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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Genome-wide transcriptional profiling of Clostridium perfringens SM101 during sporulation extends the core of putative sporulation genes and genes determining spore properties and germination characteristics
Xiao, Y. ; Hijum, S.A.F.T. van; Abee, T. ; Wells-Bennik, M.H.J. - \ 2015
PLoS One 10 (2015)5. - ISSN 1932-6203 - 19 p.
bacillus-subtilis - dipicolinic acid - bacterial-spores - heat-resistance - ser/thr kinase - killing factor - identification - difficile - proteins - sequence
The formation of bacterial spores is a highly regulated process and the ultimate properties of the spores are determined during sporulation and subsequent maturation. A wide variety of genes that are expressed during sporulation determine spore properties such as resistance to heat and other adverse environmental conditions, dormancy and germination responses. In this study we characterized the sporulation phases of C. perfringens enterotoxic strain SM101 based on morphological characteristics, biomass accumulation (OD600), the total viable counts of cells plus spores, the viable count of heat resistant spores alone, the pH of the supernatant, enterotoxin production and dipicolinic acid accumulation. Subsequently, whole-genome expression profiling during key phases of the sporulation process was performed using DNA microarrays, and genes were clustered based on their time-course expression profiles during sporulation. The majority of previously characterized C. perfringens germination genes showed upregulated expression profiles in time during sporulation and belonged to two main clusters of genes. These clusters with up-regulated genes contained a large number of C. perfringens genes which are homologs of Bacillus genes with roles in sporulation and germination; this study therefore suggests that those homologs are functional in C. perfringens. A comprehensive homology search revealed that approximately half of the upregulated genes in the two clusters are conserved within a broad range of sporeforming Firmicutes. Another 30% of upregulated genes in the two clusters were found only in Clostridium species, while the remaining 20% appeared to be specific for C. perfringens. These newly identified genes may add to the repertoire of genes with roles in sporulation and determining spore properties including germination behavior. Their exact roles remain to be elucidated in future studies.
Diversity of acid stress resistant variants of Listeria monocytogenes and the potential role of ribosomal protein S21 encoded by rpsU
Metselaar, K.I. ; Besten, H.M.W. den; Boekhorst, J. ; Hijum, S.A.F.T. van; Zwietering, M.H. ; Abee, T. - \ 2015
Frontiers in Microbiology 6 (2015). - ISSN 1664-302X - 12 p.
high hydrostatic-pressure - gamma-aminobutyric-acid - microtiter plate assay - glutamate-decarboxylase - bacillus-subtilis - escherichia-coli - low ph - genes - strains - inactivation
The dynamic response of microorganisms to environmental conditions depends on the behavior of individual cells within the population. Adverse environments can select for stable stress resistant subpopulations. In this study, we aimed to get more insight in the diversity within Listeria monocytogenes LO28 populations, and the genetic basis for the increased resistance of stable resistant fractions isolated after acid exposure. Phenotypic cluster analysis of 23 variants resulted in three clusters and four individual variants and revealed multiple-stress resistance, with both unique and overlapping features related to stress resistance, growth, motility, biofilm formation, and virulence indicators. A higher glutamate decarboxylase activity correlated with increased acid resistance. Whole genome sequencing revealed mutations in rpsU, encoding ribosomal protein S21 in the largest phenotypic cluster, while mutations in ctsR, which were previously shown to be responsible for increased resistance of heat and high hydrostatic pressure resistant variants, were not found in the acid resistant variants. This underlined that large population diversity exists within one L. monocytogenes strain and that different adverse conditions drive selection for different variants. The finding that acid stress selects for rpsU variants provides potential insights in the mechanisms underlying population diversity of L. monocytogenes.
