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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Quantifying strain variability in modeling growth of Listeria monocytogenes
Aryani, D. ; Besten, H.M.W. den; Hazeleger, W.C. ; Zwietering, M.H. - \ 2015
International Journal of Food Microbiology 208 (2015). - ISSN 0168-1605 - p. 19 - 29.
lactic-acid concentration - minimal water activity - bacillus-cereus - escherichia-coli - microbial-growth - lag time - temperature - ph - fermentation - survival
Prediction of microbial growth kinetics can differ from the actual behavior of the target microorganisms. In the present study, the impact of strain variability on maximum specific growth rate (µmax) (h- 1) was quantified using twenty Listeria monocytogenes strains. The µmax was determined as function of four different variables, namely pH, water activity (aw)/NaCl concentration [NaCl], undissociated lactic acid concentration ([HA]), and temperature (T). The strain variability was compared to biological and experimental variabilities to determine their importance. The experiment was done in duplicate at the same time to quantify experimental variability and reproduced at least twice on different experimental days to quantify biological (reproduction) variability. For all variables, experimental variability was clearly lower than biological variability and strain variability; and remarkably, biological variability was similar to strain variability. Strain variability in cardinal growth parameters, namely pHmin, [NaCl]max, [HA]max, and Tmin was further investigated by fitting secondary growth models to the µmax data, including a modified secondary pH model. The fitting results showed that L. monocytogenes had an average pHmin of 4.5 (5–95% prediction interval (PI) 4.4–4.7), [NaCl]max of 2.0 mM (PI 1.8–2.1), [HA]max of 5.1 mM (PI 4.2–5.9), and Tmin of - 2.2 °C (PI (- 3.3)–(- 1.1)). The strain variability in cardinal growth parameters was benchmarked to available literature data, showing that the effect of strain variability explained around 1/3 or less of the variability found in literature. The cardinal growth parameters and their prediction intervals were used as input to illustrate the effect of strain variability on the growth of L. monocytogenes in food products with various characteristics, resulting in 2–4 log CFU/ml(g) difference in growth prediction between the most and least robust strains, depending on the type of food product. This underlined the importance to obtain quantitative knowledge on variability factors to realistically predict the microbial growth kinetics. Keywords: Quantitative microbiology; Growth parameters; Heterogeneity; Pathogen
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.
High yields of active Thermus thermophilus proline dehydrogenase are obtained using maltose-binding protein as a solubility tag.
Huijbers, M.M.E. ; Berkel, W.J.H. van - \ 2015
Biotechnology Journal 10 (2015)3. - ISSN 1860-6768 - p. 395 - 403.
multifunctional puta flavoprotein - escherichia-coli - purification - domain - stress - biosynthesis - oxidase - crystallization - overexpression - identification
Proline dehydrogenase (ProDH) catalyzes the FAD-dependent oxidation of proline to ¿1-pyrroline-5-carboxylate, the first step of proline catabolism in many organisms. Next to being involved in a number of physiological processes, ProDH is of interest for practical applications because the proline imino acid can serve as a building block for a wide range of peptides and antibiotics. ProDH is a membrane-associated protein and recombinant soluble forms of the enzyme have only been obtained in limited amounts. We here report on the heterologous production of ProDH from Thermus thermophilus (TtProDH) in Escherichia coli. Using maltose-binding protein as solubility tag, high yields of active holoenzyme are obtained. Native TtProDH can be produced from cleaving the purified fusion protein with trypsin. Size-exclusion chromatography shows that fused and clipped TtProDH form oligomers. Thermal stability and co-solvent tolerance indicate the conformational robustness of TtProDH. These properties together with the high yield make TtProDH attractive for industrial applications.
