- E. Born van den (1)
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- W.J.M. Engels (2)
- O. Erkus Kütahya (1)
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- D. Hahn (12)
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- M.H.N. Hoefnagel (1)
- J. Hugenholtz (9)
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- M. Starrenburg(older publications) (7)
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- M.J.C. Starrenburg (5)
- M. Starrenburg (6)
- I. Swam van (1)
- W.F.H. Sybesma (4)
- W. Sybesma (1)
- L. Tijsseling (1)
- V.A. Tzeneva (1)
- W.M. Vos de (6)
- H.B.A. Wegkamp (1)
- M.W.W. Wels (1)
- J.A. Wouters (2)
Microbial domestication signatures of Lactococcus lactis can be reproduced by experimental evolution
Bachmann, H. ; Starrenburg, M.J.C. ; Molenaar, D. ; Kleerebezem, M. ; Hylckama Vlieg, J.E.T. van - \ 2012
Genome Research 22 (2012)1. - ISSN 1088-9051 - p. 115 - 124.
complete genome sequence - escherichia-coli populations - term experimental evolution - amino-acid biosynthesis - streptococcus-thermophilus - lactobacillus-bulgaricus - dairy environment - gene inactivation - mutator alleles - subsp lactis
Experimental evolution is a powerful approach to unravel how selective forces shape microbial genotypes and phenotypes. To this date, the available examples focus on the adaptation to conditions specific to the laboratory. The lactic acid bacterium Lactococcus lactis naturally occurs on plants and in dairy environments, and it is proposed that dairy strains originate from the plant niche. Here we investigate the adaptation of a L. lactis strain isolated from a plant to a dairy niche by propagating it for 1000 generations in milk. Two out of three independently evolved strains displayed significantly increased acidification rates and biomass yields in milk. Genome resequencing, revealed six, seven, and 28 mutations in the three strains, including point mutations in loci related to amino acid biosynthesis and transport and in the gene encoding MutL, which is involved in DNA mismatch repair. Two strains lost a conjugative transposon containing genes important in the plant niche but dispensable in milk. A plasmid carrying an extracellular protease was introduced by transformation. Although improving growth rate and growth yield significantly, the plasmid was rapidly lost. Comparative transcriptome and phenotypic analyses confirmed that major physiological changes associated with improved growth in milk relate to nitrogen metabolism and the loss or down-regulation of several pathways involved in the utilization of complex plant polymers. Reproducing the transition from the plant to the dairy niche through experimental evolution revealed several genome, transcriptome, and phenotype signatures that resemble those seen in strains isolated from either niche.
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.
Supplementation with engineered Lactococcus lactis improves the folate status in deficient rats
LeBlanc, J.G. ; Sybesma, W.F.H. ; Starrenburg, M. ; Sesma, F. ; Vos, W.M. de; Giori, G.S. de; Hugenholtz, J. - \ 2010
Nutrition 26 (2010)7. - ISSN 0899-9007 - p. 835 - 841.
neural-tube defects - folic-acid - riboflavin status - plasma homocysteine - dietary-folate - bacteria - bioavailability - prevention - vitamin - propionibacteria
Objective: The aim of this study was to establish the bioavailability of different folates produced by engineered Lactococcus lactis strains using a rodent depletion-repletion bioassay. Methods: Rats were fed a folate-deficient diet, which produces a reversible subclinical folate deficiency, supplemented with different L lactis cultures that were added as the only source of folate. Three bacterial strains that overexpressed the folC, folKE, or folC + KE genes were used. These strains produce folates with different poly glutamyl tail lengths. The growth response of the rats and the concentration of folates in different organs and blood samples were monitored. Results: The folate produced by the engineered strains was able to compensate the folate depletion in the diet and showed similar bioavailability compared with commercial folic acid that is normally used for food fortification. Folate concentrations in organ and blood samples increased significantly in animals that received the folate-producing strains compared with those that did not receive bacterial supplementation. Hematologic studies also showed that administration of the L towns strains was able to revert a partial megaloblastic anemia caused by folate deficiency. No significant differences were observed in the bioavailability of folates containing different glutamyl tail lengths. Conclusion: To our knowledge, this is the first study that demonstrated that folates produced by engineered lactic acid bacteria represent a bioavailable source of this essential vitamin. (C) 2010 Elsevier Inc. All rights reserved.
