Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

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    Comparative genome analysis of Lactobacillus casei strains isolated from Actimel and Yakult products reveals marked similarities and points to a common origin
    Douillard, F.P. ; Kant, R. ; Ritari, J. ; Paulin, L. ; Palva, A. ; Vos, W.M. de - \ 2013
    Microbial Biotechnology 6 (2013)5. - ISSN 1751-7907 - p. 576 - 587.
    lactic-acid bacteria - gram-positive bacteria - rhamnosus gg - functional-analysis - cell-wall - surface-proteins - staphylococcus-aureus - controlled-trial - binding-protein - sequence
    The members of the Lactobacillus genus are widely used in the food and feed industry and show a remarkable ecological adaptability. Several Lactobacillus strains have been marketed as probiotics as they possess health-promoting properties for the host. In the present study, we used two complementary next-generation sequencing technologies to deduce the genome sequences of two Lactobacillus casei strains LcA and LcY, which were isolated from the products Actimel and Yakult, commercialized as probiotics. The LcA and LcY draft genomes have, respectively, an estimated size of 3067 and 3082 Mb and a G+ C content of 46.3%. Both strains are close to identical to each other and differ by no more than minor chromosomal re-arrangements, substitutions, insertions and deletions, as evident from the verified presence of one insertion-deletion (InDel) and only 29 single-nucleotide polymorphisms (SNPs). In terms of coding capacity, LcA and LcY are predicted to encode a comparable exoproteome, indicating that LcA and LcY are likely to establish similar interactions with human intestinal cells. Moreover, both L. casei LcA and LcY harboured a 59.6 kb plasmid that shared high similarities with plasmids found in other L. casei strains, such as W56 and BD-II. Further analysis revealed that the L. casei plasmids constitute a good evolution marker within the L. casei species. The plasmids of the LcA and LcY strains are almost identical, as testified by the presence of only three verified SNPs, and share a 3.5 kb region encoding a remnant of a lactose PTS system that is absent from the plasmids of W56 and BD-II but conserved in another smaller L. casei plasmid (pLC2W). Our observations imply that the results obtained in animal and human experiments performed with the Actimel and Yakult strains can be compared with each other as these strains share a very recent common ancestor.
    Transcriptome-based characterization of interactions between Saccharomyces cerevisiae and Lactobacillus delbrueckii subsp. bulgaricus in lactose-grown chemostat cocultures
    Mendes, F. ; Sieuwerts, S. ; Hulster, E. de; Almering, M.J. ; Luttik, M.A.H. ; Pronk, J.T. ; Smid, E.J. ; Baron, P.A. ; Daran-Lapujade, P. - \ 2013
    Applied and Environmental Microbiology 79 (2013)19. - ISSN 0099-2240 - p. 5949 - 5961.
    lactic-acid bacteria - streptococcus-thermophilus - mixed-culture - kefiran production - oenococcus-oeni - fermentation - reveals - yeasts - carbon - temperature
    Mixed populations of Saccharomyces cerevisiae yeasts and lactic acid bacteria occur in many dairy, food, and beverage fermentations, but knowledge about their interactions is incomplete. In the present study, interactions between Saccharomyces cerevisiae and Lactobacillus delbrueckii subsp. bulgaricus, two microorganisms that co-occur in kefir fermentations, were studied during anaerobic growth on lactose. By combining physiological and transcriptome analysis of the two strains in the cocultures, five mechanisms of interaction were identified. (i) Lb. delbrueckii subsp. bulgaricus hydrolyzes lactose, which cannot be metabolized by S. cerevisiae, to galactose and glucose. Subsequently, galactose, which cannot be metabolized by Lb. delbrueckii subsp. bulgaricus, is excreted and provides a carbon source for yeast. (ii) In pure cultures, Lb. delbrueckii subsp. bulgaricus grows only in the presence of increased CO2 concentrations. In anaerobic mixed cultures, the yeast provides this CO2 via alcoholic fermentation. (iii) Analysis of amino acid consumption from the defined medium indicated that S. cerevisiae supplied alanine to the bacterium. (iv) A mild but significant low-iron response in the yeast transcriptome, identified by DNA microarray analysis, was consistent with the chelation of iron by the lactate produced by Lb. delbrueckii subsp. bulgaricus. (v) Transcriptome analysis of Lb. delbrueckii subsp. bulgaricus in mixed cultures showed an overrepresentation of transcripts involved in lipid metabolism, suggesting either a competition of the two microorganisms for fatty acids or a response to the ethanol produced by S. cerevisiae. This study demonstrates that chemostat-based transcriptome analysis is a powerful tool to investigate microbial interactions in mixed populations.