Characterisation of biofilms formed by Lactobacillus plantarum WCFS1 and food spoilage isolates
Fernández Ramírez, M.D. ; Smid, E.J. ; Abee, T. ; Nierop Groot, M.N. - \ 2015
International Journal of Food Microbiology 207 (2015). - ISSN 0168-1605 - p. 23 - 29.
lactic-acid bacteria - enterococcal surface protein - listeria-monocytogenes - pseudomonas-putida - bacillus-subtilis - starter cultures - genetic-analysis - rhamnosus gg - resistance - industry
Lactobacillus plantarum has been associated with food spoilage in a wide range of products and the biofilm growth mode has been implicated as a possible source of contamination. In this study we analysed the biofilm forming capacity of L. plantarum WCFS1 and six food spoilage isolates. Biofilm formation as quantified by crystal violet staining and colony forming units was largely affected by the medium composition, growth temperature and maturation time and by strain specific features. All strains showed highest biofilm formation in Brain Heart Infusion medium supplemented with manganese and glucose. For L. plantarum biofilms the crystal violet (CV) assay, that is routinely used to quantify total biofilm formation, correlates poorly with the number of culturable cells in the biofilm. This can in part be explained by cell death and lysis resulting in CV stainable material, conceivably extracellular DNA (eDNA), contributing to the extracellular matrix. The strain to strain variation may in part be explained by differences in levels of eDNA, likely as result of differences in lysis behaviour. In line with this, biofilms of all strains tested, except for one spoilage isolate, were sensitive to DNase treatment. In addition, biofilms were highly sensitive to treatment with Proteinase K suggesting a role for proteins and/or proteinaceous material in surface colonisation. This study shows the impact of a range of environmental factors and enzyme treatments on biofilm formation capacity for selected L. plantarum isolates associated with food spoilage, and may provide clues for disinfection strategies in food industry.
Bright fluorescent Streptococcus pneumoniae for live cell imaging of host-pathogen interactions
Kjos, M. ; Aprianto, R. ; Fernandes, V.E. ; Andrew, P.W. ; Strijp, J.A.G. van; Nijland, R. ; Veening, J.W. - \ 2015
Journal of Bacteriology 197 (2015)5. - ISSN 0021-9193 - p. 807 - 818.
epithelial-cells - gene-expression - pneumococcal virulence - bacillus-subtilis - in-vivo - protein - capsule - colonization - disease - invasion
Streptococcus pneumoniae is a common nasopharyngeal resident in healthy people, but at the same time one of the major causes of infectious diseases such as pneumonia, meningitis and sepsis. The shift from commensal to pathogen and its interaction with host cells is poorly understood. One of the major limitations for research on pneumococcal-host interactions is the lack of suitable tools for live cell imaging. To address this issue, we developed a generally applicable strategy to create genetically stable, highly fluorescent bacteria. Our strategy relies on fusing superfolder green fluorescent protein (GFP) or a far-red fluorescent protein (RFP) to the abundant histone-like protein HlpA. Due to efficient translation and limited cellular diffusion of these fusions, the cells are 25-fold brighter than the currently best available imaging S. pneumoniae strain. These novel bright pneumococcal strains are fully virulent and the GFP-reporter can be used for in situ imaging in mouse tissue. We used our reporter strains to study the effect of the polysaccharide capsule, a major pneumococcal virulence factor, on different stages of infection. By dual-color live cell imaging experiments, we show that unencapsulated pneumococci adhere significantly better to human lung epithelial cells compared to encapsulated strains, in line with previous data obtained by classical approaches. We also confirm with live cell imaging that the capsule protects pneumococci from neutrophil phagocytosis, demonstrating the versatility and usability of our reporters. The described imaging tools will pave the way for live cell imaging of pneumococcal infection and help understand the mechanisms of pneumococcal pathogenesis.