Phenotypic and genomic diversity of Lactobacillus plantarum strains isolated from various environmental niches
Siezen, R.J. ; Tzeneva, V.A. ; Castioni, A. ; Wels, M.W.W. ; Phan, H.T. ; Rademaker, J.L.W. ; Starrenburg, M.J.C. ; Kleerebezem, M. ; Molenaar, D. ; Hylckama Vlieg, J.E.T. van - \ 2010
Environmental Microbiology 12 (2010)3. - ISSN 1462-2912 - p. 758 - 773.
lactic-acid bacteria - horizontal gene-transfer - streptococcus-thermophilus - starter cultures - sequence - identification - paraplantarum - evolution - pcr - differentiation
Lactobacillus plantarum is a ubiquitous microorganism that is able to colonize several ecological niches, including vegetables, meat, dairy substrates and the gastro-intestinal tract. An extensive phenotypic and genomic diversity analysis was conducted to elucidate the molecular basis of the high flexibility and versatility of this species. First, 185 isolates from diverse environments were phenotypically characterized by evaluating their fermentation and growth characteristics. Strains clustered largely together within their particular food niche, but human fecal isolates were scattered throughout the food clusters, suggesting that they originate from the food eaten by the individuals. Based on distinct phenotypic profiles, 24 strains were selected and, together with a further 18 strains from an earlier low-resolution study, their genomic diversity was evaluated by comparative genome hybridization against the reference genome of L. plantarum WCFS1. Over 2000 genes were identified that constitute the core genome of the L. plantarum species, including 121 unique L. plantarum-marker genes that have not been found in other lactic acid bacteria. Over 50 genes unique for the reference strain WCFS1 were identified that were absent in the other L. plantarum strains. Strains of the L. plantarum subspecies argentoratensis were found to lack a common set of 24 genes, organized in seven gene clusters/operons, supporting their classification as a separate subspecies. The results provide a detailed view on phenotypic and genomic diversity of L. plantarum and lead to a better comprehension of niche adaptation and functionality of the organism
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
|Exploiting natural microbial biodiversity for development of flavour starters
Hylckama Vlieg, J.E.T. van; Dijkstra, A. ; Smit, B.A. ; Engels, W.J.M. ; Rijnen, L. ; Starrenburg, M.J.C. ; Smit, G. ; Wouters, J.A. - \ 2006
In: Flavour Science: recent advances and trends / Bredie, W.L.P., Petersen, M.A., Elsevier - ISBN 9780444527424 - p. 61 - 64.
|Exploiting natural microbial diversity for development of flavour starters
Hylckama Vlieg, J.E.T. van; Dijkstra, A. ; Smit, B.A. ; Engels, W.J.M. ; Rijnen, L. ; Starrenburg, M.J.C. ; Smit, G. ; Wouters, J.A. - \ 2005
Transformation of folate-consuming Lactobacillus gasseri into a folate producer
Wegkamp, H.B.A. ; Starrenburg, M. ; Vos, W.M. de; Hugenholtz, J. ; Sybesma, W.F.H. - \ 2004
Applied and Environmental Microbiology 70 (2004)5. - ISSN 0099-2240 - p. 3146 - 3148.
lactic-acid bacteria - lactococcus-lactis - folic-acid - gene - expression - cloning
Five genes essential for folate biosynthesis in Lactococcus lactis were cloned on a broad-host-range lactococcal vector and were transferred to the folate auxotroph Lactobacillus gasseri. As a result L. gasseri changed from a folate consumer to a folate producer. This principle can be used to increase folate levels in many fermented food products.
