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.

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Bridging domains : a comparison between information processing in Archaea and Eukarya
Koning, B. de - \ 2015
University. Promotor(en): John van der Oost, co-promotor(en): Stan Brouns. - Wageningen : Wageningen University - ISBN 9789462572379 - 154
archaea - transfer rna - rna-polymerase - transcriptieregulatie - biosynthese - sulfolobus solfataricus - rna polymerase - regulation of transcription - biosynthesis

Bridging Domains

A Comparison between Information Processing in Archaea and Eukarya

Studying Information Processing

Living cells evolved complex systems to handle the flow of information both accurately and efficiently. These systems are highly comparable between the three domains of life: eukaryotes, bacteria and archaea. The central components of replication, transcription, aminoacylation, and translation are found in every living cell known today, with only relatively small deviations, despite a separation of billions of years of evolution. Archaea are unicellular, do not contain organelles, and have relatively small genomes, so are, at first sight, quite similar to their far better known prokaryotic cousins: the bacteria. Nevertheless, if it comes down to information processing, archaea are, surprisingly, more related to eukaryotes than to bacteria, both at the sequence level of RNA and proteins, and at the architecture level of key complexes as well. This makes them excellent model systems to study eukaryote-like information processing. The absence of cell specialization, less cell organization, less or even no intracellular compartmentalization, and less intensive regulation, have proven to give a clearer picture of the function of conserved key elements within these complex systems. [Chapter 2]

In this thesis, we report several attempts to elucidate functional details of some very conserved factors in information processing in S. solfataricus using recently established genetic modification techniques. S. solfataricus is a thermoacidophilic crenarchaeote that grows optimally at temperatures between 70°C and 85°C and at pH values between 2 and 3. Its genome sequence is known since 2001. Best practices have become standardized between laboratories, and the genomic toolbox includes gene knockout, overexpression systems, the availability of reporter genes, and tunable promoters.

MBF1, a highly conserved activator

MBF1 (multi-protein bridging factor 1) is reported to be a transcriptional co-activator in eukaryotes. It was shown to cross the gap between transcription regulators and the transcriptional machinery itself. MBF1 was found to be highly conserved within archaea, being present in almost all species with the key exception of marine thaumarchaeotes. However, none of the associated transcription regulators were known to be present within the archaeal domain, raising the question whether a class of other regulators was overlooked, or that archaeal MBF1 might be a transcriptional activator itself, binding to DNA directly instead of indirectly via a binding partner. Additionally one study revealed a surprising dual role of this protein: in yeast it was not only associated with transcription but contributed to translation fidelity as well. A neighbourhood analysis across the archaeal domain revealed no clear preference for either transcription or translation. Elements of both systems are equally present, especially in the well conserved neighbourhood within the crenarchaeotes. [Chapter 3]

A mbf1 disruption mutant of the S. solfataricus was made using heterozygous recombination with a suicide plasmid. Under standard laboratory growth conditions mbf1 appears to be not essential for growth, and comparing growth characteristics with its parental strain did not reveal striking differences between the two. It was observed, that the Sulfolobus mbf1 disruption mutant is much more sensitive during cultivation than its parental strain, showing sudden death during growth much more often. Being hard to quantify, this behaviour was especially observed when cultures were transferred at later stages during stationary phase or unfrozen from long term storage. But the largest difference was observed in the increased sensitivity of the mbf1 disruption mutant towards paromomycin. Paromomycin is an aminoglycoside-type antibiotic that interferes with the recognition of cognate codon-anti-codon binding within the ribosomes during translation. [Chapter 4]

A more detailed study to the molecular characteristics of the archaeal MBF1 from S. solfataricus revealed hardly any associations to the transcription machinery, but strengthened the assumed association to the translation apparatus. It was found that archaeal MBF1 consists of two domains that are structurally independent: an N-terminal zinc-ribbon, which is not conserved beyond the archaeal MBF1s, and the well conserved C-terminal HTH-domain (helix-turn-helix domain). This C-terminal HTH domain was shown to bind to the small ribosomal subunit by affinity purification, and in co-purification experiments, in which we detected the presence of archaeal MBF1 in ribosomal purifications. NMR structure comparisons confirmed that archaeal MBF1 binds to the small ribosomal subunit using its C-terminal HTH domain, whereas the N-terminal zinc-ribbon might only contribute to this interaction, but does not participate directly in binding. [Chapter 5]

Altogether, these findings made us believe that MBF1s in archaea are not associated with transcription but rather with translation. Based on the observations in yeast, and more recently its binding to polyadenylated mRNAs in different eukaryotic species, and, against the backdrop that the protein domain that binds to the small ribosomal subunit in S. solfataricus is highly conserved across the archaeao-eukaryotic lineage, it is tempting to speculate that the eukaryotic MBF1 plays a comparable role in the translation process in eukaryotes as well.