    Multifactorial diversity sustains microbial community stability
    Erkus, O. ; Jager, V.C.L. de; Spus, M. ; Alen-Boerrigter, I.J. van; Rijswijck, I.M.H. van; Hazelwood, L. ; Janssen, P.W. ; Hijum, S.A.F.T. van; Kleerebezem, M. ; Smid, E.J. - \ 2013
    ISME Journal 7 (2013)11. - ISSN 1751-7362 - p. 2126 - 2136.
    lactic-acid bacteria - complete genome sequence - lactococcus-lactis - dairy environment - subsp lactis - raw-milk - cremoris - plasmids - cheese - identification
    Maintenance of a high degree of biodiversity in homogeneous environments is poorly understood. A complex cheese starter culture with a long history of use was characterized as a model system to study simple microbial communities. Eight distinct genetic lineages were identified, encompassing two species: Lactococcus lactis and Leuconostoc mesenteroides. The genetic lineages were found to be collections of strains with variable plasmid content and phage sensitivities. Kill-the-winner hypothesis explaining the suppression of the fittest strains by density-dependent phage predation was operational at the strain level. This prevents the eradication of entire genetic lineages from the community during propagation regimes (back-slopping), stabilizing the genetic heterogeneity in the starter culture against environmental uncertainty
    Comparative genomic and functional analysis of 100 Lactobacillus rhamnosus strains and their comparison with strain GG
    Douillard, F.P. ; Ribbera, A. ; Kant, R. ; Pietilä, T.E. ; Järvinen, H.M. ; Messing, M. ; Randazzo, C.L. ; Paulin, L. ; Laine, P.K. ; Ritari, J. ; Caggia, C. ; Lähteinen, T. ; Brouns, S.J.J. ; Satokari, R.M. ; Ossowski, I. von; Reunanen, J. ; Palva, A. ; Vos, W.M. de - \ 2013
    Plos Genetics 9 (2013)8. - ISSN 1553-7404
    lactic-acid bacteria - intestinal epithelial-cells - placebo-controlled trial - streptococcus-thermophilus - species identification - salmonella-typhimurium - gastrointestinal-tract - adaptive immunity - atopic disease - in-vitro
    Lactobacillus rhamnosus is a lactic acid bacterium that is found in a large variety of ecological habitats, including artisanal and industrial dairy products, the oral cavity, intestinal tract or vagina. To gain insights into the genetic complexity and ecological versatility of the species L. rhamnosus, we examined the genomes and phenotypes of 100 L. rhamnosus strains isolated from diverse sources. The genomes of 100 L. rhamnosus strains were mapped onto the L. rhamnosus GG reference genome. These strains were phenotypically characterized for a wide range of metabolic, antagonistic, signalling and functional properties. Phylogenomic analysis showed multiple groupings of the species that could partly be associated with their ecological niches. We identified 17 highly variable regions that encode functions related to lifestyle, i.e. carbohydrate transport and metabolism, production of mucus-binding pili, bile salt resistance, prophages and CRISPR adaptive immunity. Integration of the phenotypic and genomic data revealed that some L. rhamnosus strains possibly resided in multiple niches, illustrating the dynamics of bacterial habitats. The present study showed two distinctive geno-phenotypes in the L. rhamnosus species. The geno-phenotype A suggests an adaptation to stable nutrient-rich niches, i.e. milk-derivative products, reflected by the alteration or loss of biological functions associated with antimicrobial activity spectrum, stress resistance, adaptability and fitness to a distinctive range of habitats. In contrast, the geno-phenotype B displays adequate traits to a variable environment, such as the intestinal tract, in terms of nutrient resources, bacterial population density and host effects
    Transcriptome signatures of class I and III stress response deregulation in Lactobacillus plantarum reveal pleiotropic adaptation
    Bokhorst-van de Veen, H. van; Bongers, R.S. ; Wels, M. ; Bron, P.A. ; Kleerebezem, M. - \ 2013
    Microbial Cell Factories 12 (2013)1. - ISSN 1475-2859 - 15 p.
    gram-positive bacteria - heat-shock response - lactic-acid bacteria - bacillus-subtilis - listeria-monocytogenes - gastrointestinal-tract - low gc - streptococcus-pneumoniae - comparative genomics - helicobacter-pylori
    Background - To cope with environmental challenges bacteria possess sophisticated defense mechanisms that involve stress-induced adaptive responses. The canonical stress regulators CtsR and HrcA play a central role in the adaptations to a plethora of stresses in a variety of organisms. Here, we determined the CtsR and HrcA regulons of the lactic acid bacterium Lactobacillus plantarum WCFS1 grown under reference (28°C) and elevated (40°C) temperatures, using ctsR, hrcA, and ctsR-hrcA deletion mutants. Results - While the maximum specific growth rates of the mutants and the parental strain were similar at both temperatures (0.33¿±¿0.02 h-1 and 0.34¿±¿0.03 h-1, respectively), DNA microarray analyses revealed that the CtsR or HrcA deficient strains displayed altered transcription patterns of genes encoding functions involved in transport and binding of sugars and other compounds, primary metabolism, transcription regulation, capsular polysaccharide biosynthesis, as well as fatty acid metabolism. These transcriptional signatures enabled the refinement of the gene repertoire that is directly or indirectly controlled by CtsR and HrcA of L. plantarum. Deletion of both regulators, elicited transcriptional changes of a large variety of additional genes in a temperature-dependent manner, including genes encoding functions involved in cell-envelope remodeling. Moreover, phenotypic assays revealed that both transcription regulators contribute to regulation of resistance to hydrogen peroxide stress. The integration of these results allowed the reconstruction of CtsR and HrcA regulatory networks in L. plantarum, highlighting the significant intertwinement of class I and III stress regulons. Conclusions - Taken together, our results enabled the refinement of the CtsR and HrcA regulatory networks in L. plantarum, illustrating the complex nature of adaptive stress responses in this bacterium
    Probiotics can generate FoxP3 T-cell responses in the small intestine and simultaneously inducing CD4 and CD8 T cell activation in the large intestine.
