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PubMed2004

Inhibition of a methicillin-resistant Staphylococcus aureus strain in Afuega'l Pitu cheese by the nisin Z-producing strain Lactococcus lactis subsp. lactis IPLA 729.

Methicillin-resistant Staphylococcus aureus strains are a potential threat for food safety because foodborne illness caused by methicillin-resistant Staphylococcus aureus has been reported even though these strains were only associated with nosocomial infections until recently. This article focuses on the inhibitory effect of the nisin Z-producing strain Lactococcus lactis subsp. lactis IPLA 729 on the growth of Staphylococcus aureus CECT 4013, a methicillin-resistant strain. S. aureus was inhibited by the presence of the nisin producer IPLA 729 in buffered Trypticase soy broth, milk, and Afuega'l Pitu cheese, an acid-coagulated cheese manufactured in Asturias, Northern Spain. A reduction of 3.66 log units was observed in Trypticase soy broth at the end of the incubation period. In milk, viable counts of S. aureus were undetectable or were reduced by 2.16 log units in 24 h depending on the initial inoculum (1.8 x 10(4) and 7.2 x 10(6) CFU/ml). The staphylococcal strain was also undetected in test cheeses in which the nisin Z producer was present whereas 2 log units were detected in control cheeses at the end of ripening.

Anti-Infective Agents↗
PubMed2004

Antimicrobial potential of immobilized Lactococcus lactis subsp. lactis ATCC 11454 against selected bacteria.

Immobilization of living cells of lactic acid bacteria could be an alternative or complementary method of immobilizing organic acids and bacteriocins and inhibit undesirable bacteria in foods. This study evaluated the inhibition potential of immobilized Lactococcus lactis subsp. lactis ATCC 11454 on selected bacteria by a modified method of the agar spot test. L. lactis was immobilized in calcium alginate (1 to 2%)-whey protein concentrate (0 and 1%) beads. The antimicrobial potential of immobilized L. lactis was evaluated in microbiological media against pathogenic bacteria (Escherichia coli, Salmonella, and Staphylococcus aureus) or Pseudomonas putida, a natural meat contaminant, and against seven gram-positive bacteria used as indicator strains. Results obtained in this study indicated that immobilized L. lactis inhibited the growth of S. aureus, Enterococcus faecalis, Enterococcus faecium, Lactobacillus curvatus, Lactobacillus sakei, Kocuria varians, and Pediococcus acidilactici. Only 4 h of incubation at 35 degrees C resulted in a clear inhibition zone around the beads that increased with time. With the addition of 10 mM of a chelating agent (EDTA) to the media, results showed growth inhibition of E. coli; however, P. putida and Salmonella Typhi were unaffected by this treatment. These results indicate that immobilized lactic acid bacteria strains can be successfully used to produce nisin and inhibit bacterial growth in semisolid synthetic media.

Alginates↗
PubMed2006

First report of Streptococcus agalactiae and Lactococcus garvieae from a wild bottlenose dolphin (Tursiops truncatus).

The isolation and characterization of two bacterial species, Streptococcus agalactiae and Lactococcus garvieae, previously unreported in wild marine mammals are described from a freshly dead bottlenose dolphin, Tursiops truncatus, from Kuwait Bay, Kuwait, in September 2001. Conventional and rapid identification systems were used to determine that isolates from muscle and kidney were S. agalactiae and L. garvieae, respectively. The isolates were gram-positive, catalase-negative, oxidase-negative, nonhemolytic cocci. The S. agalactiae was serotyped to group antigen B, whereas the L. garvieae could not be assigned to any serogroup. These Kuwait isolates displayed considerable homogeneity with corresponding American Type Culture Collection (ATCC) type isolates. Although the dolphin S. agalactiae isolate was nonhemolytic, it was biochemically similar to S. agalactiae isolated from mullet sampled in the concurrent Kuwait Bay fish kill. Some biochemical heterogeneity was observed between the dolphin isolates and corresponding mammalian ATCC type isolates, especially with Voges Proskauer, alanine-phenylanaline-proline arylamidase, and alpha-galactosidase tests. Nile tilapia, Oreochromis niloticus, experimentally infected with the dolphin S. agalactiae and L. garvieae isolates experienced 90% and 0% mortalities, respectively. This is the first isolation of S. agalactiae and L. garvieae from a wild marine mammal, and the microbial characteristics established here provide pertinent information for the future isolation of these bacteria.

