contrast to the wild-type strain, directly demonstrating the involvement of RibU in riboflavin uptake. FMN and the toxic riboflavin analogue roseoflavin were shown to inhibit riboflavin uptake and are likely to be RibU substrates. FMN transport by RibU is consistent with the observed transcriptional regulation of the ribGBAH operon by external FMN. The presented transport data are consistent with a uniport mechanism for riboflavin translocation and provide the first detailed molecular and functional analysis of a bacterial protein involved in riboflavin transport.

Amino Acid Sequence↗
PubMed2005

Production of xylitol from D-xylose by recombinant Lactococcus lactis.

The D-xylose reductase from Pichia stipitis CBS 5773 and the xylose transporter from Lactobacillus brevis ATCC 8287 were expressed in active form in Lactococcus lactis NZ9800. Xylitol production was investigated using non-growing recombinant cells in high cell-density under microaerobic conditions in the presence of xylose and glucose. Besides xylose, the recombinant strain with xylose reductase activity reduced l-arabinose and D-ribose in significant extent to the corresponding pentitols. The ratio of xylitol produced per glucose consumed was almost 10-fold higher under glucose limitation than the ratio in the presence of excess initial glucose. The co-expression of the xylose transporter with the xylose reductase did not increase the efficiency of xylitol production appreciably when compared to the strain in which only the xylose reductase gene was expressed. A fed-batch experiment with high initial xylose concentration (160 gl(-1)) under glucose limitation was carried out using the strain co-expressing xylose reductase and xylose transporter genes. The xylitol yield from xylose was 1.0 mol mol(-1) and the ratio of xylitol produced per glucose consumed was 2.5 mol mol(-1). The volumetric productivity was 2.72 gl(-1)h(-1) at 20 h. Of the xylose initially present, 34% was consumed. Analysis of the fermentation metabolites revealed a shift from homolactic to mixed acid fermentation at early stages of the experiment.

Aldehyde Reductase↗
PubMed1996

Nucleotide sequence and analysis of pWC1, a pC194-type rolling circle replicon in Lactococcus lactis.

A 2.8-kb cryptic plasmid showing no homology to either pFX3 (rolling circle, pE194-type) or pCI305 (theta-type) lactococcal replicons was identified in Lactococcus lactis subsp. cremoris 2204. The plasmid, pWC1, was compatible with both pCI3340 (a pCI305 derivative) and pFX3 in L. lactis subsp. cremoris 2204. Sequence analysis of pWC1 showed one major ORF encoding a protein with a deduced size of 316 amino acids (aa). Database comparisons showed that the protein was distinct from the pFX- and pCI-type replication proteins (less than 21% aa identity), but shared significant homology (up to 57% aa identity) with the replication proteins from a different group of rolling circle plasmids (pC194-type) commonly found in gram-positive bacteria. A pC194-type rolling circle plasmid has not been previously described in L. lactis. Further sequence analysis showed a conserved double-stranded origin of replication in pWC1 preceded by a large (118-bp) direct repeat. The chloramphenicol-resistance gene from pC194 was inserted into a nonessential region of pWC1 to give pCP12. The host range of pCP12 included Streptococcus thermophilus, Enterococcus faecalis, and Staphylococcus aureus, but not Escherichia coli. Both pCP12 and to a lesser extent pWC1 generated single-stranded DNA (ssDNA) in L. lactis. A possible single-stranded origin of replication was identified by sequence analysis of pWC1 and by comparing levels of ssDNA produced by pCP12 deletion derivatives. The pWC1 replicon may be a useful addition to other replicons currently available for vector construction.

Amino Acid Sequence↗
PubMed2005

Roles of thioredoxin reductase during the aerobic life of Lactococcus lactis.

Thiol-disulfide bond balance is generally maintained in bacteria by thioredoxin reductase-thioredoxin and/or glutathione-glutaredoxin systems. Some gram-positive bacteria, including Lactococcus lactis, do not produce glutathione, and the thioredoxin system is presumed to be essential. We constructed an L. lactis trxB1 mutant. The mutant was obtained under anaerobic conditions in the presence of dithiothreitol (DTT). Unexpectedly, the trxB1 mutant was viable without DTT and under aerated static conditions, thus disproving the essentiality of this system. Aerobic growth of the trxB1 mutant did not require glutathione, also ruling out the need for this redox maintenance system. Proteomic analyses showed that known oxidative stress defense proteins are induced in the trxB1 mutant. Two additional effects of trxB1 were not previously reported in other bacteria: (i) induction of proteins involved in fatty acid or menaquinone biosynthesis, indicating that membrane synthesis is part of the cellular response to a redox imbalance, and (ii) alteration of the isoforms of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GapB). We determined that the two GapB isoforms in L. lactis differed by the oxidation state of catalytic-site cysteine C152. Unexpectedly, a decrease specific to the oxidized, inactive form was observed in the trxB1 mutant, possibly because of proteolysis of oxidized GapB. This study showed that thioredoxin reductase is not essential in L. lactis and that its inactivation triggers induction of several mechanisms acting at the membrane and metabolic levels. The existence of a novel redox function that compensates for trxB1 deficiency is suggested.

Aerobiosis↗
PubMed1992

Identification and characterization of two bacteriocin-producing strains of Lactococcus lactis isolated from vegetables.

Isolated from mixed salad and fermented carrots, 123 strains of lactic acid bacteria were screened for bacteriocin production. Two strains, D53 and 23, identified as Lactococcus lactis by DNA-DNA hybridizations, produced heat stable bacteriocins which were resistant to trypsin and pepsin, but were inactivated by alpha-chymotrypsin and proteinase K. The bacteriocins were active from pH 2 to 9 and inhibited species of Listeria, Lactobacillus, Lactococcus, Pediococcus, Leuconostoc, Carnobacterium, Bacillus and Staphylococcus. Strain D53 produced bacteriocin at pH values of 4.5-8.0 and from 10 to 37 degrees C.

Anti-Bacterial Agents↗
PubMed2006

Conversion of Lactococcus lactis cell envelope proteinase specificity by partial allele exchange.

AIMS: To determine whether conversion of lactocepin substrate binding regions by gene replacement can alter lactocepin specificity in Lactococcus lactis starter bacteria without affecting other important strain properties. METHODS AND RESULTS: We utilized two-step gene replacement to convert substrate-binding determinants in the L. lactis prtP genes encoding group h (bitter) lactocepin in two industrial strains into the corresponding group b (nonbitter) variant. Analysis of lactocepin activity toward alpha(s1)-casein (f 1-23) by reversed-phase high-pressure liquid chromatography demonstrated enzyme specificity among isogenic derivatives had been altered in a manner that was consistent with predicted amino acid substitutions in substrate binding regions. Milk acidification properties of some mutants were not statistically different (P > 0.05) from wild-type parent strains, and strain propensity for autolysis was also not significantly (P > 0.05) changed. CONCLUSIONS: Conversion of lactocepin substrate binding regions by allele exchange can effectively alter lactocepin specificity in industrial strains of L. lactis without significantly affecting other important strain properties. SIGNIFICANCE AND IMPACT OF THE STUDY: Methodology outlined in this study can be used to alter lactocepin specificity in commercial starter cultures with a propensity for bitter flavour defect, and prtP derivatives developed by this approach should be suitable for commercial application.

Alleles↗