Bacillus cereus ATCC 14579 RpoN (Sigma 54) is a Pleiotropic Regulator of Growth, Carbohydrate, Metabolism, Motility, Biofilm Formation and Toxin Production
Hayrapetyan, H. ; Tempelaars, M.H. ; Nierop Groot, M.N. ; Abee, T. - \ 2015
PLoS One 10 (2015)8. - ISSN 1932-6203 - 19 p.
listeria-monocytogenes - escherichia-coli - sigma(54)-dependent genes - transcriptional activator - enterococcus-faecalis - mesentericin y105 - microarray data - pts permease - subtilis - thuringiensis
Sigma 54 is a transcriptional regulator predicted to play a role in physical interaction of bacteria with their environment, including virulence and biofilm formation. In order to study the role of Sigma 54 in Bacillus cereus, a comparative transcriptome and phenotypic study was performed using B. cereus ATCC 14579 WT, a markerless rpoN deletion mutant, and its complemented strain. The mutant was impaired in many different cellular functions including low temperature and anaerobic growth, carbohydrate metabolism, sporulation and toxin production. Additionally, the mutant showed lack of motility and biofilm formation at air-liquid interphase, and this correlated with absence of flagella, as flagella staining showed only WT and complemented strain to be highly flagellated. Comparative transcriptome analysis of cells harvested at selected time points during growth in aerated and static conditions in BHI revealed large differences in gene expression associated with loss of phenotypes, including significant down regulation of genes in the mutant encoding enzymes involved in degradation of branched chain amino acids, carbohydrate transport and metabolism, flagella synthesis and virulence factors. Our study provides evidence for a pleiotropic role of Sigma 54 in B. cereus supporting its adaptive response and survival in a range of conditions and environments.
Negative Epistasis and Evolvability in TEM-1 ß-Lactamase- The Thin Line between an Enzyme's Conformational Freedom and Disorder
Dellus-Gur, E. ; Elias, M. ; Caselli, E. ; Prati, F. ; Salverda, M.L.M. ; Visser, J.A.G.M. de; Fraser, J.S. ; Tawfik, D.S. - \ 2015
Journal of Molecular Biology 427 (2015)14. - ISSN 0022-2836 - p. 2396 - 2409.
empirical fitness landscapes - antibiotic-resistance - protein structures - escherichia-coli - substrate-specificity - sequence space - sign epistasis - omega-loop - trade-offs - evolution
Epistasis is a key factor in evolution since it determines which combinations of mutations provide adaptive solutions and which mutational pathways toward these solutions are accessible by natural selection. There is growing evidence for the pervasiveness of sign epistasis—a complete reversion of mutational effects, particularly in protein evolution—yet its molecular basis remains poorly understood. We describe the structural basis of sign epistasis between G238S and R164S, two adaptive mutations in TEM-1 ß-lactamase— an enzyme that endows antibiotics resistance. Separated by 10 Å, these mutations initiate two separate trajectories toward increased hydrolysis rates and resistance toward second and third-generation cephalosporins antibiotics. Both mutations allow the enzyme's active site to adopt alternative conformations and accommodate the new antibiotics. By solving the corresponding set of crystal structures, we found that R164S causes local disorder whereas G238S induces discrete conformations. When combined, the mutations in 238 and 164 induce local disorder whereby nonproductive conformations that perturb the enzyme's catalytic preorganization dominate. Specifically, Asn170 that coordinates the deacylating water molecule is misaligned, in both the free form and the inhibitor-bound double mutant. This local disorder is not restored by stabilizing global suppressor mutations and thus leads to an evolutionary cul-de-sac. Conformational dynamism therefore underlines the reshaping potential of protein's structures and functions but also limits protein evolvability because of the fragility of the interactions networks that maintain protein structures
Dynamic Response of Pseudomonas putida S12 to Sudden Addition of Toluene and the Potential Role of the Solvent Tolerance Gene trgI
Volkers, R.J.M. ; Snoek, L.B. ; Ruijssenaars, H.J. ; Winde, J.H. de - \ 2015
PLoS One 10 (2015)7. - ISSN 1932-6203
organic-solvents - molecular characterization - transcriptional regulator - escherichia-coli - fatty-acid - bacteria - stress - metabolism - aeruginosa - efflux
