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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    Comprehensive insights into transcriptional adaptation of intracellular mycobacteria by microbe-enriched dual RNA sequencing
    Rienksma, R.A. ; Suarez Diez, M. ; Mollenkopf, H.J. ; Dolganov, G.M. ; Dorhoi, A. ; Schoolnik, G.K. ; Martins Dos Santos, V.A.P. ; Kaufmann, S. ; Schaap, P.J. ; Gengenbacher, M. - \ 2015
    BMC Genomics 16 (2015). - ISSN 1471-2164 - 31 p.
    tuberculosis gene-expression - human macrophage infection - complete genome sequence - cholesterol catabolism - regulated genes - messenger-rna - acyl-coenzyme - in-vitro - host - pathogen
    BackgroundThe human pathogen Mycobacterium tuberculosis has the capacity to escape eradication by professional phagocytes. During infection, M. tuberculosis resists the harsh environment of phagosomes and actively manipulates macrophages and dendritic cells to ensure prolonged intracellular survival. In contrast to other intracellular pathogens, it has remained difficult to capture the transcriptome of mycobacteria during infection due to an unfavorable host-to-pathogen ratio.ResultsWe infected the human macrophage-like cell line THP-1 with the attenuated M. tuberculosis surrogate M. bovis Bacillus Calmette¿Guérin (M. bovis BCG). Mycobacterial RNA was up to 1000-fold underrepresented in total RNA preparations of infected host cells. We employed microbial enrichment combined with specific ribosomal RNA depletion to simultaneously analyze the transcriptional responses of host and pathogen during infection by dual RNA sequencing. Our results confirm that mycobacterial pathways for cholesterol degradation and iron acquisition are upregulated during infection. In addition, genes involved in the methylcitrate cycle, aspartate metabolism and recycling of mycolic acids were induced. In response to M. bovis BCG infection, host cells upregulated de novo cholesterol biosynthesis presumably to compensate for the loss of this metabolite by bacterial catabolism.ConclusionsDual RNA sequencing allows simultaneous capture of the global transcriptome of host and pathogen, during infection. However, mycobacteria remained problematic due to their relatively low number per host cell resulting in an unfavorable bacterium-to-host RNA ratio. Here, we use a strategy that combines enrichment for bacterial transcripts and dual RNA sequencing to provide the most comprehensive transcriptome of intracellular mycobacteria to date. The knowledge acquired into the pathogen and host pathways regulated during infection may contribute to a solid basis for the deployment of novel intervention strategies to tackle infection
    Use of non-growing Lactococcus lactis cell suspensions for production of volatile metabolites with direct relevance for flavour formation during dairy fermentations
    Bunt, B. van de; Bron, P.A. ; Sijtsma, L. ; Vos, W.M. de; Hugenholtz, J. - \ 2014
    Microbial Cell Factories 13 (2014). - ISSN 1475-2859 - 9 p.
    amino-acid catabolism - complete genome sequence - aroma compounds - cheddar cheese - lactobacillus-helveticus - streptococcus-lactis - proteolytic systems - alpha-ketoglutarate - l-leucine - bacteria
    Background Lactococcus lactis is a lactic acid bacterium that has been used for centuries in the production of a variety of cheeses, as these bacteria rapidly acidify milk and greatly contribute to the flavour of the fermentation end-products. After a short growth phase during cheese ripening L. lactis enters an extended non-growing state whilst still strongly contributing to amino acid-derived flavour formation. Here, a research approach is presented that allows investigation of strain- and amino acid-specific flavour formation during the non-growing state. Results Non-growing cells of five selected L. lactis strains were demonstrated to degrade amino acids into flavour compounds that are relevant in food fermentations and differs greatly from production of flavour compounds using growing cells. As observed earlier in other research set-ups and with other microorganisms, addition of NADH, a-ketoglutarate and pyridoxal-5-phosphate was demonstrated to be essential for optimal flavour formation, suggesting that intracellular pools of these substrates are too low for the significant production of the flavour compounds. Production of flavours during the non-growing phase strongly depends on the individual amino acids that were supplied, on the presence of other amino acids (mixtures versus single compounds), and on the strain used. Moreover, we observed that the plasmid-free model strains L. lactis MG1363 and IL1403 produce relatively low amounts of flavour components under the various conditions tested. Conclusions By using this simplified and rapid approach to study flavour formation by non-growing lactic acid bacteria, lengthy ripening periods are no longer required to assess the capacity of strains to produce flavours in the long, non-growing state of dairy fermentation. In addition, this method also provides insight into the conversion of single amino acids versus the conversion of a mixture of amino acids as produced during protein degradation. The generated results are complementary to earlier generated datasets using growing cells, allowing assessment of the full flavour forming potential of strains used as starter cultures in industrial food fermentation processes.
