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Sample records for acid bacterium lactococcus

  1. The Complete Genome Sequence of the Lactic Acid Bacterium Lactococcus lactis ssp. lactis IL1403

    PubMed Central

    Bolotin, Alexander; Wincker, Patrick; Mauger, Stéphane; Jaillon, Olivier; Malarme, Karine; Weissenbach, Jean; Ehrlich, S. Dusko; Sorokin, Alexei

    2001-01-01

    Lactococcus lactis is a nonpathogenic AT-rich gram-positive bacterium closely related to the genus Streptococcus and is the most commonly used cheese starter. It is also the best-characterized lactic acid bacterium. We sequenced the genome of the laboratory strain IL1403, using a novel two-step strategy that comprises diagnostic sequencing of the entire genome and a shotgun polishing step. The genome contains 2,365,589 base pairs and encodes 2310 proteins, including 293 protein-coding genes belonging to six prophages and 43 insertion sequence (IS) elements. Nonrandom distribution of IS elements indicates that the chromosome of the sequenced strain may be a product of recent recombination between two closely related genomes. A complete set of late competence genes is present, indicating the ability of L. lactis to undergo DNA transformation. Genomic sequence revealed new possibilities for fermentation pathways and for aerobic respiration. It also indicated a horizontal transfer of genetic information from Lactococcus to gram-negative enteric bacteria of Salmonella-Escherichia group. [The sequence data described in this paper has been submitted to the GenBank data library under accession no. AE005176.] PMID:11337471

  2. Genome Sequence of the Lactic Acid Bacterium Lactococcus lactis subsp. lactis TOMSC161, Isolated from a Nonscalded Curd Pressed Cheese

    PubMed Central

    Velly, H.; Abraham, A.-L.; Loux, V.; Delacroix-Buchet, A.; Fonseca, F.; Bouix, M.

    2014-01-01

    Lactococcus lactis is a lactic acid bacterium used in the production of many fermented foods, such as dairy products. Here, we report the genome sequence of L. lactis subsp. lactis TOMSC161, isolated from nonscalded curd pressed cheese. This genome sequence provides information in relation to dairy environment adaptation. PMID:25377704

  3. Complete genome sequence of Lactococcus lactis IO-1, a lactic acid bacterium that utilizes xylose and produces high levels of L-lactic acid.

    PubMed

    Kato, Hiroaki; Shiwa, Yuh; Oshima, Kenshiro; Machii, Miki; Araya-Kojima, Tomoko; Zendo, Takeshi; Shimizu-Kadota, Mariko; Hattori, Masahira; Sonomoto, Kenji; Yoshikawa, Hirofumi

    2012-04-01

    We report the complete genome sequence of Lactococcus lactis IO-1 (= JCM7638). It is a nondairy lactic acid bacterium, produces nisin Z, ferments xylose, and produces predominantly L-lactic acid at high xylose concentrations. From ortholog analysis with other five L. lactis strains, IO-1 was identified as L. lactis subsp. lactis.

  4. Complete Genome Sequence of the Prototype Lactic Acid Bacterium Lactococcus lactis subsp. cremoris MG1363▿

    PubMed Central

    Wegmann, Udo; O'Connell-Motherway, Mary; Zomer, Aldert; Buist, Girbe; Shearman, Claire; Canchaya, Carlos; Ventura, Marco; Goesmann, Alexander; Gasson, Michael J.; Kuipers, Oscar P.; van Sinderen, Douwe; Kok, Jan

    2007-01-01

    Lactococcus lactis is of great importance for the nutrition of hundreds of millions of people worldwide. This paper describes the genome sequence of Lactococcus lactis subsp. cremoris MG1363, the lactococcal strain most intensively studied throughout the world. The 2,529,478-bp genome contains 81 pseudogenes and encodes 2,436 proteins. Of the 530 unique proteins, 47 belong to the COG (clusters of orthologous groups) functional category “carbohydrate metabolism and transport,” by far the largest category of novel proteins in comparison with L. lactis subsp. lactis IL1403. Nearly one-fifth of the 71 insertion elements are concentrated in a specific 56-kb region. This integration hot-spot region carries genes that are typically associated with lactococcal plasmids and a repeat sequence specifically found on plasmids and in the “lateral gene transfer hot spot” in the genome of Streptococcus thermophilus. Although the parent of L. lactis MG1363 was used to demonstrate lysogeny in Lactococcus, L. lactis MG1363 carries four remnant/satellite phages and two apparently complete prophages. The availability of the L. lactis MG1363 genome sequence will reinforce its status as the prototype among lactic acid bacteria through facilitation of further applied and fundamental research. PMID:17307855

  5. Lactococcus piscium: a psychrotrophic lactic acid bacterium with bioprotective or spoilage activity in food-a review.

    PubMed

    Saraoui, T; Leroi, F; Björkroth, J; Pilet, M F

    2016-10-01

    The genus Lactococcus comprises 12 species, some known for decades and others more recently described. Lactococcus piscium, isolated in 1990 from rainbow trout, is a psychrotrophic lactic acid bacterium, probably disregarded because most of the strains are unable to grow at 30°C. During the last 10 years, this species has been isolated from a large variety of food: meat, seafood and vegetables, mostly packed under vacuum (VP) or modified atmosphere (MAP) and stored at chilled temperature. Recently, culture-independent techniques used for characterization of microbial ecosystems have highlighted the importance of Lc. piscium in food. Its role in food spoilage varies according to the strain and the food matrix. However, most studies have indicated that Lc. piscium spoils meat, whereas it does not degrade the sensory properties of seafood. Lactococcus piscium strains have a large antimicrobial spectrum, including Gram-positive and negative bacteria. In various seafoods, some strains have a protective effect against spoilage and can extend the sensory shelf-life of the products. They can also inhibit the growth of Listeria monocytogenes, by a cell-to-cell contact-dependent. This article reviews the physiological and genomic characteristics of Lc. piscium and discusses its spoilage or protective activities in food.

  6. Genome Sequence and Transcriptome Analysis of Meat-Spoilage-Associated Lactic Acid Bacterium Lactococcus piscium MKFS47

    PubMed Central

    Johansson, Per; Laine, Pia; Smolander, Olli-Pekka; Sonck, Matti; Rahkila, Riitta; Jääskeläinen, Elina; Paulin, Lars; Auvinen, Petri; Björkroth, Johanna

    2015-01-01

    Lactococcus piscium is a psychrotrophic lactic acid bacterium and is known to be one of the predominant species within spoilage microbial communities in cold-stored packaged foods, particularly in meat products. Its presence in such products has been associated with the formation of buttery and sour off-odors. Nevertheless, the spoilage potential of L. piscium varies dramatically depending on the strain and growth conditions. Additional knowledge about the genome is required to explain such variation, understand its phylogeny, and study gene functions. Here, we present the complete and annotated genomic sequence of L. piscium MKFS47, combined with a time course analysis of the glucose catabolism-based transcriptome. In addition, a comparative analysis of gene contents was done for L. piscium MKFS47 and 29 other lactococci, revealing three distinct clades within the genus. The genome of L. piscium MKFS47 consists of one chromosome, carrying 2,289 genes, and two plasmids. A wide range of carbohydrates was predicted to be fermented, and growth on glycerol was observed. Both carbohydrate and glycerol catabolic pathways were significantly upregulated in the course of time as a result of glucose exhaustion. At the same time, differential expression of the pyruvate utilization pathways, implicated in the formation of spoilage substances, switched the metabolism toward a heterofermentative mode. In agreement with data from previous inoculation studies, L. piscium MKFS47 was identified as an efficient producer of buttery-odor compounds under aerobic conditions. Finally, genes and pathways that may contribute to increased survival in meat environments were considered. PMID:25819958

  7. Genomic features of Lactococcus lactis IO-1, a lactic acid bacterium that utilizes xylose and produces high levels of L-lactic acid.

    PubMed

    Shimizu-Kadota, Mariko; Kato, Hiroaki; Shiwa, Yuh; Oshima, Kenshiro; Machii, Miki; Araya-Kojima, Tomoko; Zendo, Takeshi; Hattori, Masahira; Sonomoto, Kenji; Yoshikawa, Hirofumi

    2013-01-01

    Lactococcus lactis IO-1 (JCM7638) produces L-lactic acid predominantly when grown at high xylose concentrations, and its utilization is highly desired in the green plastics industry. Therefore it is worthwhile studying its genomic traits. In this study, we focused on (i) genes of possible horizontal transfer derivation (prophages, the nisin-sucrose transposon, and several restriction-modification systems), and (ii) genes for the synthetic pathways of amino acids and vitamins in the IO-1 genome. In view of the results of this analysis, we consider their meanings in strain IO-1.

  8. Hyaluronic acid production by recombinant Lactococcus lactis.

    PubMed

    Chien, Liang-Jung; Lee, Cheng-Kang

    2007-11-01

    Microbial hyaluronic acid (HA), commonly produced by pathogenic Streptococcus, was made possible to be produced by a generally recognized as safe Lactococcus lactis by coexpressing HA synthase and uridine diphosphate-glucose dehydrogenase (UDP-GlcDH) of Streptococcus equi subsp. zooepidemicus in a nisin-controlled expression (NICE) system. With scarce expressed HA synthase alone, the constructed recombinant L. lactis (LL-NA) strain could produce HA with a concentration about 0.08 g/l in the M17 medium supplemented with 1% (w/v) glucose. In contrast to HA synthase, UDP-GlcDH of Streptococcus could be overexpressed in the NICE system. With coexpression of heterologous UDP-GlcDH with HA synthase, the constructed LL-NAB strain grew slightly slower to a concentration about 10% lower that of the LL-NA strain. However, the HA concentration produced was enhanced about eightfold to 0.65 g/l.

  9. Deciphering a unique biotin scavenging pathway with redundant genes in the probiotic bacterium Lactococcus lactis

    PubMed Central

    Zhang, Huimin; Wang, Qingjing; Fisher, Derek J.; Cai, Mingzhu; Chakravartty, Vandana; Ye, Huiyan; Li, Ping; Solbiati, Jose O.; Feng, Youjun

    2016-01-01

    Biotin protein ligase (BPL) is widespread in the three domains of the life. The paradigm BPL is the Escherichia coli BirA protein, which also functions as a repressor for the biotin biosynthesis pathway. Here we report that Lactococcus lactis possesses two different orthologues of birA (birA1_LL and birA2_LL). Unlike the scenario in E. coli, L. lactis appears to be auxotrophic for biotin in that it lacks a full biotin biosynthesis pathway. In contrast, it retains two biotin transporter-encoding genes (bioY1_LL and bioY2_LL), suggesting the use of a scavenging strategy to obtain biotin from the environment. The in vivo function of the two L. lactis birA genes was judged by their abilities to complement the conditional lethal E. coli birA mutant. Thin-layer chromatography and mass spectroscopy assays demonstrated that these two recombinant BirA proteins catalyze the biotinylation reaction of the acceptor biotin carboxyl carrier protein (BCCP), through the expected biotinoyl-AMP intermediate. Gel shift assays were used to characterize bioY1_LL and BirA1_LL. We also determined the ability to uptake 3H-biotin by L. lactis. Taken together, our results deciphered a unique biotin scavenging pathway with redundant genes present in the probiotic bacterium L. lactis. PMID:27161258

  10. Draft Genome Sequences of 24 Lactococcus lactis Strains

    PubMed Central

    Backus, Lennart; Wels, Michiel; Boekhorst, Jos; Dijkstra, Annereinou R.; Beerthuyzen, Marke; Kelly, William J.; Siezen, Roland J.; van Hijum, Sacha A. F. T.

    2017-01-01

    ABSTRACT The lactic acid bacterium Lactococcus lactis is widely used for the production of fermented dairy products. Here, we present the draft genome sequences of 24 L. lactis strains isolated from different environments and geographic locations. PMID:28360177

  11. Draft Genome Sequences of 11 Lactococcus lactis subsp. cremoris Strains

    PubMed Central

    Backus, Lennart; Boekhorst, Jos; Dijkstra, Annereinou; Beerthuyzen, Marke; Siezen, Roland J.; Bachmann, Herwig; van Hijum, Sacha A. F. T.

    2017-01-01

    ABSTRACT The lactic acid bacterium Lactococcus lactis is widely used for the fermentation of dairy products. Here, we present the draft genome sequences of 11 L. lactis subsp. cremoris strains isolated from different environments. PMID:28302789

  12. Dynamic modeling of lactic acid fermentation metabolism with Lactococcus lactis.

    PubMed

    Oh, Euhlim; Lu, Mingshou; Park, Changhun; Park, Changhun; Oh, Han Bin; Lee, Sang Yup; Lee, Jinwon

    2011-02-01

    A dynamic model of lactic acid fermentation using Lactococcus lactis was constructed, and a metabolic flux analysis (MFA) and metabolic control analysis (MCA) were performed to reveal an intensive metabolic understanding of lactic acid bacteria (LAB). The parameter estimation was conducted with COPASI software to construct a more accurate metabolic model. The experimental data used in the parameter estimation were obtained from an LC-MS/ MS analysis and time-course simulation study. The MFA results were a reasonable explanation of the experimental data. Through the parameter estimation, the metabolic system of lactic acid bacteria can be thoroughly understood through comparisons with the original parameters. The coefficients derived from the MCA indicated that the reaction rate of L-lactate dehydrogenase was activated by fructose 1,6-bisphosphate and pyruvate, and pyruvate appeared to be a stronger activator of L-lactate dehydrogenase than fructose 1,6-bisphosphate. Additionally, pyruvate acted as an inhibitor to pyruvate kinase and the phosphotransferase system. Glucose 6-phosphate and phosphoenolpyruvate showed activation effects on pyruvate kinase. Hexose transporter was the strongest effector on the flux through L-lactate dehydrogenase. The concentration control coefficient (CCC) showed similar results to the flux control coefficient (FCC).

  13. CXCR4(+)-targeted protein nanoparticles produced in the food-grade bacterium Lactococcus lactis.

    PubMed

    Cano-Garrido, Olivia; Céspedes, María Virtudes; Unzueta, Ugutz; Saccardo, Paolo; Roldán, Mònica; Sánchez-Chardi, Alejandro; Cubarsi, Rafael; Vázquez, Esther; Mangues, Ramon; García-Fruitós, Elena; Villaverde, Antonio

    2016-09-01

    Lactococcus lactis is a Gram-positive (endotoxin-free) food-grade bacteria exploited as alternative to Escherichia coli for recombinant protein production. We have explored here for the first time the ability of this platform as producer of complex, self-assembling protein materials. Biophysical properties, cell penetrability and in vivo biodistribution upon systemic administration of tumor-targeted protein nanoparticles produced in L. lactis have been compared with the equivalent material produced in E. coli. Protein nanoparticles have been efficiently produced in L. lactis, showing the desired size, internalization properties and biodistribution. In vitro and in vivo data confirm the potential and robustness of the production platform, pointing out L. lactis as a fascinating cell factory for the biofabrication of protein materials intended for therapeutic applications.

  14. Cyclopropanation of membrane unsaturated fatty acids is not essential to the acid stress response of Lactococcus lactis subsp. cremoris.

    PubMed

    To, Thi Mai Huong; Grandvalet, Cosette; Tourdot-Maréchal, Raphaëlle

    2011-05-01

    Cyclopropane fatty acids (CFAs) are synthetized in situ by the transfer of a methylene group from S-adenosyl-L-methionine to a double bond of unsaturated fatty acid chains of membrane phospholipids. This conversion, catalyzed by the Cfa synthase enzyme, occurs in many bacteria and is recognized to play a key role in the adaptation of bacteria in response to a drastic perturbation of the environment. The role of CFAs in the acid tolerance response was investigated in the lactic acid bacterium Lactococcus lactis MG1363. A mutant of the cfa gene was constructed by allelic exchange. The cfa gene encoding the Cfa synthase was cloned and introduced into the mutant to obtain the complemented strain for homologous system studies. Data obtained by gas chromatography (GC) and GC-mass spectrometry (GC-MS) validated that the mutant could not produce CFA. The CFA levels in both the wild-type and complemented strains increased upon their entry to stationary phase, especially with acid-adapted cells or, more surprisingly, with ethanol-adapted cells. The results obtained by performing quantitative reverse transcription-PCR (qRT-PCR) experiments showed that transcription of the cfa gene was highly induced by acidity (by 10-fold with cells grown at pH 5.0) and by ethanol (by 9-fold with cells grown with 6% ethanol) in comparison with that in stationary phase. Cell viability experiments were performed after an acidic shock on the mutant strain, the wild-type strain, and the complemented strain, as a control. The higher viability level of the acid-adapted cells of the three strains after 3 h of shock proved that the cyclopropanation of unsaturated fatty acids is not essential for L. lactis subsp. cremoris survival under acidic conditions. Moreover, fluorescence anisotropy data showed that CFA itself could not maintain the membrane fluidity level, particularly with ethanol-grown cells.

  15. Cyclopropanation of Membrane Unsaturated Fatty Acids Is Not Essential to the Acid Stress Response of Lactococcus lactis subsp. cremoris ▿

    PubMed Central

    To, Thi Mai Huong; Grandvalet, Cosette; Tourdot-Maréchal, Raphaëlle

    2011-01-01

    Cyclopropane fatty acids (CFAs) are synthetized in situ by the transfer of a methylene group from S-adenosyl-l-methionine to a double bond of unsaturated fatty acid chains of membrane phospholipids. This conversion, catalyzed by the Cfa synthase enzyme, occurs in many bacteria and is recognized to play a key role in the adaptation of bacteria in response to a drastic perturbation of the environment. The role of CFAs in the acid tolerance response was investigated in the lactic acid bacterium Lactococcus lactis MG1363. A mutant of the cfa gene was constructed by allelic exchange. The cfa gene encoding the Cfa synthase was cloned and introduced into the mutant to obtain the complemented strain for homologous system studies. Data obtained by gas chromatography (GC) and GC-mass spectrometry (GC-MS) validated that the mutant could not produce CFA. The CFA levels in both the wild-type and complemented strains increased upon their entry to stationary phase, especially with acid-adapted cells or, more surprisingly, with ethanol-adapted cells. The results obtained by performing quantitative reverse transcription-PCR (qRT-PCR) experiments showed that transcription of the cfa gene was highly induced by acidity (by 10-fold with cells grown at pH 5.0) and by ethanol (by 9-fold with cells grown with 6% ethanol) in comparison with that in stationary phase. Cell viability experiments were performed after an acidic shock on the mutant strain, the wild-type strain, and the complemented strain, as a control. The higher viability level of the acid-adapted cells of the three strains after 3 h of shock proved that the cyclopropanation of unsaturated fatty acids is not essential for L. lactis subsp. cremoris survival under acidic conditions. Moreover, fluorescence anisotropy data showed that CFA itself could not maintain the membrane fluidity level, particularly with ethanol-grown cells. PMID:21421775

  16. An in vitro investigation of Lactococcus lactis antagonizing cariogenic bacterium Streptococcus mutans.

    PubMed

    Tong, Zhongchun; Zhou, Lin; Li, Jie; Kuang, Rong; Lin, Yuan; Ni, Longxing

    2012-04-01

    The present study tested the antagonism between Lactococcus lactis and Streptococcus mutans and evaluated the feasibility of the application of L. lactis for the inhibition of S. mutans in the oral cavity. Competition assays on plates were employed to determine whether L. lactis antagonises S. mutans under different nutritional conditions, and real-time reverse-transcriptase PCR was used to evaluate the effects of metabolites of S. mutans on the bacteriocin nisin genes in L. lactis. Furthermore, the colonisation and effects of L. lactis on the surface of a tooth were examined by scanning electron microscopy. L. lactis competitively inhibited S. mutans growth under nutritional deficiency, and the metabolites of S. mutans, including several exogenous molecular signals, enhanced the expression of genes related to nisin synthesis, nisA, nisB and nisI. Additionally, L. lactis effectively colonised the surface of tooth enamel, which showed substantially less decay with L. lactis adhesion compared to S. mutans adhesion. These findings suggest avenues of research into a new strategy to reduce major cariogenic S. mutans adhesion on the surfaces of teeth. Copyright © 2011 Elsevier Ltd. All rights reserved.

  17. Respiration Capacity of the Fermenting Bacterium Lactococcus lactis and Its Positive Effects on Growth and Survival†

    PubMed Central

    Duwat, Patrick; Sourice, Sophie; Cesselin, Bénédicte; Lamberet, Gilles; Vido, Karin; Gaudu, Philippe; Le Loir, Yves; Violet, Florent; Loubière, Pascal; Gruss, Alexandra

    2001-01-01

    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. PMID:11443085

  18. Sulfur Amino Acid Metabolism and Its Control in Lactococcus lactis IL1403

    PubMed Central

    Sperandio, Brice; Polard, Patrice; Ehrlich, Dusko S.; Renault, Pierre; Guédon, Eric

    2005-01-01

    Cysteine and methionine availability influences many processes in the cell. In bacteria, transcription of the specific genes involved in the synthesis of these two amino acids is usually regulated by different mechanisms or regulators. Pathways for the synthesis of cysteine and methionine and their interconversion were experimentally determined for Lactococcus lactis, a lactic acid bacterium commonly found in food. A new gene, yhcE, was shown to be involved in methionine recycling to cysteine. Surprisingly, 18 genes, representing almost all genes of these pathways, are under the control of a LysR-type activator, FhuR, also named CmbR. DNA microarray experiments showed that FhuR targets are restricted to this set of 18 genes clustered in seven transcriptional units, while cysteine starvation modifies the transcription level of several other genes potentially involved in oxidoreduction processes. Purified FhuR binds a 13-bp box centered 46 to 53 bp upstream of the transcriptional starts from the seven regulated promoters, while a second box with the same consensus is present upstream of the first binding box, separated by 8 to 10 bp. O-Acetyl serine increases FhuR binding affinity to its binding boxes. The overall view of sulfur amino acid metabolism and its regulation in L. lactis indicates that CysE could be a master enzyme controlling the activity of FhuR by providing its effector, while other controls at the enzymatic level appear to be necessary to compensate the absence of differential regulation of the genes involved in the interconversion of methionine and cysteine and other biosynthesis genes. PMID:15901700

  19. Expression of PprI from Deinococcus radiodurans Improves Lactic Acid Production and Stress Tolerance in Lactococcus lactis

    PubMed Central

    Dong, Xiangrong; Tian, Bing; Dai, Shang; Li, Tao; Guo, Linna; Tan, Zhongfang; Jiao, Zhen; Jin, Qingsheng; Wang, Yanping; Hua, Yuejin

    2015-01-01

    PprI is a general switch protein that regulates the expression of certain proteins involved in pathways of cellular resistance in the extremophilic bacterium Deinococcus radiodurans. In this study, we transformed pprI into Lactococcus lactis strain MG1363 using the lactococcal shuttle vector pMG36e and investigated its effects on the tolerance and lactic acid production of L. lactis while under stress. PprI was stably expressed in L. lactis as confirmed by western blot assays. L. lactis expressing PprI exhibited significantly improved resistance to oxidative stress and high osmotic pressure. This enhanced cellular tolerance to stressors might be due to the regulation of resistance-related genes (e.g., recA, recO, sodA, and nah) by pprI. Moreover, transformed L. lactis demonstrated increased lactic acid production, attributed to enhanced lactate dehydrogenase activity. These results suggest that pprI can improve the tolerance of L. lactis to environmental stresses, and this transformed bacterial strain is a promising candidate for industrial applications of lactic acid production. PMID:26562776

  20. Expression of PprI from Deinococcus radiodurans Improves Lactic Acid Production and Stress Tolerance in Lactococcus lactis.

    PubMed

    Dong, Xiangrong; Tian, Bing; Dai, Shang; Li, Tao; Guo, Linna; Tan, Zhongfang; Jiao, Zhen; Jin, Qingsheng; Wang, Yanping; Hua, Yuejin

    2015-01-01

    PprI is a general switch protein that regulates the expression of certain proteins involved in pathways of cellular resistance in the extremophilic bacterium Deinococcus radiodurans. In this study, we transformed pprI into Lactococcus lactis strain MG1363 using the lactococcal shuttle vector pMG36e and investigated its effects on the tolerance and lactic acid production of L. lactis while under stress. PprI was stably expressed in L. lactis as confirmed by western blot assays. L. lactis expressing PprI exhibited significantly improved resistance to oxidative stress and high osmotic pressure. This enhanced cellular tolerance to stressors might be due to the regulation of resistance-related genes (e.g., recA, recO, sodA, and nah) by pprI. Moreover, transformed L. lactis demonstrated increased lactic acid production, attributed to enhanced lactate dehydrogenase activity. These results suggest that pprI can improve the tolerance of L. lactis to environmental stresses, and this transformed bacterial strain is a promising candidate for industrial applications of lactic acid production.

  1. Cooperation between Lactococcus lactis and nonstarter lactobacilli in the formation of cheese aroma from amino acids.

    PubMed

    Kieronczyk, Agnieszka; Skeie, Siv; Langsrud, Thor; Yvon, Mireille

    2003-02-01

    In Gouda and Cheddar type cheeses the amino acid conversion to aroma compounds, which is a major process for aroma formation, is essentially due to lactic acid bacteria (LAB). In order to evaluate the respective role of starter and nonstarter LAB and their interactions in cheese flavor formation, we compared the catabolism of phenylalanine, leucine, and methionine by single strains and strain mixtures of Lactococcus lactis subsp. cremoris NCDO763 and three mesophilic lactobacilli. Amino acid catabolism was studied in vitro at pH 5.5, by using radiolabeled amino acids as tracers. In the presence of alpha-ketoglutarate, which is essential for amino acid transamination, the lactobacillus strains degraded less amino acids than L. lactis subsp. cremoris NCDO763, and produced mainly nonaromatic metabolites. L. lactis subsp. cremoris NCDO763 produced mainly the carboxylic acids, which are important compounds for cheese aroma. However, in the reaction mixture containing glutamate, only two lactobacillus strains degraded amino acids significantly. This was due to their glutamate dehydrogenase (GDH) activity, which produced alpha-ketoglutarate from glutamate. The combination of each of the GDH-positive lactobacilli with L. lactis subsp. cremoris NCDO763 had a beneficial effect on the aroma formation. Lactobacilli initiated the conversion of amino acids by transforming them mainly to keto and hydroxy acids, which subsequently were converted to carboxylic acids by the Lactococcus strain. Therefore, we think that such cooperation between starter L. lactis and GDH-positive lactobacilli can stimulate flavor development in cheese.

  2. Cooperation between Lactococcus lactis and Nonstarter Lactobacilli in the Formation of Cheese Aroma from Amino Acids

    PubMed Central

    Kieronczyk, Agnieszka; Skeie, Siv; Langsrud, Thor; Yvon, Mireille

    2003-01-01

    In Gouda and Cheddar type cheeses the amino acid conversion to aroma compounds, which is a major process for aroma formation, is essentially due to lactic acid bacteria (LAB). In order to evaluate the respective role of starter and nonstarter LAB and their interactions in cheese flavor formation, we compared the catabolism of phenylalanine, leucine, and methionine by single strains and strain mixtures of Lactococcus lactis subsp. cremoris NCDO763 and three mesophilic lactobacilli. Amino acid catabolism was studied in vitro at pH 5.5, by using radiolabeled amino acids as tracers. In the presence of α-ketoglutarate, which is essential for amino acid transamination, the lactobacillus strains degraded less amino acids than L. lactis subsp. cremoris NCDO763, and produced mainly nonaromatic metabolites. L. lactis subsp. cremoris NCDO763 produced mainly the carboxylic acids, which are important compounds for cheese aroma. However, in the reaction mixture containing glutamate, only two lactobacillus strains degraded amino acids significantly. This was due to their glutamate dehydrogenase (GDH) activity, which produced α-ketoglutarate from glutamate. The combination of each of the GDH-positive lactobacilli with L. lactis subsp. cremoris NCDO763 had a beneficial effect on the aroma formation. Lactobacilli initiated the conversion of amino acids by transforming them mainly to keto and hydroxy acids, which subsequently were converted to carboxylic acids by the Lactococcus strain. Therefore, we think that such cooperation between starter L. lactis and GDH-positive lactobacilli can stimulate flavor development in cheese. PMID:12570989

  3. Engineering of EPA/DHA omega-3 fatty acid production by Lactococcus lactis subsp. cremoris MG1363.

    PubMed

    Amiri-Jami, Mitra; Lapointe, Gisele; Griffiths, Mansel W

    2014-04-01

    Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been shown to be of major importance in human health. Therefore, these essential polyunsaturated fatty acids have received considerable attention in both human and farm animal nutrition. Currently, fish and fish oils are the main dietary sources of EPA/DHA. To generate sustainable novel sources for EPA and DHA, the 35-kb EPA/DHA synthesis gene cluster was isolated from a marine bacterium, Shewanella baltica MAC1. To streamline the introduction of the genes into food-grade microorganisms such as lactic acid bacteria, unnecessary genes located upstream and downstream of the EPA/DHA gene cluster were deleted. Recombinant Escherichia coli harboring the 20-kb gene cluster produced 3.5- to 6.1-fold more EPA than those carrying the 35-kb DNA fragment coding for EPA/DHA synthesis. The 20-kb EPA/DHA gene cluster was cloned into a modified broad-host-range low copy number vector, pIL252m (4.7 kb, Ery) and expressed in Lactococcus lactis subsp. cremoris MG1363. Recombinant L. lactis produced DHA (1.35 ± 0.5 mg g(-1) cell dry weight) and EPA (0.12 ± 0.04 mg g(-1) cell dry weight). This is believed to be the first successful cloning and expression of EPA/DHA synthesis gene cluster in lactic acid bacteria. Our findings advance the future use of EPA/DHA-producing lactic acid bacteria in such applications as dairy starters, silage adjuncts, and animal feed supplements.

  4. Hyaluronic acid production is enhanced by the additional co-expression of UDP-glucose pyrophosphorylase in Lactococcus lactis.

    PubMed

    Prasad, Shashi Bala; Jayaraman, Guhan; Ramachandran, K B

    2010-03-01

    Hyaluronic acid (HA) production was metabolically engineered in Lactococcus lactis by introducing the HA synthetic machinery from the has operon of the pathogenic bacterium Streptococcus zooepidemicus. This study shows that the insertion of uridine diphosphate (UDP)-glucose pyrophosphorylase (hasC) gene in addition to the HA synthase (hasA) and UDP-glucose dehydrogenase (hasB) genes has a significant impact on increasing HA production. The recombinant L. lactis NZ9000 strain transformed with the plasmid pSJR2 (co-expressing hasA and hasB genes only) produced a maximum of 107 mg/l HA in static flask experiments with varying initial glucose concentrations, while the corresponding experiments with the transformant SJR3 (co-expressing hasA, hasB, and hasC genes) gave a maximum yield of 234 mg/l HA. The plasmid cloned with the insertion of the full has operon comprising of five different genes (hasA, hasB, hasC, hasD, and hasE) exhibited structural instability. The HA yield was further enhanced in batch bioreactor experiments with controlled pH and aeration, and a maximum of 1.8 g/l HA was produced by the SJR3 culture.

  5. Activation of the Diacetyl/Acetoin Pathway in Lactococcus lactis subsp. lactis bv. diacetylactis CRL264 by Acidic Growth▿ †

    PubMed Central

    García-Quintáns, Nieves; Repizo, Guillermo; Martín, Mauricio; Magni, Christian; López, Paloma

    2008-01-01

    Lactococcus lactis subsp. lactis bv. diacetylactis strains are aroma-producing organisms used in starter cultures for the elaboration of dairy products. This species is essentially a fermentative microorganism, which cometabolizes glucose and citrate to yield aroma compounds through the diacetyl/acetoin biosynthetic pathway. Our previous results have shown that under acidic growth Lactococcus bv. diacetylactis CRL264 expresses coordinately the genes responsible for citrate transport and its conversion into pyruvate. In the present work the impact of acidic growth on glucose, citrate, and pyruvate metabolism of Lactococcus bv. diacetylactis CRL264 has been investigated by proteomic analysis. The results indicated that acid growth triggers the conversion of citrate, but not glucose, into α-acetolactate via pyruvate. Moreover, they showed that low pH has no influence on levels of lactate dehydrogenase and pyruvate dehydrogenase. Therefore, the influence of external pH on regulation of the diacetyl/acetoin biosynthetic pathway in Lactococcus bv. diacetylactis CRL264 has been analyzed at the transcriptional level. Expression of the als, aldB, aldC, and butBA genes encoding the enzymes involved in conversion of pyruvate into aroma compounds has been investigated by primer extension, reverse transcription-PCR analysis, and transcriptional fusions. The results support that this biosynthetic pathway is induced at the transcriptional level by acidic growth conditions, presumably contributing to lactococcal pH homeostasis by synthesis of neutral compounds and by decreasing levels of pyruvate. PMID:18245243

  6. Chemically defined media and auxotrophy of the prolific l-lactic acid producer Lactococcus lactis IO-1.

    PubMed

    Machii, Miki; Watanabe, Satoru; Zendo, Takeshi; Chibazakura, Taku; Sonomoto, Kenji; Shimizu-Kadota, Mariko; Yoshikawa, Hirofumi

    2013-05-01

    Two chemically defined media, CDM-1G and CDM-1X, that use glucose and xylose as carbon sources, respectively, were prepared for Lactococcus lactis strain IO-1. The maximal cell density at 600 nm in CDM-1G exceeded 2. Omission growth experiments indicated that IO-1 is auxotrophic for 2 vitamins and 6 amino acids.

  7. An aminotransferase from Lactococcus lactis initiates conversion of amino acids to cheese flavor compounds.

    PubMed Central

    Yvon, M; Thirouin, S; Rijnen, L; Fromentier, D; Gripon, J C

    1997-01-01

    The enzymatic degradation of amino acids in cheese is believed to generate aroma compounds and therefore to be involved in the complex process of cheese flavor development. In lactococci, transamination is the first step in the degradation of aromatic and branched-chain amino acids which are precursors of aroma compounds. Here, the major aromatic amino acid aminotransferase of a Lactococcus lactis subsp. cremoris strain was purified and characterized. The enzyme transaminates the aromatic amino acids, leucine, and methionine. It uses the ketoacids corresponding to these amino acids and alpha-ketoglutarate as amino group acceptors. In contrast to most bacterial aromatic aminotransferases, it does not act on aspartate and does not use oxaloacetate as second substrate. It is essential for the transformation of aromatic amino acids to flavor compounds. It is a pyridoxal 5'-phosphate-dependent enzyme and is composed of two identical subunits of 43.5 kDa. The activity of the enzyme is optimal between pH 6.5 and 8 and between 35 and 45 degrees C, but it is still active under cheese-ripening conditions. PMID:9023921

  8. Complete Genome Sequence of Lactococcus piscium CNCM I-4031, a Bioprotective Strain for Seafood Products

    PubMed Central

    Marché, Laurent; Saraoui, Taous; Remenant, Benoit; Zagorec, Monique; Prévost, Hervé; Delbarre-Ladrat, Christine; Leroi, Françoise

    2017-01-01

    ABSTRACT Lactococcus piscium CNCM I-4031 is a psychotrophic foodborne lactic acid bacterium showing potential interest for the biopreservation of seafood products due to its inhibition properties toward pathogenic and spoilage bacteria. The analysis of its genome will provide a better understanding of the mechanisms of interaction between these bacteria. PMID:28126939

  9. Antagonistic lactic acid bacteria isolated from goat milk and identification of a novel nisin variant Lactococcus lactis

    PubMed Central

    2014-01-01

    Background The raw goat milk microbiota is considered a good source of novel bacteriocinogenic lactic acid bacteria (LAB) strains that can be exploited as an alternative for use as biopreservatives in foods. The constant demand for such alternative tools justifies studies that investigate the antimicrobial potential of such strains. Results The obtained data identified a predominance of Lactococcus and Enterococcus strains in raw goat milk microbiota with antimicrobial activity against Listeria monocytogenes ATCC 7644. Enzymatic assays confirmed the bacteriocinogenic nature of the antimicrobial substances produced by the isolated strains, and PCR reactions detected a variety of bacteriocin-related genes in their genomes. Rep-PCR identified broad genetic variability among the Enterococcus isolates, and close relations between the Lactococcus strains. The sequencing of PCR products from nis-positive Lactococcus allowed the identification of a predicted nisin variant not previously described and possessing a wide inhibitory spectrum. Conclusions Raw goat milk was confirmed as a good source of novel bacteriocinogenic LAB strains, having identified Lactococcus isolates possessing variations in their genomes that suggest the production of a nisin variant not yet described and with potential for use as biopreservatives in food due to its broad spectrum of action. PMID:24521354

  10. Antagonistic lactic acid bacteria isolated from goat milk and identification of a novel nisin variant Lactococcus lactis.

    PubMed

    Perin, Luana Martins; Nero, Luís Augusto

    2014-02-12

    The raw goat milk microbiota is considered a good source of novel bacteriocinogenic lactic acid bacteria (LAB) strains that can be exploited as an alternative for use as biopreservatives in foods. The constant demand for such alternative tools justifies studies that investigate the antimicrobial potential of such strains. The obtained data identified a predominance of Lactococcus and Enterococcus strains in raw goat milk microbiota with antimicrobial activity against Listeria monocytogenes ATCC 7644. Enzymatic assays confirmed the bacteriocinogenic nature of the antimicrobial substances produced by the isolated strains, and PCR reactions detected a variety of bacteriocin-related genes in their genomes. Rep-PCR identified broad genetic variability among the Enterococcus isolates, and close relations between the Lactococcus strains. The sequencing of PCR products from nis-positive Lactococcus allowed the identification of a predicted nisin variant not previously described and possessing a wide inhibitory spectrum. Raw goat milk was confirmed as a good source of novel bacteriocinogenic LAB strains, having identified Lactococcus isolates possessing variations in their genomes that suggest the production of a nisin variant not yet described and with potential for use as biopreservatives in food due to its broad spectrum of action.

  11. Enhance nisin yield via improving acid-tolerant capability of Lactococcus lactis F44

    PubMed Central

    Zhang, Jian; Caiyin, Qinggele; Feng, Wenjing; Zhao, Xiuli; Qiao, Bin; Zhao, Guangrong; Qiao, Jianjun

    2016-01-01

    Traditionally, nisin was produced industrially by using Lactococcus lactis in the neutral fermentation process. However, nisin showed higher activity in the acidic environment. How to balance the pH value for bacterial normal growth and nisin activity might be the key problem. In this study, 17 acid-tolerant genes and 6 lactic acid synthetic genes were introduced in L. lactis F44, respectively. Comparing to the 2810 IU/mL nisin yield of the original strain F44, the nisin titer of the engineered strains over-expressing hdeAB, ldh and murG, increased to 3850, 3979 and 4377 IU/mL, respectively. These engineered strains showed more stable intracellular pH value during the fermentation process. Improvement of lactate production could partly provide the extra energy for the expression of acid tolerance genes during growth. Co-overexpression of hdeAB, murG, and ldh(Z) in strain F44 resulted in the nisin titer of 4913 IU/mL. The engineered strain (ABGL) could grow on plates with pH 4.2, comparing to the surviving pH 4.6 of strain F44. The fed-batch fermentation showed nisin titer of the co-expression L. lactis strain could reach 5563 IU/mL with lower pH condition and longer cultivation time. This work provides a novel strategy of constructing robust strains for use in industry process. PMID:27306587

  12. Identification of a novel operon in Lactococcus lactis encoding three enzymes for lactic acid synthesis: phosphofructokinase, pyruvate kinase, and lactate dehydrogenase.

    PubMed Central

    Llanos, R M; Harris, C J; Hillier, A J; Davidson, B E

    1993-01-01

    The discovery of a novel multicistronic operon that encodes phosphofructokinase, pyruvate kinase, and lactate dehydrogenase in the lactic acid bacterium Lactococcus lactis is reported. The three genes in the operon, designated pfk, pyk, and ldh, contain 340, 502, and 325 codons, respectively. The intergenic distances are 87 bp between pfk and pyk and 117 bp between pyk and ldh. Plasmids containing pfk and pyk conferred phosphofructokinase and pyruvate kinase activity, respectively, on their host. The identity of ldh was established previously by the same approach (R. M. Llanos, A. J. Hillier, and B. E. Davidson, J. Bacteriol. 174:6956-6964, 1992). Each of the genes is preceded by a potential ribosome binding site. The operon is expressed in a 4.1-kb transcript. The 5' end of the transcript was determined to be a G nucleotide positioned 81 bp upstream from the pfk start codon. The pattern of codon usage within the operon is highly biased, with 11 unused amino acid codons. This degree of bias suggests that the operon is highly expressed. The three proteins encoded on the operon are key enzymes in the Embden-Meyerhoff pathway, the central pathway of energy production and lactic acid synthesis in L. lactis. For this reason, we have called the operon the las (lactic acid synthesis) operon. Images PMID:8478320

  13. Stability of active prophages in industrial Lactococcus lactis strains in the presence of heat, acid, osmotic, oxidative and antibiotic stressors.

    PubMed

    Ho, Chun-Hoong; Stanton-Cook, Mitchell; Beatson, Scott A; Bansal, Nidhi; Turner, Mark S

    2016-03-02

    Lactococcus lactis is a starter bacterium commonly used in cheese making where it has an important role in acid-mediated curd formation as well as the development of flavour compounds. Industrial L. lactis strains can harbour one or more inducible prophages which when induced can affect cell growth and possibly lead to cell lysis. This is undesirable during growth and fermentation, but can beneficially lead to faster release of enzymes during cheese ripening. Lactococci can encounter multiple stress inducing conditions during the production of cheese, such as low and high temperatures, low pH, high osmotic pressure and long-term incubation. In this study, we tested the effect of these industrial stressors on prophage induction in two cheese making L. lactis subsp. cremoris strains (ASCC890049 and ASCC890310) as well as the laboratory strain L. lactis MG1363. Firstly, in order to identify inducible prophages in these strains we exposed them to the prophage inducing chemical mitomycin C (MMC) for 1 and 2h and then subjected the total genomic DNA to next-generation Illumina sequencing. Mapping of sequence reads back to the genome sequences revealed regions which contained a much higher fold coverage indicating DNA replication. These regions were amplified by up to 332-fold per cell (relative to the control tufA gene) and were identified as having similarities to different subgroups of P335 phages including MG-5, TP901-1, ul36.k1, bIL286, TP712 and BK5-T. Next, quantitative PCR was used to confirm the strong induction of prophages by MMC and then determine the copy number of the inducible prophages following exposure to various growth inhibitory levels of HCl, lactic acid, high temperature, NaCl, hydrogen peroxide and bacitracin. With the exception of a slight induction (2 to 4-fold) with hydrogen peroxide and long-term incubation after 21days in one industrial strain, none of the other stressors induced prophage DNA replication. These findings show that the repression

  14. Spoilage potential of psychrotrophic lactic acid bacteria (LAB) species: Leuconostoc gelidum subsp. gasicomitatum and Lactococcus piscium, on sweet bell pepper (SBP) simulation medium under different gas compositions.

    PubMed

    Pothakos, Vasileios; Nyambi, Clarice; Zhang, Bao-Yu; Papastergiadis, Antonios; De Meulenaer, Bruno; Devlieghere, Frank

    2014-05-16

    Sweet bell peppers are a significant constituent of retail, chilled-stored and packaged food products like fresh salads, marinades and ready-to-eat (RTE) meals. Previously, through general screening of the Belgian market and by means of source tracking analysis in a plant manufacturing minimally processed, vegetable salads the susceptibility of fresh-cut sweet bell peppers to lactic acid bacterium (LAB) contamination was substantiated. The determination of the metabolic profiles of Leuconostoc gelidum subsp. gasicomitatum and Lactococcus piscium, two major psychrotrophic, spoilage-related LAB species, on sweet bell pepper (SBP) simulation medium under different packaging conditions - 1.) vacuum: 100% N2, 2.) air: 21% O2, 79% N2, 3.) MAP1: 30% CO2, 70% N2 and 4.) MAP2: 50% O2, 50% CO2 - facilitated a better understanding of the spoilage potential of these microbes as well as the presumptive contribution of O2 in the spectrum of produced volatile organic compounds (VOCs) associated with poor organoleptic properties of food products. Generally, none of the applied gas compositions inhibited the growth of the 4 L. gelidum subsp. gasicomitatum isolates, however the presence of O2 resulted in buttery off-odors by inducing primarily the accumulation of diacetyl and pungent "vinegar" smell due to acetic acid. The 3 tested isolates of L. piscium varied greatly among their growth dynamics and inhibition at MAP2. They exhibited either weak spoilage profile or very offensive metabolism confirming significant intraspecies diversity.

  15. Metabolome analysis of milk fermented by γ-aminobutyric acid-producing Lactococcus lactis.

    PubMed

    Hagi, Tatsuro; Kobayashi, Miho; Nomura, Masaru

    2016-02-01

    γ-Aminobutyric acid (GABA) is one of the most important functional components in fermented foods because of its physiological functions, such as neurotransmission and antihypertensive activities. However, little is known about components other than GABA in GABA-rich fermented foods. A metabolomic approach offers an opportunity to discover bioactive and flavor components in fermented food. To find specific components in milk fermented with GABA-producing Lactococcus lactis 01-7, we compared the components found in GABA-rich fermented milk with those found in control milk fermented without GABA production using capillary electrophoresis time-of-flight mass spectrometry. A principal component analysis score plot showed a clear differentiation between the control milk fermented with L. lactis 01-1, which does not produce GABA, and GABA-rich milk fermented with a combination of L. lactis strains 01-1 and 01-7. As expected, the amount of GABA in GABA-rich fermented milk was much higher (1,216-fold) than that of the control milk. Interestingly, the amount of Orn was also much higher (27-fold) than that of the control milk. Peptide analysis showed that levels of 6 putative angiotensin-I-converting enzyme (ACE)-inhibitory peptides were also higher in the GABA-rich fermented milk. Furthermore, ACE-inhibitory activity of GABA-rich fermented milk tended to be higher than that of the control milk. These results indicate that the GABA-producing strain 01-7 provides fermented milk with other functional components in addition to GABA. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  16. Endocarditis caused by Lactococcus cremoris.

    PubMed

    Halldórsdóttir, Halla D; Haraldsdóttir, Vilhelmina; Bödvarsson, Asgeir; Thorgeirsson, Gestur; Kristjánsson, Már

    2002-01-01

    We describe a case of subacute endocarditis due to Lactococcus cremoris associated with consumption of unpasteurized milk. Treatment with amoxicillin-clavulanic acid and subsequently penicillin resulted in prompt sterilization of this patient's bloodstream and full recovery.

  17. Fatty acid membrane composition and activation of glycine-betaine transport in Lactococcus lactis subjected to osmotic stress.

    PubMed

    Guillot, A; Obis, D; Mistou, M Y

    2000-04-10

    Lactococcus lactis subsp. cremoris NCDO763 accumulates glycine-betaine (betaine) when submitted to an osmotic stress with NaCl. Betaine transport activity increases with the extent of the osmotic upshock but also with growth temperature, and supplementation of the medium by Tween-80. Fatty acid analysis of the lipid fraction of L. lactis NCDO763 reveals significant modifications of the fatty acid composition of the membrane when cells are submitted to osmotic stress, high temperature or Tween-80 medium supplementation. The main modification in L. lactis membrane fatty acid composition in response to high osmolality is the increase of Cyclopropane Fatty Acid (CFA) deltaC19:0, whereas Unsaturated/Saturated ratio remains unchanged.

  18. 21 CFR 184.1985 - Aminopeptidase enzyme preparation derived from lactococcus lactis.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... bacterium Lactococcus lactis (previously named Streptococcus lactis). The preparation contains the enzyme... preparation is produced by pure culture fermentation. (b) The ingredient meets the specifications for...

  19. 21 CFR 184.1985 - Aminopeptidase enzyme preparation derived from lactococcus lactis.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... derived from the nonpathogenic and nontoxicogenic bacterium Lactococcus lactis (previously named... fermentation. (b) The ingredient meets the specifications for enzyme preparations in the Food Chemicals...

  20. 21 CFR 184.1985 - Aminopeptidase enzyme preparation derived from lactococcus lactis.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... derived from the nonpathogenic and nontoxicogenic bacterium Lactococcus lactis (previously named... fermentation. (b) The ingredient meets the specifications for enzyme preparations in the Food Chemicals...

  1. 21 CFR 184.1985 - Aminopeptidase enzyme preparation derived from lactococcus lactis.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... derived from the nonpathogenic and nontoxicogenic bacterium Lactococcus lactis (previously named... fermentation. (b) The ingredient meets the specifications for enzyme preparations in the Food Chemicals...

  2. 21 CFR 184.1985 - Aminopeptidase enzyme preparation derived from lactococcus lactis.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... derived from the nonpathogenic and nontoxicogenic bacterium Lactococcus lactis (previously named... fermentation. (b) The ingredient meets the specifications for enzyme preparations in the Food Chemicals...

  3. Sec-Mediated Secretion of Bacteriocin Enterocin P by Lactococcus lactis

    PubMed Central

    Herranz, Carmen; Driessen, Arnold J. M.

    2005-01-01

    Most lactic acid bacterium bacteriocins utilize specific leader peptides and dedicated machineries for secretion. In contrast, the enterococcal bacteriocin enterocin P (EntP) contains a typical signal peptide that directs its secretion when heterologously expressed in Lactococcus lactis. Signal peptide mutations and the SecA inhibitor azide blocked secretion. These observations demonstrate that EntP is secreted by the Sec translocase. PMID:15812026

  4. Ratio of intracellular precursors concentration and their flux influences hyaluronic acid molecular weight in Streptococcus zooepidemicus and recombinant Lactococcus lactis.

    PubMed

    Badle, Sneh Sanjay; Jayaraman, Guhan; Ramachandran, K B

    2014-07-01

    HA molecular weight variation in Streptococcus zooepidemicus and two recombinant Lactococcus lactis strains were investigated by chemostat experiments and metabolic flux analysis (MFA). The study showed that intracellular flux ratio of UDP-GlcUA to UDP-GlcNAc correlated directly with HA molecular weight, for all the three strains. The ratio of intracellular concentration of these HA precursors also exhibited a similar trend. Phosphoglucoisomerase activity and glucose flux towards lactic acid formation were found to be the major bottlenecks for HA production in all the three strains. The study suggests that environmental conditions and genetic manipulations that balance the intracellular flux and HA precursors concentrations will result in increased molecular weight. Copyright © 2014 Elsevier Ltd. All rights reserved.

  5. Design of aqueous two-phase systems for purification of hyaluronic acid produced by metabolically engineered Lactococcus lactis.

    PubMed

    Rajendran, Vivek; Puvendran, Kirubhakaran; Guru, Bharath Raja; Jayaraman, Guhan

    2016-02-01

    Hyaluronic acid has a wide range of biomedical applications and its commercial value is highly dependent on its purity and molecular weight. This study highlights the utility of aqueous two-phase separation as a primary recovery step for hyaluronic acid and for removal of major protein impurities from fermentation broths. Metabolically engineered cultures of a lactate dehydrogenase mutant strain of Lactococcus lactis (L. lactis NZ9020) were used to produce high-molecular-weight hyaluronic acid. The cell-free fermentation broth was partially purified using a polyethylene glycol/potassium phosphate system, resulting in nearly 100% recovery of hyaluronic acid in the salt-rich bottom phase in all the aqueous two-phase separation experiments. These experiments were optimized for maximum removal of protein impurities in the polyethylene glycol rich top phase. The removal of protein impurities resulted in substantial reduction of membrane fouling in the subsequent diafiltration process, carried out with a 300 kDa polyether sulfone membrane. This step resulted in considerable purification of hyaluronic acid, without any loss in recovery and molecular weight. Diafiltration was followed by an adsorption step to remove minor impurities and achieve nearly 100% purity. The final hyaluronic acid product was characterized by Fourier-transform IR and NMR spectroscopy, confirming its purity.

  6. Production of spent mushroom substrate hydrolysates useful for cultivation of Lactococcus lactis by dilute sulfuric acid, cellulase and xylanase treatment.

    PubMed

    Qiao, Jian-Jun; Zhang, Yan-Fei; Sun, Li-Fan; Liu, Wei-Wei; Zhu, Hong-Ji; Zhang, Zhijun

    2011-09-01

    Spent mushroom substrate (SMS) was treated with dilute sulfuric acid followed by cellulase and xylanase treatment to produce hydrolysates that could be used as the basis for media for the production of value added products. A L9 (3(4)) orthogonal experiment was performed to optimize the acid treatment process. Pretreatment with 6% (w/w) dilute sulfuric acid at 120°C for 120 min provided the highest reducing sugar yield of 267.57 g/kg SMS. No furfural was detected in the hydrolysates. Exposure to 20PFU of cellulase and 200 XU of xylanase per gram of pretreated SMS at 40°C resulted in the release of 79.85 g/kg or reducing sugars per kg acid pretreated SMS. The dilute sulfuric acid could be recycled to process fresh SMS four times. SMS hydrolysates neutralized with ammonium hydroxide, sodium hydroxide, or calcium hydroxide could be used as the carbon source for cultivation of Lactococcus lactis subsp. lactis W28 and a cell density of 2.9×10(11)CFU/mL could be obtained. The results provide a foundation for the development of value-added products based on SMS. Copyright © 2011 Elsevier Ltd. All rights reserved.

  7. Influence of carbohydrate starvation and arginine on culturability and amino acid utilization of lactococcus lactis subsp. lactis.

    PubMed

    Stuart, M R; Chou, L S; Weimer, B C

    1999-02-01

    Two strains of Lactococcus lactis subsp. lactis were used to determine the influence of lactose and arginine on viability and amino acid use during carbohydrate starvation. Lactose provided energy for logarithmic-phase growth, and amino acids such as arginine provided energy after carbohydrate exhaustion. Survival time, cell numbers, and ATP concentrations increased with the addition of arginine to the basal medium. By the onset of lactose exhaustion, the concentrations of glycine-valine and glutamate had decreased by as much as 67% in L. lactis ML3, whereas the serine concentration increased by 97% during the same period. When no lactose was added, the concentrations of these amino acids remained constant. Similar trends were observed for L. lactis 11454. Without lactose or arginine, L. lactis ML3 was nonculturable on agar but was viable after 2 days, as measured by fluorescent viability stains and intracellular ATP levels. However, L. lactis 11454 without lactose or arginine remained culturable for at least 14 days. These data suggest that lactococci become viable but nonculturable in response to carbohydrate depletion. Additionally, these data indicate that amino acids other than arginine facilitate the survival of L. lactis during carbohydrate starvation.

  8. Relative Rates of Amino Acid Import via the ABC Transporter GlnPQ Determine the Growth Performance of Lactococcus lactis

    PubMed Central

    Fulyani, Faizah; Schuurman-Wolters, Gea K.; Slotboom, Dirk-Jan

    2015-01-01

    ABSTRACT The GlnPQ transporter from Lactococcus lactis has the remarkable feature of having two substrate-binding domains (SBDs) fused to the N terminus of the transmembrane domain (TMD), and thus four SBDs are present in the homodimeric complex. Although X-ray structures and ligand binding data are available for both SBDs, little is known of how different amino acids compete with each other for transport via GlnPQ. Here we show GlnPQ has a broader substrate specificity than previously thought, with the ability to take up asparagine, glutamine, and glutamic acid, albeit via different routes and with different affinities. Asparagine and glutamine compete with each other at the level of binding to SBD1 and SBD2 (with differences in dissociation constant), but at the same time SBD1 and SBD2 compete with each other at the level of interaction with the translocator domain (with differences in affinity constant and rate of transport). Although glutamine transport via SBD1 is outcompeted by physiological concentrations of asparagine, SBD2 ensures high rates of import of the essential amino acid glutamine. Taken together, this study demonstrates that even in the presence of competing asparagine concentrations, GlnPQ has a high capacity to transport glutamine, which matches the high needs of the cell for glutamine and glutamate. IMPORTANCE GlnPQ is an ATP-binding cassette (ABC) transporter for glutamine, glutamic acid, and asparagine. The system is essential in various Gram-positive bacteria, including L. lactis and several pathogens. Here we show how the amino acids compete with each other for binding to the multiple SBDs of GlnPQ and how these SBDs compete with each other for substrate delivery to the transporter. Overall, our results show that GlnPQ has evolved to transport diverse substrates via different paths and to optimally acquire the abundant and essential amino acid glutamine. PMID:26553850

  9. Identification, Cloning, and Characterization of a Lactococcus lactis Branched-Chain α-Keto Acid Decarboxylase Involved in Flavor Formation

    PubMed Central

    Smit, Bart A.; van Hylckama Vlieg, Johan E. T.; Engels, Wim J. M.; Meijer, Laura; Wouters, Jan T. M.; Smit, Gerrit

    2005-01-01

    The biochemical pathway for formation of branched-chain aldehydes, which are important flavor compounds derived from proteins in fermented dairy products, consists of a protease, peptidases, a transaminase, and a branched-chain α-keto acid decarboxylase (KdcA). The activity of the latter enzyme has been found only in a limited number of Lactococcus lactis strains. By using a random mutagenesis approach, the gene encoding KdcA in L. lactis B1157 was identified. The gene for this enzyme is highly homologous to the gene annotated ipd, which encodes a putative indole pyruvate decarboxylase, in L. lactis IL1403. Strain IL1403 does not produce KdcA, which could be explained by a 270-nucleotide deletion at the 3′ terminus of the ipd gene encoding a truncated nonfunctional decarboxylase. The kdcA gene was overexpressed in L. lactis for further characterization of the decarboxylase enzyme. Of all of the potential substrates tested, the highest activity was observed with branched-chain α-keto acids. Moreover, the enzyme activity was hardly affected by high salinity, and optimal activity was found at pH 6.3, indicating that the enzyme might be active under cheese ripening conditions. PMID:15640202

  10. Chromosomal integration of hyaluronic acid synthesis (has) genes enhances the molecular weight of hyaluronan produced in Lactococcus lactis.

    PubMed

    Hmar, Rothangmawi Victoria; Prasad, Shashi Bala; Jayaraman, Guhan; Ramachandran, Kadathur B

    2014-12-01

    Microbial production of hyaluronic acid (HA) is an attractive substitute for extraction of this biopolymer from animal tissues. Natural producers such as Streptococcus zooepidemicus are potential pathogens; therefore, production of HA by recombinant bacteria that are generally recognized as safe (GRAS) organisms is a viable alternative that is being extensively explored. However, plasmid-based expression systems for HA production by recombinant bacteria have the inherent disadvantage of reduced productivity because of plasmid instability. To overcome this problem, the HA synthesis genes (hasA-hasB and hasA-hasB-hasC) from has-operon of S. zooepidemicus were integrated into the chromosome of Lactococcus lactis by site-directed, double-homologous recombination developing strains VRJ2AB and VRJ3ABC. The chromosomal integration stabilized the genes and obviated the instability observed in plasmid-expressed recombinant strains. The genome-integrated strains produced higher molecular weight (3.5-4 million Dalton [MDa]) HA compared to the plasmid-expressed strains (2 MDa). High molecular weight HA was produced when the intracellular concentration of uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) and uridine diphosphate-glucuronic acid (UDP-GlcUA) was almost equal and hasA to hasB ratio was low. This work suggests an optimal approach to obtain high molecular weight HA in recombinant strains.

  11. Expression of the sweet-tasting plant protein brazzein in Escherichia coli and Lactococcus lactis: a path toward sweet lactic acid bacteria.

    PubMed

    Berlec, Ales; Jevnikar, Zala; Majhenic, Andreja Canzek; Rogelj, Irena; Strukelj, Borut

    2006-11-01

    Brazzein is an intensely sweet-tasting plant protein with good stability, which makes it an attractive alternative to sucrose. A brazzein gene has been designed, synthesized, and expressed in Escherichia coli at 30 degrees C to yield brazzein in a soluble form and in considerable quantity. Antibodies have been produced using brazzein fused to His-tag. Brazzein without the tag was sweet and resembled closely the taste of its native counterpart. The brazzein gene was also expressed in Lactococcus lactis, using a nisin-controlled expression system, to produce sweet-tasting lactic acid bacteria. The low level of expression was detected with anti-brazzein antibodies. Secretion of brazzein into the medium has not led to significant yield increase. Surprisingly, optimizing the codon usage for Lactococcus lactis led to a decrease in the yield of brazzein.

  12. Heterologous expression of Lactobacillus casei RecO improved the multiple-stress tolerance and lactic acid production in Lactococcus lactis NZ9000 during salt stress.

    PubMed

    Wu, Chongde; Zhang, Juan; Du, Guocheng; Chen, Jian

    2013-09-01

    The aim of this study was to investigate the effect of nisin-inducible RecO expression on the stress tolerance of Lactococcus lactis NZ9000. RecO protein from Lactobacillus casei Zhang was introduced into Lactococcus lactis NZ9000 by using a nisin-inducible expression system. The recombinant strain (NZ-RecO) exhibited higher growth performances and survival rate compared with the control strain (NZ-Vector) under stress conditions. In addition, the NZ-RecO strain exhibited 1.37-, 1.41-, and 1.42-fold higher biomass, lactate production, lactate productivity, compared with the corresponding values for NZ-Vector during NaCl-stressed condition. Analysis of lactate dehydrogenase (LDH) activity showed that the production of RecO maintained the stability of LDH during salt stress. These results suggest that overproduction of RecO improved the multiple-stress tolerance and lactic acid production in Lactococcus lactis NZ9000 during salt stress. Results presented in this study may help to enhance the industrial utility of lactic acid bacteria. Copyright © 2013 Elsevier Ltd. All rights reserved.

  13. Improved Acid Stress Survival of Lactococcus lactis Expressing the Histidine Decarboxylation Pathway of Streptococcus thermophilus CHCC1524*

    PubMed Central

    Trip, Hein; Mulder, Niels L.; Lolkema, Juke S.

    2012-01-01

    Degradative amino acid decarboxylation pathways in bacteria generate secondary metabolic energy and provide resistance against acid stress. The histidine decarboxylation pathway of Streptococcus thermophilus CHCC1524 was functionally expressed in the heterologous host Lactococcus lactis NZ9000, and the benefits of the newly acquired pathway for the host were analyzed. During growth in M17 medium in the pH range of 5–6.5, a small positive effect was observed on the biomass yield in batch culture, whereas no growth rate enhancement was evident. In contrast, a strong benefit for the engineered L. lactis strain was observed in acid stress survival. In the presence of histidine, the pathway enabled cells to survive at pH values as low as 3 for at least 2 h, conditions under which the host cells were rapidly dying. The flux through the histidine decarboxylation pathway in cells grown at physiological pH was under strict control of the electrochemical proton gradient (pmf) across the membrane. Ionophores that dissipated the membrane potential (ΔΨ) and/or the pH gradient (ΔpH) strongly increased the flux, whereas the presence of glucose almost completely inhibited the flux. Control of the pmf over the flux was exerted by both ΔΨ and ΔpH and was distributed over the transporter HdcP and the decarboxylase HdcA. The control allowed for a synergistic effect between the histidine decarboxylation and glycolytic pathways in acid stress survival. In a narrow pH range around 2.5 the synergism resulted in a 10-fold higher survival rate. PMID:22351775

  14. Improved acid stress survival of Lactococcus lactis expressing the histidine decarboxylation pathway of Streptococcus thermophilus CHCC1524.

    PubMed

    Trip, Hein; Mulder, Niels L; Lolkema, Juke S

    2012-03-30

    Degradative amino acid decarboxylation pathways in bacteria generate secondary metabolic energy and provide resistance against acid stress. The histidine decarboxylation pathway of Streptococcus thermophilus CHCC1524 was functionally expressed in the heterologous host Lactococcus lactis NZ9000, and the benefits of the newly acquired pathway for the host were analyzed. During growth in M17 medium in the pH range of 5-6.5, a small positive effect was observed on the biomass yield in batch culture, whereas no growth rate enhancement was evident. In contrast, a strong benefit for the engineered L. lactis strain was observed in acid stress survival. In the presence of histidine, the pathway enabled cells to survive at pH values as low as 3 for at least 2 h, conditions under which the host cells were rapidly dying. The flux through the histidine decarboxylation pathway in cells grown at physiological pH was under strict control of the electrochemical proton gradient (pmf) across the membrane. Ionophores that dissipated the membrane potential (ΔΨ) and/or the pH gradient (ΔpH) strongly increased the flux, whereas the presence of glucose almost completely inhibited the flux. Control of the pmf over the flux was exerted by both ΔΨ and ΔpH and was distributed over the transporter HdcP and the decarboxylase HdcA. The control allowed for a synergistic effect between the histidine decarboxylation and glycolytic pathways in acid stress survival. In a narrow pH range around 2.5 the synergism resulted in a 10-fold higher survival rate.

  15. Heterologous expression of glycoside hydrolase family 2 and 42 β-galactosidases of lactic acid bacteria in Lactococcus lactis.

    PubMed

    Schwab, Clarissa; Sørensen, Kim I; Gänzle, Michael G

    2010-10-01

    This study characterized a glycoside hydrolase family 42 (GH42) β-galactosidase of Lactobacillus acidophilus (LacA) and compared lactose hydrolysis, hydrolysis of oNPG, pNPG and pNPG-analogues and galactooligosaccharides (GOSs) formation to GH2 β-galactosidases of Streptococcus thermophilus (LacZ type), Lactobacillus plantarum and Leuconostoc mesenteroides subsp. cremoris (both LacLM type). Beta-galactosidases were heterologously expressed in Lactococcus lactis using a p170 derived promoter; experiments were performed with L. lactis crude cell extract (CCE). The novel GH42 β-galactosidase of Lb. acidophilus had lower activity on lactose, oNPG and pNPG but higher relative activity on pNP analogues compared to GH2 β-galactosidases, and did not transgalactosylate at high lactose concentrations. Temperature and pH optima for lactose hydrolysis varied between GH2 β-galactosidases. oNPG and pNPG were the preferred substrates for hydrolysis; in comparison, activity on pNPG-analogues was less than 1.5%. GH2 β-galactosidases formed structurally similar GOS with varying preferences. The diversity of lactic acid bacteria β-galactosidase activity in L. lactis CCE can be exploited in future nutritional or therapeutic applications. Copyright © 2010 Elsevier GmbH. All rights reserved.

  16. Improvement in lactic acid production from starch using alpha-amylase-secreting Lactococcus lactis cells adapted to maltose or starch.

    PubMed

    Okano, Kenji; Kimura, Sakurako; Narita, Junya; Fukuda, Hideki; Kondo, Akihiko

    2007-07-01

    To achieve direct and efficient lactic acid production from starch, a genetically modified Lactococcus lactis IL 1403 secreting alpha-amylase, which was obtained from Streptococcus bovis 148, was constructed. Using this strain, the fermentation of soluble starch was achieved, although its rate was far from efficient (0.09 g l(-1) h(-1) lactate). High-performance liquid chromatography revealed that maltose accumulated during fermentation, and this was thought to lead to inefficient fermentation. To accelerate maltose consumption, starch fermentation was examined using L. lactis cells adapted to maltose instead of glucose. This led to a decrease in the amount of maltose accumulation in the culture, and, as a result, a more rapid fermentation was accomplished (1.31 g l(-1) h(-1) lactate). Maximum volumetric lactate productivity was further increased (1.57 g l(-1) h(-1) lactate) using cells adapted to starch, and a high yield of lactate (0.89 g of lactate per gram of consumed sugar) of high optical purity (99.2% of L: -lactate) was achieved. In this study, we propose a new approach to lactate production by alpha-amylase-secreting L. lactis that allows efficient fermentation from starch using cells adapted to maltose or starch before fermentation.

  17. α-Acetolactate synthase of Lactococcus lactis contributes to pH homeostasis in acid stress conditions.

    PubMed

    Zuljan, Federico A; Repizo, Guillermo D; Alarcon, Sergio H; Magni, Christian

    2014-10-01

    Lactic Acid Bacteria (LAB) are recognized as safe microorganisms with the capacity to improve the quality of dairy products. When the LAB Lactococcus lactis is employed as starter for the production of fermented foods, high quantities of important aroma compounds such as diacetyl are generated by means of the diacetyl/acetoin pathway. Our previous results obtained with L. lactis strains report that this pathway is activated under acidic conditions. In this study, we describe the metabolism of pyruvate, a diacetyl/acetoin precursor, and its contribution to pH homeostasis in this microorganism. L lactis strain IL1403 is able to cometabolize pyruvate and glucose at low pH, producing lactate, acetate as well as diacetyl/acetoin compounds. In contrast, the als defective strain, which is incapable of producing C4 compounds, appeared sensitive to pyruvate under acidic conditions rendering it unable to grow. Accordingly, the als-mutant strain showed a simultaneous inability to alkalinize internal and external media. These results demonstrate that the decarboxylation reactions associated to the diacetyl/acetoin pathway represent a competitive advantage in a condition of intracellular pyruvate accumulation during growth at low pH. Interestingly, a genomic comparative analysis shows that this pathway has been conserved in L. lactis during the domestication of different strains. Also, our analysis shows that the recent acquisition of the cit cluster required for citrate metabolism, which contributes to diacetyl/acetoin production as well, is the specific feature of the biovar. diacetylactis. In this regard, we present for first time genetic evidence supporting the proposal made by Passerini et al. (2013) who postulated that the expression "biovar. citrate" should be more appropriate to define this specific industrial strain. Copyright © 2014. Published by Elsevier B.V.

  18. Membrane Protein Production in Lactococcus lactis for Functional Studies.

    PubMed

    Seigneurin-Berny, Daphne; King, Martin S; Sautron, Emiline; Moyet, Lucas; Catty, Patrice; André, François; Rolland, Norbert; Kunji, Edmund R S; Frelet-Barrand, Annie

    2016-01-01

    Due to their unique properties, expression and study of membrane proteins in heterologous systems remains difficult. Among the bacterial systems available, the Gram-positive lactic bacterium, Lactococcus lactis, traditionally used in food fermentations, is nowadays widely used for large-scale production and functional characterization of bacterial and eukaryotic membrane proteins. The aim of this chapter is to describe the different possibilities for the functional characterization of peripheral or intrinsic membrane proteins expressed in Lactococcus lactis.

  19. Complete Genome Sequence of a thermotolerant sporogenic lactic acid bacterium, Bacillus coagulans strain 36D1

    PubMed Central

    Rhee, Mun Su; Moritz, Brélan E.; Xie, Gary; Glavina del Rio, T.; Dalin, E.; Tice, H.; Bruce, D.; Goodwin, L.; Chertkov, O.; Brettin, T.; Han, C.; Detter, C.; Pitluck, S.; Land, Miriam L.; Patel, Milind; Ou, Mark; Harbrucker, Roberta; Ingram, Lonnie O.; Shanmugam, K. T.

    2011-01-01

    Bacillus coagulans is a ubiquitous soil bacterium that grows at 50-55 °C and pH 5.0 and ferments various sugars that constitute plant biomass to L (+)-lactic acid. The ability of this sporogenic lactic acid bacterium to grow at 50-55 °C and pH 5.0 makes this organism an attractive microbial biocatalyst for production of optically pure lactic acid at industrial scale not only from glucose derived from cellulose but also from xylose, a major constituent of hemicellulose. This bacterium is also considered as a potential probiotic. Complete genome sequence of a representative strain, B. coagulans strain 36D1, is presented and discussed. PMID:22675583

  20. Complete Genome Sequence of a thermotolerant sporogenic lactic acid bacterium, Bacillus coagulans strain 36D1

    SciTech Connect

    Xie, Gary; Dalin, Eileen; Tice, Hope; Chertkov, Olga; Land, Miriam L

    2011-01-01

    Bacillus coagulans is a ubiquitous soil bacterium that grows at 50-55 C and pH 5.0 and fer-ments various sugars that constitute plant biomass to L (+)-lactic acid. The ability of this sporogenic lactic acid bacterium to grow at 50-55 C and pH 5.0 makes this organism an attractive microbial biocatalyst for production of optically pure lactic acid at industrial scale not only from glucose derived from cellulose but also from xylose, a major constituent of hemi-cellulose. This bacterium is also considered as a potential probiotic. Complete genome squence of a representative strain, B. coagulans strain 36D1, is presented and discussed.

  1. Complete Genome Sequence of a thermotolerant sporogenic lactic acid bacterium, Bacillus coagulans strain 36D1

    SciTech Connect

    Rhee, Mun Su; Moritz, Brelan E.; Xie, Gary; Glavina Del Rio, Tijana; Dalin, Eileen; Tice, Hope; Bruce, David; Goodwin, Lynne A.; Chertkov, Olga; Brettin, Thomas S; Han, Cliff; Detter, J. Chris; Pitluck, Sam; Land, Miriam L; Patel, Milind; Ou, Mark; Harbrucker, Roberta; Ingram, Lonnie O.; Shanmugam, Keelnathan T.

    2011-01-01

    Bacillus coagulans is a ubiquitous soil bacterium that grows at 50-55 C and pH 5.0 and fer- ments various sugars that constitute plant biomass to L (+)-lactic acid. The ability of this spo- rogenic lactic acid bacterium to grow at 50-55 C and pH 5.0 makes this organism an attrac- tive microbial biocatalyst for production of optically pure lactic acid at industrial scale not only from glucose derived from cellulose but also from xylose, a major constituent of hemi- cellulose. This bacterium is also considered as a potential probiotic. Complete genome se- quence of a representative strain, B. coagulans strain 36D1, is presented and discussed.

  2. Complete Genome Sequence of a thermotolerant sporogenic lactic acid bacterium, Bacillus coagulans strain 36D1.

    PubMed

    Rhee, Mun Su; Moritz, Brélan E; Xie, Gary; Glavina Del Rio, T; Dalin, E; Tice, H; Bruce, D; Goodwin, L; Chertkov, O; Brettin, T; Han, C; Detter, C; Pitluck, S; Land, Miriam L; Patel, Milind; Ou, Mark; Harbrucker, Roberta; Ingram, Lonnie O; Shanmugam, K T

    2011-12-31

    Bacillus coagulans is a ubiquitous soil bacterium that grows at 50-55 °C and pH 5.0 and ferments various sugars that constitute plant biomass to L (+)-lactic acid. The ability of this sporogenic lactic acid bacterium to grow at 50-55 °C and pH 5.0 makes this organism an attractive microbial biocatalyst for production of optically pure lactic acid at industrial scale not only from glucose derived from cellulose but also from xylose, a major constituent of hemicellulose. This bacterium is also considered as a potential probiotic. Complete genome sequence of a representative strain, B. coagulans strain 36D1, is presented and discussed.

  3. Identification of the Leucine-to-2-Methylbutyric Acid Catabolic Pathway of Lactococcus lactis† ‡

    PubMed Central

    Ganesan, Balasubramanian; Dobrowolski, Piotr; Weimer, Bart C.

    2006-01-01

    Nutrient starvation and nonculturability in bacteria lead to changes in metabolism not found during the logarithmic phase. Substrates alternate to those used during growth are metabolized in these physiological states, yielding secondary metabolites. In firmicutes and actinobacteria, amino acid catabolic pathways are induced during starvation and nonculturability. Examination of lactococci showed that the population entered a nonculturable state after carbohydrate depletion and was incapable of growth on solid media; however, the cells gained the ability to produce branched-chain fatty acids from amino acids. Gene expression profiling and in silico pathway analysis coupled with nuclear magnetic resonance spectroscopy were used to delineate the leucine catabolic pathway. Lactococci produced acetic and propionic acid during logarithmic growth and starvation. At the onset of nonculturability, 2-methylbutyric acid was produced via hydroxymethyl-glutaryl-coenzyme A (CoA) and acetyl-CoA, along with ATP and oxidation/reduction precursors. Gene expression profiling and genome sequence analysis showed that lactococci contained redundant genes for branched-chain fatty acid production that were regulated by an unknown mechanism linked to carbon metabolism. This work demonstrated the ability of a firmicute to induce new metabolic capabilities in the nonculturable state for producing energy and intermediates needed for transcription and translation. Phylogenetic analyses showed that homologues of these enzymes and their functional motifs were widespread across the domains of life. PMID:16751541

  4. Endohyphal Bacterium Enhances Production of Indole-3-Acetic Acid by a Foliar Fungal Endophyte

    PubMed Central

    Hoffman, Michele T.; Gunatilaka, Malkanthi K.; Wijeratne, Kithsiri; Gunatilaka, Leslie; Arnold, A. Elizabeth

    2013-01-01

    Numerous plant pathogens, rhizosphere symbionts, and endophytic bacteria and yeasts produce the important phytohormone indole-3-acetic acid (IAA), often with profound effects on host plants. However, to date IAA production has not been documented among foliar endophytes -- the diverse guild of primarily filamentous Ascomycota that live within healthy, above-ground tissues of all plant species studied thus far. Recently bacteria that live within hyphae of endophytes (endohyphal bacteria) have been detected, but their effects have not been studied previously. Here we show not only that IAA is produced in vitro by a foliar endophyte (here identified as Pestalotiopsis aff. neglecta, Xylariales), but that IAA production is enhanced significantly when the endophyte hosts an endohyphal bacterium (here identified as Luteibacter sp., Xanthomonadales). Both the endophyte and the endophyte/bacterium complex appear to rely on an L-tryptophan dependent pathway for IAA synthesis. The bacterium can be isolated from the fungus when the symbiotic complex is cultivated at 36°C. In pure culture the bacterium does not produce IAA. Culture filtrate from the endophyte-bacterium complex significantly enhances growth of tomato in vitro relative to controls and to filtrate from the endophyte alone. Together these results speak to a facultative symbiosis between an endophyte and endohyphal bacterium that strongly influences IAA production, providing a new framework in which to explore endophyte-plant interactions. PMID:24086270

  5. Draft Genome Sequence of Lactococcus lactis subsp. lactis Strain YF11

    PubMed Central

    Du, Yuhui; Song, Lifu; Feng, Wenjing; Pei, Guangsheng; Zheng, Ping; Yu, Zhichao; Sun, Jibin

    2013-01-01

    Lactococcus lactis subsp. lactis strain YF11 is a food preservative bacterium with a high capacity to produce nisin. Here, we announce the draft genome sequence of Lactococcus lactis subsp. lactis YF11 (2,527,433 bp with a G+C content of 34.81%). PMID:23929487

  6. Effect of tannic acid on the transcriptome of the soil bacterium Pseudomonas protegens Pf-5

    USDA-ARS?s Scientific Manuscript database

    Tannins are plant-produced organic compounds that are found in soils, are able to sequester iron, and have antimicrobial properties. We studied the effect of tannic acid on the molecular physiology of the soil-inhabiting biocontrol bacterium Pseudomonas protegens Pf-5 (formerly Pseudomonas fluoresce...

  7. First Insights into the Genome of the Amino Acid-Metabolizing Bacterium Clostridium litorale DSM 5388

    PubMed Central

    Poehlein, Anja; Alghaithi, Hamed S.; Chandran, Lenin; Chibani, Cynthia M.; Davydova, Elena; Dhamotharan, Karthikeyan; Ge, Wanwan; Gutierrez-Gutierrez, David A.; Jagirdar, Advait; Khonsari, Bahar; Nair, Kamal Prakash P. R.

    2014-01-01

    Clostridium litorale is a Gram-positive, rod-shaped, and spore-forming bacterium, which is able to use amino acids such as glycine, sarcosine, proline, and betaine as single carbon and energy sources via Stickland reactions. The genome consists of a circular chromosome (3.41 Mb) and a circular plasmid (27 kb). PMID:25081264

  8. Catalytic activity of tripeptidase from Lactococcus lactis to which amino acid substitution was introduced according to natural mutation.

    PubMed

    Mori, Sumiko; Kaneko, Satoshi; Kasumi, Takafumi

    2004-05-01

    Four mutations observed between tripeptidases from Lactococcus lactis subsp. lactis and subsp. cremoris were introduced one by one to the corresponding points in wild-type tripeptidase from L. lactis subsp. lactis. The k(cat) values of four resultant mutants were analyzed and discussed in stereographical terms. Change in catalytic activity appeared to be related to the sequential and steric location of mutation point within the enzyme protein, even though no drastic change was observed with one point mutation.

  9. Mesophilic Lactic Acid Bacteria Diversity Encountered in Brazilian Farms Producing Milk with Particular Interest in Lactococcus lactis Strains.

    PubMed

    Luiz, L M P; Chuat, V; Madec, M N; Araújo, E A; de Carvalho, A F; Valence, F

    2016-10-01

    The milk produced in regions with different traditions in Brazil is used for artisanal product production, which is characterized by different sensorial characteristics. This study aimed to identify the bacterial ecosystem of farms located in a traditional dairy region in the state of Minas Gerais and to characterize Lactococcus lactis strains, the species of interest in this study, using a multilocus sequence typing (MLST) protocol and pulsed-field gel electrophoresis (PFGE) technique. Samples were collected from raw milk and dairy environment from six farms. A total of 50 isolates were analyzed using 16S rRNA sequencing and species-specific PCR. Five genera were identified: Lactobacillus, Leuconostoc, Lactococcus, Enterococcus, and Staphylococcus, from ten different species. MLST (with six housekeeping genes) and PFGE (with SmaI endonuclease) were used for the characterization of 20 isolates of Lactococcus lactis from a dairy collection in this study. Both methods revealed a high clonal diversity of strains with a higher discriminatory level for PFGE (15 pulsotypes), compared to MLST (12 ST). This study contributes to the preservation of the Brazilian dairy heritage and provides insights into a part of the LAB population found in raw milk and dairy environment.

  10. Anaerobic Degradation of Cyanuric Acid, Cysteine, and Atrazine by a Facultative Anaerobic Bacterium

    PubMed Central

    Jessee, J. A.; Benoit, R. E.; Hendricks, A. C.; Allen, G. C.; Neal, J. L.

    1983-01-01

    A facultative anaerobic bacterium that rapidly degrades cyanuric acid (CA) was isolated from the sediment of a stream that received industrial wastewater effluent. CA decomposition was measured throughout the growth cycle by using a high-performance liquid chromatography assay, and the concomitant production of ammonia was also measured. The bacterium used CA or cysteine as a major, if not the sole, carbon and energy source under anaerobic, but not aerobic, conditions in a defined medium. The cell yield was greatly enhanced by the simultaneous presence of cysteine and CA in the medium. Cysteine was preferentially used rather than CA early in the growth cycle, but all of the CA was used without an apparent lag after the cysteine was metabolized. Atrazine was also degraded by this bacterium under anaerobic conditions in a defined medium. PMID:16346187

  11. An oleaginous bacterium that intrinsically accumulates long-chain free Fatty acids in its cytoplasm.

    PubMed

    Katayama, Taiki; Kanno, Manabu; Morita, Naoki; Hori, Tomoyuki; Narihiro, Takashi; Mitani, Yasuo; Kamagata, Yoichi

    2014-02-01

    Medium- and long-chain fatty acids are present in organisms in esterified forms that serve as cell membrane constituents and storage compounds. A large number of organisms are known to accumulate lipophilic materials as a source of energy and carbon. We found a bacterium, designated GK12, that intrinsically accumulates free fatty acids (FFAs) as intracellular droplets without exhibiting cytotoxicity. GK12 is an obligatory anaerobic, mesophilic lactic acid bacterium that was isolated from a methanogenic reactor. Phylogenetic analysis based on 16S rRNA gene sequences showed that GK12 is affiliated with the family Erysipelotrichaceae in the phylum Firmicutes but is distantly related to type species in this family (less than 92% similarity in 16S rRNA gene sequence). Saturated fatty acids with carbon chain lengths of 14, 16, 18, and 20 were produced from glucose under stress conditions, including higher-than-optimum temperatures and the presence of organic solvents that affect cell membrane integrity. FFAs were produced at levels corresponding to up to 25% (wt/wt) of the dry cell mass. Our data suggest that FFA accumulation is a result of an imbalance between excess membrane fatty acid biosynthesis due to homeoviscous adaptation and limited β-oxidation activity due to anaerobic growth involving lactic acid fermentation. FFA droplets were not further utilized as an energy and carbon source, even under conditions of starvation. A naturally occurring bacterium that accumulates significant amounts of long-chain FFAs with noncytotoxicity would provide useful strategies for microbial biodiesel production.