TGT, a conserved dichotomy

Another well conserved element within all three domains of life, which is involved in information processing, is the TGT (tRNA-guanine transglycosylase) family of proteins. This family of proteins shows a clear dichotomy: TGT is responsible for the exchange of guanine at the wobble position (position 34) of the anti-codon of certain tRNAs with either queuosine in eukaryotes or its precursor preQ1 in bacteria, whereas, in archaea, TGT is responsible for the exchange of guanine with preQ0 at position 15 in almost, if not all, archaeal tRNAs. PreQ0 is in a later stage converted to archaeosine by another protein that belongs to the TGT family as well.

Disruption of the tgt gene, which encodes the TGT protein in S. solfataricus, revealed that it was solely responsible for this process without any redundancy present. Like mbf1, this gene appeared to be non-essential, as this mutant was also as viable as its parental strain, and showed hardly any changes in growth characteristics. In comparison to the mbf1 disruption mutant, the tgt disruption mutant was much more stable and did not reveal the sensitivity to stationary phase. It grew slightly slower than the parental strain, especially at normal temperatures (75°C), but when temperature levels were raised (87-93°C) growth returned to almost wild-type levels. [Chapter 6]

Aiding research to the basal machinery of RNAP

Beyond doubt, the best studied, element of information processing systems is the RNAP (RNA polymerase) complex. Its basal core is present in all known life forms, and is highly conserved. The surrounding, auxiliary, and regulatory elements are less conserved, but, nevertheless, the archaeal RNAP is almost identical to the eukaryotic RNAP II complex (see figure). This high resemblance already proved beneficial, as the heterologous expression of the archaeal RNAP revealed numerous functional details about the molecular characteristics of the complex as a whole, and, in addition, revealed also an unprecedented insight in the separate subunits as this provided opportunities to tamper with the subunit composition and to modify the separate subunits themselves by introducing genetic variations.

Unfortunately, purification of homologously expressed complexes, which are expressed in archaeal systems itself, are, in contrast to ones heterologously expressed in bacterial hosts, hard to obtain, and involve a number of purification steps and therefore a substantial amount of biomass. To enable easier purification, a method was developed in which a purification tag was inserted in the genome of S. solfataricus after a gene that encodes an RNAP subunit, avoiding artificial overproduction by viral infections or heterologous expression in other less adapted hosts. In a proof of principle experiment, the enrichment an RNAP core component was proven, whereas an auxiliary element was tagged using this novel method. [Chapter 7]