Pseudomonas putida S12 is exceptionally tolerant to various organic solvents. To obtain further insight into this bacterium’s primary defence mechanisms towards these potentially harmful substances, we studied its genome wide transcriptional response to sudden addition of toluene. Global gene expression profiles were monitored for 30 minutes after toluene addition. During toluene exposure, high oxygen-affinity cytochrome c oxidase is specifically expressed to provide for an adequate proton gradient supporting solvent efflux mechanisms. Concomitantly, the glyoxylate bypass route was up-regulated, to repair an apparent toluene stress-induced redox imbalance. A knock-out mutant of trgI, a recently identified toluene-repressed gene, was investigated in order to identify TrgI function. Remarkably, upon addition of toluene the number of differentially expressed genes initially was much lower in the trgI-mutant than in the wild-type strain. This suggested that after deletion of trgI cells were better prepared for sudden organic solvent stress. Before, as well as after, addition of toluene many genes of highly diverse functions were differentially expressed in trgI-mutant cells as compared to wild-type cells. This led to the hypothesis that TrgI may not only be involved in the modulation of solvent-elicited responses but in addition may affect basal expression levels of large groups of genes
Folding of influenza virus hemagglutinin in insect cells is fast and efficient
Li, X. ; Oers, M.M. van; Vlak, J.M. ; Braakman, I. - \ 2015
Journal of Biotechnology 203 (2015). - ISSN 0168-1656 - p. 77 - 83.
disulfide bond formation - endoplasmic-reticulum - quality-control - membrane glycoprotein - expression system - escherichia-coli - calnexin - vaccine - ph - oligomerization
Folding of influenza virus hemagglutinin (HA) in the endoplasmic reticulum has been well defined inmammalian cells. In different mammalian cell lines the protein follows the same folding pathway withidentical folding intermediates, but folds with very different kinetics. To examine the effect of cellularcontext on HA folding and to test to which extent insect cells would support the HA folding process,we expressed HA in Sf9 insect cells. Strikingly, in this invertebrate system HA folded faster and moreefficiently, still via the same folding intermediates as in vertebrate cells. Our results suggest that insectcells provide a highly efficient and effective folding environment for influenza virus HA and the idealproduction platform for HA (emergency) vaccines.
Two Distinct DNA Binding Modes Guide Dual Roles of a CRISPR-Cas Protein
Blosser, T.R. ; Loeff, L. ; Westra, E.R. ; Vlot, M. ; Künne, T.A. ; Sobota, M. ; Dekker, C. ; Brouns, S.J.J. ; Joo, C. - \ 2015
Molecular Cell 58 (2015)1. - ISSN 1097-2765 - p. 60 - 70.
bacterial immune-system - in-vitro reconstitution - memory b-cells - surveillance complex - crystal-structure - streptococcus-thermophilus - adaptive immunity - escherichia-coli - seed sequence - rna
Small RNA-guided protein complexes play an essential role in CRISPR-mediated immunity in prokaryotes. While these complexes initiate interference by flagging cognate invader DNA for destruction, recent evidence has implicated their involvement in new CRISPR memory formation, called priming, against mutated invader sequences. The mechanism by which the target recognition complex mediates these disparate responses-interference and priming-remains poorly understood. Using single-molecule FRET, we visualize how bona fide and mutated targets are differentially probed by E. coli Cascade. We observe that the recognition of bona fide targets is an ordered process that is tightly controlled for high fidelity. Mutated targets are recognized with low fidelity, which is featured by short-lived and PAM- and seed-independent binding by any segment of the crRNA. These dual roles of Cascade in immunity with distinct fidelities underpin CRISPR-Cas robustness, allowing for efficient degradation of bona fide targets and priming of mutated DNA targets
Limited evidence for trans-generational effects of maternal dietary supplementation with ¿-3 fatty acids on immunity in broiler chickens
Koppenol, A. ; Delezie, E. ; Parmentier, H.K. ; Buyse, J. ; Everaert, N. - \ 2015
The Veterinary Journal 203 (2015)2. - ISSN 1090-0233 - p. 244 - 249.
