Molecular and metabolic adaptations of Lactococcus lactis at near-zero growth rates
Ercan, O. ; Wels, M. ; Smid, E.J. ; Kleerebezem, M. - \ 2015
Applied and Environmental Microbiology 81 (2015)1. - ISSN 0099-2240 - p. 320 - 331.
acid bacteria - saccharomyces-cerevisiae - quantitative physiology - transcriptome analysis - streptococcus-lactis - maintenance energy - catabolite control - bacillus-subtilis - escherichia-coli - sugar catabolism
This paper describes the molecular and metabolic adaptations of Lactococcus lactis during the transition from a growing to a near-zero growth state using carbon-limited retentostat cultivation. Transcriptomic analyses revealed that metabolic patterns shifted between lactic- and mixed-acid fermentation during retentostat cultivation, which appeared to be controlled at the transcription level 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 Lactococcus 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 Lactococcus lactis to conditions supporting low-growth rates, and expands our earlier analysis of the quantitative physiology of this bacterium at near-zero growth rates towards gene regulation patterns involved in zero-growth adaptation.
Production of aroma compounds in lactic fermentations
Smid, E.J. ; Kleerebezem, M. - \ 2014
Annual Review of Food Science and Technology 5 (2014). - ISSN 1941-1413 - p. 313 - 326.
cystathionine beta-lyase - citrate transporter citp - lactococcus-lactis - acid bacteria - flavor formation - lactobacillus-plantarum - starter cultures - streptococcus-thermophilus - proteolytic systems - food fermentations
This review describes recent scientific research on the production of aroma compounds by lactic acid bacteria (LAB) in fermented food products. We discuss the various precursor molecules for the formation of aroma compounds in connection with the metabolic pathways involved. The roles of nonmetabolic properties such as cell lysis are also described in relation to aroma formation. Finally, we provide an overview of the literature on methods to steer and control aroma formation by LAB in mixed culture fermentations. We demonstrate that the technological progress made recently in high-throughput analysis methods has been driving the development of new approaches to understand, control, and steer aroma formation in (dairy) fermentation processes. This currently entails proposing new rules for designing stable, high-performance mixed cultures constituting a selection of strains, which in concert and on the basis of their individual predicted gene contents deliver the required functionalities.
Quantitative physiology of Lactococcus lactis at extreme low-growth rates
Ercan, O. ; Smid, E.J. ; Kleerebezem, M. - \ 2013
Environmental Microbiology 15 (2013)8. - ISSN 1462-2912 - p. 2319 - 2332.
maintenance energy - subsp lactis - streptococcus-cremoris - continuous-culture - product formation - stationary-phase - acid bacteria - carbohydrate starvation - enterococcus-faecalis - stress resistance
This paper describes the metabolic adaptation of Lactococcus lactis during the transition from a growing to a non-growing state using retentostat cultivation. Under retentostat cultivation, the specific growth rate decreased from 0.025 h-1 to 0.0001 h-1 in 42 days, while doubling time increased to more than 260 days. Viability of the overall culture was maintained above 90% but included approximately 20% damaged cells, which had lost their colony forming capacity on solid media. Although culture biomass and viability had reached a steady-state after 14 days of retentostat cultivation, the morphology of the cells changed from coccus-to-rod shape at later stages of retentostat cultivation, by which the cell’s surface to volume ratio was estimated to increase 2.4-fold. Furthermore, the metabolic patterns switched between homolactic and mixed-acid fermentation during the retentostat cultivation. Retentostat cultivation enabled the calculation of accurate substrate- and energy-related maintenance coefficients and biomass yields under nongrowing conditions, which were in good agreement with those calculated by extrapolation from chemostat cultivations at high dilution rates. In this study, we illustrate how retentostat cultivation allows decoupling of growth and non-growth associated processes in L. lactis, enabling the analysis of quantitative physiological responses of this bacterium to near zero-specific growth rates.
L. plantarum, L. salivarius, and L. lactis Attenuate Th2 Responses and Increase Treg Frequencies in Healthy Mice in a Strain Dependent Manner
Smelt, M.J. ; Haan, B.J. de; Bron, P.A. ; Swam, I. van; Meijerink, M. ; Wells, J. ; Faas, M.M. ; Vos, P. de - \ 2012
PLoS ONE 7 (2012)10. - ISSN 1932-6203
inflammatory-bowel-disease - lactobacillus-casei shirota - influenza-virus infection - placebo-controlled trial - complete genome sequence - blood mononuclear-cells - dendritic cells - double-blind - immune-system - acid bacteria
Many studies on probiotics are aimed at restoring immune homeostasis in patients to prevent disease recurrence or reduce immune-mediated pathology. Of equal interest is the use of probiotics in sub-clinical situations, which are characterized by reduced immune function or low-grade inflammation, with an increased risk of infection or disease as a consequence. Most mechanistic studies focus on the use of probiotics in experimental disease models, which may not be informative for these sub-clinical conditions. To gain better understanding of the effects in the healthy situation, we investigated the immunomodulatory effects of two Lactobacillus probiotic strains, i.e. L. plantarum WCFS1 and L. salivarius UCC118, and a non-probiotic lactococcus strain, i.e. L. lactis MG1363, in healthy mice. We studied the effect of these bacteria on the systemic adaptive immune system after 5 days of administration. Only L. plantarum induced an increase in regulatory CD103+ DC and regulatory T cell frequencies in the spleen. However, all three bacterial strains, including L. lactis, reduced specific splenic T helper cell cytokine responses after ex vivo restimulation. The effect on IFN-¿, IL5, IL10, and IL17 production by CD4+ and CD8+ T cells was dependent on the strain administered. A shared observation was that all three bacterial strains reduced T helper 2 cell frequencies. We demonstrate that systemic immunomodulation is not only observed after treatment with probiotic organisms, but also after treatment with non-probiotic bacteria. Our data demonstrate that in healthy mice, lactobacilli can balance T cell immunity in favor of a more regulatory status, via both regulatory T cell dependent and independent mechanisms in a strain dependent manner.