Alternating-laser excitation: single-molecule FRET and beyond
Hohlbein, J.C. ; Craggs, T.D. ; Cordes, T. - \ 2014
Chemical Society Reviews 43 (2014). - ISSN 0306-0012 - p. 1156 - 1171.
resonance energy-transfer - multiparameter fluorescence detection - probability-distribution analysis - pulsed interleaved excitation - dna-polymerase-i - photon distribution analysis - nano-positioning system - promoter open complex - rna-polymerase - conformatio
The alternating-laser excitation (ALEX) scheme continues to expand the possibilities of fluorescence-based assays to study biological entities and interactions. Especially the combination of ALEX and single-molecule Förster Resonance Energy Transfer (smFRET) has been very successful as ALEX enables the sorting of fluorescently labelled species based on the number and type of fluorophores present. ALEX also provides a convenient way of accessing the correction factors necessary for determining accurate molecular distances. Here, we provide a comprehensive overview of the concept and current applications of ALEX and we explicitly discuss how to obtain fully corrected distance information across the entire FRET range. We also present new ideas for applications of ALEX which will push the limits of smFRET-based experiments in terms of temporal and spatial resolution for the study of complex biological systems.
Genome-Wide Prediction and Validation of Sigma70 Promoters in Lactobacillus plantarum WCFS1
Todt, T.J. ; Wels, M. ; Bongers, R.S. ; Siezen, R.J. ; Hijum, S.A.F.T. van; Kleerebezem, M. - \ 2012
PLoS One 7 (2012)9. - ISSN 1932-6203
transcription start sites - escherichia-coli - rna-polymerase - sequences - identification - dna - conservation - compilation - metabolism - initiation
Background - In prokaryotes, sigma factors are essential for directing the transcription machinery towards promoters. Various sigma factors have been described that recognize, and bind to specific DNA sequence motifs in promoter sequences. The canonical sigma factor s70 is commonly involved in transcription of the cell's housekeeping genes, which is mediated by the conserved s70 promoter sequence motifs. In this study the s70-promoter sequences in Lactobacillus plantarum WCFS1 were predicted using a genome-wide analysis. The accuracy of the transcriptionally-active part of this promoter prediction was subsequently evaluated by correlating locations of predicted promoters with transcription start sites inferred from the 5'-ends of transcripts detected by high-resolution tiling array transcriptome datasets. Results - To identify s70-related promoter sequences, we performed a genome-wide sequence motif scan of the L. plantarum WCFS1 genome focussing on the regions upstream of protein-encoding genes. We obtained several highly conserved motifs including those resembling the conserved s70-promoter consensus. Position weight matrices-based models of the recovered s70-promoter sequence motif were employed to identify 3874 motifs with significant similarity (p-value
CRISPR immunity relies on the consecutive binding and degradation of negatively supercoiled invader DNA by Cascade and Cas3
Westra, E.R. ; Erp, P.B.G. ; Künne, T. ; Wong, S.P. ; Staals, R.H.J. ; Seegers, C.L.C. ; Bollen, S. ; Jore, M.M. ; Semenova, E. ; Severinov, K. ; Vos, W.M. de; Dame, R.T. ; Vries, R. de; Brouns, S.J.J. ; Oost, J. van der - \ 2012
Molecular Cell 46 (2012)5. - ISSN 1097-2765 - p. 595 - 605.
rna-polymerase - complex - prokaryotes - mechanism - protein - bacteriophage - resistance - sequence - defense - system
The prokaryotic CRISPR/Cas immune system is based on genomic loci that contain incorporated sequence tags from viruses and plasmids. Using small guide RNA molecules, these sequences act as a memory to reject returning invaders. Both the Cascade ribonucleoprotein complex and the Cas3 nuclease/helicase are required for CRISPR interference in Escherichia coli, but it is unknown how natural target DNA molecules are recognized and neutralized by their combined action. Here we show that Cascade efficiently locates target sequences in negatively supercoiled DNA, but only if these are flanked by a protospacer-adjacent motif (PAM). PAM recognition by Cascade exclusively involves the crRNA-complementary DNA strand. After Cascade-mediated R loop formation, the Cse1 subunit recruits Cas3, which catalyzes nicking of target DNA through its HD-nuclease domain. The target is then progressively unwound and cleaved by the joint ATP-dependent helicase activity and Mg(2+)-dependent HD-nuclease activity of Cas3, leading to complete target DNA degradation and invader neutralization
Creation of a Nonspreading Rift Valley Fever Virus
Kortekaas, J.A. ; Oreshkova, N.D. ; Cobos-Jeménez, V. ; Vloet, R.P.M. ; Potgieter, C. ; Moormann, R.J.M. - \ 2011
Journal of Virology 85 (2011)23. - ISSN 0022-538X - p. 12622 - 12630.