    GtfA and GtfB are both required for protein O-glycosylation in Lactobacillus plantarum
    Lee, I.C. ; Swam, I.I. van; Tomita, S. ; Morsomme, P. ; Rolain, T. ; Hols, P. ; Kleerebezem, M. ; Bron, P.A. - \ 2014
    Journal of Bacteriology 196 (2014)9. - ISSN 0021-9193 - p. 1671 - 1682.
    complete genome sequence - lactic-acid bacteria - escherichia-coli - campylobacter-jejuni - acidophilus ncfm - epithelial-cells - surface protein - rhamnosus gg - glycoproteins - binding
    Acm2, the major autolysin of Lactobacillus plantarum WCFS1, was recently found to be O-glycosylated with N-acetylhexosamine, likely N-acetylglucosamine (GlcNAc). In this study, we set out to identify the glycosylation machinery by employing a comparative genomics approach to identify Gtf1 homologues, which are involved in fimbria-associated protein 1 (Fap1) glycosylation in Streptococcus parasanguinis. This in silico approach resulted in the identification of 6 candidate L. plantarum WCFS1 genes with significant homology to Gtf1, namely, tagE1 to tagE6. These candidate genes were targeted by systematic gene deletion, followed by assessment of the consequences on glycosylation of Acm2. We observed a changed mobility of Acm2 on SDS-PAGE in the tagE5E6 deletion strain, while deletion of other tagE genes resulted in Acm2 mobility comparable to that of the wild type. Subsequent mass spectrometry analysis of excised and in-gel-digested Acm2 confirmed the loss of glycosylation on Acm2 in the tagE5E6 deletion mutant, whereas a lectin blot using GlcNAc-specific succinylated wheat germ agglutinin (sWGA) revealed that besides Acm2, tagE5E6 deletion also abolished all but one other sWGA-reactive, protease-sensitive signal. Only complementation of both tagE5 and tagE6 restored those sWGA lectin signals, establishing that TagE5 and TagE6 are both required for the glycosylation of Acm2 as well as the vast majority of other sWGA-reactive proteins. Finally, sWGA lectin blotting experiments using a panel of 8 other L. plantarum strains revealed that protein glycosylation is a common feature in L. plantarum strains. With the establishment of these enzymes as protein glycosyltransferases, we propose to rename TagE5 and TagE6 as GtfA and GtfB, respectively.
    Functional Identification of Conserved Residues Involved in Lactobacillus rhamnosus Strain GG Sortase Specificity and Pilus Biogenesis
    Douillard, F.P. ; Rasinkangas, P. ; Ossowski, I. von; Reunanen, J. ; Palva, A. ; Vos, W.M. de - \ 2014
    Journal of Biological Chemistry 289 (2014)22. - ISSN 0021-9258 - p. 15764 - 15775.
    complete genome sequence - gram-positive bacteria - enterococcus-faecium isolate - group-b streptococcus - lactic-acid bacteria - lactococcus-lactis - corynebacterium-diphtheriae - bacillus-anthracis - pilin subunit - reveals pili
    In Gram-positive bacteria, sortase-dependent pili mediate the adhesion of bacteria to host epithelial cells and play a pivotal role in colonization, host signaling, and biofilm formation. Lactobacillus rhamnosus strain GG, a well known probiotic bacterium, also displays on its cell surface mucus-binding pilus structures, along with other LPXTG surface proteins, which are processed by sortases upon specific recognition of a highly conserved LPXTG motif. Bioinformatic analysis of all predicted LPXTG proteins encoded by the L. rhamnosus GG genome revealed a remarkable conservation of glycine residues juxtaposed to the canonical LPXTG motif. Here, we investigated and defined the role of this so-called triple glycine (TG) motif in determining sortase specificity during the pilus assembly and anchoring. Mutagenesis of the TG motif resulted in a lack or an alteration of the L. rhamnosus GG pilus structures, indicating that the TG motif is critical in pilus assembly and that they govern the pilin-specific and housekeeping sortase specificity. This allowed us to propose a regulatory model of the L. rhamnosus GG pilus biogenesis. Remarkably, the TG motif was identified in multiple pilus gene clusters of other Gram-positive bacteria, suggesting that similar signaling mechanisms occur in other, mainly pathogenic, species.