    Smelt, M.J. ; Haan, B.J. de; Bron, P.A. ; Swam, I. van; Meijerink, M. ; Wells, J. ; Faas, M.M. ; Vos, P. de - \ 2013
    PLoS ONE 8 (2013)7. - ISSN 1932-6203
    inflammatory-bowel-disease - influenza-virus infection - cd103(+) dendritic cells - complete genome sequence - lactic-acid bacteria - lactobacillus-plantarum - double-blind - in-vitro - maintaining remission - ulcerative-colitis
    Most studies on probiotics aim to restore intestinal homeostasis to reduce immune-pathology in disease. Of equal importance are studies on how probiotics might prevent or delay disease in healthy individuals. However, knowledge on mechanisms of probiotic actions in healthy individuals is scarce. To gain more insight in how different bacterial strains may modulate the healthy intestinal immune system, we investigated the effect of the food derived bacterial strains L. plantarum WCFS1, L. salivarius UCC118, and L. lactis MG1363, on the intestinal regulatory immune phenotype in healthy mice. All three bacterial strains induced an upregulation of activity and numbers of CD11c(+) MHCII(+) DCs in the immune-sampling Peyer's Patches. Only L. salivarius UCC118 skewed towards an immune regulatory phenotype in the small intestinal lamina propria (SILP). The effects were different in the large intestine lamina propria. L. salivarius UCC118 induced activation in both CD4 and CD8 positive T-cells while L. plantarum WCFS1 induced a more regulatory phenotype. Moreover, L. plantarum WCFS1 decreased the Th1/Th2 ratio in the SILP. Also L. lactis MG1363 had immunomodulatory effects. L. lactis MG1363 decreased the expression of the GATA-3 and T-bet in the SILP. As our data show that contradictory effects may occur in different parts of the gut, it is recommended to study effects of probiotic in different sites in the intestine. Our strain-specific results suggest that unspecified application of probiotics may not be very effective. Our data also indicate that selection of specific probiotic strain activities on the basis of responses in healthy mice may be a promising strategy to specifically stimulate or suppress immunity in specific parts of the intestine
    Glucosinolate content of blanched cabbage (Brassica oleracea var. capitata) fermented by the probiotic strain Lactobacillus paracasei LMG-P22043
    Kruse, I. ; Valerio, F. ; Lonigro, S.L. ; Candia, S. de; Verkerk, R. ; Dekker, M. ; Lavermicocca, P. - \ 2013
    Food Research International 54 (2013)1. - ISSN 0963-9969 - p. 706 - 710.
    plant-derived biomolecules - lactic-acid bacteria - white cabbage - isothiocyanates - vegetables - health - cancer - food - components - pathogens
    Conventional fermentation of cabbage like in sauerkraut production leads to a complete elimination of glucosinolates (GSs). In order to retain GSs in fermented cabbage, the effect of a thermal treatment (blanching) followed by fermentation (4% brine at 25 °C) by the probiotic strain Lactobacillus paracasei LMG P22043, was investigated. After 71 h fermentation the probiotic blanched cabbage still contained 27.2 ± 2.3 µmol/100 g GSs, corresponding to the 35% of the total GSs before fermentation. A final count of L. paracasei of 8.26 ± 1.2 log10 CFU/g, and a final pH of 4.12 ± 0.1 were reached. After 30 days of refrigerated vacuum packed storage, 23.7 ± 1.5 µmol/100 g of GSs still persisted. In the control cabbage (blanched and not inoculated with L. paracasei) no fermentation occurred and as a result final pH was 6.10 ± 0.21, leading to a product not suitable for storage and consumption. Compared to traditional sauerkraut the final product has the advantage of containing a high content of phytochemicals in combination with a high count of live probiotic bacterial cells.