north-american mosquitos - mammalian-cells - rna-polymerase - expression - rescue - vaccine - protein - particles - cdna - gene
Rift Valley fever virus (RVFV) is a mosquito-borne zoonotic bunyavirus of the genus Phlebovirus and a serious human and veterinary pathogen. RVFV contains a three-segmented RNA genome, which is comprised of the large (L), medium (M), and small (S) segments. The proteins that are essential for genome replication are encoded by the L and S segments, whereas the structural glycoproteins are encoded by the M segment. We have produced BHK replicon cell lines (BHK-Rep) that maintain replicating L and S genome segments. Transfection of BHK-Rep cells with a plasmid encoding the structural glycoproteins results in the efficient production of RVFV replicon particles (RRPs). To facilitate monitoring of infection, the NSs gene was replaced with an enhanced green fluorescent protein gene. RRPs are infectious for both mammalian and insect cells but are incapable of autonomous spreading, rendering their application outside biosafety containment completely safe. We demonstrate that a single intramuscular vaccination with RRPs protects mice from a lethal dose of RVFV and show that RRPs can be used for rapid virus neutralization tests that do not require biocontainment facilities. The methods reported here will greatly facilitate vaccine and drug development as well as fundamental studies on RVFV biology. Moreover, it may be possible to develop similar systems for other members of the bunyavirus family as well.
The logicome of environmental bacteria: merging catabolic and regulatory events with Boolean formalisms
Silva-Rocha, R. ; Tamames, J. ; Martins Dos Santos, V.A.P. ; Lorenzo, V. de - \ 2011
Environmental Microbiology 13 (2011)9. - ISSN 1462-2912 - p. 2389 - 2402.
pseudomonas-putida kt2440 - integration host factor - sigma(54)-dependent promoter pu - scale metabolic reconstruction - upper-pathway operon - tol plasmid pww0 - aromatic-compounds - transcriptional regulation - escherichia-coli - rna-polymerase
The regulatory and metabolic networks that rule biodegradation of pollutants by environmental bacteria are wired to the rest of the cellular physiology through both transcriptional factors and intermediary signal molecules. In this review, we examine some formalisms for describing catalytic/regulatory circuits of this sort and advocate the adoption of Boolean logic for combining transcriptional and enzymatic occurrences in the same biological system. As an example, we show how known regulatory and metabolic actions that bring about biodegradation of m-xylene by Pseudomonas putida mt-2 can be represented as clusters of binary operations and then reconstructed as a digital network. Despite the many simplifications, Boolean tools still capture the gross behaviour of the system even in the absence of kinetic constants determined experimentally. On this basis, we argue that still with a limited volume of data binary formalisms allow us to penetrate the raison d'être of extant regulatory and metabolic architectures
Linking genes to microbial growth kinetics: an integrated biochemical systems engineering approach
Koutinas, M. ; Kiparissides, A. ; Silva-Rocha, R. ; Lam, M.C. ; Martins Dos Santos, V.A.P. ; Lorenzo, V. de; Pistikopoulos, E.N. ; Mantalaris, A. - \ 2011
Metabolic Engineering 13 (2011)4. - ISSN 1096-7176 - p. 401 - 413.
pseudomonas tol plasmid - escherichia-coli - rna-polymerase - regulatory networks - pu promoter - putida mt-2 - host factor - in-vivo - transcription - activation
The majority of models describing the kinetic properties of a microorganism for a given substrate are unstructured and empirical. They are formulated in this manner so that the complex mechanism of cell growth is simplified. Herein, a novel approach for modelling microbial growth kinetics is proposed, linking biomass growth and substrate consumption rates to the gene regulatory programmes that control these processes. A dynamic model of the TOL (pWW0) plasmid of Pseudomonas putida mt-2 has been developed, describing the molecular interactions that lead to the transcription of the upper and meta operons, known to produce the enzymes for the oxidative catabolism of m-xylene. The genetic circuit model was combined with a growth kinetic model decoupling biomass growth and substrate consumption rates, which are expressed as independent functions of the rate-limiting enzymes produced by the operons. Estimation of model parameters and validation of the model's predictive capability were successfully performed in batch cultures of mt-2 fed with different concentrations of m-xylene, as confirmed by relative mRNA concentration measurements of the promoters encoded in TOL. The growth formation and substrate utilisation patterns could not be accurately described by traditional Monod-type models for a wide range of conditions, demonstrating the critical importance of gene regulation for the development of advanced models closely predicting complex bioprocesses. In contrast, the proposed strategy, which utilises quantitative information pertaining to upstream molecular events that control the production of rate-limiting enzymes, predicts the catabolism of a substrate and biomass formation and could be of central importance for the design of optimal bioprocesses
