Showing papers on "Lactococcus lactis published in 2002"

Diversity, Dynamics, and Activity of Bacterial Communities during Production of an Artisanal Sicilian Cheese as Evaluated by 16S rRNA Analysis

Abstract: The diversity and dynamics of the microbial communities during the manufacturing of Ragusano cheese, an artisanal cheese produced in Sicily (Italy), were investigated by a combination of classical and culture-independent approaches. The latter included PCR, reverse transcriptase-PCR (RT-PCR), and denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes (rDNA). Bacterial and Lactobacillus group-specific primers were used to amplify the V6 to V8 and V1 to V3 regions of the 16S rRNA gene, respectively. DGGE profiles from samples taken during cheese production indicated dramatic shifts in the microbial community structure. Cloning and sequencing of rDNA amplicons revealed that mesophilic lactic acid bacteria (LAB), including species of Leuconostoc, Lactococcus lactis, and Macrococcus caseolyticus were dominant in the raw milk, while Streptococcus thermophilus prevailed during lactic fermentation. Other thermophilic LAB, especially Lactobacillus delbrueckii and Lactobacillus fermentum, also flourished during ripening. Comparison of the rRNA-derived patterns obtained by RT-PCR to the rDNA DGGE patterns indicated a substantially different degree of metabolic activity for the microbial groups detected. Identification of cultivated LAB isolates by phenotypic characterization and 16S rDNA analysis indicated a variety of species, reflecting to a large extent the results obtained from the 16S rDNA clone libraries, with the significant exception of the Lactobacillus delbrueckii species, which dominated in the ripening cheese but was not detected by cultivation. The present molecular approaches combined with culture can effectively describe the complex ecosystem of natural fermented dairy products, giving useful information for starter culture design and preservation of artisanal fermented food technology.

Lactic acid bacteria and yeasts in kefir grains and kefir made from them.

Abstract: In an investigation of the changes in the microflora along the pathway: kefir grains (A)→kefir made from kefir grains (B)→kefir made from kefir as inoculum (C), the following species of lactic acid bacteria (83–90%) of the microbial count in the grains) were identified: Lactococcus lactis subsp lactis, Streptococcus thermophilus, Lactobacillus delbrueckii subsp bulgaricus, Lactobacillus helveticus, Lactobacillus casei subsp pseudoplantarum and Lactobacillus brevis Yeasts (10–17%) identified were Kluyveromyces marxianus var lactis, Saccharomyces cerevisiae, Candida inconspicua and Candida maris In the microbial population of kefir grains and kefir made from them the homofermentative lactic streptococci (52–65% and 79–86%, respectively) predominated Within the group of lactobacilli, the homofermentative thermophilic species L delbrueckii subsp bulgaricus and L helveticus (70–87% of the isolated bacilli) predominated Along the pathway A→B→C, the streptococcal proportion in the total kefir microflora increased by 26–30% whereas the lactobacilli decreased by 13–23% K marxianus var lactis was permanently present in kefir grains and kefirs, whereas the dominant lactose-negative yeast in the total yeast flora of the kefir grains dramatically decreased in kefir C Journal of Industrial Microbiology & Biotechnology (2002) 28, 1–6 DOI: 101038/sj/jim/7000186

A PCR-based method for identification of lactobacilli at the genus level

Abstract: We developed a polymerase chain reaction (PCR)-based method for the identification of lactobacilli at the genus level. One specific primer, LbLMA1-rev, was designed by analysing similarities between the nucleotide sequence of the spacer between the 16S and 23S rRNA genes in a number of Lactobacillus strains. Amplification with LbLMA1-rev and R16-1, a universal primer, generated a PCR product for 23 Lactobacillus species. Electrophoresis did not reveal any discrete bands when Escherichia coli, Lactococcus lactis, Leuconostoc mesenteroides, Streptococcus thermophilus, Carnobacterium pissicola, Pediococcus pentosaceus, Bifidobacterium bifidum, Weissella confusa, Enterococcus hirae, Staphylococcus aureus or Listeria monocytogenes DNA were used as template.