n-3 fatty-acids - red-blood-cells - antibody-responses - escherichia-coli - serum - system - immunoglobulin - proliferation - inflammation - performance
The aim of the present study was to investigate whether the immune response of broiler chickens is modulated by including different omega-3 (¿-3) polyunsaturated fatty acids (PUFAs) in the maternal diet. Broiler breeder hens (n¿=¿120 birds per group) were fed one of four diets, differing in the ratios of n-6:n-3 PUFAs and eicosapentaenoic acid (EPA):docosahexaenoic acid (DHA). At 28 weeks of age, the eggs produced were incubated to obtain 720 chicks (n¿=¿180 per group). All broiler chicks were fed a control diet and were vaccinated against Newcastle disease virus (NDV). Blood samples were taken at different time points after immunisation with human serum albumin (HuSA) in Freund's adjuvant to determine the acute phase response, antibody response and cytokine production. Addition of EPA to the maternal diet was associated with greater ovotransferrin concentrations post-immunisation, compared to other groups. Altering the ratios of n-6:n-3 PUFA or EPA:DHA in the maternal diet did not affect the offspring in terms of production of caeruloplasmin, a1-acid glycoprotein, interleukin (IL)-1ß, IL-6, IL-12 or tumour necrosis factor (TNF)-a. Dietary manipulation of the maternal diet did not influence the specific antibody response to HuSA or NDV, nor did it alter the levels of natural antibody binding to keyhole limpet haemocyanin in the offspring. Thus, maternal supplementation with n-3 PUFAs played a minor role in perinatal programming of the immune response of broiler chickens.
Quantifying variabiltiy on thermal resistance of Listeria monocytogenes
Chandra Aryani, D. ; Besten, H.M.W. den; Hazeleger, W.C. ; Zwietering, M.H. - \ 2015
International Journal of Food Microbiology 193 (2015). - ISSN 0168-1605 - p. 130 - 138.
heat-resistance - escherichia-coli - salmonella-typhimurium - inactivation parameters - physiological-state - bacillus-cereus - salt stress - shock - growth - acid
Knowledge of the impact of strain variability and growth history on thermal resistance is needed to provide a realistic prediction and an adequate design of thermal treatments. In the present study, apart from quantifying strain variability on thermal resistance of Listeria monocytogenes, also biological variability and experimental variability were determined to prioritize their importance. Experimental variability was defined as the repeatability of parallel experimental replicates and biological variability was defined as the reproducibility of biologically independent reproductions. Furthermore, the effect of growth history was quantified. The thermal inactivation curves of 20 L. monocytogenes strains were fitted using the modified Weibull model, resulting in total 360 D-value estimates. The D-value ranged from 9 to 30 min at 55 °C; from 0.6 to 4 min at 60 °C; and from 0.08 to 0.6 min at 65 °C. The estimated z-values of all strains ranged from 4.4 to 5.7 °C. The strain variability was ten times higher than the experimental variability and four times higher than the biological variability. Furthermore, the effect of growth history on thermal resistance variability was not significantly different from that of strain variability and was mainly determined by the growth phase.
Genomic, proteomic and biochemical analysis of the organohalide respiratory pathway in Desulfitobacterium dehalogenans
Kruse, T. ; Pas, B.A. van de; Atteia, A. ; Krab, K. ; Hagen, W.R. ; Goodwin, L. ; Chain, P. ; Boeren, S. ; Maphosa, F. ; Schraa, G. ; Vos, W.M. de; Oost, J. van der; Smidt, H. ; Stams, A.J.M. - \ 2015
Journal of Bacteriology 197 (2015)5. - ISSN 0021-9193 - p. 893 - 904.