  12. Effect of Tannic Acid on the Transcriptome of the Soil Bacterium Pseudomonas protegens Pf-5

    PubMed Central

    Lim, Chee Kent; Penesyan, Anahit; Hassan, Karl A.

    2013-01-01

    Tannins are a diverse group of plant-produced, polyphenolic compounds with metal-chelating and antimicrobial properties that are prevalent in many soils. Using transcriptomics, we determined that tannic acid, a form of hydrolysable tannin, broadly affects the expression of genes involved in iron and zinc homeostases, sulfur metabolism, biofilm formation, motility, and secondary metabolite biosynthesis in the soil- and rhizosphere-inhabiting bacterium Pseudomonas protegens Pf-5. PMID:23435890

  13. Metabolic and Transcriptional Analysis of Acid Stress in Lactococcus lactis, with a Focus on the Kinetics of Lactic Acid Pools

    PubMed Central

    Carvalho, Ana Lúcia; Turner, David L.; Fonseca, Luís L.; Solopova, Ana; Catarino, Teresa; Kuipers, Oscar P.; Voit, Eberhard O.; Neves, Ana Rute; Santos, Helena

    2013-01-01

    The effect of pH on the glucose metabolism of non-growing cells of L. lactis MG1363 was studied by in vivo NMR in the range 4.8 to 6.5. Immediate pH effects on glucose transporters and/or enzyme activities were distinguished from transcriptional/translational effects by using cells grown at the optimal pH of 6.5 or pre-adjusted to low pH by growth at 5.1. In cells grown at pH 5.1, glucose metabolism proceeds at a rate 35% higher than in non-adjusted cells at the same pH. Besides the upregulation of stress-related genes (such as dnaK and groEL), cells adjusted to low pH overexpressed H+-ATPase subunits as well as glycolytic genes. At sub-optimal pHs, the total intracellular pool of lactic acid reached approximately 500 mM in cells grown at optimal pH and about 700 mM in cells grown at pH 5.1. These high levels, together with good pH homeostasis (internal pH always above 6), imply intracellular accumulation of the ionized form of lactic acid (lactate anion), and the concomitant export of the equivalent protons. The average number, n, of protons exported with each lactate anion was determined directly from the kinetics of accumulation of intra- and extracellular lactic acid as monitored online by 13C-NMR. In cells non-adjusted to low pH, n varies between 2 and 1 during glucose consumption, suggesting an inhibitory effect of intracellular lactate on proton export. We confirmed that extracellular lactate did not affect the lactate: proton stoichiometry. In adjusted cells, n was lower and varied less, indicating a different mix of lactic acid exporters less affected by the high level of intracellular lactate. A qualitative model for pH effects and acid stress adaptation is proposed on the basis of these results. PMID:23844205

  14. Metabolic and transcriptional analysis of acid stress in Lactococcus lactis, with a focus on the kinetics of lactic acid pools.

    PubMed

    Carvalho, Ana Lúcia; Turner, David L; Fonseca, Luís L; Solopova, Ana; Catarino, Teresa; Kuipers, Oscar P; Voit, Eberhard O; Neves, Ana Rute; Santos, Helena

    2013-01-01

    The effect of pH on the glucose metabolism of non-growing cells of L. lactis MG1363 was studied by in vivo NMR in the range 4.8 to 6.5. Immediate pH effects on glucose transporters and/or enzyme activities were distinguished from transcriptional/translational effects by using cells grown at the optimal pH of 6.5 or pre-adjusted to low pH by growth at 5.1. In cells grown at pH 5.1, glucose metabolism proceeds at a rate 35% higher than in non-adjusted cells at the same pH. Besides the upregulation of stress-related genes (such as dnaK and groEL), cells adjusted to low pH overexpressed H(+)-ATPase subunits as well as glycolytic genes. At sub-optimal pHs, the total intracellular pool of lactic acid reached approximately 500 mM in cells grown at optimal pH and about 700 mM in cells grown at pH 5.1. These high levels, together with good pH homeostasis (internal pH always above 6), imply intracellular accumulation of the ionized form of lactic acid (lactate anion), and the concomitant export of the equivalent protons. The average number, n, of protons exported with each lactate anion was determined directly from the kinetics of accumulation of intra- and extracellular lactic acid as monitored online by (13)C-NMR. In cells non-adjusted to low pH, n varies between 2 and 1 during glucose consumption, suggesting an inhibitory effect of intracellular lactate on proton export. We confirmed that extracellular lactate did not affect the lactate: proton stoichiometry. In adjusted cells, n was lower and varied less, indicating a different mix of lactic acid exporters less affected by the high level of intracellular lactate. A qualitative model for pH effects and acid stress adaptation is proposed on the basis of these results.

  15. Constructing a recombinant hyaluronic acid biosynthesis operon and producing food-grade hyaluronic acid in Lactococcus lactis.

    PubMed

    Sheng, Juzheng; Ling, Peixue; Wang, Fengshan

    2015-02-01

    Hyaluronic acid (HA), a natural high molecular weight polysaccharide, is produced by Streptococcus zooepidemicus. However, Streptococcus has several drawbacks including its potential to produce exotoxins, so there is demand for an alternative HA source. Here, a recombinant HA biosynthesis operon, as well as the HA biosynthesis operon of S. zooepidemicus were introduced into L. lactis using the nisin-controlled expression system, respectively. HA was successfully synthesized by recombinant L. lactis. Furthermore, overexpression of the endogenous enzymes directing the synthesis of precursor sugars was effective at increasing HA production, and increasing the supply of UDP-activated monosaccharide donors aided synthesis of monodisperse HA polysaccharides. Besides GRAS host strain (L. lactis) and NICE system, the selecting marker (lacF gene) of the recombinant strain is also food grade. Therefore, HA produced by recombinant L. lactis overcomes the problems associated with Streptococcus and provides a source of food-grading HA appropriate for widespread biotechnological applications.

  16. A partial proteome reference map of the wine lactic acid bacterium Oenococcus oeni ATCC BAA-1163.

    PubMed

    Mohedano, María de la Luz; Russo, Pasquale; de Los Ríos, Vivian; Capozzi, Vittorio; Fernández de Palencia, Pilar; Spano, Giuseppe; López, Paloma

    2014-02-26

    Oenococcus oeni is the main lactic acid bacterium that carries out the malolactic fermentation in virtually all red wines and in some white and sparkling wines. Oenococcus oeni possesses an array of metabolic activities that can modify the taste and aromatic properties of wine. There is, therefore, industrial interest in the proteins involved in these metabolic pathways and related transport systems of this bacterium. In this work, we report the characterization of the O. oeni ATCC BAA-1163 proteome. Total and membrane protein preparations from O. oeni were standardized and analysed by two-dimensional gel electrophoresis. Using tandem mass spectrometry, we identified 224 different spots corresponding to 152 unique proteins, which have been classified by their putative function and subjected to bioinformatics analysis.

  17. Enhanced free fatty acid production by codon-optimized Lactococcus lactis acyl-ACP thioesterase gene expression in Escherichia coli using crude glycerol.

    PubMed

    Lee, Sunhee; Park, Soohyun; Park, Chulhwan; Pack, Seung Pil; Lee, Jinwon

    2014-12-01

    Fatty acid production and composition are determined by the type of acyl-acyl carrier protein thioesterases (acyl-ACP TEs) expressed in Escherichia coli. Bacterial acyl-ACP TEs from Lactococcus lactis (SGJS47), Enterococcus faecalis (SGJS49), and Burkholderia cepacia (SGJS50) were codon-optimized and expressed in E. coli for enhanced fatty acid production. Samples were extracted at the lag, log, and stationary phases of cell growth, and gene expression levels of the codon optimized acy-ACP TEs as well as fatty acid production were monitored. At 24h after initiation of gene expression, the OPLlTE expression level and fatty acid production in SGJS47 increased up to 15.8-fold and 3.2-fold compared to the control and other recombinant strains, respectively. Additionally, in SGJS47, improvement in free fatty acid (FFA) composition, high-specificity production of short-chain fatty acids (C8, C10) and unsaturated fatty acids (C16:1) was achieved in crude glycerol medium condition. Compared with control strain, the percentage of FFAs (C8 and C10) was enhanced by approximately 16- to 21-fold, C16:1 FFA ratio increased approximately 18-fold. Observation of codon-optimized acyl-ACP TE genes expression level in E. coli may be useful for understanding mechanisms towards improving fatty acid production. Engineered strains have the potential to overproduce specific FFAs and thereby reduce the cost of fatty acid production by using industrially inexpensive carbon sources. Copyright © 2014 Elsevier Inc. All rights reserved.

  18. Mechanism of biosynthesis of unsaturated fatty acids in Pseudomonas sp. strain E-3, a psychrotrophic bacterium.

    PubMed Central

    Wada, M; Fukunaga, N; Sasaki, S

    1989-01-01

    Biosynthesis of palmitic, palmitoleic, and cis-vaccenic acids in Pseudomonas sp. strain E-3 was investigated with in vitro and in vivo systems. [1-14C]palmitic acid was aerobically converted to palmitoleate and cis-vaccenate, and the radioactivities on their carboxyl carbons were 100 and 43%, respectively, of the total radioactivity in the fatty acids. Palmitoyl coenzyme A desaturase activity was found in the membrane fraction. [1-14C]stearic acid was converted to octadecenoate and C16 fatty acids. The octadecenoate contained oleate and cis-vaccenate, but only oleate was produced in the presence of cerulenin. [1-14C]lauric acid was aerobically converted to palmitate, palmitoleate, and cis-vaccenate. Under anaerobic conditions, palmitate (62%), palmitoleate (4%), and cis-vaccenate (34%) were produced from [1-14C]acetic acid, while they amounted to 48, 39, and 14%, respectively, under aerobic conditions. In these incorporation experiments, 3 to 19% of the added radioactivity was detected in released 14CO2, indicating that part of the added fatty acids were oxidatively decomposed. Partially purified fatty acid synthetase produced saturated and unsaturated fatty acids with chain lengths of C10 to C18. These results indicated that both aerobic and anaerobic mechanisms for the synthesis of unsaturated fatty acid are operating in this bacterium. PMID:2753856

  19. Efficient production of l-lactic acid from hydrolysate of Jerusalem artichoke with immobilized cells of Lactococcus lactis in fibrous bed bioreactors.

    PubMed

    Shi, Zhouming; Wei, Peilian; Zhu, Xiangcheng; Cai, Jin; Huang, Lei; Xu, Zhinan

    2012-10-10

    Hydrolysate of Jerusalem artichoke was applied for the production of l-lactic acid by immobilized Lactococcus lactis cells in a fibrous bed bioreactor system. Preliminary experiments had indicated that the high quality hydrolysate, which was derived from the 40 min acid treatment at 95 °C and pH 1.8, was sufficient to support the cell growth and synthesis of l-lactic acid. With the addition of 5 g/l yeast extract, the fermentative performance of free cell system was evidently improved. After the basal settlement of hydrolysate based fermentation, the batch mode and the fed-batch mode fermentation were carried out in the free cell system and the fibrous bed bioreactor system, respectively. In all cases the immobilized cells presented the superior ability to produce l-lactic acid. The comparison of batch mode and fed-batch mode also indicated that the growth-limiting feeding strategy could reduce the lag phase of fermentation process and enhance the production of l-lactic acid. The achieved maximum concentration of l-lactic acid was 142 g/l in the fed-batch mode. Subsequent repeated-batch fermentation of the fibrous bed bioreactor system had further exhibited the persistence and stability of this system for the high production of l-lactic acid in a long term. Our work suggested the great potential of the fibrous bed bioreactor system and hydrolysate of J. artichoke in the economical production of l-lactic acid at industrial scale. Copyright © 2012 Elsevier Inc. All rights reserved.

  20. [Stearic acid methyl ether: a new extracellular metabolite of the obligate methylotrophic bacterium Methylophilus quaylei].

    PubMed

    Terekhova, E A; Stepicheva, N A; Pshenichnikova, A B; Shvets, V I

    2010-01-01

    Methyl esters of fatty acids, free fatty acids, and hydrocarbons were found in the culture liquid and in the cellular lipids of the obligate methylotrophic bacterium Methylophilus quaylei under optimal growth conditions and osmotic stress. The main extracellular hydrophobic metabolite was methyl stearate. Exogenous free fatty acids C16-C18 and their methyl esters stimulated the M. quaylei growth and survivability, as well as production of exopolysaccharide under osmotic and oxidative stress, playing the role of growth factors and adaptogens. The order of hydrophobic supplements according to the ability to stimulate bacterial growth is C18 : 1 > C18 : 0 > C16 : 0 > methyl oleate > methyl stearate > no supplements > C14: 0 > C12 : 0. The mechanism underlying the protective action of fatty acids and their methyl esters is discussed.

  1. The ABC-Type Multidrug Resistance Transporter LmrCD Is Responsible for an Extrusion-Based Mechanism of Bile Acid Resistance in Lactococcus lactis▿

    PubMed Central

    Zaidi, Arsalan Haseeb; Bakkes, Patrick J.; Lubelski, Jacek; Agustiandari, Herfita; Kuipers, Oscar P.; Driessen, Arnold J. M.

    2008-01-01

    Upon prolonged exposure to cholate and other toxic compounds, Lactococcus lactis develops a multidrug resistance phenotype that has been attributed to an elevated expression of the heterodimeric ABC-type multidrug transporter LmrCD. To investigate the molecular basis of bile acid resistance in L. lactis and to evaluate the contribution of efflux-based mechanisms in this process, the drug-sensitive L. lactis NZ9000 ΔlmrCD strain was challenged with cholate. A resistant strain was obtained that, compared to the parental strain, showed (i) significantly improved resistance toward several bile acids but not to drugs, (ii) morphological changes, and (iii) an altered susceptibility to antimicrobial peptides. Transcriptome and transport analyses suggest that the acquired resistance is unrelated to elevated transport activity but, instead, results from a multitude of stress responses, changes to the cell envelope, and metabolic changes. In contrast, wild-type cells induce the expression of lmrCD upon exposure to cholate, whereupon the cholate is actively extruded from the cells. Together, these data suggest a central role for an efflux-based mechanism in bile acid resistance and implicate LmrCD as the main system responsible in L. lactis. PMID:18790870

  2. Tolerance of the nanocellulose-producing bacterium Gluconacetobacter xylinus to lignocellulose-derived acids and aldehydes.

    PubMed

    Zhang, Shuo; Winestrand, Sandra; Chen, Lin; Li, Dengxin; Jönsson, Leif J; Hong, Feng

    2014-10-08

    Lignocellulosic biomass serves as a potential alternative feedstock for production of bacterial nanocellulose (BNC), a high-value-added product of bacteria such as Gluconacetobacter xylinus. The tolerance of G. xylinus to lignocellulose-derived inhibitors (formic acid, acetic acid, levulinic acid, furfural, and 5-hydroxymethylfurfural) was investigated. Whereas 100 mM formic acid completely suppressed the metabolism of G. xylinus, 250 mM of either acetic acid or levulinic acid still allowed glucose metabolism and BNC production to occur. Complete suppression of glucose utilization and BNC production was observed after inclusion of 20 and 30 mM furfural and 5-hydroxymethylfurfural, respectively. The bacterium oxidized furfural and 5-hydroxymethylfurfural to furoic acid and 5-hydroxymethyl-2-furoic acid, respectively. The highest yields observed were 88% for furoic acid/furfural and 76% for 5-hydroxymethyl-2-furoic acid/5-hydroxymethylfurfural. These results are the first demonstration of the capability of G. xylinus to tolerate lignocellulose-derived inhibitors and to convert furan aldehydes.

  3. Multi-omics approach to study the growth efficiency and amino acid metabolism in Lactococcus lactis at various specific growth rates.

    PubMed

    Lahtvee, Petri-Jaan; Adamberg, Kaarel; Arike, Liisa; Nahku, Ranno; Aller, Kadri; Vilu, Raivo

    2011-02-24

    Lactococcus lactis is recognised as a safe (GRAS) microorganism and has hence gained interest in numerous biotechnological approaches. As it is fastidious for several amino acids, optimization of processes which involve this organism requires a thorough understanding of its metabolic regulations during multisubstrate growth. Using glucose limited continuous cultivations, specific growth rate dependent metabolism of L. lactis including utilization of amino acids was studied based on extracellular metabolome, global transcriptome and proteome analysis. A new growth medium was designed with reduced amino acid concentrations to increase precision of measurements of consumption of amino acids. Consumption patterns were calculated for all 20 amino acids and measured carbon balance showed good fit of the data at all growth rates studied. It was observed that metabolism of L. lactis became more efficient with rising specific growth rate in the range 0.10-0.60 h(-1), indicated by 30% increase in biomass yield based on glucose consumption, 50% increase in efficiency of nitrogen use for biomass synthesis, and 40% reduction in energy spilling. The latter was realized by decrease in the overall product formation and higher efficiency of incorporation of amino acids into biomass. L. lactis global transcriptome and proteome profiles showed good correlation supporting the general idea of transcription level control of bacterial metabolism, but the data indicated that substrate transport systems together with lower part of glycolysis in L. lactis were presumably under allosteric control. The current study demonstrates advantages of the usage of strictly controlled continuous cultivation methods combined with multi-omics approach for quantitative understanding of amino acid and energy metabolism of L. lactis which is a valuable new knowledge for development of balanced growth media, gene manipulations for desired product formation etc. Moreover, collected dataset is an excellent

  4. Variations in the Degree of d-Alanylation of Teichoic Acids in Lactococcus lactis Alter Resistance to Cationic Antimicrobials but Have No Effect on Bacterial Surface Hydrophobicity and Charge▿

    PubMed Central

    Giaouris, Efstathios; Briandet, Romain; Meyrand, Mickael; Courtin, Pascal; Chapot-Chartier, Marie-Pierre

    2008-01-01

    An increase of the degree of d-alanylation of teichoic acids in Lactococcus lactis resulted in a significant increase of bacterial resistance toward the cationic antimicrobials nisin and lysozyme, whereas the absence of d-alanylation led to a decreased resistance toward the same compounds. In contrast, the same variations of the d-alanylation degree did not modify bacterial cell surface charge and hydrophobicity. Bacterial adhesion to polystyrene and glass surfaces was not modified either. PMID:18539809

  5. Promoting acid resistance and nisin yield of Lactococcus lactis F44 by genetically increasing D-Asp amidation level inside cell wall.

    PubMed

    Hao, Panlong; Liang, Dongmei; Cao, Lijie; Qiao, Bin; Wu, Hao; Caiyin, Qinggele; Zhu, Hongji; Qiao, Jianjun

    2017-08-01

    Nisin fermentation by Lactococcus lactis requires a low pH to maintain a relatively higher nisin activity. However, the acidic environment will result in cell arrest, and eventually decrease the relative nisin production. Hence, constructing an acid-resistant L. lactis is crucial for nisin harvest in acidic nisin fermentation. In this paper, the first discovery of the relationship between D-Asp amidation-associated gene (asnH) and acid resistance was reported. Overexpression of asnH in L. lactis F44 (F44A) resulted in a sevenfold increase in survival capacity during acid shift (pH 3) and enhanced nisin desorption capacity compared to F44 (wild type), which subsequently contributed to higher nisin production, reaching 5346 IU/mL, 57.0% more than that of F44 in the fed-batch fermentation. Furthermore, the engineered F44A showed a moderate increase in D-Asp amidation level (from 82 to 92%) compared to F44. The concomitant decrease of the negative charge inside the cell wall was detected by a newly developed method based on the nisin adsorption amount onto cell surface. Meanwhile, peptidoglycan cross-linkage increased from 36.8% (F44) to 41.9% (F44A), and intracellular pH can be better maintained by blocking extracellular H(+) due to the maintenance of peptidoglycan integrity, which probably resulted from the action of inhibiting hydrolases activity. The inference was further supported by the acmC-overexpression strain F44C, which was characterized by uncontrolled peptidoglycan hydrolase activity. Our results provided a novel strategy for enhancing nisin yield through cell wall remodeling, which contributed to both continuous nisin synthesis and less nisin adsorption in acidic fermentation (dual enhancement).

  6. Niizalactams A-C, Multicyclic Macrolactams Isolated from Combined Culture of Streptomyces with Mycolic Acid-Containing Bacterium.

    PubMed

    Hoshino, Shotaro; Okada, Masahiro; Wakimoto, Toshiyuki; Zhang, Huiping; Hayashi, Fumiaki; Onaka, Hiroyasu; Abe, Ikuro

    2015-12-24

    A terrestrial bacterium, Streptomyces sp. NZ-6, produced niizalactams A-C (1-3), unprecedented di- and tricyclic macrolactams, by coculturing with the mycolic acid-containing bacterium Tsukamurella pulmonis TP-B0596. Their complete structures, including absolute configurations, were elucidated on the basis of spectroscopic data and chemical derivatization. Their unique skeletons are proposed to be biosynthesized from a common 26-membered macrolactam intermediate by SN2 cyclization or an intramolecular Diels-Alder reaction.

  7. UvrA expression of Lactococcus lactis NZ9000 improve multiple stresses tolerance and fermentation of lactic acid against salt stress.

    PubMed

    Moghaddam, Taher Khakpour; Zhang, Juan; Du, Guocheng

    2017-03-01

    Lactococcus lactis is subjected to several stressful conditions during industrial fermentation including oxidation, heating and cooling, acid, high osmolarity/dehydration and starvation. DNA lesion is a major cause of genetic instability in L. lactis that usually occurs at a low frequency, but it is greatly enhanced by environmental stresses. DNA damages produced by these environmental stresses are thought to induce DNA double-strand breaks, leading to illegitimate recombination. Nucleotide excision repair (NER) protein UvrA suppresses multiple stresses-induced illegitimate recombination. UvrA protein can survive a coincident condition of environmental harsh conditions, multiple stress factors supposedly encountered in the host and inducing UvrA in L. lactis. In this study the expression of UvrA and growth performance and viability of control strain L. lactisVector and recombinant strain L. lactisUvrA under multiple stress conditions were determined. The recombinants strain had 30.70 and 52.67% higher growth performances when subjected to acidic and osmotic stresses conditions. In addition, the L. lactisUvrA strain showed 1.85-, 1.65-, and 2.40-fold higher biomass, lactate production, and lactate productivity, compared with the corresponding values for L. lactisVector strain during the osmotic stress. Results demonstrated NER system is involved in adaptation to various stress conditions and suggested that cells with a compromised UvrA as DNA repair system have an enhanced protection behavior in L. lactis NZ9000 against DNA damage.

  8. Cyclopropanation of unsaturated fatty acids and membrane rigidification improve the freeze-drying resistance of Lactococcus lactis subsp. lactis TOMSC161.

    PubMed

    Velly, H; Bouix, M; Passot, S; Penicaud, C; Beinsteiner, H; Ghorbal, S; Lieben, P; Fonseca, F

    2015-01-01

    This work aimed at characterizing the biochemical and biophysical properties of the membrane of Lactococcus lactis TOMSC161 cells during fermentation at different temperatures, in relation to their freeze-drying and storage resistance. Cells were cultivated at two different temperatures (22 and 30 °C) and were harvested at different growth phases (from the middle exponential phase to the late stationary phase). Bacterial membranes were characterized by determining the fatty acid composition, the lipid phase transition, and the membrane fluidity. Cultivability and acidification activity losses of L. lactis were quantified after freezing, drying, and 3 months of storage. The direct measurement of membrane fluidity by fluorescence anisotropy was linked to lipid composition, and it was established that the cyclopropanation of unsaturated fatty acids with concomitant membrane rigidification during growth led to an increase in the freeze-drying and storage resistance of L. lactis. As expected, cultivating cells at a lower fermentation temperature than the optimum growth temperature induced a homeoviscous adaptation that was demonstrated by a lowered lipid phase transition temperature but that was not related to any improvement in freeze-drying resistance. L. lactis TOMSC161 was therefore able to develop a combined biochemical and biophysical response at the membrane level during fermentation. The ratio of cyclic fatty acids to unsaturated fatty acids (CFA/UFA) appeared to be the most relevant parameter associated with membrane rigidification and cell resistance to freeze-drying and storage. This study increased our knowledge about the physiological mechanisms that explain the resistance of lactic acid bacteria (LAB) to freeze-drying and storage stresses and demonstrated the relevance of complementary methods of membrane characterization.

  9. Influence of yeast and lactic acid bacterium on the constituent profile of soy sauce during fermentation.

    PubMed

    Harada, Risa; Yuzuki, Masanobu; Ito, Kotaro; Shiga, Kazuki; Bamba, Takeshi; Fukusaki, Eiichiro

    2017-02-01

    Soy sauce is a Japanese traditional seasoning composed of various constituents that are produced by various microbes during a long-term fermentation process. Due to the complexity of the process, the investigation of the constituent profile during fermentation is difficult. Metabolomics, the comprehensive study of low molecular weight compounds in biological samples, is thought to be a promising strategy for deep understanding of the constituent contribution to food flavor characteristics. Therefore, metabolomics is suitable for the analysis of soy sauce fermentation. Unfortunately, only few and unrefined studies of soy sauce fermentation using metabolomics approach have been reported. Therefore, we investigated changes in low molecular weight hydrophilic and volatile compounds of soy sauce using gas chromatography/mass spectrometry (GC/MS)-based non-targeted metabolic profiling. The data were analyzed by statistical analysis to evaluate influences of yeast and lactic acid bacterium on the constituent profile. Consequently, our results suggested a novel finding that lactic acid bacterium affected the production of several constituents such as cyclotene, furfural, furfuryl alcohol and methional in the soy sauce fermentation process.

  10. Sequencing of the tyrosine decarboxylase cluster of Lactococcus lactis IPLA 655 and the development of a PCR method for detecting tyrosine decarboxylating lactic acid bacteria.

    PubMed

    Fernández, María; Linares, Daniel M; Alvarez, Miguel A

    2004-11-01

    The enzymatic decarboxylation of tyrosine produces tyramine, the most abundant biogenic amine in dairy products-especially in cheeses. The screening of lactic acid bacteria isolated from different artisanal cheeses and a number of microbial collections identified 22 tyramine-producing strains belonging to different genera. The Lactococcus lactis strain IPLA 655 was selected, and the genes encoding a putative tyrosyl tRNA synthetase, a tyrosine decarboxylase (tdcA), and a tyrosine-tyramine antiporter, found together as a cluster, were sequenced. The disruption of tdcA yielded a strain unable to produce tyramine. Comparison of the L. lactis IPLA 655 tdcA gene with database tdcA sequences led to the design of two primers for use in a PCR method that identified potential tyramine-producing strains. The proposed method can use purified DNA, isolated colonies, milk, curd, and even cheese as a template. Molecular tools for the rapid detection of tyramine-producing bacteria at any time during the fermentation process could help prevent tyramine accumulation in fermented foods. The proposed technique could be of great use to the food industry.

  11. Eubacterium rangiferina, a novel usnic acid-resistant bacterium from the reindeer rumen.

    PubMed

    Sundset, Monica A; Kohn, Alexandra; Mathiesen, Svein D; Praesteng, Kirsti E

    2008-08-01

    Reindeer are able to eat and utilize lichens as an important source of energy and nutrients. In the current study, the activities of antibiotic secondary metabolites including usnic, antranoric, fumarprotocetraric, and lobaric acid commonly found in lichens were tested against a collection of 26 anaerobic rumen bacterial isolates from reindeer (Rangifer tarandus tarandus) using the agar diffusion method. The isolates were identified based on their 16S ribosomal ribonucleic acid (rRNA) gene sequences. Usnic acid had a potent antimicrobial effect against 25 of the isolates, belonging to Clostridiales, Enterococci, and Streptococci. Isolates of Clostridia and Streptococci were also susceptible to atranoric and lobaric acid. However, one isolate (R3_91_1) was found to be resistant to usnic, antranoric, fumarprotocetraric, and lobaric acid. R3_91_1 was also seen invading and adhering to lichen particles when grown in a liquid anaerobic culture as demonstrated by transmission electron microscopy. This was a Gram-negative, nonmotile rod (0.2-0.7 x 2.0-3.5 microm) with a deoxyribonucleic acid G + C content of 47.0 mol% and main cellular fatty acids including 15:0 anteiso-dimethyl acetal (DMA), 16:0 iso-fatty acid methyl ester (FAME), 13:0 iso-3OH FAME, and 17:0 anteiso-FAME, not matching any of the presently known profiles in the MIDI database. Combined, the phenotypic and genotypic traits including the 16S rRNA gene sequence show that R3_91_1 is a novel species inside the order Clostridiales within the family Lachnospiraceae, for which we propose the name Eubacterium rangiferina. This is the first record of a rumen bacterium able to tolerate and grow in the presence of usnic acid, indicating that the rumen microorganisms in these animals have adapted mechanisms to deal with lichen secondary metabolites, well known for their antimicrobial and toxic effects.

  12. Eubacterium rangiferina, a novel usnic acid-resistant bacterium from the reindeer rumen

    NASA Astrophysics Data System (ADS)

    Sundset, Monica A.; Kohn, Alexandra; Mathiesen, Svein D.; Præsteng, Kirsti E.

    2008-08-01

    Reindeer are able to eat and utilize lichens as an important source of energy and nutrients. In the current study, the activities of antibiotic secondary metabolites including usnic, antranoric, fumarprotocetraric, and lobaric acid commonly found in lichens were tested against a collection of 26 anaerobic rumen bacterial isolates from reindeer ( Rangifer tarandus tarandus) using the agar diffusion method. The isolates were identified based on their 16S ribosomal ribonucleic acid (rRNA) gene sequences. Usnic acid had a potent antimicrobial effect against 25 of the isolates, belonging to Clostridiales, Enterococci, and Streptococci. Isolates of Clostridia and Streptococci were also susceptible to atranoric and lobaric acid. However, one isolate (R3_91_1) was found to be resistant to usnic, antranoric, fumarprotocetraric, and lobaric acid. R3_91_1 was also seen invading and adhering to lichen particles when grown in a liquid anaerobic culture as demonstrated by transmission electron microscopy. This was a Gram-negative, nonmotile rod (0.2-0.7 × 2.0-3.5 μm) with a deoxyribonucleic acid G + C content of 47.0 mol% and main cellular fatty acids including 15:0 anteiso-dimethyl acetal (DMA), 16:0 iso-fatty acid methyl ester (FAME), 13:0 iso-3OH FAME, and 17:0 anteiso-FAME, not matching any of the presently known profiles in the MIDI database. Combined, the phenotypic and genotypic traits including the 16S rRNA gene sequence show that R3_91_1 is a novel species inside the order Clostridiales within the family Lachnospiraceae, for which we propose the name Eubacterium rangiferina. This is the first record of a rumen bacterium able to tolerate and grow in the presence of usnic acid, indicating that the rumen microorganisms in these animals have adapted mechanisms to deal with lichen secondary metabolites, well known for their antimicrobial and toxic effects.

  13. Genome Sequence Analysis of the Naphthenic Acid Degrading and Metal Resistant Bacterium Cupriavidus gilardii CR3

    PubMed Central

    Xiao, Jingfa; Hao, Lirui; Crowley, David E.; Zhang, Zhewen; Yu, Jun; Huang, Ning; Huo, Mingxin; Wu, Jiayan

    2015-01-01

    Cupriavidus sp. are generally heavy metal tolerant bacteria with the ability to degrade a variety of aromatic hydrocarbon compounds, although the degradation pathways and substrate versatilities remain largely unknown. Here we studied the bacterium Cupriavidus gilardii strain CR3, which was isolated from a natural asphalt deposit, and which was shown to utilize naphthenic acids as a sole carbon source. Genome sequencing of C. gilardii CR3 was carried out to elucidate possible mechanisms for the naphthenic acid biodegradation. The genome of C. gilardii CR3 was composed of two circular chromosomes chr1 and chr2 of respectively 3,539,530 bp and 2,039,213 bp in size. The genome for strain CR3 encoded 4,502 putative protein-coding genes, 59 tRNA genes, and many other non-coding genes. Many genes were associated with xenobiotic biodegradation and metal resistance functions. Pathway prediction for degradation of cyclohexanecarboxylic acid, a representative naphthenic acid, suggested that naphthenic acid undergoes initial ring-cleavage, after which the ring fission products can be degraded via several plausible degradation pathways including a mechanism similar to that used for fatty acid oxidation. The final metabolic products of these pathways are unstable or volatile compounds that were not toxic to CR3. Strain CR3 was also shown to have tolerance to at least 10 heavy metals, which was mainly achieved by self-detoxification through ion efflux, metal-complexation and metal-reduction, and a powerful DNA self-repair mechanism. Our genomic analysis suggests that CR3 is well adapted to survive the harsh environment in natural asphalts containing naphthenic acids and high concentrations of heavy metals. PMID:26301592

  14. Genome Sequence Analysis of the Naphthenic Acid Degrading and Metal Resistant Bacterium Cupriavidus gilardii CR3.

    PubMed

    Wang, Xiaoyu; Chen, Meili; Xiao, Jingfa; Hao, Lirui; Crowley, David E; Zhang, Zhewen; Yu, Jun; Huang, Ning; Huo, Mingxin; Wu, Jiayan

    2015-01-01

    Cupriavidus sp. are generally heavy metal tolerant bacteria with the ability to degrade a variety of aromatic hydrocarbon compounds, although the degradation pathways and substrate versatilities remain largely unknown. Here we studied the bacterium Cupriavidus gilardii strain CR3, which was isolated from a natural asphalt deposit, and which was shown to utilize naphthenic acids as a sole carbon source. Genome sequencing of C. gilardii CR3 was carried out to elucidate possible mechanisms for the naphthenic acid biodegradation. The genome of C. gilardii CR3 was composed of two circular chromosomes chr1 and chr2 of respectively 3,539,530 bp and 2,039,213 bp in size. The genome for strain CR3 encoded 4,502 putative protein-coding genes, 59 tRNA genes, and many other non-coding genes. Many genes were associated with xenobiotic biodegradation and metal resistance functions. Pathway prediction for degradation of cyclohexanecarboxylic acid, a representative naphthenic acid, suggested that naphthenic acid undergoes initial ring-cleavage, after which the ring fission products can be degraded via several plausible degradation pathways including a mechanism similar to that used for fatty acid oxidation. The final metabolic products of these pathways are unstable or volatile compounds that were not toxic to CR3. Strain CR3 was also shown to have tolerance to at least 10 heavy metals, which was mainly achieved by self-detoxification through ion efflux, metal-complexation and metal-reduction, and a powerful DNA self-repair mechanism. Our genomic analysis suggests that CR3 is well adapted to survive the harsh environment in natural asphalts containing naphthenic acids and high concentrations of heavy metals.

  15. Quantitative analysis of growth and volatile fatty acid production by the anaerobic ruminal bacterium Megasphaera elsdenii T81

    USDA-ARS?s Scientific Manuscript database

    Megaspheara elsdenii T81 grew on either DL-lactate or D-glucose at similar rates (0.85 per h), but displayed major differences in the fermentation of these substrates. Lactate was fermented at up to 210-mM concentration to yield acetic, propionic, butyric, and valeric acids. The bacterium was able t...

  16. The effects of a vegetable-derived probiotic lactic acid bacterium on the immune response.

    PubMed

    Chon, Heeson; Choi, Byungryul

    2010-04-01

    The objective of this study was to investigate the probiotic properties of the fermented vegetable derived lactic acid bacterium, L. plantarum. L. plantarum 10hk2 showed antibacterial activity against pathogenic bacteria and immunomodulating effects on murine macrophage cell lines. RAW 264.7 cells stimulated with viable cells of this probiotic strain increased the amounts of pro-inflammatory mediators such as IL-1beta, IL-6 and TNF-alpha, as well as the anti-inflammatory mediator, IL-10. ICR mice fed with viable cells of L. plantarum 10hk2 had reduced numbers of enteric Salmonella and Shigella species in comparison to controls from 2 weeks after supplementation, and this effect was observed for up to 4 weeks. The findings of this study suggest that this specific lactic acid bacterial strain, which is derived from vegetable fermentation, holds great promise for use in probiotics and as a food additive since it can reduce the number of some pathogenic bacteria through production of lactic acids.

  17. Analysis of the amino acid residues involved in the thermal properties of the monomeric isocitrate dehydrogenases of the psychrophilic bacterium Colwellia maris and the mesophilic bacterium Azotobacter vinelandii.

    PubMed

    Kurihara, Takayuki; Takada, Yasuhiro

    2012-01-01

    Cold-adapted monomeric isocitrate dehydrogenase of a psychrophilic bacterium, Colwellia maris, (CmIDH) showed a high degree of amino acid sequential identity (69.5%) to a mesophilic nitrogen-fixing bacterium, Azotobacter vinelandii, (AvIDH). In this study, three Ala residues of CmIDH and the corresponding Pro residues of AvIDH were exchanged by site-directed mutagenesis, and several properties of single, double, and triple mutants of the two enzymes were investigated. The mutated CmIDHs, which replaced Ala719 with Pro, showed increased activity and elevation of the optimum temperature and thermostability for activity. In contrast, mutants of AvIDH, in which Pro717 was replaced by Ala, decreased the thermostability for activity. These results indicate that Ala719 of CmIDH and Pro717 of AvIDH are involved in thermostability. On the other hand, mutated CmIDH, in which Ala710 was replaced by Pro, and the corresponding AvIDH mutant, which replaced Pro708 with Ala, showed higher and lower specific activity than the corresponding wild-type enzymes, suggesting that Pro708 of AvIDH is involved in its high catalytic ability. Furthermore, the exchange mutations between Ala740 in CmIDH and the corresponding Pro738 in AvIDH resulted in decreased and increased thermostability for CmIDH and AvIDH activity respectively, suggesting that the native Ala740 and Pro738 residues make the enzymes thermostable and thermolabile.

  18. A lactic acid bacterium isolated from kimchi ameliorates intestinal inflammation in DSS-induced colitis.

    PubMed

    Park, Jin-Soo; Joe, Inseong; Rhee, Paul Dong; Jeong, Choon-Soo; Jeong, Gajin

    2017-04-01

    Some species of lactic acid bacteria have been shown to be beneficial in inflammatory bowel disease (IBD). In the present study, a strain of lactic acid bacterium (Lactobacillus paracasei LS2) was isolated from the Korean food, kimchi, and was shown to inhibit the development of experimental colitis induced by dextran sulfate sodium (DSS). To investigate the role of LS2 in IBD, mice were fed DSS in drinking water for seven days along with LS2 bacteria which were administered intragastrically to some of the mice, while phosphate-buffered saline (PBS) was administered to others (the controls). The administration of LS2 reduced body weight loss and increased survival, and disease activity indexes (DAI) and histological scores indicated that the severity of colitis was significantly reduced. The production of inflammatory cytokines and myeloperoxidase (MPO) activity also decreased. Flow cytometry analysis showed that the number of Th1 (IFN-γ) population cells was significantly reduced in the LS2-administered mice compared with the controls. The administration of LS2 induced the increase of CD4(+)FOXP3(+) Treg cells, which are responsible for IL-10. Numbers of macrophages (CD11b(+) F4/80(+)), and neutrophils (CD11b(+) Gr-1(+)) among lamina propria lymphocytes (LPL) were also reduced. These results indicate that LS2 has an anti-inflammatory effect and ameliorates DSS-induced colitis.

  19. Lactobacillus formosensis sp. nov., a lactic acid bacterium isolated from fermented soybean meal.

    PubMed

    Chang, Chi-huan; Chen, Yi-sheng; Lee, Tzu-tai; Chang, Yu-chung; Yu, Bi

    2015-01-01

    A Gram-reaction-positive, catalase-negative, facultatively anaerobic, rod-shaped lactic acid bacterium, designated strain S215(T), was isolated from fermented soybean meal. The organism produced d-lactic acid from glucose without gas formation. 16S rRNA gene sequencing results showed that strain S215(T) had 98.74-99.60 % sequence similarity to the type strains of three species of the genus Lactobacillus (Lactobacillus farciminis BCRC 14043(T), Lactobacillus futsaii BCRC 80278(T) and Lactobacillus crustorum JCM 15951(T)). A comparison of two housekeeping genes, rpoA and pheS, revealed that strain S215(T) was well separated from the reference strains of species of the genus Lactobacillus. DNA-DNA hybridization results indicated that strain S215(T) had DNA related to the three type strains of species of the genus Lactobacillus (33-66 % relatedness). The DNA G+C content of strain S215(T) was 36.2 mol%. The cell walls contained peptidoglycan of the d-meso-diaminopimelic acid type and the major fatty acids were C18 : 1ω9c, C16 : 0 and C19 : 0 cyclo ω10c/C19 : 1ω6c. Phenotypic and genotypic features demonstrated that the isolate represents a novel species of the genus Lactobacillus, for which the name Lactobacillus formosensis sp. nov. is proposed. The type strain is S215(T) ( = NBRC 109509(T) = BCRC 80582(T)).

  20. A Highly Stable d-Amino Acid Oxidase of the Thermophilic Bacterium Rubrobacter xylanophilus

    PubMed Central

    Furukawara, Makoto; Omae, Keishi; Tadokoro, Namiho; Saito, Yayoi; Abe, Katsumasa; Kera, Yoshio

    2014-01-01

    d-Amino acid oxidase (DAO) is a biotechnologically attractive enzyme that can be used in a variety of applications, but its utility is limited by its relatively poor stability. A search of a bacterial genome database revealed a gene encoding a protein homologous to DAO in the thermophilic bacterium Rubrobacter xylanophilus (RxDAO). The recombinant protein expressed in Escherichia coli was a monomeric protein containing noncovalently bound flavin adenine dinucleotide as a cofactor. This protein exhibited oxidase activity against neutral and basic d-amino acids and was significantly inhibited by a DAO inhibitor, benzoate, but not by any of the tested d-aspartate oxidase (DDO) inhibitors, thus indicating that the protein is DAO. RxDAO exhibited higher activities and affinities toward branched-chain d-amino acids, with the highest specific activity toward d-valine and catalytic efficiency (kcat/Km) toward d-leucine. Substrate inhibition was observed in the case of d-tyrosine. The enzyme had an optimum pH range and temperature of pH 7.5 to 10 and 65°C, respectively, and was stable between pH 5.0 and pH 8.0, with a T50 (the temperature at which 50% of the initial enzymatic activity is lost) of 64°C. No loss of enzyme activity was observed after a 1-week incubation period at 30°C. This enzyme was markedly inactivated by phenylmethylsulfonyl fluoride but not by thiol-modifying reagents and diethyl pyrocarbonate, which are known to inhibit certain DAOs. These results demonstrated that RxDAO is a highly stable DAO and suggested that this enzyme may be valuable for practical applications, such as the determination and quantification of branched-chain d-amino acids, and as a scaffold to generate a novel DAO via protein engineering. PMID:25217016

  1. Recombinant Lactococcus lactis fails to secrete bovine chymosine

    PubMed Central

    Luerce, Tessália Diniz; Azevedo, Marcela Santiago Pacheco; LeBlanc, Jean Guy; Azevedo, Vasco; Miyoshi, Anderson; Pontes, Daniela Santos

    2014-01-01

    Bovine chymosin is an important milk-clotting agent used in the manufacturing of cheeses. Currently, the production of recombinant proteins by genetically modified organisms is widespread, leading to greatly reduced costs. Lactococcus (L.) lactis, the model lactic acid bacterium, was considered a good candidate for heterologous chymosin production for the following reasons: (1) it is considered to be a GRAS (generally regarded as safe) microorganism, (2) only one protease is present on its surface, (3) it can secrete proteins of different sizes, and (4) it allows for the direct production of protein in fermented food products. Thus, three genetically modified L. lactis strains were constructed to produce and target the three different forms of bovine chymosin, prochymosin B, chymosin A and chymosin B to the extracellular medium. Although all three proteins were stably produced in L. lactis, none of the forms were detected in the extracellular medium or showed clotting activity in milk. Our hypothesis is that this secretion deficiency and lack of clotting activity can be explained by the recombinant protein being attached to the cell envelope. Thus, the development of other strategies is necessary to achieve both production and targeting of chymosin in L. lactis, which could facilitate the downstream processing and recovery of this industrially important protein. PMID:25482140

  2. Recombinant Lactococcus lactis fails to secrete bovine chymosine.

    PubMed

    Luerce, Tessália Diniz; Azevedo, Marcela Santiago Pacheco; LeBlanc, Jean Guy; Azevedo, Vasco; Miyoshi, Anderson; Pontes, Daniela Santos

    2014-01-01

    Bovine chymosin is an important milk-clotting agent used in the manufacturing of cheeses. Currently, the production of recombinant proteins by genetically modified organisms is widespread, leading to greatly reduced costs. Lactococcus (L.) lactis, the model lactic acid bacterium, was considered a good candidate for heterologous chymosin production for the following reasons: (1) it is considered to be a GRAS (generally regarded as safe) microorganism, (2) only one protease is present on its surface, (3) it can secrete proteins of different sizes, and (4) it allows for the direct production of protein in fermented food products. Thus, three genetically modified L. lactis strains were constructed to produce and target the three different forms of bovine chymosin, prochymosin B, chymosin A and chymosin B to the extracellular medium. Although all three proteins were stably produced in L. lactis, none of the forms were detected in the extracellular medium or showed clotting activity in milk. Our hypothesis is that this secretion deficiency and lack of clotting activity can be explained by the recombinant protein being attached to the cell envelope. Thus, the development of other strategies is necessary to achieve both production and targeting of chymosin in L. lactis, which could facilitate the downstream processing and recovery of this industrially important protein.

  3. Fermentation Products of Solvent Tolerant Marine Bacterium Moraxella spp. MB1 and Its Biotechnological Applications in Salicylic Acid Bioconversion

    PubMed Central

    Wahidullah, Solimabi; Naik, Deepak N.; Devi, Prabha

    2013-01-01

    As part of a proactive approach to environmental protection, emerging issues with potential impact on the environment is the subject of ongoing investigation. One emerging area of environmental research concerns pharmaceuticals like salicylic acid, which is the main metabolite of various analgesics including aspirin. It is a common component of sewage effluent and also an intermediate in the degradation pathway of various aromatic compounds which are introduced in the marine environment as pollutants. In this study, biotransformation products of salicylic acid by seaweed, Bryopsis plumosa, associated marine bacterium, Moraxella spp. MB1, have been investigated. Phenol, conjugates of phenol and hydroxy cinnamic acid derivatives (coumaroyl, caffeoyl, feruloyl and trihydroxy cinnamyl) with salicylic acid (3–8) were identified as the bioconversion products by electrospray ionization mass spectrometry. These results show that the microorganism do not degrade phenolic acid but catalyses oxygen dependent transformations without ring cleavage. The degradation of salicylic acid is known to proceed either via gentisic acid pathway or catechol pathway but this is the first report of biotransformation of salicylic acid into cinnamates, without ring cleavage. Besides cinnamic acid derivatives (9–12), metabolites produced by the bacterium include antimicrobial indole (13) and β-carbolines, norharman (14), harman (15) and methyl derivative (16), which are beneficial to the host and the environment. PMID:24391802

  4. Fermentation products of solvent tolerant marine bacterium Moraxella spp. MB1 and its biotechnological applications in salicylic acid bioconversion.

    PubMed

    Wahidullah, Solimabi; Naik, Deepak N; Devi, Prabha

    2013-01-01

    As part of a proactive approach to environmental protection, emerging issues with potential impact on the environment is the subject of ongoing investigation. One emerging area of environmental research concerns pharmaceuticals like salicylic acid, which is the main metabolite of various analgesics including aspirin. It is a common component of sewage effluent and also an intermediate in the degradation pathway of various aromatic compounds which are introduced in the marine environment as pollutants. In this study, biotransformation products of salicylic acid by seaweed, Bryopsis plumosa, associated marine bacterium, Moraxella spp. MB1, have been investigated. Phenol, conjugates of phenol and hydroxy cinnamic acid derivatives (coumaroyl, caffeoyl, feruloyl and trihydroxy cinnamyl) with salicylic acid (3-8) were identified as the bioconversion products by electrospray ionization mass spectrometry. These results show that the microorganism do not degrade phenolic acid but catalyses oxygen dependent transformations without ring cleavage. The degradation of salicylic acid is known to proceed either via gentisic acid pathway or catechol pathway but this is the first report of biotransformation of salicylic acid into cinnamates, without ring cleavage. Besides cinnamic acid derivatives (9-12), metabolites produced by the bacterium include antimicrobial indole (13) and β-carbolines, norharman (14), harman (15) and methyl derivative (16), which are beneficial to the host and the environment.

  5. Generation of Food-Grade Recombinant Lactic Acid Bacterium Strains by Site-Specific Recombination

    PubMed Central

    Martín, M. Cruz; Alonso, Juan C.; Suárez, Juan E.; Alvarez, Miguel A.

    2000-01-01

    The construction of a delivery and clearing system for the generation of food-grade recombinant lactic acid bacterium strains, based on the use of an integrase (Int) and a resolvo-invertase (β-recombinase) and their respective target sites (attP-attB and six, respectively) is reported. The delivery system contains a heterologous replication origin and antibiotic resistance markers surrounded by two directly oriented six sites, a multiple cloning site where passenger DNA could be inserted (e.g., the cI gene of bacteriophage A2), the int gene, and the attP site of phage A2. The clearing system provides a plasmid-borne gene encoding β-recombinase. The nonreplicative vector-borne delivery system was transformed into Lactobacillus casei ATCC 393 and, by site-specific recombination, integrated as a single copy in an orientation- and Int-dependent manner into the attB site present in the genome of the host strain. The transfer of the clearing system into this strain, with the subsequent expression of the β-recombinase, led to site-specific DNA resolution of the non-food-grade DNA. These methods were validated by the construction of a stable food-grade L. casei ATCC 393-derived strain completely immune to phage A2 infection during milk fermentation. PMID:10831443

  6. Rhizobium hidalgonense sp. nov., a nodule endophytic bacterium of Phaseolus vulgaris in acid soil.

    PubMed

    Yan, Jun; Yan, Hui; Liu, Li Xue; Chen, Wen Feng; Zhang, Xiao Xia; Verástegui-Valdés, Myrthala M; Wang, En Tao; Han, Xiao Zeng

    2017-01-01

    One Gram-negative, aerobic, motile, rod-shaped bacterium, designated as FH14(T), was isolated from nodules of Phaseolus vulgaris grown in Hidalgo State of Mexico. Results based upon 16S rRNA gene (≥99.8 % similarities to known species), concatenated sequence (recA, atpD and glnII) analysis of three housekeeping genes (≤93.4 % similarities to known species) and average nucleotide identity (ANI) values of genome sequence (ranged from 87.6 to 90.0 % to related species) indicated the distinct position of strain FH14(T) within the genus Rhizobium. In analyses of symbiotic genes, only nitrogen fixation gene nifH was amplified that had nucleotide sequence identical to those of the bean-nodulating strains in R. phaseoli and R. vallis, while nodulation gene nodC gene was not amplified. The failure of nodulation to its original host P. vulgaris and other legumes evidenced the loss of its nodulation capability. Strain FH14(T) contained summed feature 8 (C18:1 ω6c/C18:1 ω7c, 59.96 %), C16:0 (10.6 %) and summed feature 2 (C12:0 aldehyde/unknown 10.928, 10.24 %) as the major components of cellular fatty acids. Failure to utilize alaninamide, and utilizing L-alanine, L-asparagine and γ-amino butyric acid as carbon source, distinguished the strain FH14(T) from the type strains for the related species. The genome size and DNA G+C content of FH14(T) were 6.94 Mbp and 60.8 mol %, respectively. Based on those results, a novel specie in Rhizobium, named Rhizobium hidalgonense sp. nov., was proposed, with FH14(T) (=HAMBI 3636(T) = LMG 29288(T)) as the type strain.

  7. Mycolic Acid Containing Bacterium Stimulates Tandem Cyclization of Polyene Macrolactam in a Lake Sediment Derived Rare Actinomycete.

    PubMed

    Hoshino, Shotaro; Okada, Masahiro; Awakawa, Takayoshi; Asamizu, Shumpei; Onaka, Hiroyasu; Abe, Ikuro

    2017-09-15

    Two novel macrolactams, dracolactams A and B, were identified from a combined-culture of Micromonospora species and a mycolic-acid containing bacterium (MACB). Their structures and stereochemistries were completely assigned, based on spectroscopic analyses and chemical derivatization. Both dracolactams were probably generated from a common macrolactam precursor produced by the Micromonospora species. In this combined-culture system, MACB is likely to activate cryptic oxidase genes in the Micromonospora species and induce the downstream polyene macrolactam cyclization.

  8. Complete genome sequence of the probiotic lactic acid bacterium Lactobacillus acidophilus NCFM

    PubMed Central

    Altermann, Eric; Russell, W. Michael; Azcarate-Peril, M. Andrea; Barrangou, Rodolphe; Buck, B. Logan; McAuliffe, Olivia; Souther, Nicole; Dobson, Alleson; Duong, Tri; Callanan, Michael; Lick, Sonja; Hamrick, Alice; Cano, Raul; Klaenhammer, Todd R.

    2005-01-01

    Lactobacillus acidophilus NCFM is a probiotic bacterium that has been produced commercially since 1972. The complete genome is 1,993,564 nt and devoid of plasmids. The average GC content is 34.71% with 1,864 predicted ORFs, of which 72.5% were functionally classified. Nine phage-related integrases were predicted, but no complete prophages were found. However, three unique regions designated as potential autonomous units (PAUs) were identified. These units resemble a unique structure and bear characteristics of both plasmids and phages. Analysis of the three PAUs revealed the presence of two R/M systems and a prophage maintenance system killer protein. A spacers interspersed direct repeat locus containing 32 nearly perfect 29-bp repeats was discovered and may provide a unique molecular signature for this organism. In silico analyses predicted 17 transposase genes and a chromosomal locus for lactacin B, a class II bacteriocin. Several mucus- and fibronectin-binding proteins, implicated in adhesion to human intestinal cells, were also identified. Gene clusters for transport of a diverse group of carbohydrates, including fructooligosaccharides and raffinose, were present and often accompanied by transcriptional regulators of the lacI family. For protein degradation and peptide utilization, the organism encoded 20 putative peptidases, homologs for PrtP and PrtM, and two complete oligopeptide transport systems. Nine two-component regulatory systems were predicted, some associated with determinants implicated in bacteriocin production and acid tolerance. Collectively, these features within the genome sequence of L. acidophilus are likely to contribute to the organisms' gastric survival and promote interactions with the intestinal mucosa and microbiota. PMID:15671160

  9. Lactococcus nasutitermitis sp. nov. isolated from a termite gut.

    PubMed

    Yan Yang, Shu; Zheng, Ying; Huang, Zhou; Min Wang, Xue; Yang, Hong

    2016-01-01

    Bacterial strain M19T was isolated from the gut of a wood-feeding termite, Nasutitermes hainanensis. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain M19T was related to members of the genus Lactococcus, with sequence similarities ranging from 84.8 to 95.5 %. Comparison of housekeeping gene ropB sequences revealed that strain M19T was well separated from Lactococcus fujiensis JCM 16395T and Lactococcus hircilactis 117T. The isolate was Gram-stain-positive, catalase-negative and non-motile. Cells were coccoid or ovoid-shaped, and occurred singly, in pairs or as short chains. Growth of strain M19T occurred at 10-40 °C and at pH 5.0-7.5. The DNA G+C content of strain M19T was 39.6 mol% and the major fatty acids were C16 : 0, cyclo-C19 : 0ω8c, C18 : 1ω9c, summed feature 7 and summed feature 8. Based on the phylogenetic, chemotaxonomic and phenotypic data presented, strain M19T represents a novel species of the genus Lactococcus, for which the name Lactococcus nasutitermitis sp. nov. is proposed. The type strain is M19T ( = CGMCC 1.15204T = NBRC 111537T).

  10. Identification and Characterization of a New 7-Aminocephalosporanic Acid Deacetylase from Thermophilic Bacterium Alicyclobacillus tengchongensis.

    PubMed

    Ding, Jun-Mei; Yu, Ting-Ting; Han, Nan-Yu; Yu, Jia-Lin; Li, Jun-Jun; Yang, Yun-Juan; Tang, Xiang-Hua; Xu, Bo; Zhou, Jun-Pei; Tang, Hong-Zhi; Huang, Zun-Xi

    2015-11-02

    Deacetylation of 7-aminocephalosporanic acid (7-ACA) at position C-3 provides valuable starting material for producing semisynthetic β-lactam antibiotics. However, few enzymes have been characterized in this process before now. Comparative analysis of the genome of the thermophilic bacterium Alicyclobacillus tengchongensis revealed a hypothetical protein (EstD1) with typical esterase features. The EstD1 protein was functionally cloned, expressed, and purified from Escherichia coli BL21(DE3). It indeed displayed esterase activity, with optimal activity at around 65°C and pH 8.5, with a preference for esters with short-chain acyl esters (C2 to C4). Sequence alignment revealed that EstD1 is an SGNH hydrolase with the putative catalytic triad Ser15, Asp191, and His194, which belongs to carbohydrate esterase family 12. EstD1 can hydrolyze acetate at the C-3 position of 7-aminocephalosporanic acid (7-ACA) to form deacetyl-7-ACA, which is an important starting material for producing semisynthetic β-lactam antibiotics. EstD1 retained more than 50% of its initial activity when incubated at pH values ranging from 4 to 11 at 65°C for 1 h. To the best of our knowledge, this enzyme is a new SGNH hydrolase identified from thermophiles that is able to hydrolyze 7-ACA. Deacetyl cephalosporins are highly valuable building blocks for the industrial production of various kinds of semisynthetic β-lactam antibiotics. These compounds are derived mainly from 7-ACA, which is obtained by chemical or enzymatic processes from cephalosporin C. Enzymatic transformation of 7-ACA is the main method because of the adverse effects chemical deacylation brought to the environment. SGNH hydrolases are widely distributed in plants. However, the tools for identifying and characterizing SGNH hydrolases from bacteria, especially from thermophiles, are rather limited. Here, our work demonstrates that EstD1 belongs to the SGNH family and can hydrolyze acetate at the C-3 position of 7-ACA. Moreover

  11. Identification and Characterization of a New 7-Aminocephalosporanic Acid Deacetylase from Thermophilic Bacterium Alicyclobacillus tengchongensis

    PubMed Central

    Ding, Jun-Mei; Yu, Ting-Ting; Han, Nan-Yu; Yu, Jia-Lin; Li, Jun-Jun; Yang, Yun-Juan; Tang, Xiang-Hua; Xu, Bo; Zhou, Jun-Pei

    2015-01-01

    ABSTRACT Deacetylation of 7-aminocephalosporanic acid (7-ACA) at position C-3 provides valuable starting material for producing semisynthetic β-lactam antibiotics. However, few enzymes have been characterized in this process before now. Comparative analysis of the genome of the thermophilic bacterium Alicyclobacillus tengchongensis revealed a hypothetical protein (EstD1) with typical esterase features. The EstD1 protein was functionally cloned, expressed, and purified from Escherichia coli BL21(DE3). It indeed displayed esterase activity, with optimal activity at around 65°C and pH 8.5, with a preference for esters with short-chain acyl esters (C2 to C4). Sequence alignment revealed that EstD1 is an SGNH hydrolase with the putative catalytic triad Ser15, Asp191, and His194, which belongs to carbohydrate esterase family 12. EstD1 can hydrolyze acetate at the C-3 position of 7-aminocephalosporanic acid (7-ACA) to form deacetyl-7-ACA, which is an important starting material for producing semisynthetic β-lactam antibiotics. EstD1 retained more than 50% of its initial activity when incubated at pH values ranging from 4 to 11 at 65°C for 1 h. To the best of our knowledge, this enzyme is a new SGNH hydrolase identified from thermophiles that is able to hydrolyze 7-ACA. IMPORTANCE Deacetyl cephalosporins are highly valuable building blocks for the industrial production of various kinds of semisynthetic β-lactam antibiotics. These compounds are derived mainly from 7-ACA, which is obtained by chemical or enzymatic processes from cephalosporin C. Enzymatic transformation of 7-ACA is the main method because of the adverse effects chemical deacylation brought to the environment. SGNH hydrolases are widely distributed in plants. However, the tools for identifying and characterizing SGNH hydrolases from bacteria, especially from thermophiles, are rather limited. Here, our work demonstrates that EstD1 belongs to the SGNH family and can hydrolyze acetate at the C-3 position of

  12. Desulfurella amilsii sp. nov., a novel acidotolerant sulfur-respiring bacterium isolated from acidic river sediments.

    PubMed

    Florentino, Anna P; Brienza, Claudio; Stams, Alfons J M; Sánchez-Andrea, Irene

    2016-03-01

    A novel acidotolerant and moderately thermophilic sulfur-reducing bacterium was isolated from sediments of the Tinto River (Spain), an extremely acidic environment. Strain TR1T stained Gram-negative, and was obligately anaerobic, non-spore-forming and motile. Cells were short rods (1.5-2 × 0.5-0.7 μm), appearing singly or in pairs. Strain TR1T was catalase-negative and slightly oxidase-positive. Urease activity and indole formation were absent, but gelatin hydrolysis was present. Growth was observed at 20-52 °C with an optimum close to 50 °C, and a pH range of 3-7 with optimum between pH 6 and 6.5. Yeast extract was essential for growth, but extra vitamins were not required. In the presence of sulfur, strain TR1T grew with acetate, formate, lactate, pyruvate, stearate, arginine and H2/CO2. All substrates were completely oxidized and H2S and CO2 were the only metabolic products detected. Besides elemental sulfur, thiosulfate was used as an electron acceptor. The isolate also grew by disproportionation of elemental sulfur. The predominant cellular fatty acids were saturated components: C16 : 0, anteiso-C17 : 0 and C18 : 0. The only quinone component detected was menaquinone MK-7(H2). The G+C content of the genomic DNA was 34 mol%. The isolate is affiliated to the genus Desulfurella of the class Deltaproteobacteria, sharing 97 % 16S rRNA gene sequence similarity with the four species described in the genus Desulfurella. Considering the distinct physiological and phylogenetic characteristics, strain TR1T represents a novel species within the genus Desulfurella, for which the name Desulfurella amilsii sp. nov. is proposed. The type strain is TR1T ( = DSM 29984T = JCM 30680T).

  13. Influence of Artisan Bakery- or Laboratory-Propagated Sourdoughs on the Diversity of Lactic Acid Bacterium and Yeast Microbiotas

    PubMed Central

    Minervini, Fabio; Lattanzi, Anna; De Angelis, Maria; Gobbetti, Marco

    2012-01-01

    Seven mature type I sourdoughs were comparatively back-slopped (80 days) at artisan bakery and laboratory levels under constant technology parameters. The cell density of presumptive lactic acid bacteria and related biochemical features were not affected by the environment of propagation. On the contrary, the number of yeasts markedly decreased from artisan bakery to laboratory propagation. During late laboratory propagation, denaturing gradient gel electrophoresis (DGGE) showed that the DNA band corresponding to Saccharomyces cerevisiae was no longer detectable in several sourdoughs. Twelve species of lactic acid bacteria were variously identified through a culture-dependent approach. All sourdoughs harbored a certain number of species and strains, which were dominant throughout time and, in several cases, varied depending on the environment of propagation. As shown by statistical permutation analysis, the lactic acid bacterium populations differed among sourdoughs propagated at artisan bakery and laboratory levels. Lactobacillus plantarum, Lactobacillus sakei, and Weissella cibaria dominated in only some sourdoughs back-slopped at artisan bakeries, and Leuconostoc citreum seemed to be more persistent under laboratory conditions. Strains of Lactobacillus sanfranciscensis were indifferently found in some sourdoughs. Together with the other stable species and strains, other lactic acid bacteria temporarily contaminated the sourdoughs and largely differed between artisan bakery and laboratory levels. The environment of propagation has an undoubted influence on the composition of sourdough yeast and lactic acid bacterium microbiotas. PMID:22635989

  14. The putrescine biosynthesis pathway in Lactococcus lactis is transcriptionally regulated by carbon catabolic repression, mediated by CcpA.

    PubMed

    Linares, Daniel M; del Río, Beatriz; Ladero, Victor; Redruello, Begoña; Martín, María Cruz; Fernández, María; Alvarez, Miguel A

    2013-07-01

    Lactococcus lactis is the lactic acid bacterium most widely used by the dairy industry as a starter for the manufacture of fermented products such as cheese and buttermilk. However, some strains produce putrescine from agmatine via the agmatine deiminase (AGDI) pathway. The proteins involved in this pathway, including those necessary for agmatine uptake and conversion into putrescine, are encoded by the aguB, aguD, aguA and aguC genes, which together form an operon. This paper reports the mechanism of regulation of putrescine biosynthesis in L. lactis. It is shown that the aguBDAC operon, which contains a cre site at the promoter of aguB (the first gene of the operon), is transcriptionally regulated by carbon catabolic repression (CCR) mediated by the catabolite control protein CcpA. Copyright © 2013 Elsevier B.V. All rights reserved.

  15. Molecular and Metabolic Adaptations of Lactococcus lactis at Near-Zero Growth Rates

    PubMed Central

    Ercan, Onur; Wels, Michiel; Smid, Eddy J.

    2014-01-01

    This paper describes the molecular and metabolic adaptations of Lactococcus lactis during the transition from a growing to a near-zero growth state by using carbon-limited retentostat cultivation. Transcriptomic analyses revealed that metabolic patterns shifted between lactic- and mixed-acid fermentations during retentostat cultivation, which appeared to be controlled at the level of transcription of the corresponding pyruvate dissipation-encoding genes. During retentostat cultivation, cells continued to consume several amino acids but also produced specific amino acids, which may derive from the conversion of glycolytic intermediates. We identify a novel motif containing CTGTCAG in the upstream regions of several genes related to amino acid conversion, which we propose to be the target site for CodY in L. lactis KF147. Finally, under extremely low carbon availability, carbon catabolite repression was progressively relieved and alternative catabolic functions were found to be highly expressed, which was confirmed by enhanced initial acidification rates on various sugars in cells obtained from near-zero-growth cultures. The present integrated transcriptome and metabolite (amino acids and previously reported fermentation end products) study provides molecular understanding of the adaptation of L. lactis to conditions supporting low growth rates and expands our earlier analysis of the quantitative physiology of this bacterium at near-zero growth rates toward gene regulation patterns involved in zero-growth adaptation. PMID:25344239

  16. Protein secretion in Lactococcus lactis : an efficient way to increase the overall heterologous protein production

    PubMed Central

    Le Loir, Yves; Azevedo, Vasco; Oliveira, Sergio C; Freitas, Daniela A; Miyoshi, Anderson; Bermúdez-Humarán, Luis G; Nouaille, Sébastien; Ribeiro, Luciana A; Leclercq, Sophie; Gabriel, Jane E; Guimaraes, Valeria D; Oliveira, Maricê N; Charlier, Cathy; Gautier, Michel; Langella, Philippe

    2005-01-01

    Lactococcus lactis, the model lactic acid bacterium (LAB), is a food grade and well-characterized Gram positive bacterium. It is a good candidate for heterologous protein delivery in foodstuff or in the digestive tract. L. lactis can also be used as a protein producer in fermentor. Many heterologous proteins have already been produced in L. lactis but only few reports allow comparing production yields for a given protein either produced intracellularly or secreted in the medium. Here, we review several works evaluating the influence of the localization on the production yields of several heterologous proteins produced in L. lactis. The questions of size limits, conformation, and proteolysis are addressed and discussed with regard to protein yields. These data show that i) secretion is preferable to cytoplasmic production; ii) secretion enhancement (by signal peptide and propeptide optimization) results in increased production yield; iii) protein conformation rather than protein size can impair secretion and thus alter production yields; and iv) fusion of a stable protein can stabilize labile proteins. The role of intracellular proteolysis on heterologous cytoplasmic proteins and precursors is discussed. The new challenges now are the development of food grade systems and the identification and optimization of host factors affecting heterologous protein production not only in L. lactis, but also in other LAB species. PMID:15631634

  17. A Virulent Phage Infecting Lactococcus garvieae, with Homology to Lactococcus lactis Phages

    PubMed Central

    Eraclio, Giovanni; Tremblay, Denise M.; Lacelle-Côté, Alexia; Labrie, Simon J.; Fortina, Maria Grazia

    2015-01-01

    A new virulent phage belonging to the Siphoviridae family and able to infect Lactococcus garvieae strains was isolated from compost soil. Phage GE1 has a prolate capsid (56 by 38 nm) and a long noncontractile tail (123 nm). It had a burst size of 139 and a latent period of 31 min. Its host range was limited to only two L. garvieae strains out of 73 tested. Phage GE1 has a double-stranded DNA genome of 24,847 bp containing 48 predicted open reading frames (ORFs). Putative functions could be assigned to only 14 ORFs, and significant matches in public databases were found for only 17 ORFs, indicating that GE1 is a novel phage and its genome contains several new viral genes and encodes several new viral proteins. Of these 17 ORFs, 16 were homologous to deduced proteins of virulent phages infecting the dairy bacterium Lactococcus lactis, including previously characterized prolate-headed phages. Comparative genome analysis confirmed the relatedness of L. garvieae phage GE1 to L. lactis phages c2 (22,172 bp) and Q54 (26,537 bp), although its genome organization was closer to that of phage c2. Phage GE1 did not infect any of the 58 L. lactis strains tested. This study suggests that phages infecting different lactococcal species may have a common ancestor. PMID:26407890

  18. Enterococcus faecium QU 50: a novel thermophilic lactic acid bacterium for high-yield l-lactic acid production from xylose.

    PubMed

    Abdel-Rahman, Mohamed Ali; Tashiro, Yukihiro; Zendo, Takeshi; Sakai, Kenji; Sonomoto, Kenji

    2015-01-01

    Production of optically pure lactic acid from lignocellulosic material for commercial purposes is hampered by several difficulties, including heterofermentation of pentose sugars and high energy consumption by mesophilic lactic acid bacteria. Here, we report a novel lactic acid bacterium, strain QU 50, that has the potential to produce optically pure l-lactic acid (≥99.2%) in a homofermentative manner from xylose under thermophilic conditions. Strain QU 50 was isolated from Egyptian fertile soil and identified as Enterococcus faecium QU 50 by analyzing its sugar fermentation pattern and 16S rRNA gene sequence. Enterococcus faecium QU 50 fermented xylose efficiently to produce lactic acid over wide pH (6.0-10.0) and temperature ranges (30-52°C), with a pH of 6.5 and temperature of 50°C being optimal. To our knowledge, this is the first report of homofermentative lactic acid production from xylose by a thermophilic lactic acid bacterium.

  19. DNA Macroarray Profiling of Lactococcus lactis subsp. lactis IL1403 Gene Expression during Environmental Stresses†

    PubMed Central

    Xie, Yi; Chou, Lan-szu; Cutler, Adele; Weimer, Bart

    2004-01-01

    This report describes the use of an oligonucleotide macroarray to profile the expression of 375 genes in Lactococcus lactis subsp. lactis IL1403 during heat, acid, and osmotic stress. A set of known stress-associated genes in IL1403 was used as the internal control on the array. Every stress response was accurately detected using the macroarray, compared to data from previous reports. As a group, the expression patterns of the investigated metabolic genes were significantly altered by heat, acid, and osmotic stresses. Specifically, 13 to 18% of the investigated genes were differentially expressed in each of the environmental stress treatments. Interestingly, the methionine biosynthesis pathway genes (metA-metB1 and metB2-cysK) were induced during heat shock, but methionine utilization genes, such as metK, were induced during acid stress. These data provide a possible explanation for the differences between acid tolerance mechanisms of L. lactis strains IL1403 and MG1363 reported previously. Several groups of transcriptional responses were common among the stress treatments, such as repression of peptide transporter genes, including the opt operon (also known as dpp) and dtpT. Reduction of peptide transport due to environmental stress will have important implications in the cheese ripening process. Although stress responses in lactococci were extensively studied during the last decade, additional information about this bacterium was gained from the use of this metabolic array. PMID:15528540

  20. Identification and Analysis of a Novel Group of Bacteriophages Infecting the Lactic Acid Bacterium Streptococcus thermophilus

    PubMed Central

    McDonnell, Brian; Mahony, Jennifer; Neve, Horst; Hanemaaijer, Laurens; Noben, Jean-Paul; Kouwen, Thijs

    2016-01-01

    ABSTRACT We present the complete genome sequences of four members of a novel group of phages infecting Streptococcus thermophilus, designated here as the 987 group. Members of this phage group appear to have resulted from genetic exchange events, as evidenced by their “hybrid” genomic architecture, exhibiting DNA sequence relatedness to the morphogenesis modules of certain P335 group Lactococcus lactis phages and to the replication modules of S. thermophilus phages. All four identified members of the 987 phage group were shown to elicit adsorption affinity to both their cognate S. thermophilus hosts and a particular L. lactis starter strain. The receptor binding protein of one of these phages (as a representative of this novel group) was defined using an adsorption inhibition assay. The emergence of a novel phage group infecting S. thermophilus highlights the continuous need for phage monitoring and development of new phage control measures. IMPORTANCE Phage predation of S. thermophilus is an important issue for the dairy industry, where viral contamination can lead to fermentation inefficiency or complete fermentation failure. Genome information and phage-host interaction studies of S. thermophilus phages, particularly those emerging in the marketplace, are an important part of limiting the detrimental impact of these viruses in the dairy environment. PMID:27316953

  1. Cis-9-octadecenoic acid from the rhizospheric bacterium Stenotrophomonas maltophilia BJ01 shows quorum quenching and anti-biofilm activities.

    PubMed

    Singh, Vijay Kumar; Kavita, Kumari; Prabhakaran, Rathish; Jha, Bhavanath

    2013-01-01

    Quorum quenching (QQ) is an effective approach for the prevention of bacterial infections involving biofilms. This study reports the QQ and anti-biofilm activities of a rhizospheric bacterium identified as Stenotrophomonas maltophilia BJ01. The QQ activity was demonstrated using Chromobacterium violaceum CV026 as a biosensor. A maximum of 95% reduction in violacein production, a quorum sensing-regulated behavior, was observed. Gas chromatography-mass spectroscopy of the extract showed that the active compound was cis-9-octadecenoic acid, which was confirmed by electronspray ionization-mass spectroscopy data. The extract also inhibited biofilm formation of Pseudomonas aeruginosa ATCC 9027 without affecting its growth. Scanning electron and atomic force microscopy showed architectural disruption of the biofilm when treated with the extract. This is the first report of the QQ and anti-biofilm activities of cis-9-octadecenoic acid isolated from any bacterium. It may have the potential to combat detrimental infections with P. aeruginosa. Further validation is required for any possible medical application.

  2. Fractionation of carbon isotopes in biosynthesis of fatty acids by a piezophilic bacterium Moritella japonica strain DSK1

    NASA Astrophysics Data System (ADS)

    Fang, Jiasong; Uhle, Maria; Billmark, Kaycie; Bartlett, Douglas H.; Kato, Chaki

    2006-04-01

    We examined stable carbon isotope fractionation in biosynthesis of fatty acids of a piezophilic bacterium Moritella japonica strain DSK1. The bacterium was grown to stationary phase at pressures of 0.1, 10, 20, and 50 MPa in media prepared using sterile-filtered natural seawater supplied with glucose as the sole carbon source. Strain DSK1 synthesized typical bacterial fatty acids (C 14-19 saturated, monounsaturated, and cyclopropane fatty acids) as well as long-chain polyunsaturated fatty acids (PUFA) (20:6 ω3). Bacterial cell biomass and individual fatty acids exhibited consistent pressure-dependent carbon isotope fractionations relative to glucose. The observed Δδ FA-glucose (-1.0‰ to -11.9‰) at 0.1 MPa was comparable to or slightly higher than fractionations reported in surface bacteria. However, bulk biomass and fatty acids became more depleted in 13C with pressure. Average carbon isotope fractionation (Δδ FA-glucose) at high pressures was much higher than that for surface bacteria: -15.7‰, -15.3‰, and -18.3‰ at 10, 20, and 50 MPa, respectively. PUFA were more 13C depleted than saturated and monounsaturated fatty acids at all pressures. The observed isotope effects may be ascribed to the kinetics of enzymatic reactions that are affected by hydrostatic pressure and to biosynthetic pathways that are different for short-chain and long-chain fatty acids. A simple quantitative calculation suggests that in situ piezophilic bacterial contribution of polyunsaturated fatty acids to marine sediments is nearly two orders of magnitude higher than that of marine phytoplankton and that the carbon isotope imprint of piezophilic bacteria can override that of surface phytoplankton. Our results have important implications for marine biogeochemistry. Depleted fatty acids reported in marine sediments and the water column may be derived simply from piezophilic bacteria resynthesis of organic matter, not from bacterial utilization of a 13C-depleted carbon source (i

  3. Recycling of carbon dioxide and acetate as lactic acid by the hydrogen-producing bacterium Thermotoga neapolitana.

    PubMed

    d'Ippolito, Giuliana; Dipasquale, Laura; Fontana, Angelo

    2014-09-01

    The heterotrophic bacterium Thermotoga neapolitana produces hydrogen by fermentation of sugars. Under capnophilic (carbon dioxide requiring) conditions, the process is preferentially associated with the production of lactic acid, which, as shown herein, is synthesized by reductive carboxylation of acetyl coenzyme A. The enzymatic coupling is dependent on the carbon dioxide stimulated activity of heterotetrameric pyruvate:ferredoxin oxidoreductase. Under the same culture conditions, T. neapolitana also operates the unfavorable synthesis of lactic acid from an exogenous acetate supply. This process, which requires carbon dioxide (or carbonate) and an unknown electron donor, allows for the conversion of carbon dioxide into added-value chemicals without biomass deconstruction. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. The amino acid sequence of the cytochrome c-554(547) from the chemolithotrophic bacterium Thiobacillus neapolitanus.

    PubMed Central

    Ambler, R P; Meyer, T E; Trudinger, P A; Kamen, M D

    1985-01-01

    An amino acid sequence is proposed for the cytochrome c-554(547) from the bacterium Thiobacillus neapolitanus N.C.I.B. 8539). It consists of a polypeptide chain of 91 residues, with a pair of haem-attachment cysteine residues at positions 15 and 18. There is similarity in sequence with each of the halves of the sequence of the dihaem cytochromes c4 and with a cytochrome c-554(548) from a halophilic strain of Paracoccus. Detailed evidence for the amino acid sequence of the protein has been deposited as Supplementary Publication SUP 50127 (11 pages) at the British Library (Lending Division), Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1985) 225, 5. PMID:2988504

  5. Influence of phenolic compounds on the growth and arginine deiminase system in a wine lactic acid bacterium.

    PubMed

    Alberto, María R; de Nadra, María C Manca; Arena, Mario E

    2012-01-01

    The influence of seven phenolic compounds, normally present in wine, on the growth and arginine deiminase system (ADI) of Lactobacillus hilgardii X1B, a wine lactic acid bacterium, was established. This system provides energy for bacterial growth and produces citrulline that reacts with ethanol forming the carcinogen ethyl carbamate (EC), found in some wines. The influence of phenolic compounds on bacterial growth was compound dependent. Growth and final pH values increased in presence of arginine. Arginine consumption decreased in presence of protocatechuic and gallic acids (31 and 17%, respectively) and increased in presence of quercetin, rutin, catechin and the caffeic and vanillic phenolic acids (between 10 and 13%, respectively). ADI enzyme activities varied in presence of phenolic compounds. Rutin, quercetin and caffeic and vanillic acids stimulated the enzyme arginine deiminase about 37-40%. Amounts of 200 mg/L gallic and protocatechuic acids inhibited the arginine deiminase enzyme between 53 and 100%, respectively. Ornithine transcarbamylase activity was not modified at all concentrations of phenolic compounds. As gallic and protocatechuic acids inhibited the arginine deiminase enzyme that produces citrulline, precursor of EC, these results are important considering the formation of toxic compounds.

  6. Influence of phenolic compounds on the growth and arginine deiminase system in a wine lactic acid bacterium

    PubMed Central

    Alberto, María R.; de Nadra, María C. Manca; Arena, Mario E.

    2012-01-01

    The influence of seven phenolic compounds, normally present in wine, on the growth and arginine deiminase system (ADI) of Lactobacillus hilgardii X1B, a wine lactic acid bacterium, was established. This system provides energy for bacterial growth and produces citrulline that reacts with ethanol forming the carcinogen ethyl carbamate (EC), found in some wines. The influence of phenolic compounds on bacterial growth was compound dependent. Growth and final pH values increased in presence of arginine. Arginine consumption decreased in presence of protocatechuic and gallic acids (31 and 17%, respectively) and increased in presence of quercetin, rutin, catechin and the caffeic and vanillic phenolic acids (between 10 and 13%, respectively). ADI enzyme activities varied in presence of phenolic compounds. Rutin, quercetin and caffeic and vanillic acids stimulated the enzyme arginine deiminase about 37–40%. Amounts of 200 mg/L gallic and protocatechuic acids inhibited the arginine deiminase enzyme between 53 and 100%, respectively. Ornithine transcarbamylase activity was not modified at all concentrations of phenolic compounds. As gallic and protocatechuic acids inhibited the arginine deiminase enzyme that produces citrulline, precursor of EC, these results are important considering the formation of toxic compounds. PMID:24031815

  7. Purification, characterization, gene cloning and nucleotide sequencing of D: -stereospecific amino acid amidase from soil bacterium: Delftia acidovorans.