Effects of cultivation conditions on folate production by lactic acid bacteria
Sybesma, W. ; Starrenburg, M. ; Tijsseling, L. ; Hoefnagel, M.H.N. ; Hugenholtz, J. - \ 2003
Applied and Environmental Microbiology 69 (2003)8. - ISSN 0099-2240 - p. 4542 - 4548.
gtp cyclohydrolase-i - neural-tube defects - folic-acid - streptococcus-lactis - lactococcus-lactis - enzymatic-synthesis - purification - homocysteine - biosynthesis - strain
A variety of lactic acid bacteria were screened for their ability to produce folate intracellularly and/or extracellularly. Lactococcus lactis, Streptococcus thermophilus, and Leuconostoc spp. all produced folate, while most Lactobacillus spp., with the exception of Lactobacillus plantarum, were not able to produce folate. Folate production was further investigated in L. lactis as a model organism for metabolic engineering and in S. thermophilus for direct translation to (dairy) applications. For both these two lactic acid bacteria, an inverse relationship was observed between growth rate and folate production. When cultures were grown at inhibitory concentrations of antibiotics or salt or when the bacteria were subjected to low growth rates in chemostat cultures, folate levels in the cultures were increased relative to cell mass and (lactic) acid production. S. thermophilus excreted more folate than L. lactis, presumably as a result of differences in the number of glutamyl residues of the folate produced. In S. thermophilus 5,10-methenyl and 5-formyl tetrahydrofolate were detected as the major folate derivatives, both containing three glutamyl residues, while in L. lactis 5,10-methenyl and 10-formyl tetrahydrofolate were found, both with either four, five, or six glutamyl residues. Excretion of folate was stimulated at lower pH in S. thermophilus, but pH had no effect on folate excretion by L. lactis. Finally, several environmental parameters that influence folate production in these lactic acid bacteria were observed; high external pH increased folate production and the addition of p-aminobenzoic acid stimulated folate production, while high tyrosine concentrations led to decreased folate biosynthesis
Increased production of folate by metabolic engineering of Lactococcus lactis
Sybesma, W.F.H. ; Starrenburg, M. ; Kleerebezem, M. ; Mierau, I. ; Vos, W.M. de; Hugenholtz, J. - \ 2003
Applied and Environmental Microbiology 69 (2003). - ISSN 0099-2240 - p. 3069 - 3076.
controlled gene-expression - folic-acid - streptococcus-thermophilus - dihydropteroate synthase - subsp cremoris - cloning - bacteria - disease - homocysteine - inhibition
The dairy starter bacterium Lactococcus lactis is able to synthesize folate and accumulates large amounts of folate, predominantly in the polyglutamyl form. Only small amounts of the produced folate are released in the extracellular medium. Five genes involved in folate biosynthesis were identified in a folate gene cluster in L. lactis MG1363: folA, folB, folKE, folP, and folC. The gene folKE encodes the biprotein 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase and GTP cyclohydrolase I. The overexpression of folKE in L. lactis was found to increase the extracellular folate production almost 10-fold, while the total folate production increased almost 3-fold. The controlled combined overexpression of folKE and folC, encoding polyglutamyl folate synthetase, increased the retention of folate in the cell. The cloning and overexpression of folA, encoding dihydrofolate reductase, decreased the folate production twofold, suggesting a feedback inhibition of reduced folates on folate biosynthesis.
Controlles modulation of folate polyglutamyl tail length by metabolic engineering of Lactococcus lactis
Sybesma, W.F.H. ; Born, E. van den; Starrenburg, M. ; Mierau, I. ; Kleerebezem, M. ; Vos, W.M. de; Hugenholtz, J. - \ 2003
Applied and Environmental Microbiology 69 (2003). - ISSN 0099-2240 - p. 7101 - 7107.
gamma-glutamyl hydrolase - lactobacillus-casei - folic-acid - in-vitro - disease - identification - bacteria - cloning - foods - risk
The dairy starter bacterium Lactococcus lactis is able to synthesize folate and accumulates >90% of the produced folate intracellularly, predominantly in the polyglutamyl form. Approximately 10% of the produced folate is released into the environment. Overexpression of folC in L. lactis led to an increase in the length of the polyglutamyl tail from the predominant 4, 5, and 6 glutamate residues in wild-type cells to a maximum of 12 glutamate residues in the folate synthetase overproducer and resulted in a complete retention of folate in the cells. Overexpression of folKE, encoding the bifunctional protein 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase and GTP-cyclohydrolase I, resulted in reduction of the average polyglutamyl tail length, leading to enhanced excretion of folate. By simultaneous overexpression of folKE and folC, encoding the enzyme folate synthetase or polyglutamyl folate synthetase, the average polyglutamyl tail length was increased, again resulting in normal wild-type distribution of folate. The production of bioavailable monoglutamyl folate and almost complete release of folate from the bacterium was achieved by expressing the gene for gamma-glutamyl hydrolase from human or rat origin. These engineering studies clearly establish the role of the polyglutamyl tail length in intracellular retention of the folate produced. Also, the potential application of engineered food microbes producing folates with different tail lengths is discussed.