Characterization of the genetic locus responsible for the production of ABP-118, a novel bacteriocin produced by the probiotic bacterium Lactobacillus salivarius subsp. salivarius UCC118

Abstract: ABP-118, a small heat-stable bacteriocin produced by Lactobacillus salivarius subsp. salivarius UCC118, a strain isolated from the ileal–caecal region of the human gastrointestinal tract, was purified to homogeneity. Using reverse genetics, a DNA fragment specifying part of ABP-118 was identified on a 10769 bp chromosomal region. Analysis of this region revealed that ABP-118 was a Class IIb two-peptide bacteriocin composed of Abp118α, which exhibited the antimicrobial activity, and Abp118β, which enhanced the antimicrobial activity. The gene conferring strain UCC118 immunity to the action of ABP-118, abpIM, was identified downstream of the abp118β gene. Located further downstream of abp118β, several ORFs were identified whose deduced proteins resembled those of proteins involved in bacteriocin regulation and secretion. Heterologous expression of ABP-118 was achieved in Lactobacillus plantarum, Lactococcus lactis and Bacillus cereus. In addition, the abp118 locus encoded an inducing peptide, AbpIP, which was shown to play a role in the regulation of ABP-118 production. This novel bacteriocin is, to the authors’ knowledge, the first to be isolated from a known human probiotic bacterium and to be characterized at the genetic level.

Metabolic engineering of lactic acid bacteria, the combined approach: kinetic modelling, metabolic control and experimental analysis

Abstract: Everyone who has ever tried to radically change metabolic fluxes knows that it is often harder to determine which enzymes have to be modified than it is to actually implement these changes. In the more traditional genetic engineering approaches ’bottle-necks’ are pinpointed using qualitative, intuitive approaches, but the alleviation of suspected ’rate-limiting’ steps has not often been successful. Here the authors demonstrate that a model of pyruvate distribution in Lactococcus lactis based on enzyme kinetics in combination with metabolic control analysis clearly indicates the key control points in the flux to acetoin and diacetyl, important flavour compounds. The model presented here (available at http://jjj.biochem.sun.ac.za/wcfs.html) showed that the enzymes with the greatest effect on this flux resided outside the acetolactate synthase branch itself. Experiments confirmed the predictions of the model, i.e. knocking out lactate dehydrogenase and overexpressing NADH oxidase increased the flux through the acetolactate synthase branch from 0 to 75% of measured product formation rates.

Acid- and multistress-resistant mutants of Lactococcus lactis : identification of intracellular stress signals.

Abstract: Lactococcus lactis growth is accompanied by lactic acid production, which results in acidification of the medium and arrest of cell multiplication. Despite growth limitation at low pH, there is evidence that lactococci do have inducible responses to an acid pH. In order to characterize the genes involved in acid tolerance responses, we selected acid-resistant insertional mutants of the L. lactis strain MG1363. Twenty-one independent characterized mutants were affected in 18 different loci, some of which are implicated in transport systems or base metabolism. None of these genes was identified previously as involved in lactococcal acid tolerance. The various phenotypes obtained by acid stress selection allowed us to define four classes of mutants, two of which comprise multistress-resistant strains. Our results reveal that L. lactis has several means of protecting itself against low pH, at least one of which results in multiple stress resistance. In particular, intracellular phosphate and guanine nucleotide pools, notably (p)ppGpp, are likely to act as signals that determine the level of lactococcal stress response induction. Our results provide a link between the physiological state of the cell and the level of stress tolerance and establish a role for the stringent response in acid stress response regulation.

Inhibition of Listeria innocua in cheddar cheese by addition of nisin Z in liposomes or by in situ production in mixed culture.