In vitro and in vivo characterization of DNA delivery using recombinant Lactococcus lactis expressing a mutated form of L. monocytogenes Internalin A
Azevedo, M. de; Karczewski, J. ; Lefevre, F. ; Azevedo, V. ; Miyoshi, A. ; Wells, J. ; Langella, P. ; Chatel, J.M. - \ 2012
BMC Microbiology 12 (2012). - ISSN 1471-2180
mammalian epithelial-cells - listeria-monocytogenes - genetic immunization - mucosal vaccination - viral vectors - acid bacteria - plasmid dna - vaccines - therapy - fibronectin
Background The use of food-grade Lactic Acid Bacteria (LAB) as DNA delivery vehicles represents an attractive strategy to deliver DNA vaccines at the mucosal surfaces as they are generally regarded as safe (GRAS). We previously showed that either native Lactococcus lactis (LL) or recombinant invasive LL expressing Fibronectin Binding Protein A of Staphylococcus aureus (LL-FnBPA+) or Internalin A of Listeria monocytogenes (LL-InlA+), were able to deliver and trigger DNA expression by epithelial cells, either in vitro or in vivo. InlA does not bind to its receptor, the murine E-cadherin, thus limiting the use of LL-InlA+ in in vivo murine models. Moreover, FnBPA binds to its receptors, integrins, via fibronectin introducing another limiting factor. In order to avoid the limitations of LL-InlA+ and LL-FnBPA+, a new L. lactis strain was engineered to produce a previously described mutated form of InlA (LL-mInlA+) allowing the binding of mInlA on murine E-cadherin. Results After showing the expression of mInLA at the surface of LL-mInlA+ strain, in vitro gentamycin survival assay in Caco-2 cells showed that LL-mInlA+ is 1000 times more invasive than LL. LL-mInlA+ invasivity was also validated by fluorescence microscopy. LL and LL-mInlA+ were transformed with pValacBLG, a plasmid containing the cDNA of bovine ß-Lactoglobulin (BLG), resulting in strains LL-BLG and LL-mInlA+BLG. The plasmid transfer in vitro using LL-mInlA+BLG was increased 10 times compared to LL-BLG. Moreover, the number of mice producing BLG in isolated enterocytes after oral administration of LL-mInlA+BLG in vivo was slightly higher than after oral administration of LL-BLG. Conclusions We confirmed in this study that the production of mInlA at the surface of L. lactis is a promising strategy for plasmid transfer in vitro and in vivo.
Characterization of Fen-Daqu Through Multivariate Statistical Analysis of H-1 NMR Spectroscopic Data
Van-Diep, L. ; Zheng, X. ; Ma, K. ; Chen, J.Y. ; Han, B.Z. ; Nout, M.J.R. - \ 2011
Journal of the Institute of Brewing 117 (2011)4. - ISSN 0046-9750 - p. 516 - 522.
magnetic-resonance-spectroscopy - principal component analysis - acid bacteria - fermentation - behaviors - starter - liquor - beer
J. Inst. Brew. 117(4), 516-522, 2011 Fen liquor is typical of Chinese light-flavour liquor (alcoholic spirit), which is fermented from sorghum with Fen-Daqu powder. Fen-Daqu is a saccharifying agent and fermentation starter in this fermentation process and in Fen traditional vinegar. To investigate the changes of biochemical components in Fen-Daqu during the incubation, samples at seven incubation stages were analyzed by H-1 nuclear magnetic resonance (NMR) spectrometry and principal component analysis (PCA). This revealed clear separation of the samples obtained from different incubation stages in the principal component plots by combining PC1 and PC2, which cumulatively accounted for 93.27% of the variance. The major compounds that contributed to discrimination were acetate/alanine, arginine, ascorbate, betaine, choline, ethanol, fructose, galactose, glucose, glucitol, glycerate, lactate, maltose, mannitol, phenylalanine, proline, propylene glycol, threonine and tryptophan. These compounds were regarded as the representative metabolites or biomarkers characteristic for each incubation stage and were related with microbiological changes of importance for quality control in Fen-Daqu production.