    Construction and Characterization of a Recombinant Invertebrate Iridovirus
    Ozgen, A. ; Muratoglu, H. ; Demirbag, Z. ; Vlak, J.M. ; Oers, M.M. van; Nalcacioglu, R. - \ 2014
    Virus Research 189 (2014). - ISSN 0168-1702 - p. 286 - 292.
    chilo-iridescent-virus - complete genome sequence - membrane-protein chimera - major capsid protein - vaccinia virus - proteomic analysis - dna polymerase - blackfly larva - cell-surface - host-range
    Chilo iridescent virus (CIV), officially named Insect iridescent virus 6 (IIV6), is the type species of the genus Iridovirus (family Iridoviridae). In this paper we constructed a recombinant CIV, encoding the green fluorescent protein (GFP). This recombinant can be used to investigate viral replication dynamics. We showed that homologous recombination is a valid method to make CIV gene knockouts and to insert foreign genes. The CIV 157L gene, putatively encoding a non-functional inhibitor of apoptosis (IAP), was chosen as target for foreign gene insertion. The gfp open reading frame preceded by the viral mcp promoter was inserted into the 157L locus by homologous recombination in Anthonomus grandis BRL-AG-3A cells. Recombinant virus (rCIV-¿157L-gfp) was purified by successive rounds of plaque purification. All plaques produced by the purified recombinant virus emitted green fluorescence due to the presence of GFP. One-step growth curves for recombinant and wild-type CIV were similar and the recombinant was fully infectious in vivo. Hence, CIV157L can be inactivated without altering the replication kinetics of the virus. Consequently, the CIV 157L locus can be used as a site for insertion of foreign DNA, e.g. to modify viral properties for insect biocontrol.
    The arable ecosystem as battleground for emergence of new human pathogens
    Overbeek, L.S. van; Doorn, J. van; Wichers, J.H. ; Amerongen, A. van; Roermund, H.J.W. van; Willemsen, P.T.J. - \ 2014
    Frontiers in Microbiology 5 (2014). - ISSN 1664-302X - 17 p.
    escherichia-coli o157-h7 - enterica serovar typhimurium - manure-amended soil - horizontal gene-transfer - complete genome sequence - pseudomonas-aeruginosa strains - complete nucleotide-sequence - hemolytic-uremic syndrome - fresh-cut lettuce - salmonella-enteric
    Disease incidences related to Escherichia coli and Salmonella enterica infections by consumption of (fresh) vegetables, sprouts, and occasionally fruits made clear that these pathogens are not only transmitted to humans via the “classical” routes of meat, eggs, and dairy products, but also can be transmitted to humans via plants or products derived from plants. Nowadays, it is of major concern that these human pathogens, especially the ones belonging to the taxonomical family of Enterobacteriaceae, become adapted to environmental habitats without losing their virulence to humans. Adaptation to the plant environment would lead to longer persistence in plants, increasing their chances on transmission to humans via consumption of plant-derived food. One of the mechanisms of adaptation to the plant environment in human pathogens, proposed in this paper, is horizontal transfer of genes from different microbial communities present in the arable ecosystem, like the ones originating from soil, animal digestive track systems (manure), water and plants themselves. Genes that would confer better adaptation to the phytosphere might be genes involved in plant colonization, stress resistance and nutrient acquisition and utilization. Because human pathogenic enterics often were prone to genetic exchanges via phages and conjugative plasmids, it was postulated that these genetic elements may be hold key responsible for horizontal gene transfers between human pathogens and indigenous microbes in agroproduction systems. In analogy to zoonosis, we coin the term phytonosis for a human pathogen that is transmitted via plants and not exclusively via animals.
