scholarly journals Task Distribution between Acetate and Acetoin Pathways To Prolong Growth inLactococcus lactisunder Respiration Conditions

2018 ◽  
Vol 84 (18) ◽  
Author(s):  
Bénédicte Cesselin ◽  
Christel Garrigues ◽  
Martin B. Pedersen ◽  
Célia Roussel ◽  
Alexandra Gruss ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Stationary Phase ◽  
Lactococcus Lactis ◽  
Time Course ◽  
Biomass Yield ◽  
Fine Tuning ◽  
Ph Homeostasis ◽  
Content Type ◽  
Growth And Survival ◽  
Bacterial Physiology

ABSTRACTLactococcus lactisis the main bacterium used for food fermentation and is a candidate for probiotic development. In addition to fermentation growth, supplementation with heme under aerobic conditions activates a cytochrome oxidase, which promotes respiration metabolism. In contrast to fermentation, in which cells consume energy to produce mainly lactic acid, respiration metabolism dramatically changes energy metabolism, such that massive amounts of acetic acid and acetoin are produced at the expense of lactic acid. Our goal was to investigate the metabolic changes that correlate with significantly improved growth and survival during respiration growth. Using transcriptional time course analyses, mutational analyses, and promoter-reporter fusions, we uncover two main pathways that can explain the robust growth and stability of respiration cultures. First, the acetate pathway contributes to biomass yield in respiration without affecting medium pH. Second, the acetoin pathway allows cells to cope with internal acidification, which directly affects cell density and survival in stationary phase. Our results suggest that manipulation of these pathways will lead to fine-tuning respiration growth, with improved yield and stability.IMPORTANCELactococcus lactisis used in food and biotechnology industries for its capacity to produce lactic acid, aroma, and proteins. This species grows by fermentation or by an aerobic respiration metabolism when heme is added. Whereas fermentation leads mostly to lactic acid production, respiration produces acetate and acetoin. Respiration growth leads to greatly improved bacterial growth and survival. Our study aims at deciphering mechanisms of respiration metabolism that have a major impact on bacterial physiology. Our results showed that two metabolic pathways (acetate and acetoin) are key elements of respiration. The acetate pathway contributes to biomass yield. The acetoin pathway is needed for pH homeostasis, which affects metabolic activities and bacterial viability in stationary phase. This study clarifies key metabolic elements that are required to maintain the growth advantage conferred by respiration metabolism and has potential uses in strain optimization for industrial and biomedical applications.

scholarly journals Spatial Distribution of Lactococcus lactis Colonies Modulates the Production of Major Metabolites during the Ripening of a Model Cheese

2015 ◽  
Vol 82 (1) ◽  
pp. 202-210 ◽  
Author(s):  
Clémentine Le Boucher ◽  
Valérie Gagnaire ◽  
Valérie Briard-Bion ◽  
Julien Jardin ◽  
Marie-Bernadette Maillard ◽  
...  
Keyword(s):  
Spatial Distribution ◽  
Lactococcus Lactis ◽  
Time Course ◽  
Bacterial Population ◽  
Spatial Distributions ◽  
Content Type ◽  
Cheese Ripening ◽  
Volatile Metabolites ◽  
Small Colony ◽  
Exchange Surface

ABSTRACTIn 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 aLactococcus lactisstrain 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 ofL. lactismodulated 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.

scholarly journals The KupA and KupB Proteins of Lactococcus lactis IL1403 Are Novel c-di-AMP Receptor Proteins Responsible for Potassium Uptake

2019 ◽  
Vol 201 (10) ◽  
Author(s):  
Ingrid M. Quintana ◽  
Johannes Gibhardt ◽  
Asan Turdiev ◽  
Elke Hammer ◽  
Fabian M. Commichau ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacterium ◽  
Lactococcus Lactis ◽  
Second Messenger ◽  
Cell Factory ◽  
Content Type ◽  
Potassium Homeostasis ◽  
Rna Molecules ◽  
High Affinity ◽  
Potassium Transporters