Biological control as a strategy to reduce the impact of mycotoxins in peanuts, grapes and cereals in Argentina
Chulze, S.N. ; Palazzini, J.M. ; Torres, A.M. ; Barros, G. ; Ponsone, M.L. ; Geisen, R. ; Schmidt-Heydt, M. ; Köhl, J. - \ 2015
Food Additives & Contaminants. Pt. A, Chemistry, Analysis, Control, Exposure & Risk Assessment 32 (2015)4. - ISSN 1944-0049 - p. 471 - 479.
fusarium head blight - potential biocontrol agents - wine-producing vineyards - zea-mays l. - aspergillus-flavus - aflatoxin contamination - gibberella-zeae - aureobasidium-pullulans - bacillus-subtilis - field application
Mycotoxins including aflatoxins, deoxynivalenol, fumonisins and ochratoxin A are among the main fungal secondary metabolites detected as natural contaminants in South America in different commodities such as peanuts (aflatoxins), cereals (deoxynivalenol and fumonisins) or grapes (ochratoxin A). Different strategies including crop rotation, tillage practices, fungicide application and planting less susceptible cultivars are used in order to reduce the impact of these mycotoxins in both food and feed chains. The development of fungicide resistance in many fungal pathogens as well as rising of public concern on the risks associated with pesticide use led to the search for alternative environmentally friendly methods. Biological control of plant pathogens and toxigenic fungi offers an alternative that can complement chemical control in the frame of an integrated pest management to reduce the impact of mycotoxins in the food and feed chains. The advances made in Argentina on reducing the impact of toxigenic fungi and mycotoxins in peanut, grapes and cereals using the biocontrol strategy are summarised. Native bacteria, yeasts and filamentous fungi have been selected to evaluate them as potential biocontrol agents. Field trials showed that Bacillus subtilis RC 218 and Brevibacillus sp. RC 263 were effective at reducing deoxynivalenol accumulation in wheat. The application of Clonostachys rosea isolates on wheat stubble reduced Fusarium colonisation on the stubble. Bacillus amyloliquefaciens and Microbacterium oleovorans showed good activity to control both Fusarium verticillioides growth and the accumulation of fumonisins at pre-harvest stage in maize. Control of toxigenic Aspergillus flavus and aflatoxin accumulation in peanuts was achieved using a native atoxigenic Aspergillus flavus strain based on competitive exclusion of the toxigenic strains. Kluyveromyces thermotolerans strains were used as biocontrol agents to reduce the impact of Aspergillus section Nigri and ochratoxin A accumulation in grapes
Molecular and metabolic adaptations of Lactococcus lactis at near-zero growth rates
Ercan, O. ; Wels, M. ; Smid, E.J. ; Kleerebezem, M. - \ 2015
Applied and Environmental Microbiology 81 (2015)1. - ISSN 0099-2240 - p. 320 - 331.
acid bacteria - saccharomyces-cerevisiae - quantitative physiology - transcriptome analysis - streptococcus-lactis - maintenance energy - catabolite control - bacillus-subtilis - escherichia-coli - sugar catabolism
This paper describes the molecular and metabolic adaptations of Lactococcus lactis during the transition from a growing to a near-zero growth state using carbon-limited retentostat cultivation. Transcriptomic analyses revealed that metabolic patterns shifted between lactic- and mixed-acid fermentation during retentostat cultivation, which appeared to be controlled at the transcription level of the corresponding pyruvate-dissipation encoding genes. During retentostat cultivation, cells continued to consume several amino acids, but also produced specific amino acids, which may derive from the conversion of glycolytic intermediates. We identify a novel motif containing CTGTCAG, in the upstream regions of several genes related to amino acid conversion, which we propose to be the target site for CodY in Lactococcus lactis KF147. Finally, under extremely low carbon availability, carbon catabolite repression was progressively relieved and alternative catabolic functions were found to be highly expressed, which was confirmed by enhanced initial acidification rates on various sugars in cells obtained from near-zero growth cultures. The present integrated transcriptome and metabolite (amino acids and previously reported fermentation end-products) study provides molecular understanding of the adaptation of Lactococcus lactis to conditions supporting low-growth rates, and expands our earlier analysis of the quantitative physiology of this bacterium at near-zero growth rates towards gene regulation patterns involved in zero-growth adaptation.