    Development and evaluation of Taqman assays for the differentiation of Dickeya (sub)species
    Wolf, J.M. van der; Haas, B.H. de; Hoof, R.A. van; Haan, E.G. de; Bovenkamp, G.W. van den - \ 2014
    European Journal of Plant Pathology 138 (2014)4. - ISSN 0929-1873 - p. 695 - 709.
    complete genome sequence - syn. erwinia-chrysanthemi - potato - strains - pcr - relatedness - polymerase - clade - crops - hosts
    TaqMan assays were developed for the detection of seven Dickeya species, namely D. dianthicola, D. dadantii, D. paradisiaca, D. chrysanthemi, D. zeae, D. dieffenbachiae and D. solani. Sequences of the gene coding for dnaX were used for the design of primers and probes. In studies with axenic cultures of bacteria, the assays were highly specific and only reacted with strains of the target species, and not with non-target bacteria, including those belonging to other Dickeya species and other genera. The detection thresholds for DNA extracted from pure cultures of target strains ranged from 10 to 100 fg. The TaqMan assays for D. dianthicola and D. solani were more extensively evaluated as part of a method validation procedure. The threshold level for target bacteria added to a potato peel extract diluted ten-times in a semi-selective broth, was strain dependent and ranged from 1,000 to 100,000 cfu/ml. The coefficients of variation for repeatability and reproducibility were low and results were largely independent of the type of substrate, i.e. potato tuber or carnation leaf extracts. However, during routine testing of seed potatoes, false-positive reactions were found with the assay for D. solani. The use of the TaqMan assays for inspection of plant propagation material, ecological studies and studies on the effect of control strategies in disease management strategies is discussed
    Overview of organohalide-respiring bacteria and a proposal for a classification system for reductive dehalogenases
    Hug, L.A. ; Maphosa, F. ; Leys, D. ; Loffler, F.E. ; Smidt, H. ; Edwards, E.A. ; Adrian, L. - \ 2013
    Philosophical Transactions of the Royal Society B. Biological sciences 368 (2013)1616. - ISSN 0962-8436
    dehalococcoides sp strain - vinyl-chloride reductase - desulfitobacterium-frappieri pcp-1 - strictly anaerobic bacterium - multiple sequence alignment - complete genome sequence - best-fit models - dehalospirillum-multivorans - enrichment culture - dehalobacter-re
    Organohalide respiration is an anaerobic bacterial respiratory process that uses halogenated hydrocarbons as terminal electron acceptors during electron transport-based energy conservation. This dechlorination process has triggered considerable interest for detoxification of anthropogenic groundwater contaminants. Organohalide-respiring bacteria have been identified from multiple bacterial phyla, and can be categorized as obligate and non-obligate organohalide respirers. The majority of the currently known organohalide-respiring bacteria carry multiple reductive dehalogenase genes. Analysis of a curated set of reductive dehalogenases reveals that sequence similarity and substrate specificity are generally not correlated, making functional prediction from sequence information difficult. In this article, an orthologue-based classification system for the reductive dehalogenases is proposed to aid integration of new sequencing data and to unify terminology.
    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
    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
    The quest for probiotic effector molecules - Unraveling strain specificity at the molecular level
    Lee, I.C. ; Tomita, S. ; Kleerebezem, M. ; Bron, P.A. - \ 2013
    Pharmacological Research 69 (2013)1. - ISSN 1043-6618 - p. 61 - 74.