Animals↗
PubMed1999

Determination of the phosphorylated sugars of the Embden-Meyerhoff-Parnas pathway in Lactococcus lactis using a fast sampling technique and solid phase extraction.

An experimental procedure for the determination of intracellular concentrations of the phosphorylated sugars in the lactic acid bacterium Lactococcus lactis is presented. The first step of the procedure is a rapid sampling of a small volume of the growth medium into 60% (v/v) methanol precooled to -35 degrees C, bringing about a fast and complete stop of all metabolic activity. In contrast to yeast the metabolites leak out of the cells when these are brought into contact with methanol and are present in the medium and in the biomass after the quenching. A liquid-liquid extraction with chloroform at -25 degrees C ensures a total permeability of the cellular membrane towards the metabolites of interest as well as the inactivation of enzymes liable to alter their levels. The final step of the procedure consists in a solid phase extraction using columns with a high affinity for phosphorylated components. The internal standard was recovered to an extent of 85-95%.

Bacteriological Techniques↗
PubMedDate not supplied

Exopolysaccharides produced by Lactococcus lactis: from genetic engineering to improved rheological properties?

Over the last years, important advances have been made in the study of the production of exopolysaccharides (EPS) by several lactic acid bacteria, including Lactococcus lactis. From different EPS-producing lactococcal strains the specific eps gene clusters have been characterised. They contain eps genes, which are involved in EPS repeating unit synthesis, export, polymerisation, and chain length determination. The function of the glycosyltransferase genes has been established and the availability of these genes opened the way to EPS engineering. In addition to the eps genes, biosynthesis of EPS requires a number of housekeeping genes that are involved in the metabolic pathways leading to the EPS-building blocks, the nucleotide sugars. The identification and characterisation of several of these housekeeping genes (galE, galU, rfbABCD) allows the design of metabolic engineering strategies that should lead to increased EPS production levels by L. lactis. Finally, model development has been initiated in order to predict the physicochemical consequences of the addition of a EPS to a product.

Genes, Bacterial↗
PubMedDate not supplied

[Isolation and purification of acetolactate synthase and acetolactate decarboxylase from a Lactococcus lactis culture].

Enzymes catalyzing the synthesis and subsequent transformation of alpha-acetolactate (AcL)--acetolactate synthase (AcLS) and acetolactate decarboxylase (AcLDC)--were isolated and partially purified from the cells of lactic acid bacteria Lactococcus lactis ssp. lactis biovar. diacetylactis strain 4. The preparation of AcLS, purified 560-fold, had a specific activity of 358,300 U/mg protein (9% yield). The preparation of AcLDC, purified 4828-fold, had a specific activity of 140 U/mg protein (4.8% yield). The enzymes exhibited optimum activity at pH 6.5 and 6.0, respectively (medium, phosphate buffer). The values of apparent Km, determined for AcLS and AcLDC with pyruvate and AcL, respectively, were equal to 70 mM and 20 mM. AcLS appeared as an allosteric enzyme with low affinity for the substrate and a sigmoid dependence of the activity on the substrate concentration. In the case of AcLDC, this dependence was hyperbolic, and the affinity of the enzyme for its substrate was high (Km = 20 mM). Leucine, valine, and isoleucine were shown to be activators of AcDLC.

Acetolactate Synthase↗
PubMed2000

Characterization of OpuA, a glycine-betaine uptake system of Lactococcus lactis.

A Lactococcus lactis glycine-betaine transport system was identified by functional complementation of an Escherichia coli proP proU mutant with a gene library from L. lactis sbsp. cremoris. The cloned locus forms an operon highly homologous to opuA, encoding a glycine-betaine uptake system of Bacillus subtilis. Disruption of opuA in L. lactis abolished protection by glycine-betaine against elevated osmolarity. OpuA belongs to the so-called "ABC transporters" family, which comprise an extracellularly localized substrate-binding protein. In B. subtilis OpuA system, this binding protein is a lipoprotein, attached to the external face of the cytoplasmic membrane by its lipidic moiety. In contrast, in the L. lactis opuA operon, and in other gram-positive homologues as well, a fusion between the gene encoding the integral membrane protein and the substrate-binding protein components gave rise to a hybrid protein presumably attaching the substrate-binding protein to the surface of the cell via its covalent link to the integral membrane component. Mapping of L. lactis opuA transcription start identified one mRNA, more abundant in cells grown at elevated osmolarity. Construction of an opuA-gusA fusion confirmed that opuA transcription is directed by a promoter osmotically inducible in L. lactis. When recombined upstream from a lac transcriptional fusion in the chromosome of E. coli, the opuA promoter appeared as very strong, and only poorly stimulated by elevated osmotic pressure, suggesting the existence of a specific machinery involved in the osmotic signal transduction in L. lactis.