tetrachloroethene reductive dehalogenase - geobacter-sulfurreducens am-1 - electron-transport - escherichia-coli - signal peptides - dehalococcoides-ethenogenes - wolinella-succinogenes - methacrylate reductase - rhodospirillum-rubrum - fumarate reductase
Desulfitobacterium dehalogenans is able to grow by organohalide respiration using 3-chloro-4-hydroxyphenyl acetate (Cl-OHPA) as an electron acceptor. We used a combination of genome sequencing, biochemical analysis of redox active components and shotgun proteomics to study elements of the organohalide respiratory electron transport chain. The genome of Desulfitobacterium dehalogenans JW/IU-DC1T consists of a single circular chromosome of 4321753 bp with a GC content of 44.97%. The genome encodes 4252 genes including six rRNA operons and six predicted reductive dehalogenases. One of the reductive dehalogenases, CprA, is encoded by a well characterised cprTKZEBACD gene cluster. Redox active components were identified in concentrated suspensions of cells grown on formate and Cl-OHPA or formate and fumarate, using electron-paramagnetic-resonance (EPR), visible spectroscopy, and high-performance–liquid-chromatography (HPLC) analysis of membrane extracts. In cell suspensions, these components were reduced upon addition of formate and oxidized after addition of Cl-OHPA indicating involvement in organohalide respiration. Genome analysis revealed genes that likely encode the identified components of the electron transport chain from formate to fumarate or Cl-OHPA. Data presented here suggest that the first part of the electron transport chain from formate to fumarate or Cl-OHPA is shared. Electrons are channelled from an outward facing formate dehydrogenase via menaquinones to a fumarate reductase located at the cytoplasmic face of the membrane. When Cl-OHPA is the terminal electron acceptor, electrons are transferred from menaquinones to outward facing CprA, via an yet unidentified membrane complex, and potentially an extracellular flavoprotein acting as an electron shuttle between the quinol dehydrogenase membrane complex and CprA.
Production of inflammatory mediators and extracellular traps by carp macrophages and neutrophils in response to lipopolysaccharide and/or interferon-¿2
Pijanowski, L. ; Scheer, M.H. ; Verburg-van Kemenade, B.M.L. ; Chadzinska, M.K. - \ 2015
Fish and Shellfish Immunology 42 (2015)2. - ISSN 1050-4648 - p. 473 - 482.
toll-like receptors - cyprinus-carpio - signaling pathways - expression analysis - escherichia-coli - immune-response - lps recognition - gene-expression - fish - l.
Neutrophilic granulocytes and macrophages are crucial for the innate immune response against infections. They migrate into the focus of inflammation, where they efficiently bind, engulf and kill bacteria by proteolytic enzymes, antimicrobial peptides, reactive oxygen (ROS) and nitrogen (RNS) species. Moreover, activated neutrophils and macrophages can form extracellular traps (ETs). Fish neutrophils and macrophages are morphologically, histochemically, and functionally similar to their mammalian counterparts, but their significance for regulation of inflammatory responses and pathogen killing needs further elucidation. We compared the activity of head kidney monocytes/macrophages and neutrophilic granulocytes of common carp and established that upon lipopolysaccharide stimulation, not only neutrophils, but also carp monocytes/macrophages release extracellular DNA and are capable to form macrophage extracellular traps (METs). To clarify whether many specific LPS functions reported for piscine phagocytes might be due to impurities in the commonly used LPS preparations we studied expression of inflammatory mediators, release of DNA, ROS and RNS in cells stimulated with LPS or its highly purified form (pLPS). Also IFN-¿2 stimulation and its synergism with LPS/pLPS in stimulating expression of pro-inflammatory mediators was studied. Results substantiate that a classical stimulation of TLR4 by LPS may indeed be absent in carp as most of the classically reported LPS effects are abolished or diminished when pLPS is used. Interestingly, we also observed a potent IL-10 expression in neutrophilic granulocytes upon LPS stimulation, which, apart from their pro-inflammatory function, clearly indicates a role in restrictive control of the inflammatory reaction.