    Challenges in translational research on probiotic lactobacilli: from in vitro assays to clinical trials
    Meijerink, M. ; Mercenier, A.M.E. ; Wells, J. - \ 2013
    Beneficial Microbes 4 (2013)1. - ISSN 1876-2883 - p. 83 - 100.
    placebo-controlled trial - lactic-acid bacteria - regulatory t-cells - blood mononuclear-cells - intestinal epithelial-cells - irritable-bowel-syndrome - influenza-virus infection - randomized controlled-trial - tight junction proteins - host-microbiota dialog
    Beneficial effects of certain probiotic strains have been established in the treatment and prevention of various immune and intestinal disorders in humans, including allergic diseases, chronic inflammatory diseases and diarrhoea. The proposed mechanisms underlying the immunomodulatory effects of probiotics in humans are not understood in precise detail but include enhancement of intestinal barrier function, altered epithelial signalling, competition with pathogens and effects on immune cells and immunity depending on the probiotic strain. The publication of controversial or inconclusive probiotic studies in humans highlights the need for a better understanding of the mechanisms and improved strain selection criteria. This review focuses on the immunomodulatory properties of lactobacilli and bifidobacteria in vitro and in vivo, current knowledge concerning the mechanisms in vivo and challenges in translational research on probiotics. A better understanding of the molecular mechanisms of probiotics, the effect of probiotic mixtures versus single strains, the effect of formulation of probiotics and the fate of ingested probiotics should help to clarify the value of immune assays as selection criteria for probiotics.
    Effects of dietary arabinoxylan-oligosaccharides (AXOS) and endogenous probiotics on the growth performance, non-specific immunity and gut micrbiota of juvenile Siberian sturgeon (Acipenser baerii)
    Geraylou, Z. ; Souffreau, C. ; Rurangwa, E. ; Meester, L. de; Courtin, C.M. ; Delcour, J.A. ; Buyse, J. ; Ollevier, F. - \ 2013
    Fish and Shellfish Immunology 35 (2013)3. - ISSN 1050-4648 - p. 766 - 775.
    lactic-acid bacteria - bacillus-subtilis - lactococcus-lactis - disease resistance - intestinal microbiota - scophthalmus-maximus - labeo-rohita - in-vitro - fish - supplementation
    We investigated the effects of administration of putative endogenous probiotics Lactococcus lactis spp. lactis or Bacillus circulans, alone and in combination with arabinoxylan-oligosaccharides (AXOS), a new class of candidate prebiotics, in juvenile Siberian sturgeon (Acipenser baerii). Eight experimental diets were tested: basal diet (Diet 1), basal diet supplemented with 2% AXOS (Diet 2), or L. lactis ST G81 (Diet 3), L. lactis ST G45 (Diet 4), B. circulans ST M53 (Diet 5), L. lactis ST G81 + 2% AXOS (Diet 6), L. lactis ST G45 + 2% AXOS (Diet 7), B. circulans ST M53 + 2% AXOS (Diet 8). After four weeks, growth performance and feed conversion ratio significantly improved in fish fed diet 7. Innate immune responses of fish were boosted with both AXOS and probiotic diets, however synergistic effects of AXOS and probiotic diets were only observed for phagocytic and alternative complement activity. Phagocytic and respiratory burst activity of fish macrophage increased in fish fed diet 2 and 7, while humoral immune responses only increased in fish fed diet 7. Pyrosequencing analysis (16S rDNA) of the hindgut microbiota demonstrated that AXOS improved the colonization or/and growth capacity of L. lactis, as a higher relative abundance of L. lactis was observed in fish receiving diet 7. However, no observable colonization of B. circulans was found in the hindgut of fish fed diet 5 or 8, containing this bacterium. The dietary L. lactis ST G45 + 2% AXOS caused significant alterations in the intestinal microbiota by significantly decreasing in bacterial diversity, demonstrated by the fall in richness and Shannon diversity, and improved growth performance and boosted immune responses of Siberian sturgeon.
    Using recombinant Lactococci as an approach to dissect the immunomodulating capacity of surface piliation in probiotic Lactobacillus rhamnosus GG
    Ossowski, I. von; Pietilä, T.E. ; Rintahaka, J. ; Nummenmaa, E. ; Mäkinen, V.M. ; Reunanen, J. ; Satokari, R.M. ; Vos, W.M. de; Palva, I. ; Palva, A. - \ 2013
    PLoS ONE 8 (2013)5. - ISSN 1932-6203
    lactic-acid bacteria - intestinal epithelial-cells - functional-analysis - dendritic cells - gastrointestinal-tract - dependent mechanism - protein-production - oral consumption - adhesion - pili
    Primarily arising from their well understood beneficial health effects, many lactobacilli strains are considered good candidates for use as probiotics in humans and animals. Lactobacillar probiosis can itself be best typified by the Lactobacillus rhamnosus GG strain, which, with its well-documented clinical benefits, has emerged as one of the most widely used probiotics in the food and health-supplement industries. Even so, many facets of its molecular mechanisms and limitations as a beneficial commensal bacterium still remain to be thoroughly explored and dissected. Because L. rhamnosus GG is one of only a few such strains exhibiting surface piliation (called SpaCBA), we sought to examine whether this particular type of cell-surface appendage has a discernible immunomodulating capacity and is able to trigger targeted responses in human immune-related cells. Thus, presented herein for this study, we recombinantly engineered Lactococcus lactis to produce native (and pilin-deleted) SpaCBA pili that were assembled in a structurally authentic form and anchored to the cell surface, and which had retained mucus-binding functionality. By using these recombinant lactococcal constructs, we were able to demonstrate that the SpaCBA pilus can be a contributory factor in the activation of Toll-like receptor 2-dependent signaling in HEK cells as well as in the modulation of pro- and anti-inflammatory cytokine (TNF-a, IL-6, IL-10, and IL-12) production in human monocyte-derived dendritic cells. From these data, we suggest that the recombinant-expressed and surface-anchored SpaCBA pilus, given its projected functioning in the gut environment, might be viewed as a new microbe-associated molecular pattern (MAMP)-like modulator of innate immunity. Accordingly, our study has brought some new insight to the molecular immunogenicity of the SpaCBA pilus, thus opening the way to a better understanding of its possible role in the multifaceted nature of L. rhamnosus GG probiosis within the human gut
    Microbe-microbe interactions in mixed culture food fermentations
    Smid, E.J. ; Lacroix, C. - \ 2013
    Current Opinion in Biotechnology 24 (2013)2. - ISSN 0958-1669 - p. 148 - 154.