ATP-Binding Cassette Transporters↗
PubMed1999

The role of Escherichia coli RNase E and RNase III in the processing of the citQRP operon mRNA from Lactococcus lactis biovar diacetylactis.

Citrate transport in Lactococcus lactis biovar diacetylactis (L. diacetylactis) is catalyzed by citrate permease P (CitP), which is encoded by the plasmidic citP gene. Two partial overlapping open reading frames citQ and citR are located upstream of citP. These two genes, together with citP, constitute the citQRPoperon. In this report it was shown that in L. diacetylactis and Escherichia coli, cit mRNA is subject to the same specific cleavages at a complex secondary structure which includes the central region of citQ and the 5'-end of citR. The role of ribonucleases in the fate of the cit mRNA processing was investigated in E. coli RNase mutant strains. The results obtained indicate that both endoribonucleases RNase E and RNase III are involved in the generation of mRNA processed species. RNase E is responsible for the major cleavages detected within citQ and upstream of citR, whereas RNase III cleaves citR within its ribosomal binding site. Preliminary results indicate the existence of a RNaselll-like enzyme in L. diacetylactis. Based on these results, a model for the role of cit mRNA processing in the expression of citP is presented.

Bacterial Proteins↗
PubMed2001

[Fusion expression of a peptide antibiotic-apidaecin gene in Lactococcus lactis].

The ubiquitin fusion of apidaecin was expressed in Lactococcus lactis, using a novel nisin-inducible expression system. After induction, a specific band could be detected in the extracts of the host strain by Tricine-SDS-PAGE and Western blotting. Production of the fusion was up to 7.2% of the total soluble protein of the host strain. While the fusion was cut by ubiquitin specific protease-UBP1, the product had distinct antibacterial activity.

Amino Acid Sequence↗
PubMedDate not supplied

[Isolation, purification and properties of acetolactate synthase from cultured Lactococcus lactis].

Acetolactate synthase catalyzing the synthesis of alpha-acetolactate was isolated from lactic acid bacteria Lactococcus lactis subsp. lactis biovar. diacetylactis 4 and purified. Acetolactate synthase was shown to be an allosteric enzyme with low affinity for the substrate: the Km for pyruvate was 70 mM. The curve relating the dependence of enzyme activity on pyruvate concentration had a sigmoid shape. The enzyme activity persisted for 24 h in the presence of stabilizers, pyruvate, and thiamine pyrophosphate. Acetolactate synthase had the pH optimums of 5.8 and 6.5-7.0 in acetate and phosphate buffers, respectively. The temperature optimum for this enzyme was 38-40 degrees C at pH 6.5. The molecular weight of acetolactate synthase was 150 kDa. In Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed that the enzyme consisted of three identical subunits with a molecular weight of 55 kDa.

Acetolactate Synthase↗
PubMed2001

Regulation of pyruvate metabolism in Lactococcus lactis depends on the imbalance between catabolism and anabolism.

Two strains of Lactococcus lactis ssp. cremoris, MG 1820 and MG 1363, which differed by the presence or absence of the lactose plasmid, respectively, were cultivated in batch-mode fermentation on lactose as carbon substrate. A correlation between the rate of sugar consumption, the growth rate, and the type of metabolism was observed. The MG 1820 strain grew rapidly on lactose and homolactic fermentation occurred. The major regulating factor was the NADH/NAD(+) ratio proportional to the catabolic flux, which inhibited glyceraldehyde-3-phosphate dehydrogenase activity. This control led to an increase in metabolite concentration upstream of this enzyme, glyceraldehyde-3-phosphate and dihydroxyacetone-phosphate, and inhibition of pyruvate formate lyase activity, while lactate dehydrogenase was strongly activated by the high coenzyme ratio. The contrary was observed during growth of the MG 1363 strain. Further investigation during growth of L. lactis ssp. lactis NCDO 2118 on galactose as carbon substrate and on various culture media enabling the growth rate to proceed at various rates demonstrated that the relative flux between catabolism and anabolism was the critical regulating parameter rather than the rate of glycolysis itself. In a minimal medium, where anabolism was strongly limited, the rate of sugar consumption was reduced to a low value to avoid carbon and energy waste. Despite this low sugar consumption rate, the catabolic flux was in excess relative to the anabolic capability and the NADH/NAD+ ratio was high, typical of a situation of nonlimiting catabolism leading to a homolactic metabolism.