Baculovirus-induced tree-top disease: how extended is the role of egt as a gene for the extended phenotype?
Ros, V.I.D. ; Houte, S. van; Hemerik, L. ; Oers, M.M. van - \ 2015
Molecular Ecology 24 (2015)1. - ISSN 0962-1083 - p. 249 - 258.
spodoptera-exigua larvae - udp-glucosyl transferase - escherichia-coli - lepidopteran host - trichoplusia-ni - deletion - nucleopolyhedrovirus - behavior - insect - infection
Many parasites alter host behaviour to enhance their chance of transmission. Recently, the ecdysteroid UDP-glucosyl transferase (egt) gene from the baculovirus Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV) was identified to induce tree-top disease in L. dispar larvae. Infected gypsy moth larvae died at elevated positions (hence the term tree-top disease), which is thought to promote dissemination of the virus to lower foliage. It is, however, unknown whether egt has a conserved role among baculoviruses in inducing tree-top disease. Here, we studied tree-top disease induced by the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) in two different host insects, Trichoplusia ni and Spodoptera exigua, and we investigated the role of the viral egt gene therein. AcMNPV induced tree-top disease in both T. ni and S. exigua larvae, although in S. exigua a moulting-dependent effect was seen. Those S. exigua larvae undergoing a larval moult during the infection process died at elevated positions, while larvae that did not moult after infection died at low positions. For both T. ni and S. exigua, infection with a mutant AcMNPV lacking egt did not change the position where the larvae died. We conclude that egt has no highly conserved role in inducing tree-top disease in lepidopteran larvae. The conclusion that egt is a ‘gene for an extended phenotype’ is therefore not generally applicable for all baculovirus–host interactions. We hypothesize that in some baculovirus–host systems (including LdMNPV in L. dispar), an effect of egt on tree-top disease can be observed through indirect effects of egt on moulting-related climbing behaviour.
The Rsm regulon of plant growth-promoting Pseudomonas fluorescens SS101: role of small RNAs in regulation of lipopeptide biosynthesis
Song, C. ; Voort, M. van der; Mortel, J. van de; Hassan, K.A. ; Elbourne, L.D.H. ; Paulsen, I.T. ; Loper, J.E. ; Raaijmakers, J.M. - \ 2015
Microbial Biotechnology 8 (2015)2. - ISSN 1751-7907 - p. 296 - 310.
signal-transduction pathway - escherichia-coli - phytophthora-infestans - bacterial genomes - soluble-rnas - fumarase c - aeruginosa - identification - cha0 - recognition
The rhizobacterium Pseudomonas fluorescens SS101 inhibits growth of oomycete and fungal pathogens, and induces resistance in plants against pathogens and insects. To unravel regulatory pathways of secondary metabolite production in SS101, we conducted a genome-wide search for sRNAs and performed transcriptomic analyses to identify genes associated with the Rsm (repressor of secondary metabolites) regulon. In silico analysis led to the identification of 16 putative sRNAs in the SS101 genome. In frame deletion of the sRNAs rsmY and rsmZ showed that the Rsm system regulates the biosynthesis of the lipopeptide massetolide A and involves the two repressor proteins RsmA and RsmE, with the LuxR-type transcriptional regulator MassAR as their most likely target. Transcriptome analyses of the rsmYZ mutant further revealed that genes associated with iron acquisition, motility and chemotaxis were significantly upregulated, whereas genes of the type VI secretion system were downregulated. Comparative transcriptomic analyses showed that most, but not all, of the genes controlled by RsmY/RsmZ are also controlled by the GacS/GacA two-component system. We conclude that the Rsm regulon of P.¿fluorescens SS101 plays a critical role in the regulation of lipopeptide biosynthesis and controls the expression of other genes involved in motility, competition and survival in the plant rhizosphere.