Abstract: The effect of addition of purified nisin Z in liposomes to cheese milk and of in situ production of nisin Z by Lactococcus lactis subsp. lactis biovar diacetylactis UL719 in the mixed starter on the inhibition of Listeria innocua in cheddar cheese was evaluated during 6 months of ripening. A cheese mixed starter culture containing Lactococcus lactis subsp. lactis biovar diacetylactis UL719 was selected for high-level nisin Z and acid production. Experimental cheddar cheeses were produced on a pilot scale, using the selected starter culture, from milk with added L. innocua (105 to 106 CFU/ml). Liposomes with purified nisin Z were prepared from proliposome H and added to cheese milk prior to renneting to give a final concentration of 300 IU/g of cheese. The nisin Z-producing strain and nisin Z-containing liposomes did not significantly affect cheese production and gross chemical composition of the cheeses. Immediately after cheese production, 3- and 1.5-log-unit reductions in viable counts of L. innocua were obtained in cheeses with encapsulated nisin and the nisinogenic starter, respectively. After 6 months, cheeses made with encapsulated nisin contained less than 10 CFU of L. innocua per g and 90% of the initial nisin activity, compared with 104 CFU/g and only 12% of initial activity in cheeses made with the nisinogenic starter. This study showed that encapsulation of nisin Z in liposomes can provide a powerful tool to improve nisin stability and inhibitory action in the cheese matrix while protecting the cheese starter from the detrimental action of nisin during cheese production.

Two different pathways for D-xylose metabolism and the effect of xylose concentration on the yield coefficient of L-lactate in mixed-acid fermentation by the lactic acid bacterium Lactococcus lactis IO-1.

Abstract: In lactic acid bacteria, pentoses are metabolized via the phosphoketolase pathway, which catalyzes the cleavage of D-xylulose-5-phosphate to equimolar amounts of glyceraldehyde 3-phosphate and acetylphosphate. Hence the yield coefficient of lactate from pentose does not exceed 1.0 mol/mol, while that of Lactococcus lactis IO-1(JCM7638) at high D-xylose concentrations often exceeds the theoretical value. This suggests that, in addition to the phosphoketolase pathway, L. lactisIO-1 may possess another metabolic pathway that produces only lactic acid from xylose. In the present study, the metabolism of xylose in L. lactisIO-1 was deduced from the product formation and enzyme activities of L. lactisIO-1 in batch culture and continuous culture. During cultivation with xylose concentrations above ca. 50 g/l, the yield coefficient of L-lactate exceeded 1.0 mol/mol while those of acetate, formate and ethanol were very low. At xylose concentrations less than 5 g/l, acetate, formate and ethanol were produced with yield coefficients of about 1.0 mol/mol, while L-lactate was scarcely produced. In cells grown at high xylose concentrations, a marked decrease in the specific activities of phosphoketolase and pyruvate formate lyase (PFL), and an increase in those of transketolase and transaldolase were observed. These results indicate that in L. lactisIO-1 xylose may be catabolized by two different pathways, the phosphoketolase pathway yielding acetate, formate and ethanol, and the pentose phosphate (PP)/glycolytic pathway which converts xylose to L-lactate only. Furthermore, it was deduced that the change in the xylose concentration in the culture medium shifts xylulose 5-phosphate metabolism between the phosphoketolase pathway and the PP/glycolytic pathway in L. lactisIO-1, and pyruvate metabolism between cleavage to acetyl-CoA and formic acid by PFL and the reduction to L-lactate by lactate dehydrogenase.

Evaluation of Immunomodulatory Effects of Lactic Acid Bacteria in Turbot (Scophthalmus maximus)