Cell Division↗
PubMedDate not supplied

[Stability of Lactococcus lactis phages treated with sodium hypochlorite and during storage].

Survival of lytic bacteriophages active against Lactococcus lactis ssp. lactis and ssp. cremoris was determined after treatment with sodium hypochlorite and during storage at 4 degrees C. Three phages were isolated from dairy plants in Argentina (ARG) and the other phages were isolated in the United States of America (US). All of them represent phages that infected cheese manufacture industries and belong to different morphological or serological groups. These phages showed higher survival in M17 broth, buffered with sodium glycerophosphate, than in trypteine soy broth (TSB). Phage populations did not decrease significantly during 14 weeks in M17 broth, whereas in TSB the titers of phage suspensions began to decline around 9 days. In addition, the effect of sodium hypochlorite was more marked in broth than in milk. A higher surviving fraction was obtained in milk, even when tenfold higher concentrations of chlorine were used. The effect of hypochlorite on phages of the same serological group was quite similar and independent of phage morphology. However, phage 137-1, which belongs to other serological group, showed lower resistance to sodium hypochlorite. Comparing the hypochlorite inactivation for ARG and US phages, it was observed that they have their own inactivation values, independently of their origin and morphological group. Long periods of time and high concentrations of chlorine were necessary to reduce the surviving fraction in milk. This indicates that hypochlorite concentrations and times of contact can be critical for the efficiency of the operative sanitization processes.

Animals↗
PubMed2002

Optimization of nisin production by Lactococcus lactis.

The production of nisin by batch culture of Lactococcus lactis ATCC 11454 in MRS broth (pH 6.5), as treated in 30 assays, that were set up by a fractional factorial design of two levels (2[4-1]), was improved. The minimum and maximum concentrations of sucrose (5.0-12.5 g/L), asparagine (7.5-75 g/L), potassium phosphate (6.0-18.0 g/L), and Tween-80 (1.0-6.6 g/L) were added to MRS broth. The best nisin activities ranged from 1.5 x 10(4) to 1.8 x 10(4) arbitrary units (AU)/mL for the maximum levels of sucrose, asparagine, and monobasic potassium phosphate, and for the minimum concentration of Tween-80. The best following proportions between nutrients were adopted as optimum for maximum specific nisin productivity of about 6.0 mg/mg of dry cell weight (related to 2.5 mg of pure nisin preparation with a specific activity of 1.0 x 10(5) AU/mL): C/N = 0.17, C/P = 0.69, N/P = 4.17 (C = sucrose, N = asparagine, P = phosphate, T = Tween-80).

Asparagine↗
PubMed2002

Genome plasticity in Lactococcus lactis.

Comparative genome analyses contribute significantly to our understanding of bacterial evolution and indicate that bacterial genomes are constantly evolving structures. The gene content and organisation of chromosomes of lactic acid bacteria probably result from a strong evolutionary pressure toward optimal growth of these microorganisms in milk. The genome plasticity of Lactococcus lactis was evaluated at inter- and intrasubspecies levels by different experimental approaches. Comparative genomics showed that the lactococcal genomes are not highly plastic although large rearrangements (a.o. deletions, inversions) can occur. Experimental genome shuffling using a new genetic strategy based on the Cre-loxP recombination system revealed that two domains are under strong constraints acting to maintain the original chromosome organisation: a large region around the replication origin, and a smaller one around the putative terminus of replication. Future knowledge of the rules leading to an optimal genome organisation could facilitate the definition of new strategies for industrial strain improvement.

Gene Rearrangement↗
PubMed2002

Experimental determination of control of glycolysis in Lactococcus lactis.

The understanding of control of metabolic processes requires quantitative studies of the importance of the different enzymatic steps for the magnitude of metabolic fluxes and metabolite concentrations. An important element in such studies is the modulation of enzyme activities in small steps above and below the wild-type level. We review a genetic approach that is well suited for both Metabolic Optimization and Metabolic Control Analysis and studies on the importance of a number of glycolytic enzymes for metabolic fluxes in Lactococcus lactis. The glycolytic enzymes phosphofructokinase (PEK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), pyruvate kinase (PYK) and lactate dehydrogenase (LDH) are shown to have no significant control on the glycolytic flux in exponentially growing cells of L. lactis MG1363. Introduction of an uncoupled ATPase activity results in uncoupling of glycolysis from biomass production. With MG1363 growing in defined medium supplemented with glucose, the ATP demanding processes do not have a significant control on the glycolytic flux; it appears that glycolysis is running at maximal rate. It is likely that the flux control is distributed over many enzymes in L. lactis, but it cannot yet be excluded that one of the remaining glycolytic steps is a rate-limiting step for the glycolytic flux.