    wall teichoic-acid - lactobacillus-rhamnosus gg - alanyl-lipoteichoic acid - complete genome sequence - gram-positive bacteria - antibiotic-associated diarrhea - bacillus-subtilis w23 - cell-wall - staphylococcus-aureus - dendritic-cell
    Pharmaceutical agents are widely applied for the treatment of gastrointestinal (and systemic) disorders and their role as modulators of host cell responses is relatively well characterized. By contrast, we are only beginning to understand the molecular mechanisms by which health-promoting, probiotic bacteria act as host cell modulators. The last decade has seen a rapid development of the genomics field for the widely applied probiotic genus Lactobacillus, and nowadays dozens of full genome sequences are available, as well as sophisticated post genomic and genetic engineering tools. This development has enabled comparative (functional) genomics approaches to identify the bacterial effector molecules involved in molecular communication with the host system that may underlie the probiotic effects observed. These efforts can also be complemented with dedicated mutagenesis approaches to eliminate or alter these effector molecules, followed by assessment of the host interaction consequences thereof, allowing the elucidation of the molecular mechanisms involved in probiotic health effects. Many of these approaches have pinpointed that the Lactobacillus cell envelope contains several effector molecules that are pivotal in the direct signaling capacity of these bacteria that underlies their immunomodulatory effects, including lipoteichoic acid, peptidoglycan, and (glyco)proteins. Moreover, the cell envelope contains several compounds such as wall teichoic acid and capsular polysaccharides that may not be involved in direct signaling to the host cell, but still affect signaling through shielding of other bacterial effector molecules. Initial structural studies revealed subtle strain- and species-specific biochemical differences in the canonical cell envelope compounds that are involved in these host interactions. These biochemical variations include the degree and positioning of d-alanyl and glycosyl substitution in lipoteichoic acids, and acetylation of peptidoglycan. Furthermore, specific peptides derived from peptidoglycan and envelope associated (glyco)proteins were recently identified as potent immunomodulators. The latter findings are exciting in the light of the possibility of more pharmacological application of these bioactive probiotic molecules, and especially cost-effective production and targeted delivery of bioactive peptides seems to emerge as a feasible strategy to harness this knowledge.
    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.
    Lactobacillus plantarum possesses the capability for wall teichoic acid backbone alditol switching
    Bron, P.A. ; Tomita, S. ; Swam, I. van; Remus, D.M. ; Meijerink, M. ; Wels, M. ; Okada, S. ; Wells, J. ; Kleerebezem, M. - \ 2012
    Microbial Cell Factories 11 (2012). - ISSN 1475-2859
    aureus nasal colonization - complete genome sequence - toll-like receptor-2 - staphylococcus-aureus - bacillus-subtilis - lipoteichoic acid - cell-wall - peptidoglycan - biosynthesis - glycerol
    Background - Specific strains of Lactobacillus plantarum are marketed as health-promoting probiotics. The role and interplay of cell-wall compounds like wall- and lipo-teichoic acids (WTA and LTA) in bacterial physiology and probiotic-host interactions remain obscure. L. plantarum WCFS1 harbors the genetic potential to switch WTA backbone alditol, providing an opportunity to study the impact of WTA backbone modifications in an isogenic background. Results - Through genome mining and mutagenesis we constructed derivatives that synthesize alternative WTA variants. The mutants were shown to completely lack WTA, or produce WTA and LTA that lack D-Ala substitution, or ribitol-backbone WTA instead of the wild-type glycerol-containing backbone. DNA micro-array experiments established that the tarIJKL gene cluster is required for the biosynthesis of this alternative WTA backbone, and suggest ribose and arabinose are precursors thereof. Increased tarIJKL expression was not observed in any of our previously performed DNA microarray experiments, nor in qRT-PCR analyses of L. plantarum grown on various carbon sources, leaving the natural conditions leading to WTA backbone alditol switching, if any, to be identified. Human embryonic kidney NF-¿B reporter cells expressing Toll like receptor (TLR)-2/6 were exposed to purified WTAs and/or the TA mutants, indicating that WTA is not directly involved in TLR-2/6 signaling, but attenuates this signaling in a backbone independent manner, likely by affecting the release and exposure of immunomodulatory compounds such as LTA. Moreover, human dendritic cells did not secrete any cytokines when purified WTAs were applied, whereas they secreted drastically decreased levels of the pro-inflammatory cytokines IL-12p70 and TNF-a after stimulation with the WTA mutants as compared to the wild-type. Conclusions - The study presented here correlates structural differences in WTA to their functional characteristics, thereby providing important information aiding to improve our understanding of molecular host-microbe interactions and probiotic functionality
    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.
    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.