High-resolution amplified fragment length polymorphism typing of Lactococcus lactis strains enables identification of genetic markers for subspecies-related phenotypes
Erkus Kütahya, O. ; Starrenburg, M.J.C. ; Rademaker, J.L.W. ; Klaassen, C.H.W. ; Hylckama Vlieg, J.E.T. van; Smid, E.J. ; Kleerebezem, M. - \ 2011
Applied and Environmental Microbiology 77 (2011)15. - ISSN 0099-2240 - p. 5192 - 5198.
complete genome sequence - acid bacteria - streptococcus-lactis - genus lactococcus - cremoris - aflp - diversity - dairy - nov
A high-resolution amplified fragment length polymorphism (AFLP) methodology was developed to achieve the delineation of closely related Lactococcus lactis strains. The differentiation depth of 24 enzyme-primer-nucleotide combinations was experimentally evaluated to maximize the number of polymorphisms. The resolution depth was confirmed by performing diversity analysis on 82 L. lactis strains, including both closely and distantly related strains with dairy and nondairy origins. Strains clustered into two main genomic lineages of L. lactis subsp. lactis and L. lactis subsp. cremoris type-strain-like genotypes and a third novel genomic lineage rooted from the L. lactis subsp. lactis genomic lineage. Cluster differentiation was highly correlated with small-subunit rRNA homology and multilocus sequence analysis (MLSA) studies. Additionally, the selected enzyme-primer combination generated L. lactis subsp. cremoris phenotype-specific fragments irrespective of the genotype. These phenotype-specific markers allowed the differentiation of L. lactis subsp. lactis phenotype from L. lactis subsp. cremoris phenotype strains within the same L. lactis subsp. cremoris type-strain-like genomic lineage, illustrating the potential of AFLP for the generation of phenotype-linked genetic markers.
In vitro evaluation of gastrointestinal survival of Lactobacillus amylovorus DSM 16698 alone and combined with galactooligosaccharides, milk and/or Bifidobacterium animalis subsp. lactis Bb-12
Martinez, R.C.R. ; Anynaou, A.E. ; Albrecht, S.A. ; Schols, H.A. ; Martinis, E.C.P. de; Zoetendal, E.G. ; Venema, K. ; Saad, S.M.I. ; Smidt, H. - \ 2011
International Journal of Food Microbiology 149 (2011)2. - ISSN 0168-1605 - p. 152 - 158.
bile-salt hydrolase - galacto-oligosaccharides - probiotic lactobacillus - transit tolerance - intestinal-cells - weaning piglets - acid bacteria - prebiotics - tract - acidophilus
Probiotic properties of Lactobacillus amylovorus DSM 16698 were previously demonstrated in piglets. Here, its potential as a human probiotic was studied in vitro, using the TIM-1 system, which is fully validated to simulate the human upper gastrointestinal tract. To evaluate the effect of the food matrix composition on the survival of L. amylovorus DSM 16698 in TIM-1, the microorganism was inoculated alone or with prebiotic galactooligosaccharides (GOS), partially skimmed milk (PSM) and/or commercial probiotic Bifidobacterium animalis subsp. lactis Bb-12 (Bb-12). Samples were collected from TIM-1 for six hours, at one-hour intervals and L. amylovorus populations were enumerated on MRS agar plates with confirmation of identity of selected isolates by randomly amplified polymorphic DNA (RAPD) fingerprinting. The cumulative survival for L. amylovorus alone (control) was 30% at the end of the experiment (t = 6 h). Co-administration of L. amylovorus with GOS, PSM and/or Bb-12 increased its survival in comparison with the control significantly from the 4th hour after ingestion onwards (P <0.05). Furthermore, by the use of High Performance Anion Exchange Chromatography, both L. amylovorus and Bb-12 were observed to promptly degrade GOS compounds in samples collected from TIM-1, as assessed at t = 2 h. Hence, food matrix composition interfered with survival and growth of L. amylovorus during passage through TIM-1, providing leads towards optimization of probiotic properties in vivo.
Hydrolysis of ß-casein by the cell-envelope-located PI-type protease of Lactococcus lactis: A modelling approach
Munoz-Tamayo, R. ; Groot, J. de; Bakx, E.J. ; Wierenga, P.A. ; Gruppen, H. ; Zwietering, M.H. ; Sijtsma, L. - \ 2011
International Dairy Journal 21 (2011)10. - ISSN 0958-6946 - p. 755 - 762.
acid bacteria - kappa-casein - streptococcus-cremoris - enzymatic-hydrolysis - milk - kinetics - proteolysis - strains - peptide - system
Lactic acid bacteria possess extracellular proteases that hydrolyze milk proteins. This work aimed to describe mathematically the hydrolysis of intact ß-casein by the PI-type protease of Lactococcus lactis, using a mutant strain that lacks the oligopeptide transport system. Experiments were performed under a broad range of initial protein concentrations (17–196 µm), at constant enzyme concentration or at constant initial enzyme/substrate ratio. Hydrolysis of the intact ß-casein was monitored and quantified. Four kinetic functions were evaluated to describe the hydrolysis: First-order, nth-order, Michaelis–Menten, and competitive inhibition kinetics. The hydrolysis rate was found to depend on the initial protein concentration, due to the micellisation behaviour of ß-casein. This effect was accounted for by modifying the kinetic functions. The modified competitive inhibition model provided the lowest mean square error. This model has only three parameters and described the hydrolysis of intact ß-casein effectively for a broad range of initial conditions.