Gene Expression Regulation, Bacterial↗
PubMed2002

Respiration capacity and consequences in Lactococcus lactis.

We recently reported that the well-studied fermenting bacterium Lactococcus lactis could grow via a respirative metabolism in the presence of oxygen when a heme source is present. Respiration induces profound changes in L. lactis metabolism, and improvement of oxygen tolerance and long-term survival. Compared to usual fermentation conditions, biomass is approximately doubled by the end of growth, acid production is reduced, and large amounts of normally minor end products accumulate. Lactococci grown via respiration survive markedly better after long-term storage than fermenting cells. We suggest that growth and survival of lactococci are optimal under respiration-permissive conditions, and not under fermentation conditions as previously supposed. Our results reveal the uniqueness of the L. lactis respiration model. The well-studied 'aerobic' bacteria express multiple terminal cytochrome oxidases, which assure respiration all throughout growth; they also synthesize their own heme. In contrast, the L. lactis cydAB genes encode a single cytochrome oxidase (bd), and heme must be provided. Furthermore, cydAB genes mediate respiration only late in growth. Thus, lactococci exit the lag phase via fermentation even if heme is present, and start respiration in late exponential phase. Our results suggest that the spectacularly improved survival is in part due to reduced intracellular oxidation during respiration. We predict that lactococcal relatives like the Enterococci, and some Lactobacilli, which have reported respiration potential, will display improved survival under respiration-permissive conditions.

Electron Transport Complex IV↗
PubMed2002

In situ delivery of cytokines by genetically engineered Lactococcus lactis.

The development of novel approaches that allow for accurate targeting of therapeutics to the bowel mucosa is a priority in the research on inflammatory bowel disease. We have engineered Lactococcus lactis to secrete soluble, fully active, correctly processed cytokines. We have used these live, recombinant strains for the in situ delivery of mouse interleukin (mIL)-2, -6 and -10 at airway mucosa or mucosa of the colon. Strains that secrete mIL-2 or mIL-6 and produce TTFC intracellular show a higher level of anti-TTFC induction in mice following intranasal inoculation. We showed that mIL-10 producing L. lactis can prevent and cure enterocolitis in mice. The daily ingestion of this strain leads to the prevention of colitis in IL-10 -/- 129 Sv/Ev mice. The repeated addition of DSS to the drinking water of Balb/c mice leads to the induction of chronic colitis with a typical mean histological score of five points. Subsequent daily treatment with 10(8) IL-10 producing L. lactis reduced the inflammation to a score of approximately 1 in 40% of the treated mice, which is a status equal to that of healthy control mice. Most other animals from the treated group only showed minor patchy remnants of the inflammation. Killing of the IL-10 producing bacteria by UV irradiation immediately prior to inoculation abrogates this therapeutic effect. Therefore it can be attributed to the active in vivo delivery of IL-10. We have further documented this by demonstrating in situ de novo synthesis of IL-10 in the colon of IL-10 -/- mice.

Animals↗
PubMed2002

Gene regulation in Lactococcus lactis: the gap between predicted and characterized regulators.

The genome sequence of Lactococcus lactis IL 1403 was previously determined with high quality, allowing a reliable determination of the potential ORFs present in the genome. It encodes 2310 proteins, and 138 of them were assigned as potential regulators, half of which being further classified by their similarity to known protein families. Among these regulators, most could have a direct role as transcriptional regulators, while the others may have less well defined functions in transcriptional regulation or more general functions, such as the GTP binding protein family. Current knowledge related to the regulators controlling gene expression in L. lactis will be confronted to data obtained in other bacteria. For example, comparison of the L. lactis regulators with those of B. subtilis reveals many orthologous regulators and also some clear differences in the type of regulator used in the two bacteria. Further comparison of the role and the effectors of orthologous regulators shows that direct transposition of a 'heterologous model' does not allow to build a reliable regulatory network in L. lactis. Moreover, many L. lactis regulators have functions that could not be proposed by transposition of the knowledge currently available in other bacteria. A considerable amount of work will be necessary to assess the function of L. lactis regulators and build a comprehensive model of the regulatory network. This would provide invaluable information on L. lactis biology and the way this bacterium interacts with the environment.

Bacterial Proteins↗