Bacterial Histidine Kinases as Novel Antibacterial Drug Targets
Bem, A.E. ; Velikova, N.R. ; Pellicer, M.T. ; Baarlen, P. van; Marina, A. ; Wells, J.M. - \ 2015
Acs Chemical Biology 10 (2015)1. - ISSN 1554-8929 - p. 213 - 224.
2-component signal-transduction - structure-based discovery - staphylococcus-aureus - mycobacterium-tuberculosis - escherichia-coli - multidrug-resistance - response regulator - vancomycin resistance - antibiotic-resistance - streptococcus-mutans
Bacterial histidine kinases (HKs) are promising targets for novel antibacterials. Bacterial HKs are part of bacterial two-component systems (TCSs), the main signal transduction pathways in bacteria, regulating various processes including virulence, secretion systems and antibiotic resistance. In this review, we discuss the biological importance of TCSs and bacterial HKs for the discovery of novel antibacterials, as well as published TCS and HK inhibitors that can be used as a starting point for structure-based approaches to develop novel antibacterials.
Physiological and cell morphology adaptation of Bacillus subtilis at near-zero specific growth rates: a transcriptome analysis
Overkamp, W. ; Ercan, O. ; Herber, M. ; Maris, A.J. van; Kleerebezem, M. ; Kuipers, O.P. - \ 2015
Environmental Microbiology 17 (2015)2. - ISSN 1462-2912 - p. 346 - 363.
stationary-phase mutagenesis - limiting bacterial-growth - general stress-response - gram-positive bacteria - escherichia-coli - slow growth - stringent response - maintenance energy - continuous-culture - population heterogeneity
Nutrient scarcity is a common condition in nature, but the resulting extremely low growth rates (below 0.025 h-1) are an unexplored research area in B. subtilis. To understand microbial life in natural environments, studying the adaptation of B. subtilis to near-zero growth conditions is relevant. To this end, a chemostat modified for culturing an asporogenous B. subtilis sigF mutant strain at extremely low growth rates (also named a retentostat) was set up and biomass accumulation, culture viability, metabolite production and cell morphology were analysed. During retentostat culturing the specific growth rate decreased to a minimum of 0.00006 h-1, corresponding to a doubling time of 470 days. The energy distribution between growth- and maintenance-related processes showed that a state of near-zero growth was reached. Remarkably, a filamentous cell morphology emerged, suggesting that cell separation is impaired under near-zero growth conditions. To evaluate the corresponding molecular adaptations to extremely low specific growth, transcriptome changes were analysed. These revealed that cellular responses to near-zero growth conditions share several similarities with those of cells during the stationary phase of batch-growth. However, fundamental differences between these two non-growing states are apparent by their high viability and absence of stationary phase mutagenesis under near-zero growth conditions.
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.
Performance assessment of food safety management systems in animal-based food companies in view of their context characteristics: A European study
Luning, P.A. ; Kirezieva, K. ; Hagelaar, G. ; Rovira, J. ; Uyttendaele, M. ; Jacxsens, L. - \ 2015
Food Control 49 (2015). - ISSN 0956-7135 - p. 11 - 22.
critical control point - hazard analysis - processing plants - united-states - microbial-contamination - haccp implementation - escherichia-coli - pasteurized milk - quality - meat
Recurrently the question arises if efforts in food safety management system (FSMS) have resulted in effective systems in animal-based food production systems. The aim of this study was to gain an insight in the performance of FSMS in European animal-based food production companies in view of their typical context characteristics. Hundred European companies (from Belgium, Spain, The Netherlands, Greece, Italy, and Hungary) varying in company size, and producing different types of fresh and processed animal-derived products (dairy, beef/lamb, poultry, and pork) were assed using a diagnostic instrument. Results indicated that most participating companies adapted adequately their food safety management systems to the riskiness of their context characteristics resulting in rather good safety output scores. Only a small group have overall basic systems and operate in a moderate or moderate-high risk context, which was reflected in lower safety output scores. Companies tend to invest first in the control strategies whereas assurance activities such as verification and validation seem to require more time and effort to achieve advanced levels. Our study demonstrated that also small and medium enterprises managed to have advanced systems, and achieve a good safety output. However, their typical organisational characteristics such as less resources (educated staff, laboratory facilities, time), more restricted formalisation (restricted use of procedures and formal meetings), limited information systems, but more stable workforce, might require more tailored support from government and/or branch organisations to develop towards advanced systems in the case of high-risk products and processes. More in-depth studies to successful SMEs could give insight in best practices to improve FSMS performance.