    PubMed

    Hongpattarakere, Tipparat; Komeda, Hidenobu; Asano, Yasuhisa

    2005-12-01

    The D-amino acid amidase-producing bacterium was isolated from soil samples using an enrichment culture technique in medium broth containing D-phenylalanine amide as a sole source of nitrogen. The strain exhibiting the strongest activity was identified as Delftia acidovorans strain 16. This strain produced intracellular D-amino acid amidase constitutively. The enzyme was purified about 380-fold to homogeneity and its molecular mass was estimated to be about 50 kDa, on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme was active preferentially toward D-amino acid amides rather than their L-counterparts. It exhibited strong amino acid amidase activity toward aromatic amino acid amides including D-phenylalanine amide, D-tryptophan amide and D-tyrosine amide, yet it was not specifically active toward low-molecular-weight D-amino acid amides such as D-alanine amide, L-alanine amide and L-serine amide. Moreover, it was not specifically active toward oligopeptides. The enzyme showed maximum activity at 40 degrees C and pH 8.5 and appeared to be very stable, with 92.5% remaining activity after the reaction was performed at 45 degrees C for 30 min. However, it was mostly inactivated in the presence of phenylmethanesulfonyl fluoride or Cd2+, Ag+, Zn2+, Hg2+ and As3+ . The NH2 terminal and internal amino acid sequences of the enzyme were determined; and the gene was cloned and sequenced. The enzyme gene damA encodes a 466-amino-acid protein (molecular mass 49,860.46 Da); and the deduced amino acid sequence exhibits homology to the D-amino acid amidase from Variovorax paradoxus (67.9% identity), the amidotransferase A subunit from Burkholderia fungorum (50% identity) and other enantioselective amidases.

  8. Unusual fatty acid compositions of the hyperthermophilic archaeon Pyrococcus furiosus and the bacterium Thermotoga maritima.

    PubMed Central

    Carballeira, N M; Reyes, M; Sostre, A; Huang, H; Verhagen, M F; Adams, M W

    1997-01-01

    The fatty acid compositions of the hyperthermophilic microorganisms Thermotoga maritima and Pyrococcus furiosus were studied and compared. A total of 37 different fatty acids were identified in T. maritima, including the novel 13,14-dimethyloctacosanedioic acid. In contrast, a total of 18 different fatty acids were characterized, as minor components, in P. furiosus, and these included saturated, monounsaturated, and dicarboxylic acids. This is the first report of fatty acids from an archaeon. PMID:9098079

  9. The highly tolerant acetic acid bacterium Gluconacetobacter europaeus adapts to the presence of acetic acid by changes in lipid composition, morphological properties and PQQ-dependent ADH expression.

    PubMed

    Trcek, Janja; Jernejc, Katarina; Matsushita, Kazunobu

    2007-07-01

    The strain of acetic acid bacterium, Gluconacetobacter europaeus V3, previously isolated from industrial vinegar-producing bioreactor, tolerates extremely high acetic acid concentrations of up to 10% (v/v). Increased concentration of acetic acid changed the total fatty acid composition of cells by increasing the concentration of a major unsaturated fatty acid, the cis-vaccenic acid. Among the phospholipids, the most significant change was observed for phosphatidylglycerol with 7.3-fold increase and phosphatidylethanolamin with 2.7-fold decrease in the presence of 3% (v/v) of acetic acid. The sizes of cells analyzed with scanning electron microscopy changed from short to long rods in the presence of acetic acid. The cells were covered with spongy layer. The increase of acetic acid concentration from 1 to 2% (v/v) induced the expression of PQQ-dependent alcohol dehydrogenase, but the regulation could not be demonstrated at the transcriptional level. All together, our results suggest that Ga. europaeus activates several adaptive mechanisms to resist the stress of acetic acid.

  10. Fractionation of Carbon Isotopes in Biosynthesis of Fatty Acids by A Piezophilic Bacterium Moritella Japonica DSK1

    NASA Astrophysics Data System (ADS)

    Fang, J.; Uhle, M.; Bartlett, D.; Kato, C.

    2005-12-01

    We examined stable carbon isotope fractionation in biosynthesis of fatty acids of a piezophilic bacterium Moritella japonica DSK1. DSK1 was grown to stationary phase at pressures of 0.1, 10, 20, and 50 MPa in media prepared using natural seawater supplied with glucose with the sole carbon source. DSk1 synthesized typical bacterial fatty acids (C14-19 saturated, monounsaturated, and cyclopropane fatty acids) as well as long-chain polyunsaturated fatty acids (PUFA) (20:6ω3). Bacterial cell biomass and individual fatty acids exhibited consistent pressure-dependent carbon isotope fractionations relative to glucose. The observed ΔδFA-glucose (-1.0 to -11.9%) at 0.1 MPa was comparable to or slightly higher than fractionations reported on surface bacteria. However, Bulk biomass and fatty acids became more depleted in 13C with pressure. Average carbon isotope fractionation ΔδFA-glucose) at high pressures was much higher than that for surface bacteria: -15.7, -15.3, and -18.3‰ at 10, 20, and 50 MPa, respectively. PUFA were more 13C depleted than saturated and monounsaturated fatty acids at all pressures. The observed isotope effects may be ascribed to the kinetics of enzymatic reactions affected by hydrostatic pressure and to different biosynthetic pathways for short-chain and long-chain fatty acids. Our results have important implications for marine biogeochemistry. The 13C depleted fatty acids in marine sediments and water column may be derived simply from piezophilic bacteria resynthesis of organic matter, not from bacterial utilization of a 13C-depleted carbon source (i.e., methane). The interpretation of carbon isotope signatures of marine lipids must be based on principles derived from piezophilic bacteria.

  11. Modeling of the Competitive Growth of Listeria monocytogenes and Lactococcus lactis in Vegetable Broth

    PubMed Central

    Breidt, Frederick; Fleming, Henry P.

    1998-01-01

    Current mathematical models used by food microbiologists do not address the issue of competitive growth in mixed cultures of bacteria. We developed a mathematical model which consists of a system of nonlinear differential equations describing the growth of competing bacterial cell cultures. In this model, bacterial cell growth is limited by the accumulation of protonated lactic acid and decreasing pH. In our experimental system, pure and mixed cultures of Lactococcus lactis and Listeria monocytogenes were grown in a vegetable broth medium. Predictions of the model indicate that pH is the primary factor that limits the growth of L. monocytogenes in competition with a strain of L. lactis which does not produce the bacteriocin nisin. The model also predicts the values of parameters that affect the growth and death of the competing populations. Further development of this model will incorporate the effects of additional inhibitors, such as bacteriocins, and may aid in the selection of lactic acid bacterium cultures for use in competitive inhibition of pathogens in minimally processed foods. PMID:9726854

  12. Nisin production by a mixed-culture system consisting of Lactococcus lactis and Kluyveromyces marxianus.

    PubMed

    Shimizu, H; Mizuguchi, T; Tanaka, E; Shioya, S

    1999-07-01

    To control the pH during antimicrobial peptide (nisin) production by a lactic acid bacterium, Lactococcus lactis subsp. lactis (ATCC11454), a novel method involving neither addition of alkali nor a separation system such as a ceramic membrane filter and electrodialyzer was developed. A mixed culture of L. lactis and Kluyveromyces marxianus, which was isolated from kefir grains, was utilized in the developed system. The interaction between lactate production by L. lactis and its assimilation by K. marxianus was used to control the pH. To utilize the interaction of these microorganisms to maintain high-level production of nisin, the kinetics of growth of, and production of lactate, acetate, and nisin by, L. lactis were investigated. The kinetics of growth of and lactic acid consumption by K. marxianus were also investigated. Because the pH of the medium could be controlled by the lactate consumption of K. marxianus and the specific lactate consumption rate of K. marxianus could be controlled by changing the dissolved oxygen (DO) concentration, a cascade pH controller coupled with DO control was developed. As a result, the pH was kept constant because the lactate level was kept low and nisin accumulated in the medium to a high level compared with that attained using other pH control strategies, such as with processes lacking pH control and those in which pH is controlled by addition of alkali.

  13. Nisin Production by a Mixed-Culture System Consisting of Lactococcus lactis and Kluyveromyces marxianus

    PubMed Central

    Shimizu, Hiroshi; Mizuguchi, Taiji; Tanaka, Eiji; Shioya, Suteaki

    1999-01-01

    To control the pH during antimicrobial peptide (nisin) production by a lactic acid bacterium, Lactococcus lactis subsp. lactis (ATCC11454), a novel method involving neither addition of alkali nor a separation system such as a ceramic membrane filter and electrodialyzer was developed. A mixed culture of L. lactis and Kluyveromyces marxianus, which was isolated from kefir grains, was utilized in the developed system. The interaction between lactate production by L. lactis and its assimilation by K. marxianus was used to control the pH. To utilize the interaction of these microorganisms to maintain high-level production of nisin, the kinetics of growth of, and production of lactate, acetate, and nisin by, L. lactis were investigated. The kinetics of growth of and lactic acid consumption by K. marxianus were also investigated. Because the pH of the medium could be controlled by the lactate consumption of K. marxianus and the specific lactate consumption rate of K. marxianus could be controlled by changing the dissolved oxygen (DO) concentration, a cascade pH controller coupled with DO control was developed. As a result, the pH was kept constant because the lactate level was kept low and nisin accumulated in the medium to a high level compared with that attained using other pH control strategies, such as with processes lacking pH control and those in which pH is controlled by addition of alkali. PMID:10388714

  14. Inhibition mechanism of Listeria monocytogenes by a bioprotective bacteria Lactococcus piscium CNCM I-4031.

    PubMed

    Saraoui, Taous; Fall, Papa Abdoulaye; Leroi, Françoise; Antignac, Jean-Philippe; Chéreau, Sylvain; Pilet, Marie France

    2016-02-01

    Listeria monocytogenes is a pathogenic Gram positive bacterium and the etiologic agent of listeriosis, a severe food-borne disease. Lactococcus piscium CNCM I-4031 has the capacity to prevent the growth of L. monocytogenes in contaminated peeled and cooked shrimp. To investigate the inhibititory mechanism, a chemically defined medium (MSMA) based on shrimp composition and reproducing the inhibition observed in shrimp was developed. In co-culture at 26 °C, L. monocytogenes was reduced by 3-4 log CFU g(-1) after 24 h. We have demonstrated that the inhibition was not due to secretion of extracellular antimicrobial compounds as bacteriocins, organic acids and hydrogen peroxide. Global metabolomic fingerprints of these strains in pure culture were assessed by liquid chromatography coupled with high resolution mass spectrometry. Consumption of glucose, amino-acids, vitamins, nitrogen bases, iron and magnesium was measured and competition for some molecules could be hypothesized. However, after 24 h of co-culture, when inhibition of L. monocytogenes occurred, supplementation of the medium with these compounds did not restore its growth. The inhibition was observed in co-culture but not in diffusion chamber when species were separated by a filter membrane. Taken together, these data indicate that the inhibition mechanism of L. monocytogenes by L. piscium is cell-to-cell contact-dependent.

  15. Identification of an Arachidonic Acid-Producing Bacterium and Description of Kineococcus arachidonicus sp. nov.

    SciTech Connect

    Fliermans, C.B.

    2001-05-15

    The identification of bacterial with the ability to produce polyunsaturated fatty acids as been limited almost exclusively to gram-negative, psychrophilic, marine microorganisms. Here we describe a new gram-type-positive bactgerium, strain SRS30216T, that produces the polyunsaturated fatty acid, arachidonic acid, and is neither psychrophilic nor a marine isolate.

  16. Influence of commercial inactivated yeast derivatives on the survival of probiotic bacterium Lactobacillus rhamnosus HN001 in an acidic environment.

    PubMed

    Toh, Mingzhan; Liu, Shao Quan

    2017-12-01

    This study evaluated the influence of three inactivated yeast derivatives (IYDs) used in wine production, namely OptiRed(®), OptiWhite(®) and Noblesse(®), on the viability of the probiotic strain Lactobacillus rhamnosus HN001 in an acidic environment. Addition of the IYDs at 3 g/L significantly enhanced the survival of the probiotic bacteria by 2.75-4.05 log cycles after 10-h exposure in a pH 3.0 buffer. Acid stress assay with IYD components obtained after centrifugation and filtration revealed that water-soluble compounds were responsible for improving the acid tolerance of L. rhamnosus HN001 for all three preparations. Differences in protective effect amongst the IYDs on L. rhamnosus HN001 were observed when permeates and retentates of the water-soluble extracts, obtained through ultrafiltration with a 2 kDa membrane, were assayed against the lactic acid bacterium. Chemical analysis of the water-soluble components suggests that low molecular weight polysaccharides, specific free amino acids and/or antioxidants in the 2 kDa permeates could have contributed to the enhanced survival of L. rhamnosus HN001 during acid stress. The contrast amongst the 2 kDa retentates' viability enhancing property may have been attributed to the differences in size and structure of the higher molecular weight carbohydrates and proteins, as the survival of the probiotic did not relate to the concentration of these compounds. These results suggests that oenological IYDs could potentially be applied to probiotic foods for enhancing the acid tolerance of the beneficial microorganisms, and consequently prolonging the shelf life of these products.

  17. Isolation and characterization of Halomonas sp. strain IMPC, a p-coumaric acid-metabolizing bacterium that decarboxylates other cinnamic acids under hypersaline conditions.

    PubMed

    Abdelkafi, Slim; Labat, Marc; Casalot, Laurence; Chamkha, Mohamed; Sayadi, Sami

    2006-02-01

    A moderately halophilic, mesophilic, Gram-negative, motile, nonsporulating bacterium, designated strain IMPC, was isolated from a table-olive fermentation rich in aromatic compounds, after enrichment on p-coumaric acid under halophilic conditions. Strain IMPC was able to degrade p-coumaric acid. p-hydroxybenzaldehyde and p-hydroxybenzoic acid were detected as breakdown products from p-coumaric acid. Protocatechuic acid was identified as the final aromatic product of p-coumaric acid catabolism before ring fission. Strain IMPC transformed various cinnamic acids with substituent H, OH, CH(3) or OCH(3) in the para- and/or meta-position of the aromatic ring to the corresponding benzoic acids, indicating a specific selection. A beta-oxidation pathway was proposed for these transformations. Phylogenetic analysis of the 16S rRNA gene revealed that this isolate was a member of the genus Halomonas. Strain IMPC was closely related to Halomonas elongata ATCC 33173(T)and Halomonas eurihalina ATCC 49336(T).

  18. Lactococcus lactis ssp. lactis as Potential Functional
Starter Culture

    PubMed Central

    Cvrtila, Jelena; Topić, Ivana; Delaš, Frane; Markov, Ksenija

    2014-01-01

    Summary The aim of this study is to identify and characterise potential autochthonous functional starter cultures in homemade horsemeat sausage. The dominant microflora in the samples of horsemeat sausage were lactic acid bacteria (LAB), followed by micrococci. Among the LAB, Lactococcus lactis ssp. lactis and Lactobacillus plantarum were the dominant species, and since the first is not common in fermented sausages, we characterised it as a potential functional starter culture. Lactococcus lactis ssp. lactis produced a significant amount of lactic acid, displayed good growth capability at 12, 18 and 22 °C, growth in the presence of 5% NaCl, good viability after lyophilisation and in simulated gastric and small intestinal juice, antimicrobial activity against test pathogens, and good adhesive properties in vitro. PMID:27904322

  19. A CMP-N-acetylneuraminic acid synthetase purified from a marine bacterium, Photobacterium leiognathi JT-SHIZ-145.

    PubMed

    Kajiwara, Hitomi; Mine, Toshiki; Miyazaki, Tatsuo; Yamamoto, Takeshi

    2011-01-01

    A cytidine 5'-monophospho-N-acetylneuraminic acid (CMP-Neu5Ac) synthetase was found in a crude extract prepared from Photobacterium leiognathi JT-SHIZ-145, a marine bacterium that also produces a β-galactoside α2,6-sialyltransferase. The CMP-Neu5Ac synthetase was purified from the crude extract of the cells by a combination of anion-exchange and gel filtration column chromatography. The purified enzyme migrated as a single band (60 kDa) on sodium dodecylsulfate-polyacrylamide gel electrophoresis. The activity of the enzyme was maximal at 35 °C at pH 9.0, and the synthetase required Mg(2+) for activity. Although these properties are similar to those of other CMP-Neu5Ac synthetases isolated from bacteria, this synthetase produced not only CMP-Neu5Ac from cytidine triphosphate and Neu5Ac, but also CMP-N-glycolylneuraminic acid from cytidine triphosphate and N-glycolylneuraminic acid, unlike CMP-Neu5Ac synthetase purified from Escherichia coli.

  20. Elongation of exogenous fatty acids by the bioluminescent bacterium Vibrio harveyi

    SciTech Connect

    Byers, D.M.

    1989-01-01

    Bioluminescent bacteria require myristic acid (C14:0) to produce the myristaldehyde substrate of the light-emitting luciferase reaction. Since both endogenous and exogenous C14:0 can be used for this purpose, the metabolism of exogenous fatty acids by luminescent bacteria has been investigated. Both Vibrio harveyi and Vibrio fischeri incorporated label from (1-14C)myristic acid (C14:0) into phospholipid acyl chains as well as into CO2. In contrast, Photobacterium phosphoreum did not exhibit phospholipid acylation or beta-oxidation using exogenous fatty acids. Unlike Escherichia coli, the two Vibrio species can directly elongate fatty acids such as octanoic (C8:0), lauric (C12:0), and myristic acid, as demonstrated by radio-gas liquid chromatography. The induction of bioluminescence in late exponential growth had little effect on the ability of V. harveyi to elongate fatty acids, but it did increase the amount of C14:0 relative to C16:0 labeled from (14C)C8:0. This was not observed in a dark mutant of V. harveyi that is incapable of supplying endogenous C14:0 for luminescence. Cerulenin preferentially decreased the labeling of C16:0 and of unsaturated fatty acids from all 14C-labeled fatty acid precursors as well as from (14C)acetate, suggesting that common mechanisms may be involved in elongation of fatty acids from endogenous and exogenous sources. Fatty acylation of the luminescence-related synthetase and reductase enzymes responsible for aldehyde synthesis exhibited a chain-length preference for C14:0, which also was indicated by reverse-phase thin-layer chromatography of the acyl groups attached to these enzymes. The ability of V. harveyi to activate and elongate exogenous fatty acids may be related to an adaptive requirement to metabolize intracellular C14:0 generated by the luciferase reaction during luminescence development.

  1. Lactococcus garvieae carries a chromosomally encoded pentapeptide repeat protein that confers reduced susceptibility to quinolones in Escherichia coli producing a cytotoxic effect.

    PubMed

    Gibello, Alicia; Díaz de Alba, Paula; Blanco, M Mar; Machuca, Jesus; Cutuli, M Teresa; Rodríguez-Martínez, José Manuel

    2014-09-01

    This study characterises a chromosomal gene of Lactococcus garvieae encoding a pentapeptide repeat protein designated as LgaQnr. This gene has been implicated in reduced susceptibility to quinolones in this bacterium, which is of relevance to both veterinary and human medicine. All of the L. garvieae isolates analysed were positive for the lgaqnr gene. The expression of lgaqnr in Escherichia coli reduced the susceptibility to quinolones, producing an adverse effect. The reduced susceptibility to ciprofloxacin was 16-fold in E. coli ATCC 25922 and 32-fold in E. coli DH10B, compared to the control strains. The minimum inhibitory concentration of nalidixic acid was also increased 4 or 5-fold. The effect of the expression of lgaqnr in E. coli was investigated by electron microscopy and was observed to affect the structure of the cell and the inner membrane of the recombinant cells.

  2. Statistical optimization of cell disruption techniques for releasing intracellular X-prolyl dipeptidyl aminopeptidase from Lactococcus lactis spp. lactis.

    PubMed

    Üstün-Aytekin, Özlem; Arısoy, Sevda; Aytekin, Ali Özhan; Yıldız, Ece

    2016-03-01

    X-prolyl dipeptidyl aminopeptidase (PepX) is an intracellular enzyme from the Gram-positive bacterium Lactococcus lactis spp. lactis NRRL B-1821, and it has commercial importance. The objective of this study was to compare the effects of several cell disruption methods on the activity of PepX. Statistical optimization methods were performed for two cavitation methods, hydrodynamic (high-pressure homogenization) and acoustic (sonication), to determine the more appropriate disruption method. Two level factorial design (2FI), with the parameters of number of cycles and pressure, and Box-Behnken design (BBD), with the parameters of cycle, sonication time, and power, were used for the optimization of the high-pressure homogenization and sonication methods, respectively. In addition, disruption methods, consisting of lysozyme, bead milling, heat treatment, freeze-thawing, liquid nitrogen, ethylenediaminetetraacetic acid (EDTA), Triton-X, sodium dodecyl sulfate (SDS), chloroform, and antibiotics, were performed and compared with the high-pressure homogenization and sonication methods. The optimized values of high-pressure homogenization were one cycle at 130 MPa providing activity of 114.47 mU ml(-1), while sonication afforded an activity of 145.09 mU ml(-1) at 28 min with 91% power and three cycles. In conclusion, sonication was the more effective disruption method, and its optimal operation parameters were manifested for the release of intracellular enzyme from a L. lactis spp. lactis strain, which is a Gram-positive bacterium.

  3. Hyaluronate lyase activity of Streptococcus suis serotype 2 and modulatory effects of hyaluronic acid on the bacterium's virulence properties.

    PubMed

    Haas, Bruno; Vaillancourt, Katy; Bonifait, Laetitia; Gottschalk, Marcelo; Grenier, Daniel

    2015-11-26

    Streptococcus suis serotype 2 is a major swine pathogen and zoonotic agent worldwide causing mainly meningitis and septicemia. Hyaluronate lyases are enzymes that degrade hyaluronic acid, a major constituent of animal tissues, and have been reported as virulence factors in various bacterial species. Since the hyaluronate lyase of S. suis has been considered ambiguously as a virulence factor, we screened 50 isolates from the three major clonal complexes found in North America (sequence type [ST] 1, ST25, and ST28) known to differ in their degree of virulence in order to link the presence or absence of this activity with the degree of virulence. Moreover, the effect of exogenous hyaluronic acid on S. suis virulence factor gene expression and the pro-inflammatory response of brain macrovascular endothelial cells (BMEC) was also investigated. We found that all but one ST1 isolates (high virulence) were devoid of hyaluronate lyase activity whereas all ST25 (intermediate virulence) and ST28 (low virulence) isolates possessed the activity. A 2 bp insertion was responsible for the lack of activity in ST1 strains. Since the most virulent isolates did not degrade hyaluronic acid, this tissue component may be found during the infectious process. Therefore, we investigated its effect on S. suis and host cells. Hyaluronic acid was found to modulate S. suis adhesion to BMEC, to increase S. suis virulence factor expression, and to enhance pro-inflammatory cytokine secretion by BMEC. These findings suggest that S. suis hyaluronate lyase does not represent a critical virulence factor in its active form. However, exogenous hyaluronic acid that is likely to interact with S. suis and host cells during the course of infection appears to modulate several virulence determinants of the bacterium, in addition to promote inflammation.

  4. Effect of short-chain acids on the carboxymethylcellulase activity of the ruminal bacterium Ruminococcus albus.

    PubMed

    Paggi, R A; Fay, J P

    2004-01-01

    The addition of 100-300 mmol/L of acetic, propionic, butyric or lactic acids (short-chain acids), or of acetic, propionic, and butyric acids (volatile fatty acids, VFA) mixtures increased the degradation of carboxymethyl cellulose (CMC) by R. albus (7.5 to 46 and 6 to 39 %, respectively). Differences among individual acids were observed at 300 mmol/L whereas VFA mixtures differed at 100 mmol/L. When assayed at the same concentration, CMCase activity was increased less by NaCl than by the short-chain acids, whereas ethylene glycol decreased the activity. Since osmolarity and/or ionic strength changes in the medium cannot completely account for the observed increases of carboxymethylcellulase (CMCase) activity, it is suggested that the anions of short-chain acids produce changes in the reaction media polarity that contribute to the effects observed. Alterations in the media could also bring about conformational changes in CMCase leading to increased rates of reaction and subsequent increases in CMC degradation. Finally, explanations for the observed phenomena based on the direct effect of the compounds tested on the cellulosome complex, its domains, and/or its component enzymes are proposed.

  5. Tryptophan, thiamine and indole-3-acetic acid exchange between Chlorella sorokiniana and the plant growth-promoting bacterium Azospirillum brasilense.

    PubMed

    Palacios, Oskar A; Gomez-Anduro, Gracia; Bashan, Yoav; de-Bashan, Luz E

    2016-06-01

    During synthetic mutualistic interactions between the microalga Chlorella sorokiniana and the plant growth-promoting bacterium (PGPB) Azospirillum brasilense, mutual exchange of resources involved in producing and releasing the phytohormone indole-3-acetic acid (IAA) by the bacterium, using tryptophan and thiamine released by the microalga, were measured. Although increased activities of tryptophan synthase in C. sorokiniana and indole pyruvate decarboxylase (IPDC) in A. brasilense were observed, we could not detect tryptophan or IAA in the culture medium when both organisms were co-immobilized. This indicates that no extra tryptophan or IAA is produced, apart from the quantities required to sustain the interaction. Over-expression of the ipdC gene occurs at different incubation times: after 48 h, when A. brasilense was immobilized alone and grown in exudates of C. sorokiniana and at 96 h, when A. brasilense was co-immobilized with the microalga. When A. brasilense was cultured in exudates of C. sorokiniana, increased expression of the ipdC gene, corresponding increase in activity of IPDC encoded by the ipdC gene, and increase in IAA production were measured during the first 48 h of incubation. IAA production and release by A. brasilense was found only when tryptophan and thiamine were present in a synthetic growth medium (SGM). The absence of thiamine in SGM yielded no detectable IAA. In summary, this study demonstrates that C. sorokiniana can exude sufficient tryptophan and thiamine to allow IAA production by a PGPB during their interaction. Thiamine is essential for IAA production by A. brasilense and these three metabolites are part of a communication between the two microorganisms. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  6. Development of plasmid vector and electroporation condition for gene transfer in sporogenic lactic acid bacterium, Bacillus coagulans.

    PubMed

    Rhee, Mun Su; Kim, Jin-Woo; Qian, Yilei; Ingram, L O; Shanmugam, K T

    2007-07-01

    Bacillus coagulans is a sporogenic lactic acid bacterium that ferments glucose and xylose, major components of plant biomass, a potential feedstock for cellulosic ethanol. The temperature and pH for optimum rate of growth of B. coagulans (50 to 55 degrees C, pH 5.0) are very similar to that of commercially developed fungal cellulases (50 degrees C; pH 4.8). Due to this match, simultaneous saccharification and fermentation (SSF) of cellulose to products by B. coagulans is expected to require less cellulase than needed if the SSF is conducted at a sub-optimal temperature, such as 30 degrees C, the optimum for yeast, the main biocatalyst used by the ethanol industry. To fully exploit B. coagulans as a platform organism, we have developed an electroporation method to transfer plasmid DNA into this genetically recalcitrant bacterium. We also constructed a B. coagulans/E. coli shuttle vector, plasmid pMSR10 that contains the rep region from a native plasmid (pMSR0) present in B. coagulans strain P4-102B. The native plasmid, pMSR0 (6823bp), has 9 ORFs, and replicates by rolling-circle mode of replication. Plasmid pNW33N, developed for Geobacillus stearothermophilus, was also transformed into this host and stably maintained while several other Bacillus/Escherichia coli shuttle vector plasmids were not transformed into B. coagulans. The transformation efficiency of B. coagulans strain P4-102B using the plasmids pNW33N or pMSR10 was about 1.5x10(16) per mole of DNA. The availability of shuttle vectors and an electroporation method is expected to aid in genetic and metabolic engineering of B. coagulans.

  7. Use of induction promoters to regulate hyaluronan synthase and UDP-glucose-6-dehydrogenase of Streptococcus zooepidemicus expression in Lactococcus lactis: a case study of the regulation mechanism of hyaluronic acid polymer.

    PubMed

    Sheng, J Z; Ling, P X; Zhu, X Q; Guo, X P; Zhang, T M; He, Y L; Wang, F S

    2009-07-01

    To determine the effects of the ratios of hyaluronan synthase expression level to precursor sugar UDP-GlcA biosynthesis ability on the molecular weight (MW) of hyaluronic acid (HA) in recombinant Lactococcus lactis. The genes szHasA (hyaluronan synthase gene) and szHasB (UDP-glucose-6-dehydrogenase gene) of Streptococcus zooepidemicus were introduced into L. lactis under the control of nisA promoter and lacA promoter respectively, resulting in a dual-plasmid controlled expression system. The effects of the ratios of hyaluronan synthase expression level to the precursor sugar UDP-GlcA biosynthesis ability under different induction concentration collocations with nisin and lactose on the MW of HA in recombinant L. lactis were determined. The results showed that the final weight-average molecular weight () of HA correlated with the relative ratios of HasA (hyaluronan synthase) expression level to the concentration of UDP-GlcA. Regulating the relative ratios of HasA expression level to the precursor sugar biosynthesis ability was an efficient method to control the size of HA. This study put forward a guide to establish an efficacious way to control the size of HA in fermentation.

  8. Cell Wall Anchoring of the Campylobacter Antigens to Lactococcus lactis

    PubMed Central

    Kobierecka, Patrycja A.; Olech, Barbara; Książek, Monika; Derlatka, Katarzyna; Adamska, Iwona; Majewski, Paweł M.; Jagusztyn-Krynicka, Elżbieta K.; Wyszyńska, Agnieszka K.

    2016-01-01

    Campylobacter jejuni is the most frequent cause of human food-borne gastroenteritis and chicken meat is the main source of infection. Recent studies showed that broiler chicken immunization against Campylobacter should be the most efficient way to lower the number of human infections by this pathogen. Induction of the mucosal immune system after oral antigen administration should provide protective immunity to chickens. In this work we tested the usefulness of Lactococcus lactis, the most extensively studied lactic acid bacterium, as a delivery vector for Campylobacter antigens. First we constructed hybrid protein – CjaA antigen presenting CjaD peptide epitopes on its surface. We showed that specific rabbit anti-rCjaAD serum reacted strongly with both CjaA and CjaD produced by a wild type C. jejuni strain. Next, rCjaAD and CjaA were fused to the C-terminus of the L. lactis YndF containing the LPTXG motif. The genes expressing these proteins were transcribed under control of the L. lactis Usp45 promoter and their products contain the Usp45 signal sequences. This strategy ensures a cell surface location of both analyzed proteins, which was confirmed by immunofluorescence assay. In order to evaluate the impact of antigen location on vaccine prototype efficacy, a L. lactis strain producing cytoplasm-located rCjaAD was also generated. Animal experiments showed a decrease of Campylobacter cecal load in vaccinated birds as compared with the control group and showed that the L. lactis harboring the surface-exposed rCjaAD antigen afforded greater protection than the L. lactis producing cytoplasm-located rCjaAD. To the best of our knowledge, this is the first attempt to employ Lactic Acid Bacteria (LAB) strains as a mucosal delivery vehicle for chicken immunization. Although the observed reduction of chicken colonization by Campylobacter resulting from vaccination was rather moderate, the experiments showed that LAB strains can be considered as an alternative vector to

  9. Bile acids are new products of a marine bacterium, Myroides sp. strain SM1.

    PubMed

    Maneerat, Suppasil; Nitoda, Teruhiko; Kanzaki, Hiroshi; Kawai, Fusako

    2005-06-01

    Strain SM1 was isolated as a biosurfactant-producing microorganism from seawater and presumptively identified as Myroides sp., based on morphology, biochemical characteristics and 16S rDNA sequence. The strain produced surface-active compounds in marine broth, which were purified, using emulsification activity for n-hexadecane as an indicator. The purified compounds were identified by thin-layer chromatography, (1)H- and (13)C-NMR spectra and fast atom bombardment mass spectrometry as cholic acid, deoxycholic acid and their glycine conjugates. Type strains of the genus Myroides, M. odoratus JCM7458 and M. odoramitimus JCM7460, also produced these compounds. Myroides sp. strain SM1 possessed a biosynthetic route to cholic acid from cholesterol. Thus, bile acids were found as new products of prokaryotic cells, genus Myroides.

  10. A plant pathogenic bacterium exploits the tricarboxylic acid cycle metabolic pathway of its insect vector.

    PubMed

    Killiny, Nabil; Nehela, Yasser; Hijaz, Faraj; Vincent, Christopher I

    2017-06-08

    Huanglongbing in citrus is caused by a phloem-limited, uncultivable, gram-negative α-proteobacterium, Candidatus Liberibacter asiaticus (CLas). CLas is transmitted by the phloem-sucking insect, Diaphorina citri (Hemiptera: Liviidae), in a persistent, circulative, and propagative manner. In this study, we investigated the metabolomic and respiration rates changes in D. citri upon infection with CLas using gas chromatography-mass spectrometry (GC-MS) and gas exchange analysis. The level of glycine, L-serine, L-threonine, and gamma-amino butyric acid were higher in CLas-infected D. citri, while L-proline, L-aspartic acid, and L-pyroglutamic acid were lower in CLas-infected D. citri compared with the control. Citric acid was increased in CLas-infected D. citri, whereas malic and succinic acids were reduced. Interestingly, most of the reduced metabolites such as malate, succinate, aspartate, and L-proline are required for the growth of CLas. The increase in citric acid, serine, and glycine indicated that CLas induced glycolysis and the tricarboxylic acid cycle (TCA) in its vector. In agreement with the GC-MS results, the gene expression results also indicated that glycolysis and TCA were induced in CLas-infected D. citri and this was accompanied with an increases in respiration rate. Phosphoric acid and most of the sugar alcohols were higher in CLas-infected D. citri, indicating a response to the biotic stress or cell damage. Only slight increases in the levels of few sugars were observed in CLas-infected D. citri, which indicated that sugars are tightly regulated by D. citri. Our results indicated that CLas induces nutrient and energetic stress in its host insect. This study may provide some insights into the mechanism of colonization of CLas in its vector.

  11. Complete genome sequence of Enterococcus mundtii QU 25, an efficient L-(+)-lactic acid-producing bacterium.

    PubMed

    Shiwa, Yuh; Yanase, Hiroaki; Hirose, Yuu; Satomi, Shohei; Araya-Kojima, Tomoko; Watanabe, Satoru; Zendo, Takeshi; Chibazakura, Taku; Shimizu-Kadota, Mariko; Yoshikawa, Hirofumi; Sonomoto, Kenji

    2014-08-01

    Enterococcus mundtii QU 25, a non-dairy bacterial strain of ovine faecal origin, can ferment both cellobiose and xylose to produce l-lactic acid. The use of this strain is highly desirable for economical l-lactate production from renewable biomass substrates. Genome sequence determination is necessary for the genetic improvement of this strain. We report the complete genome sequence of strain QU 25, primarily determined using Pacific Biosciences sequencing technology. The E. mundtii QU 25 genome comprises a 3 022 186-bp single circular chromosome (GC content, 38.6%) and five circular plasmids: pQY182, pQY082, pQY039, pQY024, and pQY003. In all, 2900 protein-coding sequences, 63 tRNA genes, and 6 rRNA operons were predicted in the QU 25 chromosome. Plasmid pQY024 harbours genes for mundticin production. We found that strain QU 25 produces a bacteriocin, suggesting that mundticin-encoded genes on plasmid pQY024 were functional. For lactic acid fermentation, two gene clusters were identified-one involved in the initial metabolism of xylose and uptake of pentose and the second containing genes for the pentose phosphate pathway and uptake of related sugars. This is the first complete genome sequence of an E. mundtii strain. The data provide insights into lactate production in this bacterium and its evolution among enterococci.

  12. Leucine responsive regulatory protein is involved in methionine metabolism and polyamine homeostasis in acetic acid bacterium Komagataeibacter europaeus.

    PubMed

    Ishii, Yuri; Akasaka, Naoki; Sakoda, Hisao; Hidese, Ryota; Fujiwara, Shinsuke

    2017-08-31

    The leucine responsive regulatory protein (Lrp) is a global transcription factor that regulates the expression of genes involved in amino acid metabolism. To identify metabolic pathways and related genes under the control of Lrp in the acetic acid bacterium Komagataeibacter europaeus, the Kelrp null mutant (KGMA7110), which requires supplementation of all 20 amino acids for normal growth, was cultivated in minimal media containing or lacking particular amino acids. The results confirmed that KGMA7110 was auxotrophic for methionine and its catabolites S-adenosylmethionine (SAM) and spermidine (SPD). Quantitative reverse-transcription PCR analysis revealed lower metK (SAM synthetase) and mdtI (SPD efflux pump) expression in KGMA7110 than in wild-type KGMA0119. By contrast, these genes were significantly up-regulated in the Kelrp mutant lacking the putative C-terminal ligand-sensing domain (KGMA7203), indicating abnormal regulation of target genes by the KeLrp variant in KGMA7203. KGMA7110 (0.69±0.27 μM) and KGMA7203 (4.90±0.61 μM) excreted lower and higher quantities of SPD, respectively, than KGMA0119 (2.28±0.26 μM). This was attributed to imbalanced carbon flow caused by Kelrp disruption that respectively attenuated and stimulated metK and mdtI expression. These findings indicate that KeLrp plays a key role in SAM biosynthesis and intracellular polyamine homeostasis in K. europaeus. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  13. Characterization of a nitroreductase with selective nitroreduction properties in the food and intestinal lactic acid bacterium Lactobacillus plantarum WCFS1.

    PubMed

    Guillén, Hugo; Curiel, Jose Antonio; Landete, José María; Muñoz, Rosario; Herraiz, Tomás

    2009-11-11

    Nitroreductases reduce nitroaromatic compounds and other oxidants in living organisms, having interesting implications in environmental and human health. A putative nitrobenzoate reductase encoding gene (lp_0050) was recently annotated in the completed DNA sequence of lactic acid bacterium Lactobacillus plantarum WCFS1 strain. In this research, this L. plantarum gene was cloned and expressed, and the corresponding protein (PnbA) was biochemically characterized. This L. plantarum PnbA reductase is a 216 amino acid residue FMN-flavoprotein, which exhibits 23% identity with Pseudomonas putida and Ralstonia eutropha nitroreductases and <11% identity with those from enterobacteria such as E. cloacae . This reductase also showed 32-43% identity (65-72% similarity) to predicted PnbA proteins from other lactic acid bacteria. It utilized a wide range of electron acceptors including dichlorophenolindophenol (DCPIP), nitroblue tetrazolium (NBT), ferricyanide, and quinones (menadione, benzoquinone), but not pyridinium cations (paraquat and N-methyl-beta-carbolines), and it was inhibited by dicoumarol and diphenyliodonium. HPLC-MS and spectroscopic data showed that it specifically catalyzed the reduction of the 4-nitroaromatic group to the corresponding hydroxylamine in the presence of NAD(P)H. Kinetics parameters (V(max) and K(m)) showed a higher efficiency for the reduction of 2,4-dinitrobenzoate than for the reduction of 4-nitrobenzoate. It was chemoselective for the reduction of 4-nitrobenzoates, being unable to reduce other nitroaromatics. Then, L. plantarum PnbA reductase might be more specific than other microbial nitroreductases that reduce a wider range of nitroaromatic compounds. The physiological and functional role of nitroreductases remain unknown; however, their presence in lactic acid bacteria widely occurring in foods and the human intestinal tract should be of further interest.

  14. Characterization and Antibacterial Potential of Lactic Acid Bacterium Pediococcus pentosaceus 4I1 Isolated from Freshwater Fish Zacco koreanus

    PubMed Central

    Bajpai, Vivek K.; Han, Jeong-Ho; Rather, Irfan A.; Park, Chanseo; Lim, Jeongheui; Paek, Woon Kee; Lee, Jong Sung; Yoon, Jung-In; Park, Yong-Ha

    2016-01-01

    This study was undertaken to characterize a lactic acid bacterium 4I1, isolated from the freshwater fish, Zacco koreanus. Morphological, biochemical, and molecular characterization of 4I1 revealed it to be Pediococcus pentosaceus 4I1. The cell free supernatant (CFS) of P. pentosaceus 4I1 exhibited significant (p < 0.05) antibacterial effects (inhibition zone diameters: 16.5–20.4 mm) against tested foodborne pathogenic bacteria with MIC and MBC values of 250–500 and 500–1,000 μg/mL, respectively. Further, antibacterial action of CFS of P. pentosaceus 4I1 against two selected bacteria Staphylococcus aureus KCTC-1621 and Escherichia coli O157:H7 was determined in subsequent assays. The CFS of P. pentosaceus 4I1 revealed its antibacterial action against S. aureus KCTC-1621 and E. coli O157:H7 on membrane integrity as confirmed by a reduction in cell viability, increased potassium ion release (900 and 800 mmol/L), reduced absorption at 260-nm (3.99 and 3.77 OD), and increased relative electrical conductivity (9.9 and 9.7%), respectively. Gas chromatography–mass spectrometry (GC–MS) analysis of the CFS of P. pentosaceus 4I1 resulted in the identification of seven major compounds, which included amino acids, fatty acids and organic acids. Scanning electron microscopic-based morphological analysis further confirmed the antibacterial effect of CFS of P. pentosaceus 4I1 against S. aureus KCTC-1621 and E. coli O157:H7. In addition, the CFS of P. Pentosaceus 4I1 displayed potent inhibitory effects on biofilms formation by S. aureus KCTC-1621 and E. coli O157:H7. The study indicates the CFS of P. pentosaceus 4I1 offers an alternative means of controlling foodborne pathogens. PMID:28066360

  15. Novel Antibacterial Activity of Lactococcus Lactis Subspecies Lactis Z11 Isolated from Zabady

    PubMed Central

    Enan, Gamal; Abdel-Shafi, Seham; Ouda, Sahar; Negm, Sally

    2013-01-01

    The purpose of this study was to select and characterize a probiotic bacterium with distinctive antimicrobial activities. In this respect, Lactococcus lactis subspecies lactis Z11 (L. lactis Z11) isolated from Zabady (Arabian yoghurt) inhibited other strains of lactic acid bacteria and some food-born pathogens including Listeria monocytogenes, Bacillus cereus and staphylococcus aureus. The inhibitory activity of cell free supernatant (CFS) of L. lactis Z11 isolated from zabady was lost by proteolytic enzymes, heat resistant. Consequently, the active substance(s) of CFS was characterized as a bacteriocin. This bacteriocin has been shown to consist of protein but has no lipidic or glucidic moieties in its active molecule. Its activity was stable in the pH range 2.0 to 7.0 and was not affected by organic solvents. The L. lactis Z11 bacteriocin was produced in CFS throughout the mide to the late exponential phase of growth of the producer organism and maximum bacteriocin production was obtained at initial pH 6.5 at incubation temperature of about 30°C. PMID:24151453

  16. Staphylococcus aureus Virulence Expression Is Impaired by Lactococcus lactis in Mixed Cultures▿ †

    PubMed Central

    Even, Sergine; Charlier, Cathy; Nouaille, Sébastien; Ben Zakour, Nouri L.; Cretenet, Marina; Cousin, Fabien J.; Gautier, Michel; Cocaign-Bousquet, Muriel; Loubière, Pascal; Le Loir, Yves

    2009-01-01

    Staphylococcus aureus is responsible for numerous food poisonings due to the production of enterotoxins by strains contaminating foodstuffs, especially dairy products. Several parameters, including interaction with antagonistic flora such as Lactococcus lactis, a lactic acid bacterium widely used in the dairy industry, can modulate S. aureus proliferation and virulence expression. We developed a dedicated S. aureus microarray to investigate the effect of L. lactis on staphylococcal gene expression in mixed cultures. This microarray was used to establish the transcriptomic profile of S. aureus in mixed cultures with L. lactis in a chemically defined medium held at a constant pH (6.6). Under these conditions, L. lactis hardly affected S. aureus growth. The expression of most genes involved in the cellular machinery, carbohydrate and nitrogen metabolism, and stress responses was only slightly modulated: a short time lag in mixed compared to pure cultures was observed. Interestingly, the induction of several virulence factors and regulators, including the agr locus, sarA, and some enterotoxins, was strongly affected. This work clearly underlines the complexity of L. lactis antagonistic potential for S. aureus and yields promising leads for investigations into nonantibiotic biocontrol of this major pathogen. PMID:19429556

  17. Antiviral Effects of Lactococcus lactis on Feline Calicivirus, A Human Norovirus Surrogate.

    PubMed

    Aboubakr, Hamada A; El-Banna, Amr A; Youssef, Mohammed M; Al-Sohaimy, Sobhy A A; Goyal, Sagar M

    2014-12-01

    Foodborne viruses, particularly human norovirus (NV) and hepatitis virus type A, are a cause of concern for public health making it necessary to explore novel and effective techniques for prevention of foodborne viral contamination, especially in minimally processed and ready-to-eat foods. This study aimed to determine the antiviral activity of a probiotic lactic acid bacterium (LAB) against feline calicivirus (FCV), a surrogate of human NV. Bacterial growth medium filtrate (BGMF) of Lactococcus lactis subsp. lactis LM0230 and its bacterial cell suspension (BCS) were evaluated separately for their antiviral activity against FCV grown in Crandell-Reese feline kidney (CRFK) cells. No significant antiviral effect was seen when CRFK cells were pre-treated with either BGMF (raw or pH 7-adjusted BGMF) or BCS. However, pre-treatment of FCV with BGMF and BCS resulted in a reduction in virus titers of 1.3 log10 tissue culture infectious dose (TCID)50 and 1.8 log10 TCID50, respectively. The highest reductions in FCV infectivity were obtained when CRFK cells were co-treated with FCV and pH 7-adjusted BGMF or with FCV and BCS (7.5 log10 TCID50 and 6.0 log10 TCID50, respectively). These preliminary results are encouraging and indicate the need for continued studies on the role of probiotics and LAB on inactivation of viruses in various types of foods.

  18. [Screening and identification of indoleacetic acid producing endophytic bacterium in Panax ginseng].

    PubMed

    Jiang, Yun; Tian, Lei; Chen, Chang-qing; Zhang, Guan-jun; Li, Tong; Chen, Jing-xiu; Wang, Xue

    2015-01-01

    Endophytic bacteria which was producing indoleacetic acid was screened from Panax ginseng by using the Salkowski method. The active strain was also tested for its ability of nitrogen fixation by using the Ashby agar plates, the PKV plates and quantitative analysis of Mo-Sb-Ascrobiology acid colorimetry was used to measure its ability of phosphate solubilization, for its ability of potassium solubilization the silicate medium and flame spectrophotometry was used, for its ability of producing siderophores the method detecting CAS was used, for its ability of producing ACC deaminase the Alpha ketone butyric acid method was applied. And the effect on promoting growth of seed by active strain was tested. The results showed that the indoleacetic acid producing strain of JJ5-2 was obtained from 118 endophytes, which the content of indoleacetic acid was 10.2 mg x L(-1). The JJ5-2 strain also had characteristics of phosphate and potassium solubilization, nitrogen fixation, producing siderophores traits, and the promoting germination of ginseng seeds. The JJ5-2 strain was identified as Bacillus thuringiensis by analyzing morphology, physiological and biochemical properties and 16S rRNA gene sequences.

  19. Simultaneous saccharification and high titer lactic acid fermentation of corn stover using a newly isolated lactic acid bacterium Pediococcus acidilactici DQ2.

    PubMed

    Zhao, Kai; Qiao, Qingan; Chu, Deqiang; Gu, Hanqi; Dao, Thai Ha; Zhang, Jian; Bao, Jie

    2013-05-01

    A lactic acid bacterium with high tolerance of temperature and lignocellulose derived inhibitor was isolated and characterized as Pediococcus acidilactici DQ2. The strain used in the simultaneous saccharification and fermentation (SSF) for high titer lactic acid production at the high solids loading of corn stover. Corn stover was pretreated using the dry sulphuric acid pretreatment, followed by a biological detoxification to remove the inhibitors produced in the pretreatment. The bioreactor with a novel helical impeller was used to the SSF operation of the pretreated and biodetoxified corn stover. The results show that a typical SSF operation at 48 °C, pH 5.5, and near 30% (w/w) solids loading in both 5 and 50 L bioreactors was demonstrated. The lactic acid titer, yield, and productivity reached 101.9 g/L, 77.2%, and 1.06 g/L/h, respectively. The result provided a practical process option for cellulosic lactic acid production using virgin agriculture lignocellulose residues. Copyright © 2012 Elsevier Ltd. All rights reserved.

  20. Gluconic acid production and phosphate solubilization by the plant growth-promoting bacterium Azospirillum spp.

    NASA Astrophysics Data System (ADS)

    Rodriguez, Hilda; Gonzalez, Tania; Goire, Isabel; Bashan, Yoav

    2004-11-01

    In vitro gluconic acid formation and phosphate solubilization from sparingly soluble phosphorus sources by two strains of the plant growth-promoting bacteria A. brasilense (Cd and 8-I) and one strain of A. lipoferum JA4 were studied. Strains of A. brasilense were capable of producing gluconic acid when grown in sparingly soluble calcium phosphate medium when their usual fructose carbon source is amended with glucose. At the same time, there is a reduction in pH of the medium and release of soluble phosphate. To a greater extent, gluconic acid production and pH reduction were observed for A. lipoferum JA4. For the three strains, clearing halos were detected on solid medium plates with calcium phosphate. This is the first report of in vitro gluconic acid production and direct phosphate solubilization by A. brasilense and the first report of P solubilization by A. lipoferum. This adds to the very broad spectrum of plant growth-promoting abilities of this genus.

  1. Gluconacetobacter kakiaceti sp. nov., an acetic acid bacterium isolated from a traditional Japanese fruit vinegar.

    PubMed

    Iino, Takao; Suzuki, Rei; Tanaka, Naoto; Kosako, Yoshimasa; Ohkuma, Moriya; Komagata, Kazuo; Uchimura, Tai

    2012-07-01

    Two novel acetic acid bacteria, strains G5-1(T) and I5-1, were isolated from traditional kaki vinegar (produced from fruits of kaki, Diospyros kaki Thunb.), collected in Kumamoto Prefecture, Japan. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strains G5-1(T) and I5-1 formed a distinct subline in the genus Gluconacetobacter and were closely related to Gluconacetobacter swingsii DST GL01(T) (99.3% 16S rRNA gene sequence similarity). The isolates showed 96-100% DNA-DNA relatedness with each other, but <53% DNA-DNA relatedness with closely related members of the genus Gluconacetobacter. The isolates could be distinguished from closely related members of the genus Gluconacetobacter by not producing 2- and 5-ketogluconic acids from glucose, producing cellulose, growing without acetic acid and with 30% (w/v) d-glucose, and producing acid from sugars and alcohols. Furthermore, the genomic DNA G+C contents of strains G5-1(T) and I5-1 were a little higher than those of their closest phylogenetic neighbours. On the basis of the phenotypic characteristics and phylogenetic position, strains G5-1(T) and I5-1 are assigned to a novel species, for which the name Gluconacetobacter kakiaceti sp. nov. is proposed; the type strain is G5-1(T) (=JCM 25156(T)=NRIC 0798(T)=LMG 26206(T)).

  2. Isolation of a 2-picolinic acid-assimilating bacterium and its proposed degradation pathway.

    PubMed

    Zheng, Chunli; Wang, Qiaorui; Ning, Yanli; Fan, Yurui; Feng, Shanshan; He, Chi; Zhang, Tian C; Shen, Zhenxing

    2017-09-06

    Burkholderia sp. ZD1, aerobically utilizes 2-picolinic acid as a source of carbon, nitrogen and energy, was isolated. ZD1 completely degraded 2-picolinic acid when the initial concentrations ranged from 25 to 300mg/L. Specific growth rate (μ) and specific consumption rate (q) increased continually in the concentration range of 25-100mg/L, and then declined. Based on the Haldane model and Andrew's model, μmax and qmax were calculated as 3.9 and 16.5h(-1), respectively. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) was used to determine the main intermediates in the degradation pathway. Moreover, attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) was innovatively used to deduce the ring cleavage mechanism of N-heterocycle of 2-picolinic acid. To our knowledge, this is the first report on not only the utilization of 2-picolinic acid by a Burkholderia sp., but also applying FT-ICR-MS and ATR-FTIR for exploring the biodegradation pathway of organic compounds. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Heterologous expression of Brucella abortus GroEL heat-shock protein in Lactococcus lactis

    PubMed Central

    Miyoshi, Anderson; Bermúdez-Humarán, Luis G; Ribeiro, Luciana A; Le Loir, Yves; Oliveira, Sérgio C; Langella, Philippe; Azevedo, Vasco

    2006-01-01

    Background Brucella abortus is a facultative intracellular pathogen that mainly infects cattle and humans. Current vaccines rely on live attenuated strains of B. abortus, which can revert to their pathogenic status and thus are not totally safe for use in humans. Therefore, the development of mucosal live vaccines using the food-grade lactic acid bacterium, Lactococcus lactis, as an antigen delivery vector, is an attractive alternative and a safer vaccination strategy against B. abortus. Here, we report the construction of L. lactis strains genetically modified to produce B. abortus GroEL heat-shock protein, a candidate antigen, in two cellular locations, intracellular or secreted. Results Only the secreted form of GroEL was stably produced in L. lactis, suggesting a detrimental effect of GroEL protein when intracellularly produced in this bacterium. Only trace amounts of mature GroEL were detected in the supernatant fraction of induced lactococcal cultures, and the GroEL precursor remained stacked in the cell fraction. Attempts to raise the secretion yields were made, but even when GroEL was fused to a synthetic propeptide, secretion of this antigen was not improved. Conclusion We found that L. lactis is able to produce, and to secrete, a stable form of GroEL into the extracellular medium. Despite the low secretion efficiency of GroEL, which suggest that this antigen interacts with the cell envelope of L. lactis, secretion seems to be the best way to achieve both production and protein yields, regardless of cellular location. The L. lactis strain secreting GroEL has potential for in vivo immunization. PMID:16556312

  4. Gluconacetobacter maltaceti sp. nov., a novel vinegar producing acetic acid bacterium.

    PubMed

    Slapšak, Nina; Cleenwerck, Ilse; De Vos, Paul; Trček, Janja

    2013-02-01

    Comparison of HaeIII- and HpaII-restriction profiles of PCR-amplified 16S-23S rDNA ITS regions of Gluconacetobacter sp. LMG 1529(T) and SKU 1109 with restriction profiles of reference strains of acetic acid bacteria described by Trček and Teuber [34] revealed the same but unique restriction profiles for LMG 1529(T) and SKU 1109. Further analyses of nearly complete 16S rRNA gene sequences, nearly complete 16S-23S rDNA ITS sequences, as well as concatenated partial sequences of the housekeeping genes dnaK, groEL and rpoB, allocated both strains to a single phylogenetic cluster well separated from the other species of the genus Gluconacetobacter. DNA-DNA hybridizations confirmed their novel species identity by 73% DNA-DNA relatedness between both strains, and values below the species level (<70%) between SKU 1109 and the type strains of the closest phylogenetic neighbors. The classification of strains LMG 1529(T) and SKU 1109 into a single novel species was confirmed also by AFLP and (GTG)(5)-PCR DNA fingerprinting data, as well as by phenotypic data. Strains LMG 1529(T) and SKU 1109 can be differentiated from their closely related Gluconacetobacter species, Gluconacetobacter entanii and Gluconacetobacter hansenii, by their ability to form 2-keto-d-gluconic acid from d-glucose, their ability to use d-mannitol, d-gluconate and glycerol as carbon source and form acid from d-fructose, and their ability to grow without acetic acid. The major fatty acid of LMG 1529(T) and SKU 1109 is C(18:1ω7c) (60.2-64.8%). The DNA G+C content of LMG 1529(T) and SKU 1109 is 62.5 and 63.3mol% respectively. The name Gluconacetobacter maltaceti sp. nov. is proposed. The type strain is LMG 1529(T) (=NBRC 14815(T)=NCIMB 8752(T)).

  5. Bombella intestini gen. nov., sp. nov., an acetic acid bacterium isolated from bumble bee crop.

    PubMed

    Li, Leilei; Praet, Jessy; Borremans, Wim; Nunes, Olga C; Manaia, Célia M; Cleenwerck, Ilse; Meeus, Ivan; Smagghe, Guy; De Vuyst, Luc; Vandamme, Peter

    2015-01-01

    In the frame of a bumble bee gut microbiota study, acetic acid bacteria (AAB) were isolated using a combination of direct isolation methods and enrichment procedures. MALDI-TOF MS profiling of the isolates and a comparison of these profiles with profiles of established AAB species identified most isolates as Asaia astilbis or as 'Commensalibacter intestini', except for two isolates (R-52486 and LMG 28161(T)) that showed an identical profile. A nearly complete 16S rRNA gene sequence of strain LMG 28161(T) was determined and showed the highest pairwise similarity to Saccharibacter floricola S-877(T) (96.5%), which corresponded with genus level divergence in the family Acetobacteraceae. Isolate LMG 28161(T) was subjected to whole-genome shotgun sequencing; a 16S-23S rRNA internal transcribed spacer (ITS) sequence as well as partial sequences of the housekeeping genes dnaK, groEL and rpoB were extracted for phylogenetic analyses. The obtained data confirmed that this isolate is best classified into a new genus in the family Acetobacteraceae. The DNA G+C content of strain LMG 28161(T) was 54.9 mol%. The fatty acid compositions of isolates R-52486 and LMG 28161(T) were similar to those of established AAB species [with C18:1ω7c (43.1%) as the major component], but the amounts of fatty acids such as C19:0 cyclo ω8c, C14:0 and C14:0 2-OH enabled to differentiate them. The major ubiquinone was Q-10. Both isolates could also be differentiated from the known genera of AAB by means of biochemical characteristics, such as their inability to oxidize ethanol to acetic acid, negligible acid production from melibiose, and notable acid production from d-fructose, sucrose and d-mannitol. In addition, they produced 2-keto-d-gluconate, but not 5-keto-d-gluconate from d-glucose. Therefore, the name Bombella intestini gen nov., sp. nov. is proposed for this new taxon, with LMG 28161(T) ( =DSM 28636(T) =R-52487(T)) as the type strain of the type species.

  6. Oxidation of nitrapyrin to 6-chloropicolinic acid by the ammonia-oxidizing bacterium nitrosomonas europaea

    SciTech Connect

    Vannelli, T.; Hooper, A.B.

    1992-07-01

    Suspensions of Nitrosomonas europaea catalyzed the oxidation of the commercial nitrification inhibitor nitrapyrin (2-chloro-6-(trichloromethyl)-pyridine). Rapid oxidation of nitrapyrin (at a concentration of 10 microM) required the concomitant oxidation of ammonia, hydroxylamine, or hydrazine. The turnover rate was highest in the presence of 10 mM ammonia (0.8 nmol of nitrapyrin per min/mg of protein). The product of the reaction was 6-chloropicolinic acid. By the use of (18)O2, it was shown that one of the oxygens in 6-chloropicolinic acid came from diatomic oxygen and that the other came from water. Approximately 13% of the radioactivity of (2,6-(14)C) nitrapyrin was shown to bind to cells. Most (94%) of the latter was bound indiscriminately to membrane proteins. The nitrapyrin bound to membrane proteins may account for the observed inactivation of ammonia oxidation. (Copyright (c) 1992, American Society for Microbiology.)

  7. Lactobacillus ghanensis sp. nov., a motile lactic acid bacterium isolated from Ghanaian cocoa fermentations.

    PubMed

    Nielsen, Dennis S; Schillinger, Ulrich; Franz, Charles M A P; Bresciani, José; Amoa-Awua, Wisdom; Holzapfel, Wilhelm H; Jakobsen, Mogens

    2007-07-01

    Three Gram-positive, catalase-negative, motile, rod-shaped strains, designated L486, L489(T) and L499, were isolated from fermenting cocoa. These organisms produced DL-lactic acid from glucose without gas formation. Ammonia was not produced from arginine. Acid was produced from amygdalin, D-cellobiose, aesculin, D-fructose, D-glucose, D-galactose, D-mannitol, D-mannose, N-acetylglucosamine, L-rhamnose, sucrose, salicin and D-trehalose. The cell walls contained peptidoglycan of the d-meso-diaminopimelic acid type. A 16S rRNA gene sequence analysis revealed that the isolates belong phylogenetically to the genus Lactobacillus and are closely related to Lactobacillus nagelii, Lactobacillus vini and Lactobacillus satsumensis. Low DNA-DNA reassociation values were obtained between the isolates and the phylogenetically closest neighbours. On the basis of the genetic and phenotypic results, the isolates are considered to represent a novel species, for which the name Lactobacillus ghanensis is proposed. The type strain is L489(T) (=DSM 18630(T)=CCUG 53453(T)).

  8. Biodegradation of Picolinic Acid by a Newly Isolated Bacterium Alcaligenes faecalis Strain JQ135.

    PubMed

    Qiu, Jiguo; Zhang, Junjie; Zhang, Yanting; Wang, Yuhong; Tong, Lu; Hong, Qing; He, Jian

    2017-04-01

    We isolated a bacterial strain JQ135 from municipal wastewater, which was capable of efficiently degrading picolinic acid (PA). Based on the physico-biochemical characteristics and 16S rDNA analysis, strain JQ135 was identified as Alcaligenes faecalis. In addition, strain JQ135 produced an orange pigment when cultured in the Luria-Bertani medium, which is different from the previously reported strains of A. faecalis. During the degradation of PA by the resting strain JQ135 cells, only one intermediate, 6-hydroxypicolinic acid (6HPA), was detected by ultraviolet spectrophotometry, high-pressure liquid chromatography, and liquid chromatography-mass spectrometry. A random transposon mutagenesis library of strain JQ135 was constructed. One mutant, Mut-G31, could convert PA into 6HPA without further degradation. The disrupted gene (orf2) was amplified from Mut-G31, and its product showed 32% identity to the 3-deoxy-D-manno-octulosonic acid kinase (KdkA) from Haemophilus influenzae. Results from complementation analysis confirmed that GTG was the initiation codon of the kdkA-like orf2, and that it was essential for PA biodegradation by strain JQ135. This study provides the first genetic evidence for the bacterial degradation of PA.

  9. Energy metabolism of a unique acetic acid bacterium, Asaia bogorensis, that lacks ethanol oxidation activity.

    PubMed

    Ano, Yoshitaka; Toyama, Hirohide; Adachi, Osao; Matsushita, Kazunobu

    2008-04-01

    Acetic acid bacteria (AAB) are known as a vinegar producer on account of their ability to accumulate a high concentration of acetic acid due to oxidative fermentation linking the ethanol oxidation respiratory chain. Reactions in oxidative fermentation cause poor growth because a large amount of the carbon source is oxidized incompletely and the harmful oxidized products are accumulated almost stoichiometrically in the culture medium during growth, but a newly identified AAB, Asaia, has shown unusual properties, including scanty acetic acid production and rapid growth, as compared with known AAB as Acetobacter, Gluconobacter, and Gluconacetobacter. To understand these unique properties of Asaia in more detail, the respiratory chain and energetics of this strain were investigated. It was found that Asaia lacks quinoprotein alcohol dehydrogenase, but has other sugar and sugar alcohol-oxidizing enzymes specific to the respiratory chain of Gluconobacter, especially quinoprotein glycerol dehydrogenase. It was also found that Asaia has a cyanide-sensitive cytochrome bo(3)-type ubiquinol oxidase as sole terminal oxidase in the respiratory chain, and that it exhibits a higher H(+)/O ratio.

  10. Sporolactobacillus vineae sp. nov., a spore-forming lactic acid bacterium isolated from vineyard soil.

    PubMed

    Chang, Young-Hyo; Jung, Min Young; Park, In-Soon; Oh, Hee-Mock

    2008-10-01

    Two spore-forming, facultatively anaerobic, lactic acid bacteria, strains SL153(T) and SL1153, were isolated from vineyard soil in Korea. Cells of both strains were slightly curved, Gram-positive, motile rods that measured between 1 and 4 mum in length and were approximately 0.5 mum in diameter. Strains SL153(T) and SL1153 fermented glucose, fructose, mannose and sorbitol, but were negative for nitrate reduction, catalase and oxidase. The predominant cellular fatty acids of the two isolates were iso-C(15 : 0), anteiso-C(15 : 0) and anteiso-C(17 : 0). meso-Diaminopimelic acid, glucose, mannose and galactose were determined in their whole-cell hydrolysates. 16S rRNA gene sequences from the two strains were almost identical (99.9 %) and they could be placed in the genus Sporolactobacillus according to phylogenetic analysis. The species most closely related to SL153(T) were Sporolactobacillus inulinus and Sporolactobacillus terrae with 16S rRNA gene similarities of 95.7 and 95.5 %, respectively, with the type strains. Levels of DNA-DNA relatedness between strain SL153(T) and the type strains of S. inulinus, S. terrae and Sporolactobacillus kofuensis were 18.5, 18.0 and 17.0 %, respectively. On the basis of the phylogenetic (16S rRNA gene), chemotaxonomic and phenotypic evidence given in this study, it is proposed that strains SL153(T) and SL1153 should be assigned to the genus Sporolactobacillus as representatives of the novel species Sporolactobacillus vineae sp. nov. The type strain is SL153(T) (=KCTC 5376(T)=JCM 14637(T)).

  11. The effect of humic acid on uptake/adsorption of copper by a marine bacterium and two marine ciliates.

    PubMed

    Lores, E M; Snyder, R A; Pennock, J R

    1999-01-01

    The effect of humic acid (HA) on Cu uptake by a bacterium and two bacterivorus ciliates was investigated. The presence of HA resulted in a statistically significant (p<0.001) decrease in Cu associated with bacteria that were exposed to 67 microg Cu L(-1). Complexation of Cu appears to lower the availability of Cu with respect to bacterial cell surface binding and uptake. For ciliates, 10 mg HA L(-1) significantly reduced uptake of Cu by Uronema, but did not reduce uptake of Cu by Pleuronema. Uronema exposed to 67 microg Cu L(-1) accumulated 54% less Cu when 10 mg HA L(-1) was present (0.50 pg ciliate(-1) vs 0.23 pg ciliate(-1)). Uronema feeding on V. natriegens, took up less than half as much Cu as unfed Uronema when exposed to Cu without HA (0.41 pg Cu fed ciliate(-1) vs 0.86 pg Cu unfed ciliate(-1), but only 40% less when exposed to Cu and HA (0.31 pg Cu fed ciliate(-1) vs 0.51 pg Cu unfed ciliate(-1)). The lower % reduction attributable to fed ciliates in the presence of HA suggests that some of the Cu associated with HA is available through trophic processes.

  12. Reduction of Cr(VI) under acidic conditions by the facultative Fe(III)-reducing bacterium Acidiphilium cryptum

    SciTech Connect

    David E. Cummings; Scott Fendorf; Rajesh K. Sani; Brent M. Peyton; Timothy S. Magnuson

    2007-01-01

    The potential for biological reduction of Cr(VI) under acidic conditions was evaluated with the acidophilic, facultatively metal-reducing bacterium Acidiphilium cryptum strain JF-5 to explore the role of acidophilic microorganisms in the Cr cycle in low-pH environments. An anaerobic suspension of washed A. cryptum cells rapidly reduced 50 M Cr(VI) at pH 3.2; biological reduction was detected from pH 1.7-4.7. The reduction product, confirmed by XANES analysis, was entirely Cr(III) that was associated predominantly with the cell biomass (70-80%) with the residual residing in the aqueous phase. Reduction of Cr(VI) showed a pH optimum similar to that for growth and was inhibited by 5 mM HgCl2, suggesting that the reaction was enzyme-mediated. Introduction of O2 into the reaction medium slowed the reduction rate only slightly, whereas soluble Fe(III) (as ferric sulfate) increased the rate dramatically, presumably by the shuttling of electrons from bioreduced Fe(II) to Cr(VI) in a coupled biotic-abiotic cycle. Starved cells could not reduce Cr(VI) when provided as sole electron acceptor, indicating that Cr(VI) reduction is not an energy-conserving process in A. cryptum. We speculate, rather, that Cr(VI) reduction is used here as a detoxification mechanism.

  13. Viability and dose-response studies on the effects of the immunoenhancing lactic acid bacterium Lactobacillus rhamnosus in mice.

    PubMed

    Gill, H S; Rutherfurd, K J

    2001-08-01

    Previous studies have indicated that the lactic acid bacterium Lactobacillus rhamnosus HN001 can enhance immune function in mice, following oral delivery. However, the influence of bacterial cell viability on immunoenhancement, and the optimum dose of HN001 required for this effect, have not been determined. In the present study, both live and heat-killed preparations of L. rhamnosus HN001 were shown to enhance the phagocytic activity of blood and peritoneal leucocytes in mice, at a dose of 109 micro-organisms daily. In contrast, only live HN001 enhanced gut mucosal antibody responses to cholera toxin vaccine. Feeding mice with 107 viable HN001/d for 14 d was shown to enhance the phagocytic capacity of blood leucocytes, with incremental enhancement observed at 109 and 1011 daily doses. In contrast, a minimum dose of 109 viable HN001/d was required to enhance the phagocytic activity of peritoneal leucocytes, and no further increment was observed with 1011 daily. This study demonstrates that L. rhamnosus HN001 exhibits dose-dependent effects on the phagocytic defence system of mice, and suggests that while the innate cellular immune system is responsive to killed forms of food-borne bacteria, specific gut mucosal immunity may only be stimulated by live forms.

  14. Distribution and Functions of Phosphotransferase System Genes in the Genome of the Lactic Acid Bacterium Oenococcus oeni

    PubMed Central

    Jamal, Zohra; Miot-Sertier, Cécile; Thibau, François; Dutilh, Lucie; Lonvaud-Funel, Aline; Ballestra, Patricia; Le Marrec, Claire

    2013-01-01

    Oenococcus oeni, the lactic acid bacterium primarily responsible for malolactic fermentation in wine, is able to grow on a large variety of carbohydrates, but the pathways by which substrates are transported and phosphorylated in this species have been poorly studied. We show that the genes encoding the general phosphotransferase proteins, enzyme I (EI) and histidine protein (HPr), as well as 21 permease genes (3 isolated ones and 18 clustered into 6 distinct loci), are highly conserved among the strains studied and may form part of the O. oeni core genome. Additional permease genes differentiate the strains and may have been acquired or lost by horizontal gene transfer events. The core pts genes are expressed, and permease gene expression is modulated by the nature of the bacterial growth substrate. Decryptified O. oeni cells are able to phosphorylate glucose, cellobiose, trehalose, and mannose at the expense of phosphoenolpyruvate. These substrates are present at low concentrations in wine at the end of alcoholic fermentation. The phosphotransferase system (PTS) may contribute to the perfect adaptation of O. oeni to its singular ecological niche. PMID:23524676

  15. Lactic acid bacterium and yeast microbiotas of sixteen French traditional sourdoughs.

    PubMed

    Lhomme, Emilie; Lattanzi, Anna; Dousset, Xavier; Minervini, Fabio; De Angelis, Maria; Lacaze, Guylaine; Onno, Bernard; Gobbetti, Marco

    2015-12-23

    Sixteen sourdoughs (FS1-FS16) used for the manufacture of traditional French breads were characterized by strongly acid conditions (median value of pH 3.5). The concentration of free amino acids (FAA) was highly variable, due to different proteolytic activity of flour used for back slopping and of dominant microorganisms. Median value of cell density of lactic acid bacteria (LAB) was 9.2 log CFU/g. The ratio between LAB and yeasts ranged from 10,000:1 to 10:1. According to the culture-dependent method and 16S metagenetics, Lactobacillus sanfranciscensis was the dominant species in French sourdoughs. FS5 and FS15, propagated according to protocols including one back slopping step at 14 °C, were the only exceptions. High positive correlations were found between L. sanfranciscensis, temperature of back slopping and FAA. The results of this study highlighted the broad adaptability of L. sanfranciscensis to very acid sourdough. Besides species frequently encountered (e.g., Lactobacillus parabrevis/Lactobacillus hammesii, Lactobacillus plantarum and Leuconostoc mesenteroides), first Lactobacillus xiangfangensis (FS5) and Lactobacillus diolivorans (FS15) were found in sourdough. As determined by RAPD-PCR analyses, the sourdough samples showed a different number of strains, ranging from 5 (FS9, FS11 and FS15) to 12 (FS1 and FS13), meaning a highly variable bacterial diversity. Cluster analysis showed that different sourdoughs, especially when propagated in the same bakery, may harbor similar strains. Except for L. plantarum (FS5) and Ln. mesenteroides (FS3), all the dominant species were detected by both 16S metagenetics and culture-dependent method. Yeast diversity was lower than LAB. Except for FS4 (solely dominated by Kazachstania servazzii), yeast microbiota of French sourdoughs was dominated by Saccharomyces cerevisiae. Strains isolated in this study could be a useful base for developing new basic researches on physiology, metabolism, and intraspecific diversity of L

  16. Inoculation of sugarcane with Pantoea sp. increases amino acid contents in shoot tissues; serine, alanine, glutamine and asparagine permit concomitantly ammonium excretion and nitrogenase activity of the bacterium.

    PubMed

    Loiret, F G; Grimm, B; Hajirezaei, M R; Kleiner, D; Ortega, E

    2009-07-15

    Pantoea sp. is an endophytic nitrogen-fixing bacterium isolated from sugarcane tissues. The aim of the present study was to determine the contents of amino acids in sugarcane as a result of inoculation of nodes and nodal roots with Pantoea sp. strain 9C and to evaluate the effects of amino acids on growth, nitrogenase activity and ammonium excretion of the bacterium. Content of almost all amino acids increased in 30-day-old plantlets by root inoculation. The most abundant amino acids in shoot tissues were asparagine and proline, and those in nodal roots were asparagine, proline, aspartic acid, glutamic acid and serine. The bacterium was able to grow on all tested amino acids except histidine, isoleucine and leucine. Nitrogenase Pantoea sp. was partially inhibited by 1, 2 or 5mmolL(-1) and completely inhibited by 10mmolL(-1) of NH(4)(+) in the media. Pantoea sp. showed nitrogenase activity in 5mmolL(-1) of serine, asparagine, threonine, alanine, proline, tyrosine, valine, methionine, lysine, phenylalanine, cysteine, tryptophan, citrulline and ornithine. Pantoea sp. did not excrete ammonium when it grew in vivo conditions favoring nitrogen fixation; however, ammonium was detected in the supernatant when 5mmolL(-1) asparagine, aspartic acid, alanine, serine or glutamine was added to the medium. The highest ammonium concentration in the supernatant was detected, when Pantoea grew on serine. Ammonium in the supernatant and nitrogenase activity were only detectable concomitantly when the medium was supplemented with serine, alanine, glutamine or asparagine. We discuss roles of amino acids on plant-bacteria interaction during the colonization of sugarcane plants.

  17. Microbacter margulisiae gen. nov., sp. nov., a propionigenic bacterium isolated from sediments of an acid rock drainage pond.

    PubMed

    Sánchez-Andrea, Irene; Sanz, Jose Luis; Stams, Alfons J M

    2014-12-01

    A novel anaerobic propionigenic bacterium, strain ADRI(T), was isolated from sediment of an acid rock drainage environment (Tinto River, Spain). Cells were small (0.4-0.6×1-1.7 µm), non-motile and non-spore-forming rods. Cells possessed a Gram-negative cell-wall structure and were vancomycin-resistant. Strain ADRI(T) utilized yeast extract and various sugars as substrates and formed propionate, lactate and acetate as major fermentation products. The optimum growth temperature was 30 °C and the optimum pH for growth was pH 6.5, but strain ADRI(T) was able to grow at a pH as low as 3.0. Oxidase, indole formation, and urease and catalase activities were negative. Aesculin and gelatin were hydrolysed. The predominant cellular fatty acids of strain ADRI(T) were anteiso-C15 : 0 (30.3 %), iso-C15 : 0 (29.2 %) and iso-C17 : 0 3-OH (14.9 %). Major menaquinones were MK-8 (52 %) and MK-9 (48 %). The genomic DNA G+C content was 39.9 mol%. Phylogenetically, strain ADRI(T) was affiliated to the family Porphyromonadaceae of the phylum Bacteroidetes. The most closely related cultured species were Paludibacter propionicigenes with 16S rRNA gene sequence similarity of 87.5 % and several species of the genus Dysgonomonas (similarities of 83.5-85.4 % to the type strains). Based on the distinctive ecological, phenotypic and phylogenetic characteristics of strain ADRI(T), a novel genus and species, Microbacter margulisiae gen. nov., sp. nov., is proposed. The type strain is ADRI(T) ( = JCM 19374(T) = DSM 27471(T)).

  18. Application of ethylene diamine tetra acetic acid degrading bacterium Burkholderia cepacia on biotreatment process.

    PubMed

    Chen, Wei-Ting; Shen, Shu-Min; Shu, Chi-Min

    2015-10-01

    Ethylene diamine tetra acetic acid (EDTA), the effluent of secondary biotreatment units, can be properly biodegraded by Burkholderia cepacia. Through batch degradation of EDTA, the raw wastewater of EDTA was controlled at 50 mg/L, and then nutrients was added in diluted wastewater to cultivate activated sludge, which the ratio of composition is depicted as "COD:N:P:Fe = 100:5:1:0.5". After 27 days, the removal efficiency of Fe-EDTA and COD was 100% and 92.0%, correspondingly. At the continuous process, the raw wastewater of EDTA was dictated at 166 mg/L before adding nutrients to cultivate activated sludge, in which the ratio of composition did also follow with batch process. After 22 days, the removal efficiency of Fe-EDTA and COD for experimental group was 71.46% and 62.58%, correspondingly. The results showed that the batch process was more suited for EDTA biodegradation.

  19. An engineered bacterium auxotrophic for an unnatural amino acid: a novel biological containment system

    PubMed Central

    2015-01-01

    Biological containment is a genetic technique that programs dangerous organisms to grow only in the laboratory and to die in the natural environment. Auxotrophy for a substance not found in the natural environment is an ideal biological containment. Here, we constructed an Escherichia coli strain that cannot survive in the absence of the unnatural amino acid 3-iodo-L-tyrosine. This synthetic auxotrophy was achieved by conditional production of the antidote protein against the highly toxic enzyme colicin E3. An amber stop codon was inserted in the antidote gene. The translation of the antidote mRNA was controlled by a translational switch using amber-specific 3-iodo-L-tyrosine incorporation. The antidote is synthesized only when 3-iodo-L-tyrosine is present in the culture medium. The viability of this strain rapidly decreased with less than a 1 h half-life after removal of 3-iodo-L-tyrosine, suggesting that the decay of the antidote causes the host killing by activated colicin E3 in the absence of this unnatural amino acid. The contained strain grew 1.5 times more slowly than the parent strains. The escaper frequency was estimated to be 1.4 mutations (95% highest posterior density 1.1–1.8) per 105 cell divisions. This containment system can be constructed by only plasmid introduction without genome editing, suggesting that this system may be applicable to other microbes carrying toxin-antidote systems similar to that of colicin E3. PMID:26401457

  20. Desulfosporosinus acididurans sp. nov.: an acidophilic sulfate-reducing bacterium isolated from acidic sediments.

    PubMed

    Sánchez-Andrea, Irene; Stams, Alfons J M; Hedrich, Sabrina; Ňancucheo, Ivan; Johnson, D Barrie

    2015-01-01

    Three strains of sulfate-reducing bacteria (M1(T), D, and E) were isolated from acidic sediments (White river and Tinto river) and characterized phylogenetically and physiologically. All three strains were obligately anaerobic, mesophilic, spore-forming straight rods, stained Gram-negative and displayed variable motility during active growth. The pH range for growth was 3.8-7.0, with an optimum at pH 5.5. The temperature range for growth was 15-40 °C, with an optimum at 30 °C. Strains M1(T), D, and E used a wide range of electron donors and acceptors, with certain variability within the different strains. The nominated type strain (M1(T)) used ferric iron, nitrate, sulfate, elemental sulfur, and thiosulfate (but not arsenate, sulfite, or fumarate) as electron acceptors, and organic acids (formate, lactate, butyrate, fumarate, malate, and pyruvate), alcohols (glycerol, methanol, and ethanol), yeast extract, and sugars (xylose, glucose, and fructose) as electron donors. It also fermented some substrates such as pyruvate and formate. Strain M1(T) tolerated up to 50 mM ferrous iron and 10 mM aluminum, but was inhibited by 1 mM copper. On the basis of phenotypic, phylogenetic, and genetic characteristics, strains M1(T), D, and E represent a novel species within the genus Desulfosporosinus, for which the name Desulfosporosinus acididurans sp. nov. is proposed. The type strain is M1(T) (=DSM 27692(T) = JCM 19471(T)). Strain M1(T) was the first acidophilic SRB isolated, and it is the third described species of acidophilic SRB besides Desulfosporosinus acidiphilus and Thermodesulfobium narugense.

  1. Iron sources used by the nonpathogenic lactic acid bacterium Lactobacillus sakei as revealed by electron energy loss spectroscopy and secondary-ion mass spectrometry.

    PubMed

    Duhutrel, Philippe; Bordat, Christian; Wu, Ting-Di; Zagorec, Monique; Guerquin-Kern, Jean-Luc; Champomier-Vergès, Marie-Christine

    2010-01-01

    Lactobacillus sakei is a lactic acid bacterium naturally found on meat. Although it is generally acknowledged that lactic acid bacteria are rare species in the microbial world which do not have iron requirements, the genome sequence of L. sakei 23K has revealed quite complete genetic equipment dedicated to transport and use of this metal. Here, we aimed to investigate which iron sources could be used by this species as well as their role in the bacterium's physiology. Therefore, we developed a microscopy approach based on electron energy loss spectroscopy (EELS) analysis and nano-scale secondary-ion mass spectrometry (SIMS) in order to analyze the iron content of L. sakei cells. This revealed that L. sakei can use iron sources found in its natural ecosystem, myoglobin, hemoglobin, hematin, and transferrin, to ensure long-term survival during stationary phase. This study reveals that analytical image methods (EELS and SIMS) are powerful complementary tools for investigation of metal utilization by bacteria.

  2. Production of L-lactic Acid from Biomass Wastes Using Scallop Crude Enzymes and Novel Lactic Acid Bacterium

    NASA Astrophysics Data System (ADS)

    Yanagisawa, Mitsunori; Nakamura, Kanami; Nakasaki, Kiyohiko

    In the present study, biomass waste raw materials including paper mill sludge, bamboo, sea lettuce, and shochu residue (from a distiller) and crude enzymes derived from inedible and discarded scallop parts were used to produce L-lactic acid for the raw material of biodegradable plastic poly-lactic acid. The activities of cellulase and amylase in the crude enzymes were 22 and 170units/L, respectively, and L-lactic acid was produced from every of the above mentioned biomass wastes, by the method of liquid-state simultaneous saccharification and fermentation (SSF) . The L-lactic acid concentrations produced from sea lettuce and shochu residue, which contain high concentration of starch were 3.6 and 9.3g/L, respectively, and corresponded to greater than 25% of the conversion of glucans contained in these biomass wastes. Furthermore, using the solid state SSF method, concentrations as high as 13g/L of L-lactic acid were obtained from sea lettuce and 26g/L were obtained from shochu residue.