Abstract: In the present work, the effects of several lactic acid bacteria on the immune response of turbot (Scophthalmus maximus) macrophages have been studied both in vitro and in vivo. Out of six lactic acid bacterial strains tested, only heat-killed Lactococcus lactis significantly increased the turbot head kidney macrophage chemiluminescent (CL) response after 24 h of incubation. Nitric oxide (NO) was also significantly enhanced by this bacterium after 72 h of incubation with either viable (10 3 and 10 6 cells/ml) or heat-killed (10 6 cells/ml) bacteria. Viable Leuconostoc mesenteroides (10 6 cells/ml) was also capable of significantly increasing NO production. Since L. lactis proved to be the strain with more effects on the host immune function, further in vivo and in vitro experiments were conducted with this bacterium. The in vitro capacity of L. lactis to adhere to turbot intestinal mucus was positively confirmed. When orally administered, L. lactis significantly increased the macrophage CL response and the serum NO concentration after 7 days of daily administration. The antibacterial effect of the extracellular products from the six LAB strains against the fish-pathogenic bacterium Vibrio anguillarum was also demonstrated in vitro. The increased intensification of aquaculture has led to a high number of disease outbreaks with an increasing range of pathogens. Consequently, the extensive use of broad-spectrum antibiotics in aquaculture has led, as in other fields, to drug resistance problems (32). In order to improve health and welfare in the rearing of these animals, several alternatives such as improved husbandry, nutrition, and water quality; lower stocking densities; and use of vaccines, nonspecific immunostimulants (7), and bacterial probiotics such as lactic acid bacteria (LAB) (9, 26) have been proposed. Probiotics are defined as microbial dietary adjuvants that beneficially affect the host physiology by modulating mucosal and systemic immunity, as well as improving nutritional and microbial balance in the intestinal tract (23). The role of LAB within the digestive tract of endothermic animals and humans has been extensively studied (14, 18, 28), while only a few studies have demonstrated that LAB are part of the normal intestinal microflora in larvae and juvenile fish (25). Most studies with probiotics conducted to date with fish have been undertaken with strains isolated and selected from aquatic environments and cultured animals. The main bacteria tested belonged to the genera Bacillus, Vibrio, and Pseudomonas (9, 22). Few authors have tested in vivo the protection conferred by probiotics in fish experimentally infected with pathogens. Gastesoupe (8) found that the Lactobacillus or Carnobacterium strain isolated from rotifers increased the resistance of turbot larvae against a pathogenic Vibrio sp. Gildberg et al. (10) demonstrated that Carnobacterium divergens decreased the mortality rate of Atlantic cod fry challenged with Vibrio anguillarum but not the mortality of salmon fry challenged with Aeromonas hydrophila. Robertson et al. (27) reported that another strain of Carnobacterium, administered to fingerlings and fry of Atlantic salmon, reduced the mortality caused by Aeromonas salmonicida, Vibrio ordallii, and Yersinia ruckeri but not V. anguillarum. Although the role of LAB as immunomodulators improving nonspecific defenses is well known for mammals (11, 16, 17), this role, as far as we know, still has to be shown for fish. In the present work, we have studied for the first time the effects of several LAB on the nonspecific immune response of turbot (Scophthalmus maximus). The in vitro effect of viable or heat-killed bacteria on the release of reactive radicals (both nitrogen and oxygen radicals) by turbot head kidney (HK) macrophages was determined. In the case of Lactococcus lactis, in vivo experiments to determine its effect on nonspecific immune functions were also performed, as well as experiments to determine its capacity for binding turbot intestinal mucus. Furthermore, in vitro inhibitory activities of filtered LAB culture supernatants were assayed against the pathogenic bacterium V. anguillarum.

Production and Targeting of the Brucella abortus Antigen L7/L12 in Lactococcus lactis: a First Step towards Food-Grade Live Vaccines against Brucellosis