Time-resolved genetic responses of Lactococcus lactis to a dairy environment
Bachmann, H. ; Wilt, L. de; Kleerebezem, M. ; Hylckama Vlieg, J.E.T. van - \ 2010
Environmental Microbiology 12 (2010)5. - ISSN 1462-2912 - p. 1260 - 1270.
flavor formation - acid bacteria - lactobacillus-plantarum - stationary-phase - stress responses - regulated genes - amino-acids - expression - cheese - identification
Lactococcus lactis is one of main bacterial species found in mixed dairy starter cultures for the production of semi-hard cheese. Despite the appreciation that mixed cultures are essential for the eventual properties of the manufactured cheese the vast majority of studies on L. lactis were carried out in laboratory media with a pure culture. In this study we applied an advanced recombinant in vivo expression technology (R-IVET) assay in combination with a high-throughput cheese-manufacturing protocol for the identification and subsequent validation of promoter sequences specifically induced during the manufacturing and ripening of cheese. The system allowed gene expression measurements in an undisturbed product environment without the use of antibiotics and in combination with a mixed strain starter culture. The utilization of bacterial luciferase as reporter enabled the real-time monitoring of gene expression in cheese for up to 200 h after the cheese-manufacturing process was initiated. The results revealed a number of genes that were clearly induced in cheese such as cysD, bcaP, dppA, hisC, gltA, rpsE, purL, amtB as well as a number of hypothetical genes, pseudogenes and notably genetic elements located on the non-coding strand of annotated open reading frames. Furthermore genes that are likely to be involved in interactions with bacteria used in the mixed strain starter culture were identified
Regulatory phenotyping reveals important diversity within the species Lactococcus lactis
Bachmann, H. ; Starrenburg, M. ; Dijkstra, A. ; Molenaar, D. ; Kleerebezem, M. ; Rademaker, J.L.W. ; Hylckama Vlieg, J.E.T. van - \ 2009
Applied and Environmental Microbiology 75 (2009)17. - ISSN 0099-2240 - p. 5687 - 5694.
escherichia-coli - flavor formation - streptococcus-cremoris - natural diversity - gene inactivation - parallel changes - acid bacteria - cheese - evolution - subsp
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
High-throughput identification and validation of in situ-expressed genes of Lactococcus lactis
Bachmann, H. ; Kleerebezem, M. ; Hylckama Vlieg, J.E.T. van - \ 2008
Applied and Environmental Microbiology 74 (2008)15. - ISSN 0099-2240 - p. 4727 - 4736.
lactobacillus-plantarum genes - complete genome sequence - streptococcus-lactis - escherichia-coli - functional-characterization - temporal expression - marker removal - acid bacteria - lox sites - plasmid
Understanding the functional response of bacteria to their natural environment is one of the current challenges in microbiology. Over the past decades several techniques have been developed to study gene expression in complex natural habitats. Most of these methods, however, are laborious, and validation of results under in situ conditions is cumbersome. Here we report the improvement of the recombinase-based in vivo expression technology (R-IVET) by the implementation of two additional reporter genes. The first one is an alpha-galactosidase gene (melA), which facilitates the rapid identification of in vivo-induced genes. Second, the bacterial luciferase genes (luxAB) are transcriptionally coupled to the resolvase gene, which allows rapid validation and characterization of in vivo-induced genes. The system is implemented and validated in the industrially important lactic acid bacterium Lactococcus lactis. We demonstrate the applicability of the advanced R-IVET system by the identification and validation of lactococcal promoter elements that are induced in minimal medium compared to the commonly used rich laboratory medium M17. R-IVET screening led to the identification of 19 promoters that predominantly control expression of genes involved in amino acid and nucleotide metabolism and in transport functions. Furthermore, the luciferase allows high-resolution transcription analysis and enabled the identification of complex medium constituents and specific molecules involved in promoter control. Rapid target validation exemplifies the high-throughput potential of the extended R-IVET system. The system can be applied to other bacterial species, provided that the reporter genes used are functional in the organism of interest
Mucosal delivery of a pneumococcal vaccine using lactococcus lactis affords protection against respiratory infection
Hanniffy, S.B. ; Carter, A.T. ; Hitchin, E. ; Wells, J. - \ 2007
The Journal of Infectious Diseases 195 (2007)2. - ISSN 0022-1899 - p. 185 - 193.
surface protein-a - human alveolar macrophages - cellular immune-responses - toxin fragment-c - streptococcus-pneumoniae - acid bacteria - conjugate vaccines - oral immunization - heterologous pspa - lung infection
Background - Economical and effective vaccines against Streptococcus pneumoniae (pneumococcus) are needed for implementation in poorer countries where the disease burden is highest. Here, we evaluated Lactococcus lactis intracellularly producing the pneumococcal surface protein A (PspA) as a mucosal vaccine in conferring protection against pneumococcal disease. Methods - Mice were intranasally (inl) immunized with the lactococcal vaccine. Control groups were also immunized with similar amounts of recombinant PspA administered inl or subcutaneously with alum. PspA-specific antibodies in serum samples and lung lavage fluids were measured before challenge in intraperitoneal sepsis and inl respiratory-infection models of pneumococcal disease. Results - The lactococcal vaccine afforded better protection against respiratory challenge with pneumococcus than did vaccination with purified antigen given inl or by injection with alum. This finding was associated with a shift toward a Th1-mediated immune response characterized by reduced antibody titers to the PspA antigen. In the sepsis model, the lactococcal vaccine afforded resistance to disease on a par with that obtained with the injected vaccine, demonstrating its efficacy against different forms of pneumococcal disease. Conclusion - Given the safety profile of L. lactis, there is considerable potential to develop a pneumococcal vaccine for use in humans and to broaden this approach to combat other major pathogens
Generation of a membrane potential by Lactococcus lactis through aerobic electron transport
Brooijmans, R.J.W. ; Poolman, B. ; Schuurman-Wolters, G.K. ; Vos, W.M. de; Hugenholtz, J. - \ 2007
Journal of Bacteriology 189 (2007)14. - ISSN 0021-9193 - p. 5203 - 5209.