  3. Neoasaia chiangmaiensis gen. nov., sp. nov., a novel osmotolerant acetic acid bacterium in the alpha-Proteobacteria.

    PubMed

    Yukphan, Pattaraporn; Malimas, Taweesak; Potacharoen, Wanchern; Tanasupawat, Somboon; Tanticharoen, Morakot; Yamada, Yuzo

    2005-10-01

    An acetic acid bacterium, designated as isolate AC28(T), was isolated from a flower of red ginger (khing daeng in Thai; Alpinia purpurata) collected in Chiang Mai, Thailand, at pH 3.5 by use of a glucose/ethanol/acetic acid (0.3%, w/v) medium. A phylogenetic tree based on 16S rRNA gene sequences for 1,376 bases showed that isolate AC28(T) constituted a cluster along with the type strain of Kozakia baliensis. However, the isolate formed an independent cluster in a phylogenetic tree based on 16S-23S rDNA internal transcribed spacer (ITS) region sequences for 586 bases. Pair-wise sequence similarities of the isolate in 16S rRNA gene sequences for 1,457 bases were 93.0-88.3% to the type strains of Asaia, Kozakia, Swaminathania, Acetobacter, Gluconobacter, Gluconacetobacter, Acidomonas, and Saccharibacter species. Restriction analysis of 16S-23S rDNA ITS regions discriminated isolate AC28(T) from the type strains of Asaia and Kozakia species. Cells were non-motile. Colonies were pink, shiny, and smooth. The isolate produced acetic acid from ethanol. Oxidation of acetate and lactate was negative. The isolate grew on glutamate agar and mannitol agar. Growth was positive on 30% D-glucose (w/v) and in the presence of 0.35% acetic acid (w/v), but not in the presence of 1.0% KNO(3) (w/v). Ammoniac nitrogen was hardly assimilated on a glucose medium or a mannitol medium. Production of dihydroxyacetone from glycerol was weakly positive. The isolate did not produce a levan-like polysaccharide on a sucrose medium. Major isoprenoid quinone was Q-10. DNA base composition was 63.1 mol% G+C. On the basis of the results obtained, Neoasaia gen. nov. was proposed with Neoasaia chiangmaiensis sp. nov. The type strain was isolate AC28(T) (=BCC 15763(T) =NBRC 101099(T)).

  4. Aminivibrio pyruvatiphilus gen. nov., sp. nov., an anaerobic, amino-acid-degrading bacterium from soil of a Japanese rice field.

    PubMed

    Honda, Takuya; Fujita, Takashi; Tonouchi, Akio

    2013-10-01

    A novel anaerobic bacterium that could ferment amino acids and organic acids was isolated from an anaerobic, propionate-oxidizing enrichment culture originating from soil of a rice field in Japan. Cells of the isolate, designated strain 4F6E(T), were Gram-staining-negative, oxidase- and catalase-negative, non-spore-forming, vibrio-shaped, motile rods (0.8×2.0-2.5 µm) with two or three lateral flagella. Growth occurred at 20-42 °C (optimum at 37-40 °C), at pH 6.4-8.4 (optimum at pH 7.3) and at 0-1.5 % (w/v) NaCl (optimum at 0-0.5 %). Good growth occurred on glycine, serine, cysteine, pyruvate and citrate, whereas poor growth was observed on threonine, glutamine, L-malate, α-ketoglutarate, peptone and Casamino acids. In co-culture with the hydrogen-utilizing methanogen Methanobacterium formicicum JCM 10132(T), strain 4F6E(T) oxidized alanine, valine, leucine, isoleucine, methionine, aspartate, glutamate, histidine, asparagine and fumarate. Yeast extract was required for growth. The G+C content of genomic DNA was 61.9 mol%. A phylogenetic analysis based on comparison of the 16S rRNA gene sequence showed that the type strains of Fretibacterium fastidiosum, Aminobacterium colombiense and Aminobacterium mobile, members of the family Synergistaceae, were the closest relatives of strain 4F6E(T), with low sequence similarities (89.3, 89.5 and 86.2 %, respectively). Strain 4F6E(T) contained iso-C13 : 0 (24.43 %), iso-C15 : 0 (16.47 %) and C19 : 1ω11c/C19 : 1ω9c (16.32 %) as the major fatty acids, which differed from those of F. fastidiosum, Aminobacterium colombiense and Aminobacterium mobile. On the basis of phenotypic, chemotaxonomic and phylogenetic differences between strain 4F6E(T) and the type strains of F. fastidiosum and Aminobacterium species, we propose that strain 4F6E(T) represents a novel genus and species, Aminivibrio pyruvatiphilus gen. nov., sp. nov. The type strain of Aminivibrio pyruvatiphilus is strain 4F6E(T) (

  5. Lactobacillus vini sp. nov., a wine lactic acid bacterium homofermentative for pentoses.

    PubMed

    Rodas, Ana María; Chenoll, Empar; Macián, M Carmen; Ferrer, Sergi; Pardo, Isabel; Aznar, Rosa

    2006-03-01

    Six strains with more than 99.5 % 16S rRNA gene sequence similarity, identical internal spacer region profiles and restriction analysis of the amplified 16S rRNA gene patterns were isolated from fermenting grape musts during independent studies carried out in France and Spain many years apart. Strains are Gram-positive, motile, facultatively anaerobic rods that do not exhibit catalase activity and have the ability to utilize pentose sugars (ribose and/or l-arabinose), although they are homofermentative bacteria. Strains ferment pentoses exclusively yielding lactic acid as the end product. A broad set of molecular techniques has been applied to characterize these strains and the results show a high degree of genotypical congruence, sharing identical profiles with 16S rRNA-based techniques. Phylogenetic analysis based on 16S rRNA gene sequences placed these strains within the genus Lactobacillus, closely related to Lactobacillus mali, Lactobacillus nagelii and Lactobacillus satsumensis (with approximately 95 % sequence similarity). DNA-DNA hybridization experiments confirmed the independent status at the species level of these fermenting grape-musts strains. Phenotypically they can be distinguished from the closest relatives by several traits such as growth temperatures and fermentation of carbohydrates. The name Lactobacillus vini sp. nov. is proposed, with strain Mont 4T (= DSM 20605T = CECT 5924T) as the type strain.

  6. Enterococcus bulliens sp. nov., a novel lactic acid bacterium isolated from camel milk.

    PubMed

    Kadri, Zaina; Spitaels, Freek; Cnockaert, Margo; Praet, Jessy; El Farricha, Omar; Swings, Jean; Vandamme, Peter

    2015-11-01

    Four lactic acid bacteria isolates obtained from fresh dromedary camel milk produced in Dakhla, a city in southern Morocco, were characterised in order to determine their taxonomic position. The four isolates had highly similar MALDI-TOF MS and RAPD fingerprints and identical 16S rRNA gene sequences. Comparative sequence analysis revealed that the 16S rRNA gene sequence of the four isolates was most similar to that of Enterococcus sulfureus ATCC 49903(T) and Enterococcus italicus DSM 15952(T) (99.33 and 98.59% similarity, respectively). However, sequence analysis of the phenylalanyl-tRNA synthase (pheS), RNA polymerase (rpoA) and ATP synthase (atpA) genes revealed that the taxon represented by strain LMG 28766(T) was well separated from E. sulfureus LMG 13084(T) and E. italicus LMG 22039(T), which was further confirmed by DNA-DNA hybridization values that were clearly below the species demarcation threshold. The novel taxon was easily differentiated from its nearest neighbour species through sequence analysis of protein encoding genes, MALDI-TOF mass spectrometry and multiple biochemical tests, but had a similar percentage G+C content of about 39%. We therefore propose to formally classify these isolates as Enterococcus bulliens sp. nov., with LMG 28766(T) (=CCMM B1177(T)) as the type strain.

  7. Asaia krungthepensis sp. nov., an acetic acid bacterium in the alpha-Proteobacteria.

    PubMed

    Yukphan, Pattaraporn; Potacharoen, Wanchern; Tanasupawat, Somboon; Tanticharoen, Morakot; Yamada, Yuzo

    2004-03-01

    Three bacterial strains were isolated from flowers collected in Bangkok, Thailand, by an enrichment-culture approach for acetic acid bacteria. Phylogenetic analysis based on 16S rRNA gene sequences showed that the isolates were located in the lineage of the genus Asaia but constituted a cluster separate from the type strains of Asaia bogorensis and Asaia siamensis. The DNA base composition of the isolates was 60.2-60.5 mol% G+C, with a range of 0.3 mol%. The isolates constituted a taxon separate from Asaia bogorensis and Asaia siamensis on the basis of DNA-DNA relatedness. The isolates had morphological, physiological, biochemical and chemotaxonomic characteristics similar to those of the type strains of Asaia bogorensis and Asaia siamensis, but the isolates grew on maltose. The major ubiquinone was Q(10). On the basis of the results obtained, the name Asaia krungthepensis sp. nov. is proposed for the isolates. The type strain is isolate AA08(T) (=BCC 12978(T)=TISTR 1524(T)=NBRC 100057(T)=NRIC 0535(T)), which had a DNA G+C content of 60.3 mol% and was isolated from a heliconia flower ('paksaasawan' in Thai; Heliconia sp.) collected in Bangkok, Thailand.

  8. Isolation and characterisation of lactic acid bacterium for effective fermentation of cellobiose into optically pure homo L-(+)-lactic acid.

    PubMed

    Abdel-Rahman, Mohamed Ali; Tashiro, Yukihiro; Zendo, Takeshi; Shibata, Keisuke; Sonomoto, Kenji

    2011-02-01

    Effective utilisation of cellulosic biomasses for economical lactic acid production requires a microorganism with potential ability to utilise efficiently its major components, glucose and cellobiose. Amongst 631 strains isolated from different environmental samples, strain QU 25 produced high yields of l-(+)-lactic acid of high optical purity from cellobiose. The QU 25 strain was identified as Enterococcus mundtii based on its sugar fermentation pattern and 16S rDNA sequence. The production of lactate by fermentation was optimised for the E. mundtii QU25 strain. The optimal pH and temperature for batch culturing were found to be 7.0°C and 43°C, respectively. E. mundtii QU 25 was able to metabolise a mixture of glucose and cellobiose simultaneously without apparent carbon catabolite repression. Moreover, under the optimised culture conditions, production of optically pure l-lactic acid (99.9%) increased with increasing cellobiose concentrations. This indicates that E. mundtii QU 25 is a potential candidate for effective lactic acid production from cellulosic hydrolysate materials.

  9. Rewiring Lactococcus lactis for Ethanol Production

    PubMed Central

    Dehli, Tore; Jensen, Peter Ruhdal

    2013-01-01

    Lactic acid bacteria (LAB) are known for their high tolerance toward organic acids and alcohols (R. S. Gold, M. M. Meagher, R. Hutkins, and T. Conway, J. Ind. Microbiol. 10:45–54, 1992) and could potentially serve as platform organisms for production of these compounds. In this study, we attempted to redirect the metabolism of LAB model organism Lactococcus lactis toward ethanol production. Codon-optimized Zymomonas mobilis pyruvate decarboxylase (PDC) was introduced and expressed from synthetic promoters in different strain backgrounds. In the wild-type L. lactis strain MG1363 growing on glucose, only small amounts of ethanol were obtained after introducing PDC, probably due to a low native alcohol dehydrogenase activity. When the same strains were grown on maltose, ethanol was the major product and lesser amounts of lactate, formate, and acetate were formed. Inactivating the lactate dehydrogenase genes ldhX, ldhB, and ldh and introducing codon-optimized Z. mobilis alcohol dehydrogenase (ADHB) in addition to PDC resulted in high-yield ethanol formation when strains were grown on glucose, with only minor amounts of by-products formed. Finally, a strain with ethanol as the sole observed fermentation product was obtained by further inactivating the phosphotransacetylase (PTA) and the native alcohol dehydrogenase (ADHE). PMID:23377945

  10. Rewiring Lactococcus lactis for ethanol production.

    PubMed

    Solem, Christian; Dehli, Tore; Jensen, Peter Ruhdal

    2013-04-01

    Lactic acid bacteria (LAB) are known for their high tolerance toward organic acids and alcohols (R. S. Gold, M. M. Meagher, R. Hutkins, and T. Conway, J. Ind. Microbiol. 10:45-54, 1992) and could potentially serve as platform organisms for production of these compounds. In this study, we attempted to redirect the metabolism of LAB model organism Lactococcus lactis toward ethanol production. Codon-optimized Zymomonas mobilis pyruvate decarboxylase (PDC) was introduced and expressed from synthetic promoters in different strain backgrounds. In the wild-type L. lactis strain MG1363 growing on glucose, only small amounts of ethanol were obtained after introducing PDC, probably due to a low native alcohol dehydrogenase activity. When the same strains were grown on maltose, ethanol was the major product and lesser amounts of lactate, formate, and acetate were formed. Inactivating the lactate dehydrogenase genes ldhX, ldhB, and ldh and introducing codon-optimized Z. mobilis alcohol dehydrogenase (ADHB) in addition to PDC resulted in high-yield ethanol formation when strains were grown on glucose, with only minor amounts of by-products formed. Finally, a strain with ethanol as the sole observed fermentation product was obtained by further inactivating the phosphotransacetylase (PTA) and the native alcohol dehydrogenase (ADHE).

  11. Isolation, characterization, and amino acid sequences of auracyanins, blue copper proteins from the green photosynthetic bacterium Chloroflexus aurantiacus

    NASA Technical Reports Server (NTRS)

    McManus, J. D.; Brune, D. C.; Han, J.; Sanders-Loehr, J.; Meyer, T. E.; Cusanovich, M. A.; Tollin, G.; Blankenship, R. E.

    1992-01-01

    Three small blue copper proteins designated auracyanin A, auracyanin B-1, and auracyanin B-2 have been isolated from the thermophilic green gliding photosynthetic bacterium Chloroflexus aurantiacus. All three auracyanins are peripheral membrane proteins. Auracyanin A was described previously (Trost, J. T., McManus, J. D., Freeman, J. C., Ramakrishna, B. L., and Blankenship, R. E. (1988) Biochemistry 27, 7858-7863) and is not glycosylated. The two B forms are glycoproteins and have almost identical properties to each other, but are distinct from the A form. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis apparent monomer molecular masses are 14 (A), 18 (B-2), and 22 (B-1) kDa. The amino acid sequences of the B forms are presented. All three proteins have similar absorbance, circular dichroism, and resonance Raman spectra, but the electron spin resonance signals are quite different. Laser flash photolysis kinetic analysis of the reactions of the three forms of auracyanin with lumiflavin and flavin mononucleotide semiquinones indicates that the site of electron transfer is negatively charged and has an accessibility similar to that found in other blue copper proteins. Copper analysis indicates that all three proteins contain 1 mol of copper per mol of protein. All three auracyanins exhibit a midpoint redox potential of +240 mV. Light-induced absorbance changes and electron spin resonance signals suggest that auracyanin A may play a role in photosynthetic electron transfer. Kinetic data indicate that all three proteins can donate electrons to cytochrome c-554, the electron donor to the photosynthetic reaction center.

  12. Systemic immunity-enhancing effects in healthy subjects following dietary consumption of the lactic acid bacterium Lactobacillus rhamnosus HN001.

    PubMed

    Sheih, Y H; Chiang, B L; Wang, L H; Liao, C K; Gill, H S

    2001-04-01

    To determine the effects of the probiotic lactic acid bacterium, Lactobacillus rhamnosus HN001, on natural cellular immunity when delivered orally in normal low-fat milk (LFM) or lactose-hydrolyzed low-fat milk (LFM-LH). A three stage, pre-post intervention trial, spanning nine weeks. Taipei Medical College Hospital, Taipei, Taiwan. Fifty-two healthy middle-aged and elderly volunteers (17 males, 35 females; median age 63.5, range 44-80). Stage 1 (run-in diet): 25 g/200 mL reconstituted LFM powder, twice daily for 3 weeks. Stage 2 (probiotic intervention): LFM or LFM-LH, supplemented with 10(9) CFUs/g L. rhamnosus HN001 in each case, for 3 weeks. Stage 3 (wash-out): LFM for 3 weeks. In vitro phagocytic capacity of peripheral blood polymorphonuclear (PMN) leukocytes; in vitro tumoricidal activity of natural killer (NK) leukocytes. Immunological responses were unaffected by the run-in diet of LFM alone. In contrast, the relative proportion of PMN cells showing phagocytic activity increased by 19% and 15%, respectively, following consumption of HN001 in either LFM or LFM-LH; the relative level of NK cell tumor killing activity increased by 71% and 147%. In most cases these levels declined following cessation, but remained above baseline. Dietary consumption of L. rhamnosus HN001, in a base of low-fat milk or lactose-hydrolyzed low-fat milk, appears to enhance systemic cellular immune responses and may be useful as a dietary supplement to boost natural immunity.

  13. Isolation, characterization, and amino acid sequences of auracyanins, blue copper proteins from the green photosynthetic bacterium Chloroflexus aurantiacus

    NASA Technical Reports Server (NTRS)

    McManus, J. D.; Brune, D. C.; Han, J.; Sanders-Loehr, J.; Meyer, T. E.; Cusanovich, M. A.; Tollin, G.; Blankenship, R. E.

    1992-01-01

    Three small blue copper proteins designated auracyanin A, auracyanin B-1, and auracyanin B-2 have been isolated from the thermophilic green gliding photosynthetic bacterium Chloroflexus aurantiacus. All three auracyanins are peripheral membrane proteins. Auracyanin A was described previously (Trost, J. T., McManus, J. D., Freeman, J. C., Ramakrishna, B. L., and Blankenship, R. E. (1988) Biochemistry 27, 7858-7863) and is not glycosylated. The two B forms are glycoproteins and have almost identical properties to each other, but are distinct from the A form. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis apparent monomer molecular masses are 14 (A), 18 (B-2), and 22 (B-1) kDa. The amino acid sequences of the B forms are presented. All three proteins have similar absorbance, circular dichroism, and resonance Raman spectra, but the electron spin resonance signals are quite different. Laser flash photolysis kinetic analysis of the reactions of the three forms of auracyanin with lumiflavin and flavin mononucleotide semiquinones indicates that the site of electron transfer is negatively charged and has an accessibility similar to that found in other blue copper proteins. Copper analysis indicates that all three proteins contain 1 mol of copper per mol of protein. All three auracyanins exhibit a midpoint redox potential of +240 mV. Light-induced absorbance changes and electron spin resonance signals suggest that auracyanin A may play a role in photosynthetic electron transfer. Kinetic data indicate that all three proteins can donate electrons to cytochrome c-554, the electron donor to the photosynthetic reaction center.

  14. Soil acidity determines the effectiveness of an organic amendment and a native bacterium for increasing soil stabilisation in semiarid mine tailings.

    PubMed

    Carrasco, L; Caravaca, F; Azcón, R; Roldán, A

    2009-01-01

    Unstable mine tailings are vulnerable to water and air erosion, so it is important to promote their surface stabilisation in order to avoid the spread of heavy metals. In a greenhouse experiment, we assessed the effect of the addition of Aspergillus niger-treated sugar beet waste and inoculation with a native bacterium, Bacillus cereus, on the stabilisation of soil aggregates of two acidic, semiarid mine tailings, with different acidity degree, during watering and drying periods. Organic amendment raised the pH of both the moderately and highly acidic tailings, whereas the bacterial inoculation increased this parameter in the former. Only the amendment addition increased soil water-soluble carbon in both tailings compared with their controls, under either watering or drying conditions. Both the amendment and B. cereus enhanced water-soluble carbohydrates. Both treatments increased dehydrogenase activity and aggregate stability, particularly in the moderately acidic tailing under drying conditions. After soil drying, aggregate stability was increased by the amendment (about 66% higher than the control soil) and by the bacterium (about 45% higher than the control soil) in the moderately acidic tailing. The effectiveness of these treatments as structure-stabilisation methods for degraded, semiarid mine ecosystems appears to be restricted to tailings of moderate acidity.

  15. Alterations in membrane phospholipid fatty acids of Gram-positive piezotolerant bacterium Sporosarcina sp. DSK25 in response to growth pressure.

    PubMed

    Wang, Jiani; Li, Jiangtao; Dasgupta, Shamik; Zhang, Li; Golovko, Mikhail Y; Golovko, Svetlana A; Fang, Jiasong

    2014-04-01

    Pressure is an important thermodynamic property of the ocean and the deep biosphere that affects microbial physiology and biochemistry. Here, we report on our investigation of the response of Gram-positive piezotolerant bacterium Sporosarcina sp. DSK25 to hydrostatic pressure. Strain DSK25 responded in an adaptive manner to upshifts of growth pressure and showed systematic changes in phospholipid fatty acids. As the pressure increased from 0.1 to 10 MPa (Megapascal), unsaturated fatty acids in DSK25 increased from 21.7 to 31.1% of total fatty acids, while the level of iso- and anteiso-branched fatty acids remained unchanged. At higher pressures (30, 50, and 60 MPa), the amount of unsaturated fatty acids decreased, and that of anteiso-branched fatty acids increased from 34.4 to 49.9% at the expense of iso-branched fatty acids. For the first time, two polyunsaturated fatty acids (PUFA), 18:2n-6 and 18:2n-x, with the latter having much higher abundance than the former, were identified in DSK25. The concentration of the PUFA increased with growth pressure. These results indicate the involvement of unsaturated and methyl-branched fatty acids in the modulation of bacteria membrane fluidity and function over environmentally relevant parameter (pressure). Piezotolerant bacterium Sporosarcina sp. DSK25 appears to utilize two regulatory mechanisms for adaptation to high pressure, a rapid-responding mechanism on transient scale, expressed as increased biosynthesis of monounsaturated fatty acids, and a long-term adaptation mechanism in increased synthesis of anteiso-branched and polyunsaturated fatty acids. Our results further suggest that Gram-positive piezophilic bacteria respond differently than Gram-negative bacteria in adaptation to high pressure.

  16. Description of Paralactobacillus selangorensis gen. nov., sp. nov., a new lactic acid bacterium isolated from chili bo, a Malaysian food ingredient.

    PubMed

    Leisner, J J; Vancanneyt, M; Goris, J; Christensen, H; Rusul, G

    2000-01-01

    Paralactobacillus selangorensis gen. nov., sp. nov. is described. This organism, isolated from a Malaysian food ingredient called chili bo, is an obligatory homofermentative, rod-shaped lactic acid bacterium. The G+C content is 46.1-46.2+/-0.3 mol%. Earlier 16S rRNA studies showed that this organism constitutes a new taxon distantly related to the Lactobacillus casei-Pediococcus group. A phenotypic description that distinguishes Paralactobacillus selangorensis from other genera of lactic acid bacteria is presented. The type strain of Paralactobacillus selangorensis is LMG 17710T.

  17. Development of Recombinant Lactococcus lactis Displaying Albumin-Binding Domain Variants against Shiga Toxin 1 B Subunit

    PubMed Central

    Zadravec, Petra; Marečková, Lucie; Petroková, Hana; Hodnik, Vesna; Perišić Nanut, Milica; Anderluh, Gregor; Štrukelj, Borut; Malý, Petr; Berlec, Aleš

    2016-01-01

    Infections with shiga toxin-producing bacteria, like enterohemorrhagic Escherichia coli and Shigella dysenteriae, represent a serious medical problem. No specific and effective treatment is available for patients with these infections, creating a need for the development of new therapies. Recombinant lactic acid bacterium Lactococcus lactis was engineered to bind Shiga toxin by displaying novel designed albumin binding domains (ABD) against Shiga toxin 1 B subunit (Stx1B) on their surface. Functional recombinant Stx1B was produced in Escherichia coli and used as a target for selection of 17 different ABD variants (named S1B) from the ABD scaffold-derived high-complex combinatorial library in combination with a five-round ribosome display. Two most promising S1Bs (S1B22 and S1B26) were characterized into more details by ELISA, surface plasmon resonance and microscale thermophoresis. Addition of S1Bs changed the subcellular distribution of Stx1B, completely eliminating it from Golgi apparatus most likely by interfering with its retrograde transport. All ABD variants were successfully displayed on the surface of L. lactis by fusing to the Usp45 secretion signal and to the peptidoglycan-binding C terminus of AcmA. Binding of Stx1B by engineered lactococcal cells was confirmed using flow cytometry and whole cell ELISA. Lactic acid bacteria prepared in this study are potentially useful for the removal of Shiga toxin from human intestine. PMID:27606705

  18. Lactococcus petauri sp. nov., isolated from an abscess of a sugar glider.

    PubMed

    Goodman, Laura B; Lawton, Marie R; Franklin-Guild, Rebecca J; Anderson, Renee R; Schaan, Lynn; Thachil, Anil J; Wiedmann, Martin; Miller, Claire B; Alcaine, Samuel D; Kovac, Jasna

    2017-09-25

    A strain of lactic acid bacteria, designated 159469T, isolated from a facial abscess in a sugar glider, was characterized genetically and phenotypically. Cells of the strain were Gram-stain-positive, coccoid and catalase-negative. Morphological, physiological and phylogenetic data indicated that the isolate belongs to the genus Lactococcus. Strain 159469T was closely related to Lactococcus garvieae ATCC 43921T, showing 95.86 and 98.08 % sequence similarity in 16S rRNA gene and rpoB gene sequences, respectively. Furthermore, a pairwise average nucleotide identity blast (ANIb) value of 93.54 % and in silico DNA-DNA hybridization value of 50.7  % were determined for the genome of strain 159469T, when compared with the genome of the type strain of Lactococcus garvieae. Based on the data presented here, the isolate represents a novel species of the genus Lactococcus, for which the name Lactococcus petauri sp. nov. is proposed. The type strain is 159469T (=LMG 30040T=DSM 104842T).

  19. Leuconostoc gelidum and Leuconostoc gasicomitatum strains dominated the lactic acid bacterium population associated with strong slime formation in an acetic-acid herring preserve.

    PubMed

    Lyhs, Ulrike; Koort, Joanna M K; Lundström, Hanna-Saara; Björkroth, K Johanna

    2004-01-15

    Spoilage characterised by strong slime and gas formation affected some manufacture lots of an acetic-acid Baltic herring (Culpea haerengus membras) preserve after few weeks of storage at 0-6 degrees C. The product consisted of herring filets in acetic acid marinade containing sugar, salt, allspice and carrot slices. Microbiological analyses of the spoiled product showed high lactic acid bacterium (LAB) levels ranging from 4.5x10(8) to 2.4x10(9) CFU/g. Yeasts were not detected in any of the herring samples. Since LAB contaminants are seldom associated with fresh fish, LAB populations associated with marinade ingredients (carrots, allspice) were also analyzed. The highest LAB levels exceeding 10(7) CFU/g were detected in equilibrium modified atmosphere packaged baby carrots whereas the levels detected in the allspice samples did not exceed 4.3x10(5). A total of 176 randomly selected LAB isolates originating from herring, carrot and allspice samples were further identified to species level using a 16 and 23S rRNA gene RFLP (ribotyping) database. Leuconostoc gelidum and Leuconostoc gasicomitatum strains dominated both in the spoiled herring and carrot samples. These species are heterofermentative-producing CO(2) from glucose and they also produce dextran from sucrose. Inoculation of some commercial-herring products with spoilage-associated L. gelidum and L. gasicomitatum strains verified that these strains have the capability of producing slime and gas in herring preserves although slime formation was not as strong as in the original samples. Since L. gelidum and L. gasicomitatum strains were commonly detected in carrots, carrot slices used for the fish marinade were considered to be the probable source of these specific spoilage organisms.

  20. Growth Phase-Dependent Proteomes of the Malaysian Isolated Lactococcus lactis Dairy Strain M4 Using Label-Free Qualitative Shotgun Proteomics Analysis

    PubMed Central

    Yap, Theresa Wan Chen; Rabu, Amir; Abu Bakar, Farah Diba; Abdul Rahim, Raha; Mahadi, Nor Muhammad; Illias, Rosli Md.

    2014-01-01

    Lactococcus lactis is the most studied mesophilic fermentative lactic acid bacterium. It is used extensively in the food industry and plays a pivotal role as a cell factory and also as vaccine delivery platforms. The proteome of the Malaysian isolated L. lactis M4 dairy strain, obtained from the milk of locally bred cows, was studied to elucidate the physiological changes occurring between the growth phases of this bacterium. In this study, ultraperformance liquid chromatography nanoflow electrospray ionization tandem mass spectrometry (UPLC- nano-ESI-MSE) approach was used for qualitative proteomic analysis. A total of 100 and 121 proteins were identified from the midexponential and early stationary growth phases, respectively, of the L. lactis strain M4. During the exponential phase, the most important reaction was the generation of sufficient energy, whereas, in the early stationary phase, the metabolic energy pathways decreased and the biosynthesis of proteins became more important. Thus, the metabolism of the cells shifted from energy production in the exponential phase to the synthesis of macromolecules in the stationary phase. The resultant proteomes are essential in providing an improved view of the cellular machinery of L. lactis during the transition of growth phases and hence provide insight into various biotechnological applications. PMID:24982972

  1. Growth phase-dependent proteomes of the Malaysian isolated Lactococcus lactis dairy strain M4 using label-free qualitative shotgun proteomics analysis.

    PubMed

    Yap, Theresa Wan Chen; Rabu, Amir; Abu Bakar, Farah Diba; Rahim, Raha Abdul; Mahadi, Nor Muhammad; Illias, Rosli Md; Murad, Abdul Munir Abdul

    2014-01-01

    Lactococcus lactis is the most studied mesophilic fermentative lactic acid bacterium. It is used extensively in the food industry and plays a pivotal role as a cell factory and also as vaccine delivery platforms. The proteome of the Malaysian isolated L. lactis M4 dairy strain, obtained from the milk of locally bred cows, was studied to elucidate the physiological changes occurring between the growth phases of this bacterium. In this study, ultraperformance liquid chromatography nanoflow electrospray ionization tandem mass spectrometry (UPLC- nano-ESI-MS(E)) approach was used for qualitative proteomic analysis. A total of 100 and 121 proteins were identified from the midexponential and early stationary growth phases, respectively, of the L. lactis strain M4. During the exponential phase, the most important reaction was the generation of sufficient energy, whereas, in the early stationary phase, the metabolic energy pathways decreased and the biosynthesis of proteins became more important. Thus, the metabolism of the cells shifted from energy production in the exponential phase to the synthesis of macromolecules in the stationary phase. The resultant proteomes are essential in providing an improved view of the cellular machinery of L. lactis during the transition of growth phases and hence provide insight into various biotechnological applications.

  2. [Isolation, identification and oxidizing characterization of an iron-sulfur oxidizing bacterium LY01 from acid mine drainage].

    PubMed

    Liu, Yu-jiao; Yang, Xin-ping; Wang, Shi-mei; Liang, Yin

    2013-05-01

    An acidophilic iron-sulfur oxidizing bacterium LY01 was isolated from acid mine drainage of coal in Guizhou Province, China. Strain LY01 was identified as Acidithiobacillusferrooxidans by morphological and physiological characteristics, and phylogenetic analysis of its 16S rRNA gene sequence. Strain LY01 was able to grow using ferrous ion (Fe2+), elemental sulfur (S0) and pyrite as sole energy source, respectively, but significant differences in oxidation efficiency and bacterial growth were observed when different energy source was used. When strain LY01 was cultured in 9K medium with 44.2 g x L(-1) FeSO4.7H2O as the substrate, the oxidation efficiency of Fe2+ was 100% in 30 h and the cell number of strain LY01 reached to 4.2 x 10(7) cell x mL(-1). When LY01 was cultured in 9K medium with 10 g x L(-1) S0 as the substrate, 6.7% S0 oxidation efficiency, 2001 mg x L(-1) SO4(2-) concentration and 8.9 x 10(7) cell x mL(-1) cell number were observed in 21 d respectively. When LY01 was cultured with 30 g x L(-1) pyrite as the substrate, the oxidation efficiency of pyrite, SO4(2-) concentration and cell number reached 10%, 4443 mg x L(-1) and 3.4 x 10(8) cell x mL(-1) respectively in 20 d. The effects of different heavy metals (Ni2+, Pb2+) on oxidation activity of strain LY01 cultured with pyrite were investigated. Results showed that the oxidation activity of strain LY01 was inhibited to a certain extent with the addition of Ni2+ at 10-100 mg x L(-1) to the medium, but the addition of 10-100 mg x L(-1) Pb2+ had no effect on LY01 activity.

  3. Thermosyntropha lipolytica gen. nov., sp. nov., a lipolytic, anaerobic, alkalitolerant, thermophilic bacterium utilizing short- and long-chain fatty acids in syntrophic coculture with a methanogenic archaeum.

    PubMed

    Svetlitshnyi, V; Rainey, F; Wiegel, J

    1996-10-01

    Three strains of an anaerobic thermophilic organoheterotrophic lipolytic alkalitolerant bacterium, Thermosyntropha lipolytica gen. nov., sp. nov. (type strain JW/VS-265T; DSM 11003), were isolated from alkaline hot springs of Lake Bogoria (Kenya). The cells were nonmotile, non-spore forming, straight or slightly curved rods. At 60 degrees C the pH range for growth determined at 25 degrees C [pH25 degrees C] was 7.15 to 9.5, with an optimum between 8.1 and 8.9 (pH60 degrees C of 7.6 and 8.1). At a pH25 degrees C of 8.5 the temperature range for growth was from 52 to 70 degrees C, with an optimum between 60 and 66 degrees C. The shortest doubling time was around 1 h. In pure culture the bacterium grew in a mineral base medium supplemented with yeast extract, tryptone, Casamino Acids, betaine, and crotonate as carbon sources, producing acetate as a major product and constitutively a lipase. During growth in the presence of olive oil, free long-chain fatty acids were accumulated in the medium but the pure culture could not utilize olive oil, triacylglycerols, short- and long-chain fatty acids, and glycerol for growth. In syntrophic coculture (Methanobacterium strain JW/VS-M29) the lipolytic bacteria grew on triacylglycerols and linear saturated and unsaturated fatty acids with 4 to 18 carbon atoms, but glycerol was not utilized. Fatty acids with even numbers of carbon atoms were degraded to acetate and methane, while from odd-numbered fatty acids 1 mol of propionate per mol of fatty acid was additionally formed. 16S rDNA sequence analysis identified Syntrophospora and Syntrophomonas spp. as closest phylogenetic neighbors.

  4. Use of murine models to detect the allergenicity of genetically modified Lactococcus lactis NZ9000/pNZPNK.

    PubMed

    Chiang, Shen-Shih; Liu, Chin-Feng; Ku, Ting-Wei; Mau, Jeng-Leun; Lin, Hsin-Tang; Pan, Tzu-Ming

    2011-04-27

    By introducing aprN into Lactococcus lactis NZ9000, the genetically modified L. lactis NZ9000/pNZPNK successfully expressed the nattokinase. The safety assessment of this novel strain was based on allergenicity of pepsin digestion stability and murine model serologic identity. Subjecting to the GM strain and host to pepsin digestion, the soluble fractions and cell debris were fast degraded completely. Feeding with ovalbumin resulted in significantly higher production of IgG1 and IgE as compared to that of L. lactis NZ9000/pNZPNK or L. lactis NZ9000. Further, the serum IgG2a level increased dose-dependently at week 2 and induced immune reaction toward Th1 pathway. Secretion of cytokines IL-4 and IL-10 fed with lactococci was significantly lower than that of the OVA group. L. lactis NZ9000/pNZPNK did not increase the proliferation of type 2 helper T cells in spleen or induce allergenicity in BALB/c mice. On the basis of the results, the new GM lactic acid bacterium is regarded as safe to use.

  5. Construction of a new shuttle vector for DNA delivery into mammalian cells using non-invasive Lactococcus lactis.

    PubMed

    Yagnik, Bhrugu; Padh, Harish; Desai, Priti

    2016-04-01

    Use of food grade Lactococcus lactis (L. lactis) is fast emerging as a safe alternative for delivery of DNA vaccine. To attain efficient DNA delivery, L. lactis, a non-invasive bacterium is converted to invasive strain either by expressing proteins like Internalin A (InlA) or Fibronectin binding protein A (FnBPA) or through chemical treatments. However the safety status of invasive L. lactis is questionable. In the present report, we have shown that non-invasive L. lactis efficiently delivered the newly constructed reporter plasmid pPERDBY to mammalian cells without any chemical enhancers. The salient features of the vector are; I) Ability to replicate in two different hosts; Escherichia coli (E. coli) and Lactic Acid Bacteria (LAB), II) One of the smallest reporter plasmid for DNA vaccine, III) Enhanced Green Fluorescence Protein (EGFP) linked to Multiple Cloning Site (MCS), IV) Immunostimulatory CpG motifs functioning as an adjuvant. Expression of EGFP in pPERDBY transfected CHO-K1 and Caco-2 cells demonstrates its functionality. Non-invasive r-L. lactis was found efficient in delivering pPERDBY to Caco-2 cells. The in vitro data presented in this article supports the hypothesis that in the absence of invasive proteins or relevant chemical treatment, L. lactis was found efficient in delivering DNA to mammalian cells.

  6. Investigation of Associations of Yarrowia lipolytica, Staphylococcus xylosus, and Lactococcus lactis in Culture as a First Step in Microbial Interaction Analysis▿ †

    PubMed Central

    Mansour, S.; Bailly, J.; Landaud, S.; Monnet, C.; Sarthou, A. S.; Cocaign-Bousquet, M.; Leroy, S.; Irlinger, F.; Bonnarme, P.

    2009-01-01

    The interactions that may occur between microorganisms in different ecosystems have not been adequately studied yet. We investigated yeast-bacterium interactions in a synthetic medium using different culture associations involving the yeast Yarrowia lipolytica 1E07 and two bacteria, Staphylococcus xylosus C2a and Lactococcus lactis LD61. The growth and biochemical characteristics of each microorganism in the different culture associations were studied. The expression of genes related to glucose, lactate, and amino acid catabolism was analyzed by reverse transcription followed by quantitative PCR. Our results show that the growth of Y. lipolytica 1E07 is dramatically reduced by the presence of S. xylosus C2a. As a result of a low amino acid concentration in the medium, the expression of Y. lipolytica genes involved in amino acid catabolism was downregulated in the presence of S. xylosus C2a, even when L. lactis was present in the culture. Furthermore, the production of lactate by both bacteria had an impact on the lactate dehydrogenase gene expression of the yeast, which increased up to 30-fold in the three-species culture compared to the Y. lipolytica 1E07 pure culture. S. xylosus C2a growth dramatically decreased in the presence of Y. lipolytica 1E07. The growth of lactic acid bacteria was not affected by the presence of S. xylosus C2a or Y. lipolytica 1E07, although the study of gene expression showed significant variations. PMID:19684166

  7. Thermosyntropha lipolytica gen. nov., sp. nov., a lipolytic, anaerobic, alkalitolerant, thermophilic bacterium utilizing short- and long-chain fatty acids in syntrophic coculture with a methanogenic archaeum

    SciTech Connect

    Svetlitshnyi, V.; Wiegel, J.; Rainey, F.

    1996-10-01

    Three strains of an anaerobic thermophilic organoheterotrophic lipolytic alkalitolerant bacterium, Thermosyntropha lipolytica gen. nov., sp. nov. (type strain JW/VS-264{sup T}; DSM 11003) were isolated from alkaline hot springs of Lake Bogoria (Kenya). The cells were nonmotile, non-spore forming, straight or slightly curved rods. At 60{degrees}C, the pH range for growth determined at 25{degrees}C [pH{sup 25{degrees}C}] was 7.15 to 9.5, with an optimum between 8.1 and 8.9 (pH{sup 60{degrees}C} of 7.6 and 8.1). At a pH{sup 25{degrees}C} of 8.5 temperature range for growth was from 52 to 70{degrees}C, with an optimum between 60 and 66{degrees}C. The shortest doubling time was around 1 h. In pure culture the bacterium grew in a mineral base medium supplemented with yeast extract, tryptone, Casamino Acids, betaine, and crotonate as carbon sources, producing acetate as a major product and constitutively a lipase. During growth in the presence of olive oil, free long-chain fatty acids were accumulated in the medium but the pure culture syntrophic coculture (Methanobacterium strain JW/VS-M29) the lipolytic bacteria grew on triacylglycerols and linear saturated and unsaturated fatty acids with 4 to 18 carbon atoms, but glycerol was not utilized. Fatty acids with even numbers of carbon atoms were degraded to acetate and methane, while from odd-numbered fatty acids 1 mol of propionate per mol of fatty acid was additionally formed. 16S rDNA sequence analysis identified Syntrophospora and Syntrophomonas spp. as closest phylogenetic neighbors.

  8. Protein turnover forms one of the highest maintenance costs in Lactococcus lactis.

    PubMed

    Lahtvee, Petri-Jaan; Seiman, Andrus; Arike, Liisa; Adamberg, Kaarel; Vilu, Raivo

    2014-07-01

    Protein turnover plays an important role in cell metabolism by regulating metabolic fluxes. Furthermore, the energy costs for protein turnover have been estimated to account for up to a third of the total energy production during cell replication and hence may represent a major limiting factor in achieving either higher biomass or production yields. This work aimed to measure the specific growth rate (μ)-dependent abundance and turnover rate of individual proteins, estimate the ATP cost for protein production and turnover, and compare this with the total energy balance and other maintenance costs. The lactic acid bacteria model organism Lactococcus lactis was used to measure protein turnover rates at μ = 0.1 and 0.5 h(-1) in chemostat experiments. Individual turnover rates were measured for ~75% of the total proteome. On average, protein turnover increased by sevenfold with a fivefold increase in growth rate, whilst biomass yield increased by 35%. The median turnover rates found were higher than the specific growth rate of the bacterium, which suggests relatively high energy consumption for protein turnover. We found that protein turnover costs alone account for 38 and 47% of the total energy produced at μ = 0.1 and 0.5 h(-1), respectively, and gene ontology groups Energy metabolism and Translation dominated synthesis costs at both growth rates studied. These results reflect the complexity of metabolic changes that occur in response to changes in environmental conditions, and signify the trade-off between biomass yield and the need to produce ATP for maintenance processes.

  9. Monte-Carlo Modeling of the Central Carbon Metabolism of Lactococcus lactis: Insights into Metabolic Regulation

    PubMed Central

    Murabito, Ettore; Verma, Malkhey; Bekker, Martijn; Bellomo, Domenico; Westerhoff, Hans V.; Teusink, Bas; Steuer, Ralf

    2014-01-01

    Metabolic pathways are complex dynamic systems whose response to perturbations and environmental challenges are governed by multiple interdependencies between enzyme properties, reactions rates, and substrate levels. Understanding the dynamics arising from such a network can be greatly enhanced by the construction of a computational model that embodies the properties of the respective system. Such models aim to incorporate mechanistic details of cellular interactions to mimic the temporal behavior of the biochemical reaction system and usually require substantial knowledge of kinetic parameters to allow meaningful conclusions. Several approaches have been suggested to overcome the severe data requirements of kinetic modeling, including the use of approximative kinetics and Monte-Carlo sampling of reaction parameters. In this work, we employ a probabilistic approach to study the response of a complex metabolic system, the central metabolism of the lactic acid bacterium Lactococcus lactis, subject to perturbations and brief periods of starvation. Supplementing existing methodologies, we show that it is possible to acquire a detailed understanding of the control properties of a corresponding metabolic pathway model that is directly based on experimental observations. In particular, we delineate the role of enzymatic regulation to maintain metabolic stability and metabolic recovery after periods of starvation. It is shown that the feedforward activation of the pyruvate kinase by fructose-1,6-bisphosphate qualitatively alters the bifurcation structure of the corresponding pathway model, indicating a crucial role of enzymatic regulation to prevent metabolic collapse for low external concentrations of glucose. We argue that similar probabilistic methodologies will help our understanding of dynamic properties of small-, medium- and large-scale metabolic networks models. PMID:25268481

  10. Monte-Carlo modeling of the central carbon metabolism of Lactococcus lactis: insights into metabolic regulation.

    PubMed

    Murabito, Ettore; Verma, Malkhey; Bekker, Martijn; Bellomo, Domenico; Westerhoff, Hans V; Teusink, Bas; Steuer, Ralf

    2014-01-01

    Metabolic pathways are complex dynamic systems whose response to perturbations and environmental challenges are governed by multiple interdependencies between enzyme properties, reactions rates, and substrate levels. Understanding the dynamics arising from such a network can be greatly enhanced by the construction of a computational model that embodies the properties of the respective system. Such models aim to incorporate mechanistic details of cellular interactions to mimic the temporal behavior of the biochemical reaction system and usually require substantial knowledge of kinetic parameters to allow meaningful conclusions. Several approaches have been suggested to overcome the severe data requirements of kinetic modeling, including the use of approximative kinetics and Monte-Carlo sampling of reaction parameters. In this work, we employ a probabilistic approach to study the response of a complex metabolic system, the central metabolism of the lactic acid bacterium Lactococcus lactis, subject to perturbations and brief periods of starvation. Supplementing existing methodologies, we show that it is possible to acquire a detailed understanding of the control properties of a corresponding metabolic pathway model that is directly based on experimental observations. In particular, we delineate the role of enzymatic regulation to maintain metabolic stability and metabolic recovery after periods of starvation. It is shown that the feedforward activation of the pyruvate kinase by fructose-1,6-bisphosphate qualitatively alters the bifurcation structure of the corresponding pathway model, indicating a crucial role of enzymatic regulation to prevent metabolic collapse for low external concentrations of glucose. We argue that similar probabilistic methodologies will help our understanding of dynamic properties of small-, medium- and large-scale metabolic networks models.

  11. PpiA, a Surface PPIase of the Cyclophilin Family in Lactococcus lactis

    PubMed Central

    Trémillon, Nicolas; Morello, Eric; Llull, Daniel; Mazmouz, Rabia; Gratadoux, Jean-Jacques; Guillot, Alain; Chapot-Chartier, Marie-Pierre; Monlezun, Laura; Solé, Véronique; Ginisty, Hervé; Poquet, Isabelle

    2012-01-01

    Background Protein folding in the envelope is a crucial limiting step of protein export and secretion. In order to better understand this process in Lactococcus lactis, a lactic acid bacterium, genes encoding putative exported folding factors like Peptidyl Prolyl Isomerases (PPIases) were searched for in lactococcal genomes. Results In L. lactis, a new putative membrane PPIase of the cyclophilin subfamily, PpiA, was identified and characterized. ppiA gene was found to be constitutively expressed under normal and stress (heat shock, H2O2) conditions. Under normal conditions, PpiA protein was synthesized and released from intact cells by an exogenously added protease, showing that it was exposed at the cell surface. No obvious phenotype could be associated to a ppiA mutant strain under several laboratory conditions including stress conditions, except a very low sensitivity to H2O2. Induction of a ppiA copy provided in trans had no effect i) on the thermosensitivity of an mutant strain deficient for the lactococcal surface protease HtrA and ii) on the secretion and stability on four exported proteins (a highly degraded hybrid protein and three heterologous secreted proteins) in an otherwise wild-type strain background. However, a recombinant soluble form of PpiA that had been produced and secreted in L. lactis and purified from a culture supernatant displayed both PPIase and chaperone activities. Conclusions Although L. lactis PpiA, a protein produced and exposed at the cell surface under normal conditions, displayed a very moderate role in vivo, it was found, as a recombinant soluble form, to be endowed with folding activities in vitro. PMID:22442694

  12. Microbiology neutralization of zearalenone using Lactococcus lactis and Bifidobacterium sp.

    PubMed

    Król, A; Pomastowski, P; Rafińska, K; Railean-Plugaru, V; Walczak, J; Buszewski, B

    2017-08-29

    The aim of the study was to neutralize zearalenone by lactic acid bacteria (LAB) such as Lactococcus lactis and Bifidobacterium sp. and investigate the mechanism of zearalenone (ZEA) binding. Neutralization of ZEA by LAB was confirmed by identification of binding kinetics and spectroscopic studies such as Fourier transform infrared spectroscopy (FT-IR) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The obtained results showed that the kinetic process of zearalenone binding to L. lactis is not homogeneous but is expressed with an initial rapid stage with about 90% of ZEA biosorption and with a much slower second step. In case of Bifidobacterium sp., the neutralization process is homogeneous; the main stage can be described with about 88% of ZEA biosorption. MALDI-TOF-MS measurements and FTIR analysis confirmed the uptake of zearalenone molecules by bacterial species. Moreover, the assessment of dead and live lactic acid bacteria cells after zearalenone treatment was performed using fluorescence microscopy. Graphical abstract Microbiology neutralization of zearalenone using Lactococcus lactis and Bifidobacterium sp. was confirmed by identification of binding kinetics and spectroscopic studies such as FT-IR spectroscopy and MALDI-TOF-MS spectrometry. The mechanism of ZEA binding was also investigated.

  13. Identification of Lactococcus-Specific Bacteriocins Produced by Lactococcal Isolates, and the Discovery of a Novel Bacteriocin, Lactococcin Z.

    PubMed

    Ishibashi, Naoki; Seto, Hiromi; Koga, Shoko; Zendo, Takeshi; Sonomoto, Kenji

    2015-09-01

    Lactic acid bacteria that produce Lactococcus-specific bacteriocins were isolated and identified as Lactococcus lactis from fresh corn or lettuce. Among them, four isolates were identified as lactococcin Q producers. Seven isolates showed antimicrobial activity against a lactococcin Q producer, L. lactis QU 4, as well as against nisin Z and lacticin Q producers belonging to L. lactis. Strain QU 7 was selected as a standard strain and showed no cross-immunity to lactococcin Q or other lactococcal bacteriocins. The bacteriocin produced by strain QU 7 was purified in three chromatographic steps, and its molecular mass was determined to be 5041.35 Da. The amino acid sequence analysis revealed that it is a novel class IId bacteriocin, referred to as lactococcin Z. It consisted of 45 amino acid residues. The lczA gene encoding the prepeptide of lactococcin Z showed homology to lactococcins A, B, and M. Thus, this report demonstrates a new example of Lactococcus-specific bacteriocins.

  14. Towards Enhanced Galactose Utilization by Lactococcus lactis▿

    PubMed Central

    Neves, Ana R.; Pool, Wietske A.; Solopova, Ana; Kok, Jan; Santos, Helena; Kuipers, Oscar P.

    2010-01-01

    Accumulation of galactose in dairy products due to partial lactose fermentation by lactic acid bacteria yields poor-quality products and precludes their consumption by individuals suffering from galactosemia. This study aimed at extending our knowledge of galactose metabolism in Lactococcus lactis, with the final goal of tailoring strains for enhanced galactose consumption. We used directed genetically engineered strains to examine galactose utilization in strain NZ9000 via the chromosomal Leloir pathway (gal genes) or the plasmid-encoded tagatose 6-phosphate (Tag6P) pathway (lac genes). Galactokinase (GalK), but not galactose permease (GalP), is essential for growth on galactose. This finding led to the discovery of an alternative route, comprising a galactose phosphotransferase system (PTS) and a phosphatase, for galactose dissimilation in NZ9000. Introduction of the Tag6P pathway in a galPMK mutant restored the ability to metabolize galactose but did not sustain growth on this sugar. The latter strain was used to prove that lacFE, encoding the lactose PTS, is necessary for galactose metabolism, thus implicating this transporter in galactose uptake. Both PTS transporters have a low affinity for galactose, while GalP displays a high affinity for the sugar. Furthermore, the GalP/Leloir route supported the highest galactose consumption rate. To further increase this rate, we overexpressed galPMKT, but this led to a substantial accumulation of α-galactose 1-phosphate and α-glucose 1-phosphate, pointing to a bottleneck at the level of α-phosphoglucomutase. Overexpression of a gene encoding α-phosphoglucomutase alone or in combination with gal genes yielded strains with galactose consumption rates enhanced up to 50% relative to that of NZ9000. Approaches to further improve galactose metabolism are discussed. PMID:20817811

  15. Garvieacin Q, a Novel Class II Bacteriocin from Lactococcus garvieae BCC 43578

    PubMed Central

    Zendo, Takeshi; Visessanguan, Wonnop; Roytrakul, Sittiruk; Pumpuang, Laphaslada; Jaresitthikunchai, Janthima; Sonomoto, Kenji

    2012-01-01

    Lactococcus garvieae BCC 43578 produces a novel class II bacteriocin, garvieacin Q (GarQ), 70 amino acids in length and containing a 20-amino-acid N-terminal leader peptide. It is cleaved at the Gly-Gly site to generate the mature GarQ (5,339 Da), which is especially inhibitory against Listeria monocytogenes ATCC 19115 and other L. garvieae strains. PMID:22210221

  16. 10-Oxo-trans-11-octadecenoic acid generated from linoleic acid by a gut lactic acid bacterium Lactobacillus plantarum is cytoprotective against oxidative stress.

    PubMed

    Furumoto, Hidehiro; Nanthirudjanar, Tharnath; Kume, Toshiaki; Izumi, Yasuhiko; Park, Si-Bum; Kitamura, Nahoko; Kishino, Shigenobu; Ogawa, Jun; Hirata, Takashi; Sugawara, Tatsuya

    2016-04-01

    Oxidative stress is a well-known cause of multiple diseases. The nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway plays a central role in cellular antioxidative responses. In this study, we investigated the effects of novel fatty acid metabolite derivatives of linoleic acid generated by the gut lactic acid bacteria Lactobacillus plantarum on the Nrf2-ARE pathway. 10-Oxo-trans-11-octadecenoic acid (KetoC) protected HepG2 cells from cytotoxicity induced by hydrogen peroxide. KetoC also significantly increased cellular Nrf2 protein levels, ARE-dependent transcription, and the gene expression of antioxidative enzymes such as heme oxygenase-1 (HO-1), glutamate-cysteine ligase modifier subunit (GCLM), and quinone oxidoreductase 1 (NQO1) in HepG2 cells. Additionally, a single oral dose administration of KetoC also increased antioxidative gene expression and protein levels of Nrf2 and HO-1 in mouse organs. Since other fatty acid metabolites and linoleic acid did not affect cellular antioxidative responses, the cytoprotective effect of KetoC may be because of its α,β-unsaturated carbonyl moiety. Collectively, our data suggested that KetoC activated the Nrf2-ARE pathway to enhance cellular antioxidative responses in vitro and in vivo, which further suggests that KetoC may prevent multiple diseases induced by oxidative stress. Copyright © 2016. Published by Elsevier Inc.

  17. A consolidated analysis of the physiologic and molecular responses induced under acid stress in the legume-symbiont model-soil bacterium Sinorhizobium meliloti

    PubMed Central

    Draghi, W. O.; Del Papa, M. F.; Hellweg, C.; Watt, S. A.; Watt, T. F.; Barsch, A.; Lozano, M. J.; Lagares, A.; Salas, M. E.; López, J. L.; Albicoro, F. J.; Nilsson, J. F.; Torres Tejerizo, G. A.; Luna, M. F.; Pistorio, M.; Boiardi, J. L.; Pühler, A.; Weidner, S.; Niehaus, K.; Lagares, A.

    2016-01-01

    Abiotic stresses in general and extracellular acidity in particular disturb and limit nitrogen-fixing symbioses between rhizobia and their host legumes. Except for valuable molecular-biological studies on different rhizobia, no consolidated models have been formulated to describe the central physiologic changes that occur in acid-stressed bacteria. We present here an integrated analysis entailing the main cultural, metabolic, and molecular responses of the model bacterium Sinorhizobium meliloti growing under controlled acid stress in a chemostat. A stepwise extracellular acidification of the culture medium had indicated that S. meliloti stopped growing at ca. pH 6.0–6.1. Under such stress the rhizobia increased the O2 consumption per cell by more than 5-fold. This phenotype, together with an increase in the transcripts for several membrane cytochromes, entails a higher aerobic-respiration rate in the acid-stressed rhizobia. Multivariate analysis of global metabolome data served to unequivocally correlate specific-metabolite profiles with the extracellular pH, showing that at low pH the pentose-phosphate pathway exhibited increases in several transcripts, enzymes, and metabolites. Further analyses should be focused on the time course of the observed changes, its associated intracellular signaling, and on the comparison with the changes that operate during the sub lethal acid-adaptive response (ATR) in rhizobia. PMID:27404346

  18. A consolidated analysis of the physiologic and molecular responses induced under acid stress in the legume-symbiont model-soil bacterium Sinorhizobium meliloti.

    PubMed

    Draghi, W O; Del Papa, M F; Hellweg, C; Watt, S A; Watt, T F; Barsch, A; Lozano, M J; Lagares, A; Salas, M E; López, J L; Albicoro, F J; Nilsson, J F; Torres Tejerizo, G A; Luna, M F; Pistorio, M; Boiardi, J L; Pühler, A; Weidner, S; Niehaus, K; Lagares, A

    2016-07-11

    Abiotic stresses in general and extracellular acidity in particular disturb and limit nitrogen-fixing symbioses between rhizobia and their host legumes. Except for valuable molecular-biological studies on different rhizobia, no consolidated models have been formulated to describe the central physiologic changes that occur in acid-stressed bacteria. We present here an integrated analysis entailing the main cultural, metabolic, and molecular responses of the model bacterium Sinorhizobium meliloti growing under controlled acid stress in a chemostat. A stepwise extracellular acidification of the culture medium had indicated that S. meliloti stopped growing at ca. pH 6.0-6.1. Under such stress the rhizobia increased the O2 consumption per cell by more than 5-fold. This phenotype, together with an increase in the transcripts for several membrane cytochromes, entails a higher aerobic-respiration rate in the acid-stressed rhizobia. Multivariate analysis of global metabolome data served to unequivocally correlate specific-metabolite profiles with the extracellular pH, showing that at low pH the pentose-phosphate pathway exhibited increases in several transcripts, enzymes, and metabolites. Further analyses should be focused on the time course of the observed changes, its associated intracellular signaling, and on the comparison with the changes that operate during the sub lethal acid-adaptive response (ATR) in rhizobia.

  19. Structure and regulation of the omega-3 polyunsaturated fatty acid synthase genes from the deep-sea bacterium Photobacterium profundum strain SS9.

    PubMed

    Allen, Eric E; Bartlett, Douglas H

    2002-06-01

    Omega-3 polyunsaturated fatty acids (PUFAs) such as eicosapentaenoic acid (20:5n-3; EPA) and docosahexaenoic acid (22:6n-3; DHA) have been shown to be of major importance in the promotion of cardiovascular health, proper human development and the prevention of some cancers. A high proportion of bacterial isolates from low-temperature and high-pressure marine environments produce EPA or DHA. This paper presents the sequence of a 33 kbp locus from the deep-sea bacterium Photobacterium profundum strain SS9 which includes four of the five genes required for EPA biosynthesis. As with other bacterial pfa (polyunsaturated fatty acid) genes, the deduced amino acid sequences encoded by the SS9 genes reveal large multidomain proteins that are likely to catalyse EPA biosynthesis by a novel polyketide synthesis mechanism. RNase protection experiments separated the SS9 pfa genes into two transcriptional units, pfaA-C and pfaD. The pfaA transcriptional start site was identified. Cultivation at elevated hydrostatic pressure or reduced temperature did not increase pfa gene expression despite the resulting increase in percentage composition of EPA under these conditions. However, a regulatory mutant was characterized which showed both increased expression of pfaA-D and elevated EPA percentage composition. This result suggests that a regulatory factor exists which coordinates pfaA-D transcription. Additional consideration regarding the activities required for PUFA synthesis is provided together with comparative analyses of bacterial pfa genes and gene products.

  20. Production of the small heat shock protein Lo18 from Oenococcus oeni in Lactococcus lactis improves its stress tolerance.

    PubMed

    Weidmann, Stéphanie; Maitre, Magali; Laurent, Julie; Coucheney, Françoise; Rieu, Aurélie; Guzzo, Jean

    2017-04-17

    Lactococcus lactis is a lactic acid bacterium widely used in cheese and fermented milk production. During fermentation, L. lactis is subjected to acid stress that impairs its growth. The small heat shock protein (sHsp) Lo18 from the acidophilic species Oenococcus oeni was expressed in L. lactis. This sHsp is known to play an important role in protein protection and membrane stabilization in O. oeni. The role of this sHsp could be studied in L. lactis, since no gene encoding for sHsp has been detected in this species. L. lactis subsp. cremoris strain MG1363 was transformed with the pDLhsp18 plasmid, which is derived from pDL278 and contains the hsp18 gene (encoding Lo18) and its own promoter sequence. The production of Lo18 during stress conditions was checked by immunoblotting and the cellular distribution of Lo18 in L. lactis cells after heat shock was determined. Our results clearly indicated a role for Lo18 in cytoplasmic protein protection and membrane stabilization during stress. The production of sHsp in L. lactis improved tolerance to heat and acid conditions in this species. Finally, the improvement of the L. lactis survival in milk medium thanks to Lo18 was highlighted, suggesting an interesting role of this sHsp. These findings suggest that the expression of a sHsp by a L. lactis strain results in greater resistance to stress, and, can consequently enhance the performances of industrial strains. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Impact of osmotic stress on protein diffusion in Lactococcus lactis.

    PubMed

    Mika, Jacek T; Schavemaker, Paul E; Krasnikov, Victor; Poolman, Bert

    2014-11-01

    We measured translational diffusion of proteins in the cytoplasm and plasma membrane of the Gram-positive bacterium Lactococcus lactis and probed the effect of osmotic upshift. For cells in standard growth medium the diffusion coefficients for cytosolic proteins (27 and 582 kDa) and 12-transmembrane helix membrane proteins are similar to those in Escherichia coli. The translational diffusion of GFP in L. lactis drops by two orders of magnitude when the medium osmolality is increased by ∼ 1.9 Osm, and the decrease in mobility is partly reversed in the presence of osmoprotectants. We find a large spread in diffusion coefficients over the full population of cells but a smaller spread if only sister cells are compared. While in general the diffusion coefficients we measure under normal osmotic conditions in L. lactis are similar to those reported in E. coli, the decrease in translational diffusion upon osmotic challenge in L. lactis is smaller than in E. coli. An even more striking difference is that in L. lactis the GFP diffusion coefficient drops much more rapidly with volume than in E. coli. We discuss these findings in the light of differences in turgor, cell volume, crowding and cytoplasmic structure of Gram-positive and Gram-negative bacteria.

  2. Expression of biologically active murine interleukin-18 in Lactococcus lactis.

    PubMed

    Feizollahzadeh, Sadegh; Khanahmad, Hossein; Rahimmanesh, Ilnaz; Ganjalikhani-Hakemi, Mazdak; Andalib, Alireza; Sanei, Mohammad Hossein; Rezaei, Abbas

    2016-11-01

    The food-grade bacterium Lactococcus lactis is increasingly used for heterologous protein expression in therapeutic and industrial applications. The ability of L. lactis to secrete biologically active cytokines may be used for the generation of therapeutic cytokines. Interleukin (IL)-18 enhances the immune response, especially on mucosal surfaces, emphasizing its therapeutic potential. However, it is produced as an inactive precursor and has to be enzymatically cleaved for maturation. We genetically manipulated L. lactis to secrete murine IL-18. The mature murine IL-18 gene was inserted downstream of a nisin promoter in pNZ8149 plasmid and the construct was used to transform L. lactis NZ3900. The transformants were selected on Elliker agar and confirmed by restriction enzyme digestion and sequencing. The expression and secretion of IL-18 protein was verified by SDS-PAGE, western blotting and ELISA. The biological activity of recombinant IL-18 was determined by its ability to induce interferon (IFN)-γ production in L. lactis co-cultured with murine splenic T cells. The amounts of IL-18 in bacterial lysates and supernatants were 3-4 μg mL(-1) and 0.6-0.7 ng mL(-1), respectively. The successfully generated L. lactis strain that expressed biologically active murine IL-18 can be used to evaluate the possible therapeutic effects of IL-18 on mucosal surfaces.

  3. Cloacibacillus evryensis gen. nov., sp. nov., a novel asaccharolytic, mesophilic, amino-acid-degrading bacterium within the phylum 'Synergistetes', isolated from an anaerobic sludge digester.

    PubMed

    Ganesan, Akila; Chaussonnerie, Sébastien; Tarrade, Anne; Dauga, Catherine; Bouchez, Théodore; Pelletier, Eric; Le Paslier, Denis; Sghir, Abdelghani

    2008-09-01

    A novel anaerobic, mesophilic, amino-acid-utilizing bacterium, strain 158T, was isolated from an anaerobic digester of a wastewater treatment plant. Cells of strain 158T were non-motile, rod-shaped (2.0-3.0 x 0.8-1.0 microm) and stained Gram-negative. Optimal growth occurred at 37 degrees C and pH 7.0 in an anaerobic basal medium containing 1 % Casamino acids. Strain 158T fermented arginine, histidine, lysine and serine and showed growth on yeast extract, brain-heart infusion (BHI) medium and tryptone, but not on carbohydrates, organic acids or alcohols. The end products of degradation were: acetate, butyrate, H2 and CO2 from arginine; acetate, propionate, butyrate, H2 and CO2 from lysine; and acetate, propionate, butyrate, valerate, H2 and CO2 from histidine, serine, BHI medium, Casamino acids and tryptone. The DNA G+C content was 55.8 mol%. The 16S rRNA gene sequence of strain 158T showed only 92.6 % sequence similarity with that of Synergistes jonesii, the only described species of the 'Synergistes' group. The major cellular fatty acids were iso-C(15:0) (16.63 %), iso-C(15:0) 3-OH (12.41 %) and C(17:1)omega6c (9.46 %) and the polar fatty acids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and phosphatidylmonomethylamine; these fatty acid profiles did not resemble those of any recognized bacterial species. Due to the considerable differences in genotypic, phenotypic and phylogenetic characteristics between strain 158T and those of its nearest relative, it is proposed that strain 158T represents a novel species in a new genus, Cloacibacillus evryensis gen. nov., sp. nov., in the phylum 'Synergistetes'. The type strain is 158T (=DSM 19522T=JCM 14828T).

  4. The Plasmid Complement of the Cheese Isolate Lactococcus garvieae IPLA 31405 Revealed Adaptation to the Dairy Environment.

    PubMed

    Flórez, Ana Belén; Mayo, Baltasar

    2015-01-01

    Lactococcus garvieae is a lactic acid bacterium found in raw-milk dairy products as well as a range of aquatic and terrestrial environments. The plasmids in L. garvieae have received little attention compared to those of dairy Lactococcus lactis, in which the genes carried by these extrachromosomal elements are considered of adaptive value. The present work reports the sequencing and analysis of the plasmid complement of L. garvieae IPLA 31405, a strain isolated from a traditional, Spanish, starter-free cheese made from raw-milk. It consists of pLG9 and pLG42, of 9,124 and 42,240 nucleotides, respectively. Based on sequence and structural homology in the putative origin of replication (ori) region, pLG9 and pLG42 are predicted to replicate via a theta mechanism. Real-time, quantitative PCR showed the number of copies per chromosome equivalent of pLG9 and pLG42 to be around two and five, respectively. Sequence analysis identified eight complete open reading frames (orfs) in pLG9 and 36 in pLG42; these were organized into functional modules or cassettes containing different numbers of genes. These modules were flanked by complete or interrupted insertion sequence (IS)-like elements. Among the modules of pLG42 was a gene cluster encoding specific components of a phosphoenolpyruvate-phosphotransferase (PEP-PTS) system, including a phospho-β-galacosidase. The cluster showed a complete nucleotide identity respect to that in plasmids of L. lactis. Loss of pLG42 showed this to be involved in lactose assimilation. In the same plasmid, an operon encoding a type I restriction/modification (R/M) system was also identified. The specificity of this R/M system might be broadened by different R/M specificity subunits detected in pLG9 and in the bacterial chromosome. However, challenges of L. garvieae IPLA 31405 against L. lactis phages proved that the R/M system was not involved in phage resistance. Together, these results support the hypothesis that, as in L. lactis, pLG42

  5. The Plasmid Complement of the Cheese Isolate Lactococcus garvieae IPLA 31405 Revealed Adaptation to the Dairy Environment

    PubMed Central

    Flórez, Ana Belén; Mayo, Baltasar

    2015-01-01

    Lactococcus garvieae is a lactic acid bacterium found in raw-milk dairy products as well as a range of aquatic and terrestrial environments. The plasmids in L. garvieae have received little attention compared to those of dairy Lactococcus lactis, in which the genes carried by these extrachromosomal elements are considered of adaptive value. The present work reports the sequencing and analysis of the plasmid complement of L. garvieae IPLA 31405, a strain isolated from a traditional, Spanish, starter-free cheese made from raw-milk. It consists of pLG9 and pLG42, of 9,124 and 42,240 nucleotides, respectively. Based on sequence and structural homology in the putative origin of replication (ori) region, pLG9 and pLG42 are predicted to replicate via a theta mechanism. Real-time, quantitative PCR showed the number of copies per chromosome equivalent of pLG9 and pLG42 to be around two and five, respectively. Sequence analysis identified eight complete open reading frames (orfs) in pLG9 and 36 in pLG42; these were organized into functional modules or cassettes containing different numbers of genes. These modules were flanked by complete or interrupted insertion sequence (IS)-like elements. Among the modules of pLG42 was a gene cluster encoding specific components of a phosphoenolpyruvate-phosphotransferase (PEP-PTS) system, including a phospho-β-galacosidase. The cluster showed a complete nucleotide identity respect to that in plasmids of L. lactis. Loss of pLG42 showed this to be involved in lactose assimilation. In the same plasmid, an operon encoding a type I restriction/modification (R/M) system was also identified. The specificity of this R/M system might be broadened by different R/M specificity subunits detected in pLG9 and in the bacterial chromosome. However, challenges of L. garvieae IPLA 31405 against L. lactis phages proved that the R/M system was not involved in phage resistance. Together, these results support the hypothesis that, as in L. lactis, pLG42

  6. Megasphaera hexanoica sp. nov., a medium-chain carboxylic acid-producing bacterium isolated from a cow rumen.

    PubMed

    Jeon, Byoung Seung; Kim, Seil; Sang, Byoung-In

    2017-07-01

    Strain MHT, a strictly anaerobic, Gram-stain-negative, non-spore-forming, spherical coccus or coccoid-shaped microorganism, was isolated from a cow rumen during a screen for hexanoic acid-producing bacteria. The microorganism grew at 30-40 °C and pH 5.5-7.5 and exhibited production of various short- and medium-chain carboxylic acids (acetic acid, butyric acid, pentanoic acid, isobutyric acid, isovaleric acid, hexanoic acid, heptanoic acid and octanoic acid), as well as H2 and CO2 as biogas. Phylogenetic analysis based on 16S rRNA gene sequencing demonstrated that MHT represents a member of the genus Megasphaera, with the closest relatives being Megapsphaera indica NMBHI-10T (94.1 % 16S rRNA sequence similarity), Megasphaera elsdenii DSM 20460T (93.8 %) and Megasphaera paucivorans DSM 16981T (93.8 %). The major cellular fatty acids produced by MHT included C12 : 0, C16 : 0, C18 : 1cis 9, and C18 : 0, and the DNA G+C content of the MHT genome is 51.8 mol%. Together, the distinctive phenotypic and phylogenetic characteristics of MHT indicate that this microorganism represents a novel species of the genus Megasphaera, for which the name Megasphaera hexanoica sp. nov. is herein proposed. The type strain of this species is MHT (=KCCM 43214T=JCM 31403T).

  7. Purification and partial characterization of bacteriocin produced by Lactococcus lactis ssp. lactis LL171.

    PubMed

    Kumari, Archana; Akkoç, Nefise; Akçelik, Mustafa

    2012-04-01

    Lactic acid bacteria (LAB) are possessing ability to synthesize antimicrobial compounds (like bacteriocin) during their growth. In this regard, novel bacteriocin compound secreting capability of LAB isolated from Tulum Cheese in Turkey was demonstrated. The synthesized bacteriocin was purified by ammonium sulphate precipitation, dialysis and gel filtration. The molecular weight (≈3.4 kDa) of obtained bacteriocin was confirmed by SDS-PAGE, which revealed single peptide band. Molecular identification of LAB strain isolated from Tulum Cheese was conducted using 16S rDNA gene sequencing as Lactococcus lactis ssp. lactis LL171. The amino acid sequences (KKIDTRTGKTMEKTEKKIELSLKNMKTAT) of the bacteriocin from Lactococcus lactis ssp. lactis LL171 was found unique and novel than reported bacteriocins. Further, the bacteriocin was possessed the thermostable property and active at wide range of pH values from 1 to 11. Thus, bacteriocin reported in this study has the potential applications property as food preservative agent.

  8. Effects of hydrostatic pressure and temperature on the uptake and respiration of amino acids by a facultatively psychrophilic marine bacterium.

    NASA Technical Reports Server (NTRS)

    Paul, K. L.; Morita, R. Y.

    1971-01-01

    Studies of pressure and temperature effects on glutamic acid transport and utilization indicated that hydrostatic pressure and low temperature inhibit glutamate transport more than glutamate respiration. The effects of pressure on transport were reduced at temperatures near the optimum. Similar results were obtained for glycine, phenylalanine, and proline. Pressure effects on the transport systems of all four amino acids were reversible to some degree. Both proline and glutamic acid were able to protect their transport proteins against pressure damage. The data presented indicate that the uptake of amino acids by cells under pressure is inhibited, which is the cause of their inability to grow under pressure.

  9. Fatty acid DSF binds and allosterically activates histidine kinase RpfC of phytopathogenic bacterium Xanthomonas campestris pv. campestris to regulate quorum-sensing and virulence.

    PubMed

    Cai, Zhen; Yuan, Zhi-Hui; Zhang, Huan; Pan, Yue; Wu, Yao; Tian, Xiu-Qi; Wang, Fang-Fang; Wang, Li; Qian, Wei

    2017-04-01

    As well as their importance to nutrition, fatty acids (FA) represent a unique group of quorum sensing chemicals that modulate the behavior of bacterial population in virulence. However, the way in which full-length, membrane-bound receptors biochemically detect FA remains unclear. Here, we provide genetic, enzymological and biophysical evidences to demonstrate that in the phytopathogenic bacterium Xanthomonas campestris pv. campestris, a medium-chain FA diffusible signal factor (DSF) binds directly to the N-terminal, 22 amino acid-length sensor region of a receptor histidine kinase (HK), RpfC. The binding event remarkably activates RpfC autokinase activity by causing an allosteric change associated with the dimerization and histidine phosphotransfer (DHp) and catalytic ATP-binding (CA) domains. Six residues were found essential for sensing DSF, especially those located in the region adjoining to the inner membrane of cells. Disrupting direct DSF-RpfC interaction caused deficiency in bacterial virulence and biofilm development. In addition, two amino acids within the juxtamembrane domain of RpfC, Leu172 and Ala178, are involved in the autoinhibition of the RpfC kinase activity. Replacements of them caused constitutive activation of RpfC-mediated signaling regardless of DSF stimulation. Therefore, our results revealed a biochemical mechanism whereby FA activates bacterial HK in an allosteric manner, which will assist in future studies on the specificity of FA-HK recognition during bacterial virulence regulation and cell-cell communication.

  10. Syntrophus aciditrophicus sp. nov., a new anaerobic bacterium that degrades fatty acids and benzoate in syntrophic association with hydrogen-using microorganisms

    NASA Technical Reports Server (NTRS)

    Jackson, B. E.; Bhupathiraju, V. K.; Tanner, R. S.; Woese, C. R.; McInerney, M. J.

    1999-01-01

    Strain SBT is a new, strictly anaerobic, gram-negative, nonmotile, non-sporeforming, rod-shaped bacterium that degrades benzoate and certain fatty acids in syntrophic association with hydrogen/formate-using microorganisms. Strain SBT produced approximately 3 mol of acetate and 0.6 mol of methane per mol of benzoate in coculture with Methanospirillum hungatei strain JF1. Saturated fatty acids, some unsaturated fatty acids, and methyl esters of butyrate and hexanoate also supported growth of strain SBT in coculture with Desulfovibrio strain G11. Strain SBT grew in pure culture with crotonate, producing acetate, butyrate, caproate, and hydrogen. The molar growth yield was 17 +/- 1 g cell dry mass per mol of crotonate. Strain SBT did not grow with fumarate, iron(III), polysulfide, or oxyanions of sulfur or nitrogen as electron acceptors with benzoate as the electron donor. The DNA base composition of strain SBT was 43.1 mol% G+C. Analysis of the 16 S rRNA gene sequence placed strain SBT in the delta-subdivision of the Proteobacteria, with sulfate-reducing bacteria. Strain SBT was most closely related to members of the genus Syntrophus. The clear phenotypic and genotypic differences between strain SBT and the two described species in the genus Syntrophus justify the formation of a new species, Syntrophus aciditrophicus.

  11. Thermovirga lienii gen. nov., sp. nov., a novel moderately thermophilic, anaerobic, amino-acid-degrading bacterium isolated from a North Sea oil well.

    PubMed

    Dahle, Håkon; Birkeland, Nils-Kåre

    2006-07-01

    A novel anaerobic, moderately thermophilic bacterium, strain Cas60314(T), was isolated from hot oil-well production water obtained from an oil reservoir in the North Sea. The cells were Gram-negative, motile, straight rods. The salinity and pH growth optima were 2.0-3.0 % NaCl and 6.5-7.0, respectively. The optimum temperature was 58 degrees C. Strain Cas60314(T) had a fermentative type of metabolism and utilized proteinous substrates, some single amino acids and a limited number of organic acids, but not sugars, fatty acids or alcohols. Cystine and elemental sulfur were reduced to sulfide. The G+C content of the DNA was 46.6 mol%. On the basis of phenotypic and phylogenetic features, it is proposed that this isolate represents a novel genus and species with the name Thermovirga lienii gen. nov., sp. nov. within the family Syntrophomonadaceae. The proposed type strain is strain Cas60314(T) (=DSM 17291(T)=ATTC BAA-1197(T)).

  12. Syntrophus aciditrophicus sp. nov., a new anaerobic bacterium that degrades fatty acids and benzoate in syntrophic association with hydrogen-using microorganisms.

    PubMed

    Jackson, B E; Bhupathiraju, V K; Tanner, R S; Woese, C R; McInerney, M J

    1999-01-01

    Strain SBT is a new, strictly anaerobic, gram-negative, nonmotile, non-sporeforming, rod-shaped bacterium that degrades benzoate and certain fatty acids in syntrophic association with hydrogen/formate-using microorganisms. Strain SBT produced approximately 3 mol of acetate and 0.6 mol of methane per mol of benzoate in coculture with Methanospirillum hungatei strain JF1. Saturated fatty acids, some unsaturated fatty acids, and methyl esters of butyrate and hexanoate also supported growth of strain SBT in coculture with Desulfovibrio strain G11. Strain SBT grew in pure culture with crotonate, producing acetate, butyrate, caproate, and hydrogen. The molar growth yield was 17 +/- 1 g cell dry mass per mol of crotonate. Strain SBT did not grow with fumarate, iron(III), polysulfide, or oxyanions of sulfur or nitrogen as electron acceptors with benzoate as the electron donor. The DNA base composition of strain SBT was 43.1 mol% G+C. Analysis of the 16 S rRNA gene sequence placed strain SBT in the delta-subdivision of the Proteobacteria, with sulfate-reducing bacteria. Strain SBT was most closely related to members of the genus Syntrophus. The clear phenotypic and genotypic differences between strain SBT and the two described species in the genus Syntrophus justify the formation of a new species, Syntrophus aciditrophicus.

  13. Production of poly-3-hydroxyalkanoic acids by a moderately halophilic bacterium, Halomonas marina HMA 103 isolated from solar saltern of Orissa, India.

    PubMed

    Biswas, Amrita; Patra, A; Paul, A K

    2009-06-01

    Halomonas marina HMA 103 (MTCC 8968), the moderately halophilic bacterium isolated and characterized from the solar saltern of Orissa, India, grows optimally at 10% (w/v) NaCl in culture medium and is able to synthesize poly(3-hydroxybutyrate) [P(3HB)] during growth. This study is an attempt to optimize the cultural conditions for efficient production of P(3HB) by H. marina in batch cultivation. Growth of the organism under shake-flask culture using 2% (w/v) glucose resulted in P(3HB) accumulation accounting for more than 59% of cell dry weight after 50 h of incubation. The optimum P(3HB) production was attained with a combined supply of NH4Cl and yeast extract as N-source, 0.01% (w/v) phosphate, 1.5% (w/v) sulphate and 10% (w/v) NaCl. Qualitative and quantitative 1HNMR and FT-IR analysis of cells grown in alkanoic acids (C3-C6) as sole source of carbon and co-substrates revealed synthesis of PHA co-polymers composed of 3-hydroxybutyric acid and 3-hydroxyvaleric acid [P(3HB-co-3HV)]. In two-step cultivation, accumulation of the co-polymer was significantly improved (80% CDW) in glucose medium supplemented with valerate (0.1%, w/v) as co-substrate and the polymer contained 88.1 and 12.8 mol% 3HB and 3HV monomers, respectively.

  14. Fatty acid DSF binds and allosterically activates histidine kinase RpfC of phytopathogenic bacterium Xanthomonas campestris pv. campestris to regulate quorum-sensing and virulence

    PubMed Central

    Zhang, Huan; Pan, Yue; Wu, Yao; Tian, Xiu-Qi; Wang, Fang-Fang; Wang, Li

    2017-01-01

    As well as their importance to nutrition, fatty acids (FA) represent a unique group of quorum sensing chemicals that modulate the behavior of bacterial population in virulence. However, the way in which full-length, membrane-bound receptors biochemically detect FA remains unclear. Here, we provide genetic, enzymological and biophysical evidences to demonstrate that in the phytopathogenic bacterium Xanthomonas campestris pv. campestris, a medium-chain FA diffusible signal factor (DSF) binds directly to the N-terminal, 22 amino acid-length sensor region of a receptor histidine kinase (HK), RpfC. The binding event remarkably activates RpfC autokinase activity by causing an allosteric change associated with the dimerization and histidine phosphotransfer (DHp) and catalytic ATP-binding (CA) domains. Six residues were found essential for sensing DSF, especially those located in the region adjoining to the inner membrane of cells. Disrupting direct DSF-RpfC interaction caused deficiency in bacterial virulence and biofilm development. In addition, two amino acids within the juxtamembrane domain of RpfC, Leu172 and Ala178, are involved in the autoinhibition of the RpfC kinase activity. Replacements of them caused constitutive activation of RpfC-mediated signaling regardless of DSF stimulation. Therefore, our results revealed a biochemical mechanism whereby FA activates bacterial HK in an allosteric manner, which will assist in future studies on the specificity of FA-HK recognition during bacterial virulence regulation and cell-cell communication. PMID:28369120

  15. Fructose metabolism of the purple non-sulfur bacterium Rhodospirillum rubrum: effect of carbon dioxide on growth, and production of bacteriochlorophyll and organic acids.

    PubMed

    Rudolf, Christiane; Grammel, Hartmut

    2012-04-05

    During fermentative metabolism, carbon dioxide fixation plays a key role in many bacteria regarding growth and production of organic acids. The present contribution, dealing with the facultative photosynthetic bacterium Rhodospirillum rubrum, reveals not only the strong influence of ambient carbon dioxide on the fermentative break-down of fructose but also a high impact on aerobic growth with fructose as sole carbon source. Both growth rates and biomass yield increased with increasing carbon dioxide supply in chemoheterotrophic aerobic cultures. Furthermore, intracellular metabolite concentration measurements showed almost negligible concentrations of the tricarboxylic acid cycle intermediates succinate, fumarate and malate under aerobic growth, in contrast to several metabolites of the glycolysis. In addition, we present a dual phase fed-batch process, where an aerobic growth phase is followed by an anaerobic production phase. The biosynthesis of bacteriochlorophyll and the secretion of organic acids were both affected by the carbon dioxide supply, the pH value and by the cell density at the time of switching from aerobic to anaerobic conditions. The formation of pigmented photosynthetic membranes and the amount of bacteriochlorophyll were inversely correlated to the secretion of succinate. Accounting the high biotechnological potential of R. rubrum, optimization of carbon dioxide supply is important because of the favored application of fructose-containing fermentable feedstock solutions in bio-industrial processes. Copyright © 2012 Elsevier Inc. All rights reserved.