Thirty years of baculovirus-insect cell protein expression: From dark horse to mainstream technology
Oers, M.M. van; Pijlman, G.P. ; Vlak, J.M. - \ 2015
Journal of General Virology 96 (2015)1. - ISSN 0022-1317 - p. 6 - 23.
nuclear polyhedrosis-virus - late gene-expression - human fibroblast interferon - n-glycosylation pathway - large-scale production - non-hr origin - spodoptera-frugiperda - escherichia-coli - messenger-rna - mammalian-cells
In December 1983 a seminal paper appeared on the overexpression of human interferon-ß in insect cells with a genetically engineered baculovirus. The finding that baculoviruses produce massive amounts of two proteins (polyhedrin and p10) by means of two very strong promoters and that the corresponding genes are dispensable for virus propagation in insect cells was crucial in the development of this expression system. During the next 30 years major improvements have been achieved over the original baculovirus expression vector (BEV) system, facilitating the engineering of the baculovirus vectors, the modification of the sugar moieties of glycoproteins expressed in insect cells, and the scale-up of the cell culture process. To date, thousands of recombinant proteins have been produced in this successful expression system, including several protein-based human and veterinary vaccines that are currently on the market. Viral vectors based on adeno-associated virus are being produced using recombinant baculovirus technology and the first gene therapy treatment based on this method has been registered. Specially adapted baculovirus expression vectors are used to deliver and express heterologous genes in mammalian cells and may find applications for gene therapy and cancer treatment in the future. The purpose of this paper is to highlight the 30-years 'anniversary' of this expression system by summarizing the fundamental research that allowed the development of this expression system and by indicating the major technological advances since 1983. Finally, attention will be paid to the future challenges to further optimize this amazing technology.
Mode of action of Bacillus licheniformis pectin methylesterase on highly methylesterified and acetylated pectins
Remoroza, C.A. ; Wagenknecht, M. ; Buchholt, H.C. ; Moerschbacher, B.M. ; Gruppen, H. ; Schols, H.A. - \ 2015
Carbohydrate Polymers 115 (2015). - ISSN 0144-8617 - p. 540 - 550.
sugar-beet pectins - aspergillus-niger - esterified oligogalacturonides - environmental-conditions - structural features - de-esterification - escherichia-coli - methyl esterase - degradation - calcium
A gene encoding a putative pectinesterase from Bacillus licheniformis DSM13 was cloned and expressed in Escherichia coli. The resulting recombinant enzyme (BliPME) was purified and characterized as a pectin methylesterase. The enzyme showed maximum activity at pH 8.0 and 50 °C. BliPME is able to release up to 100% of the methylesters from lime pectin (DM 34–76 ¿ DM 0) and up to 73% of all methylesters from SBPs (DM 30–73 ¿ DM 14). BliPME efficiently de-methylesterifies lemon pectins and SBPs in a blockwise manner and is quite tolerant towards the acetyl groups present within the SBPs. Detailed analysis of the BliPME-modified pectins using HILIC–MSn and the classical calcium reactivity measurement showed that the enzyme generates pectins with low methylesterification (lime and SBP) and high acetyl content (SBP) while creating blocks of nonmethylesterified galacturonic acid residues. The high activity of BliPME towards highly methylesterified and acetylated pectins makes this novel esterase more efficient in removing methylesters from highly esterified beet pectin compared to other PMEs, e.g. Aspergillus niger PME.
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