ABSTRACT Cyclic di-AMP (c-di-AMP) is a second messenger involved in diverse metabolic processes, including osmolyte uptake, cell wall homeostasis, and antibiotic and heat resistance. In Lactococcus lactis, a lactic acid bacterium which is used in the dairy industry and as a cell factory in biotechnological processes, the only reported interaction partners of c-di-AMP are the pyruvate carboxylase and BusR, the transcription regulator of the busAB operon for glycine betaine uptake. However, recent studies uncovered a major role of c-di-AMP in the control of potassium homeostasis, and potassium is the signal that triggers c-di-AMP synthesis. In this study, we have identified KupA and KupB, which belong to the Kup/HAK/KT family, as novel c-di-AMP binding proteins. Both proteins are high-affinity potassium transporters, and their transport activities are inhibited by binding of c-di-AMP. Thus, in addition to the well-studied Ktr/Trk potassium channels, KupA and KupB represent a second class of potassium transporters that are subject to inhibition by c-di-AMP. IMPORTANCE Potassium is an essential ion in every living cell. Even though potassium is the most abundant cation in cells, its accumulation can be toxic. Therefore, the level of potassium has to be tightly controlled. In many Gram-positive bacteria, the second messenger cyclic di-AMP plays a key role in the control of potassium homeostasis by binding to potassium transporters and regulatory proteins and RNA molecules. In the lactic acid bacterium Lactococcus lactis, none of these conserved c-di-AMP-responsive molecules are present. In this study, we demonstrate that the KupA and KupB proteins of L. lactis IL1403 are high-affinity potassium transporters and that their transport activity is inhibited by the second messenger c-di-AMP.

scholarly journals Natural DNA Transformation Is Functional in Lactococcus lactis subsp. cremoris KW2

2017 ◽  
Vol 83 (16) ◽  
Author(s):  
Blandine David ◽  
Amandine Radziejwoski ◽  
Frédéric Toussaint ◽  
Laetitia Fontaine ◽  
Marie Henry de Frahan ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Lactococcus Lactis ◽  
Natural Transformation ◽  
Single Point ◽  
Adaptor Protein ◽  
Single Point Mutation ◽  
Dna Transformation ◽  
Dna Uptake ◽  
Genetic Traits ◽  
Content Type

ABSTRACT Lactococcus lactis is one of the most commonly used lactic acid bacteria in the dairy industry. Activation of competence for natural DNA transformation in this species would greatly improve the selection of novel strains with desired genetic traits. Here, we investigated the activation of natural transformation in L. lactis subsp. cremoris KW2, a strain of plant origin whose genome encodes the master competence regulator ComX and the complete set of proteins usually required for natural transformation. In the absence of knowledge about competence regulation in this species, we constitutively overproduced ComX in a reporter strain of late competence phase activation and showed, by transcriptomic analyses, a ComX-dependent induction of all key competence genes. We further demonstrated that natural DNA transformation is functional in this strain and requires the competence DNA uptake machinery. Since constitutive ComX overproduction is unstable, we alternatively expressed comX under the control of an endogenous xylose-inducible promoter. This regulated system was used to successfully inactivate the adaptor protein MecA and subunits of the Clp proteolytic complex, which were previously shown to be involved in ComX degradation in streptococci. In the presence of a small amount of ComX, the deletion of mecA, clpC, or clpP genes markedly increased the activation of the late competence phase and transformability. Altogether, our results report the functionality of natural DNA transformation in L. lactis and pave the way for the identification of signaling mechanisms that trigger the competence state in this species. IMPORTANCE Lactococcus lactis is a lactic acid bacterium of major importance, which is used as a starter species for milk fermentation, a host for heterologous protein production, and a delivery platform for therapeutic molecules. Here, we report the functionality of natural transformation in L. lactis subsp. cremoris KW2 by the overproduction of the master competence regulator ComX. The developed procedure enables a flexible approach to modify the chromosome with single point mutation, sequence insertion, or sequence replacement. These results represent an important step for the genetic engineering of L. lactis that will facilitate the design of strains optimized for industrial applications. This will also help to discover natural regulatory mechanisms controlling competence in the genus Lactococcus.

scholarly journals Contribution of Lactococcus lactis Reducing Properties to the Downregulation of a Major Virulence Regulator in Staphylococcus aureus, theagrSystem