electrochemical proton gradient - complete genome sequence - escherichia-coli - cytochrome bd - acid bacteria - streptococcus-faecalis - bacillus-subtilis - adenosine-triphosphatase - respiration metabolism - environmental heme
Lactococcus lactis, a facultative anaerobic lactic acid bacterium, is known to have an increased growth yield when grown aerobically in the presence of heme. We have now established the presence of a functional, proton motive force-generating electron transfer chain (ETC) in L. lactis under these conditions. Proton motive force generation in whole cells was measured using a fluorescent probe (3',3'-dipropylthiadicarbocyanine), which is sensitive to changes in membrane potential (). Wild-type cells, grown aerobically in the presence of heme, generated a even in the presence of the F1-Fo ATPase inhibitor N,N'-dicyclohexylcarbodiimide, while a cytochrome bd-negative mutant strain (CydA) did not. We also observed high oxygen consumption rates by membrane vesicles prepared from heme-grown cells, compared to CydA cells, upon the addition of NADH. This demonstrates that NADH is an electron donor for the L. lactis ETC and demonstrates the presence of a membrane-bound NADH-dehydrogenase. Furthermore, we show that the functional respiratory chain is present throughout the exponential and late phases of growth.
Expression of plant flavour genes in Lactococcus lactis
Hernández, I. ; Molenaar, D. ; Beekwilder, M.J. ; Bouwmeester, H.J. ; Hylckama Vlieg, J.E.T. van - \ 2007
Applied and Environmental Microbiology 73 (2007)5. - ISSN 0099-2240 - p. 1544 - 1552.
alcohol acyl-transferase - escherichia-coli - acid bacteria - transfer-rna - heterologous expression - terpenoid metabolism - functional-analysis - molecular-cloning - dna microarrays - volatile esters
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
Using Lactococcus lactis for glutathione overproduction
Li, Y. ; Hugenholtz, J. ; Sybesma, W. ; Abee, T. ; Molenaar, D. - \ 2005
Applied Microbiology and Biotechnology 67 (2005)1. - ISSN 0175-7598 - p. 83 - 90.
escherichia-coli-b - gamma-glutamylcysteine synthetase - molecular-weight thiols - acid bacteria - saccharomyces-cerevisiae - nucleotide-sequence - oxidative stress - gene - expression - metabolism
Glutathione and -glutamylcysteine were produced in Lactococcus lactis using a controlled expression system and the genes gshA and gshB from Escherichia coli encoding the enzymes -glutamylcysteine synthetase and glutathione synthetase. High levels of -glutamylcysteine were found in strains growing on chemically defined medium and expressing either gshA alone or both gshA and gshB. As anticipated, glutathione was found in a strain expressing gshA and gshB. The level of glutathione production could be increased by addition of the precursor amino acid cysteine to the medium. The addition of cysteine led to an increased activity of glutathione synthetase, which is remarkable because the amino acid is not a substrate of this enzyme. The final intracellular glutathione concentration attained was 358 nmol mg¿1 protein, which is the highest concentration reported for a bacterium, demonstrating the suitability of engineered L. lactis for fine-chemical production and as a model for studies of the impact of glutathione on flavour formation and other properties of food.
Complete sequences of four plasmids of Lactococcus lactis subsp cremoris SK11 reveal extensive adaptation to the dairy environment
Siezen, R.J. ; Renckens, B. ; Swam, I. van; Peters, S. ; Kranenburg, R. van; Kleerebezem, M. ; Vos, W.M. de - \ 2005
Applied and Environmental Microbiology 71 (2005)12. - ISSN 0099-2240 - p. 8371 - 8382.
oligopeptide transport-system - complete genome sequence - group-ii intron - acid bacteria - transcriptional analysis - lactate-dehydrogenase - nucleotide-sequence - molecular analysis - serine proteinase - escherichia-coli
Lactococcus lactis strains are known to carry plasmids encoding industrially important traits. L. lactis subsp. cremoris SK11 is widely used by the dairy industry in cheese making. Its complete plasmid complement was sequenced and found to contain the plasmids pSK11A (10,372 bp), pSK11B (13,332 bp), pSK11L (47,165 bp), and pSK11P (75,814 bp). Six highly homologous repB-containing replicons were found, all belonging to the family of lactococcal theta-type replicons. Twenty-three complete insertion sequence elements segment the plasmids into numerous modules, many of which can be identified as functional units or containing functionally related genes. Plasmid-encoded functions previously known to reside on L. lactis SK11 plasmids were now mapped in detail, e.g., lactose utilization (lacR-lacABCDFEGX), the proteolytic system (prtM-prtP, pepO, pepF), and the oligopeptide permease system (oppDFBCA). Newly identified plasmid-encoded functions could facilitate the uptake of various cations, while the pabA and pabB genes could be essential for folate biosynthesis. A competitive advantage could be obtained by using the putative flavin adenine dinucleotide-dependent D-lactate dehydrogenase and oxalate:formate antiporter for enhanced ATP synthesis, while the activity of the predicted -acetolactate decarboxylase may contribute to the formation of an additional electron sink. Various stress response proteins are plasmid encoded, which could enhance strain robustness. A substantial number of these "adaptation" genes have not been described before on L. lactis plasmids. Moreover, several genes were identified for the first time in L. lactis, possibly reflecting horizontal gene transfer
Unity in organisation and regulation of catabolic operons in Lactobacillus plantarum, Lactococcus lactis and Listeria monocytogenes
Andersson, U. ; Molenaar, D. ; Radstrom, P. ; Vos, W.M. de - \ 2005
Systematic and Applied Microbiology 28 (2005)3. - ISSN 0723-2020 - p. 187 - 195.