    lactic-acid bacteria - cheese - genomics - growth - propionibacteria - communication - lactobacilli - probiotics - consortia - milk
    Most known natural and industrial food fermentation processes are driven by either simple or complex communities of microorganisms. Obviously, these fermenting microbes will not only interact with the fermentable substrate but also with each other. These microbe–microbe interactions are complex but thought to be crucial for obtaining the desired product characteristics. Microbial interactions are mediated through a variety of molecular and physiological mechanisms. Examples of interaction mechanisms which have an impact on the outcome of food fermentation processes will be discussed. Finally, the technological and scientific challenges associated with the production and propagation of complex mixed starter cultures are briefly addressed. Research on the composition and functionality of complex microbial consortia is gaining momentum and will open new avenues for controlling and improving food fermentation processes, and developing new applications for mixed cultures.
    Comparative Genomic and Functional Analysis of Lactobacillus casei and Lactobacillus rhamnosus Strains Marketed as Probiotics
    Douillard, F.P. ; Ribbera, A. ; Järvinen, H.M. ; Kant, R. ; Pietilä, T.E. ; Randazzo, C.L. ; Paulin, L. ; Laine, P.K. ; Caggia, C. ; Ossowski, I. von; Reunanen, J. ; Satokari, R. ; Salminen, S. ; Palva, A. ; Vos, W.M. de - \ 2013
    Applied and Environmental Microbiology 79 (2013)6. - ISSN 0099-2240 - p. 1923 - 1933.
    lactic-acid bacteria - binding-protein - in-vitro - carbohydrate-metabolism - sequence - gg - adhesion - bl23 - stress - mucus
    Four Lactobacillus strains were isolated from marketed probiotic products, including L. rhamnosus strains from Vifit (Friesland Campina) and Idoform (Ferrosan) and L. casei strains from Actimel (Danone) and Yakult (Yakult Honsa Co.). Their genomes and phenotypes were characterized and compared in detail with L. casei strain BL23 and L. rhamnosus strain GG. Phenotypic analysis of the new isolates indicated differences in carbohydrate utilization between L. casei and L. rhamnosus strains, which could be linked to their genotypes. The two isolated L. rhamnosus strains had genomes that were virtually identical to that of L. rhamnosus GG, testifying to their genomic stability and integrity in food products. The L. casei strains showed much greater genomic heterogeneity. Remarkably, all strains contained an intact spaCBA pilus gene cluster. However, only the L. rhamnosus strains produced mucus-binding SpaCBA pili under the conditions tested. Transcription initiation mapping demonstrated that the insertion of an iso-IS30 element upstream of the pilus gene cluster in L. rhamnosus strains but absent in L. casei strains had constituted a functional promoter driving pilus gene expression. All L. rhamnosus strains triggered an NF-¿B response via Toll-like receptor 2 (TLR2) in a reporter cell line, whereas the L. casei strains did not or did so to a much lesser extent. This study demonstrates that the two L. rhamnosus strains isolated from probiotic products are virtually identical to L. rhamnosus GG and further highlights the differences between these and L. casei strains widely marketed as probiotics, in terms of genome content, mucus-binding and metabolic capacities, and host signaling capabilities
    Metabolic shifts: a fitness perspective for microbial cell factories
    Goel, A. ; Wortel, M.T. ; Molenaar, D. ; Teusink, B. - \ 2012
    Biotechnology Letters 34 (2012)12. - ISSN 0141-5492 - p. 2147 - 2160.
    lactic-acid bacteria - heterologous protein secretion - escherichia-coli - saccharomyces-cerevisiae - lactococcus-lactis - bacillus-subtilis - pyruvate metabolism - overflow metabolism - aerobic glycolysis - glucose-metabolism
    Performance of industrial microorganisms as cell factories is limited by the capacity to channel nutrients to desired products, of which optimal production usually requires careful manipulation of process conditions, or strain improvement. The focus in process improvement is often on understanding and manipulating the regulation of metabolism. Nonetheless, one encounters situations where organisms are remarkably resilient to further optimization or their properties become unstable. Therefore it is important to understand the origin of these apparent limitations to find whether and how they can be improved. We argue that by considering fitness effects of regulation, a more generic explanation for certain behaviour can be obtained. In this view, apparent process limitations arise from trade-offs that cells faced as they evolved to improve fitness. A deeper understanding of such trade-offs using a systems biology approach can ultimately enhance performance of cell factories.