Passaging of a Newcastle disease virus pigeon variant in chickens results in selection of viruses with mutations in the polymerase complex enhancing virus replication and virulence
Dortmans, J.C.F.M. ; Rottier, P.J.M. ; Koch, G. ; Peeters, B.P.H. - \ 2011
Journal of General Virology 92 (2011)2. - ISSN 0022-1317 - p. 336 - 345.
avian paramyxovirus type-1 - fusion protein - great-britain - cleavage site - influenza-virus - molecular-basis - rna-polymerase - pmv-1 viruses - pathogenicity - host
Some Newcastle disease virus (NDV) variants isolated from pigeons (pigeon paramyxovirus type 1; PPMV-1) do not show their full virulence potential for domestic chickens but may become virulent upon spread in these animals. In this study we examined the molecular changes responsible for this gain of virulence by passaging a low-pathogenic PPMV-1 isolate in chickens. Complete genome sequencing of virus obtained after 1, 3 and 5 passages showed the increase in virulence was not accompanied by changes in the fusion protein – a well known virulence determinant of NDV – but by mutations in the L and P replication proteins. The effect of these mutations on virulence was confirmed by means of reverse genetics using an infectious cDNA clone. Acquisition of three amino acid mutations, two in the L protein and one in the P protein, significantly increased virulence as determined by intracerebral pathogenicity index tests in day-old chickens. The mutations enhanced virus replication in vitro and in vivo and increased the plaque size in infected cell culture monolayers. Furthermore, they increased the activity of the viral replication complex as determined by an in vitro minigenome replication assay. Our data demonstrate that PPMV-1 replication in chickens results in mutations in the polymerase complex rather than the viral fusion protein, and that the virulence level of pigeon paramyxoviruses is directly related to the activity of the viral replication complex.
The regulatory logic of m-xylene biodegradation by Pseudomonas putida mt-2 exposed by dynamic modelling of the principal node Ps/Pr of the TOL plasmid
Koutinas, M. ; Lam, M.C. ; Kiparissides, A. ; Silva-Rocha, R. ; Godinho, M. ; Livingston, A.G. ; Pistikopoulos, E.N. ; Lorenzo, V. de; Martins Dos Santos, V.A.P. ; Mantalaris, A. - \ 2010
Environmental Microbiology 12 (2010)6. - ISSN 1462-2912 - p. 1705 - 1718.
global sensitivity-analysis - escherichia-coli - rna-polymerase - pu promoter - catabolite repression - transcription - xylr - activation - pathway - translation
P>The structure of the extant transcriptional control network of the TOL plasmid pWW0 born by Pseudomonas putida mt-2 for biodegradation of m-xylene is far more complex than one would consider necessary from a mere engineering point of view. In order to penetrate the underlying logic of such a network, which controls a major environmental cleanup bioprocess, we have developed a dynamic model of the key regulatory node formed by the Ps/Pr promoters of pWW0, where the clustering of control elements is maximal. The model layout was validated with batch cultures estimating parameter values and its predictive capability was confirmed with independent sets of experimental data. The model revealed how regulatory outputs originated in the divergent and overlapping Ps/Pr segment, which expresses the transcription factors XylS and XylR respectively, are computed into distinct instructions to the upper and lower catabolic xyl operons for either simultaneous or stepwise consumption of m-xylene and/or succinate. In this respect, the model reveals that the architecture of the Ps/Pr is poised to discriminate the abundance of alternative and competing C sources, in particular m-xylene versus succinate. The proposed framework provides a first systemic understanding of the causality and connectivity of the regulatory elements that shape this exemplary regulatory network, facilitating the use of model analysis towards genetic circuit optimization.
Molecular characterization of factors involved in regulation of archaeal translation
Blombach, F. - \ 2010
University. Promotor(en): John van der Oost, co-promotor(en): Stan Brouns. - [S.l. : S.n. - ISBN 9789085856436 - 142
ribosomen - translatie - genexpressie - bacteriën - transcriptie - rna-polymerase - archaea - transcriptieregulatie - ribosomes - translation - gene expression - bacteria - transcription - rna polymerase - regulation of transcription