Metabolic engineering of lactic acid bacteria, the combined approach: kinetic modelling, metabolic control and experimental analysis
Hoefnagel, M.H.N. ; Starrenburg, M.J.C. ; Martens, D.E. ; Hugenholtz, J. ; Kleerenbezem, M. ; Swam, I. van; Bongers, R. - \ 2002
Microbiology 148 (2002). - ISSN 1350-0872 - p. 1003 - 1013.
Everyone who has ever tried to radically change metabolic fluxes knows that it is often harder to determine which enzymes have to be modified than it is to actually implement these changes. In the more traditional genetic engineering approaches ’bottle-necks’ are pinpointed using qualitative, intuitive approaches, but the alleviation of suspected ’rate-limiting’ steps has not often been successful. Here the authors demonstrate that a model of pyruvate distribution in Lactococcus lactis based on enzyme kinetics in combination with metabolic control analysis clearly indicates the key control points in the flux to acetoin and diacetyl, important flavour compounds. The model presented here (available at http://jjj.biochem.sun.ac.za/wcfs.html) showed that the enzymes with the greatest effect on this flux resided outside the acetolactate synthase branch itself. Experiments confirmed the predictions of the model, i.e. knocking out lactate dehydrogenase and overexpressing NADH oxidase increased the flux through the acetolactate synthase branch from 0 to 75% of measured product formation rates.
|Control of internal pH in lactic acid bacteria.
Hugenholtz, J. ; Starrenburg, M. ; Breeuwer, P. ; Abee, T. - \ 1996
In: FEMS 5th Symp. on Lactic acid bacteria: genetics, metabolism and applications, Veldhoven, NL - p. G47 - G47.
|Metabolic engineering of Lactococcus lactis: Influence of overproduction of ß-acetolactate synthase in strains deficient in lactate dehydrogenase as a function of culture conditions.
Platteeuw, C. ; Hugenholtz, J. ; Starrenburg, M. ; Alen-Boerrigter, I. van; Vos, W.M. de - \ 1995
Applied and Environmental Microbiology 61 (1995). - ISSN 0099-2240 - p. 3967 - 3971.
|Metabolic engineering of Lactococci: efficient conversion of pyruvate to acetoin and diacetyl.
Platteeuw, C. ; Hugenholtz, J. ; Starrenburg, M. ; Vos, W.M. de - \ 1994
In: Abstract 5th Neth. Congr. Biotechnology. Neth. Biotechnol. Soc. Amsterdam (1994) PK-17
Extraction of ribosomal RNA from soil for detection of Frankia with oligonucleotide probes.
Hahn, D. ; Kester, R. ; Starrenburg, M.J.C. ; Akkermans, A.D.L. - \ 1990
Archives of Microbiology 154 (1990). - ISSN 0302-8933 - p. 329 - 335.
|Oligonucleotide probes that hybridize with rRNA as a tool to study Frankia strains in root nodules.
Hahn, D. ; Starrenburg, M.J.C. ; Akkermans, A.D.L. - \ 1990
Applied and Environmental Microbiology 56 (1990). - ISSN 0099-2240 - p. 1342 - 1346.
Growth increment of Alnus glutinosa upon dual inoculation with effective and ineffective Frankia strains.
Hahn, D. ; Starrenburg, M.J.C. ; Akkermans, A.D.L. - \ 1990
Plant and Soil 122 (1990). - ISSN 0032-079X - p. 121 - 127.
|Oligonucleotide probes against rRNA in Frankia ecology.
Hahn, D. ; Starrenburg, M.J.C.S. ; Akkermans, A.D.L. - \ 1989
Forum Mikrobiologie 12 (1989). - p. 64 - 64.
|Application of DNA probes against effective and in effective Frankia strains for identification of nodule symbionts of Alnus glutinosa.
Hahn, D. ; Starrenburg, M.J.C. ; Akkermans, A.D.L. - \ 1988
In: Abstract Int. Symp. Forest tree physiology. Nancy, France (1988) 34, P4