2014 ◽  
Vol 80 (22) ◽  
pp. 7028-7035 ◽  
Author(s):  
Sébastien Nouaille ◽  
Lucie Rault ◽  
Sophie Jeanson ◽  
Pascal Loubière ◽  
Yves Le Loir ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Lactic Acid ◽  
Lactococcus Lactis ◽  
Response Regulator ◽  
Food Poisoning ◽  
Accessory Gene ◽  
Content Type ◽  
Accessory Gene Regulator ◽  
Virulence Regulator ◽  
Reducing Properties

ABSTRACTStaphylococcus aureusis a major cause of food poisoning outbreaks associated with dairy products, because of the ingestion of preformed enterotoxins. The biocontrol ofS. aureususing lactic acid bacteria (LAB) offers a promising opportunity to fight this pathogen while respecting the product ecosystem. We had previously established the ability ofLactococcus lactis, a lactic acid bacterium widely used in the dairy industry, to downregulate a major staphylococcal virulence regulator, the accessory gene regulator (agr) system, and, as a consequence,agr-controlled enterotoxins. In the present paper, we have shown that the oxygen-independent reducing properties ofL. lactiscontribute toagrdownregulation. Neutralizing lactococcal reduction by adding potassium ferricyanide or maintaining the oxygen pressure constant at 50% releasedagrdownregulation in the presence ofL. lactis. This downregulation still occurred in anS. aureus srrAmutant, indicating that the staphylococcal respiratory response regulator SrrAB was not the only component in the signaling pathway. Therefore, this study clearly demonstrates the ability ofL. lactisreducing properties to interfere with the expression ofS. aureusvirulence, thus highlighting this general property of LAB as a lever to control the virulence expression of this major pathogen in a food context and beyond.

scholarly journals Versatile Cas9-Driven Subpopulation Selection Toolbox forLactococcus lactis

2018 ◽  
Vol 84 (8) ◽  
Author(s):  
Simon van der Els ◽  
Jennelle K. James ◽  
Michiel Kleerebezem ◽  
Peter A. Bron
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Lactococcus Lactis ◽  
Mobile Genetic Elements ◽  
Broad Host Range ◽  
Content Type ◽  
Genetic Elements ◽  
Wide Range ◽  
Dairy Fermentation ◽  
Selection Of

ABSTRACTCRISPR-Cas9 technology has been exploited for the removal or replacement of genetic elements in a wide range of prokaryotes and eukaryotes. Here, we describe the extension of the Cas9 application toolbox to the industrially important dairy speciesLactococcus lactis. The Cas9 expression vector pLABTarget, encoding theStreptocccus pyogenesCas9 under the control of a constitutive promoter, was constructed, allowing plug and play introduction of short guide RNA (sgRNA) sequences to target specific genetic loci. Introduction of apepN-targeting derivative of pLABTarget intoL. lactisstrain MG1363 led to a strong reduction in the number of transformants obtained, which did not occur in apepNdeletion derivative of the same strain, demonstrating the specificity and lethality of the Cas9-mediated double-strand breaks in the lactococcal chromosome. Moreover, the same pLABTarget derivative allowed the selection of apepNdeletion subpopulation from its corresponding single-crossover plasmid integrant precursor, accelerating the construction and selection of gene-specific deletion derivatives inL. lactis. Finally, pLABTarget, which contained sgRNAs designed to target mobile genetic elements, allowed the effective curing of plasmids, prophages, and integrative conjugative elements (ICEs). These results establish that pLABTarget enables the effective exploitation of Cas9 targeting inL. lactis, while the broad-host-range vector used suggests that this toolbox could readily be expanded to other Gram-positive bacteria.IMPORTANCEMobile genetic elements inLactococcus lactisand other lactic acid bacteria (LAB) play an important role in dairy fermentation, having both positive and detrimental effects during the production of fermented dairy products. The pLABTarget vector offers an efficient cloning platform for Cas9 application in lactic acid bacteria. Targeting Cas9 toward mobile genetic elements enabled their effective curing, which is of particular interest in the context of potentially problematic prophages present in a strain. Moreover, Cas9 targeting of other mobile genetic elements enables the deciphering of their contribution to dairy fermentation processes and further establishment of their importance for product characteristics.