Impact of interspecific interactions on antimicrobial activity among soil bacteria
Tyc, O. ; Berg, M. van den; Gerards, S. ; Veen, J.A. van; Raaijmakers, J.M. ; Boer, W. de; Garbeva, P. - \ 2014
Frontiers in Microbiology 5 (2014). - ISSN 1664-302X
community composition - bacillus-subtilis - gene-expression - antibiotics - diversity - rhizosphere - inhibition - environment - resistance - reveals
Certain bacterial species produce antimicrobial compounds only in the presence of a competing species. However, little is known on the frequency of interaction-mediated induction of antibiotic compound production in natural communities of soil bacteria. Here we developed a high-throughput method to screen for the production of antimicrobial activity by monocultures and pair-wise combinations of 146 phylogenetically different bacteria isolated from similar soil habitats. Growth responses of two human pathogenic model organisms, Escherichia coli WA321 and Staphylococcus aureus 533R4, were used to monitor antimicrobial activity. From all isolates, 33% showed antimicrobial activity only in monoculture and 42% showed activity only when tested in interactions. More bacterial isolates were active against S. aureus than against E. coli. The frequency of interaction-mediated induction of antimicrobial activity was 6% (154 interactions out of 2798) indicating that only a limited set of species combinations showed such activity. The screening revealed also interaction-mediated suppression of antimicrobial activity for 22% of all combinations tested. Whereas all patterns of antimicrobial activity (non-induced production, induced production and suppression) were seen for various bacterial classes, interaction-mediated induction of antimicrobial activity was more frequent for combinations of Flavobacteria and alpha- Proteobacteria. The results of our study give a first indication on the frequency of interference competitive interactions in natural soil bacterial communities which may forms a basis for selection of bacterial groups that are promising for the discovery of novel, cryptic antibiotics.
The Ccpa regulon of Streptococcus suis reveals novel insights into the regulation of the streptococcal central carbon metabolism by binding of CcpA to two distinct binding motifs.
Willenborg, J. ; Greeff, A. de; Jarek, M. ; Valentin-Weigand, P. ; Goethe, R. - \ 2014
Molecular Microbiology 92 (2014)1. - ISSN 0950-382X - p. 61 - 83.
sugar phosphotransferase system - bacillus-subtilis - catabolite repression - virulence - bacteria - phosphoenolpyruvate - expression - pathogen - hpr - phosphocarrier
Streptococcus suis (S.¿suis) is a neglected zoonotic streptococcus causing fatal diseases in humans and in pigs. The transcriptional regulator CcpA (catabolite control protein A) is involved in the metabolic adaptation to different carbohydrate sources and virulence of S.¿suis and other pathogenic streptococci. In this study, we determined the DNA binding characteristics of CcpA and identified the CcpA regulon during growth of S.¿suis. Electrophoretic mobility shift analyses showed promiscuous DNA binding of CcpA to cognate cre sites in vitro. In contrast, sequencing of immunoprecipitated chromatin revealed two specific consensus motifs, a pseudo-palindromic cre motif (WWGAAARCGYTTTCWW) and a novel cre2 motif (TTTTYHWDHHWWTTTY), within the regulatory elements of the genes directly controlled by CcpA. Via these elements CcpA regulates expression of genes involved in carbohydrate uptake and conversion, and in addition in important metabolic pathways of the central carbon metabolism, like glycolysis, mixed-acid fermentation, and the fragmentary TCA cycle. Furthermore, our analyses provide evidence that CcpA regulates the genes of the central carbon metabolism by binding either the pseudo-palindromic cre motif or the cre2 motif in a HPr(Ser)~P independent conformation.