  16. Syntrophus aciditrophicus sp. nov., a new anaerobic bacterium that degrades fatty acids and benzoate in syntrophic association with hydrogen-using microorganisms

    NASA Technical Reports Server (NTRS)

    Jackson, B. E.; Bhupathiraju, V. K.; Tanner, R. S.; Woese, C. R.; McInerney, M. J.

    1999-01-01

    Strain SBT is a new, strictly anaerobic, gram-negative, nonmotile, non-sporeforming, rod-shaped bacterium that degrades benzoate and certain fatty acids in syntrophic association with hydrogen/formate-using microorganisms. Strain SBT produced approximately 3 mol of acetate and 0.6 mol of methane per mol of benzoate in coculture with Methanospirillum hungatei strain JF1. Saturated fatty acids, some unsaturated fatty acids, and methyl esters of butyrate and hexanoate also supported growth of strain SBT in coculture with Desulfovibrio strain G11. Strain SBT grew in pure culture with crotonate, producing acetate, butyrate, caproate, and hydrogen. The molar growth yield was 17 +/- 1 g cell dry mass per mol of crotonate. Strain SBT did not grow with fumarate, iron(III), polysulfide, or oxyanions of sulfur or nitrogen as electron acceptors with benzoate as the electron donor. The DNA base composition of strain SBT was 43.1 mol% G+C. Analysis of the 16 S rRNA gene sequence placed strain SBT in the delta-subdivision of the Proteobacteria, with sulfate-reducing bacteria. Strain SBT was most closely related to members of the genus Syntrophus. The clear phenotypic and genotypic differences between strain SBT and the two described species in the genus Syntrophus justify the formation of a new species, Syntrophus aciditrophicus.

  17. Growth and gas formation by Lactobacillus wasatchensis, a novel obligatory heterofermentative nonstarter lactic acid bacterium, in Cheddar-style cheese made using a Streptococcus thermophilus starter.

    PubMed

    Ortakci, Fatih; Broadbent, Jeffery R; Oberg, Craig J; McMahon, Donald J

    2015-11-01

    A novel slow-growing, obligatory heterofermentative, nonstarter lactic acid bacterium (NSLAB), Lactobacillus wasatchensis WDC04, was studied for growth and gas production in Cheddar-style cheese made using Streptococcus thermophilus as the starter culture. Cheesemaking trials were conducted using S. thermophilus alone or in combination with Lb. wasatchensis deliberately added to cheese milk at a level of ~10(4) cfu/mL. Resulting cheeses were ripened at 6 or 12°C. At d 1, starter streptococcal numbers were similar in both cheeses (~10(9) cfu/g) and fast-growing NSLAB lactobacilli counts were below detectable levels (<10(2) cfu/g). As expected, Lactobacillus wasatchensis counts were 3×10(5) cfu/g in cheeses inoculated with this bacterium and below enumeration limits in the control cheese. Starter streptococci decreased over time at both storage temperatures but declined more rapidly at 12°C, especially in cheese also containing Lb. wasatchensis. Populations of fast-growing NSLAB and the slow-growing Lb. wasatchensis reached 5×10(7) and 2×10(8) cfu/g, respectively, after 16 wk of storage at 12°C. Growth of NSLAB coincided with a reduction in galactose concentration in the cheese from 0.6 to 0.1%. Levels of galactose at 6°C had similar decrease. Gas formation and textural defects were only observed in cheese with added Lb. wasatchensis ripened at 12°C. Use of S. thermophilus as starter culture resulted in galactose accumulation that Lb. wasatchensis can use to produce CO2, which contributes to late gas blowing in Cheddar-style cheeses, especially when the cheese is ripened at elevated temperature.

  18. Lysinibacillus endophyticus sp. nov., an indole-3-acetic acid producing endophytic bacterium isolated from corn root (Zea mays cv. Xinken-5).

    PubMed

    Yu, Jiang; Guan, Xuejiao; Liu, Chongxi; Xiang, Wensheng; Yu, Zhenhua; Liu, Xiaobing; Wang, Guanghua

    2016-10-01

    A Gram-positive, aerobic, motile, rod-shaped bacterium, designated strain C9(T), was isolated from surface sterilised corn roots (Zea mays cv. Xinken-5) and found to be able to produce indole-3-acetic acid. A polyphasic taxonomic study was carried out to determine the status of strain C9(T). The major cellular fatty acids were found to contain iso-C15:0, anteiso-C15:0 and anteiso-C17:0, and the only menaquinone was identified as MK-7. The polar lipid profile was found to contain diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, two unidentified phospholipids and an unidentified lipid. The cell wall peptidoglycan was found to be of the A4α L-Lys-D-Asp type and the whole cell sugar was found to be glucose. Phylogenetic analysis based on the 16S rRNA gene sequence showed that strain C9(T) belongs to the genus Lysinibacillus and is closely related to Lysinibacillus chungkukjangi NBRC 108948(T) (98.1 % similarity) and Lysinibacillus sinduriensis DSM 27595(T) (98.0 %). However, the low levels of DNA-DNA relatedness and some differential phenotypic characteristics allowed the strain to be distinguished from its close relatives. Therefore, it is concluded that strain C9(T) represents a novel species of the genus Lysinibacillus, for which the name Lysinibacillus endophyticus sp. nov. is proposed. The type strain is C9(T) (=DSM 100506(T) = CGMCC 1.15291(T)).

  19. Favourable effects of eicosapentaenoic acid on the late step of the cell division in a piezophilic bacterium, Shewanella violacea DSS12, at high-hydrostatic pressures.

    PubMed

    Kawamoto, Jun; Sato, Takako; Nakasone, Kaoru; Kato, Chiaki; Mihara, Hisaaki; Esaki, Nobuyoshi; Kurihara, Tatsuo

    2011-08-01

    Shewanella violacea DSS12, a deep-sea bacterium, produces eicosapentaenoic acid (EPA) as a component of membrane phospholipids. Although various isolates from the deep sea, such as Photobacterium profundum SS9, Colwellia psychrerythraea 34H and various Shewanella strains, produce EPA- or docosahexaenoic acid-containing phospholipids, the physiological role of these polyunsaturated fatty acids remains unclear. In this article, we illustrate the physiological importance of EPA for high-pressure adaptation in strain DSS12 with the help of an EPA-deficient mutant (DSS12(pfaA)). DSS12(pfaA) showed significant growth retardation at 30 MPa, but not at 0.1 MPa. We also found that DSS12(pfaA) grown at 30 MPa forms filamentous cells. When an EPA-containing phospholipid (sn-1-oleoly-sn-2-eicosapentaenoyl phosphatidylethanolamine) was supplemented, the growth retardation and the morphological defect of DSS12(pfaA) were suppressed, indicating that the externally added EPA-containing phospholipid compensated for the loss of endogenous EPA. In contrast, the addition of an oleic acid-containing phospholipid (sn-1,2-dioleoyl phosphatidylethanolamine) did not affect the growth and the morphology of the cells. Immunofluorescent microscopic analysis with anti-FtsZ antibody revealed a number of Z-rings and separated nucleoids in DSS12(pfaA) grown at 30 MPa. These results demonstrate the physiological importance of EPA for the later step of Z-ring formation of S. violacea DSS12 under high-pressure conditions.

  20. Nucleotide and deduced amino acid sequences of a subtilisin-like serine protease from a deep-sea bacterium, Alkalimonas collagenimarina AC40(T).

    PubMed

    Kurata, Atsushi; Uchimura, Kohsuke; Shimamura, Shigeru; Kobayashi, Tohru; Horikoshi, Koki

    2007-11-01

    The acpI gene encoding an alkaline protease (AcpI) from a deep-sea bacterium, Alkalimonas collagenimarina AC40(T), was shotgun-cloned and sequenced. It had a 1,617-bp open reading frame encoding a protein of 538 amino acids. Based on analysis of the deduced amino acid sequence, AcpI is a subtilisin-like serine protease belonging to subtilase family A. It consists of a prepropeptide, a catalytic domain, and a prepeptidase C-terminal domain like other serine proteases from the genera Pseudomonas, Shewanella, Alteromonas, and Xanthomonas. Heterologous expression of the acpI gene in Escherichia coli cells yielded a 28-kDa recombinant AcpI (rAcpI), suggesting that both the prepropeptide and prepeptidase C-terminal domains were cleaved off to give the mature form. Analysis of N-terminal and C-terminal amino acid sequences of purified rAcpI showed that the mature enzyme would be composed of 273 amino acids. The optimal pH and temperature for the caseinolytic activity of the purified rAcpI were 9.0-9.5 and 45 degrees C in 100 mM glycine-NaOH buffer. Calcium ions slightly enhanced the enzyme activity and stability. The enzyme favorably hydrolyzed gelatin, collagen, and casein. AcpI from A. collagenimarina AC40(T) was also purified from culture broth, and its molecular mass was around 28 kDa, indicating that the cleavage manner of the enzyme is similar to that in E. coli cells.

  1. The Ll.LtrB intron from Lactococcus lactis excises as circles in vivo: insights into the group II intron circularization pathway.

    PubMed

    Monat, Caroline; Quiroga, Cecilia; Laroche-Johnston, Felix; Cousineau, Benoit

    2015-07-01

    Group II introns are large ribozymes that require the assistance of intron-encoded or free-standing maturases to splice from their pre-mRNAs in vivo. They mainly splice through the classical branching pathway, being released as RNA lariats. However, group II introns can also splice through secondary pathways like hydrolysis and circularization leading to the release of linear and circular introns, respectively. Here, we assessed in vivo splicing of various constructs of the Ll.LtrB group II intron from the Gram-positive bacterium Lactococcus lactis. The study of excised intron junctions revealed, in addition to branched intron lariats, the presence of perfect end-to-end intron circles and alternatively circularized introns. Removal of the branch point A residue prevented Ll.LtrB excision through the branching pathway but did not hinder intron circle formation. Complete intron RNA circles were found associated with the intron-encoded protein LtrA forming nevertheless inactive RNPs. Traces of double-stranded head-to-tail intron DNA junctions were also detected in L. lactis RNA and nucleic acid extracts. Some intron circles and alternatively circularized introns harbored variable number of non-encoded nucleotides at their splice junction. The presence of mRNA fragments at the splice junction of some intron RNA circles provides insights into the group II intron circularization pathway in bacteria.

  2. Development of a new DNA vaccine based on mycobacterial ESAT-6 antigen delivered by recombinant invasive Lactococcus lactis FnBPA+.

    PubMed

    Pereira, Vanessa Bastos; Saraiva, Tessália Diniz Luerce; Souza, Bianca Mendes; Zurita-Turk, Meritxell; Azevedo, Marcela Santiago Pacheco; De Castro, Camila Prósperi; Mancha-Agresti, Pamela; Dos Santos, Janete Soares Coelho; Santos, Ana Cristina Gomes; Faria, Ana Maria Caetano; Leclercq, Sophie; Azevedo, Vasco; Miyoshi, Anderson

    2015-02-01

    The use of the food-grade bacterium Lactococcus lactis as a vehicle for the oral delivery of DNA vaccine plasmids constitutes a promising strategy for vaccination. The delivery of DNA plasmids into eukaryotic cells is of critical importance for subsequent DNA expression and effectiveness of the vaccine. In this context, the use of the recombinant invasive L. lactis FnBPA+ (fibronectin-binding protein A) strain for the oral delivery of the eukaryotic expression vector vaccination using lactic acid bacteria (pValac), coding for the 6-kDa early secreted antigenic target (ESAT-6) gene of Mycobacterium tuberculosis, could represent a new DNA vaccine strategy against tuberculosis. To this end, the ESAT-6 sequence was cloned into the pValac vector; the L. lactis fibronectin-binding protein A (FnBPA)+ (pValac:ESAT-6) strain was obtained, and its immunological profile was checked in BALB/c mice. This strain was able to significantly increase interferon gamma (IFN-γ) production in spleen cells, showing a systemic T helper 1 (Th1) cell response. The mice also showed a significant increase in specific secretory immunoglobulin A (sIgA) production in colon tissue and fecal extracts. Thus, this is the first time that L. lactis has been used to deliver a plasmid DNA harboring a gene that encodes an antigen against tuberculosis through mucous membranes.

  3. Fatty acids in bacterium Dietzia sp. grown on simple and complex hydrocarbons determined as FAME by GC-MS.

    PubMed

    Hvidsten, Ina; Mjøs, Svein Are; Bødtker, Gunhild; Barth, Tanja

    2015-09-01

    The influence of growth substrates on the fatty acids produced by Dietzia sp. A14101 has been studied to investigate how qualitative and semi-quantitative information on fatty acids correlates with the ability of this strain to access and utilize a wide range of water-immiscible HC-substrates by modifying the FA content and thus also the properties of the cellular membrane. After incubation on different substrates and media, the profiles of fatty acids (FA) were analyzed by gas chromatography and mass spectrometry (GC-MS). The equivalent chain length (ECL) index calibration system was employed to identify FA. The effect of each substrate on the cell surface charge and on the hydrophobicity of the cellular membrane was also investigated. The results indicate that the variation of the content of saturated fatty acids (SAT-FA) versus mono-unsaturated fatty acids (MUFA) was found to be the most pronounced while branched FA exhibited much less variation in spite of different substrate regimes. The regulation of the ratio of SAT-FA and MUFA seems to be coupled with the regulation of the charge and hydrophobicity of the outer cellular surface. The exposure to a water immiscible substrate led to the development of the negative cellular surface charge, production of carotenoid-type pigments and increased hydrophobicity of the cellular surface. The specific aspects of the adaptation mechanism could have implications for bioremediation and/or (M)EOR applications.

  4. Immunization against Leishmania major Infection Using LACK- and IL-12-Expressing Lactococcus lactis Induces Delay in Footpad Swelling

    PubMed Central

    Hugentobler, Felix; Yam, Karen K.; Gillard, Joshua; Mahbuba, Raya; Olivier, Martin; Cousineau, Benoit

    2012-01-01

    Background Leishmania is a mammalian parasite affecting over 12 million individuals worldwide. Current treatments are expensive, cause severe side effects, and emerging drug resistance has been reported. Vaccination is the most cost-effective means to control infectious disease but currently there is no vaccine available against Leishmaniasis. Lactococcus lactis is a non-pathogenic, non-colonizing Gram-positive lactic acid bacterium commonly used in the dairy industry. Recently, L. lactis was used to express biologically active molecules including vaccine antigens and cytokines. Methodology/Principal findings We report the generation of L. lactis strains expressing the protective Leishmania antigen, LACK, in the cytoplasm, secreted or anchored to the bacterial cell wall. L. lactis was also engineered to secrete biologically active single chain mouse IL-12. Subcutaneous immunization with live L. lactis expressing LACK anchored to the cell wall and L. lactis secreting IL-12 significantly delayed footpad swelling in Leishmania major infected BALB/c mice. The delay in footpad swelling correlated with a significant reduction of parasite burden in immunized animals compared to control groups. Immunization with these two L. lactis strains induced antigen-specific multifunctional TH1 CD4+ and CD8+ T cells and a systemic LACK-specific TH1 immune response. Further, protection in immunized animals correlated with a Leishmania-specific TH1 immune response post-challenge. L. lactis secreting mouse IL-12 was essential for directing immune responses to LACK towards a protective TH1 response. Conclusions/Significance This report demonstrates the use of L. lactis as a live vaccine against L. major infection in BALB/c mice. The strains generated in this study provide the basis for the development of an inexpensive and safe vaccine against the human parasite Leishmania. PMID:22348031

  5. Expression of a hepatitis A virus antigen in Lactococcus lactis and Escherichia coli and evaluation of its immunogenicity.

    PubMed

    Berlec, Aleš; Malovrh, Tadej; Zadravec, Petra; Steyer, Andrej; Ravnikar, Matjaž; Sabotič, Jerica; Poljšak-Prijatelj, Mateja; Štrukelj, Borut

    2013-05-01

    An epidemic shift in Hepatitis A virus (HAV) infection has been observed in recent years in rapidly developing countries, with increasing numbers of severe adult cases which has led to renewed interest in vaccination. Our approach in vaccine development uses recombinant expression of the highly immunogenic HAV antigen VP1-P2a in food-grade lactic acid bacterium Lactococcus lactis and in Escherichia coli. We used genetic constructs that enable nisin-controlled expression of the antigen in L. lactis in three different forms: (a) intracellularly, (b) on the bacterial surface and (c) on the bacterial surface fused with the fragment of the E. coli flagellin molecule that can act as a molecular adjuvant. Expression of the two surface forms of the antigen was achieved in L. lactis, and the resulting antigen-displaying bacteria were administered orally to mice. Half the animals in each of the two groups developed specific IgGs, with titers increasing over time and reaching 1:422 without flagellin and 1:320 with flagellin. A much higher titer 1:25,803 was observed with the parenterally administered antigen, which was purified from E. coli. With the latter, a significant mucosal IgA response was also observed. Despite significant titers, the IgGs elicited with oral or parenteral administration could not prevent HAV from infecting cells in a virus neutralization assay, suggesting that the antibodies cannot recognize viral surface epitopes. Nevertheless, orally administered HAV antigen expressed in L. lactis elicited significant systemic humoral immune response showing the feasibility for development of effective HAV vaccine for mucosal delivery.

  6. Biological degradation of 4-chlorobenzoic acid by a PCB-metabolizing bacterium through a pathway not involving (chloro)catechol.

    PubMed

    Adebusoye, Sunday A

    2017-02-01

    Cupriavidus sp. strain SK-3, previously isolated on polychlorinated biphenyl mixtures, was found to aerobically utilize a wide spectrum of substituted aromatic compounds including 4-fluoro-, 4-chloro- and 4-bromobenzoic acids as a sole carbon and energy source. Other chlorobenzoic acid (CBA) congeners such as 2-, 3-, 2,3-, 2,5-, 3,4- and 3,5-CBA were all rapidly transformed to respective chlorocatechols (CCs). Under aerobic conditions, strain SK-3 grew readily on 4-CBA to a maximum concentration of 5 mM above which growth became impaired and yielded no biomass. Growth lagged significantly at concentrations above 3 mM, however chloride elimination was stoichiometric and generally mirrored growth and substrate consumption in all incubations. Experiments with resting cells, cell-free extracts and analysis of metabolite pools suggest that 4-CBA was metabolized in a reaction exclusively involving an initial hydrolytic dehalogenation yielding 4-hydroxybenzoic acid, which was then hydroxylated to protocatechuic acid (PCA) and subsequently metabolized via the β-ketoadipate pathway. When strain SK-3 was grown on 4-CBA, there was gratuitous induction of the catechol-1,2-dioxygenase and gentisate-1,2-dioxygenase pathways, even if both were not involved in the metabolism of the acid. While activities of the modified ortho- and meta-cleavage pathways were not detectable in all extracts, activity of PCA-3,4-dioxygenase was over ten-times higher than those of catechol-1,2- and gentisate-1,2-dioxygenases. Therefore, the only reason other congeners were not utilized for growth was the accumulation of CCs, suggesting a narrow spectrum of the activity of enzymes downstream of benzoate-1,2-dioxygenase, which exhibited affinity for a number of substituted analogs, and that the metabolic bottlenecks are either CCs or catabolites of the modified ortho-cleavage metabolic route.

  7. Diversity of the Lactic Acid Bacterium and Yeast Microbiota in the Switch from Firm- to Liquid-Sourdough Fermentation

    PubMed Central

    Di Cagno, Raffaella; Pontonio, Erica; Buchin, Solange; De Angelis, Maria; Lattanzi, Anna; Valerio, Francesca; Calasso, Maria

    2014-01-01

    Four traditional type I sourdoughs were comparatively propagated (28 days) under firm (dough yield, 160) and liquid (dough yield, 280) conditions to mimic the alternative technology options frequently used for making baked goods. After 28 days of propagation, liquid sourdoughs had the lowest pH and total titratable acidity (TTA), the lowest concentrations of lactic and acetic acids and free amino acids, and the most stable density of presumptive lactic acid bacteria. The cell density of yeasts was the highest in liquid sourdoughs. Liquid sourdoughs showed simplified microbial diversity and harbored a low number of strains, which were persistent. Lactobacillus plantarum dominated firm sourdoughs over time. Leuconostoc lactis and Lactobacillus brevis dominated only some firm sourdoughs, and Lactobacillus sanfranciscensis persisted for some time only in some firm sourdoughs. Leuconostoc citreum persisted in all firm and liquid sourdoughs, and it was the only species detected in liquid sourdoughs at all times; it was flanked by Leuconostoc mesenteroides in some sourdoughs. Saccharomyces cerevisiae, Candida humilis, Saccharomyces servazzii, Saccharomyces bayanus-Kazachstania sp., and Torulaspora delbrueckii were variously identified in firm and liquid sourdoughs. A total of 197 volatile components were identified through purge and trap–/solid-phase microextraction–gas chromatography-mass spectrometry (PT–/SPME–GC-MS). Aldehydes, several alcohols, and some esters were at the highest levels in liquid sourdoughs. Firm sourdoughs mainly contained ethyl acetate, acetic acid, some sulfur compounds, and terpenes. The use of liquid fermentation would change the main microbial and biochemical features of traditional baked goods, which have been manufactured under firm conditions for a long time. PMID:24632249

  8. Diversity of the lactic acid bacterium and yeast microbiota in the switch from firm- to liquid-sourdough fermentation.

    PubMed

    Di Cagno, Raffaella; Pontonio, Erica; Buchin, Solange; De Angelis, Maria; Lattanzi, Anna; Valerio, Francesca; Gobbetti, Marco; Calasso, Maria

    2014-05-01

    Four traditional type I sourdoughs were comparatively propagated (28 days) under firm (dough yield, 160) and liquid (dough yield, 280) conditions to mimic the alternative technology options frequently used for making baked goods. After 28 days of propagation, liquid sourdoughs had the lowest pH and total titratable acidity (TTA), the lowest concentrations of lactic and acetic acids and free amino acids, and the most stable density of presumptive lactic acid bacteria. The cell density of yeasts was the highest in liquid sourdoughs. Liquid sourdoughs showed simplified microbial diversity and harbored a low number of strains, which were persistent. Lactobacillus plantarum dominated firm sourdoughs over time. Leuconostoc lactis and Lactobacillus brevis dominated only some firm sourdoughs, and Lactobacillus sanfranciscensis persisted for some time only in some firm sourdoughs. Leuconostoc citreum persisted in all firm and liquid sourdoughs, and it was the only species detected in liquid sourdoughs at all times; it was flanked by Leuconostoc mesenteroides in some sourdoughs. Saccharomyces cerevisiae, Candida humilis, Saccharomyces servazzii, Saccharomyces bayanus-Kazachstania sp., and Torulaspora delbrueckii were variously identified in firm and liquid sourdoughs. A total of 197 volatile components were identified through purge and trap-/solid-phase microextraction-gas chromatography-mass spectrometry (PT-/SPME-GC-MS). Aldehydes, several alcohols, and some esters were at the highest levels in liquid sourdoughs. Firm sourdoughs mainly contained ethyl acetate, acetic acid, some sulfur compounds, and terpenes. The use of liquid fermentation would change the main microbial and biochemical features of traditional baked goods, which have been manufactured under firm conditions for a long time.

  9. Specificity of Milk Peptide Utilization by Lactococcus lactis

    PubMed Central

    Juillard, Vincent; Guillot, Alain; Le Bars, Dominique; Gripon, Jean-Claude

    1998-01-01

    To study the substrate specificity of the oligopeptide transport system of Lactococcus lactis for its natural substrates, the growth of L. lactis MG1363 was studied in a chemically defined medium containing milk peptides or a tryptic digest of αs2-casein as the source of amino acids. Peptides were separated into acidic, neutral, and basic pools by solid-phase extraction or by cation-exchange liquid chromatography. Their ability to sustain growth and the time course of their utilization demonstrated the preferential use of hydrophobic basic peptides with molecular masses ranging between 600 and 1,100 Da by L. lactis MG1363 and the inability to use large, acidic peptides. These peptide utilization preferences reflect the substrate specificity of the oligopeptide transport system of the strain, since no significant cell lysis was inferred. Considering the free amino acid content of milk and these findings on peptide utilization, it was demonstrated that the cessation of growth of L. lactis MG1363 in milk was due to deprivation of leucine and methionine. PMID:9546157

  10. The Antisense RNA Approach: a New Application for In Vivo Investigation of the Stress Response of Oenococcus oeni, a Wine-Associated Lactic Acid Bacterium

    PubMed Central

    Darsonval, Maud; Msadek, Tarek; Alexandre, Hervé

    2015-01-01

    Oenococcus oeni is a wine-associated lactic acid bacterium mostly responsible for malolactic fermentation in wine. In wine, O. oeni grows in an environment hostile to bacterial growth (low pH, low temperature, and ethanol) that induces stress response mechanisms. To survive, O. oeni is known to set up transitional stress response mechanisms through the synthesis of heat stress proteins (HSPs) encoded by the hsp genes, notably a unique small HSP named Lo18. Despite the availability of the genome sequence, characterization of O. oeni genes is limited, and little is known about the in vivo role of Lo18. Due to the lack of genetic tools for O. oeni, an efficient expression vector in O. oeni is still lacking, and deletion or inactivation of the hsp18 gene is not presently practicable. As an alternative approach, with the goal of understanding the biological function of the O. oeni hsp18 gene in vivo, we have developed an expression vector to produce antisense RNA targeting of hsp18 mRNA. Recombinant strains were exposed to multiple stresses inducing hsp18 gene expression: heat shock and acid shock. We showed that antisense attenuation of hsp18 affects O. oeni survival under stress conditions. These results confirm the involvement of Lo18 in heat and acid tolerance of O. oeni. Results of anisotropy experiments also confirm a membrane-protective role for Lo18, as previous observations had already suggested. This study describes a new, efficient tool to demonstrate the use of antisense technology for modulating gene expression in O. oeni. PMID:26452552

  11. The amino acid sequence of the zinc-requiring beta-lactamase II from the bacterium Bacillus cereus 569.

    PubMed

    Ambler, R P; Daniel, M; Fleming, J; Hermoso, J M; Pang, C; Waley, S G

    1985-09-23

    The amino acid sequence of the zinc-requiring beta-lactamase II from Bacillus cereus strain 569 has been determined. It consists of a single polypeptide chain of 227 residues. It is the only example so far fully characterized of a class B beta-lactamase, and is structurally and mechanistically distinct from both the widely distributed class A beta-lactamases (such as the Escherichia coli RTEM enzyme) and from the chromosomally encoded class C enzymes from Gram-negative bacteria.

  12. Aminiphilus circumscriptus gen. nov., sp. nov., an anaerobic amino-acid-degrading bacterium from an upflow anaerobic sludge reactor.

    PubMed

    Díaz, C; Baena, S; Fardeau, M-L; Patel, B K C

    2007-08-01

    Strain ILE-2(T) was isolated from an upflow anaerobic sludge bed reactor treating brewery wastewater. The motile, non-sporulating, slightly curved cells (2-4 x 0.1 microm) stained Gram-negative and grew optimally at 42 degrees C and pH 7.1 with 0.5 % NaCl. The strain required yeast extract for growth and fermented Casamino acids, peptone, isoleucine, arginine, lysine, alanine, valine, glutamate, histidine, glutamine, methionine, malate, fumarate, glycerol and pyruvate to acetate, propionate and minor amounts of branched-chain fatty acids. Carbohydrates, formate, acetate, propionate, butyrate, isovalerate, methanol, ethanol, 1-propanol, butanol, lactate, succinate, starch, casein, gelatin, xylan and a number of other amino acids were not utilized. The DNA G+C content of strain ILE-2(T) was 52.7 mol%. 16S rRNA gene sequence analysis revealed that ILE-2(T) was distantly related to members of the genera Aminobacterium (83 % similarity) and Aminomonas (85 % similarity) in the family Syntrophomonadaceae, order Clostridiales, phylum Firmicutes. On the basis of the results of our polyphasic analysis, strain ILE-2(T) represents a novel species and genus within the family Syntrophomonadaceae, for which the name Aminiphilus circumscriptus gen. nov., sp. nov. is proposed. The type strain of Aminiphilus circumscriptus is ILE-2(T) (=DSM 16581(T) =JCM 14039(T)).

  13. Diversity of Lactic Acid Bacteria Associated with Fish and the Fish Farm Environment, Established by Amplified rRNA Gene Restriction Analysis▿

    PubMed Central

    Michel, Christian; Pelletier, Claire; Boussaha, Mekki; Douet, Diane-Gaëlle; Lautraite, Armand; Tailliez, Patrick

    2007-01-01

    Lactic acid bacteria have become a major source of concern for aquaculture in recent decades. In addition to true pathogenic species of worldwide significance, such as Streptococcus iniae and Lactococcus garvieae, several species have been reported to produce occasional fish mortalities in limited geographic areas, and many unidentifiable or ill-defined isolates are regularly isolated from fish or fish products. To clarify the nature and prevalence of different fish-associated bacteria belonging to the lactic acid bacterium group, a collection of 57 isolates of different origins was studied and compared with a set of 22 type strains, using amplified rRNA gene restriction analysis (ARDRA). Twelve distinct clusters were delineated on the basis of ARDRA profiles and were confirmed by sequencing of sodA and 16S rRNA genes. These clusters included the following: Lactococcus raffinolactis, L. garvieae, Lactococcus l., S. iniae, S. dysgalactiae, S. parauberis, S. agalactiae, Carnobacterium spp., the Enterococcus “faecium” group, a heterogeneous Enterococcus-like cluster comprising indiscernible representatives of Vagococcus fluvialis or the recently recognized V. carniphilus, V. salmoninarum, and Aerococcus spp. Interestingly, the L. lactis and L. raffinolactis clusters appeared to include many commensals of fish, so opportunistic infections caused by these species cannot be disregarded. The significance for fish populations and fish food processing of three or four genetic clusters of uncertain or complex definition, namely, Aerococcus and Enterococcus clusters, should be established more accurately. PMID:17337536

  14. Functional and Morphological Adaptation to Peptidoglycan Precursor Alteration in Lactococcus lactis*

    PubMed Central

    Deghorain, Marie; Fontaine, Laetitia; David, Blandine; Mainardi, Jean-Luc; Courtin, Pascal; Daniel, Richard; Errington, Jeff; Sorokin, Alexei; Bolotin, Alexander; Chapot-Chartier, Marie-Pierre; Hallet, Bernard; Hols, Pascal

    2010-01-01

    Cell wall peptidoglycan assembly is a tightly regulated process requiring the combined action of multienzyme complexes. In this study we provide direct evidence showing that substrate transformations occurring at the different stages of this process play a crucial role in the spatial and temporal coordination of the cell wall synthesis machinery. Peptidoglycan substrate alteration was investigated in the Gram-positive bacterium Lactococcus lactis by substituting the peptidoglycan precursor biosynthesis genes of this bacterium for those of the vancomycin-resistant bacterium Lactobacillus plantarum. A set of L. lactis mutant strains in which the normal d-Ala-ended precursors were partially or totally replaced by d-Lac-ended precursors was generated. Incorporation of the altered precursor into the cell wall induced morphological changes arising from a defect in cell elongation and cell separation. Structural analysis of the muropeptides confirmed that the activity of multiple enzymes involved in peptidoglycan synthesis was altered. Optimization of this altered pathway was necessary to increase the level of vancomycin resistance conferred by the utilization of d-Lac-ended peptidoglycan precursors in the mutant strains. The implications of these findings on the control of bacterial cell morphogenesis and the mechanisms of vancomycin resistance are discussed. PMID:20525686

  15. Inhibitory Effect of Lactococcus lactis HY 449 on Cariogenic Biofilm.

    PubMed

    Kim, Young-Jae; Lee, Sung-Hoon

    2016-11-28

    Dental caries is caused by cariogenic biofilm, an oral biofilm including Streptococcus mutans. Recently, the prevention of dental caries using various probiotics has been attempted. Lactococcus lactis HY 449 is a probiotic bacterium. The aim of this study was to investigate the effect of L. lactis HY 449 on cariogenic biofilm and to analyze its inhibitory mechanisms. Cariogenic biofilm was formed in the presence or absence of L. lactis HY 449 and L. lactis ATCC 19435, and analyzed with a confocal laser microscope. The formation of cariogenic biofilm was reduced in cultures spiked with both L. lactis strains, and L. lactis HY 449 exhibited more inhibitory effects than L. lactis ATCC 19435. In order to analyze and to compare the inhibitory mechanisms, the antibacterial activity of the spent culture medium from both L. lactis strains against S. mutans was investigated, and the expression of glucosyltransferases (gtfs) of S. mutans was then analyzed by real-time RT-PCR. In addition, the sucrose fermentation ability of both L. lactis strains was examined. Both L. lactis strains showed antibacterial activity and inhibited the expression of gtfs, and the difference between both strains did not show. In the case of sucrose-fermenting ability, L. lactis HY 449 fermented sucrose but L. lactis ATCC 19435 did not. L. lactis HY 449 inhibited the uptake of sucrose and the gtfs expression of S. mutans, whereby the development of cariogenic biofilm may be inhibited. In conclusion, L. lactis HY 449 may be a useful probiotic for the prevention of dental caries.

  16. Genes but Not Genomes Reveal Bacterial Domestication of Lactococcus Lactis

    PubMed Central

    Passerini, Delphine; Beltramo, Charlotte; Coddeville, Michele; Quentin, Yves; Ritzenthaler, Paul

    2010-01-01

    Background The population structure and diversity of Lactococcus lactis subsp. lactis, a major industrial bacterium involved in milk fermentation, was determined at both gene and genome level. Seventy-six lactococcal isolates of various origins were studied by different genotyping methods and thirty-six strains displaying unique macrorestriction fingerprints were analyzed by a new multilocus sequence typing (MLST) scheme. This gene-based analysis was compared to genomic characteristics determined by pulsed-field gel electrophoresis (PFGE). Methodology/Principal Findings The MLST analysis revealed that L. lactis subsp. lactis is essentially clonal with infrequent intra- and intergenic recombination; also, despite its taxonomical classification as a subspecies, it displays a genetic diversity as substantial as that within several other bacterial species. Genome-based analysis revealed a genome size variability of 20%, a value typical of bacteria inhabiting different ecological niches, and that suggests a large pan-genome for this subspecies. However, the genomic characteristics (macrorestriction pattern, genome or chromosome size, plasmid content) did not correlate to the MLST-based phylogeny, with strains from the same sequence type (ST) differing by up to 230 kb in genome size. Conclusion/Significance The gene-based phylogeny was not fully consistent with the traditional classification into dairy and non-dairy strains but supported a new classification based on ecological separation between “environmental” strains, the main contributors to the genetic diversity within the subspecies, and “domesticated” strains, subject to recent genetic bottlenecks. Comparison between gene- and genome-based analyses revealed little relationship between core and dispensable genome phylogenies, indicating that clonal diversification and phenotypic variability of the “domesticated” strains essentially arose through substantial genomic flux within the dispensable genome

  17. High genetic diversity among strains of the unindustrialized lactic acid bacterium Carnobacterium maltaromaticum in dairy products as revealed by multilocus sequence typing.

    PubMed

    Rahman, Abdur; Cailliez-Grimal, Catherine; Bontemps, Cyril; Payot, Sophie; Chaillou, Stéphane; Revol-Junelles, Anne-Marie; Borges, Frédéric

    2014-07-01

    Dairy products are colonized with three main classes of lactic acid bacteria (LAB): opportunistic bacteria, traditional starters, and industrial starters. Most of the population structure studies were previously performed with LAB species belonging to these three classes and give interesting knowledge about the population structure of LAB at the stage where they are already industrialized. However, these studies give little information about the population structure of LAB prior their use as an industrial starter. Carnobacterium maltaromaticum is a LAB colonizing diverse environments, including dairy products. Since this bacterium was discovered relatively recently, it is not yet commercialized as an industrial starter, which makes C. maltaromaticum an interesting model for the study of unindustrialized LAB population structure in dairy products. A multilocus sequence typing scheme based on an analysis of fragments of the genes dapE, ddlA, glpQ, ilvE, pyc, pyrE, and leuS was applied to a collection of 47 strains, including 28 strains isolated from dairy products. The scheme allowed detecting 36 sequence types with a discriminatory index of 0.98. The whole population was clustered in four deeply branched lineages, in which the dairy strains were spread. Moreover, the dairy strains could exhibit a high diversity within these lineages, leading to an overall dairy population with a diversity level as high as that of the nondairy population. These results are in agreement with the hypothesis according to which the industrialization of LAB leads to a diversity reduction in dairy products.

  18. Desulfosporosinus acidiphilus sp. nov.: a moderately acidophilic sulfate-reducing bacterium isolated from acid mining drainage sediments : New taxa: Firmicutes (Class Clostridia, Order Clostridiales, Family Peptococcaceae).

    PubMed

    Alazard, Didier; Joseph, Manon; Battaglia-Brunet, Fabienne; Cayol, Jean-Luc; Ollivier, Bernard

    2010-05-01

    An obligately anaerobic, spore-forming, acidophilic sulfate-reducing bacterium, strain SJ4(T), was isolated from an acid mining effluent decantation pond sediment sample (pH around 3.0). Cells were Gram negative, non-motile, curved rods occurring singly. Strain SJ4(T) grew at pH 3.6-5.5 with an optimum at pH 5.2. Strain SJ4(T) utilized H(2), lactate, pyruvate, glycerol, glucose, and fructose as electron donors. Lactate and glucose were weakly used. Sulfate was used as electron acceptors, but not sulfite, elemental sulfur, arsenate (V), and fumarate. The G + C content of genomic DNA was 42.3 mol% (HPLC). 16S rRNA gene sequence analysis indicated that strain SJ4(T) belonged to the genus Desulfosporosinus within the family Peptococcaceae in the phylum Firmicutes. The level of 16S rRNA gene sequence similarity with other Desulfosporosinus species was 94.7-96.2%, D. orientis DSM 765(T) (similarity of 96.2%) and D. auripigmenti DSM 13351(T) (similarity of 95%) being its closest relatives. DNA-DNA relatedness values with D. orientis and D. auripigmenti were 16.5 and 31.8%, respectively. On the basis of phenotypic, phylogenetic, and genetic characteristics, strain SJ4(T) represents a novel species within the genus Desulfosporosinus, for which the name Desulfosporosinus acidiphilus sp. nov. is proposed. The type strain is SJ4(T) (=DSM 22704(T) = JCM 16185(T)).

  19. Identification of a 4-deoxy-L-erythro-5-hexoseulose uronic acid reductase, FlRed, in an alginolytic bacterium Flavobacterium sp. strain UMI-01.

    PubMed

    Inoue, Akira; Nishiyama, Ryuji; Mochizuki, Shogo; Ojima, Takao

    2015-01-16

    In alginate-assimilating bacteria, alginate is depolymerized to unsaturated monosaccharide by the actions of endolytic and exolytic alginate lyases (EC 4.2.2.3 and EC 4.2.2.11). The monosaccharide is non-enzymatically converted to 4-deoxy-L-ery thro-5-hexoseulose uronic acid (DEH), then reduced to 2-keto-3-deoxy-D-gluconate (KDG) by a specific reductase, and metabolized through the Entner-Doudoroff pathway. Recently, the NADPH-dependent reductase A1-R that belongs to short-chain dehydrogenases/reductases (SDR) superfamily was identified as the DEH-reductase in Sphingomonas sp. A1. We have subsequently noticed that an SDR-like enzyme gene, flred, occurred in the genome of an alginolytic bacterium Flavobacterium sp. strain UMI-01. In the present study, we report on the deduced amino-acid sequence of flred and DEH-reducing activity of recombinant FlRed. The deduced amino-acid sequence of flred comprised 254 residues and showed 34% amino-acid identities to that of A1-R from Sphingomonas sp. A1 and 80%-88% to those of SDR-like enzymes from several alginolytic bacteria. Common sequence motifs of SDR-superfamily enzymes, e.g., the catalytic tetrad Asn-Lys-Tyr-Ser and the cofactor-binding sequence Thr-Gly-x-x-x-Gly-x-Gly in Rossmann fold, were completely conserved in FlRed. On the other hand, an Arg residue that determined the NADPH-specificity of Sphingomonas A1-R was replaced by Glu in FlRed. Thus, we investigated cofactor-preference of FlRed using a recombinant enzyme. As a result, the recombinant FlRed (recFlRed) was found to show high specificity to NADH. recFlRed exhibited practically no activity toward variety of aldehyde, ketone, keto ester, keto acid and aldose substrates except for DEH. On the basis of these results, we conclude that FlRed is the NADH-dependent DEH-specific SDR of Flavobacterium sp. strain UMI-01.

  20. Aureispira marina gen. nov., sp. nov., a gliding, arachidonic acid-containing bacterium isolated from the southern coastline of Thailand.

    PubMed

    Hosoya, Shoichi; Arunpairojana, Vullapa; Suwannachart, Chatrudee; Kanjana-Opas, Akkharawit; Yokota, Akira

    2006-12-01

    Three strains of gliding bacteria, 24(T), 62 and 71, isolated from a marine sponge and algae from the southern coastline of Thailand, were studied using a polyphasic approach to clarify their taxonomic positions. A phylogenetic analysis based on 16S rRNA gene sequences showed that the three isolates formed a distinct lineage within the family 'Saprospiraceae' of the phylum Bacteroidetes and were related to members of the genus Saprospira. The G+C contents of the isolates were in the range 38-39 mol%. The major respiratory quinone was MK-7. The predominant cellular fatty acids were 20 : 4omega6c (arachidonic acid), 16 : 0 and iso-17 : 0. On the basis of morphological, physiological and chemotaxonomic characteristics, together with DNA-DNA hybridization data and 16S rRNA gene sequences, the isolates represent a novel species of a novel genus, for which the name Aureispira marina gen. nov., sp. nov. is proposed. The type strain of Aureispira marina is 24(T) (=IAM 15389(T)=TISTR 1719(T)).

  1. Characterization of lactose utilization and β-galactosidase in Lactobacillus brevis KB290, the hetero-fermentative lactic acid bacterium.

    PubMed

    Honda, Hiroyuki; Yajima, Nobuhiro; Saito, Tadao

    2012-12-01

    Unlike dairy lactic acid bacteria, Lactobacillus brevis cannot ferment milk. We characterized the lactose utilization by L. brevis KB290. In a carbohydrate fermentation assay using API 50 CHL, we showed during 7 days L. brevis did not ferment lactose. L. brevis grew to the stationary phase in 2 weeks in MRS broth containing lactose as the carbon source. L. brevis slowly consumed the lactose in the medium. L. brevis hydrolyzed lactose and a lactose analog, o-nitrophenyl-β-D-galactopyranoside (ONPGal). This β-galactosidase activity for ONPGal was not repressed by glucose, galactose, fructose, xylose, or maltose showing the microorganism may not have carbon catabolite repression. We purified the L. brevis β-galactosidase using ammonium sulfate precipitation and several chromatographies. The enzyme's molecular weight is estimated at 72 and 37 kDa using SDS-PAGE analysis. The N-terminal amino acid sequence of the larger protein was 90 % similar to the sequence of the putative β-galactosidase (YP_796339) and the smaller protein was identical to the sequence of the putative β-galactosidase (YP_796338) in L. brevis ATCC367. This suggests the enzyme is a heterodimeric β-galactosidase. The specific activity of the purified enzyme for lactose is 55 U/mg. We speculate inhibition of lactose transport delays the lactose utilization in L. brevis KB290.

  2. Lactococcus lactis BFE920 activates the innate immune system of olive flounder (Paralichthys olivaceus), resulting in protection against Streptococcus iniae infection and enhancing feed efficiency and weight gain in large-scale field studies.

    PubMed

    Kim, Daniel; Beck, Bo Ram; Heo, Saet-Byeol; Kim, Jungjoon; Kim, Hyun Duk; Lee, Sun-Min; Kim, Youngchan; Oh, So Young; Lee, Kyungro; Do, HyungKi; Lee, KwanHee; Holzapfel, Wilhelm H; Song, Seong Kyu

    2013-11-01

    The protective effect of a food-grade lactic acid bacterium Lactococcus lactis BFE920 against disease of olive flounder (Paralichthys olivaceus) cultivated on a large scale was studied. Initially, antimicrobial activity of L. lactis against several fish pathogens was evaluated in vitro; the probiotic showed strong antibacterial activity against Streptococcus iniae, Streptococcus parauberis and Enterococcus viikkiensis, and moderate activity against Lactococcus garviae. When olive flounders were fed for two weeks with experimental diets containing varying concentrations of L. lactis (1 × 10(6), 5 × 10(6), 2.5 × 10(7) and 1.25 × 10(8) CFU/g feed), all the experimental feed groups showed 68-77% survival upon challenge with S. iniae. A field-scale feeding trial with L. lactis dietary supplement was conducted in a local fish farm (n = 12,000) for three months, and disease resistance, innate immune parameters and growth performance were evaluated. The average weight gain and feed efficiency were increased up to 6.8% and 8.5%, respectively. At the end of the feeding trial, the olive flounders were challenged with S. iniae. The L. lactis-fed group was protected from S. iniae challenge with a 66% survival rate. This disease protection is due to the flounder's innate immunity activated by the L. lactis administration: increased lysosomal activities and production of IL-12 and IFN-γ. These data clearly indicated that L. lactis BFE920 may be developed as a functional feed additive for protection against diseases, and for enhancement of feed efficiency and weight gain in olive flounder farming.

  3. Genome-Scale Model of Streptococcus thermophilus LMG18311 for Metabolic Comparison of Lactic Acid Bacteria▿ †

    PubMed Central

    Pastink, Margreet I.; Teusink, Bas; Hols, Pascal; Visser, Sanne; de Vos, Willem M.; Hugenholtz, Jeroen

    2009-01-01

    In this report, we describe the amino acid metabolism and amino acid dependency of the dairy bacterium Streptococcus thermophilus LMG18311 and compare them with those of two other characterized lactic acid bacteria, Lactococcus lactis and Lactobacillus plantarum. Through the construction of a genome-scale metabolic model of S. thermophilus, the metabolic differences between the three bacteria were visualized by direct projection on a metabolic map. The comparative analysis revealed the minimal amino acid auxotrophy (only histidine and methionine or cysteine) of S. thermophilus LMG18311 and the broad variety of volatiles produced from amino acids compared to the other two bacteria. It also revealed the limited number of pyruvate branches, forcing this strain to use the homofermentative metabolism for growth optimization. In addition, some industrially relevant features could be identified in S. thermophilus, such as the unique pathway for acetaldehyde (yogurt flavor) production and the absence of a complete pentose phosphate pathway. PMID:19346354

  4. Probiotic assessment of Enterococcus durans 6HL and Lactococcus lactis 2HL isolated from vaginal microflora.

    PubMed

    Nami, Yousef; Abdullah, Norhafizah; Haghshenas, Babak; Radiah, Dayang; Rosli, Rozita; Khosroushahi, Ahmad Yari

    2014-08-01

    Forty-five lactic acid bacteria (LAB) were isolated from the vaginal specimens of healthy fertile women, and the identities of the bacteria were confirmed by sequencing of their 16S rDNA genes. Among these bacteria, only four isolates were able to resist and survive in low pH, bile salts and simulated in vitro digestion conditions. Lactococcus lactis 2HL, Enterococcus durans 6HL, Lactobacillus acidophilus 36YL and Lactobacillus plantarum 5BL showed the best resistance to these conditions. These strains were evaluated further to assess their ability to adhere to human intestinal Caco-2 cells. Lactococcus lactis 2HL and E. durans 6HL were the most adherent strains. In vitro tests under neutralized pH proved the antimicrobial activity of both strains. Results revealed that the growth of Escherichia coli O26, Staphylococcus aureus and Shigella flexneri was suppressed by both LAB strains. The antibiotic susceptibility tests showed that these strains were sensitive to all nine antibiotics: vancomycin, tetracycline, ampicillin, penicillin, gentamicin, erythromycin, clindamycin, sulfamethoxazole and chloramphenicol. These data suggest that E. durans 6HL and Lactococcus lactis 2HL could be examined further for their useful properties and could be developed as new probiotics.

  5. Deduced amino acid sequence, functional expression, and unique enzymatic properties of the form I and form II ribulose bisphosphate carboxylase/oxygenase from the chemoautotrophic bacterium Thiobacillus denitrificans.

    PubMed Central

    Hernandez, J M; Baker, S H; Lorbach, S C; Shively, J M; Tabita, F R

    1996-01-01

    The cbbL cbbS and cbbM genes of Thiobacillus denitrificans, encoding form I and form II ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO), respectively, were found to complement a RubisCO-negative mutant of Rhodobacter sphaeroides to autotrophic growth. Endogenous T. denitrificans promoters were shown to function in R. sphaeroides, resulting in high levels of cbbL cbbS and cbbM expression in the R. sphaeroides host. This expression system provided high levels of both T. denitrificans enzymes, each of which was highly purified. The deduced amino acid sequence of the form I enzyme indicated that the large subunit was closely homologous to previously sequenced form I RubisCO enzymes from sulfur-oxidizing bacteria. The form I T. denitrificans enzyme possessed a very low substrate specificity factor and did not exhibit fallover, and yet this enzyme showed a poor ability to recover from incubation with ribulose 1,5-bisphosphate. The deduced amino acid sequence of the form II T. denitrificans enzyme resembled those of other form II RubisCO enzymes. The substrate specificity factor was characteristically low, and the lack of fallover and the inhibition by ribulose 1,5-bisphosphate were similar to those of form II RubisCO obtained from nonsulfur purple bacteria. Both form I and form II RubisCO from T. denitrificans possessed high KCO2 values, suggesting that this organism might suffer in environments containing low levels of dissolved CO2. These studies present the initial description of the kinetic properties of form I and form II RubisCO from a chemoautotrophic bacterium that synthesizes both types of enzyme. PMID:8550452

  6. Influence of nitrogen substrates and substrate C:N ratios on the nitrogen isotopic composition of amino acids from the marine bacterium Vibrio harveyi

    NASA Astrophysics Data System (ADS)

    Maki, K.; Ohkouchi, N.; Chikaraishi, Y.; Fukuda, H.; Miyajima, T.; Nagata, T.

    2014-09-01

    Nitrogen (N) isotopic compositions of individual hydrolysable amino acids (δ15NAAs) in N pools have been increasingly used for trophic position assessment and evaluation of sources and transformation processes of organic matter in marine environments. However, there are limited data about variability in δ15NAAs patterns and how this variability influences marine bacteria, an important mediator of trophic transfer and organic matter transformation. We explored whether marine bacterial δ15NAAs profiles change depending on the type and C:N ratio of the substrate. The δ15NAAs profile of a marine bacterium, Vibrio harveyi, was examined using medium containing either glutamate, alanine or ammonium as the N source [substrate C:N ratios (range, 3 to 20) were adjusted with glucose]. The data were interpreted as a reflection of isotope fractionations associated with de novo synthesis of amino acids by bacteria. Principal component analysis (PCA) using the δ15N offset values normalized to glutamate + glutamine δ15N revealed that δ15NAAs profiles differed depending on the N source and C:N ratio of the substrate. High variability in the δ15N offset of alanine and valine largely explained this bacterial δ15NAAs profile variability. PCA was also conducted using bacterial and phytoplankton (cyanobacteria and eukaryotic algae) δ15NAAs profile data reported previously. The results revealed that bacterial δ15NAAs patterns were distinct from those of phytoplankton. Therefore, the δ15NAAs profile is a useful indicator of biochemical responses of bacteria to changes in substrate conditions, serving as a potentially useful method for identifying organic matter sources in marine environments.

  7. Deduced amino acid sequence, functional expression, and unique enzymatic properties of the form I and form II ribulose bisphosphate carboxylase/oxygenase from the chemoautotrophic bacterium Thiobacillus denitrificans.

    PubMed

    Hernandez, J M; Baker, S H; Lorbach, S C; Shively, J M; Tabita, F R

    1996-01-01

    The cbbL cbbS and cbbM genes of Thiobacillus denitrificans, encoding form I and form II ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO), respectively, were found to complement a RubisCO-negative mutant of Rhodobacter sphaeroides to autotrophic growth. Endogenous T. denitrificans promoters were shown to function in R. sphaeroides, resulting in high levels of cbbL cbbS and cbbM expression in the R. sphaeroides host. This expression system provided high levels of both T. denitrificans enzymes, each of which was highly purified. The deduced amino acid sequence of the form I enzyme indicated that the large subunit was closely homologous to previously sequenced form I RubisCO enzymes from sulfur-oxidizing bacteria. The form I T. denitrificans enzyme possessed a very low substrate specificity factor and did not exhibit fallover, and yet this enzyme showed a poor ability to recover from incubation with ribulose 1,5-bisphosphate. The deduced amino acid sequence of the form II T. denitrificans enzyme resembled those of other form II RubisCO enzymes. The substrate specificity factor was characteristically low, and the lack of fallover and the inhibition by ribulose 1,5-bisphosphate were similar to those of form II RubisCO obtained from nonsulfur purple bacteria. Both form I and form II RubisCO from T. denitrificans possessed high KCO2 values, suggesting that this organism might suffer in environments containing low levels of dissolved CO2. These studies present the initial description of the kinetic properties of form I and form II RubisCO from a chemoautotrophic bacterium that synthesizes both types of enzyme.

  8. Marinilactibacillus piezotolerans sp. nov., a novel marine lactic acid bacterium isolated from deep sub-seafloor sediment of the Nankai Trough.

    PubMed

    Toffin, Laurent; Zink, Klaus; Kato, Chiaki; Pignet, Patricia; Bidault, Adeline; Bienvenu, Nadège; Birrien, Jean-Louis; Prieur, Daniel

    2005-01-01

    A piezotolerant, mesophilic, marine lactic acid bacterium (strain LT20T) was isolated from a deep sub-seafloor sediment core collected at Nankai Trough, off the coast of Japan. Cells were Gram-positive, rod-shaped, non-sporulating and non-motile. The NaCl concentration range for growth was 0-120 g l(-1), with the optimum at 10-20 g l(-1). The temperature range for growth at pH 7.0 was 4-50 degrees C, with the optimum at 37-40 degrees C. The optimum pH for growth was 7.0-8.0. The optimum pressure for growth was 0.1 MPa with tolerance up to 30 MPa. The main cellular phospholipids were phosphatidylglycerols (25 %), diphosphatidylglycerols (34 %) and a group of compounds tentatively identified as ammonium-containing phosphatidylserines (32 %); phosphatidylethanolamines (9 %) were minor components. The fatty acid composition was dominated by side chains of 16 : 0, 14 : 0 and 16 : 1. The G+C content of the genomic DNA was 42 mol%. On the basis of 16S rRNA gene sequence analysis and the secondary structure of the V6 region, this organism was found to belong to the genus Marinilactibacillus and was closely related to Marinilactibacillus psychrotolerans M13-2(T) (99 %), Marinilactibacillus sp. strain MJYP.25.24 (99 %) and Alkalibacterium olivapovliticus strain ww2-SN4C (97 %). Despite the high similarity between their 16S rRNA gene sequences (99 %), the DNA-DNA hybridization levels were less than 20 %. On the basis of physiological and genetic characteristics, it is proposed that this organism be classified as a novel species, Marinilactibacillus piezotolerans sp. nov. The type strain is LT20T (=DSM 16108T=JCM 12337T).

  9. Aminobacterium thunnarium sp. nov., a mesophilic, amino acid-degrading bacterium isolated from an anaerobic sludge digester, pertaining to the phylum Synergistetes.

    PubMed

    Hamdi, Olfa; Ben Hania, Wajdi; Postec, Anne; Bouallagui, Hassib; Hamdi, Moktar; Bonin, Patricia; Ollivier, Bernard; Fardeau, Marie-Laure

    2015-02-01

    A new Gram-staining-positive, non-sporulating, mesophilic, amino acid-degrading anaerobic bacterium, designated strain OTA 102(T), was isolated from an anaerobic sequencing batch reactor treating wastewater from cooking tuna. The cells were curved rods (0.6-2.5×0.5 µm) and occurred singly or in pairs. The strain was motile by means of one lateral flagellum. Strain OTA 102(T) grew at temperatures between 30 and 45 °C (optimum 40 °C), between pH 6.0 and 8.4 (optimum pH 7.2) and NaCl concentrations between 1 and 5 % (optimum 2 %, w/v). Strain OTA 102(T) required yeast extract for growth. Serine, threonine, glycine, cysteine, citrate, fumarate, α-ketoglutarate and pyruvate were fermented. When co-cultured with Methanobacterium formicicum as the hydrogen scavenger, strain OTA 102(T) oxidized alanine, valine, leucine, isoleucine, aspartate, tyrosine, methionine, histidine and asparagine. The genomic DNA G+C content of strain OTA 102(T) was 41.7 mol%. The main fatty acid was iso-C15 : 0. Phylogenetic analysis of the 16S rRNA gene sequence indicated that strain OTA 102(T) was related to Aminobacterium colombiense and Aminobacterium mobile (95.5 and 95.2 % similarity, respectively), of the phylum Synergistetes. On the basis of phylogenetic, genetic and physiological characteristics, strain OTA 102(T) is proposed to represent a novel species of the genus Aminobacterium, Aminobacterium thunnarium sp. nov. The type strain is OTA 102(T) ( = DSM 27500(T) = JCM 19320(T)).

  10. Genetic response to bacteriophage infection in Lactococcus lactis reveals a four-strand approach involving induction of membrane stress proteins, D-alanylation of the cell wall, maintenance of proton motive force, and energy conservation.

    PubMed

    Fallico, Vincenzo; Ross, R Paul; Fitzgerald, Gerald F; McAuliffe, Olivia

    2011-11-01

    In this study, whole-genome microarrays were used to gain insights into the global molecular response of Lactococcus lactis subsp. lactis IL1403 at an early stage of infection with the lytic phage c2. The bacterium differentially regulated the expression of 61 genes belonging to 14 functional categories, including cell envelope processes (12 genes), regulatory functions (11 genes), and carbohydrate metabolism (7 genes). The nature of these genes suggests a complex response involving four main mechanisms: (i) induction of membrane stress proteins, (ii) d-alanylation of cell wall lipoteichoic acids (LTAs), (iii) maintenance of the proton motive force (PMF), and (iv) energy conservation. The phage presence is sensed as a membrane stress in L. lactis subsp. lactis IL1403, which activated a cell wall-targeted response probably orchestrated by the concerted action of membrane phage shock protein C-like homologues, the global regulator SpxB, and the two-component system CesSR. The bacterium upregulated genes (ddl and dltABCD) responsible for incorporation of d-alanine esters into LTAs, an event associated with increased resistance to phage attack in Gram-positive bacteria. The expression of genes (yshC, citE, citF) affecting both PMF components was also regulated to restore the physiological PMF, which was disrupted following phage infection. While mobilizing the response to the phage-mediated stress, the bacterium activated an energy-saving program by repressing growth-related functions and switching to anaerobic respiration, probably to sustain the PMF and the overall cell response to phage. To our knowledge, this represents the first detailed description in L. lactis of the molecular mechanisms involved in the host response to the membrane perturbations mediated by phage infection.

  11. Vinegar production from post-distillation slurry deriving from rice shochu production with the addition of caproic acid-producing bacteria consortium and lactic acid bacterium.

    PubMed

    Yuan, Hua-Wei; Tan, Li; Chen, Hao; Sun, Zhao-Yong; Tang, Yue-Qin; Kida, Kenji

    2017-07-22

    To establish a zero emission process, the post-distillation slurry of a new type of rice shochu (NTRS) was used for the production of health promoting vinegar. Since the NTRS post-distillation slurry contained caproic acid and lactic acid, the effect of these two organic acids on acetic acid fermentation was first evaluated. Based on these results, Acetobacter aceti CICC 21684 was selected as a suitable strain for subsequent production of vinegar. At the laboratory scale, acetic acid fermentation of the NTRS post-distillation slurry in batch mode resulted in an acetic acid concentration of 41.9 g/L, with an initial ethanol concentration of 40 g/L, and the acetic acid concentration was improved to 44.5 g/L in fed-batch mode. Compared to the NTRS post-distillation slurry, the vinegar product had higher concentrations of free amino acids and inhibition of angiotensin I converting enzyme activity. By controlling the volumetric oxygen transfer coefficient to be similar to that of the laboratory scale production, 45 g/L of acetic acid was obtained at the pilot scale, using a 75-L fermentor with a working volume of 40 L, indicating that vinegar production can be successfully scaled up. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  12. Insertion sequence elements in Lactococcus garvieae.

    PubMed

    Eraclio, Giovanni; Ricci, Giovanni; Fortina, Maria Grazia

    2015-01-25

    Insertion sequences are the simplest intracellular Mobile Genetic Elements which can occur in very high numbers in prokaryotic genomes, where they play an important evolutionary role by promoting genome plasticity. As such, the studies on the diversity and distribution of insertion sequences in genomes not yet investigated can contribute to improve the knowledge on a bacterial species and to identify new transposable elements. The present work describes the occurrence of insertion sequences in Lactococcus garvieae, an opportunistic emerging zoonotic and human pathogen, also associated with different food matrices. To date, no insertion elements have been described for L. garvieae in the IS element database. The analysis of the twelve published L. garvieae genomes identified 15 distinct insertion sequences that are members of the IS3, IS982, IS6, IS21 and IS256 families, including five new elements. Most of the insertion sequences in L. garvieae show substantial homology to the Lactococcus lactis elements, suggesting the movement of IS between these two species phylogenetically closely related. ISLL6 elements belonging to IS3 family were most abundant, with several copies distributed in 9 of the 12 genomes analyzed. An alignment analysis of two complete genomes carrying multi-copies of this insertion sequence indicates a possible involvement of ISLL6 in chromosomal rearrangement.

  13. Hematite Reduction Buffers Acid Generation and Enhances Nutrient Uptake by a Fermentative Iron Reducing Bacterium, Orenia metallireducens Strain Z6.

    PubMed

    Dong, Yiran; Sanford, Robert A; Chang, Yun-Juan; McInerney, Michael J; Fouke, Bruce W

    2017-01-03

    Fermentative iron-reducing organisms have been identified in a variety of environments. Instead of coupling iron reduction to respiration, they have been consistently observed to use ferric iron minerals as an electron sink for fermentation. In the present study, a fermentative iron reducer, Orenia metallireducens strain Z6, was shown to use iron reduction to enhance fermentation not only by consuming electron equivalents, but also by generating alkalinity that effectively buffers the pH. Fermentation of glucose by this organism in the presence of a ferric oxide mineral, hematite (Fe2O3), resulted in enhanced glucose decomposition compared with fermentation in the absence of an iron source. Parallel evidence (i.e., genomic reconstruction, metabolomics, thermodynamic analyses, and calculation of electron transfer) suggested hematite reduction as a proton-consuming reaction effectively consumed acid produced by fermentation. The buffering effect of hematite was further supported by a greater extent of glucose utilization by strain Z6 in media with increasing buffer capacity. Such maintenance of a stable pH through hematite reduction for enhanced glucose fermentation complements the thermodynamic interpretation of interactions between microbial iron reduction and other biogeochemical processes. This newly discovered feature of iron reducer metabolism also has significant implications for groundwater management and contaminant remediation by providing microbially mediated buffering systems for the associated microbial and/or chemical reactions.

  14. Effects of phenazine-1-carboxylic acid on the biology of the plant-pathogenic bacterium Xanthomonas oryzae pv. oryzae.

    PubMed

    Xu, Shu; Pan, Xiayan; Luo, Jianying; Wu, Jian; Zhou, Zehua; Liang, Xiaoyu; He, Yawen; Zhou, Mingguo

    2015-01-01

    Xanthomonas oryzae pv. oryzae (Xoo) is the casual agent of bacterial blight, which is one of the most serious diseases of rice. The antibiotic phenazine-1-carboxylic acid (PCA), which is primarily produced by Pseudomonas spp., was found and previously reported very effective against Xoo. However, the biological effects of PCA on Xoo remain unclear. In this study, we found that PCA increased the accumulation of reactive oxygen species (ROS) and reduced the activities of catalase (CAT) and superoxide dismutase (SOD) in Xoo. Xoo was more sensitive to H2O2 than Xanthomonas oryzae pv. oryzicola (Xoc), and had a much lower expression of CAT genes. In addition, proteomic analysis indicated that PCA inhibited carbohydrate metabolism and nutrient uptake in Xoo, and analysis of carbon source utilization further confirmed that carbohydrate metabolism in Xoo was repressed by PCA. In conclusion, PCA acted as a redox-cycling agent that disturbed the redox balance in Xoo and reduced CAT and SOD activities, resulting in higher accumulation of ROS, altered carbohydrate metabolism, and lower energy production and nutrient uptake. Moreover, a deficient antioxidant system in Xoo made it very sensitive to PCA.

  15. Specialized adaptation of a lactic acid bacterium to the milk environment: the comparative genomics of Streptococcus thermophilus LMD-9.

    PubMed

    Goh, Yong Jun; Goin, Caitlin; O'Flaherty, Sarah; Altermann, Eric; Hutkins, Robert

    2011-08-30

    involved in amino acid transport and metabolism as well as DNA replication. The genome of S. thermophilus LMD-9 is shaped by its domestication in the dairy environment, with gene features that conferred rapid growth in milk, stress response mechanisms and host defense systems that are relevant to its industrial applications. The presence of a unique exopolysaccharide gene cluster and cell surface protein orthologs commonly associated with probiotic functionality revealed potential probiotic applications of LMD-9.

  16. Specialized adaptation of a lactic acid bacterium to the milk environment: the comparative genomics of Streptococcus thermophilus LMD-9

    PubMed Central

    2011-01-01

    overexpressed genes involved in amino acid transport and metabolism as well as DNA replication. Conclusions The genome of S. thermophilus LMD-9 is shaped by its domestication in the dairy environment, with gene features that conferred rapid growth in milk, stress response mechanisms and host defense systems that are relevant to its industrial applications. The presence of a unique exopolysaccharide gene cluster and cell surface protein orthologs commonly associated with probiotic functionality revealed potential probiotic applications of LMD-9. PMID:21995282

  17. Genome Sequence and Analysis of the Oral Bacterium Fusobacterium nucleatum Strain ATCC 25586

    PubMed Central

    Kapatral, Vinayak; Anderson, Iain; Ivanova, Natalia; Reznik, Gary; Los, Tamara; Lykidis, Athanasios; Bhattacharyya, Anamitra; Bartman, Allen; Gardner, Warren; Grechkin, Galina; Zhu, Lihua; Vasieva, Olga; Chu, Lien; Kogan, Yakov; Chaga, Oleg; Goltsman, Eugene; Bernal, Axel; Larsen, Niels; D'Souza, Mark; Walunas, Theresa; Pusch, Gordon; Haselkorn, Robert; Fonstein, Michael; Kyrpides, Nikos; Overbeek, Ross

    2002-01-01

    We present a complete DNA sequence and metabolic analysis of the dominant oral bacterium Fusobacterium nucleatum. Although not considered a major dental pathogen on its own, this anaerobe facilitates the aggregation and establishment of several other species including the dental pathogens Porphyromonas gingivalis and Bacteroides forsythus. The F. nucleatum strain ATCC 25586 genome was assembled from shotgun sequences and analyzed using the ERGO bioinformatics suite (http://www.integratedgenomics.com). The genome contains 2.17 Mb encoding 2,067 open reading frames, organized on a single circular chromosome with 27% GC content. Despite its taxonomic position among the gram-negative bacteria, several features of its core metabolism are similar to that of gram-positive Clostridium spp., Enterococcus spp., and Lactococcus spp. The genome analysis has revealed several key aspects of the pathways of organic acid, amino acid, carbohydrate, and lipid metabolism. Nine very-high-molecular-weight outer membrane proteins are predicted from the sequence, none of which has been reported in the literature. More than 137 transporters for the uptake of a variety of substrates such as peptides, sugars, metal ions, and cofactors have been identified. Biosynthetic pathways exist for only three amino acids: glutamate, aspartate, and asparagine. The remaining amino acids are imported as such or as di- or oligopeptides that are subsequently degraded in the cytoplasm. A principal source of energy appears to be the fermentation of glutamate to butyrate. Additionally, desulfuration of cysteine and methionine yields ammonia, H2S, methyl mercaptan, and butyrate, which are capable of arresting fibroblast growth, thus preventing wound healing and aiding penetration of the gingival epithelium. The metabolic capabilities of F. nucleatum revealed by its genome are therefore consistent with its specialized niche in the mouth. PMID:11889109

  18. Carboxydothermus pertinax sp. nov., a thermophilic, hydrogenogenic, Fe(III)-reducing, sulfur-reducing carboxydotrophic bacterium from an acidic hot spring.

    PubMed

    Yoneda, Yasuko; Yoshida, Takashi; Kawaichi, Satoshi; Daifuku, Takashi; Takabe, Keiji; Sako, Yoshihiko

    2012-07-01

    A novel anaerobic, Fe(III)-reducing, hydrogenogenic, carboxydotrophic bacterium, designated strain Ug1(T), was isolated from a volcanic acidic hot spring in southern Kyushu Island, Japan. Cells of the isolate were rod-shaped (1.0-3.0 µm long) and motile due to peritrichous flagella. Strain Ug1(T) grew chemolithoautotrophically on CO (100% in the gas phase) with reduction of ferric citrate, amorphous iron (III) oxide, 9,10-anthraquinone 2,6-disulfonate, thiosulfate or elemental sulfur. No carboxydotrophic growth occurred with sulfate, sulfite, nitrate or fumarate as electron acceptor. During growth on CO, H(2) and CO(2) were produced. Growth occurred on molecular hydrogen as an energy source and carbon dioxide as a sole carbon source. Growth was observed on various organic compounds under an N(2) atmosphere with the reduction of ferric iron. The temperature range for carboxydotrophic growth was 50-70 °C, with an optimum at 65 °C. The pH(25 °C) range for growth was 4.6-8.6, with an optimum between 6.0 and 6.5. The doubling time under optimum conditions using CO with ferric citrate was 1.5 h. The DNA G+C content was 42.2 mol%. Analysis of 16S rRNA gene sequences demonstrated that this strain belongs to the thermophilic carboxydotrophic bacterial genus Carboxydothermus, with sequence similarities of 94.1-96.6% to members of this genus. The isolate can be distinguished from other members of the genus Carboxydothermus by its ability to grow with elemental sulfur or thiosulfate coupled to CO oxidation. On the basis of phylogenetic analysis and unique physiological features, the isolate represents a novel species of the genus Carboxydothermus for which the name Carboxydothermus pertinax sp. nov. is proposed; the type strain of the novel species is Ug1(T) (=DSM 23698(T)=NBRC 107576(T)).

  19. Induction by a Lactic Acid Bacterium of a Population of CD4+ T Cells with Low Proliferative Capacity That Produce Transforming Growth Factor β and Interleukin-10

    PubMed Central

    von der Weid, Thierry; Bulliard, Christine; Schiffrin, Eduardo J.

    2001-01-01

    We investigated whether certain strains of lactic acid bacteria (LAB) could antagonize specific T-helper functions in vitro and thus have the potential to prevent inflammatory intestinal immunopathologies. All strains tested induced various levels of both interleukin-12 (IL-12) and IL-10 in murine splenocytes. In particular, Lactobacillus paracasei (strain NCC2461) induced the highest levels of these cytokines. Since IL-12 and IL-10 have the potential to induce and suppress Th1 functions, respectively, we addressed the impact of this bacterium on the outcome of CD4+ T-cell differentiation. For this purpose, bacteria were added to mixed lymphocyte cultures where CD4+ T-cells from naive BALB/c mice were stimulated weekly in the presence of irradiated allogeneic splenocytes. In these cultures, L. paracasei NCC2461 strongly inhibited the proliferative activity of CD4+ T cells in a dose-dependent fashion. This was accompanied by a marked decrease of both Th1 and Th2 effector cytokines, including gamma interferon, IL-4, and IL-5. In contrast, IL-10 was maintained and transforming growth factor β (TGF-β) was markedly induced in a dose-dependent manner. The bacteria were not cytotoxic, because cell viability was not affected after two rounds of stimulation. Thus, unidentified bacterial components from L. paracasei NCC2461 induced the development of a population of CD4+ T cells with low proliferative capacity that produced TGF-β and IL-10, reminiscent of previously described subsets of regulatory cells implicated in oral tolerance and gut homeostasis. PMID:11427413

  20. The Low Biomass Yields of the Acetic Acid Bacterium Acetobacter pasteurianus Are Due to a Low Stoichiometry of Respiration-Coupled Proton Translocation

    PubMed Central

    Luttik, M.; Van Spanning, R.; Schipper, D.; Van Dijken, J. P.; Pronk, J. T.

    1997-01-01

    Growth energetics of the acetic acid bacterium Acetobacter pasteurianus were studied with aerobic, ethanol-limited chemostat cultures. In these cultures, production of acetate was negligible. Carbon limitation and energy limitation were also evident from the observation that biomass concentrations in the cultures were proportional to the concentration of ethanol in the reservoir media. Nevertheless, low concentrations of a few organic metabolites (glycolate, citrate, and mannitol) were detected in culture supernatants. From a series of chemostat cultures grown at different dilution rates, the maintenance energy requirements for ethanol and oxygen were estimated at 4.1 mmol of ethanol (middot) g of biomass(sup-1) (middot) h(sup-1) and 11.7 mmol of O(inf2) (middot) g of biomass(sup-1) (middot) h(sup-1), respectively. When biomass yields were corrected for these maintenance requirements, the Y(infmax) values on ethanol and oxygen were 13.1 g of biomass (middot) mol of ethanol(sup-1) and 5.6 g of biomass (middot) mol of O(inf2)(sup-1), respectively. These biomass yields are very low in comparison with those of other microorganisms grown under comparable conditions. To investigate whether the low growth efficiency of A. pasteurianus might be due to a low gain of metabolic energy from respiratory dissimilation, (symbl)H(sup+)/O stoichiometries were estimated during acetate oxidation by cell suspensions. These experiments indicated an (symbl)H(sup+)/O stoichiometry for acetate oxidation of 1.9 (plusmn) 0.1 mol of H(sup+)/mol of O. Theoretical calculations of growth energetics showed that this low (symbl)H(sup+)/O ratio adequately explained the low biomass yield of A. pasteurianus in ethanol-limited cultures. PMID:16535681

  1. Enhancing immunity by dietary consumption of a probiotic lactic acid bacterium (Bifidobacterium lactis HN019): optimization and definition of cellular immune responses.

    PubMed

    Chiang, B L; Sheih, Y H; Wang, L H; Liao, C K; Gill, H S

    2000-11-01

    To define the cellular basis for immune enhancement by a probiotic lactic acid bacteria strain (Bifidobacterium lactis HN019); and to determine whether immune enhancement can be optimized by delivery in oligosaccharide-enriched low-fat milk. A double-blind, three-stage before-and-after intervention trial. Taipei Medical College Hospital, Taipei, Taiwan. Fifty healthy Taiwanese citizens (age range 41-81; median 60) randomly allocated to two groups. In stage 1 (run-in control stage) all subjects consumed reconstituted low-fat milk (LFM) for 3 weeks; in stage 2 (probiotic intervention) subjects consumed B. lactis in LFM (group A) or B. lactis in lactose-hydrolysed LFM (group B) for 3 weeks; in stage 3 all subjects returned to non-supplemented LFM for a further 3 weeks (washout stage). The innate immune functions of two different leucocyte types (polymorphonuclear (PMN) cells and natural killer (NK) cells) were assessed at four time points via in vitro analyses on peripheral blood samples. While consumption of LFM alone had no significant effect on immune responses, stage 2 results indicated significantly enhanced PMN cell phagocytosis and NK cell tumour killing activity following consumption of milk containing B. lactis. These increases levelled off following cessation of B. lactis consumption, but remained above the pre-treatment values. Increases in PMN and NK cell activity were greatest among subjects who consumed B. lactis in lactose-hydrolysed LFM. Dietary consumption of the probiotic bacterium B. lactis HN019 enhanced immune function of two different types of leucocytes; the degree of enhancement was increased by consuming B. lactis in an oligosaccharide-rich substrate. Financial support was provided by the New Zealand Dairy Board.

  2. Engineering trehalose synthesis in Lactococcus lactis for improved stress tolerance.

    PubMed

    Carvalho, Ana Lúcia; Cardoso, Filipa S; Bohn, Andreas; Neves, Ana Rute; Santos, Helena

    2011-06-01

    Trehalose accumulation is a common cell defense strategy against a variety of stressful conditions. In particular, our team detected high levels of trehalose in Propionibacterium freudenreichii in response to acid stress, a result that led to the idea that endowing Lactococcus lactis with the capacity to synthesize trehalose could improve the acid tolerance of this organism. To this end, we took advantage of the endogenous genes involved in the trehalose catabolic pathway of L. lactis, i.e., trePP and pgmB, encoding trehalose 6-phosphate phosphorylase and β-phosphoglucomutase, respectively, which enabled the synthesis of trehalose 6-phosphate. Given that L. lactis lacks trehalose 6-phosphate phosphatase, the respective gene, otsB, from the food-grade organism P. freudenreichii was used to provide the required activity. The trehalose yield was approximately 15% in resting cells and in mid-exponential-phase cells grown without pH control. The intracellular concentration of trehalose reached maximal values of approximately 170 mM, but at least 67% of the trehalose produced was found in the growth medium. The viability of mutant and control strains was examined after exposure to heat, cold or acid shock, and freeze-drying. The trehalose-producing strains showed improved tolerance (5- to 10-fold-higher survivability) to acid (pH 3) and cold shock (4°C); there was also a strong improvement in cell survival in response to heat shock (45°C), and no protection was rendered against dehydration. The insight provided by this work may help the design of food-grade strains optimized for the dairy industry as well as for oral drug delivery.

  3. Activities of amylase, proteinase, and lipase enzymes from Lactococcus chungangensis and its application in dairy products.

    PubMed

    Konkit, Maytiya; Kim, Wonyong

    2016-07-01

    Several enzymes are involved in the process of converting milk to lactic acid and coagulated milk to curd and, therefore, are important in dairy fermented products. Amylase, proteinase, and lipase are enzymes that play an important role in degrading milk into monomeric molecules such as oligosaccharides, amino acids, and fatty acids, which are the main molecules responsible for flavors in cheese. In the current study, we determined the amylase, proteinase, and lipase activities of Lactococcus chungangensis CAU 28(T), a bacterial strain of nondairy origin, and compared them with those of the reference strain, Lactococcus lactis ssp. lactis KCTC 3769(T), which is commonly used in the dairy industry. Lactococcus chungangensis CAU 28(T) and L. lactis ssp. lactis KCTC 3769(T) were both found to have amylase, proteinase, and lipase activities in broth culture, cream cheese, and yogurt. Notably, the proteinase and lipase activities of L. chungangensis CAU 28(T) were higher than those of L. lactis ssp. lactis KCTC 3769(T), with proteinase activity of 10.50 U/mL in tryptic soy broth and 8.64 U/mL in cream cheese, and lipase activity of 100 U/mL of tryptic soy broth, and 100 U/mL of cream cheese. In contrast, the amylase activity was low, with 5.28 U/mL in tryptic soy broth and 8.86 U/mL in cream cheese. These enzyme activities in L. chungangensis CAU 28(T) suggest that this strain has potential to be used for manufacturing dairy fermented products, even though the strain is of nondairy origin. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  4. Effects of the organic acids produced by a lactic acid bacterium in Apis mellifera colony development, Nosema ceranae control and fumagillin efficiency.

    PubMed

    Maggi, Matías; Negri, Pedro; Plischuk, Santiago; Szawarski, Nicolás; De Piano, Fiorella; De Feudis, Leonardo; Eguaras, Martín; Audisio, Carina

    2013-12-27

    The European honey bee Apis mellifera is known to be affected by many parasites and pathogens that have great impact over the insect development. Among parasites affecting bee health, Nosema ceranae is one of the main biotic factors affecting colony populations. As honey bee populations decline, interest in pathogenic and mutualistic relationships between bees and microorganisms has increased. The main goal of the current study was to assess the effect of the oral administration of the metabolites produced by Lactobacillus johnsonii CRL1647 (mainly organic acids) supplemented in syrup, on: (I) N. ceranae sporulation dynamics before and after fumagillin application, and (II) performance of A. mellifera colonies. Different experiments were conducted to evaluate the effects of these bacterial metabolites on bees: in vitro administration revealed no toxic effects against bees. Colonies fed with the lactic acids incremented their beehive population and also the amount of fat bodies per bee. Finally, the organic acids reduced the intensity of the pathogen after the second application of treatment as well as enhanced the fumagillin efficiency. This study provides important information for the development of new control substances against nosemosis. Copyright © 2013 Elsevier B.V. All rights reserved.

  5. Surface Display of the Receptor-Binding Region of the Lactobacillus brevis S-Layer Protein in Lactococcus lactis Provides Nonadhesive Lactococci with the Ability To Adhere to Intestinal Epithelial Cells

    PubMed Central

    Åvall-Jääskeläinen, Silja; Lindholm, Agneta; Palva, Airi

    2003-01-01

    Lactobacillus brevis is a promising lactic acid bacterium for use as a probiotic dietary adjunct and a vaccine vector. The N-terminal region of the S-layer protein (SlpA) of L. brevis ATCC 8287 was recently shown to mediate adhesion to various human cell lines in vitro. In this study, a surface display cassette was constructed on the basis of this SlpA receptor-binding domain, a proteinase spacer, and an autolysin anchor. The cassette was expressed under control of the nisA promoter in Lactococcus lactis NZ9000. Western blot assay of lactococcal cell wall extracts with anti-SlpA antibodies confirmed that the SlpA adhesion domain of the fusion protein was expressed and located within the cell wall layer. Whole-cell enzyme-linked immunosorbent assay and immunofluorescence microscopy verified that the SlpA adhesion-mediating region was accessible on the lactococcal cell surface. In vitro adhesion assays with the human intestinal epithelial cell line Intestine 407 indicated that the recombinant lactococcal cells had gained an ability to adhere to Intestine 407 cells significantly greater than that of wild-type L. lactis NZ9000. Serum inhibition assay further confirmed that adhesion of recombinant lactococci to Intestine 407 cells was indeed mediated by the N terminus-encoding part of the slpA gene. The ability of the receptor-binding region of SlpA to adhere to fibronectin was also confirmed with this lactococcal surface display system. These results show that, with the aid of the receptor-binding region of the L. brevis SlpA protein, the ability to adhere to gut epithelial cells can indeed be transferred to another, nonadhesive, lactic acid bacterium. PMID:12676705

  6. Surface display of the receptor-binding region of the Lactobacillus brevis S-layer protein in Lactococcus lactis provides nonadhesive lactococci with the ability to adhere to intestinal epithelial cells.