    Complete resequencing and reannotation of the Lactobacillus plantarum WCFS1 genome
    Siezen, R.J. ; Francke, C. ; Renckens, B. ; Boekhorst, L.J.S. ; Wels, M. ; Kleerebezem, M. ; Hijum, S.A.F.T. van - \ 2012
    Journal of Bacteriology 194 (2012)1. - ISSN 0021-9193 - p. 195 - 196.
    lactic-acid bacteria - gastrointestinal-tract - identification - fermentation - database - artemis - growth - genes - mice - carbohydrate
    There is growing interest in the beneficial effects of Lactobacillus plantarum on human health. The genome of L. plantarum WCFS1, first sequenced in 2001, was resequenced using Solexa technology. We identified 116 nucleotide corrections and improved function prediction for nearly 1,200 proteins, with a focus on metabolic functions and cell surface-associated proteins.
    Genome sequence of the naturally plasmid-free Lactobacillus plantarum strain NC8 (CCUG 61730)
    Axelsson, L. ; Rud, I. ; Naterstad, K. ; Blom, H. ; Renckens, B. ; Boekhorst, L.J.S. ; Kleerebezem, M. ; Hijum, S.A.F.T. van; Siezen, R.J. - \ 2012
    Journal of Bacteriology 194 (2012)9. - ISSN 0021-9193 - p. 2391 - 2392.
    inducible gene-expression - lactic-acid bacteria - sakei - fermentations - diversity - vectors - healthy - mucosa - wcfs1
    Lactobacillus plantarum is a highly versatile lactic acid bacterium found in various ecological niches, such as fermented vegetable, meat, and dairy products and the gastrointestinal tract. We sequenced the genome of L. plantarum NC8, a naturally plasmid-free strain, which has been used as a model strain in many laboratories worldwide.
    Transcriptomes reveal genetic signatures underlying physiological variations imposed by different fermentation conditions in Lactobacillus plantarum
    Bron, P.A. ; Wels, M. ; Bongers, R.S. ; Bokhorst-van de Veen, H. van; Wiersma, A. ; Overmars, L. ; Marco, M.L. ; Kleerebezem, M. - \ 2012
    PLoS ONE 7 (2012)7. - ISSN 1932-6203
    lactic-acid bacteria - complete genome sequence - lactococcus-lactis - ribonucleotide reductase - escherichia-coli - stationary-phase - microarray data - aerobic growth - diversity - pathways
    Lactic acid bacteria (LAB) are utilized widely for the fermentation of foods. In the current post-genomic era, tools have been developed that explore genetic diversity among LAB strains aiming to link these variations to differential phenotypes observed in the strains investigated. However, these genotype-phenotype matching approaches fail to assess the role of conserved genes in the determination of physiological characteristics of cultures by environmental conditions. This manuscript describes a complementary approach in which Lactobacillus plantarum WCFS1 was fermented under a variety of conditions that differ in temperature, pH, as well as NaCl, amino acid, and O2 levels. Samples derived from these fermentations were analyzed by full-genome transcriptomics, paralleled by the assessment of physiological characteristics, e.g., maximum growth rate, yield, and organic acid profiles. A data-storage and -mining suite designated FermDB was constructed and exploited to identify correlations between fermentation conditions and industrially relevant physiological characteristics of L. plantarum, as well as the associated transcriptome signatures. Finally, integration of the specific fermentation variables with the transcriptomes enabled the reconstruction of the gene-regulatory networks involved. The fermentation-genomics platform presented here is a valuable complementary approach to earlier described genotype-phenotype matching strategies which allows the identification of transcriptome signatures underlying physiological variations imposed by different fermentation conditions.