Metabolomic engineering for the microbial production of cartenoids and related products with a focus on the rare C50 carotenoids
Heider, S.A.E. ; Peters-Wendisch, P. ; Wendisch, V.F. ; Beekwilder, M.J. - \ 2014
Applied Microbiology and Biotechnology 98 (2014)10. - ISSN 0175-7598 - p. 4355 - 4368.
alga haematococcus-pluvialis - blue-light photoreception - escherichia-coli - bacterial carotenoids - biosynthetic-pathway - bacillus-subtilis - astaxanthin biosynthesis - functional-analysis - beta-carotene - corynebacterium-glutamicum
Carotenoids, a subfamily of terpenoids, are yellowtored-colored pigments synthesized by plants, fungi, algae, and bacteria. They are ubiquitous in nature and take over crucial roles in many biological processes as for example photosynthesis, vision, and the quenching of free radicals and singlet oxygen. Due to their color and their potential beneficial effects on human health, carotenoids receive increasing attention. Carotenoids can be classified due to the length of their carbon backbone. Most carotenoids have a C40 backbone, but also C30 and C50 carotenoids are known. All carotenoids are derived fromisopentenyl pyrophosphate (IPP) as a common precursor. Pathways leading to IPP as well as metabolic engineering of IPP synthesis and C40 carotenoid production have been reviewed expertly elsewhere. Since C50 carotenoids are synthesized from the C40 carotenoid lycopene, we will summarize common strategies for optimizing lycopene production and we will focus our review on the characteristics, biosynthesis, glycosylation, and overproduction of C50 carotenoids.
Carbohydrate Availability Regulates Virulence Gene Expression in Streptococcus suis
Ferrando, M.L. ; Baarlen, P. van; Orrù, G. ; Piga, R. ; Bongers, R.S. ; Wels, M. ; Greeff, A. de; Smith, H. ; Wells, J.M. - \ 2014
PLoS One 9 (2014)3. - ISSN 1932-6203 - 16 p.
group-a streptococcus - fibronectin-binding protein - bacillus-subtilis - glycogen-metabolism - escherichia-coli - epithelial-cells - mouse intestine - molecular characterization - hyaluronan concentration - catabolite repression
Streptococcus suis is a major bacterial pathogen of young pigs causing worldwide economic problems for the pig industry. S. suis is also an emerging pathogen of humans. Colonization of porcine oropharynx by S. suis is considered to be a high risk factor for invasive disease. In the oropharyngeal cavity, where glucose is rapidly absorbed but dietary a-glucans persist, there is a profound effect of carbohydrate availability on the expression of virulence genes. Nineteen predicted or confirmed S. suis virulence genes that promote adhesion to and invasion of epithelial cells were expressed at higher levels when S. suis was supplied with the a-glucan starch/pullulan compared to glucose as the single carbon source. Additionally the production of suilysin, a toxin that damages epithelial cells, was increased more than ten-fold when glucose levels were low and S. suis was growing on pullulan. Based on biochemical, bioinformatics and in vitro and in vivo gene expression studies, we developed a biological model that postulates the effect of carbon catabolite repression on expression of virulence genes in the mucosa, organs and blood. This research increases our understanding of S. suis virulence mechanisms and has important implications for the design of future control strategies including the development of anti-infective strategies by modulating animal feed composition
Regulation of reductive dehalogenase gene transcription in Dehalococcoides mccartyi
Wagner, A. ; Segler, L. ; Kleinsteuber, S. ; Sawers, G. ; Smidt, H. ; Lechner, U. - \ 2013
Philosophical Transactions of the Royal Society. Series B, Biological Sciences 368 (2013)1616. - ISSN 0962-8436
sp strain cbdb1 - vinyl-chloride reductase - escherichia-coli - desulfitobacterium-hafniense - response regulators - bacillus-subtilis - genome sequence - marr family - ethenogenes - expression