    PubMed

    Avall-Jääskeläinen, Silja; Lindholm, Agneta; Palva, Airi

    2003-04-01

    Lactobacillus brevis is a promising lactic acid bacterium for use as a probiotic dietary adjunct and a vaccine vector. The N-terminal region of the S-layer protein (SlpA) of L. brevis ATCC 8287 was recently shown to mediate adhesion to various human cell lines in vitro. In this study, a surface display cassette was constructed on the basis of this SlpA receptor-binding domain, a proteinase spacer, and an autolysin anchor. The cassette was expressed under control of the nisA promoter in Lactococcus lactis NZ9000. Western blot assay of lactococcal cell wall extracts with anti-SlpA antibodies confirmed that the SlpA adhesion domain of the fusion protein was expressed and located within the cell wall layer. Whole-cell enzyme-linked immunosorbent assay and immunofluorescence microscopy verified that the SlpA adhesion-mediating region was accessible on the lactococcal cell surface. In vitro adhesion assays with the human intestinal epithelial cell line Intestine 407 indicated that the recombinant lactococcal cells had gained an ability to adhere to Intestine 407 cells significantly greater than that of wild-type L. lactis NZ9000. Serum inhibition assay further confirmed that adhesion of recombinant lactococci to Intestine 407 cells was indeed mediated by the N terminus-encoding part of the slpA gene. The ability of the receptor-binding region of SlpA to adhere to fibronectin was also confirmed with this lactococcal surface display system. These results show that, with the aid of the receptor-binding region of the L. brevis SlpA protein, the ability to adhere to gut epithelial cells can indeed be transferred to another, nonadhesive, lactic acid bacterium.

  7. Binding Properties of Streptococcus gordonii SspA and SspB (Antigen I/II Family) Polypeptides Expressed on the Cell Surface of Lactococcus lactis MG1363

    PubMed Central

    Holmes, Ann R.; Gilbert, Christophe; Wells, Jeremy M.; Jenkinson, Howard F.

    1998-01-01

    The oral bacterium Streptococcus gordonii expresses two cell wall-associated polypeptides, designated SspA (1,542 amino acid residues) and SspB (1,462 amino acid residues), that have 70% sequence identity. These polypeptides are members of the antigen I/II family of oral streptococcal adhesins and mediate the binding of streptococci to salivary glycoproteins, collagen, and other oral microorganisms such as Actinomyces naeslundii. To determine if SspA and SspB have differential binding properties, the coding sequences of the sspA and sspB genes were cloned into expression plasmid vector pTREX1-usp45LS to generate pTREX1-sspA and pTREX1-sspB, respectively, and the Ssp polypeptides were displayed on the cell surface of Lactococcus lactis MG1363. Lactococcal cells expressing similar levels of surface SspA or SspB polypeptide were then compared for their abilities to adhere to a range of antigen I/II polypeptide substrates. More than twice as many L. lactis cells expressing SspA bound to immobilized salivary agglutinin glycoprotein (SAG) as did L. lactis cells expressing SspB. In contrast, lactococci expressing SspB adhered twice as well as lactococci producing SspA to collagen type I and to Candida albicans. The binding of A. naeslundii to lactococci was only weakly enhanced by surface expression of Ssp polypeptides. L. lactis(pTREX1-sspB) cells bound in greater numbers to SAG than did Enterococcus faecalis JH2-2 cells expressing SspB from pAM401EB-5. The results suggest that SspA and SspB have markedly different binding affinities for their oral substrates and thus may function to promote site diversity in colonization by S. gordonii. PMID:9746559

  8. Use of non-growing Lactococcus lactis cell suspensions for production of volatile metabolites with direct relevance for flavour formation during dairy fermentations.

    PubMed

    van de Bunt, Bert; Bron, Peter A; Sijtsma, Lolke; de Vos, Willem M; Hugenholtz, Jeroen

    2014-12-10

    Lactococcus lactis is a lactic acid bacterium that has been used for centuries in the production of a variety of cheeses, as these bacteria rapidly acidify milk and greatly contribute to the flavour of the fermentation end-products. After a short growth phase during cheese ripening L. lactis enters an extended non-growing state whilst still strongly contributing to amino acid-derived flavour formation. Here, a research approach is presented that allows investigation of strain- and amino acid-specific flavour formation during the non-growing state. Non-growing cells of five selected L. lactis strains were demonstrated to degrade amino acids into flavour compounds that are relevant in food fermentations and differs greatly from production of flavour compounds using growing cells. As observed earlier in other research set-ups and with other microorganisms, addition of NADH, α-ketoglutarate and pyridoxal-5-phosphate was demonstrated to be essential for optimal flavour formation, suggesting that intracellular pools of these substrates are too low for the significant production of the flavour compounds. Production of flavours during the non-growing phase strongly depends on the individual amino acids that were supplied, on the presence of other amino acids (mixtures versus single compounds), and on the strain used. Moreover, we observed that the plasmid-free model strains L. lactis MG1363 and IL1403 produce relatively low amounts of flavour components under the various conditions tested. By using this simplified and rapid approach to study flavour formation by non-growing lactic acid bacteria, lengthy ripening periods are no longer required to assess the capacity of strains to produce flavours in the long, non-growing state of dairy fermentation. In addition, this method also provides insight into the conversion of single amino acids versus the conversion of a mixture of amino acids as produced during protein degradation. The generated results are complementary to

  9. Expression of Plant Flavor Genes in Lactococcus lactis▿ †

    PubMed Central

    Hernández, Igor; Molenaar, Douwe; Beekwilder, Jules; Bouwmeester, Harro; van Hylckama Vlieg, Johan E. T.

    2007-01-01

    Lactic acid bacteria, such as Lactococcus lactis, are attractive hosts for the production of plant-bioactive compounds because of their food grade status, efficient expression, and metabolic engineering tools. Two genes from strawberry (Fragaria x ananassa), encoding an alcohol acyltransferase (SAAT) and a linalool/nerolidol synthase (FaNES), were cloned in L. lactis and actively expressed using the nisin-induced expression system. The specific activity of SAAT could be improved threefold (up to 564 pmol octyl acetate h−1 mg protein−1) by increasing the concentration of tRNA1Arg, which is a rare tRNA molecule in L. lactis. Fermentation tests with GM17 medium and milk with recombinant L. lactis strains expressing SAAT or FaNES resulted in the production of octyl acetate (1.9 μM) and linalool (85 nM) to levels above their odor thresholds in water. The results illustrate the potential of the application of L. lactis as a food grade expression platform for the recombinant production of proteins and bioactive compounds from plants. PMID:17209074

  10. A review on Lactococcus lactis: from food to factory.

    PubMed

    Song, Adelene Ai-Lian; In, Lionel L A; Lim, Swee Hua Erin; Rahim, Raha Abdul

    2017-04-04

    Lactococcus lactis has progressed a long way since its discovery and initial use in dairy product fermentation, to its present biotechnological applications in genetic engineering for the production of various recombinant proteins and metabolites that transcends the heterologous species barrier. Key desirable features of this gram-positive lactic acid non-colonizing gut bacteria include its generally recognized as safe (GRAS) status, probiotic properties, the absence of inclusion bodies and endotoxins, surface display and extracellular secretion technology, and a diverse selection of cloning and inducible expression vectors. This have made L. lactis a desirable and promising host on par with other well established model bacterial or yeast systems such as Escherichia coli, Salmonella cerevisiae and Bacillus subtilis. In this article, we review recent technological advancements, challenges, future prospects and current diversified examples on the use of L. lactis as a microbial cell factory. Additionally, we will also highlight latest medical-based applications involving whole-cell L. lactis as a live delivery vector for the administration of therapeutics against both communicable and non-communicable diseases.

  11. Encapsulated Lactococcus lactis with enhanced gastrointestinal survival for the development of folate enriched functional foods.

    PubMed

    Divya, Jayakumar Beena; Nampoothiri, Kesavan Madhavan

    2015-01-01

    Two lactic acid bacteria (LAB) isolated from cow's milk were identified as Lactococcus lactis strains and designated as L. lactis CM22 and L. lactis CM28. They were immobilised by co-encapsulation using alginate and mannitol and by hybrid entrapment with skim milk, glycerol, CaCO3 and alginate. The encapsulated cells survived better in simulated gastrointestinal conditions compared to the free cells. The percentage survival of probiotics encapsulated by hybrid entrapment method was 62.74% for L. lactis CM22 and 68% for L. lactis CM28. Studies to check their efficacy in fermentative fortification of skim milk and ice cream revealed an enhancement in folate level.

  12. Simultaneous lactic acidification and coagulation by using recombinant Lactococcus lactis strain.

    PubMed

    Raftari, M; Ghafourian, S; Abu Bakar, F

    2017-04-01

    This study was an attempt to create a novel milk clotting procedure using a recombinant bacterium capable of milk coagulation. The Rhizomucor pusillus proteinase (RPP) gene was sub-cloned into a pALF expression vector. The recombinant pALF-RPP vector was then electro-transferred into Lactococcus lactis. Finally, the milk coagulation ability of recombinant L. lactis carrying a RPP gene was evaluated. Nucleotide sequencing of DNA insertion from the clone revealed that the RPP activity corresponded to an open reading frame consisting of 1218 bp coding for a 43·45 kDa RPP protein. The RPP protein assay results indicated that the highest RPP enzyme expression with 870 Soxhlet units (SU) per ml and 7914 SU/OD were obtained for cultures which were incubated at pH 5·5 and 30°C. Interestingly, milk coagulation was observed after 205 min of inoculating milk with recombinant L. lactis carrying the RPP gene. The recombinant L. lactis carrying RPP gene has the ability to function as a starter culture for acidifying and subsequently coagulating milk by producing RPP as a milk coagulant agent. Creating a recombinant starter culture bacterium that is able to coagulate milk. It is significant because the recombinant L. lactis has the ability to work as a starter culture and milk coagulation agent. © 2016 The Society for Applied Microbiology.

  13. Lactococcus lactis catalyses electricity generation at microbial fuel cell anodes via excretion of a soluble quinone.

    PubMed

    Freguia, Stefano; Masuda, Masaki; Tsujimura, Seiya; Kano, Kenji

    2009-09-01

    Lactococcus lactis is a gram-positive, normally homolactic fermenter that is known to produce several kinds of membrane associated quinones, which are able to mediate electron transfer to extracellular electron acceptors such as Fe(3+), Cu(2+) and hexacyanoferrate. Here we show that this bacterium is also capable of performing extracellular electron transfer to anodes by utilizing at least two soluble redox mediators, as suggested by the two-step catalytic current developed. One of these two mediators was herein suggested to be 2-amino-3-dicarboxy-1,4-naphthoquinone (ACNQ), via evaluation of standard redox potential, ability of the bacterium to exploit the quinone when exogenously provided, as well as by high performance liquid chromatography coupled with UV spectrum analysis. During electricity generation, L. lactis slightly deviated from its normal homolactic metabolism by excreting acetate and pyruvate in stoichiometric amounts with respect to the electrical current. In this metabolism, the anode takes on the role of electron sink for acetogenic fermentation. The finding that L. lactis self-catalyses anodic electron transfer by excretion of redox mediators is remarkable as the mechanisms of extracellular electron transfer by pure cultures of gram-positive bacteria had previously never been elucidated.

  14. 2,4-Dichlorophenoxyacetic acid (2,4-D)- and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T)-degrading gene cluster in the soybean root-nodulating bacterium Bradyrhizobium elkanii USDA94.

    PubMed

    Hayashi, Shohei; Sano, Tomoki; Suyama, Kousuke; Itoh, Kazuhito

    2016-01-01

    Herbicides 2,4-dichlorophenoxyacetic acid (2,4-D)- and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T)-degrading Bradyrhizobium strains possess tfdAα and/or cadABC as degrading genes. It has been reported that root-nodulating bacteria belonging to Bradyrhizobium elkanii also have tfdAα and cadA like genes but lack the ability to degrade these herbicides and that the cadA genes in 2,4-D-degrading and non-degrading Bradyrhizobium are phylogenetically different. In this study, we identified cadRABCK in the genome of a type strain of soybean root-nodulating B. elkanii USDA94 and demonstrated that the strain could degrade the herbicides when cadABCK was forcibly expressed. cadABCK-cloned Escherichia coli also showed the degrading ability. Because co-spiked phenoxyacetic acid (PAA) could induce the degradation of 2,4-D in B. elkanii USDA94, the lack of degrading ability in this strain was supposed to be due to the low inducing potential of the herbicides for the degrading gene cluster. On the other hand, tfdAα from B. elkanii USDA94 showed little potential to degrade the herbicides, but it did for 4-chlorophenoxyacetic acid and PAA. The 2,4-D-degrading ability of the cad cluster and the inducing ability of PAA were confirmed by preparing cadA deletion mutant. This is the first study to demonstrate that the cad cluster in the typical root-nodulating bacterium indeed have the potential to degrade the herbicides, suggesting that degrading genes for anthropogenic compounds could be found in ordinary non-degrading bacteria. Copyright © 2016 Elsevier GmbH. All rights reserved.

  15. Bacterium-Like Particles for Efficient Immune Stimulation of Existing Vaccines and New Subunit Vaccines in Mucosal Applications

    PubMed Central

    Van Braeckel-Budimir, Natalija; Haijema, Bert Jan; Leenhouts, Kees

    2013-01-01

    The successful development of a mucosal vaccine depends critically on the use of a safe and effective immunostimulant and/or carrier system. This review describes the effectiveness and mode of action of an immunostimulating particle, derived from bacteria, used in mucosal subunit vaccines. The non-living particles, designated bacterium-like particles are based on the food-grade bacterium Lactococcus lactis. The focus of the overview is on the development of intranasal BLP-based vaccines to prevent diseases caused by influenza and respiratory syncytial virus, and includes a selection of Phase I clinical data for the intranasal FluGEM vaccine. PMID:24062748

  16. Antifungal and sprout regulatory bioactivities of phenylacetic acid, indole-3-acetic acid, and tyrosol isolated from the potato dry rot suppressive bacterium Enterobacter cloacae S11:T:07.

    PubMed

    Slininger, P J; Burkhead, K D; Schisler, D A

    2004-12-01

    Enterobacter cloacae S11: T:07 (NRRL B-21050) is a promising biological control agent that has significantly reduced both fungal dry rot disease and sprouting in laboratory and pilot potato storages. The metabolites phenylacetic acid (PAA), indole-3-acetic acid (IAA), and tyrosol (TSL) were isolated from S11:T:07 liquid cultures provided with three different growth media. The bioactivities of these metabolites were investigated via thin-layer chromatography bioautography of antifungal activity, wounded potato assays of dry rot suppressiveness, and cored potato eye assays of sprout inhibition. Relative accumulations of PAA, IAA, and TSL in cultures were nutrient dependent. For the first time, IAA, TSL, and PAA were shown to have antifungal activity against the dry rot causative pathogen Gibberella pulicaris, and to suppress dry rot infection of wounded potatoes. Disease suppression was optimal when all three metabolites were applied in combination. Dosages of IAA that resulted in disease suppression also resulted in sprout inhibition. These results suggest the potential for designing culture production and formulation conditions to achieve a dual purpose biological control agent able to suppress both dry rot and sprouting of stored potatoes.

  17. Evaluation of lactic acid bacterium fermentation products and food-grade chemicals to control Listeria monocytogenes in blue crab (Callinectes sapidus) meat.

    PubMed

    Degnan, A J; Kaspar, C W; Otwell, W S; Tamplin, M L; Luchansky, J B

    1994-09-01

    Fresh blue crab (Callinectes sapidus) meat was obtained from retail markets in Florida and sampled for viable Listeria monocytogenes. The pathogen was found in crabmeat in three of four different lots tested by enrichment and at levels of 75 CFU/g in one of the same four lots by direct plating. Next, crabmeat was steam sterilized, inoculated with a three-strain mixture of L. monocytogenes (ca. 5.5 log10 CFU/g), washed with various lactic acid bacterium fermentation products (2,000 to 20,000 arbitrary units [AU]/ml of wash) or food-grade chemicals (0.25 to 4 M), and stored at 4 degrees C. Counts of the pathogen remained relatively constant in control samples during storage for 6 days, whereas in crabmeat washed with Perlac 1911 or MicroGard (10,000 to 20,000 AU), numbers initially decreased (0.5 to 1.0 log10 unit/g) but recovered to original levels within 6 days. Numbers of L. monocytogenes cells decreased 1.5 to 2.7 log10 units/g of crabmeat within 0.04 day when washed with 10,000 to 20,000 AU of Alta 2341, enterocin 1083, or Nisin per ml. Thereafter, counts increased 0.5 to 1.6 log10 units within 6 days. After washing with food-grade chemicals, modest reductions (0.4 to 0.8 log10 unit/g) were observed with sodium acetate (4 M), sodium diacetate (0.5 or 1 M), sodium lactate (1 M), or sodium nitrite (1.5 M). However, Listeria counts in crabmeat washed with 2 M sodium diacetate decreased 2.6 log10 units/g within 6 days. In addition, trisodium phosphate reduced L. monocytogenes counts from 1.7 (0.25 M) to > 4.6 (1 M) log10 units/g within 6 days.(ABSTRACT TRUNCATED AT 250 WORDS)

  18. Evaluation of lactic acid bacterium fermentation products and food-grade chemicals to control Listeria monocytogenes in blue crab (Callinectes sapidus) meat.

    PubMed Central

    Degnan, A J; Kaspar, C W; Otwell, W S; Tamplin, M L; Luchansky, J B

    1994-01-01

    Fresh blue crab (Callinectes sapidus) meat was obtained from retail markets in Florida and sampled for viable Listeria monocytogenes. The pathogen was found in crabmeat in three of four different lots tested by enrichment and at levels of 75 CFU/g in one of the same four lots by direct plating. Next, crabmeat was steam sterilized, inoculated with a three-strain mixture of L. monocytogenes (ca. 5.5 log10 CFU/g), washed with various lactic acid bacterium fermentation products (2,000 to 20,000 arbitrary units [AU]/ml of wash) or food-grade chemicals (0.25 to 4 M), and stored at 4 degrees C. Counts of the pathogen remained relatively constant in control samples during storage for 6 days, whereas in crabmeat washed with Perlac 1911 or MicroGard (10,000 to 20,000 AU), numbers initially decreased (0.5 to 1.0 log10 unit/g) but recovered to original levels within 6 days. Numbers of L. monocytogenes cells decreased 1.5 to 2.7 log10 units/g of crabmeat within 0.04 day when washed with 10,000 to 20,000 AU of Alta 2341, enterocin 1083, or Nisin per ml. Thereafter, counts increased 0.5 to 1.6 log10 units within 6 days. After washing with food-grade chemicals, modest reductions (0.4 to 0.8 log10 unit/g) were observed with sodium acetate (4 M), sodium diacetate (0.5 or 1 M), sodium lactate (1 M), or sodium nitrite (1.5 M). However, Listeria counts in crabmeat washed with 2 M sodium diacetate decreased 2.6 log10 units/g within 6 days. In addition, trisodium phosphate reduced L. monocytogenes counts from 1.7 (0.25 M) to > 4.6 (1 M) log10 units/g within 6 days.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7944362

  19. Efficient Overproduction of Membrane Proteins in Lactococcus lactis Requires the Cell Envelope Stress Sensor/Regulator Couple CesSR

    PubMed Central

    Pinto, Joao P. C.; Kuipers, Oscar P.; Marreddy, Ravi K. R.; Poolman, Bert; Kok, Jan

    2011-01-01

    Background Membrane proteins comprise an important class of molecules whose study is largely frustrated by several intrinsic constraints, such as their hydrophobicity and added requirements for correct folding. Additionally, the complexity of the cellular mechanisms that are required to insert membrane proteins functionally in the membrane and to monitor their folding state makes it difficult to foresee the yields at which one can obtain them or to predict which would be the optimal production host for a given protein. Methods and Findings We describe a rational design approach to improve the lactic acid bacterium Lactococcus lactis as a producer of membrane proteins. Our transcriptome data shows that the two-component system CesSR, which senses cell envelope stresses of different origins, is one of the major players when L. lactis is forced to overproduce the endogenous membrane protein BcaP, a branched-chain amino acid permease. Growth of the BcaP-producing L. lactis strain and its capability to produce membrane proteins are severely hampered when the CesSR system itself or particular members of the CesSR regulon are knocked out, notably the genes ftsH, oxaA2, llmg_2163 and rmaB. Overexpressing cesSR reduced the growth defect, thus directly improving the production yield of BcaP. Applying this rationale to eukaryotic proteins, some of which are notoriously more difficult to produce, such as the medically-important presenilin complex, we were able to significantly diminish the growth defect seen in the wild-type strain and improve the production yield of the presenilin variant PS1Δ9-H6 more than 4-fold. Conclusions The results shed light into a key, and perhaps central, membrane protein quality control mechanism in L. lactis. Modulating the expression of CesSR benefited the production yields of membrane proteins from different origins. These findings reinforce L. lactis as a legitimate alternative host for the production of membrane proteins. PMID:21818275

  20. Characterization of Lactococcus lactis response to ampicillin and ciprofloxacin using surface-enhanced Raman spectroscopy.

    PubMed

    Wang, Panxue; Pang, Shintaro; Zhang, Hua; Fan, Mingtao; He, Lili

    2016-01-01

    Decades of antibiotic use or misuse has resulted in antibiotic resistance in lactic acid bacteria, a group of common culture starters and probiotic microorganisms. This has urged researchers to study how lactic acid bacteria respond to antibiotics, so as to have a better strategy to identify and predict the antibiotic-resistant bacteria. This study aimed to characterize the biochemical profiles of Lactococcus lactis responding to antibiotics using surface-enhanced Raman spectroscopy (SERS). Lactococcus lactis exposed to antibiotics was mixed with 50-nm gold nanoparticles for subsequent SERS measurements. The SERS spectra analyzed by principal component analysis showed no significant change after 30 min of antibiotic treatment, whereas distinct changes were clearly observed after 60 and 90 min of antibiotic treatment. Different antibiotics induced different spectral changes, and these changes revealed the detailed biochemical information of cellular responses. This study demonstrates that the SERS method developed not only senses the changes in the bacterial cell wall, but also reveals details of the biochemical profiles, which help us to understand how lactic acid bacteria respond to antibiotics, as well as to set a base for the detection of antibiotic susceptibility of bacteria by SERS.

  1. Anaerobic degradation of 2-aminobenzoic acid (anthranilic acid) via benzoyl-coenzyme A (CoA) and cyclohex-1-enecarboxyl-CoA in a denitrifying bacterium.

    PubMed Central

    Lochmeyer, C; Koch, J; Fuchs, G

    1992-01-01

    The enzymes catalyzing the initial reactions in the anaerobic degradation of 2-aminobenzoic acid (anthranilic acid) were studied with a denitrifying Pseudomonas sp. anaerobically grown with 2-aminobenzoate and nitrate as the sole carbon and energy sources. Cells grown on 2-aminobenzoate are simultaneously adapted to growth with benzoate, whereas cells grown on benzoate degrade 2-aminobenzoate several times less efficiently than benzoate. Evidence for a new reductive pathway of aromatic metabolism and for four enzymes catalyzing the initial steps is presented. The organism contains 2-aminobenzoate-coenzyme A ligase (2-aminobenzoate-CoA ligase), which forms 2-aminobenzoyl-CoA. 2-Aminobenzoyl-CoA is then reductively deaminated to benzoyl-CoA by an oxygen-sensitive enzyme, 2-aminobenzoyl-CoA reductase (deaminating), which requires a low potential reductant [Ti(III)]. The specific activity is 15 nmol of 2-aminobenzoyl-CoA reduced min-1 mg-1 of protein at an optimal pH of 7. The two enzymes are induced by the substrate under anaerobic conditions only. Benzoyl-CoA is further converted in vitro by reduction with Ti(III) to six products; the same products are formed when benzoyl-CoA or 2-aminobenzoyl-CoA is incubated under reducing conditions. Two of them were identified preliminarily. One product is cyclohex-1-enecarboxyl-CoA, the other is trans-2-hydroxycyclohexane-carboxyl-CoA. The complex transformation of benzoyl-CoA is ascribed to at least two enzymes, benzoyl-CoA reductase (aromatic ring reducing) and cyclohex-1-enecarboxyl-CoA hydratase. The reduction of benzoyl-CoA to alicyclic compounds is catalyzed by extracts from cells grown anaerobically on either 2-aminobenzoate or benzoate at almost the same rate (10 to 15 nmol min-1 mg-1 of protein). In contrast, extracts from cells grown anaerobically on acetate or grown aerobically on benzoate or 2-aminobenzoate are inactive. This suggests a sequential induction of the enzymes. Images PMID:1592816

  2. Prospective uses of recombinant Lactococcus lactis expressing both listeriolysin O and mutated internalin A from Listeria monocytogenes as a tool for DNA vaccination.

    PubMed

    De Azevedo, M S P; Santos Rocha, C; Pereira, V B; De Oliveira Junior, A F; De Sousa, C S; Azevedo, V; LeBlanc, J G; Chatel, J M; Miyoshi, A

    2015-12-28

    In this study, Lactococcus lactis was engineered to express mutated internalin A on its surface and to secrete large amounts of listeriolysin O (LLO) in order to improve its potential as a vehicle for DNA vaccination. Western blotting experiments demonstrated that the bacterium expressed LLO in both the cytoplasmic and extracellular compartments, with higher quantities found in the culture supernatants. A hemolytic assay showed that the recombinant strain secreted 250 ng active LLO/mg total protein. This mInlA/LLO-producing strain of L. lactis may be used as an alternative tool in DNA vaccination against a number of infectious diseases or in cancer therapy.

  3. Regulatory phenotyping reveals important diversity within the species Lactococcus lactis.

    PubMed

    Bachmann, Herwig; Starrenburg, Marjo J C; Dijkstra, Annereinou; Molenaar, Douwe; Kleerebezem, Michiel; Rademaker, Jan L W; van Hylckama Vlieg, Johan E T

    2009-09-01

    The diversity in regulatory phenotypes among a collection of 84 Lactococcus lactis strains isolated from dairy and nondairy origin was explored. The specific activities of five enzymes were assessed in cell extracts of all strains grown in two different media, a nutritionally rich broth and a relatively poor chemically defined medium. The five investigated enzymes, branched chain aminotransferase (BcaT), aminopeptidase N (PepN), X-prolyl dipeptidyl peptidase (PepX), alpha-hydroxyisocaproic acid dehydrogenase (HicDH), and esterase, are involved in nitrogen and fatty acid metabolism and catalyze key steps in the production of important dairy flavor compounds. The investigated cultures comprise 75 L. lactis subsp. lactis isolates (including 7 L. lactis subsp. lactis biovar diacetylactis isolates) and 9 L. lactis subsp. cremoris isolates. All L. lactis subsp. cremoris and 22 L. lactis subsp. lactis (including 6 L. lactis subsp. lactis biovar diacetylactis) cultures originated from a dairy environment. All other cultures originated from (fermented) plant materials and were isolated at different geographic locations. Correlation analysis of specific enzyme activities revealed significantly different regulatory phenotypes for dairy and nondairy isolates. The enzyme activities in the two investigated media were in general poorly correlated and revealed a high degree of regulatory diversity within this collection of closely related strains. To the best of our knowledge, these results represent the most extensive diversity analysis of regulatory phenotypes within a single bacterial species to date. The presented findings underline the importance of the availability of screening procedures for, e.g., industrially relevant enzyme activities in models closely mimicking application conditions. Moreover, they corroborate the notion that regulatory changes are important drivers of evolution.

  4. Identification of an anti-inflammatory protein from Faecalibacterium prausnitzii, a commensal bacterium deficient in Crohn's disease

    PubMed Central

    Chain, F.; Marquant, R.; Tailhades, J.; Miquel, S.; Carlier, L.; Bermúdez-Humarán, L. G.; Pigneur, B.; Lequin, O.; Kharrat, P.; Thomas, G.; Rainteau, D.; Aubry, C.; Breyner, N.; Afonso, C.; Lavielle, S.; Grill, J.-P.; Chassaing, G.; Chatel, J. M.; Trugnan, G.; Xavier, R.; Langella, P.

    2016-01-01

    Background Crohn's disease (CD) associated dysbiosis is characterized by a loss of Faecalibacterium prausnitzii, whose culture supernatant exerts an anti-inflammatory effect both in vitro and in vivo. However, the chemical nature of the anti-inflammatory compounds has not yet been determined. Methods Peptidomic analysis using mass spectrometry was applied to F. prausnitzii supernatant. Anti-inflammatory effects of identified peptides were tested in vitro directly on intestinal epithelial cell lines and on cell lines transfected with a plasmid construction coding for the candidate protein encompassing these peptides. In vivo, the cDNA of the candidate protein was delivered to the gut by recombinant Lactic Acid Bacteria to prevent DNBS-colitis in mice. Results The seven peptides, identified in the F. prausnitzii culture supernatants, derived from a single Microbial Anti-inflammatory Molecule (MAM), a protein of 15 kDa and comprising 53% of nonpolar residues. This last feature prevented the direct characterization of the putative anti-inflammatory activity of MAM-derived peptides. Transfection of MAM cDNA in epithelial cells led to a significant decrease in the activation of the NF-κB pathway with a dose-dependent effect. Finally, the use of a food-grade bacterium, Lactococcus lactis, delivering a plasmid encoding MAM was able to alleviate DNBS-induced colitis in mice. Conclusion A 15kDa protein with anti-inflammatory properties is produced by F. prausnitzii, a commensal bacterium involved in CD pathogenesis. This protein is able to inhibit the NF-κB pathway in intestinal epithelial cells and to prevent colitis in an animal model. PMID:26045134

  5. Effects in the use of a genetically engineered strain of Lactococcus lactis delivering in situ IL-10 as a therapy to treat low-grade colon inflammation.

    PubMed

    Martín, Rebeca; Chain, Florian; Miquel, Sylvie; Natividad, Jane M; Sokol, Harry; Verdu, Elena F; Langella, Philippe; Bermúdez-Humarán, Luis G

    2014-01-01

    Irritable bowel syndrome (IBS) is a gastrointestinal disorder characterized by chronic abdominal pain, discomfort, and bloating. Interestingly, there is now evidence of the presence of a low-grade inflammatory status in many IBS patients, including histopathological and mucosal cytokine levels in the colon, as well as the presence of IBS-like symptoms in quiescent inflammatory bowel disease (IBD). The use of a genetically engineered food-grade bacterium, such as Lactococcus lactis, secreting the anti-inflammatory cytokine IL-10 has been proven by many pre-clinical studies to be a successful therapy to treat colon inflammation. In this study, we first reproduced the recovery-recurrence periods observed in IBS-patients in a new chronic model characterized by 2 episodes of DiNitro-BenzeneSulfonic-acid (DNBS)-challenge and we tested the effects of a recombinant strain of L. lactis secreting IL-10 under a Stress-Inducible Controlled Expression (SICE) system. In vivo gut permeability, colonic serotonin levels, cytokine profiles, and spleen cell populations were then measured as readouts of a low-grade inflammation. In addition, since there is increasing evidence that gut microbiota tightly regulates gut barrier function, tight junction proteins were also measured by qRT-PCR after administration of recombinant L. lactis in DNBS-treated mice. Strikingly, oral administration of L. lactis secreting active IL-10 in mice resulted in significant protective effects in terms of permeability, immune activation, and gut-function parameters. Although genetically engineered bacteria are, for now, used only as a "proof-of-concept," our study validates the interest in the use of the novel SICE system in L. lactis to express therapeutic molecules, such as IL-10, locally at mucosal surfaces.

  6. Catechol siderophore excretion by magnetotactic bacterium Magnetospirillum magneticum AMB-1.

    PubMed

    Calugay, Ronie J; Takeyama, Haruko; Mukoyama, Daikichi; Fukuda, Yorikane; Suzuki, Takeyuki; Kanoh, Kaneo; Matsunaga, Tadashi

    2006-05-01

    Siderophore activity was detected in the culture supernatant of the magnetotactic bacterium Magnetospirillum magneticum AMB-1. Here we report the first structural elucidation of a siderophore produced by a magnetotactic bacterium. The structure of the purified compound was 3,4-dihydroxybenzoic acid as determined by nuclear magnetic resonance (NMR) and electro-spray ionization mass spectroscopy (ESI-MS).

  7. Stimulation of cadaverine production by foodborne pathogens in the presence of Lactobacillus, Lactococcus, and Streptococcus spp.

    PubMed

    Kuley, Esmeray; Balıkcı, Esra; Özoğul, Ilyas; Gökdogan, Saadet; Ozoğul, Fatih

    2012-12-01

    The effect of Lactobacillus plantarum (FI8595), Lactococcus lactis subsp. cremoris MG 1363), Lactococcus lactis subsp. lactis (IL 1403), and Streptococcus thermophilus on cadaverine and other biogenic amine production by foodborne pathogens was investigated lysine decarboxylase broth. Both of lactic acid bacteria and foodborne pathogens used (especially Staphylococcus aureus, E. coli, Lc. lactis subsp. lactis and Lb. plantarum) had an ability to convert aminoacids into biogenic amine. The conversion of lysine into cadaverine was the highest (167.11 mg/L) by Lactobacillus spp. Gram-positive bacteria generally had a greater ability to produce cadaverine with corresponding value of 46.26, 53.76, and 154.54 mg/L for Enterococcus faecalis, S. aureus, and Listeria monocytogenes, respectively. Significant variations on biogenic amine production were observed in the presence of lactic acid bacteria strains (P < 0.05). The role of lactic acid bacteria on biogenic amine production by foodborne pathogens varied depending on strains and specific amine. Cadaverine accumulation by Enterobactericeae was increased in the presence of lactic acid bacteria strains except for St. thermophilus, which induced 2-fold lower cadaverine production by S. Paratyphi A. Lc. lactis subsp. lactis and Lc. lactis subsp. cremoris induced 10-fold higher increases in histamine for E. coli and K. pneumoniae, respectively. Lactic acid bacteria resulted in strong increases in cadaverine production by P. aeruginosa, although remarkable decreases were observed for histamine, spermidine, dopamine, agmatine, and TMA in the presence of lactic acid bacteria in lysine decarboxylase broth . The result of the study showed that amine positive lactic acid bacteria strains in fermented food led to significant amine accumulation by contaminant bacteria and their accumulation in food product may be controlled by the use of proper starters with amine-negative activity. © 2012 Institute of Food Technologists®

  8. Bioaugmentation with the resin acid-degrading bacterium Zoogloea resiniphila DhA-35 to counteract pH stress in an aerated lagoon treating pulp and paper mill effluent.

    PubMed

    Yu, Zhongtang; Mohn, William W

    2002-06-01

    Efficient and reliable removal of resin acids such as dehydroabietic acid (DhA), which are the major toxicants in pulp and paper mill effluents and form pitch interfering with papermaking, is critically important to prevent toxicity discharge and failure of paper machines. Low- and high-pH stresses sometimes occur in effluent treatment systems due to the use of large amounts of acids and alkalines in the pulping processes. We found that both low- and high-pH stresses (pH 3 and 10, respectively) decreased the removal of total organic carbon and completely inhibited the removal of DhA by the biomass of an aerated lagoon treating pulp mill effluent. The pH stresses caused changes in the bacterial community structure as assessed by ribosomal intergenic spacer length polymorphism. The pH stresses greatly reduced the indigenous DhA-degrading populations in the lagoon community. Bioaugmentation with Zoogloea resiniphila DhA-35, a DhA-degrading bacterium originally isolated from a pulp mill treatment system, restored the DhA removal by both the low- and high-pH-stressed lagoon biomass. This bacterium was persistent after introduction into the lagoon microbial community, and its cellular rRNA:rDNA ratio increased during the period of DhA removal. The introduction of strain DhA-35 changed the microbial community structure, but did not adversely affect the TOC removal by the community. This study suggests that it is feasible and potentially useful to use bioaugmentation with resin-acid-degrading bacteria such as DhA-35 to restore and enhance resin acid removal by aerated lagoon microbial communities.

  9. Lactic Acid Bacteria in Durum Wheat Flour Are Endophytic Components of the Plant during Its Entire Life Cycle

    PubMed Central

    Minervini, Fabio; Celano, Giuseppe; Lattanzi, Anna; Tedone, Luigi; De Mastro, Giuseppe; De Angelis, Maria

    2015-01-01

    This study aimed at assessing the dynamics of lactic acid bacteria and other Firmicutes associated with durum wheat organs and processed products. 16S rRNA gene-based high-throughput sequencing showed that Lactobacillus, Streptococcus, Enterococcus, and Lactococcus were the main epiphytic and endophytic genera among lactic acid bacteria. Bacillus, Exiguobacterium, Paenibacillus, and Staphylococcus completed the picture of the core genus microbiome. The relative abundance of each lactic acid bacterium genus was affected by cultivars, phenological stages, other Firmicutes genera, environmental temperature, and water activity (aw) of plant organs. Lactobacilli, showing the highest sensitivity to aw, markedly decreased during milk development (Odisseo) and physiological maturity (Saragolla). At these stages, Lactobacillus was mainly replaced by Streptococcus, Lactococcus, and Enterococcus. However, a key sourdough species, Lactobacillus plantarum, was associated with plant organs during the life cycle of Odisseo and Saragolla wheat. The composition of the sourdough microbiota and the overall quality of leavened baked goods are also determined throughout the phenological stages of wheat cultivation, with variations depending on environmental and agronomic factors. PMID:26187970

  10. Lactic Acid Bacteria in Durum Wheat Flour Are Endophytic Components of the Plant during Its Entire Life Cycle.

    PubMed

    Minervini, Fabio; Celano, Giuseppe; Lattanzi, Anna; Tedone, Luigi; De Mastro, Giuseppe; Gobbetti, Marco; De Angelis, Maria

    2015-10-01

    This study aimed at assessing the dynamics of lactic acid bacteria and other Firmicutes associated with durum wheat organs and processed products. 16S rRNA gene-based high-throughput sequencing showed that Lactobacillus, Streptococcus, Enterococcus, and Lactococcus were the main epiphytic and endophytic genera among lactic acid bacteria. Bacillus, Exiguobacterium, Paenibacillus, and Staphylococcus completed the picture of the core genus microbiome. The relative abundance of each lactic acid bacterium genus was affected by cultivars, phenological stages, other Firmicutes genera, environmental temperature, and water activity (aw) of plant organs. Lactobacilli, showing the highest sensitivity to aw, markedly decreased during milk development (Odisseo) and physiological maturity (Saragolla). At these stages, Lactobacillus was mainly replaced by Streptococcus, Lactococcus, and Enterococcus. However, a key sourdough species, Lactobacillus plantarum, was associated with plant organs during the life cycle of Odisseo and Saragolla wheat. The composition of the sourdough microbiota and the overall quality of leavened baked goods are also determined throughout the phenological stages of wheat cultivation, with variations depending on environmental and agronomic factors.

  11. The zoonotic potential of Lactococcus garvieae: An overview on microbiology, epidemiology, virulence factors and relationship with its presence in foods.

    PubMed

    Gibello, Alicia; Galán-Sánchez, Fátima; Blanco, M Mar; Rodríguez-Iglesias, Manuel; Domínguez, Lucas; Fernández-Garayzábal, José F

    2016-12-01

    Lactococcus garvieae is a relevant worldwide fish pathogen affecting various farmed and wild marine and freshwater species. It has also been isolated from other animals, such as ruminants with subclinical mastitis and pigs with pneumonia. From the early 90s, L. garvieae has been associated with different human infections, mainly endocarditis. During the last five years, human infections by this bacterium appear to be increasing, likely due to the improvement in microbiological methods for bacterial identification and the alertness of this bacterium by physicians. Human L. garvieae infections have been associated with the consumption or the handling of contaminated raw fish or seafood, and recently, a genetic study showed that meat, raw milk and dairy products may also be food sources of human L. garvieae infections. However, the status of L. garvieae as a potential zoonotic bacterium is still controversial to date. In this work, we describe four new human infections by L. garvieae in elderly and inmunocompromised patients, and we show an overview on L. garvieae microbiology, epidemiology, virulence factors and relationship with its presence in foods. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Expanding the recombinant protein quality in Lactococcus lactis.

    PubMed

    Cano-Garrido, Olivia; Rueda, Fabian L; Sànchez-García, Laura; Ruiz-Ávila, Luis; Bosser, Ramon; Villaverde, Antonio; García-Fruitós, Elena

    2014-12-04

    Escherichia coli has been a main host for the production of recombinant proteins of biomedical interest, but conformational stress responses impose severe bottlenecks that impair the production of soluble, proteolytically stable versions of many protein species. In this context, emerging Generally Recognized As Safe (GRAS) bacterial hosts provide alternatives as cell factories for recombinant protein production, in which limitations associated to the use of Gram-negative microorganisms might result minimized. Among them, Lactic Acid Bacteria and specially Lactococcus lactis are Gram-positive GRAS organisms in which recombinant protein solubility is generically higher and downstream facilitated, when compared to E. coli. However, deep analyses of recombinant protein quality in this system are still required to completely evaluate its performance and potential for improvement. We have explored here the conformational quality (through specific fluorescence emission) and solubility of an aggregation-prone GFP variant (VP1GFP) produced in L. lactis. In this context, our results show that parameters such as production time, culture conditions and growth temperature have a dramatic impact not only on protein yield, but also on protein solubility and conformational quality, that are particularly favored under fermentative metabolism. Metabolic regime and cultivation temperature greatly influence solubility and conformational quality of an aggregation-prone protein in L. lactis. Specifically, the present study proves that anaerobic growth is the optimal condition for recombinant protein production purposes. Besides, growth temperature plays an important role regulating both protein solubility and conformational quality. Additionally, our results also prove the great versatility for the manipulation of this bacterial system regarding the improvement of functionality, yield and quality of recombinant proteins in this species. These findings not only confirm L. lactis as an

  13. Detection and viability of Lactococcus lactis throughout cheese ripening.

    PubMed

    Ruggirello, Marianna; Dolci, Paola; Cocolin, Luca

    2014-01-01

    Recent evidences highlighted the presence of Lactococcus lactis during late cheese ripening. For this reason, the role of this microorganism, well known as dairy starter, should be reconsidered throughout cheese manufacturing and ripening. Thus, the main objective of this study was to develop a RT-qPCR protocol for the detection, quantification and determination of the viability of L. lactis in ripened cheese samples by direct analysis of microbial nucleic acids. Standard curves were constructed for the specific quantification of L. lactis in cheese matrices and good results in terms of selectivity, correlation coefficient and efficiency were obtained. Thirty-three ripened cheeses were analyzed and, on the basis of RNA analysis, twelve samples showed 106 to 108 CFU of L. lactis per gram of product, thirteen from 103 to 105 CFU/g, and in eight cheeses, L. lactis was not detected. Traditional plating on M17 medium led to loads ranging from 105 to 109 CFU/g, including the cheese samples where no L. lactis was found by RT-qPCR. From these cheeses, none of the colonies isolated on M17 medium was identified as L. lactis species. These data could be interpreted as a lack of selectivity of M17 medium where colony growth is not always related to lactococcal species. At the same time, the absence or low abundance of L. lactis isolates on M17 medium from cheese where L. lactis was detected by RT-qPCR support the hypothesis that L. lactis starter populations are mainly present in viable but not culturable state during ripening and, for this reason, culture-dependent methods have to be supplemented with direct analysis of cheese.

  14. Mobile CRISPR/Cas-Mediated Bacteriophage Resistance in Lactococcus lactis

    PubMed Central

    Millen, Anne M.; Horvath, Philippe; Boyaval, Patrick; Romero, Dennis A.

    2012-01-01

    Lactococcus lactis is a biotechnological workhorse for food fermentations and potentially therapeutic products and is therefore widely consumed by humans. It is predominantly used as a starter microbe for fermented dairy products, and specialized strains have adapted from a plant environment through reductive evolution and horizontal gene transfer as evidenced by the association of adventitious traits with mobile elements. Specifically, L. lactis has armed itself with a myriad of plasmid-encoded bacteriophage defensive systems to protect against viral predation. This known arsenal had not included CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins), which forms a remarkable microbial immunity system against invading DNA. Although CRISPR/Cas systems are common in the genomes of closely related lactic acid bacteria (LAB), none was identified within the eight published lactococcal genomes. Furthermore, a PCR-based search of the common LAB CRISPR/Cas systems (Types I and II) in 383 industrial L. lactis strains proved unsuccessful. Here we describe a novel, Type III, self-transmissible, plasmid-encoded, phage-interfering CRISPR/Cas discovered in L. lactis. The native CRISPR spacers confer resistance based on sequence identity to corresponding lactococcal phage. The interference is directed at phages problematic to the dairy industry, indicative of a responsive system. Moreover, targeting could be modified by engineering the spacer content. The 62.8-kb plasmid was shown to be conjugally transferrable to various strains. Its mobility should facilitate dissemination within microbial communities and provide a readily applicable system to naturally introduce CRISPR/Cas to industrially relevant strains for enhanced phage resistance and prevention against acquisition of undesirable genes. PMID:23240053

  15. Detection and Viability of Lactococcus lactis throughout Cheese Ripening

    PubMed Central

    Cocolin, Luca

    2014-01-01

    Recent evidences highlighted the presence of Lactococcus lactis during late cheese ripening. For this reason, the role of this microorganism, well known as dairy starter, should be reconsidered throughout cheese manufacturing and ripening. Thus, the main objective of this study was to develop a RT-qPCR protocol for the detection, quantification and determination of the viability of L. lactis in ripened cheese samples by direct analysis of microbial nucleic acids. Standard curves were constructed for the specific quantification of L. lactis in cheese matrices and good results in terms of selectivity, correlation coefficient and efficiency were obtained. Thirty-three ripened cheeses were analyzed and, on the basis of RNA analysis, twelve samples showed 106 to 108 CFU of L. lactis per gram of product, thirteen from 103 to 105 CFU/g, and in eight cheeses, L. lactis was not detected. Traditional plating on M17 medium led to loads ranging from 105 to 109 CFU/g, including the cheese samples where no L. lactis was found by RT-qPCR. From these cheeses, none of the colonies isolated on M17 medium was identified as L. lactis species. These data could be interpreted as a lack of selectivity of M17 medium where colony growth is not always related to lactococcal species. At the same time, the absence or low abundance of L. lactis isolates on M17 medium from cheese where L. lactis was detected by RT-qPCR support the hypothesis that L. lactis starter populations are mainly present in viable but not culturable state during ripening and, for this reason, culture-dependent methods have to be supplemented with direct analysis of cheese. PMID:25503474

  16. Gene inactivation in Lactococcus lactis: histidine biosynthesis.

    PubMed Central

    Delorme, C; Godon, J J; Ehrlich, S D; Renault, P

    1993-01-01

    Lactococcus lactis strains from dairy and nondairy sources were tested for the ability to grow in the absence of histidine. Among 60 dairy strains tested, 56 required histidine, whereas only 1 of 11 nondairy strains had this requirement. Moreover, 10 of the 56 auxotrophic strains were able to grow in the presence of histidinol (Hol+), the immediate histidine precursor. This indicates that adaptation to milk often results in histidine auxotrophy. The histidine operon was detected by Southern hybridization in eight dairy auxotrophic strains tested. A large part of the histidine operon (8 kb, containing seven histidine biosynthetic genes and three unrelated open reading frames [ORFs]) was cloned from an auxotroph, which had an inactive hisD gene, as judged by its inability to grow on histidinol. Complementation analysis of three genes, hisA, hisB, and hisG, in Escherichia coli showed that they also were inactive. Sequence analysis of the cloned histidine region, which revealed 98.6% overall homology with that of the previously analyzed prototrophic strain, showed the presence of frameshift mutations in three his genes, hisC, hisG, and hisH, and two genes unrelated to histidine biosynthesis, ORF3 and ORF6. In addition, several mutations were detected in the promoter region of the operon. Northern (RNA) hybridization analysis showed a much lower amount of the his transcript in the auxotrophic strain than in the prototrophic strain. The mutations detected account for the histidine auxotrophy of the analyzed strain. Certain other dairy auxotrophic strains carry a lower number of mutations, since they were able to revert either to a Hol+ phenotype or to histidine prototrophy. Images PMID:7687248

  17. Bacteriocinogenic Lactococcus lactis subsp. lactis DF04Mi isolated from goat milk: Evaluation of the probiotic potential

    PubMed Central

    Furtado, Danielle N.; Todorov, Svetoslav D.; Landgraf, Mariza; Destro, Maria T.; Franco, Bernadette D.G.M.

    2014-01-01

    Lactic acid bacteria capable of producing bacteriocins and presenting probiotic potential open innovative technological applications in the dairy industry. In this study, a bacteriocinogenic strain (Lactococcus lactis subsp. lactis DF4Mi) was isolated from goat milk, and studied for its probiotic potential. Lc. lactis DF4Mi was resistant to acidic pH and oxbile, presented co-aggregation with Listeria monocytogenes, and was not affected by several drugs from different generic groups, being sensitive to most tested antibiotics. These properties indicate that this Lc. lactis strain can be used for enhancement of dairy foods safety and quality, in combination with potential probiotic properties. PMID:25477942

  18. Bacteriocinogenic Lactococcus lactis subsp. lactis DF04Mi isolated from goat milk: evaluation of the probiotic potential.

    PubMed

    Furtado, Danielle N; Todorov, Svetoslav D; Landgraf, Mariza; Destro, Maria T; Franco, Bernadette D G M

    2014-01-01

    Lactic acid bacteria capable of producing bacteriocins and presenting probiotic potential open innovative technological applications in the dairy industry. In this study, a bacteriocinogenic strain (Lactococcus lactis subsp. lactis DF4Mi) was isolated from goat milk, and studied for its probiotic potential. Lc. lactis DF4Mi was resistant to acidic pH and oxbile, presented co-aggregation with Listeria monocytogenes, and was not affected by several drugs from different generic groups, being sensitive to most tested antibiotics. These properties indicate that this Lc. lactis strain can be used for enhancement of dairy foods safety and quality, in combination with potential probiotic properties.

  19. From meadows to milk to mucosa - adaptation of Streptococcus and Lactococcus species to their nutritional environments.

    PubMed

    Price, Claire E; Zeyniyev, Araz; Kuipers, Oscar P; Kok, Jan

    2012-09-01

    Lactic acid bacteria (LAB) are indigenous to food-related habitats as well as associated with the mucosal surfaces of animals. The LAB family Streptococcaceae consists of the genera Lactococcus and Streptococcus. Members of the family include the industrially important species Lactococcus lactis, which has a long history safe use in the fermentative food industry, and the disease-causing streptococci Streptococcus pneumoniae and Streptococcus pyogenes. The central metabolic pathways of the Streptococcaceae family have been extensively studied because of their relevance in the industrial use of some species, as well as their influence on virulence of others. Recent developments in high-throughput proteomic and DNA-microarray techniques, in in vivo NMR studies, and importantly in whole-genome sequencing have resulted in new insights into the metabolism of the Streptococcaceae family. The development of cost-effective high-throughput sequencing has resulted in the publication of numerous whole-genome sequences of lactococcal and streptococcal species. Comparative genomic analysis of these closely related but environmentally diverse species provides insight into the evolution of this family of LAB and shows that the relatively small genomes of members of the Streptococcaceae family have been largely shaped by the nutritionally rich environments they inhabit. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  20. From Genome to Phenotype: An Integrative Approach to Evaluate the Biodiversity of Lactococcus lactis

    PubMed Central

    Laroute, Valérie; Tormo, Hélène; Couderc, Christel; Mercier-Bonin, Muriel; Le Bourgeois, Pascal; Cocaign-Bousquet, Muriel; Daveran-Mingot, Marie-Line

    2017-01-01

    Lactococcus lactis is one of the most extensively used lactic acid bacteria for the manufacture of dairy products. Exploring the biodiversity of L. lactis is extremely promising both to acquire new knowledge and for food and health-driven applications. L. lactis is divided into four subspecies: lactis, cremoris, hordniae and tructae, but only subsp. lactis and subsp. cremoris are of industrial interest. Due to its various biotopes, Lactococcus subsp. lactis is considered the most diverse. The diversity of L. lactis subsp. lactis has been assessed at genetic, genomic and phenotypic levels. Multi-Locus Sequence Type (MLST) analysis of strains from different origins revealed that the subsp. lactis can be classified in two groups: “domesticated” strains with low genetic diversity, and “environmental” strains that are the main contributors of the genetic diversity of the subsp. lactis. As expected, the phenotype investigation of L. lactis strains reported here revealed highly diverse carbohydrate metabolism, especially in plant- and gut-derived carbohydrates, diacetyl production and stress survival. The integration of genotypic and phenotypic studies could improve the relevance of screening culture collections for the selection of strains dedicated to specific functions and applications. PMID:28534821

  1. Nisin-Producing Lactococcus lactis Strains Isolated from Human Milk

    PubMed Central

    Beasley, Shea S.; Saris, Per E. J.

    2004-01-01

    Characterization by partial 16S rRNA gene sequencing, ribotyping, and green fluorescent protein-based nisin bioassay revealed that 6 of 20 human milk samples contained nisin-producing Lactococcus lactis bacteria. This suggests that the history of humans consuming nisin is older than the tradition of consuming fermented milk products. PMID:15294850

  2. Plasmid transfer via transduction from Streptococcus thermophilus to Lactococcus lactis.

    PubMed

    Ammann, Andreas; Neve, Horst; Geis, Arnold; Heller, Knut J

    2008-04-01

    Using Streptococcus thermophilus phages, plasmid transduction in Lactococcus lactis was demonstrated. The transduction frequencies were 4 orders of magnitude lower in L. lactis than in S. thermophilus. These results are the first evidence that there is phage-mediated direct transfer of DNA from S. thermophilus to L. lactis. The implications of these results for phage evolution are discussed.

  3. Validation of a model for growth of Lactococcus lactis and Listeria innocua in a structured gel system: effect of monopotassium phosphate.

    PubMed

    Antwi, M; Theys, T E; Bernaerts, K; Van Impe, J F; Geeraerd, A H

    2008-07-31

    The effect of monopotassium phosphate (KH(2)PO(4)) on the chemical environment and on growth of Listeria innocua and Lactococcus lactis in coculture were investigated in a liquid and in a gelled microbiological medium at 12 degrees C and an initial pH of 6.2. As expected, addition of KH(2)PO(4) to both the liquid and gelled media resulted in an increase in buffering capacity. This effect on buffering capacity changed the profiles of lactic acid dissociation and pH evolution. At all gelatin concentrations studied, addition of KH(2)PO(4) increased the growth rate and the stationary cell concentration of L. lactis. In addition, the growth rate of L. innocua slightly increased but, in contrast, the stationary cell concentration remained unchanged. A new class of predictive models developed previously in our research team to quantify the effect of food model gel structure on microbial growth [Antwi, M., Bernaerts, K., Van Impe, J. F., Geeraerd, A. H., 2007. Modelling the combined effect of food model system and lactic acid on L. innocua and L. lactis growth in mono- and coculture. International Journal of Food Microbiology 120, 71-84] was applied. Our analysis indicate that KH(2)PO(4) influenced the parameters of the chemical and microbiological subprocesses of the model. Nonetheless, the growth model satisfactorily predicted the stationary cell concentration when (i) the undissociated lactic acid concentrations at which L. innocua and L. lactis growth cease were chosen as previously reported, and (ii) all other parameters of the chemical and microbiological subprocesses were computed for each medium. This confirms that the undissociated lactic acid concentrations at which growth ceases is a unique property of a bacterium and does not, within our case study, depend on growth medium. The study indicates that microbial growth depends on the interplay between the individual food components which affect the physicochemical properties of the food, such as the buffering

  4. Low nitrogen stress stimulating the indole-3-acetic acid biosynthesis of Serratia sp. ZM is vital for the survival of the bacterium and its plant growth-promoting characteristic.

    PubMed

    Ouyang, Liming; Pei, Haiyan; Xu, Zhaohui

    2017-04-01

    Serratia sp. ZM is a plant growth-promoting (PGP) bacterial strain isolated from the rhizospheric soil of Populus euphratica in northwestern China. In this study, low nitrogen supply significantly stimulated the production of indole-3-acetic acid (IAA) in Serratia sp.ZM. The inoculation of the bacterium to wheat seedlings improved plant growth compared with the uninoculated group, and the stimulating effect was more prominent under low nitrogen stress. Inactivation of the predicted key gene in the IAA biosynthesis pathway impaired IAA production and significantly hampered mutant growth in poor medium. Furthermore, the IAA-deficient mutant lost the PGP effect under either normal or low nitrogen conditions in plant experiments. This study revealed the significant impact of environmental nitrogen levels on IAA production in the PGP strain and the vital effect of IAA on resistance physiology of both the bacterium and host plant. The characteristics of Serratia sp. ZM also indicated its application potential as a biofertilizer for plants, especially those suffering from poor nitrogen soil.

  5. Adaptation of Lactococcus lactis to high growth temperature leads to a dramatic increase in acidification rate

    PubMed Central

    Chen, Jun; Shen, Jing; Ingvar Hellgren, Lars; Ruhdal Jensen, Peter; Solem, Christian

    2015-01-01

    Lactococcus lactis is essential for most cheese making, and this mesophilic bacterium has its growth optimum around 30 °C. We have, through adaptive evolution, isolated a mutant TM29 that grows well up to 39 °C, and continuous growth at 40 °C is possible if pre-incubated at a slightly lower temperature. At the maximal permissive temperature for the wild-type, 38 °C, TM29 grows 33% faster and has a 12% higher specific lactate production rate than its parent MG1363, which results in fast lactate accumulation. Genome sequencing was used to reveal the mutations accumulated, most of which were shown to affect thermal tolerance. Of the mutations with more pronounced effects, two affected expression of single proteins (chaperone; riboflavin transporter), two had pleiotropic effects (RNA polymerase) which changed the gene expression profile, and one resulted in a change in the coding sequence of CDP-diglyceride synthase. A large deletion containing 10 genes was also found to affect thermal tolerance significantly. With this study we demonstrate a simple approach to obtain non-GMO derivatives of the important L. lactis that possess properties desirable by the industry, e.g. thermal robustness and increased rate of acidification. The mutations we have identified provide a genetic basis for further investigation of thermal tolerance. PMID:26388459

  6. Adaptation of Lactococcus lactis to high growth temperature leads to a dramatic increase in acidification rate.

    PubMed

    Chen, Jun; Shen, Jing; Ingvar Hellgren, Lars; Ruhdal Jensen, Peter; Solem, Christian

    2015-09-21

    Lactococcus lactis is essential for most cheese making, and this mesophilic bacterium has its growth optimum around 30 °C. We have, through adaptive evolution, isolated a mutant TM29 that grows well up to 39 °C, and continuous growth at 40 °C is possible if pre-incubated at a slightly lower temperature. At the maximal permissive temperature for the wild-type, 38 °C, TM29 grows 33% faster and has a 12% higher specific lactate production rate than its parent MG1363, which results in fast lactate accumulation. Genome sequencing was used to reveal the mutations accumulated, most of which were shown to affect thermal tolerance. Of the mutations with more pronounced effects, two affected expression of single proteins (chaperone; riboflavin transporter), two had pleiotropic effects (RNA polymerase) which changed the gene expression profile, and one resulted in a change in the coding sequence of CDP-diglyceride synthase. A large deletion containing 10 genes was also found to affect thermal tolerance significantly. With this study we demonstrate a simple approach to obtain non-GMO derivatives of the important L. lactis that possess properties desirable by the industry, e.g. thermal robustness and increased rate of acidification. The mutations we have identified provide a genetic basis for further investigation of thermal tolerance.

  7. Protective effect of clove oil-supplemented fish diets on experimental Lactococcus garvieae infection in tilapia.

    PubMed

    Rattanachaikunsopon, Pongsak; Phumkhachorn, Parichat

    2009-09-01

    The essential oils extracted from the four herbs, cinnamon (Cinnamomum verum), clove (Syzygium aromaticum), ginger (Zingiber officinale) and holy basil (Ocimum sanctum), were investigated for their antimicrobial activity and mode of action against Lactococcus garvieae, a fish pathogenic bacteria causing lactococcosis. Of all the tested oils, clove oil had the strongest inhibitory effect and exhibited a bactericidal mode of action against the pathogenic bacterium. When an intraperitoneal infection of tilapia (Oreochromis niloticus) with L. garvieae was performed, the median lethal dose (LD(50)) was determined to be 1.78x10(2) CFU/fish. For an in vivo trial, no mortality was apparent in fish fed on the fish diets supplemented with 3% (w/w) of clove oil and with 0.5% (w/w) of oxytetracycline 5 d prior to the infection with L. garvieae. These results indicate that clove oil had a protective effect on experimental L. garvieae infection in tilapia and the potential to replace antibiotics for controlling the disease.

  8. Precision genome engineering in lactic acid bacteria

    PubMed Central

    2014-01-01

    Innovative new genome engineering technologies for manipulating chromosomes have appeared in the last decade. One of these technologies, recombination mediated genetic engineering (recombineering) allows for precision DNA engineering of chromosomes and plasmids in Escherichia coli. Single-stranded DNA recombineering (SSDR) allows for the generation of subtle mutations without the need for selection and without leaving behind any foreign DNA. In this review we discuss the application of SSDR technology in lactic acid bacteria, with an emphasis on key factors that were critical to move this technology from E. coli into Lactobacillus reuteri and Lactococcus lactis. We also provide a blueprint for how to proceed if one is attempting to establish SSDR technology in a lactic acid bacterium. The emergence of CRISPR-Cas technology in genome engineering and its potential application to enhancing SSDR in lactic acid bacteria is discussed. The ability to perform precision genome engineering in medically and industrially important lactic acid bacteria will allow for the genetic improvement of strains without compromising safety. PMID:25185700

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

    PubMed

    Gansel, X; Dutreix, M; Hartke, A; Boutibonnes, P; Auffray, Y

    1993-11-01

    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.

  10. 3-mercaptopropionate dioxygenase, a cysteine dioxygenase homologue, catalyzes the initial step of 3-mercaptopropionate catabolism in the 3,3-thiodipropionic acid-degrading bacterium variovorax paradoxus.

    PubMed

    Bruland, Nadine; Wübbeler, Jan Hendrik; Steinbüchel, Alexander

    2009-01-02

    The thioether 3,3-thiodipropionic acid can be used as precursor substrate for biotechnological synthesis of 3-mercaptopropionic acid-containing polythioesters. Therefore, the hitherto unknown catabolism of this compound was elucidated to engineer novel and improved polythioester biosynthesis pathways in the future. Bacteria capable of using 3,3-thiodipropionic acid as the sole source of carbon and energy for growth were enriched from the environment. From eleven isolates, TBEA3, TBEA6, and SFWT were morphologically and physiologically characterized. Their 16 S rDNAs and other features affiliated these isolates to the beta-subgroup of the proteobacteria. Tn5::mob mutagenesis of isolate Variovorax paradoxus TBEA6 yielded ten mutants fully or partially impaired in growth on 3,3-thiodipropionic acid. Genotypic characterization of two 3,3-thiodipropionic acid-negative mutants demonstrated the involvement of a bacterial cysteine dioxygenase (EC 1.13.11.22) homologue in the further catabolism of the 3,3-thiodipropionic acid cleavage product 3-mercaptopropionic acid. Detection of 3-sulfinopropionate in the supernatant of one of these mutants during cultivation on 3,3-thiodipropionic acid as well as in vivo and in vitro enzyme assays using purified protein demonstrated oxygenation of 3-mercaptopropionic acid to 3-sulfinopropionate by this enzyme; cysteine and cysteamine were not used as substrate. Beside cysteine dioxygenase and cysteamine dioxygenase, this 3-mercaptopropionic acid dioxygenase is the third example for a thiol dioxygenase and the first report about the microbial catabolism of 3-mercaptopropionic acid. Insertion of Tn5::mob in a gene putatively coding for a family III acyl-CoA-transferase resulted in the accumulation of 3-sulfinopropionate during cultivation on 3,3-thiodipropionic acid, indicating that this compound is further metabolized to 3-sulfinopropionyl-CoA and subsequently to propionyl-CoA.

  11. Bacteriocinogenic Lactococcus lactis subsp. lactis DF04Mi isolated from goat milk: Characterization of the bacteriocin

    PubMed Central

    Furtado, Danielle N.; Todorov, Svetoslav D.; Landgraf, Mariza; Destro, Maria T.; Franco, Bernadette D.G.M.

    2014-01-01

    Lactic acid bacteria capable of producing bacteriocins and presenting probiotic potential open innovative technological applications in the dairy industry. In this study, a bacteriocinogenic strain (Lactococcus lactis subsp. lactis DF4Mi) was isolated from goat milk, and studied for its antimicrobial activity. The bacteriocin presented a broad spectrum of activity, was sensitive to proteolytic enzymes, resistant to heat and pH extremes, and not affected by the presence of SDS, Tween 20, Tween 80, EDTA or NaCl. Bacteriocin production was dependent on the components of the culture media, especially nitrogen source and salts. When tested by PCR, the bacteriocin gene presented 100% homology to nisin Z gene. These properties indicate that this L. lactis subsp. lactis DF4Mi can be used for enhancement of dairy foods safety and quality. PMID:25763065

  12. Lactococcus lactis is capable of improving the riboflavin status in deficient rats.

    PubMed

    LeBlanc, Jean Guy; Burgess, Catherine; Sesma, Fernando; de Giori, Graciela Savoy; van Sinderen, Douwe

    2005-08-01

    Lactococcus lactis is a commonly used starter strain that can be converted from a vitamin B2 consumer into a vitamin B2 'factory' by over-expressing its riboflavin biosynthesis genes. The present study was conducted to assess in a rat bioassay the response of riboflavin produced by GM or native lactic acid bacteria (LAB). The riboflavin-producing strains were able to eliminate most physiological manifestations of ariboflavinosis such as stunted growth, elevated erythrocyte glutathione reductase activation coefficient values and hepatomegalia that were observed using a riboflavin depletion-repletion model. Riboflavin status and growth rates were greatly improved when the depleted rats were fed with cultures of L. lactis that overproduced this vitamin whereas the native strain did not show the same effect. The present study is the first animal trial with food containing living bacteria that were engineered to overproduce riboflavin. These results pave the way for analysing the effect of similar riboflavin-overproducing LAB in human trials.

  13. Inflammation-related pro-apoptotic activity of exopolysaccharides isolated from Lactococcus lactis subsp. lactis.

    PubMed

    Wu, Z; Wang, G; Pan, D; Guo, Y; Zeng, X; Sun, Y; Cao, J

    2016-11-30

    Exopolysaccharides (EPS) have attracted attention recently for possible use in suppressing early stage breast cancer. In this study, a mannan EPS produced by Lactococcus lactis subsp. lactis was found to affect the production of inflammatory cytokines. EPS (300 μg/ml) can significantly enhance tumour necrosis factor alpha and inducible NO synthase release in MCF-7 cells compared to control cells in a concentration-dependent manner. Also the intracellular calcium level was found to increase with the concentration of EPS. After EPS-treatment, a significant reduction in mitochondrial potential was observed, as was nuclear condensation and cell shrinkage. These results may be helpful in further understanding the anti-tumour properties of lactic acid bacteria.

  14. Bacteriocinogenic Lactococcus lactis subsp. lactis DF04Mi isolated from goat milk: characterization of the bacteriocin.

    PubMed

    Furtado, Danielle N; Todorov, Svetoslav D; Landgraf, Mariza; Destro, Maria T; Franco, Bernadette D G M

    2014-01-01

    Lactic acid bacteria capable of producing bacteriocins and presenting probiotic potential open innovative technological applications in the dairy industry. In this study, a bacteriocinogenic strain (Lactococcus lactis subsp. lactis DF4Mi) was isolated from goat milk, and studied for its antimicrobial activity. The bacteriocin presented a broad spectrum of activity, was sensitive to proteolytic enzymes, resistant to heat and pH extremes, and not affected by the presence of SDS, Tween 20, Tween 80, EDTA or NaCl. Bacteriocin production was dependent on the components of the culture media, especially nitrogen source and salts. When tested by PCR, the bacteriocin gene presented 100% homology to nisin Z gene. These properties indicate that this L. lactis subsp. lactis DF4Mi can be used for enhancement of dairy foods safety and quality.

  15. Draft Genome Sequence of Lactococcus lactis subsp. lactis bv. diacetylactis CRL264, a Citrate-Fermenting Strain

    PubMed Central

    Zuljan, Federico; Espariz, Martín; Blancato, Victor S.; Esteban, Luis; Alarcón, Sergio

    2016-01-01

    We report the draft genome sequence of Lactococcus lactis subsp. lactis bv. diacetylactis CRL264, a natural strain isolated from artisanal cheese from northwest Argentina. L. lactis subsp. lactis bv. diacetylactis is one of the most important microorganisms used as starter culture around the world. The CRL264 strain constitutes a model microorganism in the studies on the generation of aroma compounds (diacetyl, acetoin, and 2,3-butanediol) by lactic acid bacteria. Our genome analysis shows similar genetic organization to other available genomes of L. lactis bv. diacetylactis strains. PMID:26847906

  16. Draft Genome Sequence of Lactococcus lactis subsp. lactis bv. diacetylactis CRL264, a Citrate-Fermenting Strain.

    PubMed

    Zuljan, Federico; Espariz, Martín; Blancato, Victor S; Esteban, Luis; Alarcón, Sergio; Magni, Christian

    2016-02-04

    We report the draft genome sequence of Lactococcus lactis subsp. lactis bv. diacetylactis CRL264, a natural strain isolated from artisanal cheese from northwest Argentina. L. lactis subsp. lactis bv. diacetylactis is one of the most important microorganisms used as starter culture around the world. The CRL264 strain constitutes a model microorganism in the studies on the generation of aroma compounds (diacetyl, acetoin, and 2,3-butanediol) by lactic acid bacteria. Our genome analysis shows similar genetic organization to other available genomes of L. lactis bv. diacetylactis strains. Copyright © 2016 Zuljan et al.

  17. Characterization of Garvicin ML, a Novel Circular Bacteriocin Produced by Lactococcus garvieae DCC43, Isolated from Mallard Ducks (Anas platyrhynchos)▿

    PubMed Central

    Borrero, Juan; Brede, Dag A.; Skaugen, Morten; Diep, Dzung B.; Herranz, Carmen; Nes, Ingolf F.; Cintas, Luis M.; Hernández, Pablo E.

    2011-01-01

    Lactococcus garvieae DCC43 produces a bacteriocin, garvicin ML (GarML), with a molecular mass of 6,004.2 Da. Data from de novo amino acid sequencing by tandem mass spectrometry and nucleotide sequencing by reverse genetics suggested that the bacteriocin is synthesized as a 63-amino-acid precursor with a 3-amino-acid leader peptide that is removed by cleavage. Subsequently, a covalent linkage between the N and C termini forms the mature version of this novel 60-amino-acid circular bacteriocin. PMID:21057028

  18. pSEUDO, a genetic integration standard for Lactococcus lactis.

    PubMed

    Pinto, Joao P C; Zeyniyev, Araz; Karsens, Harma; Trip, Hein; Lolkema, Juke S; Kuipers, Oscar P; Kok, Jan

    2011-09-01

    Plasmid pSEUDO and derivatives were used to show that llmg_pseudo_10 in Lactococcus lactis MG1363 and its homologous locus in L. lactis IL1403 are suitable for chromosomal integrations. L. lactis MG1363 and IL1403 nisin-induced controlled expression (NICE) system derivatives (JP9000 and IL9000) and two general stress reporter strains (NZ9000::PhrcA-GFP and NZ9000::PgroES-GFP) enabling in vivo noninvasive monitoring of cellular fitness were constructed.

  19. A Deficiency in Aspartate Biosynthesis in Lactococcus lactis subsp. lactis C2 Causes Slow Milk Coagulation†

    PubMed Central

    Wang, Hua; Yu, Weizhu; Coolbear, Tim; O’Sullivan, Dan; McKay, Larry L.

    1998-01-01

    A mutant of fast milk-coagulating (Fmc+) Lactococcus lactis subsp. lactis C2, designated L. lactis KB4, was identified. Although possessing the known components essential for utilizing casein as a nitrogen source, which include functional proteinase (PrtP) activity and oligopeptide, di- and tripeptide, and amino acid transport systems, KB4 exhibited a slow milk coagulation (Fmc−) phenotype. When the amino acid requirements of L. lactis C2 were compared with those of KB4 by use of a chemically defined medium, it was found that KB4 was unable to grow in the absence of aspartic acid. This aspartic acid requirement could also be met by aspartate-containing peptides. The addition of aspartic acid to milk restored the Fmc+ phenotype of KB4. KB4 was found to be defective in pyruvate carboxylase and thus was deficient in the ability to form oxaloacetate and hence aspartic acid from pyruvate and carbon dioxide. The results suggest that when lactococci are propagated in milk, aspartate derived from casein is unable to meet fully the nutritional demands of the lactococci, and they become dependent upon aspartate biosynthesis. PMID:9572935

  20. Stimulation of respiratory immunity by oral administration of Lactococcus lactis.

    PubMed

    Villena, Julio; Medina, Marcela; Vintiñi, Elisa; Alvarez, Susana

    2008-08-01

    This work demonstrates that non-recombinant Lactococcus lactis NZ, administered by the oral route at the proper dose, is able to improve resistance against pneumococcal infection. Lactococcus lactis NZ oral administration was able to improve pathogen lung clearance, increased survival of infected mice, and reduced lung injuries. This effect was related to an upregulation of the respiratory innate and specific immune responses. Administration of L. lactis NZ improved production of bronchoalveolar lavage (BAL) fluid TNF-alpha, enhanced recruitment of neutrophils into the alveolar spaces, and induced a higher activation of BAL phagocytes compared with the control group. Lactococcus lactis NZ administered orally stimulated the IgA cycle, increased IgA+ cells in intestine and bronchus, and improved production of BAL IL-4 and IL-10 during infection. Moreover, mice treated with L. lactis NZ showed higher levels of BAL anti-pneumococcal IgA and IgG. Taking into consideration that orally administered L. lactis NZ stimulates both the innate and the specific immune responses in the respiratory tract and that bacterial and viral antigens have been efficiently produced in this strain, L. lactis NZ is an excellent candidate for the development of an effective pneumococcal oral vaccine.

  1. Whole-Genome Sequence Analysis of Bombella intestini LMG 28161T, a Novel Acetic Acid Bacterium Isolated from the Crop of a Red-Tailed Bumble Bee, Bombus lapidarius

    PubMed Central

    Li, Leilei; Illeghems, Koen; Van Kerrebroeck, Simon; Borremans, Wim; Cleenwerck, Ilse; Smagghe, Guy; De Vuyst, Luc

    2016-01-01

    The whole-genome sequence of Bombella intestini LMG 28161T, an endosymbiotic acetic acid bacterium (AAB) occurring in bumble bees, was determined to investigate the molecular mechanisms underlying its metabolic capabilities. The draft genome sequence of B. intestini LMG 28161T was 2.02 Mb. Metabolic carbohydrate pathways were in agreement with the metabolite analyses of fermentation experiments and revealed its oxidative capacity towards sucrose, D-glucose, D-fructose and D-mannitol, but not ethanol and glycerol. The results of the fermentation experiments also demonstrated that the lack of effective aeration in small-scale carbohydrate consumption experiments may be responsible for the lack of reproducibility of such results in taxonomic studies of AAB. Finally, compared to the genome sequences of its nearest phylogenetic neighbor and of three other insect associated AAB strains, the B. intestini LMG 28161T genome lost 69 orthologs and included 89 unique genes. Although many of the latter were hypothetical they also included several type IV secretion system proteins, amino acid transporter/permeases and membrane proteins which might play a role in the interaction with the bumble bee host. PMID:27851750

  2. Genome Sequence of a Lactococcus lactis Strain Isolated from Salmonid Intestinal Microbiota

    PubMed Central

    Opazo, Rafael; Gajardo, Felipe; Ruiz, Mauricio

    2016-01-01

    Lactococcus lactis is a common inhabitant of the intestinal microbiota of salmonids, especially those in aquaculture systems. Here, we present a genome sequence of a Lactococcus lactis strain isolated from the intestinal contents of rainbow trout reared in Chile. PMID:27563049

  3. Osteomyelitis and possible endocarditis secondary to Lactococcus garvieae: a first case report

    PubMed Central

    James, P; Hardman, S.; Patterson, D.

    2000-01-01

    Although osteomyelitis is commonly caused by staphylococcal infection, the first case of a lumbar osteomyelitis secondary to Lactococcus garvieae is reported. The case was complicated by possible endocarditis of an aortic valve prosthesis.


Keywords: Lactococcus garvieae; osteomyelitis PMID:10775286

  4. Influence of triethyl phosphate on phosphatase activity in shooting range soil: Isolation of a zinc-resistant bacterium with an acid phosphatase

    DOE PAGES

    Story, Sandra; Brigmon, Robin L.

    2016-12-19

    Phosphatase-mediated hydrolysis of organic phosphate may be a viable means of stabilizing heavy metals via precipitation as a metal phosphate in bioremediation applications. We investigated the effect of triethyl phosphate (TEP) on soil microbial-phosphatase activity in a heavy-metal contaminated soil. Gaseous TEP has been used at subsurface sites for bioremediation of organic contaminants but not applied in heavy-metal contaminated areas. Little is known about how TEP affects microbial activity in soils and it is postulated that TEP can serve as a phosphate source in nutrient-poor groundwater and soil/sediments. Over a 3-week period, TEP amendment to microcosms containing heavy-metal contaminated soilmore » resulted in increased activity of soil acid-phosphatase and repression of alkaline phosphatase, indicating a stimulatory effect on the microbial population. A soil-free enrichment of microorganisms adapted to heavy-metal and acidic conditions was derived from the TEP-amended soil microcosms using TEP as the sole phosphate source and the selected microbial consortium maintained a high acid-phosphatase activity with repression of alkaline phosphatase. Addition of 5 mM zinc to soil-free microcosms had little effect on acid phosphatase but inhibited alkaline phosphatase. One bacterial member from the consortium, identified as Burkholderia cepacia sp., expressed an acid-phosphatase activity uninhibited by high concentrations of zinc and produced a soluble, indigo pigment under phosphate limitation. The pigment was produced in a phosphate-free medium and was not produced in the presence of TEP or phosphate ion, indicative of purple acid-phosphatase types that are pressed by bioavailable phosphate. Finally, these results demonstrate that TEP amendment was bioavailable and increased overall phosphatase activity in both soil and soil-free microcosms supporting the possibility of positive outcomes in bioremediation applications.« less

  5. Endosymbiosis in trypanosomatids: the genomic cooperation between bacterium and host in the synthesis of essential amino acids is heavily influenced by multiple horizontal gene transfers

    PubMed Central

    2013-01-01

    Background Trypanosomatids of the genera Angomonas and Strigomonas live in a mutualistic association characterized by extensive metabolic cooperation with obligate endosymbiotic Betaproteobacteria. However, the role played by the symbiont has been more guessed by indirect means than evidenced. Symbiont-harboring trypanosomatids, in contrast to their counterparts lacking symbionts, exhibit lower nutritional requirements and are autotrophic for essential amino acids. To evidence the symbiont’s contributions to this autotrophy, entire genomes of symbionts and trypanosomatids with and without symbionts were sequenced here. Results Analyses of the essential amino acid pathways revealed that most biosynthetic routes are in the symbiont genome. By contrast, the host trypanosomatid genome contains fewer genes, about half of which originated from different bacterial groups, perhaps only one of which (ornithine cyclodeaminase, EC:4.3.1.12) derived from the symbiont. Nutritional, enzymatic, and genomic data were jointly analyzed to construct an integrated view of essential amino acid metabolism in symbiont-harboring trypanosomatids. This comprehensive analysis showed perfect concordance among all these data, and revealed that the symbiont contains genes for enzymes that complete essential biosynthetic routes for the host amino acid production, thus explaining the low requirement for these elements in symbiont-harboring trypanosomatids. Phylogenetic analyses show that the cooperation between symbionts and their hosts is complemented by multiple horizontal gene transfers, from bacterial lineages to trypanosomatids, that occurred several times in the course of their evolution. Transfers occur preferentially in parts of the pathways that are missing from other eukaryotes. Conclusion We have herein uncovered the genetic and evolutionary bases of essential amino acid biosynthesis in several trypanosomatids with and without endosymbionts, explaining and complementing decades of

  6. Influence of triethyl phosphate on phosphatase activity in shooting range soil: Isolation of a zinc-resistant bacterium with an acid phosphatase.

    PubMed

    Story, Sandra; Brigmon, Robin L

    2017-03-01

    Phosphatase-mediated hydrolysis of organic phosphate may be a viable means of stabilizing heavy metals via precipitation as a metal phosphate in bioremediation applications. We investigated the effect of triethyl phosphate (TEP) on soil microbial-phosphatase activity in a heavy-metal contaminated soil. Gaseous TEP has been used at subsurface sites for bioremediation of organic contaminants but not applied in heavy-metal contaminated areas. Little is known about how TEP affects microbial activity in soils and it is postulated that TEP can serve as a phosphate source in nutrient-poor groundwater and soil/sediments. Over a 3-week period, TEP amendment to microcosms containing heavy-metal contaminated soil resulted in increased activity of soil acid-phosphatase and repression of alkaline phosphatase, indicating a stimulatory effect on the microbial population. A soil-free enrichment of microorganisms adapted to heavy-metal and acidic conditions was derived from the TEP-amended soil microcosms using TEP as the sole phosphate source and the selected microbial consortium maintained a high acid-phosphatase activity with repression of alkaline phosphatase. Addition of 5mM zinc to soil-free microcosms had little effect on acid phosphatase but inhibited alkaline phosphatase. One bacterial member from the consortium, identified as Burkholderia cepacia sp., expressed an acid-phosphatase activity uninhibited by high concentrations of zinc and produced a soluble, indigo pigment under phosphate limitation. The pigment was produced in a phosphate-free medium and was not produced in the presence of TEP or phosphate ion, indicative of purple acid-phosphatase types that are pressed by bioavailable phosphate. These results demonstrate that TEP amendment was bioavailable and increased overall phosphatase activity in both soil and soil-free microcosms supporting the possibility of positive outcomes in bioremediation applications. Copyright © 2016. Published by Elsevier Inc.

  7. Influence of triethyl phosphate on phosphatase activity in shooting range soil: Isolation of a zinc-resistant bacterium with an acid phosphatase

    SciTech Connect

    Story, Sandra; Brigmon, Robin L.