    Diversity and natural functions of antibiotics produced by beneficial and plant pathogenic bacteria
    Raaijmakers, J.M. ; Mazzola, M. - \ 2012
    Annual Review of Phytopathology 50 (2012). - ISSN 0066-4286 - p. 403 - 424.
    complete genome sequence - cyclic lipopeptide production - pseudomonas-fluorescens pf-5 - imaging mass-spectrometry - secondary metabolite 2,4-diacetylphloroglucinol - bacillus-amyloliquefaciens fzb42 - dose-response relationships - soil microbial community - bio
    Soil- and plant-associated environments harbor numerous bacteria that produce antibiotic metabolites with specific or broad-spectrum activities against coexisting microorganisms. The function and ecological importance of antibiotics have long been assumed to yield a survival advantage to the producing bacteria in the highly competitive but resource-limited soil environments through direct suppression. Although specific antibiotics may enhance producer persistence when challenged by competitors or predators in soil habitats, at subinhibitory concentrations antibiotics exhibit a diversity of other roles in the life history of the producing bacteria. Many processes modulated by antibiotics may be inherently critical to the producing bacterium, such as the acquisition of substrates or initiation of developmental changes that will ensure survival under stressful conditions. Antibiotics may also have roles in more complex interactions, including in virulence on host plants or in shaping the outcomes of multitrophic interactions. The innate functions of antibiotics to producing bacteria in their native ecosystem are just beginning to emerge, but current knowledge already reveals a breadth of activities well beyond the historical perspective of antibiotics as weaponry in microbial conflicts.
    Low numbers of repeat units in variable number of tandem repeats (VNTR) regions of white spot syndrome virus are correlated with disease outbreaks
    Tran Thi Tuyet, H. ; Zwart, M.P. ; Phuong, N.T. ; Jong, M.C.M. de; Vlak, J.M. - \ 2012
    Journal of Fish Diseases 35 (2012)11. - ISSN 0140-7775 - p. 817 - 826.
    repetitive dna-sequences - complete genome sequence - ribonucleotide reductase - penaeus-monodon - in-vivo - shrimp - virulence - wssv - identification - pathogenicity
    White spot syndrome virus (WSSV) is the most important pathogen in shrimp farming systems worldwide including the Mekong Delta, Vietnam. The genome of WSSV is characterized by the presence of two major 'indel regions' found at ORF14/15 and ORF23/24 (WSSV-Thailand) and three regions with variable number tandem repeats (VNTR) located in ORF75, ORF94 and ORF125. In the current study, we investigated whether or not the number of repeat units in the VNTRs correlates with virus outbreak status and/or shrimp farming practice. We analysed 662 WSSV samples from individual WSSV-infected Penaeus monodon shrimp from 104 ponds collected from two important shrimp farming regions of the Mekong Delta: Ca Mau and Bac Lieu. Using this large data set and statistical analysis, we found that for ORF94 and ORF125, the mean number of repeat units (RUs) in VNTRs was significantly lower in disease outbreak ponds than in non-outbreak ponds. Although a higher mean RU number was observed in the improved-extensive system than in the rice-shrimp or semi-intensive systems, these differences were not significant. VNTR sequences are thus not only useful markers for studying WSSV genotypes and populations, but specific VNTR variants also correlate with disease outbreaks in shrimp farming systems.
    Genetic data from avian influenza and avian paramyxoviruses generated by the European network of excellence (EPIZONE) between 2006 and 2011 - Review and recommendations for surveillance
    Dundon, W.G. ; Heidari, A. ; Fusaro, A. ; Monne, I. ; Beato, M.S. ; Cattoli, G. ; Koch, G. ; Starick, E. ; Brown, I.H. ; Aldous, E.W. ; Briand, F.X. ; Gall-Reculé, G. Le; Jestin, V. ; Jorgensen, P.H. ; Berg, M. ; Zohari, S. ; Metreveli, G. ; Munir, M. ; Stahl, K. ; Albina, E. ; Hammoumi, S. ; Gil, P. ; Servan de Almeida, R. ; Smietanka, K. ; Domanska-Blicharz, K. ; Minta, Z. ; Borm, S. van; Berg, T. van den; Martin, A.M. ; Barbieri, I. ; Capua, I. - \ 2012
    Veterinary Microbiology 154 (2012)3-4. - ISSN 0378-1135 - p. 209 - 221.