The three domains of life – Bacteria, Archaea, and Eukaryotes – can be easily distinguished based on how the genetic information is processed during transcription, translation, and (DNA) replication. Generally, Eukaryotes turned out to employ machineries for these processes that are in their essence homologous to the corresponding archaeal machineries. This prompted the idea of an “archaeal parent” at the beginning of eukaryotic evolution.
This thesis deals with various aspects of translation in the crenarchaeon Sulfolobus solfataricus and Archaea in general. Numerous proteins are directly involved in the translation process as factors regulating initiation, elongation, and termination, or as ribosomal proteins being an integral part of the ribosome, the machinery catalyzing protein synthesis. These proteins usually show a high level of conservation and a considerable part of these proteins is common to all cellular life. Other proteins aiding the translation machinery can be involved in different translation-related processes. This starts with the assembly of the ribosomal subunits from rRNA molecules and ribosomal proteins. The synthesis of ribosomal RNA by an RNA polymerase is the starting point of ribosome assembly. Eukaryotes possess RNA polymerases I and III that are specially dedicated to the transcription of ribosomal RNA and other structural RNAs, i.e. RNA molecules that are not translated into proteins but serve for instance as building blocks for the translation machinery. Archaea and Bacteria are thought to have only a single type of RNA polymerase.
Chapter 6 of this thesis describes the identification of an archaeal ortholog of RNA polymerase III subunit RPC34. This subunit was considered to be unique for eukaryotic RNA polymerase III and plays an essential role in the recruitment of the RNA polymerase to promoter-bound general transcription factors. The archaeal RPC34 ortholog is predicted to function in transcription as well. Gene synteny analysis revealed that the archaeal gene orthologs cluster with various genes involved in the modification and processing of structural RNAs. The functional separation of transcription of protein-encoding and structural RNAs in Eukaryotes might therefore date back to the “archaeal parent” of Eukaryotes, with RPC34 playing a key role therein.
As stated above, the stepwise assembly of the ribosome begins already during the transcription of rRNA, with the first ribosomal proteins binding to the growing rRNA transcript. This process is controlled by a multitude of factors and in Bacteria and Eukaryotes a major group of ribosome assembly factors are GTPases of the translation-factor related class (TRAFAC). There are only a few TRAFAC GTPases in Archaea that serve as candidates for a role in ribosome assembly, as outlined in Chapter 1. In Chapter 2, one of those archaeal TRAFAC GTPases, the HflX ortholog SsGBP from S. solfataricus was studied in structural detail. The crystal structure revealed a two-domain arrangement including a prototypic HflX-domain that probably functions as a nucleic acid binding domain. The presence of the HflX-domain influences the GTP-hydrolysing properties of the C-terminal G-domain. Overall, the enzyme shows biochemical properties similar to other translational GTPases with slow intrinsic GTPase activity and relatively low affinity for GTP.
Chapter 3 describes the interaction of SsGBP with the large ribosomal subunit. Binding
assays show that SsGBP similar to its bacterial orthologs binds to the 50S ribosomal subunit both in GDP- and GTP-bound form. Apo-SsGBP shows less stable binding. These results were somewhat unexpected as no conformational changes were observed between the GDP-bound and apo-SsGBP crystal structures whereas GTP-binding was predicted to drive major conformational rearrangements (Chapter 2). In line with the prediction derived from the SsGBP crystal structures, the HflX domain provides a major surface for the ribosome interaction of SsGBP.
In S. solfataricus the gene coding for SsGBP is co-transcribed with a gene coding for an archaeal ortholog of the eukaryotic transcription co-activator MBF1. Archaeal and eukaryotic MBF1 orthologs share a conserved helix-turn-helix domain. Chapter 4 describes the results of a genetic approach for the characterization of the mbf1 gene. It is shown that, at least under the conditions tested, mbf1 is not essential in the crenarchaeon Sulfolobus solfataricus and gene deletion caused only a mild phenotype and little change on transcriptome level. In Chapter 5 it is shown that the archaeal MBF1 shows properties that differ from the published data for its eukaryotic ortholog. While no evidence was found for an interaction with the transcription machinery, the archaeal MBF1 ortholog does bind to ribosomes engaged in translation via the 30S ribosomal subunit. Intriguingly, the helix-turn-helix domain of archaeal MBF1 provides the major binding surface for the interaction with the 30S ribosomal subunit.