The remarkable capacity of the genus Dehalococcoides to dechlorinate a multitude of different chlorinated organic compounds reflects the number and diversity of genes in the genomes of Dehalococcoides species encoding reductive dehalogenase homologues (rdh). Most of these genes are located in the vicinity of genes encoding multiple antibiotic resistance regulator (MarR)-type or two-component system regulators. Here, the transcriptional response of rdhA genes (coding for the catalytic subunit) to 2,3- and 1,3-dichlorodi-benzo-p-dioxin (DCDD) was studied in Dehalococcoides mccartyi strain CBDB1. Almost all rdhA genes were transcribed in the presence of 2,3-DCDD, albeit at different levels as shown for the transcripts of cbrA, cbdbA1453, cbdbA1624 and cbdbA1588. By contrast, 1,3-DCDD did not induce rdhA transcription. The putative MarR CbdbA1625 was heterologously produced and its ability to bind in vitro to the overlapping promoter regions of the genes cbdbA1624 and cbdbA1625 was demonstrated. To analyse regulation in vivo, single-copy transcriptional promoter-lacZ fusions of different rdhA genes and of cbdbA1625 were constructed and introduced into the heterologous host Escherichia coli, and expression levels of the fusions were measured. The cbdbA1625 gene was cloned into a vector allowing a regulation of expression by arabinose and it was transformed into the strains containing the rdh-promoter-lacZ fusion derivatives. CbdbA1625 was shown to downregulate transcription from its own promoter resulting in a 40-50% reduction in the beta-galactosidase activity, giving the first hint that it acts as a repressor.
Single Mutation in Shine-Dalgarno-Like Sequence Present in the Amino Terminal of Lactate Dehydrogenase of Plasmodium Effects the Production of an Eukaryotic Protein Expressed in a Prokaryotic System
Cicek, M. ; Mutlu, O. ; Erdemir, A. ; Ozkan, E. ; Saricay, Y. ; Turgut-Balik, D. - \ 2013
Molecular Biotechnology 54 (2013)2. - ISSN 1073-6085 - p. 602 - 608.
human malaria parasite - escherichia-coli - messenger-rna - recombinant proteins - bacillus-subtilis - ribosomal-rna - q-beta - falciparum - genome - gene
One of the most important step in structure-based drug design studies is obtaining the protein in active form after cloning the target gene. In one of our previous study, it was determined that an internal Shine-Dalgarno-like sequence present just before the third methionine at N-terminus of wild type lactate dehydrogenase enzyme of Plasmodium falciparum prevent the translation of full length protein. Inspection of the same region in P. vivax LDH, which was overproduced as an active enzyme, indicated that the codon preference in the same region was slightly different than the codon preference of wild type PfLDH. In this study, 5'-GGAGGC-3' sequence of P. vivax that codes for two glycine residues just before the third methionine was exchanged to 5'-GGAGGA-3', by mimicking P. falciparum LDH, to prove the possible effects of having an internal SD-like sequence when expressing an eukaryotic protein in a prokaryotic system. Exchange was made by site-directed mutagenesis. Results indicated that having two glycine residues with an internal SD-like sequence (GGAGGA) just before the third methionine abolishes the enzyme activity due to the preference of the prokaryotic system used for the expression. This study emphasizes the awareness of use of a prokaryotic system to overproduce an eukaryotic protein.
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
The structure of an alternative wall teichoic acid produced by a Lactobacillus plantarum WCFS1 mutant contains a 1,5-linked poly(ribitol phosphate) backbone with 2-a-D-glucosyl substitutions
Tomita, S. ; Waard, P. de; Bakx, E.J. ; Schols, H.A. ; Kleerebezem, M. ; Bron, P.A. - \ 2013
Carbohydrate Research : an international journal 370 (2013). - ISSN 0008-6215 - p. 67 - 71.