    2016-12-19

    Phosphatase-mediated hydrolysis of organic phosphate may be a viable means of stabilizing heavy metals via precipitation as a metal phosphate in bioremediation applications. We investigated the effect of triethyl phosphate (TEP) on soil microbial-phosphatase activity in a heavy-metal contaminated soil. Gaseous TEP has been used at subsurface sites for bioremediation of organic contaminants but not applied in heavy-metal contaminated areas. Little is known about how TEP affects microbial activity in soils and it is postulated that TEP can serve as a phosphate source in nutrient-poor groundwater and soil/sediments. Over a 3-week period, TEP amendment to microcosms containing heavy-metal contaminated soil resulted in increased activity of soil acid-phosphatase and repression of alkaline phosphatase, indicating a stimulatory effect on the microbial population. A soil-free enrichment of microorganisms adapted to heavy-metal and acidic conditions was derived from the TEP-amended soil microcosms using TEP as the sole phosphate source and the selected microbial consortium maintained a high acid-phosphatase activity with repression of alkaline phosphatase. Addition of 5 mM zinc to soil-free microcosms had little effect on acid phosphatase but inhibited alkaline phosphatase. One bacterial member from the consortium, identified as Burkholderia cepacia sp., expressed an acid-phosphatase activity uninhibited by high concentrations of zinc and produced a soluble, indigo pigment under phosphate limitation. The pigment was produced in a phosphate-free medium and was not produced in the presence of TEP or phosphate ion, indicative of purple acid-phosphatase types that are pressed by bioavailable phosphate. Finally, these results demonstrate that TEP amendment was bioavailable and increased overall phosphatase activity in both soil and soil-free microcosms supporting the possibility of positive outcomes in bioremediation applications.

  8. Lactococcus lactis as an adjuvant and delivery vehicle of antigens against pneumococcal respiratory infections.

    PubMed

    Medina, Marcela; Vintiñi, Elisa; Villena, Julio; Raya, Raul; Alvarez, Susana

    2010-01-01

    Most studies of Lactococcus lactis as delivery vehicles of pneumococcal antigens are focused on the effectiveness of mucosal recombinant vaccines against Streptococcus pneumoniae in animal models. At present, there are three types of pneumococcal vaccines: capsular polysaccharide pneumococcal vaccines (PPV), protein-polysaccharide conjugate pneumococcal vaccines (PCV) and protein-based pneumococcal vaccines (PBPV). Only PPV and PCV have been licensed. These vaccines, however, do not represent a definitive solution. Novel, safe and inexpensive vaccines are necessary, especially in developing countries. Probiotic microorganisms such as lactic acid bacteria (LAB) are an interesting alternative for their use as vehicles in pneumococcal vaccines due to their GRAS (Generally Recognized As Safe) status. Thus, the adjuvanticity of Lactococcus lactis by itself represents added value over the use of other bacteria, a question dealt with in this review. In addition, the expression of different pneumococcal antigens as well as the use of oral and nasal mucosal routes of administration of lactococcal vaccines is considered. The advantages of nasal live vaccines are evident; nonetheless, oral vaccines can be a good alternative when the adequate dose is used. Another point addressed here is the use of live versus inactivated vaccines. In this sense, few researchers have focused on inactivated strains to be used as vaccines against pneumoccoccus. The immunogenicity of live vaccines is better than the one afforded by inactivated ones; however, the probiotic-inactivated vaccine combination has improved this matter considerably. The progress made so far in the protective immune response induced by recombinant vaccines, the successful trials in animal models and the safety considerations of their application in humans suggest that the use of recombinant vaccines represents a good short-term option in the control of pneumococcal diseases. © 2010 Landes Bioscience

  9. Lactococcus lactis as an adjuvant and delivery vehicle of antigens against pneumococcal respiratory infections

    PubMed Central

    Vintiñi, Elisa; Villena, Julio; Raya, Raul

    2010-01-01

    Most studies of Lactococcus lactis as delivery vehicles of pneumococcal antigens are focused on the effectiveness of mucosal recombinant vaccines against Streptococcus pneumoniae in animal models. At present, there are three types of pneumococcal vaccines: capsular polysaccharide pneumococcal vaccines (PPV), protein-polysaccharide conjugate pneumococcal vaccines (PCV) and protein-based pneumococcal vaccines (PBPV). Only PPV and PCV have been licensed. These vaccines, however, do not represent a definitive solution. Novel, safe and inexpensive vaccines are necessary, especially in developing countries. Probiotic microorganisms such as lactic acid bacteria (LAB) are an interesting alternative for their use as vehicles in pneumococcal vaccines due to their GRAS (Generally Recognized As Safe) status. Thus, the adjuvanticity of Lactococcus lactis by itself represents added value over the use of other bacteria, a question dealt with in this review. In addition, the expression of different pneumococcal antigens as well as the use of oral and nasal mucosal routes of administration of lactococcal vaccines is considered. The advantages of nasal live vaccines are evident; nonetheless, oral vaccines can be a good alternative when the adequate dose is used. Another point addressed here is the use of live versus inactivated vaccines. In this sense, few researchers have focused on inactivated strains to be used as vaccines against pneumoccoccus. The immunogenicity of live vaccines is better than the one afforded by inactivated ones; however, the probiotic-inactivated vaccine combination has improved this matter considerably. The progress made so far in the protective immune response induced by recombinant vaccines, the successful trials in animal models and the safety considerations of their application in humans suggest that the use of recombinant vaccines represents a good short-term option in the control of pneumococcal diseases. PMID:21326831

  10. Draft genome sequence of Sporolactobacillus inulinus strain CASD, an efficient D-lactic acid-producing bacterium with high-concentration lactate tolerance capability.

    PubMed

    Yu, Bo; Su, Fei; Wang, Limin; Xu, Ke; Zhao, Bo; Xu, Ping

    2011-10-01

    Sporolactobacillus inulinus CASD is an efficient D-lactic acid producer with high optical purity. Here we report for the first time the draft genome sequence of S. inulinus (2,930,096 bp). The large number of annotated two-component system genes makes it possible to explore the mechanism of extraordinary lactate tolerance of S. inulinus CASD.

  11. Transcription profiling of interactions between Lactococcus lactis subsp. cremoris SK11 and Lactobacillus paracasei ATCC 334 during Cheddar cheese simulation.

    PubMed

    Desfossés-Foucault, Émilie; LaPointe, Gisèle; Roy, Denis

    2014-05-16

    The starter cultures (Lactococcus sp.) and non-starter lactic acid bacteria (mostly Lactobacillus spp.) are essential to flavor development of Cheddar cheese. The aim of this study was to elucidate the transcriptional interaction between Lactococcus lactis subsp. cremoris SK11 and Lactobacillus paracasei ATCC 334 in mixed cultures during simulated Cheddar cheese manufacture (Pearce activity test) and ripening (slurry). Reverse transcription quantitative PCR (RT-qPCR) was used to quantify the expression of 34 genes common to both bacteria and for eight genes specific to either L. lactis subsp. cremoris SK11 or L. paracasei ATCC 334. The multifactorial analysis (MFA) performed on fold change results for each gene revealed that the genes linked to stress, protein and peptide degradation as well as carbohydrate metabolism of L. paracasei ATCC 334 were especially overexpressed in mixed culture with L. lactis subsp. cremoris SK11 during the ripening simulation. For L. lactis subsp. cremoris SK11, genes coding for amino acid metabolism were more expressed during the cheese manufacture simulation, especially in single culture. These results show how complementary functions of starter and NSLAB contribute to activities useful for flavor development.

  12. Incidence of nisin Z production in Lactococcus lactis subsp. lactis TFF 221 isolated from Thai fermented foods.

    PubMed

    Rattanachaikunsopon, Pongsak; Phumkhachorn, Parichat

    2008-10-01

    Lactic acid bacteria isolated from various Thai fermented foods were screened for the presence of nisin gene by using PCR with primers specific to nisin A structural gene. Only one strain, Lactococcus lactis subsp. lactis TFF 221, isolated from kung jom, a traditional shrimp paste, was found to carry a nisin gene. The TFF 221 nisin had antimicrobial activity against not only closely related lactic acid bacteria but also some foodborne pathogens. It was heat stable and inactivated by alpha-chymotrypsin and proteinase K. Some characteristics of TFF 221 nisin were found to be very similar to those of nisin A produced by Lactococcus lactis subsp. lactis NCDO 2111. Both of them had the same antimicrobial spectrum and MICs against all indicator bacteria. However, when assayed with indicator organisms, in all cases the TFF 221 nisin produced larger zones of inhibition in agar diffusion assays than the nisin A did. Sequencing of the TFF 221 nisin gene showed that it was the natural nisin variant, nisin Z, as indicated by the substitution of asparagine residue instead of histidine at position 27. The nisin determinant in strain TFF 221 was found to be located on a conjugative transposon residing in the chromosome. The ability of the nisin produced by L. lactis subsp. lactis TFF 221 to inhibit a wide range of foodborne pathogens may be useful in improving the food safety of the fermented product, especially in the Thai environment, which suffers from perennial problems of poor food hygiene.

  13. In Situ Determination of the Intracellular pH of Lactococcus lactis and Lactobacillus plantarum during Pressure Treatment

    PubMed Central

    Molina-Gutierrez, Adriana; Stippl, Volker; Delgado, Antonio; Gänzle, Michael G.; Vogel, Rudi F.

    2002-01-01

    Hydrostatic pressure may affect the intracellular pH of microorganisms by (i) enhancing the dissociation of weak organic acids and (ii) increasing the permeability of the cytoplasmic membrane and inactivation of enzymes required for pH homeostasis. The internal pHs of Lactococcus lactis and Lactobacillus plantarum during and after pressure treatment at 200 and 300 MPa and at pH values ranging from 4.0 to 6.5 were determined. Pressure treatment at 200 MPa for up to 20 min did not reduce the viability of either strain at pH 6.5. Pressure treatment at pH 6.5 and 300 MPa reduced viable cell counts of Lactococcus lactis and Lactobacillus plantarum by 5 log after 20 and 120 min, respectively. Pressure inactivation was faster at pH 5 or 4. At ambient pressure, both strains maintained a transmembrane pH gradient of 1 pH unit at neutral pH and about 2 pH units at pH 4.0. During pressure treatment at 200 and 300 MPa, the internal pH of L. lactis was decreased to the value of the extracellular pH during compression. The same result was observed during treatment of Lactobacillus plantarum at 300 MPa. Lactobacillus plantarum was unable to restore the internal pH after a compression-decompression cycle at 300 MPa and pH 6.5. Lactococcus lactis lost the ability to restore its internal pH after 20 and 4 min of pressure treatment at 200 and 300 MPa, respectively. As a consequence, pressure-mediated stress reactions and cell death may be considered secondary effects promoted by pH and other environmental conditions. PMID:12200293

  14. Role of two amino acid residues' insertion on thermal stability of thermophilic α-amylase AMY121 from a deep sea bacterium Bacillus sp. SCSIO 15121.

    PubMed

    Li, Lizhen; Yang, Jian; Li, Jie; Long, Lijuan; Xiao, Yunzhu; Tian, Xinpeng; Wang, Fazuo; Zhang, Si

    2015-05-01

    α-Amylases from Bacillus licheniformis (BLA) and Bacillus amyloliquefaciens (BAA) are both important industrial enzymes with high similarity in structure but significant differences in thermostability. The mechanisms underlying this discrepancy are still poorly understood. Here, we investigated the role of two amino acids' insertion on the thermostability of these two group amylases. A newly obtained thermophilic amylase AMY121 was found much closer to BLA in both primary structure and enzymological properties. Two amino acids' insertion widespread among BAA group α-amylases was identified as one of the key factors leading to the thermostability differences, since thermostability of insertion mutants (AMY121-EG and AMY121-AA) from AMY121 significantly decreased, while that of deletion mutant from BAA increased. Moreover, we proposed that conformational disturbance caused by insertion mutation might weaken the calcium-binding affinity and consequently decrease the enzyme thermostability.

  15. Proteomic Signature of Lactococcus lactis NCDO763 Cultivated in Milk†

    PubMed Central

    Gitton, Christophe; Meyrand, Mickael; Wang, Juhui; Caron, Christophe; Trubuil, Alain; Guillot, Alain; Mistou, Michel-Yves

    2005-01-01

    We have compared the proteomic profiles of L. lactis subsp. cremoris NCDO763 growing in the synthetic medium M17Lac, skim milk microfiltrate (SMM), and skim milk. SMM was used as a simple model medium to reproduce the initial phase of growth of L. lactis in milk. To widen the analysis of the cytoplasmic proteome, we used two different gel systems (pH ranges of 4 to 7 and 4.5 to 5.5), and the proteins associated with the cell envelopes were also studied by two-dimensional electrophoresis. In the course of the study, we analyzed about 800 spots and identified 330 proteins by mass spectrometry. We observed that the levels of more than 50 and 30 proteins were significantly increased upon growth in SMM and milk, respectively. The large redeployment of protein synthesis was essentially associated with an activation of pathways involved in the metabolism of nitrogenous compounds: peptidolytic and peptide transport systems, amino acid biosynthesis and interconversion, and de novo biosynthesis of purines. We also showed that enzymes involved in reactions feeding the purine biosynthetic pathway in one-carbon units and amino acids have an increased level in SMM and milk. The analysis of the proteomic data suggested that the glutamine synthetase (GS) would play a pivotal role in the adaptation to SMM and milk. The analysis of glnA expression during growth in milk and the construction of a glnA-defective mutant confirmed that GS is an essential enzyme for the development of L. lactis in dairy media. This analysis thus provides a proteomic signature of L. lactis, a model lactic acid bacterium, growing in its technological environment. PMID:16269754

  16. Identification and quantification of the caproic acid-producing bacterium Clostridium kluyveri in the fermentation of pit mud used for Chinese strong-aroma type liquor production.

    PubMed

    Hu, Xiao-long; Du, Hai; Xu, Yan

    2015-12-02

    Chinese strong-aroma type liquor (CSAL) is a popular distilled alcoholic beverage in China. It is produced by a complex fermentation process that is conducted in pits in the ground. Ethyl caproate is a key flavor compound in CSAL and is thought to originate from caproic acid produced by Clostridia inhabiting the fermentation pit mud. However, the particular species of Clostridium associated with this production are poorly understood and problematic to quantify by culturing. In this study, a total of 28 closest relatives including 15 Clostridia and 8 Bacilli species in pit muds from three CSAL distilleries, were detected by culture-dependent and -independent methods. Among them, Clostridium kluyveri was identified as the main producer of caproic acid. One representative strain C. kluyveri N6 could produce caproic, butyric and octanoic acids and their corresponding ethyl esters, contributing significantly to CSAL flavor. A real time quantitative PCR assay of C. kluyveri in pit muds developed showed that a concentration of 1.79×10(7) 16S rRNA gene copies/g pit mud in LZ-old pit was approximately six times higher than that in HLM and YH pits and sixty times higher than that in LZ-new pit respectively. This method can be used to improve the management of pit mud microbiology and its impact on CSAL quality. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Characterization of the cellulose-degrading bacterium NCIMB 10462

    SciTech Connect

    Dees, C.; Scott, T.C.; Phelps, T.J.

    1995-12-31

    The gram-negative cellulase-producing bacterium NCIMB 10462 has been previously named Pseudomonas fluorescens subsp. or var. cellulose. Because of renewed interest in cellulose-degrading bacteria for use in the bioconversion of cellulose to chemical feed stocks and fuels, we re-examined the characteristics of this microorganism to determine its true metabolic potential. Metabolic and physical characterization of NCIMB 10462 revealed that this is an alkalophilic, non-fermentative, gram-negative, oxidase-positive, motile, cellulose-degrading bacterium. The aerobic substrate utilization profile of this bacterium has few characteristics consistent with a classification of P. fluorescens and a very low probability match with the genus Sphingomonas. However, total lipid analysis did not reveal that any sphingolipid bases are produced by this bacterium. NCIMB 10462 grows best aerobically, but also grows well in complex media under reducing conditions. NCIMB 10462 grows slowly under anaerobic conditions on complex media, but growth on cellulosic media occurred only under aerobic conditions. Total fatty acid analysis (MIDI) of NCIMB 10462 failed to group this bacterium with a known pseudomonas species. However, fatty acid analysis of the bacteria when grown at temperatures below 37{degrees}C suggest that the organism is a pseudomonad. Since a predominant characteristic of this bacterium is its ability to degrade cellulose, we suggest that it be called Pseudomonas cellulosa.

  18. Taxonomic characterization of the cellulose-degrading bacterium NCIB 10462

    SciTech Connect

    Dees, C.; Ringleberg, D.; Scott, T.C.; Phelps, T.

    1994-06-01

    The gram negative cellulase-producing bacterium NCIB 10462 has been previously named Pseudomonas fluorescens subsp. or var. cellulosa. Since there is renewed interest in cellulose-degrading bacteria for use in bioconversion of cellulose to chemical feed stocks and fuels, we re-examined the characteristics of this microorganism to determine its proper taxonomic characterization and to further define it`s true metabolic potential. Metabolic and physical characterization of NCIB 10462 revealed that this was an alkalophilic, non-fermentative, gram negative, oxidase positive, motile, cellulose-degrading bacterium. The aerobic substrate utilization profile of this bacterium was found to have few characteristics consistent with a classification of P. fluorescens with a very low probability match with the genus Sphingomonas. Total lipid analysis did not reveal that any sphingolipid bases are produced by this bacterium. NCIB 10462 was found to grow best aerobically but also grows well in complex media under reducing conditions. NCIB 10462 grew slowly under full anaerobic conditions on complex media but growth on cellulosic media was found only under aerobic conditions. Total fatty acid analysis (MIDI) of NCIB 10462 failed to group this bacterium with a known pseudomonas species. However, fatty acid analysis of the bacteria when grown at temperatures below 37{degrees}C suggest that the organism is a pseudomonad. Since a predominant characteristic of this bacterium is it`s ability to degrade cellulose, we suggest it be called Pseudomonas cellulosa.

  19. A Computational Study of Amensalistic Control of Listeria monocytogenes by Lactococcus lactis under Nutrient Rich Conditions in a Chemostat Setting

    PubMed Central

    Khassehkhan, Hassan; Eberl, Hermann J.

    2016-01-01

    We study a previously introduced mathematical model of amensalistic control of the foodborne pathogen Listeria monocytogenes by the generally regarded as safe lactic acid bacteria Lactococcus lactis in a chemostat setting under nutrient rich growth conditions. The control agent produces lactic acids and thus affects pH in the environment such that it becomes detrimental to the pathogen while it is much more tolerant to these self-inflicted environmental changes itself. The mathematical model consists of five nonlinear ordinary differential equations for both bacterial species, the concentration of lactic acids, the pH and malate. The model is algebraically too involved to allow a comprehensive, rigorous qualitative analysis. Therefore, we conduct a computational study. Our results imply that depending on the growth characteristics of the medium in which the bacteria are cultured, the pathogen can survive in an intermediate flow regime but will be eradicated for slower flow rates and washed out for higher flow rates. PMID:28231156

  20. Induction of heat shock proteins DnaK, GroEL, and GroES by salt stress in Lactococcus lactis.

    PubMed Central

    Kilstrup, M; Jacobsen, S; Hammer, K; Vogensen, F K

    1997-01-01

    The bacterium Lactococcus lactis has become a model organism in studies of growth physiology and membrane transport, as a result of its simple fermentative metabolism. It is also used as a model for studying the importance of specific genes and functions during life in excess nutrients, by comparison of prototrophic wild-type strains and auxotrophic domesticated (dairy) strains. In a study of the capacity of domesticated strains to perform directed responses toward various stress conditions, we have analyzed the heat and salt stress response in the established L. lactis subsp. cremoris laboratory strain MG1363, which was originally derived from a dairy strain. After two-dimensional separation of proteins, the DnaK, GroEL, and GroES heat shock proteins, the HrcA (Orf1) heat shock repressor, and the glycolytic enzymes pyruvate kinase, glyceral-dehyde-3-phosphate dehydrogenase, and phosphoglycerate kinase were identified by a combination of Western blotting and direct N-terminal amino acid sequencing of proteins from the gels. Of 400 to 500 visible proteins, 17 were induced more than twofold during heat stress. Two classes of heat stress proteins were identified from their temporal induction pattern. The fast-induced proteins (including DnaK) showed an abruptly increased rate of synthesis during the first 10 min, declining to intermediate levels after 15 min. GroEL and GroES, which also belong to this group, maintained a high rate of synthesis after 15 min. The class of slowly induced proteins exhibited a gradual increase in the rate of synthesis after the onset of stress. Unlike other organisms, all salt stress-induced proteins in L. lactis were also subjected to heat stress induction. DnaK, GroEL, and GroES showed similar temporal patterns of induction during salt stress, resembling the timing during heat stress although at a lower induction level. These data indicate an overlap between the heat shock and salt stress responses in L. lactis. PMID:9143115

  1. Transcriptome and membrane fatty acid analyses reveal different strategies for responding to permeating and non-permeating solutes in the bacterium Sphingomonas wittichii

    PubMed Central

    2011-01-01

    Background Sphingomonas wittichii strain RW1 can completely oxidize dibenzo-p-dioxins and dibenzofurans, which are persistent contaminants of soils and sediments. For successful application in soil bioremediation systems, strain RW1 must cope with fluctuations in water availability, or water potential. Thus far, however, little is known about the adaptive strategies used by Sphingomonas bacteria to respond to changes in water potential. To improve our understanding, strain RW1 was perturbed with either the cell-permeating solute sodium chloride or the non-permeating solute polyethylene glycol with a molecular weight of 8000 (PEG8000). These solutes are assumed to simulate the solute and matric components of the total water potential, respectively. The responses to these perturbations were then assessed and compared using a combination of growth assays, transcriptome profiling, and membrane fatty acid analyses. Results Under conditions producing a similar decrease in water potential but without effect on growth rate, there was only a limited shared response to perturbation with sodium chloride or PEG8000. This shared response included the increased expression of genes involved with trehalose and exopolysaccharide biosynthesis and the reduced expression of genes involved with flagella biosynthesis. Mostly, the responses to perturbation with sodium chloride or PEG8000 were very different. Only sodium chloride triggered the increased expression of two ECF-type RNA polymerase sigma factors and the differential expression of many genes involved with outer membrane and amino acid metabolism. In contrast, only PEG8000 triggered the increased expression of a heat shock-type RNA polymerase sigma factor along with many genes involved with protein turnover and repair. Membrane fatty acid analyses further corroborated these differences. The degree of saturation of membrane fatty acids increased after perturbation with sodium chloride but had the opposite effect and decreased

  2. Falcatimonas natans gen. nov., sp. nov., a strictly anaerobic, amino-acid-decomposing bacterium isolated from a methanogenic reactor of cattle waste.

    PubMed

    Watanabe, Misa; Kaku, Nobuo; Ueki, Katsuji; Ueki, Atsuko

    2016-11-01

    A strictly anaerobic bacterial strain (WN011T) was isolated from a methanogenic reactor treating waste from cattle farms. Cells of the strain were Gram-stain-negative curved rods with a polar flagellum. Spores were not produced. The optimum temperature for growth was 35-37 °C and the optimum pH was 6.7. The strain did not utilize carbohydrates as growth substrates. The strain grew in PY medium and produced acetate, butyrate, isovalerate and H2 as well as propionate and isobutyrate as minor products. Amino acids (l-isoleucine, l-leucine, l-lysine, l-serine, l-threonine and l-valine) added to PY medium enhanced growth of the strain and increased the amounts of fermentation products. Oxidase, catalase and nitrate-reducing activities were negative. Hydrogen sulfide was produced. The genomic DNA G+C content was 38.8 mol%. Compounds related to iso-C15 : 0 (fatty acid, dimethylacetal and aldehyde) were detected as predominant components by the cellular fatty acids analysis. The diagnostic diamino acid of the cell-wall peptidoglycan was meso-diaminopimelic acid. On the basis of 16S rRNA gene sequences, three clones from wastewater were very closely related to strain WN011T (up to 99.9 % sequence similarity). The most closely related described species were those in cluster XIVa of the class Clostridia such as Ruminococcus gauvreauii (93.8 % 16S rRNA gene sequence similarity), Clostridium fimetarium (93.5 %) and Clostridium bolteae(93.5 %). Based on the distinct differences in phylogenetic and phenotypic characteristics of strain WN011T from those of related species, it is concluded that strain WN011T represents a novel species of a new genus in the family Lachnospiraceae, for which the name Falcatimonas natans gen. nov., sp. nov. is proposed. The type strain of the type species is WN011T (=JCM 16476T=DSM 22923T).

  3. Dethiosulfatibacter aminovorans gen. nov., sp. nov., a novel thiosulfate-reducing bacterium isolated from coastal marine sediment via sulfate-reducing enrichment with Casamino acids.

    PubMed

    Takii, Susumu; Hanada, Satoshi; Tamaki, Hideyuki; Ueno, Yutaka; Sekiguchi, Yuji; Ibe, Akihiro; Matsuura, Katsumi

    2007-10-01

    A sulfate-reducing enrichment culture originating from coastal marine sediment of the eutrophic Tokyo Bay, Japan, was successfully established with Casamino acids as a substrate. A thiosulfate reducer, strain C/G2(T), was isolated from the enrichment culture after further enrichment with glutamate. Cells of strain C/G2(T) were non-motile rods (0.6-0.8 microm x 2.2-4.8 microm) and were found singly or in pairs and sometimes in short chains. Spores were not formed. Cells of strain C/G2(T) stained Gram-negatively, despite possessing Gram-positive cell walls. The optimum temperature for growth was 28-30 degrees C, the optimum pH was around 7.8 and the optimum salt concentration was 20-30 g l(-1). Lactate, pyruvate, serine, cysteine, threonine, glutamate, histidine, lysine, arginine, Casamino acids, peptone and yeast extract were fermented as single substrates and no sugar was used as a fermentative substrate. A Stickland reaction was observed with some pairs of amino acids. Fumarate, alanine, proline, phenylalanine, tryptophan, glutamine and aspartate were utilized only in the presence of thiosulfate. Strain C/G2(T) fermented glutamate to H2, CO2, acetate and propionate. Thiosulfate and elemental sulfur were reduced to sulfide. Sulfate, sulfite and nitrate were not utilized as electron acceptors. The growth of strain C/G2(T) on Casamino acids or glutamate was enhanced by co-culturing with Desulfovibrio sp. isolated from the original mixed culture enriched with Casamino acids. The DNA G+C content of strain C/G2(T) was 41.0 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain C/G2(T) formed a distinct cluster with species of the genus Sedimentibacter. The closest relative was Sedimentibacter hydroxybenzoicus (with a gene sequence similarity of 91 %). On the basis of its phylogenetic and phenotypic properties, strain C/G2(T) (=JCM 13356(T)=NBRC 101112(T)=DSM 17477(T)) is proposed as representing a new genus and novel species, Dethiosulfatibacter

  4. Nasal administration of Lactococcus lactis improves local and systemic immune responses against Streptococcus pneumoniae.

    PubMed

    Medina, Marcela; Villena, Julio; Salva, Susana; Vintiñi, Elisa; Langella, Philippe; Alvarez, Susana

    2008-08-01

    Lactococcus lactis NZ9000 is a non-pathogenic non-invasive bacterium extensively used for the delivery of antigens and cytokines at the mucosal level. However, there are no reports concerning the per se immunomodulatory capacity of this strain. The aim of the present study was to investigate the intrinsic immunostimulating properties of the nasal administration of L. lactis NZ9000 in a pneumococcal infection model. Mice were preventively treated with L. lactis (2, 5 or 7 days with 10(8) cells/day per mouse) and then challenged with Streptococcus pneumoniae. The local and the systemic immune responses were evaluated. Our results showed that nasal administration of L. lactis for 5 days (LLN5d) increased the clearance rate of S. pneumoniae from lung and prevented the dissemination of pneumococci into blood. This effect coincided with an upregulation of the innate and specific immune responses in both local and systemic compartments. LLN5d increased phagocyte activation in lung, blood and bone marrow, determined by NBT and peroxidase tests. Anti-pneumococcal immunoglobulin (Ig)A in bronchoalveolar lavages (BAL) and IgG in BAL and serum were increased in the LLN5d group. Lung tissue injury was reduced by LLN5d treatment as revealed by histopathological examination and albumin concentration and lactate dehydrogenase activity in BAL. The adjuvant effect of L. lactis in our infection model would be an important advantage for its use as a delivery vehicle of pneumococcal proteins and nasal immunization with recombinant L. lactis emerges as an effective route of vaccination for both systemic and mucosal immunity against pneumococcal infection.

  5. Pilus Biogenesis in Lactococcus lactis: Molecular Characterization and Role in Aggregation and Biofilm Formation

    PubMed Central

    Oxaran, Virginie; Ledue-Clier, Florence; Dieye, Yakhya; Herry, Jean-Marie; Péchoux, Christine; Meylheuc, Thierry; Briandet, Romain; Juillard, Vincent; Piard, Jean-Christophe

    2012-01-01

    The genome of Lactococcus lactis strain IL1403 harbors a putative pilus biogenesis cluster consisting of a sortase C gene flanked by 3 LPxTG protein encoding genes (yhgD, yhgE, and yhhB), called here pil. However, pili were not detected under standard growth conditions. Over-expression of the pil operon resulted in production and display of pili on the surface of lactococci. Functional analysis of the pilus biogenesis machinery indicated that the pilus shaft is formed by oligomers of the YhgE pilin, that the pilus cap is formed by the YhgD pilin and that YhhB is the basal pilin allowing the tethering of the pilus fibers to the cell wall. Oligomerization of pilin subunits was catalyzed by sortase C while anchoring of pili to the cell wall was mediated by sortase A. Piliated L. lactis cells exhibited an auto-aggregation phenotype in liquid cultures, which was attributed to the polymerization of major pilin, YhgE. The piliated lactococci formed thicker, more aerial biofilms compared to those produced by non-piliated bacteria. This phenotype was attributed to oligomers of YhgE. This study provides the first dissection of the pilus biogenesis machinery in a non-pathogenic Gram-positive bacterium. Analysis of natural lactococci isolates from clinical and vegetal environments showed pili production under standard growth conditions. The identification of functional pili in lactococci suggests that the changes they promote in aggregation and biofilm formation may be important for the natural lifestyle as well as for applications in which these bacteria are used. PMID:23236417

  6. A Novel Uncultured Bacterium of the Family Gallionellaceae: Description and Genome Reconstruction Based on the Metagenomic Analysis of Microbial Community in Acid Mine Drainage.

    PubMed

    Kadnikov, V V; Ivasenko, D A; Beletsky, A V; Mardanov, A V; Danilova, E V; Pimenov, N V; Karnachuk, O V; Ravin, N V

    2016-07-01

    Drainage waters at the metal mining areas often have low pH and high content of dissolved metals due to oxidation of sulfide minerals. Extreme conditions limit microbial diversity in- such ecosystems. A drainage water microbial community (6.5'C, pH 2.65) in an open pit at the Sherlovaya Gora polymetallic open-cast mine (Transbaikal region, Eastern Siberia, Russia) was studied using metagenomic techniques. Metagenome sequencing provided information for taxonomic and functional characterization of the micro- bial community. The majority of microorganisms belonged to a single uncultured lineage representing a new Betaproteobacteria species of the genus Gallionella. While no.acidophiles are known among the cultured members of the family Gallionellaceae, similar 16S rRNA gene sequences were detected in acid mine drain- ages. Bacteria ofthe genera Thiobacillus, Acidobacterium, Acidisphaera, and Acidithiobacillus,-which are com- mon in acid mine drainage environments, were the minor components of the community. Metagenomic data were -used to determine the almost complete (-3.4 Mb) composite genome of the new bacterial. lineage desig- nated Candidatus Gallionella acididurans ShG14-8. Genome analysis revealed that Fe(II) oxidation probably involved the cytochromes localized on the outer membrane of the cell. The electron transport chain included NADH dehydrogenase, a cytochrome bc1 complex, an alternative complex III, and cytochrome oxidases of the bd, cbb3, and bo3 types. Oxidation of reduced sulfur compounds probably involved the Sox system, sul- fide-quinone oxidoreductase, adenyl sulfate reductase, and sulfate adenyltransferase. The genes required for autotrophic carbon assimilation via the Calvin cycle were present, while no pathway for nitrogen fixation was revealed. High numbers of RND metal transporters and P type ATPases were probably responsible for resis- tance to heavy metals. The new microorganism was an aerobic chemolithoautotroph of the group of

  7. Uptake of α-Ketoglutarate by Citrate Transporter CitP Drives Transamination in Lactococcus lactis

    PubMed Central

    Pudlik, Agata M.

    2013-01-01

    Transamination is the first step in the conversion of amino acids into aroma compounds by lactic acid bacteria (LAB) used in food fermentations. The process is limited by the availability of α-ketoglutarate, which is the best α-keto donor for transaminases in LAB. Here, uptake of α-ketoglutarate by the citrate transporter CitP is reported. Cells of Lactococcus lactis IL1403 expressing CitP showed significant levels of transamination activity in the presence of α-ketoglutarate and one of the amino acids Ile, Leu, Val, Phe, or Met, while the same cells lacking CitP showed transamination activity only after permeabilization of the cell membrane. Moreover, the transamination activity of the cells followed the levels of CitP in a controlled expression system. The involvement of CitP in the uptake of the α-keto donor was further demonstrated by the increased consumption rate in the presence of l-lactate, which drives CitP in the fast exchange mode of transport. Transamination is the only active pathway for the conversion of α-ketoglutarate in IL1403; a stoichiometric conversion to glutamate and the corresponding α-keto acid from the amino acids was observed. The transamination activity by both the cells and the cytoplasmic fraction showed a remarkably flat pH profile over the range from pH 5 to pH 8, especially with the branched-chain amino acids. Further metabolism of the produced α-keto acids into α-hydroxy acids and other flavor compounds required the coupling of transamination to glycolysis. The results suggest a much broader role of the citrate transporter CitP in LAB than citrate uptake in the citrate fermentation pathway alone. PMID:23204417

  8. Brain abscess caused by Lactococcus lactis cremoris in a child.

    PubMed

    Topçu, Yasemin; Akıncı, Gülçin; Bayram, Erhan; Hız, Semra; Türkmen, Mehmet

    2011-12-01

    Lactococcus lactis cremoris infections are very rare in humans. It is recognized as a commensal organism of mucocutaneous surfaces of cattle, and is occasionally isolated from human mucocutaneous surfaces. We report a brain abscess caused by L. lactis cremoris in an immunocompetent child. A 19-month-old female patient was admitted with fever and vomiting. Brain computed tomography (CT) revealed brain abscess. L. lactis cremoris was isolated from culture of the abscess material. The patient was treated with pus drainage from brain abscess and antibiotics including vancomycin and meropenem. The patient recovered completely. To our knowledge, this is the first report of a L. lactis cremoris infection in children.

  9. Caproiciproducens galactitolivorans gen. nov., sp. nov., a bacterium capable of producing caproic acid from galactitol, isolated from a wastewater treatment plant.

    PubMed

    Kim, Byung-Chun; Seung Jeon, Byoung; Kim, Seil; Kim, Hyunook; Um, Youngsoon; Sang, Byoung-In

    2015-12-01

    A strictly anaerobic, Gram-stain-positive, non-spore-forming, rod-shaped bacterial strain, designated BS-1T, was isolated from an anaerobic digestion reactor during a study of bacteria utilizing galactitol as the carbon source. Its cells were 0.3-0.5 μm × 2-4 μm, and they grew at 35-45 °C and at pH 6.0-8.0. Strain BS-1T produced H2, CO2, ethanol, acetic acid, butyric acid and caproic acid as metabolic end products of anaerobic fermentation. Phylogenetic analysis, based on the 16S rRNA gene sequence, showed that strain BS-1T represented a novel bacterial genus within the family Ruminococcaceae, Clostridium Cluster IV. The type strains that were most closely related to strain BS-1T were Clostridium sporosphaeroides KCTC 5598T (94.5 %), Clostridium leptum KCTC 5155T (94.3 %), Ruminococcus bromii ATCC 27255T (92.1 %) and Ethanoligenens harbinense YUAN-3T (91.9 %). Strain BS-1T had 17.6 % and 20.9 % DNA-DNA relatedness values with C. sporosphaeroides DSM 1294T and C. leptum DSM 753T, respectively. The major components of the cellular fatty acids were C16 : 0 dimethyl aldehyde (DMA) (22.1 %), C16 : 0 aldehyde (14.1 %) and summed feature 11 (iso-C17 : 0 3-OH and/or C18 : 2 DMA; 10.0 %). The genomic DNA G+C content was 50.0 mol%. Phenotypic and phylogenetic characteristics allowed strain BS-1T to be clearly distinguished from other taxa of the genus Clostridium Cluster IV. On the basis of these data, the isolate is considered to represent a novel genus and novel species within Clostridium Cluster IV, for which the name Caproiciproducens galactitolivorans gen. nov., sp. nov. is proposed. The type species is BS-1T ( = JCM 30532T and KCCM 43048T).

  10. Quantitative PCR for the specific quantification of Lactococcus lactis and Lactobacillus paracasei and its interest for Lactococcus lactis in cheese samples.

    PubMed

    Achilleos, Christine; Berthier, Françoise

    2013-12-01

    The first objective of this work was to develop real-time quantitative PCR (qPCR) assays to quantify two species of mesophilic lactic acid bacteria technologically active in food fermentation, including cheese making: Lactococcus lactis and Lactobacillus paracasei. The second objective was to compare qPCR and plate counts of these two species in cheese samples. Newly designed primers efficiently amplified a region of the tuf gene from the target species. Sixty-three DNA samples from twenty different bacterial species, phylogenetically related or commonly found in raw milk and dairy products, were selected as positive and negative controls. Target DNA was successfully amplified showing a single peak on the amplicon melting curve; non-target DNA was not amplified. Quantification was linear over 5 log units (R(2) > 0.990), down to 22 gene copies/μL per well for Lc. lactis and 73 gene copies/μL per well for Lb. paracasei. qPCR efficiency ranged from 82.9% to 93.7% for Lc. lactis and from 81.1% to 99.5% for Lb. paracasei. At two stages of growth, Lc. lactis was quantified in 12 soft cheeses and Lb. paracasei in 24 hard cooked cheeses. qPCR proved to be useful for quantifying Lc. lactis, but not Lb. paracasei.

  11. Methylocystis bryophila sp. nov., a facultatively methanotrophic bacterium from acidic Sphagnum peat, and emended description of the genus Methylocystis (ex Whittenbury et al. 1970) Bowman et al. 1993.

    PubMed

    Belova, Svetlana E; Kulichevskaya, Irina S; Bodelier, Paul L E; Dedysh, Svetlana N

    2013-03-01

    A novel species is proposed for two facultatively methanotrophic representatives of the genus Methylocystis, strains H2s(T) and S284, which were isolated from an acidic (pH 4.3) Sphagnum peat-bog lake (Teufelssee, Germany) and an acidic (pH 3.8) peat bog (European North Russia), respectively. Cells of strains H2s(T) and S284 are aerobic, Gram-negative, non-motile, curved coccoids or short rods that contain an intracytoplasmic membrane system typical of type-II methanotrophs. They possess both a soluble and a particulate methane monooxygenase (MMO); the latter is represented by two isozymes, pMMO1 and pMMO2. The preferred growth substrates are methane and methanol. In the absence of C1 substrates, however, these methanotrophs are capable of slow growth on acetate. Atmospheric nitrogen is fixed by means of an aerotolerant nitrogenase. Strains H2s(T) and S284 grow between pH 4.2 and 7.6 (optimum pH 6.0-6.5) and at 8-37 °C (optimum 25-30 °C). The major fatty acids are C18 : 1ω8c, C18 : 1ω7c and C16 : 1ω7c; the major quinone is Q-8. The DNA G+C content is 62.0-62.3 mol%. Strains H2s(T) and S284 share identical 16S rRNA gene sequences, which displayed 96.6-97.3 % similarity to sequences of other taxonomically characterized members of the genus Methylocystis. Therefore, strains H2s(T) and S284 are classified as members of a novel species, for which the name Methylocystis bryophila sp. nov. is proposed; strain H2s(T) ( = DSM 21852(T)  = VKM B-2545(T)) is the type strain.

  12. Modeling the acid-base properties of bacterial surfaces: A combined spectroscopic and potentiometric study of the gram-positive bacterium Bacillus subtilis.

    PubMed

    Leone, Laura; Ferri, Diego; Manfredi, Carla; Persson, Per; Shchukarev, Andrei; Sjöberg, Staffan; Loring, John

    2007-09-15

    In this study, macroscopic and spectroscopic data were combined to develop a surface complexation model that describes the acid-base properties of Bacillus subtilis. The bacteria were freeze-dried and then resuspended in 0.1 M NaCl ionic medium. Macroscopic measurements included potentiometric acid-base titrations and electrophoretic mobility measurements. In addition, ATR-FTIR spectra of wet pastes from suspensions of Bacillus subtilis at different pH values were collected. The least-squares program MAGPIE was used to generate a surface complexation model that takes into account the presence of three acid-base sites on the surface: tripple bond COOH, tripple bond NH+, and tripple bond PO-, which were identified previously by XPS measurements. Both potentiometric titration data and ATR-FTIR spectra were used quantitatively, and electrostatic effects at the charged bacterial surface were accounted for using the constant capacitance model. The model was calculated using two different approaches: in the first one XPS data were used to constrain the ratio of the total concentrations of all three surface sites. The capacitance of the double layer, the total buffer capacity, and the deprotonation constants of the tripple bond NH+, tripple bond POH, and tripple bond COOH species were determined in the fit. A second approach is presented in which the ratio determined by XPS of the total concentrations of tripple bond NH+ to tripple bond PO- sites is relaxed. The total concentration of tripple bond PO- sites was determined in the fit, while the deprotonation constant for tripple bond POH was manually varied until the minimization led to a model which predicted an isoelectric point that resulted in consistency with electrophoretic mobility data. The model explains well the buffering capacity of Bacillus subtilis suspensions in a wide pH range (between pH=3 and pH=9) which is of considerable environmental interest. In particular, a similar quantitative use of the IR data

  13. Short communication: Presence of Lactococcus and lactococcal exopolysaccharide operons on the leaves of Pinguicula vulgaris supports the traditional source of bacteria present in Scandinavian ropy fermented milk.

    PubMed

    Porcellato, Davide; Tranvåg, Malena; Narvhus, Judith

    2016-09-01

    Some traditional Scandinavian fermented milk products have a pronounced ropy consistency due to the presence of exopolysaccharide-producing strains of Lactococcus lactis ssp. cremoris. Norwegian food folklore describes how leaves from the carnivorous plant Pinguicula vulgaris (common butterwort) may be added to milk to initiate the fermentation of the traditional fermented milk product tettemelk. However, scientific confirmation of the link between the plant and the milk product has not been previously published. In the present study, the microbiome on 20 samples of P. vulgaris leaves collected from 5 different rural geographical locations in Norway and from 4 samples of commercial tettemelk was analyzed using high-throughput sequencing methods. The leaf microbiota of P. vulgaris was dominated by Proteobacteria and Firmicutes and the genus Lactococcus was demonstrated in all leaf samples. In addition, DNA extracted from the leaf microbiome contained genes identical to those responsible for exopolysaccharide production in Lactococcus. These results confirm the traditional use of P. vulgaris as a source of bacteria for the Norwegian ropy fermented milk product tettemelk and indicate that P. vulgaris microbiomes can be a potential source of lactic acid bacteria with interesting dairy technological features.

  14. Aldehyde dehydrogenase activity in Lactococcus chungangensis: Application in cream cheese to reduce aldehyde in alcohol metabolism.

    PubMed

    Konkit, Maytiya; Choi, Woo Jin; Kim, Wonyong

    2016-03-01

    Previous studies have shown that the metabolic capability of colonic microflora may be at least as high as that of the liver or higher than that of the whole human body. Aldehyde dehydrogenase (ALDH) is an enzyme produced by these bacteria that can metabolize acetaldehyde, produce from ethanol to acetate. Lactococcus species, which is commonly used as a starter in dairy products, was recently found to possess the ALDH gene, and the activity of this enzyme was determined. In this study, the ALDH activity of Lactococcus chungangensis CAU 28(T) and 11 other type strains in the genus Lactococcus was studied. Only 5 species, 3 of dairy origin (Lactococcus lactis ssp. lactis KCTC 3769(T), Lactococcus lactis ssp. cremoris KCCM 40699(T), and Lactococcus raffinolactis DSM 20443(T)) and 2 of nondairy origin (Lactococcus fujiensis NJ317(T) and L. chungangensis CAU 28(T)), showed ALDH activity and possessed a gene encoding ALDH. All of these strains were capable of making cream cheese. Among the strains, L. chungangensis produced cream cheese that contained the highest level of ALDH and was found to reduce the level of acetaldehyde in the serum of mice. These results predict a promising role for L. chungangensis CAU28(T) to be used in cheese that can be developed as functional food.

  15. Alcohol dehydrogenase activity in Lactococcus chungangensis: application in cream cheese to moderate alcohol uptake.

    PubMed

    Konkit, Maytiya; Choi, Woo Jin; Kim, Wonyong

    2015-09-01

    Many human gastrointestinal facultative anaerobic and aerobic bacteria possess alcohol dehydrogenase (ADH) activity and are therefore capable of oxidizing ethanol to acetaldehyde. However, the ADH activity of Lactococcus spp., except Lactococcus lactis ssp. lactis, has not been widely determined, though they play an important role as the starter for most cheesemaking technologies. Cheese is a functional food recognized as an aid to digestion. In the current study, the ADH activity of Lactococcus chungangensis CAU 28(T) and 11 reference strains from the genus Lactococcus was determined. Only 5 strains, 3 of dairy origin, L. lactis ssp. lactis KCTC 3769(T), L. lactis ssp. cremoris KCCM 40699(T), and Lactococcus raffinolactis DSM 20443(T), and 2 of nondairy origin, Lactococcus fujiensis NJ317(T) and Lactococcus chungangensis CAU 28(T) KCTC 13185(T), showed ADH activity and possessed the ADH gene. All these strains were capable of making cheese, but the highest level of ADH activity was found in L. chungangensis, with 45.9nmol/min per gram in tryptic soy broth and 65.8nmol/min per gram in cream cheese. The extent that consumption of cheese, following imbibing alcohol, reduced alcohol uptake was observed by following the level of alcohol in the serum of mice. The results show a potential novel benefit of cheese as a dairy functional food. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  16. Use of Lactococcus lactis expressing pili from group B Streptococcus as a broad-coverage vaccine against streptococcal disease.

    PubMed

    Buccato, Scilla; Maione, Domenico; Rinaudo, Cira Daniela; Volpini, Gianfranco; Taddei, Anna Rita; Rosini, Roberto; Telford, John L; Grandi, Guido; Margarit, Immaculada

    2006-08-01

    Recent data indicate that the human pathogen group B Streptococcus (GBS) produces pilus-like structures encoded in genomic islands with similar organization to pathogenicity islands. On the basis of the amino acid sequence of their protein components, 3 different types of pili have been identified in GBS, at least 1 of which is present in all isolates. We recently demonstrated that recombinant pilus proteins protect mice from lethal challenge with GBS and are thus potential vaccine candidates. Here, we show that GBS pilin island 1, transferred into the nonpathogenic microorganism Lactococcus lactis, leads to pilus assembly. We also show that systemically or mucosally delivered Lactococcus expressing pilin island 1 protects mice from challenge with GBS isolates carrying pilus 1. Furthermore, lactococci engineered to express hybrid pili containing GBS pilus 1 and pilus 2 components confer protection against strains expressing either of the 2 pilus types. These data pave the way to the design of pilus-based, multivalent live vaccines against streptococcal pathogens.

  17. Determination of the carbonate dissolution mechanism of Lactococcus sp.

    NASA Astrophysics Data System (ADS)

    Yanmiş, Derya; Orhan, Furkan; Güllüce, Medine; Şahin, Fikrettin

    2017-04-01

    Magnesite, the main source for magnesium and magnesium derivatives, are also commonly used in the production of caustic, dead-burned and fused magnesia. World magnesite resources are estimated to be at 12 billion tonnes mostly located in China, Russia, North Korea, Australia and Turkey. Turkey is the second producer of the magnesite. Magnesite deposits in Turkey are sedimentary magnesite which have been formed in specific conditions as high concentrations of MgSO4 and CO2 and presence of certain organic salts or created by hot or cold dissolution connected with carbonate rocks mainly with dolomites. According to the genesis of magnesite deposits, they have some impurities as calcium, quartz, iron, etc. Impurities of magnesite, especially CaCO3, reduce its economic value and industrial usability. In our previous study, we have performed biotechnologically enrichment of magnesite by Lactococcus sp., which gave significantly important results. However, we had no information about carbonate dissolution mechanism of bacteria. Therefore, it is aimed to reveal the metabolites of Lactococcus sp. and mechanism leading to the carbonate dissolution (MgCO3 and CaCO3).

  18. Characterization of the multiple molecular mechanisms underlying RsaL control of phenazine-1-carboxylic acid biosynthesis in the rhizosphere bacterium Pseudomonas aeruginosa PA1201.

    PubMed

    Sun, Shuang; Chen, Bo; Jin, Zi-Jing; Zhou, Lian; Fang, Yun-Ling; Thawai, Chitti; Rampioni, Giordano; He, Ya-Wen

    2017-03-18

    Phenazines are important secondary metabolites that have been found to affect a broad spectrum of organisms. Two almost identical gene clusters phz1 and phz2 are responsible for phenazines biosynthesis in the rhizobacterium Pseudomonas aeruginosa PA1201. Here, we show that the transcriptional regulator RsaL is a potent repressor of phenazine-1-carboxylic acid (PCA) biosynthesis. RsaL negatively regulates phz1 expression and positively regulates phz2 expression via multiple mechanisms. First, RsaL binds to a 25-bp DNA region within the phz1 promoter to directly repress phz1 expression. Second, RsaL indirectly regulates the expression of both phz clusters by decreasing the activity of the las and pqs quorum sensing (QS) systems, and by promoting the rhl QS system. Finally, RsaL represses phz1 expression through the downstream transcriptional regulator CdpR. RsaL directly binds to the promoter region of cdpR to positively regulate its expression, and subsequently CdpR regulates phz1 expression in a negative manner. We also show that RsaL represents a new mechanism for the turnover of the QS signal molecule N-3-oxododecanoyl-homoserine lactone (3-oxo-C12-HSL). Overall, this study elucidates RsaL control of phenazines biosynthesis and indicates that a PA1201 strain harboring deletions in both the rsaL and cdpR genes could be used to improve the industrial production of PCA.

  19. Growth of Leuconostoc mesenteroides NRRL-B523 in an alkaline medium: suboptimal pH growth inhibition of a lactic acid bacterium.

    PubMed

    Wolf, Barry F; Fogler, H Scott

    2005-01-05

    Bacterial profile modification (BPM), a form of tertiary oil recovery, diverts water from the water-flooded high-permeability zone into the oil-bearing low-permeability zone. During field use, exopolymer-producing bacteria plug the high-permeability zone only in the immediate vicinity of the injection point (the near-well bore region). For effective BPM the plug must penetrate far into the formation. Slowing the specific growth rate, lengthening the lag phase, and slowing the polymerization rate are techniques that can prolong the onset of biopolymer gelation and extend the depth of the biological plug. In batch experiments, the growth of Leuconostoc mesenteroides NRRL-B523 was inhibited by the synergistic effects of high substrate loading and an alkaline pH. Exponential growth was delayed up to 190 h. It was observed that cell division was significantly retarded until the medium pH, reduced by the acid byproducts of fermentation, reached a critical value of 6.79 +/- 0.06. A mathematical model was developed to describe the relationship between specific growth rate, lag time, and medium pH.

  20. Identification and characterization of the alpha-acetolactate synthase gene from Lactococcus lactis subsp. lactis biovar diacetylactis.

    PubMed Central

    Marugg, J D; Goelling, D; Stahl, U; Ledeboer, A M; Toonen, M Y; Verhue, W M; Verrips, C T

    1994-01-01

    The conversion of 3-13C-labelled pyruvate in an acetoin-producing clone from a Lactococcus lactis subsp. lactis biovar diacetylactis strain DSM 20384 plasmid bank in Escherichia coli was studied by 13C nuclear magnetic resonance analysis. The results showed that alpha-acetolactate was the first metabolic product formed from pyruvate, whereas acetoin appeared at a much slower rate and reached only low concentrations. This alpha-acetolactate production shows that the cells express the gene for alpha-acetolactate synthase (als). Nucleotide sequence analysis identified an open reading frame encoding a protein of 554 amino acids. The deduced amino acid sequence exhibits extensive similarities to those of known alpha-acetolactate synthases from both prokaryotes and eukaryotes. The als gene is expressed on a monocistronic transcriptional unit, which is transcribed from a promoter located just upstream of the coding region. Images PMID:8017926

  1. GABA Production in Lactococcus lactis Is Enhanced by Arginine and Co-addition of Malate

    PubMed Central

    Laroute, Valérie; Yasaro, Chonthicha; Narin, Waranya; Mazzoli, Roberto; Pessione, Enrica; Cocaign-Bousquet, Muriel; Loubière, Pascal

    2016-01-01

    Lactococcus lactis NCDO 2118 was previously selected for its ability to decarboxylate glutamate to γ-aminobutyric acid (GABA), an interesting nutritional supplement able to improve mood and relaxation. Amino acid decarboxylation is generally considered as among the biochemical systems allowing lactic acid bacteria to counteracting acidic stress and obtaining metabolic energy. These strategies also include arginine deiminase pathway and malolactic fermentation but little is known about their possible interactions of with GABA production. In the present study, the effects of glutamate, arginine, and malate (i.e., the substrates of these acid-resistance pathways) on L. lactis NCDO 2118 growth and GABA production performances were analyzed. Both malate and arginine supplementation resulted in an efficient reduction of acidity and improvement of bacterial biomass compared to glutamate supplementation. Glutamate decarboxylation was limited to narrow environmental conditions (pH < 5.1) and physiological state (stationary phase). However, some conditions were able to improve GABA production or activate glutamate decarboxylation system even outside of this compass. Arginine clearly stimulated glutamate decarboxylation: the highest GABA production (8.6 mM) was observed in cultures supplemented with both arginine and glutamate. The simultaneous addition of arginine, malate, and glutamate enabled earlier GABA production (i.e., during exponential growth) at relatively high pH (6.5). As far as we know, no previous study has reported GABA production in such conditions. Although further studies are needed to understand the molecular basis of these phenomena, these results represent important keys suitable of application in GABA production processes. PMID:27458444

  2. Potential aquaculture probiont Lactococcus lactis TW34 produces nisin Z and inhibits the fish pathogen Lactococcus garvieae.

    PubMed

    Sequeiros, Cynthia; Garcés, Marisa E; Vallejo, Marisol; Marguet, Emilio R; Olivera, Nelda L

    2015-04-01

    Bacteriocin-producing Lactococcus lactis TW34 was isolated from marine fish. TW34 bacteriocin inhibited the growth of the fish pathogen Lactococcus garvieae at 5 AU/ml (minimum inhibitory concentration), whereas the minimum bactericidal concentration was 10 AU/ml. Addition of TW34 bacteriocin to L. garvieae cultures resulted in a decrease of six orders of magnitude of viable cells counts demonstrating a bactericidal mode of action. The direct detection of the bacteriocin activity by Tricine-SDS-PAGE showed an active peptide with a molecular mass ca. 4.5 kDa. The analysis by MALDI-TOF-MS detected a strong signal at m/z 2,351.2 that corresponded to the nisin leader peptide mass without the initiating methionine, whose sequence STKDFNLDLVSVSKKDSGASPR was confirmed by MS/MS. Sequence analysis of nisin structural gene confirmed that L. lactis TW34 was a nisin Z producer. This nisin Z-producing strain with probiotic properties might be considered as an alternative in the prevention of lactococcosis, a global disease in aquaculture systems.

  3. Secreted expression of Leuconostoc mesenteroides glucansucrase in Lactococcus lactis for the production of insoluble glucans.

    PubMed

    Skory, Christopher D; Côté, Gregory L

    2015-12-01

    We expressed a glucansucrase, DsrI, from Leuconostoc mesenteroides that catalyzes formation of water-insoluble glucans from sucrose using a nisin-controlled gene expression system in Lactococcus lactis. These polymers have potential for production of biodegradable gels, fibers, and films. We optimized production of DsrI using several different background vectors, signal peptides, strains, induction conditions, and bioreactor parameters to increase extracellular accumulation. Optimal production of the enzyme utilized a high-copy plasmid, pMSP3535H3, which contains a nisin immunity gene, L. lactis LM0230, and bioreactors maintained at pH 6.0 to stabilize the enzyme. We were able to significantly improve growth using the lactic acid inhibitor heme and by continuous removal of lactic acid with anion exchange resins, but enzyme production was less than the controls. The recombinant enzyme under optimized conditions accumulated in the culture medium to approximately 380 mg/L, which was over 150-fold higher compared to the native L. mesenteroides strain. Methods are also included for purification of DsrI utilizing the glucan-binding domain of the enzyme.

  4. Identification of biosynthetic intermediates of the extracellular polysaccharide viilian in Lactococcus lactis subspecies cremoris SBT 0495.

    PubMed

    Oba, T; Doesburg, K K; Iwasaki, T; Sikkema, J

    1999-04-01

    Lactococcus lactis subspecies cremoris SBT 0495 produces the phosphopolysaccharide viilian, which consists of the repeating unit beta-D-glucosyl-(1-->4)-(alpha-L-rhamnosyl-(1-->2))-(alpha-D-galac tose-1- phosphoryl-(-->3)-beta-galactosyl-(1-->4)-beta-D-glucose. A lipid extract was prepared from cells in the late exponential phase of growth and was hydrolyzed by hydrochloric acid under mild conditions to split lipid-linked intermediates in the extract into lipid and sugar moieties. Both moieties were purified by chromatographic techniques and were characterized to identify intermediates of the viilian biosynthetic pathway. A polyisoprenoid isolated from the chloroform-soluble fraction of the hydrolyzed lipid extract was identified by mass spectrometry as undecaprenol. Saccharides isolated from the water-soluble fraction of the hydrolyzed lipid extract by anion-exchange chromatography, were characterized by glycosidic linkage analysis to discriminate sugar moieties of intermediates of viilian biosynthesis from compounds liberated from cell wall components. Some oligosaccharide analogues contain a glycerol residue, suggesting that these are fragments of glycosylglycerides and/or lipoteichoic acid. Three fragments were identified to be glucose, galactosyl-(1-->4)-glucose, and rhamnosyl-(1-->2)-galactosyl-(1-->4)-glucose, which are in agreement with the structure of the repeating unit of viilian. These saccharides most likely represent the first three steps of the sequential assembly of the repeating unit of the undecaprenol assembly.

  5. Stress response in Lactococcus lactis: cloning, expression analysis, and mutation of the lactococcal superoxide dismutase gene.

    PubMed Central

    Sanders, J W; Leenhouts, K J; Haandrikman, A J; Venema, G; Kok, J

    1995-01-01

    In an analysis of the stress response of Lactococcus lactis, three proteins that were induced under low pH culture conditions were detected. One of these was identified as the lactococcal superoxide dismutase (SodA) by N-terminal amino acid sequence analysis. The gene encoding this protein, designated sodA, was cloned by the complementation of a sodA sodB Escherichia coli strain. The deduced amino acid sequence of L. lactis SodA showed the highest degree of similarity to the manganese-containing Sod (MnSod) of Bacillus stearothermophilus. A promoter upstream of the sodA gene was identified by primer extension analysis, and an inverted repeat surrounding the -35 hexanucleotide of this promoter is possibly involved in the regulation of the expression of sodA. The expression of sodA was analyzed by transcriptional fusions with a promoterless lacZ gene. The induction of beta-galactosidase activity occurred in aerated cultures. Deletion experiments revealed that a DNA fragment of more than 130 bp surrounding the promoter was needed for the induction of lacZ expression by aeration. The growth rate of an insertion mutant of sodA did not differ from that of the wild type in standing cultures but was decreased in aerated cultures. PMID:7665513

  6. Heterologous expression of the Bacillus subtilis (natto) alanine dehydrogenase in Escherichia coli and Lactococcus lactis.

    PubMed

    Ye, Wei; Huo, Guicheng; Chen, Junliang; Liu, Fei; Yin, Jingyuan; Yang, Lijie; Ma, Xiaolong

    2010-05-30

    The major objective of the present study is to change the alanine production of Lactic acid bacteria by expression of Bacillus subtilis (natto) alanine dehydrogenase (AlaDH), the gene that is not present in Lactic acid. B. subtilis AlaDH gene (ald) was cloned into a pGEX6p-1 and expressed in E. coli JM109. Its enzyme activity was 48.3U/mg at 30 degrees C and 45.2U/mg at 42 degrees C. This ald gene was then cloned into a vector pNZ8148 to generate a vector pNZ8148/ald. The same ald gene was placed downstream of the ldh promoter from Streptococcus thermophilus to generate pNZ273/ldhp/ald. The pNZ8148/ald and pNZ273/ldhp/ald were introduced separately in Lactococcus lactis NZ9000. As a result of over-expressed ald, the production of alanine detected by HPLC in L. lactis NZ9000 carrying pNZ273/ldhp/ald reached 52mug/ml, an approximately 26-fold increase compared to the parent strain L. lactis NZ9000, but not in L. lactis NZ9000 carrying pNZ8148/ald. This study would help strain improvement to be used in dairy fermentation for developing healthy yogurts with sweet taste or other fermented dairy foods. Copyright 2009 Elsevier GmbH. All rights reserved.

  7. Excess of threonine compared with serine promotes threonine aldolase activity in Lactococcus lactis IL1403.

    PubMed

    Aller, Kadri; Adamberg, Kaarel; Reile, Indrek; Timarova, Veronica; Peebo, Karl; Vilu, Raivo

    2015-05-01

    Lactococcus lactis is an important lactic acid starter for food production as well as a cell factory for production of food grade additives, among which natural flavour production is one of the main interests of food producers. Flavour production is associated with the degradation of amino acids and comprehensive studies are required to elucidate mechanisms behind these pathways. In this study using chemically defined medium, labelled substrate and steady-state cultivation, new data for the catabolism of threonine in Lc. lactis have been obtained. The biosynthesis of glycine in this organism is associated with the catabolic pathways of glucose and serine. Nevertheless, if threonine concentration in the growth environment exceeds that of serine, threonine becomes the main source for glycine biosynthesis and the utilization of serine decreases. Also, the conversion of threonine to glycine was initiated by a threonine aldolase and this was the principal pathway used for threonine degradation. As in Streptococcus thermophilus, serine hydroxymethyltransferase in Lc. lactis may possess a secondary activity as threonine aldolase. Other catabolic pathways of threonine (e.g. threonine dehydrogenase and threonine dehydratase) were not detected. © 2015 The Authors.

  8. Purification and Characterization of an Aminopeptidase from Lactococcus lactis subsp. cremoris AM2

    PubMed Central

    Neviani, E.; Boquien, C. Y.; Monnet, V.; Thanh, L. Phan; Gripon, J.-C.

    1989-01-01

    An aminopeptidase was purified from cell extracts of Lactococcus lactis subsp. cremoris AM2 by ion-exchange chromatography. After electrophoresis of the purified enzyme in the presence or absence of sodium dodecyl sulfate, one protein band was detected. The enzyme was a 300-kilodalton hexamer composed of identical subunits not linked by disulfide bridges. Activity was optimal at 40°C and pH 7 and was inhibited by classical thiol group inhibitors. The aminopeptidase hydrolyzed naphthylamide-substituted amino acids, as well as dipeptides and tripeptides. Longer protein chains such as the B chain of insulin were hydrolyzed, but at a much slower rate. The Michaelis constant (Km) and the maximal rate of hydrolysis (Vmax) were, respectively, 4.5 mM and 3,600 pkat/mg for the substrate l-histidyl-β-naphthylamide. Amino acid analysis showed that the enzyme contained low levels of hydrophobic residues. The partial N-terminal sequence of the first 19 residues of the mature enzyme was determined. Polyclonal antibodies were obtained from the purified enzyme, and after immunoblotting, there was no cross-reaction between these antibodies and other proteins in the crude extract. Images PMID:16348010

  9. Arginine metabolism in sugar deprived Lactococcus lactis enhances survival and cellular activity, while supporting flavour production.

    PubMed

    Brandsma, J B; van de Kraats, I; Abee, T; Zwietering, M H; Meijer, W C

    2012-02-01

    Flavour development in cheese is affected by the integrity of Lactococcus lactis cells. Disintegrated cells enhance for instance the enzymatic degradation of casein to free amino acids, while integer cells are needed to produce specific flavour compounds from amino acids. The impact of the cellular activity of these integer cells on flavour production remains to be elucidated. In this study we investigated whether lactose-deprived L. lactis cells that use arginine as an alternative energy source can extend cellular activity and produce more specific flavours. In cheese experiments we demonstrated that arginine metabolising cells survived about 3 times longer than non-arginine metabolising cells, which suggests prolonged cellular activity. Cellular activity and flavour production of L. lactis was further studied in vitro to enable controlled arginine supplementation. Comparable with the results found in cheese, the survival rates of in vitro incubated cells improved when arginine was metabolised. Furthermore, elongated cellular activity was reflected in 3-4-fold increased activity of flavour generating enzymes. The observed prolonged cellular activity resulted in about 2-fold higher concentrations of typical Gouda cheese flavours. These findings provide new leads for composing starter cultures that will produce specific flavour compounds. Copyright © 2011 Elsevier Ltd. All rights reserved.

  10. Production of Pediocin PA-1 by Lactococcus lactis Using the Lactococcin A Secretory Apparatus

    PubMed Central

    Horn, Nikki; Martínez, María I.; Martínez, José M.; Hernández, Pablo E.; Gasson, Michael J.; Rodríguez, Juan M.; Dodd, Helen M.

    1998-01-01

    The class II bacteriocins pediocin PA-1, from Pediococcus acidilactici, and lactococcin A, from Lactococcus lactis subsp. lactis bv. diacetylactis WM4 have a number of features in common. They are produced as precursor peptides containing similar amino-terminal leader sequences with a conserved processing site (Gly-Gly at positions −1 and −2). Translocation of both bacteriocins occurs via a dedicated secretory system. Because of the strong antilisterial activity of pediocin PA-1, its production by lactic acid bacteria strains adapted to dairy environments would considerably extend its application in the dairy industry. In this study, the lactococcin A secretory system was adapted for the expression and secretion of pediocin PA-1. A vector containing an in-frame fusion of sequences encoding the lcnA promoter, the lactococcin A leader, and the mature pediocin PA-1, was introduced into L. lactis IL1403. This strain is resistant to pediocin PA-1 and encodes a lactococcin translocation apparatus. The resulting L. lactis strains secreted a bacteriocin with an antimicrobial activity of approximately 25% of that displayed by the parental pediocin-producing P. acidilactici 347. A noncompetitive indirect enzyme-linked immunosorbent assay with pediocin PA-1-specific antibodies and amino-terminal amino acid sequencing confirmed that pediocin PA-1 was being produced by the heterologous host. PMID:9501421

  11. Non-enzymic copper reduction by menaquinone enhances copper toxicity in Lactococcus lactis IL1403.

    PubMed

    Abicht, Helge K; Gonskikh, Yulia; Gerber, Simon D; Solioz, Marc

    2013-06-01

    Lactococcus lactis possesses a pronounced extracellular Cu(2+)-reduction activity which leads to the accumulation of Cu(+) in the medium. The kinetics of this reaction were not saturable by increasing copper concentrations, suggesting a non-enzymic reaction. A copper-reductase-deficient mutant, isolated by random transposon mutagenesis, had an insertion in the menE gene, which encodes O-succinylbenzoic acid CoA ligase. This is a key enzyme in menaquinone biosynthesis. The ΔmenE mutant was deficient in short-chain menaquinones, and exogenously added menaquinone complemented the copper-reductase-deficient phenotype. Haem-induced respiration of wild-type L. lactis efficiently suppressed copper reduction, presumably by competition by the bd-type quinol oxidase for menaquinone. As expected, the ΔmenE mutant was respiration-deficient, but could be made respiration-proficient by supplementation with menaquinone. Growth of wild-type cells was more copper-sensitive than that of the ΔmenE mutant, due to the production of Cu(+) ions by the wild-type. This growth inhibition of the wild-type was strongly attenuated if Cu(+) was scavenged with the Cu(I) chelator bicinchoninic acid. These findings support a model whereby copper is non-enzymically reduced at the membrane by menaquinones. Respiration effectively competes for reduced quinones, which suppresses copper reduction. These findings highlight novel links between copper reduction, respiration and Cu(+) toxicity in L. lactis.

  12. Familial Adenomatous Polyposis Manifesting as Lactococcus Endocarditis: A Case Report and Review of the Association of Lactococcus with Underlying Gastrointestinal Disease

    PubMed Central

    Bazemore, Taylor C.; Maskarinec, Stacey A.; Zietlow, Kahli; Hendershot, Edward F.

    2016-01-01

    A 45-year-old male with a prosthetic aortic valve presented to the hospital with several months of generalized malaise. On admission, he was noted to have anemia of unclear etiology and subsequently became febrile with multiple blood cultures growing Lactococcus garvieae. Inpatient workup was concerning for infectious endocarditis (IE) secondary to Lactococcus. The patient was discharged home with appropriate antimicrobial therapy; however, he was readmitted for persistent, symptomatic anemia and underwent colonoscopy, which revealed innumerable colonic polyps consistent with Familial Adenomatous Polyposis (FAP) that was later confirmed with genetic testing. Surveillance computed tomography (CT) imaging of the aortic repair later demonstrated valve dehiscence with surrounding fluid collection; he underwent redo surgery and was found to have destruction of the aortic annulus and a large pseudoaneurysm. Histopathology of the valve prosthesis confirmed IE. It is suspected that the patient developed Lactococcus IE from enteric translocation. Review of the literature provides several reports of Lactococcus infections in association with underlying gastrointestinal disease, including colorectal cancer. Given this association, we raise the question of whether the diagnosis of Lactococcus IE should evoke suspicion and encourage evaluation for gastrointestinal pathology, as occurs with Streptococcus bovis. PMID:27818810

  13. Use of rRNA gene restriction patterns to evaluate lactic acid bacterium contamination of vacuum-packaged sliced cooked whole-meat product in a meat processing plant.

    PubMed Central

    Björkroth, K J; Korkeala, H J

    1997-01-01

    Molecular typing was applied to an in-plant lactic acid bacterium (LAB) contamination analysis of a vacuum-packaged sliced cooked whole-meat product. A total of 982 LAB isolates from the raw mass, product, and the environment at different production stages were screened by restriction endonuclease (EcoRI and HindIII) analysis. rRNA gene restriction patterns were further determined for different strains obtained from each source. These patterns were used for recognizing the spoilage-causing LAB strains from the product on the sell-by day and tracing the sources and sites of spoilage LAB contamination during the manufacture. LAB typing resulted in 71 different ribotypes, of which 27 were associated with contamination routes. Raw material was distinguished as the source of the major spoilage strains. Contamination of the product surfaces after cooking was shown to be airborne. The removal of the product from the cooking forms was localized as a major site of airborne LAB contamination. Food handlers and some surfaces in contact with the product during the manufacture were also contaminated with the spoilage strains. Some LAB strains were also able to resist cooking in the core of the product bar. These strains may have an effect on the product shelf life by contaminating the slicing machine. The air in the slicing department and adjacent cold room contained very few LAB. Surface-mediated contamination was detected during the slicing and packaging stages. Food handlers also carried strains later found in the packaged product. Molecular typing provided useful information revealing the LAB contamination sources and sites of this product. The production line will be reorganized in accordance with these results to reduce spoilage LAB contamination. PMID:9023922

  14. Use of rRNA gene restriction patterns to evaluate lactic acid bacterium contamination of vacuum-packaged sliced cooked whole-meat product in a meat processing plant.

    PubMed

    Björkroth, K J; Korkeala, H J

    1997-02-01

    Molecular typing was applied to an in-plant lactic acid bacterium (LAB) contamination analysis of a vacuum-packaged sliced cooked whole-meat product. A total of 982 LAB isolates from the raw mass, product, and the environment at different production stages were screened by restriction endonuclease (EcoRI and HindIII) analysis. rRNA gene restriction patterns were further determined for different strains obtained from each source. These patterns were used for recognizing the spoilage-causing LAB strains from the product on the sell-by day and tracing the sources and sites of spoilage LAB contamination during the manufacture. LAB typing resulted in 71 different ribotypes, of which 27 were associated with contamination routes. Raw material was distinguished as the source of the major spoilage strains. Contamination of the product surfaces after cooking was shown to be airborne. The removal of the product from the cooking forms was localized as a major site of airborne LAB contamination. Food handlers and some surfaces in contact with the product during the manufacture were also contaminated with the spoilage strains. Some LAB strains were also able to resist cooking in the core of the product bar. These strains may have an effect on the product shelf life by contaminating the slicing machine. The air in the slicing department and adjacent cold room contained very few LAB. Surface-mediated contamination was detected during the slicing and packaging stages. Food handlers also carried strains later found in the packaged product. Molecular typing provided useful information revealing the LAB contamination sources and sites of this product. The production line will be reorganized in accordance with these results to reduce spoilage LAB contamination.

  15. Lactococcus lactis produces short-chain quinones that cross-feed Group B Streptococcus to activate respiration growth.

    PubMed

    Rezaïki, Lahcen; Lamberet, Gilles; Derré, Aurélie; Gruss, Alexandra; Gaudu, Philippe

    2008-03-01

    Quinones are essential components of the respiration chain that shuttle electrons between oxidoreductases. We characterized the quinones synthesized by Lactococcus lactis, a fermenting bacterium that activates aerobic respiration when a haem source is provided. Two distinct subgroups were characterized: Menaquinones (MK) MK-8 to MK-10, considered as hallmarks of L. lactis, are produced throughout growth. MK-3 and demethylMK-3 [(D)MK-3] are newly identified and are present only late in growth. Production of (D)MK-3 was conditional on the carbon sugar and on the presence of carbon catabolite regulator gene ccpA. Electron flux driven by both (D)MK fractions was shared between the quinol oxidase and extracellular acceptors O(2), iron and, with remarkable efficiency, copper. Purified (D)MK-3, but not MK-8-10, complemented a menB defect in L. lactis. We previously showed that a respiratory metabolism is activated in Group B Streptococcus (GBS) by exogenous haem and MK, and that this activity is implicated in virulence. Here we show that growing lactococci donate (D)MK to GBS to activate respiration and stimulate growth of this opportunist pathogen. We propose that conditions favouring (D)MK production in dense microbial ecosystems, as present in the intestinal tract, could favour implantation of (D)MK-scavengers like GBS within the complex.

  16. Comparison of antigenic proteins from Lactococcus garvieae KG- and KG+ strains that are recognized by olive flounder (Paralichthys olivaceus) antibodies.

    PubMed

    Shin, Gee-Wook; Nho, Seong-Won; Park, Seong-Bin; Jang, Ho-Bin; Cha, In-Seok; Ha, Mi-Ae; Kim, Young-Rim; Dalvi, Rishikesh S; Joh, Seong-Joon; Jung, Tae-Sung

    2009-10-20

    Lactococcus garvieae is an important etiological agent of lactococcosis in various fish species including olive flounder (Paralichthys olivaceus). In this study, proteomic and immunoproteomic analyses were employed to compare the antigenic profiles of strains KG9408, MS93003, and NSS9310 strains of L. garvieae. Proteomic analysis using two-dimensional gel electrophoresis (2-DE) revealed differences in five protein spots among the different L. garvieae strains. In immunoproteomic analysis, there was a significant difference in the 2-DE immunoblot profiles of the L. garvieae strains using sera collected from fish surviving infection with either L. garvieae strains KG9408 or NSS9310. These sera reacted with 8 and 7 unique antigenic protein spots, respectively. Heat shock protein (HSP) 70 and DNA-directed RNA polymerase were among the specific antigens recognized by the anti-NSS9310 serum. In addition, the anti-NSS9310 and anti-KG9408 olive flounder sera reacted with 25 common antigenic protein spots of all the L. garvieae strains, which included elongation factor (EF)-Tu, arginine deiminase (AD), inosine-5'-monophosphate dehydrogenase (IMPD), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphomannomutase (PMM), L-lactate dehydrogenase (L-LDH), 6-phosphofructokinase and UDP-galactose 4-epimerase (UDP-galactose). Based on the present results, the 8 antigens recognized by the anti-KG9408 serum and the 25 common antigens recognized by both sera may serve as potential markers for developing an effective vaccine against this bacterium.

  17. The Pool of ADP and ATP Regulates Anaerobic Product Formation in Resting Cells of Lactococcus lactis

    PubMed Central

    Palmfeldt, Johan; Paese, Marco; Hahn-Hägerdal, Bärbel; van Niel, Ed W. J.

    2004-01-01

    Lactococcus lactis grows homofermentatively on glucose, while its growth on maltose under anaerobic conditions results in mixed acid product formation in which formate, acetate, and ethanol are formed in addition to lactate. Maltose was used as a carbon source to study mixed acid product formation as a function of the growth rate. In batch and nitrogen-limited chemostat cultures mixed acid product formation was shown to be linked to the growth rate, and homolactic fermentation occurred only in resting cells. Two of the four lactococcal strains investigated with maltose, L. lactis 65.1 and MG1363, showed more pronounced mixed acid product formation during growth than L. lactis ATCC 19435 or IL-1403. In resting cell experiments all four strains exhibited homolactic fermentation. In resting cells the intracellular concentrations of ADP, ATP, and fructose 1,6-bisphosphate were increased and the concentration of Pi was decreased compared with the concentrations in growing cells. Addition of an ionophore (monensin or valinomycin) to resting cultures of L. lactis 65.1 induced mixed acid product formation concomitant with decreases in the ADP, ATP, and fructose 1,6-bisphosphate concentrations. ADP and ATP were shown to inhibit glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, and alcohol dehydrogenase in vitro. Alcohol dehydrogenase was the most sensitive enzyme and was totally inhibited at an adenine nucleotide concentration of 16 mM, which is close to the sum of the intracellular concentrations of ADP and ATP of resting cells. This inhibition of alcohol dehydrogenase might be partially responsible for the homolactic behavior of resting cells. A hypothesis regarding the level of the ATP-ADP pool as a regulating mechanism for the glycolytic flux and product formation in L. lactis is discussed. PMID:15345435

  18. Secretory expression of a heterologous nattokinase in Lactococcus lactis.

    PubMed

    Liang, Xiaobo; Zhang, Lixin; Zhong, Jin; Huan, Liandong

    2007-05-01

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

  19. Lactococcus garvieae: a small bacteria and a big data world

    PubMed Central

    2015-01-01

    Objective To describe the importance of bioinformatics tools to analyze the big data yielded from new "omics" generation-methods, with the aim of unraveling the biology of the pathogen bacteria Lactococcus garvieae. Methods The paper provides the vision of the large volume of data generated from genome sequences, gene expression profiles by microarrays and other experimental methods that require biomedical informatics methods for management and analysis. Results The use of biomedical informatics methods improves the analysis of big data in order to obtain a comprehensive characterization and understanding of the biology of pathogenic organisms, such as L. garvieae. Conclusions The "Big Data" concepts of high volume, veracity and variety are nowadays part of the research in microbiology associated with the use of multiple methods in the "omic" era. The use of biomedical informatics methods is a requisite necessary to improve the analysis of these data. PMID:25960872

  20. Cloning, Characterization, Controlled Overexpression, and Inactivation of the Major Tributyrin Esterase Gene of Lactococcus lactis

    PubMed Central

    Fernández, Leonides; Beerthuyzen, Marke M.; Brown, Julie; Siezen, Roland J.; Coolbear, Tim; Holland, Ross; Kuipers, Oscar P.

    2000-01-01

    The gene encoding the major intracellular tributyrin esterase of Lactococcus lactis was cloned using degenerate DNA probes based on 19 known N-terminal amino acid residues of the purified enzyme. The gene, named estA, was sequenced and found to encode a protein of 258 amino acid residues. The transcription start site was mapped 233 nucleotides upstream of the start codon, and a canonical promoter sequence was identified. The deduced amino acid sequence of the estA product contained the typical GXSXG motif found in most lipases and esterases. The protein was overproduced up to 170-fold in L. lactis by use of the nisin-controlled expression system recently developed for lactic acid bacteria. The estA gene was inactivated by chromosomal integration of a temperature-sensitive integration vector. This resulted in the complete loss of esterase activity, which could then be recovered after complementation of the constructed esterase-deficient strain with the wild-type estA gene. This confirms that EstA is the main enzyme responsible for esterase activity in L. lactis. Purified recombinant enzyme showed a preference for short-chain acyl esters, surprisingly also including phospholipids. Medium- and long-acyl-chain lipids were also hydrolyzed, albeit less efficiently. Intermediate characteristics between esterases and lipases make intracellular lactococcal EstA difficult to classify in either of these two groups of esterolytic enzymes. We suggest that, in vivo, EstA could be involved in (phospho)lipid metabolism or cellular detoxification or both, as its sequence showed significant similarity to S-formylglutathione hydrolase (FGH) of Paracoccus denitrificans and human EstD (or FGH), which are part of a universal formaldehyde detoxification pathway. PMID:10742212

  1. Glucose metabolism and regulation of glycolysis in Lactococcus lactis strains with decreased lactate dehydrogenase activity.

    PubMed

    Garrigues, C; Goupil-Feuillerat, N; Cocaign-Bousquet, M; Renault, P; Lindley, N D; Loubiere, P

    2001-07-01

    The distribution of carbon flux at the pyruvate node was investigated in Lactococcus lactis under anaerobic conditions with mutant strains having decreased lactate dehydrogenase activity. Strains previously selected by random mutagenesis by H. Boumerdassi, C. Monnet, M. Desmazeaud, and G. Corrieu (Appl. Environ. Microbiol. 63, 2293-2299, 1997) were found to have single punctual mutations in the ldh gene and presented a high degree of instability. The strain L. lactis JIM 5711 in which lactate dehydrogenase activity was diminished to less than 30% of the wild type maintained homolactic metabolism. This was due to an increase in the intracellular pyruvate concentration, which ensures the maintained flux through the lactate dehydrogenase. Pyruvate metabolism was linked to the flux limitation at the level of glyceraldehyde-3-phosphate dehydrogenase, as previously postulated for the parent strain (C. Garrigues, P. Loubière, N. D. Lindley, and M. Cocaign-Bousquet (1997) J. Bacteriol. 179, 5282-5287, 1997). However, a strain (L. lactis JIM 5954) in which the ldh gene was interrupted reoriented pyruvate metabolism toward mixed metabolism (production of formate, acetate, and ethanol), though the glycolytic flux was not strongly diminished. Only limited production of acetoin occurred despite significant overflow of pyruvate. Intracellular metabolite profiles indicated that the in vivo glyceraldehyde-3-phosphate dehydrogenase activity was no longer flux limiting in the Deltaldh strain. The shift toward mixed acid fermentation was correlated with the lower intracellular trioses phosphate concentration and diminished allosteric inhibition of pyruvate formate lyase. Copyright 2001 Academic Press.