gram-positive bacteria - complete genome sequence - phosphotransferase system - bacillus-subtilis - streptococcus-lactis - lactate-dehydrogenase - acid bacteria - gene - repression - metabolism
Global regulatory circuits together with more specific local regulators play a notable role when cells are adapting to environmental changes. Lactococcus lactis is a lactic acid bacterium abundant in nature fermenting most mono- and disaccharides. Comparative genomics analysis of the operons encoding the proteins and enzymes crucial for catabolism of lactose, maltose and threhalose revealed an obvious unity in operon organisation. The local regulator of each operon was located in a divergent transcriptional direction to the rest of the operon including the transport protein-encoding genes. Furthermore, in all three operons a catabolite responsive element (CRE) site was detected inbetween the gene encoding the local regulator and one of the genes encoding ! sugar transport protein. It is evident that regardless of type of transport system and catabolic enzymes acting upon lactose, maltose and trehalose, respectively, Lc. lactis shows unity in both operon organisation and regulation of these catabolic operons. This knowledge was further extended to other catabolic operons in Lc. lactis and the two related bacteria Lactobacillus plantarum and Listeria monocytogenes. Thirty-nine catabolic operons responsible for degradation of sugars and sugar alcohols in Lc. lactis, Lb. plantarum and L. monocytogenes were investigated and the majority of those possessed the same organisation as the lactose, maltose and trehalose operons of Lc. lactis. Though, the frequency of CRE sites and their location varied among the bacteria. Both Lc. lactis and Lb. plantarum showed CRE sites in direct proximity to genes coding for proteins responsible for sugar uptake. However, in, L. monocytogenes CRE sites were not frequently found and not in the vicinity of genes encoding transport proteins, suggesting a more local mode of regulation of the catabolic operons found and/or the use of inducer control in this bacterium. © 2004 Elsevier GrnbH. All rights reserved.
Overproduction of heterologous mannitol 1-phosphatase : a key factor for engineering mannitol production by Lactococcus lactis
Wisselink, H.W. ; Moers, A.P.H.A. ; Mars, A.E. ; Hoefnagel, M.H.N. ; Vos, W.M. de; Hugenholtz, J. - \ 2005
Applied and Environmental Microbiology 71 (2005)3. - ISSN 0099-2240 - p. 1507 - 1514.
controlled gene-expression - complete genome sequence - acid bacteria - lactate-dehydrogenase - lactobacillus-plantarum - leuconostoc - deficient - protects - glucose - mesenteroides
To achieve high mannitol production by Lactococcus lactis, the mannitol 1-phosphatase gene of Eimeria tenella and the mannitol 1-phosphate dehydrogenase gene mtlD of Lactobacillus plantarum were cloned in the nisin-dependent L. lactis NICE overexpression system. As predicted by a kinetic L. lactis glycolysis model, increase in mannitol 1-phosphate dehydrogenase and mannitol 1-phosphatase activities resulted in increased mannitol production. Overexpression of both genes in growing cells resulted in glucose-mannitol conversions of 11, 21, and 27% by the L. lactis parental strain, a strain with reduced phosphofructokinase activity, and a lactate dehydrogenase-deficient strain, respectively. Improved induction conditions and increased substrate concentrations resulted in an even higher glucose-to-mannitol conversion of 50% by the lactate dehydrogenase-deficient L. lactis strain, close to the theoretical mannitol yield of 67%. Moreover, a clear correlation between mannitol 1-phosphatase activity and mannitol production was shown, demonstrating the usefulness of this metabolic engineering approach.
Metabolic Engineering of Mannitol Production in Lactococcus lactis: Influence of Overexpression of Mannitol 1-Phosphate Dehydrogenase in Different Genetic Backgrounds
Wisselink, H.W. ; Mars, A.E. ; Meer, P. van der; Eggink, G. ; Hugenholtz, J. - \ 2004
Applied and Environmental Microbiology 70 (2004)7. - ISSN 0099-2240 - p. 4286 - 4292.
complete genome sequence - lactate-dehydrogenase - lactobacillus-plantarum - acid bacteria - phosphotransferase system - expression systems - protects - nisin - phosphorylation - activation
To obtain a mannitol-producing Lactococcus lactis strain, the mannitol 1-phosphate dehydrogenase gene (mtlD) from Lactobacillus plantarum was overexpressed in a wild-type strain, a lactate dehydrogenase(LDH)-deficient strain, and a strain with reduced phosphofructokinase activity. High-performance liquid chromatography and 13C nuclear magnetic resonance analysis revealed that small amounts (
Identification and functional characterization of the Lactococcus lactis rfb operon, required for dTDP-rhamnose biosynthesis
Boels, I.C. ; Beerthuyzen, M.M. ; Kosters, M.H. ; Kaauwen, M.P.W. van; Kleerebezem, M. ; Vos, W.M. de - \ 2004
Journal of Bacteriology 186 (2004)5. - ISSN 0021-9193 - p. 1239 - 1248.