Abstract: Brucella abortus is a facultative intracellular gram-negative bacterial pathogen that infects humans and animals by entry mainly through the digestive tract. B. abortus causes abortion in pregnant cattle and undulant fever in humans. The immunogenic B. abortus ribosomal protein L7/L12 is a promising candidate antigen for the development of oral live vaccines against brucellosis, using food-grade lactic acid bacteria (LAB) as a carrier. The L7/L12 gene was expressed in Lactococcus lactis, the model LAB, under the nisin-inducible promoter. Using different signals, L7/L12 was produced in cytoplasmic, cell-wall-anchored, and secreted forms. Cytoplasmic production of L7/L12 gave a low yield, estimated at 0.5 mg/liter. Interestingly, a secretable form of this normally cytoplasmic protein via fusion with a signal peptide resulted in increased yield of L7/L12 to 3 mg/liter; secretion efficiency (SE) was 35%. A fusion between the mature moiety of the staphylococcal nuclease (Nuc) and L7/L12 further increased yield to 8 mg/liter. Fusion with a synthetic propeptide (LEISSTCDA) previously described as an enhancer for heterologous protein secretion in L. lactis (Y. Le Loir, A. Gruss, S. D. Ehrlich, and P. Langella, J. Bacteriol. 180:1895-1903, 1998) raised the yield to 8 mg/liter and SE to 50%. A surface-anchored L7/L12 form in L. lactis was obtained by fusing the cell wall anchor of Streptococcus pyogenes M6 protein to the C-terminal end of L7/L12. The fusions described allow the production and targeting of L7/L12 in three different locations in L. lactis. This is the first example of a B. abortus antigen produced in a food-grade bacterium and opens new perspectives for alternative vaccine strategies against brucellosis.

Increase of conjugated linoleic acid content in milk by fermentation with lactic acid bacteria

Abstract: The objectives of this study were to identify the factors and procedures responsible for increasing the conjugated linoleic acid (CLA) content in fermented milk. Fourteen lactic acid bacteria were screened for CLA-producing ability using sunflower oil (containing 70% linoleic acid) as a substrate. Among the screened strains, Lactococcus lactis I-01 showed the highest CLA-producing ability. The optimal concentration of sunflower oil for CLA production was 0.1 g/L in whole milk, which accounted for 0.25% of total milk fat. Our results demonstrated that CLA formation in fermented milk could be affected by numerous factors such as bacterial strain, cell number, optimal substrate concentration, and the period of incubation at neutral pH.

Glutamate dehydrogenase activity: a major criterion for the selection of flavour-producing lactic acid bacteria strains

Abstract: Lactic acid bacteria (LAB) have the enzyme potential to transform amino acids into aroma compounds that contribute greatly to cheese flavour. Generally, amino acid conversion by LAB is limited by their low production of α-ketoglutarate since this α-ketoacid is essential for the first step of the conversion. Indeed, we have demonstrated that adding exogenous α-ketoglutarate to cheese curd, as well as using a genetically modified L. lactis strain capable of producing α-ketoglutarate from glutamate, greatly increased the conversion of amino acid to potent aroma compounds in cheese. Here we report the presence of glutamate dehydrogenase (GDH) activity required for the conversion of glutamate to α-ketoglutarate in several ‘natural’ LAB strains, commonly used in cheese manufacturing. Moreover, we show that the ability of LAB to produce aroma compounds from amino acids is closely related to their GDH activity. Therefore, GDH activity appears to be a major criterion for the selection of flavour-producing LAB strains, which could be used as a starter or as an adjunct to intensify flavour formation in some cheeses.

Production of human papillomavirus type 16 E7 protein in Lactococcus lactis.

Abstract: The E7 protein of human papillomavirus type 16 was produced in Lactococcus lactis. Secretion allowed higher production yields than cytoplasmic production. In stationary phase, amounts of cytoplasmic E7 were reduced, while amounts of secreted E7 increased, suggesting a phase-dependent intracellular proteolysis. Fusion of E7 to the staphylococcal nuclease, a stable protein, resulted in a highly stable cytoplasmic protein. This work provides new candidates for development of viral screening systems and for oral vaccine against cervical cancer.

Identification and characterization of phage-resistance genes in temperate lactococcal bacteriophages.