scholarly journals Complete Genome Sequences of Lactococcus lactis subsp. lactis bv. diacetylactis FM03 and Leuconostoc mesenteroides FM06 Isolated from Cheese

2017 ◽  
Vol 5 (28) ◽  
Author(s):  
Oscar van Mastrigt ◽  
Tjakko Abee ◽  
Eddy J. Smid
Keyword(s):  
Lactococcus Lactis ◽  
Complete Genome ◽  
Leuconostoc Mesenteroides ◽  
Genome Sequences ◽  
Content Type ◽  
Growth And Survival ◽  
Lactococcus Lactis Subsp ◽  
Genes Encoding

ABSTRACT Here, the genome sequences of Lactococcus lactis subsp. lactis bv. diacetylactis FM03 and Leuconostoc mesenteroides FM06, both isolated from cheese, are presented. FM03 and FM06 contain 7 and 3 plasmids, respectively, that carry genes encoding functions important for growth and survival in dairy fermentations.

scholarly journals Functional and Genetic Characterization of the Tap Efflux Pump in Mycobacterium bovis BCG

2012 ◽  
Vol 56 (4) ◽  
pp. 2074-2083 ◽  
Author(s):  
Santiago Ramón-García ◽  
Virginie Mick ◽  
Elisa Dainese ◽  
Carlos Martín ◽  
Charles J. Thompson ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Stationary Phase ◽  
Mycobacterium Bovis ◽  
Efflux Pump ◽  
Expression Patterns ◽  
Efflux Pumps ◽  
Gene Encoding ◽  
Content Type ◽  
Active Efflux ◽  
Bacterial Physiology

ABSTRACTEfflux pumps extrude a wide variety of chemically unrelated compounds conferring multidrug resistance and participating in numerous physiological processes.Mycobacterium tuberculosispossesses many efflux pumps, and their roles in drug resistance and physiology are actively investigated. In this work we found thattapmutant cells showed changes in morphology and a progressive loss of viability upon subcultivation in liquid medium. Transcriptome analysis inMycobacterium bovisBCG revealed that disruption of theRv1258cgene, encoding the Tap efflux pump, led to an extensive change in gene expression patterns during stationary phase, with no changes during exponential growth. In stationary phase, Tap inactivation triggered a general stress response and led to a general repression of genes involved in cell wall biosynthesis, in particular the formation of the peptidoglycan; this suggested the accumulation of an unknown Tap substrate that reaches toxic concentrations during stationary phase. We also found that both disruption and overexpression oftapaltered susceptibility to many clinically approved antibiotics inM. bovisBCG. Acriflavine and tetracycline accumulation assays and carbonyl cyanidem-chlorophenylhydrazone (CCCP) potentiation experiments demonstrated that this phenotype was due to an active efflux mechanism. These findings emphasize the important role of the Tap efflux pump in bacterial physiology and intrinsic drug resistance.

Antimutagenicity of Lactic Acid Bacteria from “Idly” Against Food-Related Mutagens

1993 ◽  
Vol 56 (12) ◽  
pp. 1061-1066 ◽  
Author(s):  
NAGAPPA THYAGARAJA ◽  
AKIYOSHI HOSONO
Keyword(s):  
Lactic Acid ◽  
Amino Acid ◽  
Lactic Acid Bacteria ◽  
Stationary Phase ◽  
Time Course ◽  
Growth Stages ◽  
Enzyme Profile ◽  
Mutagenicity Assay ◽  
Enzymes Of Carbohydrate Metabolism ◽  
Different Growth Stages