DNA photolyases of Chrysodeixis chalcites nucleopolyhedrovirus are targeted to the nucleus and interact with chromosomes and mitotic spindle structures
Xu, F. ; Vlak, J.M. ; Eker, A.P.M. ; Oers, M.M. van - \ 2010
Journal of General Virology 91 (2010)4. - ISSN 0022-1317 - p. 907 - 914.
cyclobutane pyrimidine dimers - induced apoptosis - rna-polymerase - baculovirus - repair - identification - photorepair - irradiation - sequence - genome
Cyclobutane pyrimidine dimer (CPD) photolyases convert UV-induced CPDs in DNA into monomers using visible light as the energy source. Two phr genes encoding class II CPD photolyases PHR1 and PHR2 have been identified in Chrysodeixis chalcites nucleopolyhedrovirus (ChchNPV). Transient expression assays in insect cells showed that PHR1–EGFP fusion protein was localized in the nucleus. Early after transfection, PHR2–EGFP was distributed over the cytoplasm and nucleus but, over time, it became localized predominantly in the nucleus. Immunofluorescence analysis with anti-PHR2 antiserum showed that, early after transfection, non-fused PHR2 was already present mainly in the nucleus, suggesting that the fusion of PHR2 to EGFP hindered its nuclear import. Both PHR–EGFP fusion proteins strongly colocalized with chromosomes and spindle, aster and midbody structures during host-cell mitosis. When PHR2–EGFP-transfected cells were superinfected with Autographa californica multiple-nucleocapsid NPV (AcMNPV), the protein colocalized with virogenic stroma, the replication factories of baculovirus DNA. The collective data support the supposition that the PHR2 protein plays a role in baculovirus DNA repair
Suitability of PER.C6 (R) cells to generate epidemic and pandemic influenza vaccine strains by reverse genetics
Koudstaal, W. ; Hartgroves, L. ; Havenga, M. ; Legastelois, I. ; Ophorst, C. ; Sieuwerts, M. ; Zuijdgeest, D. ; Vogels, R. ; Custers, J. ; Boer-Luijtze, E.A. de; Leeuw, O.S. de; Cornelissen, A.H.M. ; Goudsmit, J. ; Barclay, W. - \ 2009
Vaccine 27 (2009)19. - ISSN 0264-410X - p. 2588 - 2593.
a virus generation - rna-polymerase - system - establishment - rescue - dna
Reverse genetics, the generation of influenza viruses from cDNA. presents a rapid method for creating vaccine strains. The technique necessitates the use of cultured cells. Due to technical and regulatory requirements, the choice of cell lines for production of human influenza vaccines is limited. PER.C6 (R) cells, among the most extensively characterized and documented cells, support growth of all influenza Viruses tested to date, and can be grown to high densities in large bioreactors in the absence of serum or micro carriers. Here, the suitability of these cells for the generation of influenza Viruses by reverse genetics was investigated. A range of viruses reflective of vaccine strains was rescued exclusively using PER.C6 cells by Various transfection methods, including an animal component-free procedure. Furthermore, a whole inactivated vaccine carrying the HA and NA segments of A/HK/156/97 (H5N1) that was both rescued from and propagated oil PER.C6 cells, conferred protection in a mouse model. Thus PER.C6 cells provide an attractive platform for generation of influenza vaccine strains via reverse genetics.
Characterization of CprK1, a CRP/FNR-type transcriptional regulator of halorespiration from Desulfitobacterium hafniense
Gabor, K. ; Verissimo, C.S. ; Cyran, B.C. ; Horst, P. Ter; Meijer, N.P. ; Smidt, H. ; Vos, W.M. de; Oost, J. van der - \ 2006
Journal of Bacteriology 188 (2006)7. - ISSN 0021-9193 - p. 2604 - 2613.
activator protein cap - escherichia-coli - gene-expression - receptor protein - reductive dehalogenase - dependent promoters - structural basis - disulfide bond - rna-polymerase - dna complex
The recently identified CprK branch of the CRP (cyclic AMP receptor protein)-FNR (fumarate and nitrate reduction regulator) family of transcriptional regulators includes proteins that activate the transcription of genes encoding proteins involved in reductive dehalogenation of chlorinated aromatic compounds. Here we report the characterization of the CprK1 protein from Desulfitobacterium hafniense, an anaerobic low-G+C gram-positive bacterium that is capable of reductive dechlorination of 3-chloro-4-hydroxyphenylacetic acid (Cl-OHPA). The gene encoding CprK1 was cloned and functionally overexpressed in Escherichia coli, and the protein was subsequently purified to homogeneity. To investigate the interaction of CprK1 with three of its predicted binding sequences (dehaloboxes), we performed in vitro DNA-binding assays (electrophoretic mobility shift assays) as well as in vivo promoter probe assays. Our results show that CprK1 binds its target dehaloboxes with high affinity (dissociation constant, 90 nM) in the presence of Cl-OHPA and that transcriptional initiation by CprK1 is influenced by deviations in the dehaloboxes from the consensus TTAAT----ATTAA sequence. A mutant CprK1 protein was created by a ValGlu substitution at a conserved position in the recognition -helix that gained FNR-type DNA-binding specificity, recognizing the TTGAT----ATCAA sequence (FNR box) instead of the dehaloboxes. CprK1 was subject to oxidative inactivation in vitro, most likely caused by the formation of an intermolecular disulfide bridge between Cys11 and Cys200. The possibility of redox regulation of CprK1 by a thiol-disulfide exchange reaction was investigated by using two CysSer mutants. Our results indicate that a Cys11-Cys200 disulfide bridge does not appear to play a physiological role in the regulation of CprK1
The alternative sigma factor sigmaB and the stress response of Bacillus cereus
Schaik, W. van - \ 2005