Abstract: The sie2009 gene, which is situated between the genes encoding the repressor and integrase, on the lysogeny module of the temperate lactococcal bacteriophage Tuc2009, was shown to mediate a phage-resistance phenotype in Lactococcus lactis against a number of bacteriophages. The Sie2009 protein is associated with the cell membrane and its expression leaves phage adsorption, transfection and plasmid transformation unaffected, but interferes with plasmid transduction, as well as phage replication. These observations indicate that this resistance is as a result of DNA injection blocking, thus representing a novel superinfection exclusion system. A polymerase chain reaction (PCR)-based strategy was used to screen a number of lactococcal strains for the presence of other prophage-encoded phage-resistance systems. This screening resulted in the identification of two such systems, without homology to sie2009, which were shown to mediate a phage-resistance phenotype similar to that conferred by sie2009. To our knowledge, this is the first description of a phage-encoded super-infection exclusion/injection blocking mechanism in the genus Lactococcus.

Antibacterial Activities of Nisin Z Encapsulated in Liposomes or Produced In Situ by Mixed Culture during Cheddar Cheese Ripening

Abstract: This study investigated both the activity of nisin Z, either encapsulated in liposomes or produced in situ by a mixed starter, against Listeria innocua, Lactococcus spp., and Lactobacillus casei subsp. casei and the distribution of nisin Z in a Cheddar cheese matrix. Nisin Z molecules were visualized using gold-labeled anti-nisin Z monoclonal antibodies and transmission electron microscopy (immune-TEM). Experimental Cheddar cheeses were made using a nisinogenic mixed starter culture, containing Lactococcus lactis subsp. lactis biovar diacetylactis UL 719 as the nisin producer and two nisin-tolerant lactococcal strains and L. casei subsp. casei as secondary flora, and ripened at 7°C for 6 months. In some trials, L. innocua was added to cheese milk at 105 to 106 CFU/ml. In 6-month-old cheeses, 90% of the initial activity of encapsulated nisin (280 ± 14 IU/g) was recovered, in contrast to only 12% for initial nisin activity produced in situ by the nisinogenic starter (300 ± 15 IU/g). During ripening, immune-TEM observations showed that encapsulated nisin was located mainly at the fat/casein interface and/or embedded in whey pockets while nisin produced by biovar diacetylactis UL 719 was uniformly distributed in the fresh cheese matrix but concentrated in the fat area as the cheeses aged. Cell membrane in lactococci appeared to be the main nisin target, while in L. casei subsp. casei and L. innocua, nisin was more commonly observed in the cytoplasm. Cell wall disruption and digestion and lysis vesicle formation were common observations among strains exposed to nisin. Immune-TEM observations suggest several modes of action for nisin Z, which may be genus and/or species specific and may include intracellular target-specific activity. It was concluded that nisin-containing liposomes can provide a powerful tool to improve nisin stability and availability in the cheese matrix.

Use of the alr Gene as a Food-Grade Selection Marker in Lactic Acid Bacteria

Abstract: Both Lactococcus lactis and Lactobacillus plantarum contain a single alr gene, encoding an alanine racemase (EC 5.1.1.1), which catalyzes the interconversion of D-alanine and L-alanine. The alr genes of these lactic acid bacteria were investigated for their application as food-grade selection markers in a heterologous complementation approach. Since isogenic mutants of both species carrying an alr deletion (Deltaalr) showed auxotrophy for D-alanine, plasmids carrying a heterologous alr were constructed and could be selected, since they complemented D-alanine auxotrophy in the L. plantarum Deltaalr and L. lactis Deltaalr strains. Selection was found to be highly stringent, and plasmids were stably maintained over 200 generations of culturing. Moreover, the plasmids carrying the heterologous alr genes could be stably maintained in wild-type strains of L. plantarum and L. lactis by selection for resistance to D-cycloserine, a competitive inhibitor of Alr (600 and 200 micro g/ml, respectively). In addition, a plasmid carrying the L. plantarum alr gene under control of the regulated nisA promoter was constructed to demonstrate that D-cycloserine resistance of L. lactis is linearly correlated to the alr expression level. Finally, the L. lactis alr gene controlled by the nisA promoter, together with the nisin-regulatory genes nisRK, were integrated into the chromosome of L. plantarum Deltaalr. The resulting strain could grow in the absence of D-alanine only when expression of the alr gene was induced with nisin.