Lactic acid bacteria from “Idly”, a traditional cereal pulse product of southern India, were evaluated for antimutagenic properties. Effect of their presence in Salmonella mutagenicity assay with foodborne mutagens like spice extracts, amino acid pyrolysates, and aflatoxins was examined. Variation of antimutagenic potential at different growth stages of these lactic acid bacteria was examined in time-course studies. The enzyme profile was examined to determine any possible relationship between antimutagenicity and enzymes in these lactic acid bacteria. Most of the lactic acid bacteria tested were found to decrease mutagenicities exerted by these mutagens significantly. Time-course studies showed that antimutagenic ability decreased in stationary phase of growth of lactic acid bacteria. There was no correlation between antimutagenicity and enzyme profile quantifying proteolytic, lipolytic, and other enzymes of carbohydrate metabolism. Lactic acid bacteria from “Idly” were found to be effective in suppressing the mutagenicities of the kinds encountered in foods.

scholarly journals Attenuated Lactococcus lactis and Surface Bacteria as Tools for Conditioning the Microbiota and Driving the Ripening of Semisoft Caciotta Cheese

2019 ◽  
Vol 86 (5) ◽  
Author(s):  
Maria Calasso ◽  
Fabio Minervini ◽  
Francesca De Filippis ◽  
Danilo Ercolini ◽  
Maria De Angelis ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Lactococcus Lactis ◽  
Lactobacillus Delbrueckii ◽  
Bacterial Strains ◽  
Multivariate Statistical ◽  
Content Type ◽  
Cheese Ripening ◽  
Brochothrix Thermosphacta ◽  
Staphylococcus Equorum ◽  
Metabolic Activities

ABSTRACT This study aimed at establishing the effects of attenuated starters and surface bacteria on various features of caciotta cheese. The cheese undergoes a ripening period during which the house microbiota contaminates the surface. Conventional cheese (the control cheese [CC]) is made using only primary starters. Primary starters and attenuated (i.e., unable to grow and synthesize lactic acid) Lactococcus lactis (Lc. lactis) subsp. lactis were used to produce caciotta cheese without (ATT cheese) or with an inoculum of surface bacteria: (i) Leuconostoc lactis (Le. lactis) (LL cheese), (ii) Vibrio casei (VC cheese), (iii) Staphylococcus equorum (SE cheese), (iv) Brochothrix thermosphacta (BX cheese), and (v) a mixture of all four (MIX cheese). Attenuated Lc. lactis increased microbial diversity during cheese ripening. At the core, attenuated starter mainly increased indigenous lactococci and Lactobacillus delbrueckii group bacteria. At the surface, the main effect was on Macrococcus caseolyticus. Autochthonous Le. lactis strains took advantage of the attenuated starter, becoming dominant. Adjunct Le. lactis positively affected Lactobacillus sakei group bacteria on the LL cheese surface. Adjunct V. casei, S. equorum, and B. thermosphacta did not become dominant. Surfaces of VC, SE, and BX cheeses mainly harbored Staphylococcus succinus. Peptidase activities were higher in cheeses made with attenuated starter than in CC, which had the lowest concentration of free amino acids. Based on the enzymatic activities of adjunct Le. lactis, LL and MIX cheeses exhibited the highest glutamate dehydrogenase, cystathionine-γ-lyase, and esterase activities. As shown by multivariate statistical analyses, LL and MIX cheeses showed the highest similarity for microbiological and biochemical features. LL and MIX cheeses received the highest scores for overall sensory acceptability. IMPORTANCE This study provides in-depth knowledge of the effects of attenuated starters and surface bacterial strains on the microbiota and related metabolic activities during cheese ripening. The use of attenuated Lc. lactis strongly impacted the microbiota assembly of caciotta cheese. This led to improved biochemical and sensory features compared to conventional cheese. Among surface bacterial strains, Le. lactis played a key role in the metabolic activities involved in cheese ripening. This resulted in an improvement of the sensory quality of caciotta cheese. The use of attenuated lactic acid bacteria and the surface adjunct Le. lactis could be a useful biotechnology to improve the flavor formation of caciotta cheese.

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

2017 ◽  
Vol 5 (11) ◽  
Author(s):  
Michiel Wels ◽  
Lennart Backus ◽  
Jos Boekhorst ◽  
Annereinou Dijkstra ◽  
Marke Beerthuyzen ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacterium ◽  
Lactococcus Lactis ◽  
Dairy Products ◽  
Draft Genome ◽  
Genome Sequences ◽  
Content Type ◽  
Lactococcus Lactis Subsp

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.

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