cell-wall - staphylococcus-aureus - bacillus-subtilis - units
A tagF1-tagF2 deletion mutant of Lactobacillus plantarum lacks poly(glycerol phosphate) polymerase activity required for glycerol-type wall teichoic acid (WTA) biosynthesis. The mutant activates an alternative genetic locus, tarIJKL, encoding the enzymes for nucleotide activation and incorporation of ribitol in the WTA backbone polymer. This alternative ribitol-type WTA backbone and its repeating unit were isolated and characterized by HPAEC, UPLC-MS, NMR spectroscopy, and MALDI-TOF MS, using synthetic molecules as references. The structure was established as 1,5-linked poly(ribitol phosphate) which was substituted at the C-2 hydroxyl group of the ribitol residue with a-D-glucosyl at a frequency of 28%.
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.
Dissecting the energy metabolism in Mycoplasma pneumoniae through genome-scale metabolic modeling
Wodke, J.A. ; Puchalka, J. ; Lluch-Senar, M. ; Marcos, J. ; Yus, E. ; Godinho, M. ; Gutierrez-Gallego, R. ; Martins Dos Santos, V.A.P. ; Serrano, L. ; Klipp, E. ; Maier, T. - \ 2013
Molecular Systems Biology 9 (2013). - ISSN 1744-4292 - 19 p.
cobra toolbox extension - flux balance models - escherichia-coli - reduced bacterium - biochemical networks - invivo measurement - bacillus-subtilis - optimal selection - genetic-analysis - membrane-lipids
Mycoplasma pneumoniae, a threatening pathogen with a minimal genome, is a model organism for bacterial systems biology for which substantial experimental information is available. With the goal of understanding the complex interactions underlying its metabolism, we analyzed and characterized the metabolic network of M. pneumoniae in great detail, integrating data from different omics analyses under a range of conditions into a constraint-based model backbone. Iterating model predictions, hypothesis generation, experimental testing, and model refinement, we accurately curated the network and quantitatively explored the energy metabolism. In contrast to other bacteria, M. pneumoniae uses most of its energy for maintenance tasks instead of growth. We show that in highly linear networks the prediction of flux distributions for different growth times allows analysis of time-dependent changes, albeit using a static model. By performing an in silico knock-out study as well as analyzing flux distributions in single and double mutant phenotypes, we demonstrated that the model accurately represents the metabolism of M. pneumoniae. The experimentally validated model provides a solid basis for understanding its metabolic regulatory mechanisms
The species-specific mode of action of the antimicrobial peptide subtilosin against Listeria monocytogenes Scott A
Kuijk, S.J.A. van; Noll, K.S. ; Chikindas, M.L. - \ 2012
Letters in Applied Microbiology 54 (2012)1. - ISSN 0266-8254 - p. 52 - 58.
bacillus-subtilis - bacteriocins - antibiotics - mechanism - pathogen - growth - acid - ph
Aims: To elucidate the molecular mechanism of action of the antimicrobial peptide subtilosin against the foodborne pathogen Listeria monocytogenes Scott A. Methods and Results: Subtilosin was purified from a culture of Bacillus amylliquefaciens. The minimal inhibitory concentration of subtilosin against L. moilocytogenes Scott A was determined by broth microdilution method. The effect of subtilosin on the transmembrane electrical potential (Ali) and pH gradient (ApH), and its ability to induce efflux of intracellular ATP, was investigated. Subtilosin fully inhibited L. monocytogencs growth at a concentration of 19 fig Subtilosin caused a partial depletion of the AT and had a similar minor effect on the ApIL There was no significant efflux of intracellular ATP. Conclusion: Subtilosin likely acts upon L. monocytogencs Scott A by perturbing the lipid bilayer of the cellular membrane and causing intracellular damage, leading to eventual cell death. Subtilosin's mode of action against L. monocytogcues Scott A differs from the one previously described for another human path()gen, Cam dnerella vaginalis. Significance and Impact of the Study: This is the first report on the specific mode of action of subtilosin against L. monocytogenes and the first report of a bacteriocin with a species specific mode of action
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.
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
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.
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