Integrated polymerase chain reaction‐based procedures for the detection and identification of species and subspecies of the Gram‐positive bacterial genus Lactococcus

Abstract: Aims: Five species of the Gram-positive bacterial genus Lactococcus (Lactococcus lactis, L. garvieae, L. plantarum, L. piscium and L. raffinolactis) are currently recognized. The aim of this work was to develop a simple approach for the identification of these species, as well as to differentiate the industrially important dairy subspecies L. lactis subsp. lactis and L. lactis subsp. cremoris. Methods and Results: Methods were devised based on specific polymerase chain reaction (PCR) amplifications that exploit differences in the sequences of the 16S ribosomal RNA genes of each species, followed by restriction enzyme cleavage of the PCR products. The techniques developed were used to characterize industrial cheese starter strains of L. lactis and the results were compared with biochemical phenotype and DNA sequence data. Conclusions: The PCR primers designed can be used simultaneously, providing a simple scheme for screening unknown isolates. Strains of L. lactis show heterogeneity in the 16S ribosomal RNA gene sequence. Significance and Impact of the Study: This work provides an integrated set of methods for differentiation and identification of lactococcal species associated with agricultural, veterinary, medical and processed food industries.

Respiration capacity and consequences in Lactococcus lactis.

Abstract: 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.

Caught in the act: the structure of phosphorylated beta-phosphoglucomutase from Lactococcus lactis.

Abstract: Phosphoglucomutases catalyze the interconversion of d-glucose 1-phosphate and d-glucose 6-phosphate, a reaction central to energy metabolism in all cells and to the synthesis of cell wall polysaccharides in bacterial cells. Two classes of phosphoglucomutases (α-PGM and β-PGM) are distinguished on the basis of their specificity for α- and β-glucose-1-phosphate. β-PGM is a member of the haloacid dehalogenase (HAD) superfamily, which includes the sarcoplasmic Ca2+-ATPase, phosphomannomutase, and phosphoserine phosphatase. β-PGM is unusual among family members in that the common phosphoenzyme intermediate exists as a stable ground-state complex in this enzyme. Herein we report, for the first time, the three-dimensional structure of a β-PGM and the first view of the true phosphoenzyme intermediate in the HAD superfamily. The crystal structure of the Mg(II) complex of phosphorylated β-phosphoglucomutase (β-PGM) from Lactococcus lactis has been determined to 2.3 A resolution by multiwavelength anomalous diffract...

The level of pyruvate-formate lyase controls the shift from homolactic to mixed-acid product formation in Lactococcus lactis.

Abstract: Regulation of pyruvate-formate lyase (PFL) activity in vivo plays a central role in the shift from homolactic to mixed-acid product formation observed during the growth of Lactococcus lactis on glucose and galactose, respectively. Characterisation of L. lactis MG1363 in anaerobic batch cultures showed that the specific in vivo activity (flux) of PFL was 4-fold higher in L. lactis cells grown with galactose, compared with cells grown with glucose. The change in the PFL flux correlated with the observed variation in the PFL enzyme level, i.e. the PFL enzyme level was 3.4-fold higher in L. lactis cells grown on galactose than in those grown on glucose. To investigate whether a variation in the level of PFL was responsible for the shift in pyruvate metabolism, L. lactis strains with altered expression of pfl were constructed. The pfl gene was expressed under the control of different constitutive promoters in L. lactis MG1363 and in the PFL-deficient strain CRM40. Strains with five different PFL levels were obtained. Variation in the PFL level markedly affected the resulting end-product formation in these strains. During growth on galactose, the flux towards mixed-acid products was to a great extent controlled by the PFL level. This demonstrates that a regulated PFL level plays a predominant role in the regulation of the metabolic shift from homolactic to mixed-acid product formation in L. lactis.