    Impact of 4 Lactobacillus plantarum capsular polysaccharide clusters on surface glycan composition and host cell signaling
    Remus, D.M. ; Kranenburg, R. van; Swam, I.I. van; Taverne, N. ; Bongers, R.S. ; Wels, M. ; Wells, J. ; Bron, P.A. ; Kleerebezem, M. - \ 2012
    Microbial Cell Factories 11 (2012)1. - ISSN 1475-2859 - 10 p.
    lactic-acid bacteria - gene-expression omnibus - streptococcus-pneumoniae - lactococcus-lactis - exopolysaccharide biosynthesis - microarray data - subsp cremoris - rhamnosus gg - identification - strains
    Background - Bacterial cell surface-associated polysaccharides are involved in the interactions of bacteria with their environment and play an important role in the communication between pathogenic bacteria and their host organisms. Cell surface polysaccharides of probiotic species are far less well described. Therefore, improved knowledge on these molecules is potentially of great importance to understand the strain-specific and proposed beneficial modes of probiotic action. Results - The Lactobacillus plantarum WCFS1 genome encodes 4 clusters of genes that are associated with surface polysaccharide production. Two of these clusters appear to encode all functions required for capsular polysaccharide formation (cps2A-J and cps4A-J), while the remaining clusters are predicted to lack genes encoding chain-length control functions and a priming glycosyl-transferase (cps1A-I and cps3A-J). We constructed L. plantarum WCFS1 gene deletion mutants that lack individual (¿cps1A-I, ¿cps2A-J, ¿cps3A-J and ¿cps4A-J) or combinations of cps clusters (¿cps1A-3J and ¿cps1A-3I, ¿cps4A-J) and assessed the genome wide impact of these mutations by transcriptome analysis. The cps cluster deletions influenced the expression of variable gene sets in the individual cps cluster mutants, but also considerable numbers of up- and down-regulated genes were shared between mutants in cps cluster 1 and 2, as well as between mutant in cps clusters 3 and 4. Additionally, the composition of overall cell surface polysaccharide fractions was altered in each mutant strain, implying that despite the apparent incompleteness of cps1A-I and cps3A-J, all clusters are active and functional in L. plantarum. The ¿cps1A-I strain produced surface polysaccharides in equal amounts as compared to the wild-type strain, while the polysaccharides were characterized by a reduced molar mass and the lack of rhamnose. The mutants that lacked functional copies of cps2A-J, cps3A-J or cps4A-J produced decreased levels of surface polysaccharides, whereas the molar mass and the composition of polysaccharides was not affected by these cluster mutations. In the quadruple mutant, the amount of surface polysaccharides was strongly reduced. The impact of the cps cluster mutations on toll-like receptor (TLR)-mediated human nuclear factor (NF)-¿B activation in host cells was evaluated using a TLR2 reporter cell line. In comparison to a L. plantarum wild-type derivative, TLR2 activation remained unaffected by the ¿cps1A-I and ¿cps3A-J mutants but appeared slightly increased after stimulation with the ¿cps2A-J and ¿cps4A-J mutants, while the ¿cps1A-3J and ¿cps1A-3J, ¿cps4A-J mutants elicited the strongest responses and clearly displayed enhanced TLR2 signaling. Conclusions - Our study reveals that modulation of surface glycan characteristics in L. plantarum highlights the role of these molecules in shielding of cell envelope embedded host receptor ligands. Although the apparently complete cps clusters (cps2A-J and cps4A-J) contributed individually to this shielding, the removal of all cps clusters led to the strongest signaling enhancement. Our findings provide new insights into cell surface glycan biosynthesis in L. plantarum, which bears relevance in the context of host-cell signaling by probiotic bacteria.
    Identification of key peptidoglycan hydrolases for morphogenesis, autolysis, and peptidoglycan composition of Lactobacillus plantarum WCFS1.
    Rolain, T. ; Bernard, E. ; Courtin, P. ; Bron, P.A. ; Kleerebezem, M. ; Chapot-Chartier, M.P. ; Hols, P. - \ 2012
    Microbial Cell Factories 11 (2012). - ISSN 1475-2859
    lactic-acid bacteria - lactococcus-lactis - n-acetylglucosaminidase - cell-wall - staphylococcus-aureus - bacillus-subtilis - murein hydrolase - gene - genome - electroporation
    Background - Lactobacillus plantarum is commonly used in industrial fermentation processes. Selected strains are also marketed as probiotics for their health beneficial effects. Although the functional role of peptidoglycan-degrading enzymes is increasingly documented to be important for a range of bacterial processes and host-microbe interactions, little is known about their functional roles in lactobacilli. This knowledge holds important potential for developing more robust strains resistant to autolysis under stress conditions as well as peptidoglycan engineering for a better understanding of the contribution of released muramyl-peptides as probiotic immunomodulators. Results - Here, we explored the functional role of the predicted peptidoglycan hydrolase (PGH) complement encoded in the genome of L. plantarum by systematic gene deletion. From twelve predicted PGH-encoding genes, nine could be individually inactivated and their corresponding mutant strains were characterized regarding their cell morphology, growth, and autolysis under various conditions. From this analysis, we identified two PGHs, the predicted N-acetylglucosaminidase Acm2 and NplC/P60 D,L-endopeptidase LytA, as key determinants in the morphology of L. plantarum. Acm2 was demonstrated to be required for the ultimate step of cell separation of daughter cells, whereas LytA appeared to be required for cell shape maintenance and cell-wall integrity. We also showed by autolysis experiments that both PGHs are involved in the global autolytic process with a dominant role for Acm2 in all tested conditions, identifying Acm2 as the major autolysin of L. plantarum WCFS1. In addition, Acm2 and the putative N-acetylmuramidase Lys2 were shown to play redundant roles in both cell separation and autolysis under stress conditions. Finally, the analysis of the peptidoglycan composition of Acm2- and LytA-deficient derivatives revealed their potential hydrolytic activities by the disappearance of specific cleavage products. Conclusion - In this study, we showed that two PGHs of L. plantarum have a predominant physiological role in a range of growth conditions. We demonstrate that the N-acetylglucosaminidase Acm2 is the major autolysin whereas the D,L-endopeptidase LytA is a key morphogenic determinant. In addition, both PGHs have a direct impact on PG structure by generating a higher diversity of cleavage products that could be of importance for interaction with the innate immune system.