controlled gene-expression - gram-positive bacteria - subsp cremoris - capsular polysaccharide - streptococcus-pneumoniae - acid bacteria - cell-wall - o-antigen - exopolysaccharide production - escherichia-coli
dTDP-rhamnose is an important precursor of cell wall polysaccharides and rhamnose-containing exopolysaccharides (EPS) in Lactococcus lactis. We cloned the rfbACBD operon from L. lactis MG1363, which comprises four genes involved in dTDP-rhamnose biosynthesis. When expressed in Escherichia coli, the lactococcal rfbACBD genes could sustain heterologous production of the Shigella flexneri O antigen, providing evidence of their functionality. Overproduction of the RfbAC proteins in L. lactis resulted in doubled dTDP-rhamnose levels, indicating that the endogenous RfbAC activities control the intracellular dTDP-rhamnose biosynthesis rate. However, RfbAC overproduction did not affect rhamnose-containing B40-EPS production levels. A nisin-controlled conditional RfbBD mutant was unable to grow in media lacking the inducer nisin, indicating that the rfb genes have an essential role in L. lactis. Limitation of RfbBD activities resulted in the production of altered EPS. The monomeric sugar of the altered EPS consisted of glucose, galactose, and rhamnose at a molar ratio of 1:0.3:0.2, which is clearly different from the ratio in the native sugar. Biophysical analysis revealed a fourfold-greater molecular mass and a twofold-smaller radius of gyration for the altered EPS, indicating that these EPS are more flexible polymers with changed viscosifying properties. This is the first indication that enzyme activity at the level of central carbohydrate metabolism affects EPS composition.
Sugar utilisation and conservation of the gal-lac gene cluster in Streptococcus thermophilus
Bogaard, P.T.C. van den; Hols, P. ; Kuipers, O.P. ; Kleerebezem, M. ; Vos, W.M. de - \ 2004
Systematic and Applied Microbiology 27 (2004)1. - ISSN 0723-2020 - p. 10 - 17.
lactose transport-system - 16s ribosomal-rna - beta-galactosidase - exopolysaccharide production - phosphotransferase systems - escherichia-coli - acid bacteria - phosphoenolpyruvate - expression - transcription
The adaptation to utilise lactose as primary carbon and energy source is a characteristic for Streptococcus thermophilus. These organisms, however only utilise the glucose moiety of lactose while the galactose moiety is excreted into the growth medium. In this study we evaluated the diversity of sugar utilisation and the conservation of the gal-lac gene cluster in a collection of 18 S. thermophilus strains isolated from a variety of sources. For this purpose analysis was performed on DNA from these isolates and the results were compared with those obtained with a strain from which the complete genome sequence has been determined. The sequence, organisation and flanking regions of the S. thermophilus gal-lac gene cluster were found to be highly conserved among all strains. The vast majority of the S. thermophilus strains were able to utilize only glucose, lactose, and sucrose as carbon sources, some strains could also utilize fructose and two of these were able to grow on galactose. Molecular characterisation of these naturally occurring Gal+ strains revealed up-mutations in the galKTE promoter that were absent in all other strains. These data support the hypothesis that the loss of the ability to ferment galactose can be attributed to the low activity of the galKTE promoter, probably as a consequence of the adaptation to milk in which the lactose levels are in excess.
Ability of Lactococcus lactis to export viral capsid antigens: a crucial step for development of live vaccines
Dieye, Y. ; Hoekman, A.J.W. ; Clier, F. ; Juillard, V. ; Boot, H.J. ; Piard, J.C. - \ 2003
Applied and Environmental Microbiology 69 (2003)12. - ISSN 0099-2240 - p. 7281 - 7288.
bursal disease virus - toxin fragment-c - acid bacteria - bacillus-subtilis - escherichia-coli - mucosal surfaces - subsp lactis - cell-wall - protein - streptococcus
Thefood grade bacterium Lactococcus lactis is a potential vehicle for protein delivery in the gastrointestinal tract. As a model, we constructed lactococcal strains producing antigens of infectious bursal disease virus (IBDV). IBDV infects chickens and causes depletion of B-lymphoid cells in the bursa of Fabricius and subsequent immunosuppression, morbidity, or acute mortality. The two major IBDV antigens, i.e., VP2 and VP3, that form the viral capsid were expressed and targeted to the cytoplasm, the cell wall, or the extracellular compartment of L. lactis. Whereas VP3 was successfully targeted to the three compartments by the use of relevant expression and export vectors, VP2 was recalcitrant to export, thus confirming the difficulty of translocating naturally nonsecreted proteins across the bacterial membrane. This defect could be partly overcome by fusing VP2 to a naturally secreted protein (the staphylococcal nuclease Nuc) that carried VP2 through the membrane. Lactococcal strains producing Nuc-VP2 and VP3 in various bacterial compartments were administered orally to chickens. The chickens did not develop any detectable immune response against VP2 and VP3 but did exhibit an immune response against Nuc when Nuc-VP2 was anchored to the cell wall of lactococci.