  2. Isolation of nisin-producing Lactococcus lactis strains from dry fermented sausages.

    PubMed

    Rodríguez, J M; Cintas, L M; Casaus, P; Horn, N; Dodd, H M; Hernández, P E; Gasson, M J

    1995-02-01

    A total of 4608 lactic acid bacteria (LAB) were isolated from 24 Spanish fermented sausages and screened for bacteriocin production. Two strains, BB24 and G18, produced bacteriocins that inhibited a broad spectrum of Gram-positive bacteria. BB24 and G18 were tentatively identified as Lactococcus lactis by carbohydrate fermentation patterns and other biochemical characteristics. The characterization of their bacteriocins suggested that both could be the well-known lantibiotic nisin. This was confirmed by PCR analysis of their genomic DNA. Nucleotide sequencing revealed that they produced nisin A. The fact that BB24 and G18 were isolated from sausages produced in two different regions of Spain suggests that nisin-producing L. lactis strains may be more widespread in meat products than previously thought. Nisin produced by L. lactis BB24 has been purified to homogeneity by a procedure that included ammonium sulphate precipitation and cation-exchange, hydrophobic-interaction and reverse-phase chromatography. The purification procedure was simple, rapid and reproducible.

  3. Lactococcus lactis KR-050L inhibit IL-6/STAT3 activation.

    PubMed

    Hwang, J T; Jang, H-J; Kim, J H; Park, C S; Kim, Y; Lim, C-H; Lee, S W; Rho, M-C

    2017-05-01

    The purpose of this study was to investigate IL-6/STAT3 inhibitory activity using lactic acid bacteria (LABs) isolated from Gajuknamu kimchi. Six LABs were isolated from Gajuknamu kimchi and identified through 16S rRNA sequencing. Among them, the culture broth of Lactococcus lactis KR-050L inhibited IL-6-induced STAT3 luciferase activity. Fifteen compounds were isolated from the EtOAc extract of culture broth though column chromatography and preparative high-performance liquid chromatography, and they were identified as 2,5-diketopipperazine structures by spectroscopic analyses (MS, (1) H- and (13) C-NMR). They also showed inhibitory activities on IL-6-induced STAT3 activation, and showed the different in activity according to the presence of a phenylalanine residue, hydroxyl groups and isometric structure. The six new LABs isolated from Gajuknamu kimchi, and Lc. lactis KR-050L was selected as candidate IL-6/STAT3 inhibitors. The activity levels of 15 2,5-DKPs isolated from Lc. lactis KR-050L were verified. This study constitutes the first attempt to isolate various LABs from Gajuknamu kimchi and to discover IL-6/STAT3 inhibitors in the EtOAc extract of Lc. lactis KR-050L culture broth. Moreover, our data provide useful biochemical information regarding the commercialization of Lc. lactis isolated from Gajuknamu kimchi as an approach to use functional foods for the treatment of various diseases via IL-6/STAT3 activation. © 2017 The Society for Applied Microbiology.

  4. Insights into new bacteriophages of Lactococcus garvieae belonging to the family Podoviridae.

    PubMed

    Ghasemi, Seyed Mahdi; Bouzari, Majid; Shaykh Baygloo, Nima; Chang, Hyo-Ihl

    2014-11-01

    Lactococcus garvieae is an emerging pathogen responsible for lactococcosis, a serious disease in trout aquaculture. The identification of new bacteriophages against L. garvieae strains may be an effective way to fight this disease and to study the pathogen's biology. Three L. garvieae phages, termed WP-1, WWP-2 and SP-2, were isolated from different environments, and their morphological features, genome restriction profiles and structural protein patterns were studied. Random cloning of HindIII-cut fragments was performed, and the fragments were partially sequenced for each phage. Although slight differences were observed by transmission electron microscopy, all of the phages had hexagonal heads and short non-contractile tails and were classified as members of the family Podoviridae. Restriction digestion analysis of the nucleic acids of the different phages revealed that the HindIII and AseI digests produced similar DNA fragment patterns. Additionally, SDS-PAGE analysis indicated that the isolated phages have similar structural proteins. The sequence BLAST results did not show any significant similarity with other previously identified phages. To the best of our knowledge, this study provides the first molecular characterization of L. garvieae phages.

  5. Isolation of halotolerant Lactococcus lactis subsp. lactis from intestinal tract of coastal fish.

    PubMed

    Itoi, Shiro; Abe, Takeshi; Washio, Sayaka; Ikuno, Erika; Kanomata, Yuna; Sugita, Haruo

    2008-01-15

    We isolated lactic acid bacteria from the intestinal tract of the pufferfish Takifugu niphobles caught in Shimoda, Shizuoka, Japan by using MRS broth prepared with 50% seawater. Additional screening was carried out using phenotypic tests such as Gram staining, cell morphology, catalase, oxidase and fermentation of glucose. Subsequently 227 isolates screened by the phenotypic tests were subjected to species-specific PCR for Lactococcus lactis, resulting in four positive isolates. The 16S rRNA gene sequences from three isolates were highly similar to that of L. lactis subsp. lactis (DNA database accession number M58837), while that of one isolate was identical to that of Leuconostoc mesenteroides (AB023246). These isolates were characterized by API 50 CH for carbohydrate fermentation and other phenotypic criteria for salt tolerance, and the characteristics were compared with those of L. lactis subsp. lactis from a cheese starter culture. The carbohydrate fermentation profiles of these isolates were characteristic of L. lactis subsp. lactis strains, whereas the tolerance of these isolates to salt was higher than that of L. lactis subsp. lactis from the cheese starter culture: the new L. lactis isolates showed high salt tolerance in MRS-agar plates containing 200% seawater or 6% sodium chloride. This is the first report of the isolation of halotolerant strains of L. lactis subsp. lactis from a marine environment.

  6. Effects of diverse environmental conditions on {phi}LC3 prophage stability in Lactococcus lactis.

    PubMed

    Lunde, Merete; Aastveit, Are Halvor; Blatny, Janet Martha; Nes, Ingolf F

    2005-02-01

    The effects of various growth conditions on spontaneous phiLC3 prophage induction in Lactococcus lactis subsp. cremoris IMN-C1814 was analyzed with a half fraction of a 4(4) factorial experimental design. The four factors included in the study were nutrient availability, acidity, osmolarity, and temperature, each applied at four levels. These environmental factors are related to the fermentation processes in the dairy industry, in which bacteriophage attacks on sensitive starter strains are a constant threat to successful fermentation processes. The frequency of spontaneous phiLC3 induction was determined by quantitative analyses of restored DNA attachment sites (attB) on the bacterial chromosomes in a population of lysogenic cells. Statistical analysis revealed that all four environmental factors tested affected phiLC3 prophage stability and that the environmental factors were involved in interactions (interactions exist when the effect of one factor depends on the level of another factor). The spontaneous phiLC3 induction frequency varied from 0.08 to 1.76%. In general, the induction frequency remained at the same rate or decreased when level 1 to 3 of the four environmental factors was applied. At level 4, which generally gave the least favorable growth conditions, the induction frequency was either unchanged, decreased, or increased, depending on the type of stress. It appeared that the spontaneous induction frequency was independent of the growth behavior of the host. It was the environmental growth conditions that were the decisive factor in induction frequency.

  7. Lactococcus lactis LMG2081 Produces Two Bacteriocins, a Nonlantibiotic and a Novel Lantibiotic.

    PubMed

    Mirkovic, Nemanja; Polovic, Natalija; Vukotic, Goran; Jovcic, Branko; Miljkovic, Marija; Radulovic, Zorica; Diep, Dzung B; Kojic, Milan

    2016-04-01

    Bacteriocin producers normally possess dedicated immunity systems to protect themselves from their own bacteriocins.Lactococcus lactis strains LMG2081 and BGBM50 are known as lactococcin G producers. However, BGBM50 was sensitive to LMG2081, which indicated that LMG2081 might produce additional bacteriocins that are not present in BGBM50. Therefore, whole-genome sequencing of the two strains was performed, and a lantibiotic operon (called lctLMG) was identified in LMG2081 but not in BGBM50. The lctLMG operon contains six open reading frames; the first three genes,lmgA ,lmgM, and lmgT, are involved in the biosynthesis and export of bacteriocin, while the other three genes,lmgF,lmgE, and lmgG, are involved in lantibiotic immunity. Mutational analysis confirmed that the lctLMG operon is responsible for the additional antimicrobial activity. Specifically, site-directed mutation within this operon rendered LMG2081 inactive toward BGBM50. Subsequent purification and electrospray ionization-time of flight mass spectrometric analysis confirmed that the lantibiotic bacteriocin called lacticin LMG is exported as a 25-amino-acid peptide. Lacticin LMG is highly similar to the lacticin 481 group. It is interesting that a bacteriocin producer produces two different classes of bacteriocins, whose operons are located in the chromosome and a plasmid.

  8. [Characteristics and identification of bacteriocins produced by Lactococcus lactis subsp. lactis 194-K].

    PubMed

    Ustiugova, E A; Timofeeva, A V; Stoianova, L G; Netrusov, A I; Katrukha, G S

    2012-01-01

    The Lactococcus lactis subsp. lactis 194-K strain has been established to be able to produce two bacteriocins, one of which was identified as the known lantibiotic nisin A, and the other 194-D bacteriocin represents a polypeptide with a 2589-Da molecular mass and comprises 20 amino acid residues. Both bacteriocins were produced in varying proportions in all of the studied nutrient media, which support the growth of the producer. Depending on the cultivation medium, the nisin A content was 380- to 1123-fold lower in the 194-K stain culture fluid than that of the 194-D peptide. In comparision to to nisin A Bacteriocin 194-D possessed a wide range of antibacterial activity and suppressed the growth of both Gram-positive and Gram-negative bacteria. An optimal medium for 194-D bacteriocin synthesis was shown to be a fermentation medium which contained yeast extract, casein hydrolysate, and potassium phosphate. The biosynthesis ofbacteriocin 194-D by the 194-K strain in these media occurred parallel to producer growth, and its maximal accumulation in the culture fluid was observed at 14-20 h of the strain's growth.

  9. Characterisation of technologically proficient wild Lactococcus lactis strains resistant to phage infection.

    PubMed

    Madera, Carmen; García, Pilar; Janzen, Thomas; Rodríguez, Ana; Suárez, Juan E

    2003-09-15

    The aim of this work was to establish whether Lactococcus lactis strains isolated from spontaneous dairy fermentations exhibited useful milk-processing capabilities and resistance to bacteriophage infection in order to be used as components in starter formulations. The 33 out of 100 isolates of L. lactis, originated from farmhouse cheeses, were found to be resistant to a collection of 34 phages belonging to the c2 and 936 groups. Six of the isolates were discarded as potential starters because they were lysogenic and other five because they produced tyramine. Plasmid and chromosomal profiles of the 22 remaining isolates allowed their classification into 16 different strains. All of these were good lactic acid producers from lactose, moderately proteolytic and, in eight cases, diacetyl production from citrate was observed. The mechanism(s) leading to the phenotype of phage resistance was identified for all the strains used in this study. Inhibition of adsorption was the most frequent one, although genetic determinants for some abortive infection systems were also detected (abiB, abiG and abiI). Frequently, more than one mechanism was present in the same strain. One of the strains, L. lactis IPLA542, was selected as a model starter for pilot fermentations. It clotted milk normally both in the absence and in the presence of phage at concentrations that completely abolished the process when promoted by a phage-susceptible strain.

  10. Sequencing and transcriptional analysis of the biosynthesis gene cluster of putrescine-producing Lactococcus lactis.

    PubMed

    Ladero, Victor; Rattray, Fergal P; Mayo, Baltasar; Martín, María Cruz; Fernández, María; Alvarez, Miguel A

    2011-09-01

    Lactococcus lactis is a prokaryotic microorganism with great importance as a culture starter and has become the model species among the lactic acid bacteria. The long and safe history of use of L. lactis in dairy fermentations has resulted in the classification of this species as GRAS (General Regarded As Safe) or QPS (Qualified Presumption of Safety). However, our group has identified several strains of L. lactis subsp. lactis and L. lactis subsp. cremoris that are able to produce putrescine from agmatine via the agmatine deiminase (AGDI) pathway. Putrescine is a biogenic amine that confers undesirable flavor characteristics and may even have toxic effects. The AGDI cluster of L. lactis is composed of a putative regulatory gene, aguR, followed by the genes (aguB, aguD, aguA, and aguC) encoding the catabolic enzymes. These genes are transcribed as an operon that is induced in the presence of agmatine. In some strains, an insertion (IS) element interrupts the transcription of the cluster, which results in a non-putrescine-producing phenotype. Based on this knowledge, a PCR-based test was developed in order to differentiate nonproducing L. lactis strains from those with a functional AGDI cluster. The analysis of the AGDI cluster and their flanking regions revealed that the capacity to produce putrescine via the AGDI pathway could be a specific characteristic that was lost during the adaptation to the milk environment by a process of reductive genome evolution.

  11. Metabolic engineering of Lactococcus lactis influence of the overproduction of lipase enzyme.

    PubMed

    Raftari, Mohammad; Ghafourian, Sobhan; Bakar, Fatimah Abu

    2013-11-01

    The dairy industry uses lipase extensively for hydrolysis of milk fat. Lipase is used in the modification of the fatty acid chain length, to enhance the flavours of various chesses. Therefore finding the unlimited source of lipase is a concern of dairy industry. Due to the importance of lipase, this study was an attempt to express the lipase from Burkholderia cepacia in Lactococcus lactis. To achieve this, a gene associated with lipase transport was amplified and subcloned in inducible pNZ8148 vector, and subsequently transformed into Lc. lactis NZ9000. The enzyme assay as well as SDS-PAGE and western blotting were carried out to analysis the recombinant lipase expression. Nucleotide sequencing of the DNA insert from the clone revealed that the lipase activity corresponded to an open reading frame consisting of 1092 bp coding for a 37·5-kDa size protein. Blue colour colonies on nile blue sulphate agar and sharp band on 37·5-kD size on SDS-PAGE and western blotting results confirm the successful expression of lipase by Lc. lactis. The protein assay also showed high expression, approximately 152·2 μg/ml.h, of lipase by recombinant Lc. lactis. The results indicate that Lc. lactis has high potential to overproduce the recombinant lipase which can be used commercially for industrially purposes.

  12. Ingestion of milk fermented by genetically modified Lactococcus lactis improves the riboflavin status of deficient rats.

    PubMed

    LeBlanc, J G; Burgess, C; Sesma, F; Savoy de Giori, G; van Sinderen, D

    2005-10-01

    Riboflavin deficiency is common in many parts of the world, particularly in developing countries. The use of riboflavin-producing strains in the production of dairy products such as fermented milks, yogurts, and cheeses is feasible and economically attractive because it would decrease the costs involved during conventional vitamin fortification and satisfy consumer demands for healthier foods. The present study was conducted to assess in a rat bioassay the response of administration of milk fermented by modified Lactococcus lactis on the riboflavin status of deficient rats. Rats were fed a riboflavin-deficient diet during 21 d after which this same diet was supplemented with milk fermented by Lactoccus lactis pNZGBAH, a strain that overproduces riboflavin during fermentation. The novel fermented product, with increased levels of riboflavin, was able to eliminate most physiological manifestations of ariboflavinosis, such as stunted growth, elevated erythrocyte glutathione reductase activation coefficient values and hepatomegaly, that were observed using a riboflavin depletion-repletion model, whereas a product fermented with a nonriboflavin-producing strain did not show similar results. A safety assessment of this modified strain was performed by feeding rodents with the modified strain daily for 4 wk. This strain caused no detectable secondary effects. These results pave the way for analyzing the effect of similar riboflavin-overproducing lactic acid bacteria in human trials. The regular consumption of products with increased levels of riboflavin could help prevent deficiencies of this essential vitamin.

  13. Molecular Characterization of a Recombinant Manganese Superoxide Dismutase from Lactococcus lactis M4

    PubMed Central

    Chor Leow, Thean; Foo, Hooi Ling; Abdul Rahim, Raha

    2014-01-01

    A superoxide dismutase (SOD) gene of Lactococcus lactis M4 was cloned and expressed in a prokaryotic system. Sequence analysis revealed an open reading frame of 621 bp which codes for 206 amino acid residues. Expression of sodA under T7 promoter exhibited a specific activity of 4967 U/mg when induced with 1 mM of isopropyl-β-D-thiogalactopyranoside. The recombinant SOD was purified to homogeneity by immobilised metal affinity chromatography and Superose 12 gel filtration chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot analyses of the recombinant SOD detected a molecular mass of approximately 27 kDa. However, the SOD was in dimer form as revealed by gel filtration chromatography. The purified recombinant enzyme had a pI of 4.5 and exhibited maximal activity at 25°C and pH 7.2. It was stable up to 45°C. The insensitivity of this lactococcal SOD to cyanide and hydrogen peroxide established that it was a MnSOD. Although it has 98% homology to SOD of L. lactis IL1403, this is the first elucidated structure of lactococcal SOD revealing active sites containing the catalytic manganese coordinated by four ligands (H-27, H-82, D-168, and H-172). PMID:24592392

  14. Analysis of T-2 Toxin Removal Factors in a Lactococcus Fermentation System.

    PubMed

    Zhou, Lang-Hua; Wang, Ya-Ling; Qiu, Mei; Shi, Qi; Sun, Li-Jun; Liao, Jian-Meng; Xu, De-Feng; Liu, Ying; Fang, Zhi-Jia; Gooneratne, Ravi

    2017-09-01

    The objective of this work was to determine the bacterial strains and factors that most efficiently degrade T-2 toxin in foods or animal feed. To determine the most efficient strain and optimal incubation times for degradation of T-2, the rate of T-2 removal by three lactic acid bacteria strains was quantified by liquid chromatography plus tandem mass spectrometry after incubation in de Man Rogosa Sharpe broth with 50 ng mL(-1) T-2 at 37°C for 96 h. Various components of the most efficient degradation strain fermentation systems were extracted, and the ability to remove T-2 was assayed. Lactococcus lactis CAMT22361 was the most efficient degradation strain for removing T-2. Yeast extract powder interfered with L. lactis CAMT22361 in the degradation process. T-2 toxin was removed by various components of the L. lactis CAMT22361 cells in the following order: nonprotein material of the extracellular fraction > protein in the extracellular fraction > whole cell ≈ cell wall > cell intracellular matrix fluid. T-2 removal rates were 54.08% ± 0.79%, 43.65% ± 0.84%, 43.09% ± 0.87%, 41.98% ± 0.8%, and 23.45% ± 0.66%, respectively. The nonprotein fraction in the extracellular fluid was most likely the key component in L. lactis CAMT22361 and hence would be the most desirable cellular component to be used to remove T-2 from food or feed.

  15. Mechanism and energetics of dipeptide transport in membrane vesicles of Lactococcus lactis.

    PubMed Central

    Smid, E J; Driessen, A J; Konings, W N

    1989-01-01

    Alanyl-alpha-glutamate transport has been studied in Lactococcus lactis ML3 cells and in membrane vesicles fused with liposomes containing beefheart cytochrome c oxidase as a proton-motive-force-generating system. The uptake of Ala-Glu observed in de-energized cells can be stimulated 26-fold upon addition of lactose. No intracellular dipeptide pool could be detected in intact cells. In fused membranes, a 40-fold accumulation of Ala-Glu was observed in response to a proton motive force. Addition of ionophores and uncouplers resulted in a rapid efflux of the accumulated dipeptide, indicating that Ala-Glu accumulation is directly coupled to the proton motive force as a driving force. Ala-Glu uptake is an electrogenic process and the dipeptide is transported in symport with two protons. In both fused membranes and intact cells the same affinity constant (0.70 mM) for Ala-Glu uptake was found. Accumulated Ala-Glu is exchangeable with externally added alanyl-glutamate, glutamyl-glutamate, and leucyl-leucine, while no exchange occurred upon addition of the amino acid glutamate or alanine. These results indicate that the Ala-Glu transport system has a broad substrate specificity. PMID:2492499

  16. Lactose-mediated carbon catabolite repression of putrescine production in dairy Lactococcus lactis is strain dependent.

    PubMed

    del Rio, Beatriz; Ladero, Victor; Redruello, Begoña; Linares, Daniel M; Fernández, Maria; Martín, Maria Cruz; Alvarez, Miguel A

    2015-06-01

    Lactococcus lactis is the lactic acid bacterial (LAB) species most widely used as a primary starter in the dairy industry. However, several strains of L. lactis produce the biogenic amine putrescine via the agmatine deiminase (AGDI) pathway. We previously reported the putrescine biosynthesis pathway in L. lactis subsp. cremoris GE2-14 to be regulated by carbon catabolic repression (CCR) via glucose but not lactose (Linares et al., 2013). The present study shows that both these sugars repress putrescine biosynthesis in L. lactis subsp. lactis T3/33, a strain isolated from a Spanish artisanal cheese. Furthermore, we demonstrated that both glucose and lactose repressed the transcriptional activity of the aguBDAC catabolic genes of the AGDI route. Finally, a screening performed in putrescine-producing dairy L. lactis strains determined that putrescine biosynthesis was repressed by lactose in all the L. lactis subsp. lactis strains tested, but in only one L. lactis subsp. cremoris strain. Given the obvious importance of the lactose-repression in cheese putrescine accumulation, it is advisable to consider the diversity of L. lactis in this sense and characterize consequently the starter cultures to select the safest strains.

  17. Lactococcus lactis LMG2081 Produces Two Bacteriocins, a Nonlantibiotic and a Novel Lantibiotic

    PubMed Central

    Mirkovic, Nemanja; Polovic, Natalija; Vukotic, Goran; Jovcic, Branko; Miljkovic, Marija; Radulovic, Zorica; Diep, Dzung B.

    2016-01-01

    Bacteriocin producers normally possess dedicated immunity systems to protect themselves from their own bacteriocins. Lactococcus lactis strains LMG2081 and BGBM50 are known as lactococcin G producers. However, BGBM50 was sensitive to LMG2081, which indicated that LMG2081 might produce additional bacteriocins that are not present in BGBM50. Therefore, whole-genome sequencing of the two strains was performed, and a lantibiotic operon (called lctLMG) was identified in LMG2081 but not in BGBM50. The lctLMG operon contains six open reading frames; the first three genes, lmgA, lmgM, and lmgT, are involved in the biosynthesis and export of bacteriocin, while the other three genes, lmgF, lmgE, and lmgG, are involved in lantibiotic immunity. Mutational analysis confirmed that the lctLMG operon is responsible for the additional antimicrobial activity. Specifically, site-directed mutation within this operon rendered LMG2081 inactive toward BGBM50. Subsequent purification and electrospray ionization–time of flight mass spectrometric analysis confirmed that the lantibiotic bacteriocin called lacticin LMG is exported as a 25-amino-acid peptide. Lacticin LMG is highly similar to the lacticin 481 group. It is interesting that a bacteriocin producer produces two different classes of bacteriocins, whose operons are located in the chromosome and a plasmid. PMID:26896142

  18. Genome-Wide Transcriptional Responses to Carbon Starvation in Nongrowing Lactococcus lactis

    PubMed Central

    Ercan, Onur; Wels, Michiel; Smid, Eddy J.

    2015-01-01

    This paper describes the transcriptional adaptations of nongrowing, retentostat cultures of Lactococcus lactis to starvation. Near-zero-growth cultures (μ = 0.0001 h−1) obtained by extended retentostat cultivation were exposed to starvation by termination of the medium supply for 24 h, followed by a recovery period of another 24 h by reinitiating the medium supply to the retentostat culture. During starvation, the viability of the culture was largely retained, and the expression of genes involved in transcription and translational machineries, cell division, and cell membrane energy metabolism was strongly repressed. Expression of these genes was largely recovered following the reinitiation of the medium supply. Starvation triggered the elevated expression of genes associated with synthesis of branched-chain amino acids, histidine, purine, and riboflavin. The expression of these biosynthesis genes was found to remain at an elevated level after reinitiation of the medium supply. In addition, starvation induced the complete gene set predicted to be involved in natural competence in L. lactis KF147, and the elevated expression of these genes was sustained during the subsequent recovery period, but our attempts to experimentally demonstrate natural transformation in these cells failed. Mining the starvation response gene set identified a conserved cis-acting element that resembles the lactococcal CodY motif in the upstream regions of genes associated with transcription and translational machineries, purine biosynthesis, and natural transformation in L. lactis, suggesting a role for CodY in the observed transcriptome adaptations to starvation in nongrowing cells. PMID:25636846

  19. Surface display of glycosylated Tyrosinase related protein-2 (TRP-2) tumour antigen on Lactococcus lactis.

    PubMed

    Kalyanasundram, Jeevanathan; Chia, Suet Lin; Song, Adelene Ai-Lian; Raha, Abdul Rahim; Young, Howard A; Yusoff, Khatijah

    2015-12-29

    The exploitation of the surface display system of food and commensal lactic acid bacteria (LAB) for bacterial, viral, or protozoan antigen delivery has received strong interest recently. The Generally Regarded as Safe (GRAS) status of the Lactococcus lactis coupled with a non-recombinant strategy of in-trans surface display, provide a safe platform for therapeutic drug and vaccine development. However, production of therapeutic proteins fused with cell-wall anchoring motifs is predominantly limited to prokaryotic expression systems. This presents a major disadvantage in the surface display system particularly when glycosylation has been recently identified to significantly enhance epitope presentation. In this study, the glycosylated murine Tyrosinase related protein-2 (TRP-2) with the ability to anchor onto the L. lactis cell wall was produced in suspension adapted Chinese Hamster Ovary (CHO-S) cells by expressing TRP-2 fused with cell wall anchoring LysM motif (cA) at the C-terminus. A total amount of 33 μg of partially purified TRP-2-cA from ~6.0 g in wet weight of CHO-S cells was purified by His-tag affinity chromatography. The purified TRP-2-cA protein was shown to be N-glycosylated and successfully anchored to the L. lactis cell wall. Thus cell surface presentation of glycosylated mammalian antigens may now permit development of novel and inexpensive vaccine platforms.

  20. Single Bacterium Detection Using Sers

    NASA Astrophysics Data System (ADS)

    Gonchukov, S. A.; Baikova, T. V.; Alushin, M. V.; Svistunova, T. S.; Minaeva, S. A.; Ionin, A. A.; Kudryashov, S. I.; Saraeva, I. N.; Zayarny, D. A.

    2016-02-01

    This work is devoted to the study of a single Staphylococcus aureus bacterium detection using surface-enhanced Raman spectroscopy (SERS) and resonant Raman spectroscopy (RS). It was shown that SERS allows increasing sensitivity of predominantly low frequency lines connected with the vibrations of Amide, Proteins and DNA. At the same time the lines of carotenoids inherent to this kind of bacterium are well-detected due to the resonance Raman scattering mechanism. The reproducibility and stability of Raman spectra strongly depend on the characteristics of nanostructured substrate, and molecular structure and size of the tested biological object.

  1. Enhancement of nisin production by Lactococcus lactis in periodically re-alkalized cultures.

    PubMed

    Guerra, Nelson Pérez; Castro, Lorenzo Pastrana

    2003-10-01

    Synthesis of nisin as well as biomass production by Lactococcus lactis subsp. lactis CECT (Colección Española de Cultivos Tipo) 539 on both hydrolysed mussel-processing waste and whey medium were followed in three fixed volume fed-batch fermentations, with re-alkalization cycles. The two cultures on mussel-processing waste (MPW) were fed with a 240 g/l concentrated glucose and with a concentrated MPW (about 100 g of glucose/l). The culture on whey was fed with a mixture of concentrated whey (48 g of total sugars/l) and a 400 g/l concentrated lactose. The three cultures were mainly characterized with higher nisin titres [49.7, 109.6 and 124.7 bacteriocin activity units (AU)/ml respectively] compared with the batch process on de Man, Rogosa and Sharpe [(1960) J. Appl. Bacteriol. 23, 130-135] medium (49.6 AU/ml), MPW (9.5 AU/ml) and whey (22.5 AU/ml) [1 AU/ml is the amount of antibacterial compound needed to obtain 50% growth inhibition (LD50) compared with control tubes]. In the three fed-batch cultures a shift from homolactic to mixed-acid fermentation was observed, and other products (acetic acid, butane-2,3-diol or ethanol) in addition to lactic acid were detectable in the medium. However, their contributions to the total antibacterial activity of the post-incubates (the cell-free culture supernatant obtained at the end of the fermentation process) of L. lactis CECT 539 against Carnobacterium piscicola CECT 4020 were very low.

  2. Immunogenicity and immunoprotection of porcine circovirus type 2 (PCV2) Cap protein displayed by Lactococcus lactis.

    PubMed

    Li, Peng-Cheng; Qiao, Xu-Wen; Zheng, Qi-Sheng; Hou, Ji-Bo

    2016-01-27

    The capsid (Cap) protein, an important immunoprotective protein of porcine circovirus type 2 (PCV2), was expressed on the cell surface of the Gram-positive food-grade bacterium, Lactococcus lactis. Cap protein was fused to the peptidoglycan binding domain (known as the protein anchor domain, PA) of the lactococcal AcmA cell-wall hydrolase. The Cap protein fusion was non-covalently rebound to the surface of non-genetically modified, non-living high-binder L. lactis cells (designated Gram-positive enhancer matrix (GEM) particles). Expression of the recombinant GEM-displaying capsid protein (GEM-PA-Cap) was verified by Western blotting and immunofluorescence and transmission electron microscopy assays. To evaluate the immunogenicity of the recombinant Cap protein (rCap), 20 PCV2-seronegative piglets were immunized with the GEM-PA-Cap subunit vaccine, GEM alone, or phosphate-buffered saline (PBS, challenge control and empty control). Each group consisted of five piglets. The results showed that the level of PCV2-specific antibodies in piglets immunized with the GEM-PA-Cap subunit vaccine was significantly higher than that of the piglets immunized with GEM alone or the control group at all the time points post-vaccination (P<0.01). After challenge with the PCV2 wild-type strain, piglets that received the GEM-PA-Cap subunit vaccine showed significantly higher average daily weight gain (DWG) and shorter fever duration than the other two groups (P<0.001). Furthermore, a significant reduction in the gross lung lesion scores and lymph node lesion scores was noted in the GEM-PA-Cap-immunized group compared with the scores of the GEM or PBS-treated group (P<0.01). The results suggest that recombinant rCap displayed by L. lactis GEM particles provided the piglets with significant immunoprotection from PCV2-associated disease. Thus, the novel GEM-PA-Cap subunit vaccine has potential to be considered an effective and safe candidate vaccine against PCV2 infection in piglets

  3. Burkholderia heleia sp. nov., a nitrogen-fixing bacterium isolated from an aquatic plant, Eleocharis dulcis, that grows in highly acidic swamps in actual acid sulfate soil areas of Vietnam.

    PubMed

    Aizawa, Tomoko; Ve, Nguyen Bao; Nakajima, Mutsuyasu; Sunairi, Michio

    2010-05-01

    Nitrogen-fixing bacteria, strains SA41(T), SA42 and SA53, were isolated from an aquatic plant, Eleocharis dulcis, that grows in highly acidic swamps (pH 2-4) in actual acid sulfate soil areas of Vietnam. The isolates were Gram-negative, aerobic, non-spore-forming, rod-shaped bacteria, having a cell width of 0.6-0.7 microm and a length of 1.5-1.7 microm. They showed good growth between pH 3.0 and 7.0, and between 17 and 37 degrees C. The organisms contained ubiquinone Q-8 as the predominant isoprenoid quinone, and C(16 : 0), C(17 : 0) cyclo, C(18 : 1) omega7c and summed feature 3 (C(16 : 1) omega7c and/or iso-C(15 : 0) 2-OH) as major fatty acids. Their fatty acid profiles are similar to those reported for other Burkholderia species. The DNA G+C content of these strains was 64 mol%. On the basis of 16S rRNA gene sequence similarity, these strains were shown to belong to the genus Burkholderia. Although their calculated 16S rRNA gene sequence similarity values to Burkholderia silvatlantica, Burkholderia mimosarum, Burkholderia ferrariae and Burkholderia tropica were 98.5, 98.2, 98.0 and 97.0 %, respectively, the isolates formed a distinct group in phylogenetic trees, and the DNA-DNA relatedness values of strain SA41(T) to these species were 39, 41, 39 and 33 %, respectively. The results of physiological and biochemical tests, including whole-cell protein pattern analysis, allowed phenotypic differentiation of these strains from the published Burkholderia species. Therefore, strains SA41(T), SA42 and SA53 represent a novel species for which the name Burkholderia heleia sp. nov. is proposed. The type strain is SA41(T) (=NBRC 101817(T)=VTCC-D6-7(T)).

  4. First Report of a Hip Prosthetic and Joint Infection Caused by Lactococcus garvieae in a Woman Fishmonger▿

    PubMed Central

    Aubin, G. G.; Bémer, P.; Guillouzouic, A.; Crémet, L.; Touchais, S.; Fraquet, N.; Boutoille, D.; Reynaud, A.; Lepelletier, D.; Corvec, S.

    2011-01-01

    We describe the first case of hip prosthetic infection due to Lactococcus garvieae. The patient, a 71-year-old woman fishmonger, developed a hip infection 7 years after total hip arthroplasty. The origin of infection was possibly due to the manipulation or intake of seafood or fish contaminated with Lactococcus garvieae. PMID:21367987

  5. First report of a hip prosthetic and joint infection caused by Lactococcus garvieae in a woman fishmonger.

    PubMed

    Aubin, G G; Bémer, P; Guillouzouic, A; Crémet, L; Touchais, S; Fraquet, N; Boutoille, D; Reynaud, A; Lepelletier, D; Corvec, S

    2011-05-01

    We describe the first case of hip prosthetic infection due to Lactococcus garvieae. The patient, a 71-year-old woman fishmonger, developed a hip infection 7 years after total hip arthroplasty. The origin of infection was possibly due to the manipulation or intake of seafood or fish contaminated with Lactococcus garvieae.

  6. Dynamics of lactic acid bacteria populations in Rioja wines by PCR-DGGE, comparison with culture-dependent methods.

    PubMed

    González-Arenzana, Lucía; López, Rosa; Santamaría, Pilar; López-Alfaro, Isabel

    2013-08-01

    Lactic acid bacteria populations of red wine samples from industrial fermentations, including two different vinification methods were studied. For this investigation, polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis was employed to supplement previous results that were obtained by culture-dependent methods. PCR-DGGE was aimed to study two targeted genes, 16S ribosomal DNA (rDNA) and rpoB, and the results were useful to evaluate the microbial populations in wine samples. Moreover, an improvement of a detection limit determined so far for DGGE analysis was obtained with the method described in this study, what made possible to identify lactic acid bacteria populations below 10(1) colony-forming unit/mL. The species Oenococcus oeni was the most frequently detected bacterium, but identifications close to species Oenococcus kitaharae and Lactococcus lactis that are not often found in wine were firstly identified in samples of this research. PCR-DGGE allowed to detect 9 out of 11 lactic acid bacteria species identified in this study (nine by PCR-16S rDNA/DGGE and four by PCR-rpoB/DGGE), while five species were detected using the modified de Man, Rogosa and Sharpe agar. Therefore, the two methods were demonstrated to be complementary. This finding suggests that analysis of the lactic acid bacteria population structure in wine should be carried out using both culture-dependent and culture-independent techniques with more than one primer pair.

  7. Molecular Description and Industrial Potential of Tn6098 Conjugative Transfer Conferring Alpha-Galactoside Metabolism in Lactococcus lactis▿ †

    PubMed Central

    Machielsen, Ronnie; Siezen, Roland J.; van Hijum, Sacha A. F. T.; van Hylckama Vlieg, Johan E. T.

    2011-01-01

    A novel 51-kb conjugative transposon of Lactococcus lactis, designated Tn6098, encoding the capacity to utilize α-galactosides such as raffinose and stachyose, was identified and characterized. Alpha-galactosides are a dominant carbon source in many plant-derived foods. Most dairy lactococcus strains are unable to use α-galactosides as a growth substrate, yet many of these strains are known to have beneficial industrial traits. Conjugal transfer of Tn6098 was demonstrated from the plant-derived donor strain L. lactis KF147 to the recipient L. lactis NZ4501, a derivative of the dairy model strain L. lactis MG1363. The integration of Tn6098 into the genome of the recipient strain was confirmed by Illumina sequencing of the transconjugant L. lactis NIZO3921. The molecular structure of the integration site was confirmed by a PCR product spanning the insertion site. A 15-bp direct repeat sequence (TTATACCATAATTAC) is present on either side of Tn6098 in the chromosome of L. lactis KF147. One copy of this sequence is also present in the L. lactis MG1363 chromosome and represents the sole integration site. Phenotypic characterization of all strains showed that the transconjugant has not only acquired the ability to grow well in soy milk, a substrate rich in α-galactosides, but also has retained the flavor-forming capabilities of the recipient strain L. lactis MG1363. This study demonstrates how (induced) conjugation can be used to exploit the beneficial industrial traits of industrial dairy lactic acid bacteria in fermentation of plant-derived substrates. PMID:21115709

  8. Expression of immune-related genes in rainbow trout (Oncorhynchus mykiss) induced by probiotic bacteria during Lactococcus garvieae infection.

    PubMed

    Pérez-Sánchez, Tania; Balcázar, José Luis; Merrifield, Daniel L; Carnevali, Oliana; Gioacchini, Giorgia; de Blas, Ignacio; Ruiz-Zarzuela, Imanol

    2011-08-01

    The aim of the present study was to investigate the effect of lactic acid bacteria (LAB) on the control of lactococcosis as well as to assess the impact of probiotics on the expression of immune-related genes in the head kidney and intestine of rainbow trout (Oncorhynchus mykiss). Lactobacillus plantarum, Lactococcus lactis and Leuconostoc mesenteroides, were administered orally at 10⁶ CFU g⁻¹ feed to fish for 36 days. Twenty-one days after the start of the feeding period, fish were challenged with Lactococcus garvieae. Only the fish fed the diet containing Lb. plantarum showed significantly (P < 0.05) improved protection against L. garvieae compared to the control. Subsequently, real-time PCR was employed to determine the mRNA levels of IL-1β, IL-8, IL-10 and TNF-α in the head kidney, and IL-8, Tlr5 and IgT in the intestine of the control and Lb. plantarum groups. IL-1β, IL-10 and TNF-α gene expression were significantly up-regulated by Lb. plantarum. Moreover, the mRNA levels of IL-10, IL-8 and IgT were significantly higher in the Lb. plantarum group after L. garvieae infection, suggesting that Lb. plantarum can stimulate the immune response of rainbow trout. PCR-DGGE revealed no detectable levels of the probiotics or the pathogen present on the distal intestinal mucosa. These findings demonstrate that direct probiotic-host interactions with the intestine are not always necessary to induce host stimulatory responses which ultimately enhance disease resistance. Furthermore, as L. garvieae did not colonise the intestinal tract, and therefore likely did not infect via this route, the antagonistic properties of the probiotic candidate towards L. garvieae were likely of little influence in mediating the improved disease resistance which could be attributed to the elevated immunological response.

  9. Analysis of hemin effect on lactate reduction in Lactococcus lactis.

    PubMed

    Nagayasu, Machiko; Wardani, Agustin Krisna; Nagahisa, Keisuke; Shimizu, Hiroshi; Shioya, Suteaki

    2007-06-01

    Lactococcus lactis is a facultative anaerobic microorganism that produces lactate as the major product, and acetate and acetoin as by-products; some strains of this species produce an antimicrobial compound, nisin. Lactate has a strong inhibitory effect on L. lactis growth. On the other hand, hemin has a suppressive effect on lactate production during L. lactis growth under aerobic condition. To achieve the optimum effect of hemin on lactate amount reduction in L. lactis ATCC11454, cultures entailing various conditions were performed with and without hemin. In the culture with hemin, L. lactis growth and lactate reduction improved compared with those in the culture without hemin; that is, lactate production was suppressed by 1.8- and 1.3-fold under batch and fed-batch cultures, respectively. In microaerobic fed-batch culture with hemin, lactate production was sufficiently suppressed. This result suggests that microaerobic fed-batch culture could be applied to the maintenance of the low lactate amount. Under this condition, metabolic shift was observed from lactate to acetoin and acetate. However, no increase in nisin production was observed even though lactate production could significantly decrease in L. lactis ATCC11454.

  10. Evolution of Lactococcus lactis phages within a cheese factory.

    PubMed

    Rousseau, Geneviève M; Moineau, Sylvain

    2009-08-01

    We have sequenced the double-stranded DNA genomes of six lactococcal phages (SL4, CB13, CB14, CB19, CB20, and GR7) from the 936 group that were isolated over a 9-year period from whey samples obtained from a Canadian cheese factory. These six phages infected the same two industrial Lactococcus lactis strains out of 30 tested. The CB14 and GR7 genomes were found to be 100% identical even though they were isolated 14 months apart, indicating that a phage can survive in a cheese plant for more than a year. The other four genomes were related but notably different. The length of the genomes varied from 28,144 to 32,182 bp, and they coded for 51 to 55 open reading frames. All five genomes possessed a 3' overhang cos site that was 11 nucleotides long. Several structural proteins were also identified by nano-high-performance liquid chromatography-tandem mass spectrometry, confirming bioinformatic analyses. Comparative analyses suggested that the most recently isolated phages (CB19 and CB20) were derived, in part, from older phage isolates (CB13 and CB14/GR7). The organization of the five distinct genomes was similar to the previously sequenced lactococcal phage genomes of the 936 group, and from these sequences, a core genome was determined for lactococcal phages of the 936 group.

  11. Characterization of Lactococcus lactis subsp. lactis isolated from surface waters.

    PubMed

    Svec, P; Sedlácek, I

    2008-01-01

    A group of nine presumptive enterococci was isolated on enterococcal selective media Slanetz-Bartley agar and/or kanamycin-esculin-azide agar during a screening of Enterococcus spp. in surface waters. All strains formed a homogeneous cluster separated from all enterococcal species using rep-PCR fingerprinting with the (GTG)5 primer but they matched fingerprints revealed by Lactococcus lactis subsp. lactis representatives. Further identification using extensive biotyping and automated ribotyping with EcoRI (RiboPrinter microbial characterization system) confirmed all strains as L. lactis subsp. lactis in full correspondence with the (GTG)5-PCR. We demonstrated that L. lactis subsp. lactis strains occur in different surface waters and can be confused with enterococci due to their positive growth on selective enterococcal media as well as positive results in tests commonly used for identification of the genus Enterococcus (esculin hydrolysis, acetoin and pyrrolidonyl arylamidase production, growth at 10 degrees C and in 6.5% NaCl). The (GTG)5-PCR fingerprinting was revealed as a reliable and fast method for the identification of L. lactis subsp lactis while automated ribotyping with EcoRI proved to be a good tool for intrasubspecies typing purposes.

  12. Prophage induction in Lactococcus lactis by the bacteriocin Lactococcin 972.

    PubMed

    Madera, Carmen; García, Pilar; Rodríguez, Ana; Suárez, Juan E; Martínez, Beatriz

    2009-01-31

    Lactococcin 972 (Lcn972) is a non-pore forming bacteriocin with a narrow spectrum of activity restricted to Lactococcus. Lcn972 inhibits the incorporation of cell wall precursors in the septum area, thereby inhibiting cell division. In this work, an additional inhibitory effect is described, namely, the induction of the lytic cycle of resident prophages in the lysogenic strain L. lactis IPLA 513. Lcn972 triggered the release of prophages in a concentration-dependent fashion. The extent of prophage induction was influenced by the physiological status of the cultures, being maximal at the early exponential growth phase. A microtiter based protocol was designed and the induction ability of several antimicrobials was compared. Prophages were activated by all cell wall biosynthesis inhibitors tested, although the levels of induction were lower than those obtained after activation of the SOS response. As far as we know, this is the first report of prophage induction by an antimicrobial peptide. Since Lcn972 is active against L. lactis strains currently used in commercial starters, promising applications for dairy fermentations are discussed.

  13. Histidine biosynthesis genes in Lactococcus lactis subsp. lactis.

    PubMed Central

    Delorme, C; Ehrlich, S D; Renault, P

    1992-01-01

    The genes of Lactococcus lactis subsp. lactis involved in histidine biosynthesis were cloned and characterized by complementation of Escherichia coli and Bacillus subtilis mutants and DNA sequencing. Complementation of E. coli hisA, hisB, hisC, hisD, hisF, hisG, and hisIE genes and the B. subtilis hisH gene (the E. coli hisC equivalent) allowed localization of the corresponding lactococcal genes. Nucleotide sequence analysis of the 11.5-kb lactococcal region revealed 14 open reading frames (ORFs), 12 of which might form an operon. The putative operon includes eight ORFs which encode proteins homologous to enzymes involved in histidine biosynthesis. The operon also contains (i) an ORF encoding a protein homologous to the histidyl-tRNA synthetases but lacking a motif implicated in synthetase activity, which suggests that it has a role different from tRNA aminoacylation, and (ii) an ORF encoding a protein that is homologous to the 3'-aminoglycoside phosphotransferases but does not confer antibiotic resistance. The remaining ORFs specify products which have no homology with proteins in the EMBL and GenBank data bases. PMID:1400209

  14. Analyses of the probiotic property and stress resistance-related genes of Lactococcus lactis subsp. lactis NCDO 2118 through comparative genomics and in vitro assays

    PubMed Central

    Saraiva, Tessália D. L.; Silva, Wanderson M.; Pereira, Ulisses P.; Campos, Bruno C.; Benevides, Leandro J.; Rocha, Flávia S.; Figueiredo, Henrique C. P.; Azevedo, Vasco; Soares, Siomar C.

    2017-01-01

    Lactococcus lactis subsp. lactis NCDO 2118 was recently reported to alleviate colitis symptoms via its anti-inflammatory and immunomodulatory activities, which are exerted by exported proteins that are not produced by L. lactis subsp. lactis IL1403. Here, we used in vitro and in silico approaches to characterize the genomic structure, the safety aspects, and the immunomodulatory activity of this strain. Through comparative genomics, we identified genomic islands, phage regions, bile salt and acid stress resistance genes, bacteriocins, adhesion-related and antibiotic resistance genes, and genes encoding proteins that are putatively secreted, expressed in vitro and absent from IL1403. The high degree of similarity between all Lactococcus suggests that the Symbiotic Islands commonly shared by both NCDO 2118 and KF147 may be responsible for their close relationship and their adaptation to plants. The predicted bacteriocins may play an important role against the invasion of competing strains. The genes related to the acid and bile salt stresses may play important roles in gastrointestinal tract survival, whereas the adhesion proteins are important for persistence in the gut, culminating in the competitive exclusion of other bacteria. Finally, the five secreted and expressed proteins may be important targets for studies of new anti-inflammatory and immunomodulatory proteins. Altogether, the analyses performed here highlight the potential use of this strain as a target for the future development of probiotic foods. PMID:28384209

  15. Enhancement of 2-methylbutanal formation in cheese by using a fluorescently tagged Lacticin 3147 producing Lactococcus lactis strain.

    PubMed

    Fernández de Palencia, Pilar; de la Plaza, Marta; Mohedano, M Luz; Martínez-Cuesta, M Carmen; Requena, Teresa; López, Paloma; Peláez, Carmen

    2004-06-15

    The amino acid conversion to volatile compounds by lactic acid bacteria is important for aroma formation in cheese. In this work, we analyzed the effect of the lytic bacteriocin Lacticin 3147 on transamination of isoleucine and further formation of the volatile compound 2-methylbutanal in cheese. The Lacticin 3147 producing strain Lactococcus lactis IFPL3593 was fluorescently tagged (IFPL3593-GFP) by conjugative transfer of the plasmid pMV158GFP from Streptococcus pneumoniae, and used as starter in cheese manufacture. Starter adjuncts were the bacteriocin-sensitive strains L. lactis T1 and L. lactis IFPL730, showing branched chain amino acid aminotransferase and alpha-keto acid decarboxylase activity, respectively. Adjunct strains were selected to complete the isoleucine conversion pathway and, hence, increase formation of 2-methylbutanal conferring aroma to the cheese. The non-bacteriocin-producing strain L. lactis IFPL359-GFP was included as starter in the control batch. Fluorescent tagging of the starter strains allowed their tracing in cheese during ripening by fluorescence microscopy and confocal scanning laser microscopy. The bacteriocin produced by L. lactis IFPL3593-GFP enhanced lysis of the adjuncts with a concomitant increase in isoleucine transamination and about a two-fold increase of the derived volatile compound 2-methylbutanal. This led to an enhancement of the cheese aroma detected by a sensory panel. The improvement of cheese flavour and aroma may be of significant importance for the dairy industry.

  16. Pellet feed adsorbed with the recombinant Lactococcus lactis BFE920 expressing SiMA antigen induced strong recall vaccine effects against Streptococcus iniae infection in olive flounder (Paralichthys olivaceus).

    PubMed

    Kim, Daniel; Beck, Bo Ram; Lee, Sun Min; Jeon, Jongsu; Lee, Dong Wook; Lee, Jae Il; Song, Seong Kyu

    2016-08-01

    The aim of this study was to develop a fish feed vaccine that provides effective disease prevention and convenient application. A lactic acid bacterium (LAB), Lactococcus lactis BFE920, was modified to express the SiMA antigen, a membrane protein of Streptococcus iniae. The antigen was engineered to be expressed under the nisin promoter, which is induced by nisin produced naturally by the host LAB. Various sizes (40 ± 3.5 g, 80 ± 2.1 g, and 221 ± 2.4 g) of olive flounder (Paralichthys olivaceus) were vaccinated by feeding the extruded pellet feed, onto which the SiMA-expressing L. lactis BFE920 (1.0 × 10(7) CFU/g) was adsorbed. Vaccine-treated feed was administered twice a day for 1 week, and priming and boosting were performed with a 1-week interval in between. The vaccinated fish had significantly elevated levels of antigen-specific serum antibodies and T cell marker mRNAs: CD4-1, CD4-2, and CD8a. In addition, the feed vaccine significantly induced T cell effector functions, such as the production of IFN-γ and activation of the transcription factor that induces its expression, T-bet. When the flounder were challenged by intraperitoneal infection and bath immersion with S. iniae, the vaccinated fish showed 84% and 82% relative percent survival (RPS), respectively. Furthermore, similar protective effects were confirmed even 3 months after vaccination in a field study (n = 4800), indicating that this feed vaccine elicited prolonged duration of immunopotency. In addition, the vaccinated flounder gained 21% more weight and required 16% less feed to gain a unit of body weight compared to the control group. The data clearly demonstrate that the L. lactis BFE920-SiMA feed vaccine has strong protective effects, induces prolonged vaccine efficacy, and has probiotic effects. In addition, this LAB-based fish feed vaccine can be easily used to target many different pathogens of diverse fish species. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Expression of Staphylococcus aureus clumping factor A in Lactococcus lactis subsp. cremoris using a new shuttle vector.

    PubMed

    Que, Y A; Haefliger, J A; Francioli, P; Moreillon, P

    2000-06-01

    Staphylococcus aureus harbors redundant adhesins mediating tissue colonization and infection. To evaluate their intrinsic role outside of the staphylococcal background, a system was designed to express them in Lactococcus lactis subsp. cremoris 1363. This bacterium is devoid of virulence factors and has a known genetic background. A new Escherichia coli-L. lactis shuttle and expression vector was constructed for this purpose. First, the high-copy-number lactococcal plasmid pIL253 was equipped with the oriColE1 origin, generating pOri253 that could replicate in E. coli. Second, the lactococcal promoters P23 or P59 were inserted at one end of the pOri253 multicloning site. Gene expression was assessed by a luciferase reporter system. The plasmid carrying P23 (named pOri23) expressed luciferase constitutively at a level 10,000 times greater than did the P59-containing plasmid. Transcription was absent in E. coli. The staphylococcal clumping factor A (clfA) gene was cloned into pOri23 and used as a model system. Lactococci carrying pOri23-clfA produced an unaltered and functional 130-kDa ClfA protein attached to their cell walls. This was indicated both by the presence of the protein in Western blots of solubilized cell walls and by the ability of ClfA-positive lactococci to clump in the presence of plasma. ClfA-positive lactococci had clumping titers (titer of 4,112) similar to those of S. aureus Newman in soluble fibrinogen and bound equally well to solid-phase fibrinogen. These experiments provide a new way to study individual staphylococcal pathogenic factors and might complement both classical knockout mutagenesis and modern in vivo expression technology and signature tag mutagenesis.

  18. Recombinant invasive Lactococcus lactis can transfer DNA vaccines either directly to dendritic cells or across an epithelial cell monolayer.

    PubMed

    de Azevedo, Marcela; Meijerink, Marjolein; Taverne, Nico; Pereira, Vanessa Bastos; LeBlanc, Jean Guy; Azevedo, Vasco; Miyoshi, Anderson; Langella, Philippe; Wells, Jerry M; Chatel, Jean-Marc

    2015-09-11

    Lactococcus lactis (L. lactis), a generally regarded as safe (GRAS) bacterium has recently been investigated as a mucosal delivery vehicle for DNA vaccines. Because of its GRAS status, L. lactis represents an attractive alternative to attenuated pathogens. Previous studies showed that eukaryotic expression plasmids could be delivered into intestinal epithelial cells (IECs) by L. lactis, or recombinant invasive strains of L. lactis, leading to heterologous protein expression. Although expression of antigens in IECs might lead to vaccine responses, it would be of interest to know whether uptake of L. lactis DNA vaccines by dendritic cells (DCs) could lead to antigen expression as they are unique in their ability to induce antigen-specific T cell responses. To test this, we incubated mouse bone marrow-derived DCs (BMDCs) with invasive L. lactis strains expressing either Staphylococcus aureus Fibronectin Binding Protein A (LL-FnBPA+), or Listeria monocytogenes mutated Internalin A (LL-mInlA+), both strains carrying a plasmid DNA vaccine (pValac) encoding for the cow milk allergen β-lactoglobulin (BLG). We demonstrated that they can transfect BMDCs, inducing the secretion of the pro-inflammatory cytokine IL-12. We also measured the capacity of strains to invade a polarized monolayer of IECs, mimicking the situation encountered in the gastrointestinal tract. Gentamycin survival assay in these cells showed that LL-mInlA+ is 100 times more invasive than L. lactis. The cross-talk between differentiated IECs, BMDCs and bacteria was also evaluated using an in vitro transwell co-culture model. Co-incubation of strains in this model showed that DCs incubated with LL-mInlA+ containing pValac:BLG could express significant levels of BLG. These results suggest that DCs could sample bacteria containing the DNA vaccine across the epithelial barrier and express the antigen.

  19. An Export-Specific Reporter Designed for Gram-Positive Bacteria: Application to Lactococcus lactis

    PubMed Central

    Poquet, Isabelle; Ehrlich, S. Dusko; Gruss, Alexandra

    1998-01-01

    The identification of exported proteins by fusion studies, while well developed for gram-negative bacteria, is limited for gram-positive bacteria, in part due to drawbacks of available export reporters. In this work, we demonstrate the export specificity and use of the Staphylococcus aureus secreted nuclease (Nuc) as a reporter for gram-positive bacteria. Nuc devoid of its export signal (called ΔSPNuc) was used to create two fusions whose locations could be differentiated. Nuclease activity was shown to require an extracellular location in Lactococcus lactis, thus demonstrating the suitability of ΔSPNuc to report protein export. The shuttle vector pFUN was designed to construct ΔSPNuc translational fusions whose expression signals are provided by inserted DNA. The capacity of ΔSPNuc to reveal and identify exported proteins was tested by generating an L. lactis genomic library in pFUN and by screening for Nuc activity directly in L. lactis. All ΔSPNuc fusions displaying a strong Nuc+ phenotype contained a classical or a lipoprotein-type signal peptide or single or multiple transmembrane stretches. The function of some of the predicted signals was confirmed by cell fractionation studies. The fusions analyzed included long (up to 455-amino-acid) segments of the exported proteins, all previously unknown in L. lactis. Homology searches indicate that several of them may be implicated in different cell surface functions, such as nutrient uptake, peptidoglycan assembly, environmental sensing, and protein folding. Our results with L. lactis show that ΔSPNuc is well suited to report both protein export and membrane protein topology. PMID:9537391

  20. Lactococcus lactis Metabolism and Gene Expression during Growth on Plant Tissues

    PubMed Central

    Golomb, Benjamin L.

    2014-01-01

    Lactic acid bacteria have been isolated from living, harvested, and fermented plant materials; however, the adaptations these bacteria possess for growth on plant tissues are largely unknown. In this study, we investigated plant habitat-specific traits of Lactococcus lactis during growth in an Arabidopsis thaliana leaf tissue lysate (ATL). L. lactis KF147, a strain originally isolated from plants, exhibited a higher growth rate and reached 7.9-fold-greater cell densities during growth in ATL than the dairy-associated strain L. lactis IL1403. Transcriptome profiling (RNA-seq) of KF147 identified 853 induced and 264 repressed genes during growth in ATL compared to that in GM17 laboratory culture medium. Genes induced in ATL included those involved in the arginine deiminase pathway and a total of 140 carbohydrate transport and metabolism genes, many of which are involved in xylose, arabinose, cellobiose, and hemicellulose metabolism. The induction of those genes corresponded with L. lactis KF147 nutrient consumption and production of metabolic end products in ATL as measured by gas chromatography-time of flight mass spectrometry (GC-TOF/MS) untargeted metabolomic profiling. To assess the importance of specific plant-inducible genes for L. lactis growth in ATL, xylose metabolism was targeted for gene knockout mutagenesis. Wild-type L. lactis strain KF147 but not an xylA deletion mutant was able to grow using xylose as the sole carbon source. However, both strains grew to similarly high levels in ATL, indicating redundancy in L. lactis carbohydrate metabolism on plant tissues. These findings show that certain strains of L. lactis are well adapted for growth on plants and possess specific traits relevant for plant-based food, fuel, and feed fermentations. PMID:25384484

  1. Lactococcus lactis metabolism and gene expression during growth on plant tissues.

    PubMed

    Golomb, Benjamin L; Marco, Maria L

    2015-01-01

    Lactic acid bacteria have been isolated from living, harvested, and fermented plant materials; however, the adaptations these bacteria possess for growth on plant tissues are largely unknown. In this study, we investigated plant habitat-specific traits of Lactococcus lactis during growth in an Arabidopsis thaliana leaf tissue lysate (ATL). L. lactis KF147, a strain originally isolated from plants, exhibited a higher growth rate and reached 7.9-fold-greater cell densities during growth in ATL than the dairy-associated strain L. lactis IL1403. Transcriptome profiling (RNA-seq) of KF147 identified 853 induced and 264 repressed genes during growth in ATL compared to that in GM17 laboratory culture medium. Genes induced in ATL included those involved in the arginine deiminase pathway and a total of 140 carbohydrate transport and metabolism genes, many of which are involved in xylose, arabinose, cellobiose, and hemicellulose metabolism. The induction of those genes corresponded with L. lactis KF147 nutrient consumption and production of metabolic end products in ATL as measured by gas chromatography-time of flight mass spectrometry (GC-TOF/MS) untargeted metabolomic profiling. To assess the importance of specific plant-inducible genes for L. lactis growth in ATL, xylose metabolism was targeted for gene knockout mutagenesis. Wild-type L. lactis strain KF147 but not an xylA deletion mutant was able to grow using xylose as the sole carbon source. However, both strains grew to similarly high levels in ATL, indicating redundancy in L. lactis carbohydrate metabolism on plant tissues. These findings show that certain strains of L. lactis are well adapted for growth on plants and possess specific traits relevant for plant-based food, fuel, and feed fermentations.

  2. Expression of food-grade phytase in Lactococcus lactis from optimized conditions in milk broth.

    PubMed

    Miao, Yuzhi; Xu, Hui; Fei, Baojin; Qiao, Dairong; Cao, Yi

    2013-07-01

    The major objective of this study was to engineer lactic acid bacteria to produce the enzyme phytase from a gene native to Bacillus subtilis GYPB04. The phytase gene (phyC) of B. subtilis GYPB04 was cloned into the plasmid pMG36e for expression in Lactococcus lactis. The enzyme activity in L. lactis cultured in GM17 broth was 20.25 U/mL at 36°C. The expressed phytase was characterized as active in a pH range of 2.0-9.0 at a temperature range of 20-80°C, with an optimum pH of 5.5-6.5 and temperature of 60°C. When cultured in food-grade milk broth, the transformed L. lactis grew to an OD(600 nm) value of 1.05 and had a phytase yield of 13.58 U/mL. In same broth under optimized conditions for cell growth and phytase production, the transformant reached an OD(600 nm) value of 1.68 and a phytase yield of 42.12 U/mL, representing approximately 1.6-fold and 3.1-fold increases, respectively, compared to growth in natural milk broth. Fermentation was scaled to 5 L under optimized conditions, and product analysis revealed a final OD(600 nm) value of 1.89 and an extracellular enzyme activity of 24.23 U/mL. The results of this study may be used in the dairy fermentation industry for the development of functional, healthy yogurts and other fermented dairy foods that provide both active phytase and viable probiotics to the consumer.

  3. Interaction between the genomes of Lactococcus lactis and phages of the P335 species

    PubMed Central

    Kelly, William J.; Altermann, Eric; Lambie, Suzanne C.; Leahy, Sinead C.

    2013-01-01

    Phages of the P335 species infect Lactococcus lactis and have been particularly studied because of their association with strains of L. lactis subsp. cremoris used as dairy starter cultures. Unlike other lactococcal phages, those of the P335 species may have a temperate or lytic lifestyle, and are believed to originate from the starter cultures themselves. We have sequenced the genome of L. lactis subsp. cremoris KW2 isolated from fermented corn and found that it contains an integrated P335 species prophage. This 41 kb prophage (Φ KW2) has a mosaic structure with functional modules that are highly similar to several other phages of the P335 species associated with dairy starter cultures. Comparison of the genomes of 26 phages of the P335 species, with either a lytic or temperate lifestyle, shows that they can be divided into three groups and that the morphogenesis gene region is the most conserved. Analysis of these phage genomes in conjunction with the genomes of several L. lactis strains shows that prophage insertion is site specific and occurs at seven different chromosomal locations. Exactly how induced or lytic phages of the P335 species interact with carbohydrate cell surface receptors in the host cell envelope remains to be determined. Genes for the biosynthesis of a variable cell surface polysaccharide and for lipoteichoic acids (LTAs) are found in L. lactis and are the main candidates for phage receptors, as the genes for other cell surface carbohydrates have been lost from dairy starter strains. Overall, phages of the P335 species appear to have had only a minor role in the adaptation of L. lactis subsp. cremoris strains to the dairy environment, and instead they appear to be an integral part of the L. lactis chromosome. There remains a great deal to be discovered about their role, and their contribution to the evolution of the bacterial genome. PMID:24009606

  4. Structural basis for the transcriptional regulation of heme homeostasis in Lactococcus lactis.

    PubMed

    Sawai, Hitomi; Yamanaka, Masaru; Sugimoto, Hiroshi; Shiro, Yoshitsugu; Aono, Shigetoshi

    2012-08-31

    Although heme is a crucial element for many biological processes including respiration, heme homeostasis should be regulated strictly due to the cytotoxicity of free heme molecules. Numerous lactic acid bacteria, including Lactococcus lactis, acquire heme molecules exogenously to establish an aerobic respiratory chain. A heme efflux system plays an important role for heme homeostasis to avoid cytotoxicity of acquired free heme, but its regulatory mechanism is not clear. Here, we report that the transcriptional regulator heme-regulated transporter regulator (HrtR) senses and binds a heme molecule as its physiological effector to regulate the expression of the heme-efflux system responsible for heme homeostasis in L. lactis. To elucidate the molecular mechanisms of how HrtR senses a heme molecule and regulates gene expression for the heme efflux system, we determined the crystal structures of the apo-HrtR·DNA complex, apo-HrtR, and holo-HrtR at a resolution of 2.0, 3.1, and 1.9 Å, respectively. These structures revealed that HrtR is a member of the TetR family of transcriptional regulators. The residue pair Arg-46 and Tyr-50 plays a crucial role for specific DNA binding through hydrogen bonding and a CH-π interaction with the DNA bases. HrtR adopts a unique mechanism for its functional regulation upon heme sensing. Heme binding to HrtR causes a coil-to-helix transition of the α4 helix in the heme-sensing domain, which triggers a structural change of HrtR, causing it to dissociate from the target DNA for derepression of the genes encoding the heme efflux system. HrtR uses a unique heme-sensing motif with bis-His (His-72 and His-149) ligation to the heme, which is essential for the coil-to-helix transition of the α4 helix upon heme sensing.

  5. Characterization of Plasmids in a Human Clinical Strain of Lactococcus garvieae

    PubMed Central

    Blanco, M. Mar; López-Campos, Guillermo H.; Cutuli, M. Teresa; Fernández-Garayzábal, José F.

    2012-01-01

    The present work describes the molecular characterization of five circular plasmids found in the human clinical strain Lactococcus garvieae 21881. The plasmids were designated pGL1-pGL5, with molecular sizes of 4,536 bp, 4,572 bp, 12,948 bp, 14,006 bp and 68,798 bp, respectively. Based on detailed sequence analysis, some of these plasmids appear to be mosaics composed of DNA obtained by modular exchange between different species of lactic acid bacteria. Based on sequence data and the derived presence of certain genes and proteins, the plasmid pGL2 appears to replicate via a rolling-circle mechanism, while the other four plasmids appear to belong to the group of lactococcal theta-type replicons. The plasmids pGL1, pGL2 and pGL5 encode putative proteins related with bacteriocin synthesis and bacteriocin secretion and immunity. The plasmid pGL5 harbors genes (txn, orf5 and orf25) encoding proteins that could be considered putative virulence factors. The gene txn encodes a protein with an enzymatic domain corresponding to the family actin-ADP-ribosyltransferases toxins, which are known to play a key role in pathogenesis of a variety of bacterial pathogens. The genes orf5 and orf25 encode two putative surface proteins containing the cell wall-sorting motif LPXTG, with mucin-binding and collagen-binding protein domains, respectively. These proteins could be involved in the adherence of L. garvieae to mucus from the intestine, facilitating further interaction with intestinal epithelial cells and to collagenous tissues such as the collagen-rich heart valves. To our knowledge, this is the first report on the characterization of plasmids in a human clinical strain of this pathogen. PMID:22768237

  6. Roles of Thioredoxin Reductase during the Aerobic Life of Lactococcus lactis

    PubMed Central

    Vido, Karin; Diemer, Hélène; Van Dorsselaer, Alain; Leize, Emmanuelle; Juillard, Vincent; Gruss, Alexandra; Gaudu, Philippe

    2005-01-01

    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. PMID:15629931

  7. Expression of the immunoreactive buckwheat major allergenic storage protein in Lactococcus lactis.

    PubMed

    Shigemori, Suguru; Yonekura, Shinichi; Sato, Takashi; Otani, Hajime; Shimosato, Takeshi

    2013-04-01

    Proteins from buckwheat (Fagopyrum esculentum) are strong allergens that can cause serious symptoms, including anaphylaxis, in patients with hypersensitivity. In this study, we successfully developed a modified lactic acid bacterial vector (pNSH) and a recombinant strain of Lactococcus lactis NZ9000 (NZ9000) that produced a major allergenic storage protein of buckwheat, Fagag1 (61.2 kDa, GenBank accession number AF152003), with or without a green fluorescent protein (GFP) tag. GFP fluorescence allows for rapid, simple, and accurate measurement of target protein expression by microscopy or fluorimetry. We describe a convenient method for production of rGFP-Fagag1 fusion and rFagag1 proteins with a good yield in an advantageous probiotic host. We found that in vitro treatment of splenocytes isolated from buckwheat crude protein-immunized mice with rFagag1 increased the expression of allergic inflammation cytokines such as IL-4, IL-13, and IL-17 F. Because it was less antigenic, rGFP-Fagag1 protein from NZ9000 might be of limited use; however, rFagag1 from NZ9000 evoked a robust response as measured by induction of IL-4 and IL-17 F expression levels. The observed allergic activity is indicative of a Th2 cell-mediated immune response and is similar to the effects induced by exposure to buckwheat crude protein. Our results suggest that expression of rFagag1 in NZ9000 may facilitate in vivo applications of this system aimed at improving the specificity of immunological responses to buckwheat allergens.

  8. Fate of Lactococcus lactis starter cultures during late ripening in cheese models.

    PubMed

    Ruggirello, Marianna; Cocolin, Luca; Dolci, Paola

    2016-10-01

    The presence of Lactococcus lactis, commonly employed as starter culture, was, recently, highlighted and investigated during late cheese ripening. Thus, the main goal of the present study was to assess the persistence and viability of this microorganism throughout manufacturing and ripening of model cheeses. Eight commercial starters, constituted of L. lactis subsp. lactis and L. lactis subsp. cremoris, were inoculated in pasteurized milk in order to manufacture miniature cheeses, ripened for six months. Samples were analysed at different steps (milk after inoculum, curd after cutting, curd after pressing and draining, cheese immediately after salting and cheese at 7, 15, 30, 60, 90, 120, 150 and 180 days of ripening) and submitted to both culture-dependent (traditional plating on M17) and -independent analysis (reverse transcription-quantitative PCR). On the basis of direct RNA analysis, L. lactis populations were detected in all miniature cheeses up to the sixth month of ripening, confirming the presence of viable cells during the whole ripening process, including late stages. Noteworthy, L. lactis was detected by RT-qPCR in cheese samples also when traditional plating failed to indicate its presence. This discrepancy could be explain with the fact that lactococci, during ripening process, enter in a stressed physiological state (viable not culturable, VNC), which might cause their inability to grow on synthetic medium despite their viability in cheese matrix. Preliminary results obtained by "resuscitation" assays corroborated this hypothesis and 2.5% glucose enrichment was effective to recover L. lactis cells in VNC state. The capability of L. lactis to persist in late ripening, and the presence of VNC cells which are known to shift their catabolism to peptides and amino acids consumption, suggests a possible technological role of this microorganism in cheese ripening with a possible impact on flavour formation.

  9. Strain-Dependent Transcriptome Signatures for Robustness in Lactococcus lactis

    PubMed Central

    Dijkstra, Annereinou R.; Alkema, Wynand; Starrenburg, Marjo J. C.; van Hijum, Sacha A. F. T.; Bron, Peter A.

    2016-01-01

    Recently, we demonstrated that fermentation conditions have a strong impact on subsequent survival of Lactococcus lactis strain MG1363 during heat and oxidative stress, two important parameters during spray drying. Moreover, employment of a transcriptome-phenotype matching approach revealed groups of genes associated with robustness towards heat and/or oxidative stress. To investigate if other strains have similar or distinct transcriptome signatures for robustness, we applied an identical transcriptome-robustness phenotype matching approach on the L. lactis strains IL1403, KF147 and SK11, which have previously been demonstrated to display highly diverse robustness phenotypes. These strains were subjected to an identical fermentation regime as was performed earlier for strain MG1363 and consisted of twelve conditions, varying in the level of salt and/or oxygen, as well as fermentation temperature and pH. In the exponential phase of growth, cells were harvested for transcriptome analysis and assessment of heat and oxidative stress survival phenotypes. The variation in fermentation conditions resulted in differences in heat and oxidative stress survival of up to five 10-log units. Effects of the fermentation conditions on stress survival of the L. lactis strains were typically strain-dependent, although the fermentation conditions had mainly similar effects on the growth characteristics of the different strains. By association of the transcriptomes and robustness phenotypes highly strain-specific transcriptome signatures for robustness towards heat and oxidative stress were identified, indicating that multiple mechanisms exist to increase robustness and, as a consequence, robustness of each strain requires individual optimization. However, a relatively small overlap in the transcriptome responses of the strains was also identified and this generic transcriptome signature included genes previously associated with stress (ctsR and lplL) and novel genes, including nan

  10. Food-grade cloning and expression system for Lactococcus lactis.

    PubMed Central

    Platteeuw, C; van Alen-Boerrigter, I; van Schalkwijk, S; de Vos, W M

    1996-01-01

    A versatile set of cloning and expression vectors has been developed for application in self-cloning and other genetic modifications of Lactococcus lactis. The expression vectors were equipped with the controlled and strong lacA promoter of the lactococcal lactose operon. In addition, the transcriptional terminator of the aminopeptidase N gene, pepN, was inserted, which in some cases increased the genetic stabilities of the vectors and the cloned DNA. The small, 0.3-kb lacF gene encoding the soluble carrier enzyme IIALac was used as a dominant selection marker in the plasmid-free L. lactis strain NZ3000 carrying an in-frame deletion of the chromosomal lacF gene. Lactose-utilizing transformants were easily selected on lactose indicator plates at high frequencies and showed a copy number of approximately 50 plasmids per cell. All vectors were stably maintained in the lacF strain NZ3000 when grown on lactose, and only the high-level expression vectors showed some instability when their host was grown on glucose-containing medium. The application potentials of the expression vectors carrying the lacF marker were determined by cloning of the promoterless Escherichia coli gusA reporter gene under control of the lacA promoter followed by analysis of its expression. While in one of the vectors this resulted in a promoter-down mutation in the -10 region of the lacA promoter, in other vectors high-level and controlled expression of the gusA gene was observed. PMID:8975595

  11. Strain-Dependent Transcriptome Signatures for Robustness in Lactococcus lactis.

    PubMed

    Dijkstra, Annereinou R; Alkema, Wynand; Starrenburg, Marjo J C; Hugenholtz, Jeroen; van Hijum, Sacha A F T; Bron, Peter A

    2016-01-01

    Recently, we demonstrated that fermentation conditions have a strong impact on subsequent survival of Lactococcus lactis strain MG1363 during heat and oxidative stress, two important parameters during spray drying. Moreover, employment of a transcriptome-phenotype matching approach revealed groups of genes associated with robustness towards heat and/or oxidative stress. To investigate if other strains have similar or distinct transcriptome signatures for robustness, we applied an identical transcriptome-robustness phenotype matching approach on the L. lactis strains IL1403, KF147 and SK11, which have previously been demonstrated to display highly diverse robustness phenotypes. These strains were subjected to an identical fermentation regime as was performed earlier for strain MG1363 and consisted of twelve conditions, varying in the level of salt and/or oxygen, as well as fermentation temperature and pH. In the exponential phase of growth, cells were harvested for transcriptome analysis and assessment of heat and oxidative stress survival phenotypes. The variation in fermentation conditions resulted in differences in heat and oxidative stress survival of up to five 10-log units. Effects of the fermentation conditions on stress survival of the L. lactis strains were typically strain-dependent, although the fermentation conditions had mainly similar effects on the growth characteristics of the different strains. By association of the transcriptomes and robustness phenotypes highly strain-specific transcriptome signatures for robustness towards heat and oxidative stress were identified, indicating that multiple mechanisms exist to increase robustness and, as a consequence, robustness of each strain requires individual optimization. However, a relatively small overlap in the transcriptome responses of the strains was also identified and this generic transcriptome signature included genes previously associated with stress (ctsR and lplL) and novel genes, including nan

  12. Characterization of lactic acid bacteria coexisting with a nisin Z producer in Tsuda-turnip pickles.

    PubMed

    Aso, Yuji; Takeda, Ai; Sato, Masako; Takahashi, Tetsuya; Yamamoto, Tatsuyuki; Yoshikiyo, Keisuke

    2008-07-01

    Bacteriocin-producing lactic acid bacteria (LAB) are believed to be associated with many types of fermented food. The present study reports the identification of lactic acid bacterium MS27 producing a bacteriocin isolated from the Tsuda-turnip pickle, which is a Japanese fermented food, and characterization of LAB coexisting with the bacteriocin producers in the Tsuda-turnip pickle. The strain MS27 was identified as Lactococcus lactis subsp. lactis based on a partial 16S rRNA gene sequence and sugar fermentation pattern analyses. Mass spectroscopy and genetic analysis revealed that it produces nisin Z. Microbial population analysis revealed that the LAB community in the Tsuda-turnip pickle comprises nisin Z-sensitive and nisin Z-insensitive LAB (nonbacteriocin producers) and nisin Z producers at population rates of 52.5%, 37.5%, and 10.0%, respectively. This revealed that Leuconostoc spp. (nisin Z insensitive) is the dominant species among LAB microflora and that nisin Z insensitivity of a bacterial strain is proportional to its ability to dominate the population in Tsuda-turnip pickles. Competitive growth assay revealed that Leuconostoc spp. considerably suppressed the bacteriocin production of L. lactis MS27. These results suggested that Leuconostoc spp. contributes to the formation of the LAB community with a wide variety of microorganisms in Tsuda-turnip pickles.

  13. A lactose fermentation product produced by Lactococcus lactis subsp. lactis, acetate, inhibits the motility of flagellated pathogenic bacteria.

    PubMed

    Nakamura, Shuichi; Morimoto, Yusuke V; Kudo, Seishi

    2015-04-01

    Many strains of lactic acid bacteria have been used for the production of probiotics. Some metabolites produced by lactic acid bacteria impair the motilities of pathogenic bacteria. Because bacterial motility is strongly associated with virulence, the metabolic activities of lactic acid bacteria are effective for suppressing bacterial infections. Here we show that lactose fermentation by Lactococcus lactis subsp. lactis inhibits the motility of Salmonella enterica serovar Typhimurium. A single-cell tracking and rotation assay for a single flagellum showed that the swimming behaviour of Salmonella was severely but transiently impaired through disruption of flagellar rotation on exposure to media cultivated with Lac. lactis. Using a pH-sensitive fluorescent protein, we observed that the intracellular pH of Salmonella was decreased because of some fermentation products of Lac. lactis. We identified acetate as the lactose fermentation product of Lac. lactis triggering the paralysis of Salmonella flagella. The motilities of Pseudomonas, Vibrio and Leptospira strains were also severely disrupted by lactose utilization by Lac. lactis. These results highlight the potential use of Lac. lactis for preventing infections by multiple bacterial species.

  14. Effect of sodium acetate on the adhesion to porcine gastric mucin in a Lactococcus lactis strain grown on fructose.

    PubMed

    Kimoto-Nira, Hiromi; Moriya, Naoko; Yamasaki, Seishi; Takenaka, Akio; Suzuki, Chise

    2016-06-01

    The association of lactic acid bacteria with mucosal surfaces plays important roles in the beneficial effects of these bacteria on human health, such as colonization of the gastrointestinal tract for pathogen antagonism. Previously, we found that the adhesion of Lactococcus lactis 7-1 to porcine gastric mucin was higher with fructose than with lactose, galactose or xylose as the carbon source. In this study, we examined the effect of growth conditions on the adhesion of strain 7-1 grown on fructose. Medium components affect the adhesion: the adhesion of strain 7-1 grown with sodium acetate was higher than that without it. The enhancement of adhesion by sodium acetate was not observed under aerobic conditions. Cellular properties grown with or without sodium acetate were characterized: strain 7-1 grown with sodium acetate had similar sugar contents, and different fatty acid composition to those grown without it. Strain 7-1 grown with sodium acetate showed significantly lower cell yield and significantly higher hydrophobicity than those grown without it, which is associated with higher adhesion. Fructose and sodium acetate are frequently used in the food industry; this study may reveal a simple way to enhance the adhesion of lactic acid bacteria by growing them with these substances. © 2015 Japanese Society of Animal Science.

  15. Spatial Distribution of Lactococcus lactis Colonies Modulates the Production of Major Metabolites during the Ripening of a Model Cheese

    PubMed Central

    Le Boucher, Clémentine; Gagnaire, Valérie; Briard-Bion, Valérie; Jardin, Julien; Maillard, Marie-Bernadette; Dervilly-Pinel, Gaud; Le Bizec, Bruno; Lortal, Sylvie; Jeanson, Sophie

    2015-01-01

    In cheese, lactic acid bacteria are immobilized at the coagulation step and grow as colonies. The spatial distribution of bacterial colonies is characterized by the size and number of colonies for a given bacterial population within cheese. Our objective was to demonstrate that different spatial distributions, which lead to differences in the exchange surface between the colonies and the cheese matrix, can influence the ripening process. The strategy was to generate cheeses with the same growth and acidification of a Lactococcus lactis strain with two different spatial distributions, big and small colonies, to monitor the production of the major ripening metabolites, including sugars, organic acids, peptides, free amino acids, and volatile metabolites, over 1 month of ripening. The monitored metabolites were qualitatively the same for both cheeses, but many of them were more abundant in the small-colony cheeses than in the big-colony cheeses over 1 month of ripening. Therefore, the results obtained showed that two different spatial distributions of L. lactis modulated the ripening time course by generating moderate but significant differences in the rates of production or consumption for many of the metabolites commonly monitored throughout ripening. The present work further explores the immobilization of bacteria as colonies within cheese and highlights the consequences of this immobilization on cheese ripening. PMID:26497453

  16. Antimicrobial susceptibilities and random amplified polymorphic DNA-PCR fingerprint characterization of Lactococcus lactis ssp. lactis and Lactococcus garvieae isolated from bovine intramammary infections.

    PubMed

    Plumed-Ferrer, C; Barberio, A; Franklin-Guild, R; Werner, B; McDonough, P; Bennett, J; Gioia, G; Rota, N; Welcome, F; Nydam, D V; Moroni, P

    2015-09-01

    In total, 181 streptococci-like bacteria isolated from intramammary infections (IMI) were submitted by a veterinary clinic to Quality Milk Production Services (QMPS, Cornell University, Ithaca, NY). The isolates were characterized by sequence analysis, and 46 Lactococcus lactis ssp. lactis and 47 Lactococcus garvieae were tested for susceptibility to 17 antibiotics. No resistant strains were found for β-lactam antibiotics widely used in clinical practice (penicillin, ampicillin, and amoxicillin), and all minimum inhibitory concentrations (MIC) were far from the resistance breakpoints. Eight strains had MIC intermediate to cefazolin. The random amplification of polymorphic DNA (RAPD)-PCR fingerprint patterns showed a slightly higher heterogeneity for Lc. lactis ssp. lactis isolates than for Lc. garvieae isolates.

  17. Comparative genome analysis of Lactococcus garvieae using a suppression subtractive hybridization library: discovery of novel DNA signatures.

    PubMed

    Kim, Wonyong; Park, Hee Kuk; Thanh, Hien Dang; Lee, Bo-Young; Shin, Jong Wook; Shin, Hyoung-Shik

    2011-12-01

    Lactococcus garvieae, the pathogenic species in the genus Lactococcus, is recognized as an emerging pathogen in fish, animals, and humans. Despite the widespread distribution and emerging clinical significance of L. garvieae, little is known about the genomic content of this microorganism. Suppression subtractive hybridization was performed to identify the genomic differences between L. garvieae and Lactococcus lactis ssp. lactis, its closest phylogenetic neighbor, and the type species of the genus Lactococcus. Twenty-seven clones were specific to L. garvieae and were highly different from Lactococcus lactis in their nucleotide and protein sequences. Lactococcus garvieae primer sets were subsequently designed for two of these clones corresponding to a pyrH gene and a novel DNA signature for application in the specific detection of L. garvieae. The primer specificities were evaluated relative to three previously described 16S rRNA gene-targeted methods using 32 Lactococcus and closely related strains. Both newly designed primer sets were highly specific to L. garvieae and performed better than did the existing primers. Our find