    B-glucans are involved in immune-modulation of THP-1
    Chanput, W. ; Reitsma, M. ; Kleinjans, L. ; Mes, J.J. ; Savelkoul, H.F.J. ; Wichers, H.J. - \ 2012
    Molecular Nutrition & Food Research 56 (2012)5. - ISSN 1613-4125 - p. 822 - 833.
    lactic-acid bacteria - factor-kappa-b - cell-line - cytokine production - interferon-gamma - dendritic cells - receptor - lentinan - mice - lipopolysaccharide
    Scope We aimed to examine different immunological aspects of ß-glucans derived from different food sources (oat, barley and shiitake) on phorbol myristate acetate (PMA)-differentiated THP-1 macrophages. Commercially purified barley ß-glucan (commercial BG) and lentinan were included to compare ß-glucans from the same origin but different degree of purity and processing. Methods and results Chemical composition and molecular weight distribution of ß-glucan samples were determined. Inflammation-related gene expression kinetics (IL-1ß, IL-8, nuclear factor kappa B [NF-¿B] and IL-10) after 3, 6 and 24 h of stimulation with 100 µg/mL ß-glucan were investigated. All tested ß-glucans mildly upregulated the observed inflammation-related genes with differential gene expression patterns. Similar gene expression kinetics, but different fold induction values, was found for the crude ß-glucan extracts and their corresponding commercial forms. Pre-incubation of THP-1 macrophages with ß-glucans prior to lipopolysaccharide (LPS) exposure decreased the induction of inflammation-related genes compared to LPS treatment. No production of nitric oxide (NO) and hydrogen peroxide (H2O2) was detected in ß-glucan stimulated THP-1 macrophages. Phagocytic activity was not different after stimulation by ß-glucan samples. Conclusion Based on these in vitro analyses, it can be concluded that the analysed ß-glucans have varying levels of immunomodulating properties, which are likely related to structure, molecular weight and compositional characteristic of ß-glucan
    Modulation of Lactobacillus plantarum gastrointestinal robustness by fermentation conditions enables identification of bacterial robustness markers
    Bokhorst-van de Veen, H. van; Lee, I. ; Marco, M.L. ; Bron, P.A. ; Kleerebezem, M. - \ 2012
    PLoS ONE 7 (2012)7. - ISSN 1932-6203 - 13 p.
    lactic-acid bacteria - stress-response - in-vitro - genetic-characterization - bile-salt - strains - wcfs1 - lactococcus - survival - reuteri
    Background - Lactic acid bacteria (LAB) are applied worldwide in the production of a variety of fermented food products. Additionally, specific Lactobacillus species are nowadays recognized for their health-promoting effects on the consumer. To optimally exert such beneficial effects, it is considered of great importance that these probiotic bacteria reach their target sites in the gut alive. Methodology/Principal Findings - In the accompanying manuscript by Bron et al. the probiotic model organism Lactobacillus plantarum WCFS1 was cultured under different fermentation conditions, which was complemented by the determination of the corresponding molecular responses by full-genome transcriptome analyses. Here, the gastrointestinal (GI) survival of the cultures produced was assessed in an in vitro assay. Variations in fermentation conditions led to dramatic differences in GI-tract survival (up to 7-log) and high robustness could be associated with low salt and low pH during the fermentations. Moreover, random forest correlation analyses allowed the identification of specific transcripts associated with robustness. Subsequently, the corresponding genes were targeted by genetic engineering, aiming to enhance robustness, which could be achieved for 3 of the genes that negatively correlated with robustness and where deletion derivatives displayed enhanced survival compared to the parental strain. Specifically, a role in GI-tract survival could be confirmed for the lp_1669-encoded AraC-family transcription regulator, involved in capsular polysaccharide remodeling, the penicillin-binding protein Pbp2A involved in peptidoglycan biosynthesis, and the Na+/H+ antiporter NapA3. Moreover, additional physiological analysis established a role for Pbp2A and NapA3 in bile salt and salt tolerance, respectively. Conclusion - Transcriptome trait matching enabled the identification of biomarkers for bacterial (gut-)robustness, which is important for our molecular understanding of GI-tract survival and could facilitate the design of culture conditions aimed to enhance probiotic culture robustness
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