Engineering of carbon distribution between glycolysis and sugar nucleobiosynthesis in Lactococcus lactis
Boels, I.C. ; Kleerebezem, M. ; Vos, W.M. de - \ 2003
Applied and Environmental Microbiology 69 (2003). - ISSN 0099-2240 - p. 1129 - 1135.
controlled gene-expression - exopolysaccharide biosynthesis - streptococcus-thermophilus - subsp cremoris - cellular phosphoglucomutase - polysaccharide formation - rheological properties - escherichia-coli - acid bacteria - cloning
We describe the effects of modulating the activities of glucokinase, phosphofructokinase, and phosphoglucomutase on the branching point between sugar degradation and the biosynthesis of sugar nucleotides involved in the production of exopolysaccharide biosynthesis by Lactococcus lactis. This was realized by using a described isogenic L. lactis mutant with reduced enzyme activities or by controlled expression of the well-characterized genes for phosphoglucomutase or glucokinase from Escherichia coli or Bacillus subtilis, respectively. The role of decreased metabolic flux was studied in L. lactis strains with decreased phosphofructokinase activities. The concomitant reduction of the activities of phosphofructokinase and other enzymes encoded by the las operon (lactate dehydrogenase and pyruvate kinase) resulted in significant changes in the concentrations of sugar-phosphates. In contrast, a >25-fold overproduction of glucokinase resulted in 7-fold-increased fructose-6-phosphate levels and 2-fold-reduced glucose-1-phosphate and glucose-6-phosphate levels. However, these increased sugar-phosphate concentrations did not affect the levels of sugar nucleotides. Finally, an similar to100-fold overproduction of phosphoglucomutase resulted in 5-fold-increased levels of both UDP-glucose and UDPgalactose. While the increased concentrations of sugar-phosphates or sugar nucleotides did not significantly affect the production of exopolysaccharides, they demonstrate the metabolic flexibility of L. lactis.
Characterization, expression, and mutation of the Lactococcus lactis galPMKTE genes, involved in galactose utilization via the Leloir pathway
Groossiord, B.P. ; Luesink, E.J. ; Vaughan, E.E. ; Arnaud, A. ; Vos, W.M. de - \ 2003
Journal of Bacteriology 185 (2003). - ISSN 0021-9193 - p. 870 - 878.
tagatose 6-phosphate pathway - streptococcus-lactis - acid bacteria - nucleotide-sequence - escherichia-coli - molecular-cloning - melibiose carrier - exopolysaccharide biosynthesis - lactose metabolism - bacillus-subtilis
A cluster containing five similarly oriented genes involved in the metabolism of galactose via the Leloir pathway in Lactococcus lactis subsp. cremoris MG1363 was cloned and characterized. The order of the genes is galPMKTE, and these genes encode a galactose permease (GalP), an aldose I-epimerase (GaIM), a galactokinase (GalK), a hexose-1-phosphate uridylyltransferase (GalT), and a UDP-glucose 4-epimerase (GaIE), respectively. This genetic organization reflects the order of the metabolic conversions during galactose utilization via the Leloir pathway. The functionality of the galP, galK, galT, and galE genes was shown by complementation studies performed with both Escherichia coli and L. lactis mutants. The GalP permease is a new member of the galactoside-pentose-hexuronide family of transporters. The capacity of GalP to transport galactose was demonstrated by using galP disruption mutant strains of L. lactis MG1363. A galK deletion was constructed by replacement recombination, and the mutant strain was not able to ferment galactose. Disruption of the galE gene resulted in a deficiency in cell separation along with the appearance of a long-chain phenotype when cells were grown on glucose as the sole carbon source. Recovery of the wild-type phenotype for the galE mutant was obtained either by genetic complementation or by addition of galactose to the growth medium.
Glutathione protects Lactococcus lactis against oxidative stress
Li, Y. ; Hugenholtz, J. ; Abee, T. ; Molenaar, D. - \ 2003
Applied and Environmental Microbiology 69 (2003)10. - ISSN 0099-2240 - p. 5739 - 5745.
escherichia-coli - saccharomyces-cerevisiae - oxidized glutathione - growth-conditions - acid bacteria - reductase - thioredoxin - purification - peroxidase - metabolism
Glutathione was found in several dairy Lactococcus lactis strains grown in M17 medium. None of these strains was able to synthesize glutathione. In chemically defined medium, L. lactis subsp. cremoris strain SK11 was able to accumulate up to similar to60 mM glutathione when this compound was added to the medium. Stationary-phase cells of strain SK11 grown in chemically defined medium supplemented with glutathione showed significantly increased resistance (up to fivefold increased resistance) to treatment with H2O2 compared to the resistance of cells without intracellular glutathione. The resistance to H2O2 treatment was found to be dependent on the accumulation of glutathione in 16 strains of L. lactis tested. We propose that by taking up glutathione, L. lactis might activate a glutathione-glutathione peroxidase-glutathione reductase system in stationary-phase cells, which catalyzes the reduction of H2O2. Glutathione reductase, which reduces oxidized glutathione, was detectable in most strains of L. lactis, but the activities of different strains were very variable. In general, the glutathione reductase activities of L. lactis subsp. lactis are higher than those of L. lactis subsp. cremoris, and the activities were much higher when strains were grown aerobically. In addition, glutathione peroxidase is detectable in strain SK11, and the level was fivefold greater when the organism was grown aerobically than when the organism was grown anaerobically. Therefore, the presence of glutathione in L. lactis could result in greater stability under storage conditions and quicker growth upon inoculation, two important attributes of successful starter cultures.