    newcastle-disease-virus - complete genome sequence - wild birds - a viruses - h5n1 viruses - phylogenetic analysis - subtype h5n1 - northern europe - domestic ducks - great-britain
    Since 2006, the members of the molecular epidemiological working group of the European “EPIZONE” network of excellence have been generating sequence data on avianinfluenza and avianparamyxoviruses from both European and African sources in an attempt to more fully understand the circulation and impact of these viruses. This review presents a timely update on the epidemiological situation of these viruses based on sequence datagenerated during the lifetime of this project in addition to data produced by other groups during the same period. Based on this information and putting it all into a European context, recommendations for continued surveillance of these important viruses within Europe are presented.
    Metagenome analysis reveals yet unexplored reductive dechlorinating potential of Dehalobacter sp. E1 growing in coculture with Sedimentibacter sp.
    Maphosa, F. ; Passel, M.W.J. van; Vos, W.M. de; Smidt, H. - \ 2012
    Environmental Microbiology Reports 4 (2012)6. - ISSN 1758-2229 - p. 604 - 616.
    desulfitobacterium-frappieri pcp-1 - complete genome sequence - dehalococcoides spp. - online tool - hafniense - tetrachloroethene - dehalogenation - vitamin-b-12 - restrictus - bacterium
    The importance of Dehalobacter species in bioremediation as dedicated degraders of chlorinated organics has been well recognized. However, still little is known about Dehalobacter's full genomic repertoires, including the genes involved in dehalogenation. Here we report the first insights into the genome sequence of Dehalobacter sp. E1 that grows in strict co-culture with Sedimentibacter sp. B4. Based on the co-culture metagenome and the genome of strain B4 (4.2¿Mbp) we estimate the genome sequence of strain E1 to be 2.6¿Mbp. Ten putative reductive dehalogenase homologue (Rdh)-encoding gene clusters were identified. One cluster has a putative tetrachloroethene Rdh-encoding gene cluster, similar to the pceABCT operon previously identified in Dehalobacter restrictus. Metagenome analysis indicated that the inability of strain E1 to synthesize cobalamin, an essential cofactor of reductive dehalogenases, is complemented by Sedimentibacter. The metagenomic exploration described here maps the extensive dechlorinating potential of Dehalobacter, and paves way for elucidation of the interactions with its co-cultured Sedimentibacter
    Pathways of sulfide oxidation by haloalkaliphilic bacteria in limited-oxygen gas lift bioreactors
    Klok, J.B. ; Bosch, P.L.F. van den; Buisman, C.J.N. ; Stams, A.J.M. ; Keesman, K.J. ; Janssen, A.J.H. - \ 2012
    Environmental Science and Technology 46 (2012)14. - ISSN 0013-936X - p. 7581 - 7586.
    sulfur-oxidizing bacteria - complete genome sequence - alkaline conditions - hydrogen-sulfide - soda lakes - mechanism - removal - reactor
    Physicochemical processes, such as the Lo-cat and Amine-Claus process, are commonly used to remove hydrogen sulfide from hydrocarbon gas streams such as landfill gas, natural gas, and synthesis gas. Biodesulfurization offers environmental advantages, but still requires optimization and more insight in the reaction pathways and kinetics. We carried out experiments with gas lift bioreactors inoculated with haloalkaliphilic sulfide-oxidizing bacteria. At oxygen-limiting levels, that is, below an O(2)/H(2)S mole ratio of 1, sulfide was oxidized to elemental sulfur and sulfate. We propose that the bacteria reduce NAD(+) without direct transfer of electrons to oxygen and that this is most likely the main route for oxidizing sulfide to elemental sulfur which is subsequently oxidized to sulfate in oxygen-limited bioreactors. We call this pathway the limited oxygen route (LOR). Biomass growth under these conditions is significantly lower than at higher oxygen levels. These findings emphasize the importance of accurate process control. This work also identifies a need for studies exploring similar pathways in other sulfide oxidizers such as Thiobacillus bacteria
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