University. Promotor(en): Tjakko Abee; Willem de Vos; Marcel Zwietering. - Wageningen : s.n. - ISBN 9085041848 - 165
bacillus cereus - stressreactie - pathogenen - voedselveiligheid - overleving - rna-polymerase - stress response - pathogens - food safety - survival - rna polymerase - cum laude
cum laude graduation (with distinction) The bacterium Bacillus cereus is responsible for a large number of cases of foodborne illness across the world. It is also an important cause of spoilage of food, in particular of milk and dairy-products. The growth and survival of B. cereus in food or during an infection is for a large part determined by to what extent the bacterium can adapt to changes in its environment. This process is known as the stress response and the protein SigmaB has an important role in it. Stress in B. cereus leads to the rapid activation of SigmaB. Subsequently, SigmaB coordinates the transcription of a set of genes, which leads to an increased resistance to stress. For example, SigmaB contributes to the growth and survival of B. cereus at low and high temperatures. It also has a role in cellular metabolism, which may indirectly also contribute to stress resistance. The knowledge obtained on the role of SigmaB in the stress response of B. cereus may contribute to the development of new, efficient, and safe methods for the production of food.
Phage display-selected single chain antibodies confer high levels of resistance against Tomato spotted wilt virus
Prins, M.W. ; Lohuis, H. ; Schots, A. ; Goldbach, R.W. - \ 2005
Journal of General Virology 86 (2005)7. - ISSN 0022-1317 - p. 2107 - 2113.
transgenic tobacco - gene-sequences - rna-polymerase - plant cytosol - fv-antibody - expression - protein - fragments - cells - tospoviruses
Rational design of antibodies targeting essential viral proteins can complement the palette of antiviral resistance strategies. Here, stable and high expression of single-chain monoclonal antibodies targeting the nucleoprotein of the economically important plant virus Tomato spotted wilt virus, a protein that is involved in multiple steps in the viral infection cycle, is reported. High cytoplasmic expression levels of three selected phage display-derived anti-viral single-chain antibodies were established. Of these antibodies, two led to high levels of resistance against this plant virus. Protoplast experiments provided evidence that the two resistance-conferring antibodies may have a different mode of action and could be combined for higher durability of resistance in the field
The alternative sigma factor SigmaB of Bacillus cereus: response to stress and role in heat adaptation
Schaik, W. van; Tempelaars, M.H. ; Wouters, J.A. ; Vos, W.M. de; Abee, T. - \ 2004
Journal of Bacteriology 186 (2004)2. - ISSN 0021-9193 - p. 316 - 325.
sigma-factor sigma(b) - gram-positive bacteria - rna-polymerase - staphylococcus-aureus - genome sequence - listeria-monocytogenes - transcription factor - lactococcus-lactis - escherichia-coli - phospholipase-c
A gene cluster encoding the alternative sigma factor sigma(B), three predicted regulators of sigma(B) (RsbV, RsbW, and RsbY), and one protein whose function is not known (Orf4) was identified in the genome sequence of the food pathogen Bacillus cereus ATCC 14579. Western blotting with polyclonal antibodies raised against sigma(B) revealed that there was 20.1-fold activation of sigma(B) after a heat shock from 30 to 42degreesC. Osmotic upshock and ethanol exposure also upregulated sigma(B), albeit less than a heat shock. When the intracellular ATP concentration was decreased by exposure to carbonyl cyanide m-chlorophenylhydrazone (CCCP), only limited increases in sigma(B) levels were observed, revealing that stress due to ATP depletion is not an important factor in sigma(B) activation in B. cereus. Analysis of transcription of the sigB operon by Northern blotting and primer extension revealed the presence of a sigma(B) -dependent promoter upstream of the first open reading frame (rsbV) of the sigB operon, indicating that transcription of sigB is autoregulated. A second sigma(B)-dependent promoter was identified upstream of the last open reading frame (orf4) of the sigB operon. Production of virulence factors and the nonhemolytic enterotoxin Nhe in a sigB null mutant was the same as in the parent strain. However, sigma(B) was found to play a role in the protective heat shock response of B. cereus. The sigB null mutant was less protected against the lethal temperature of 50degreesC by a preadaptation to 42degreesC than the parent strain was, resulting in a more-than-100-fold-reduced survival of the mutant after 40 min at 50degreesC.
Identification of sigmaB-dependent genes in Bacillus cereus by proteome and in vitro transcription analysis
Schaik, W. van; Zwietering, M.H. ; Vos, W.M. de; Abee, T. - \ 2004
Journal of Bacteriology 186 (2004)13. - ISSN 0021-9193 - p. 4100 - 4109.
listeria-monocytogenes sigma(b) - oxidative stress resistance - closely-related bacteria - lactic-acid bacteria - escherichia-coli - genome sequence - rna-polymerase - staphylococcus-aureus - anthracis pxo1 - subtilis
The alternative sigma factor sigma(B) of the food pathogen Bacillus cereus is activated upon stress exposure and plays a role in the adaptive response of vegetative cells. This study describes the identification of sigma(B)-dependent genes in B. cereus. Two-dimensional gel electrophoresis was performed with protein extracts from a sigma(B)- overproducing B. cereus strain. Nine protein spots, which were absent from the negative control, were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry or N-terminal sequencing. The sigma(B)-dependent expression of the corresponding genes was confirmed by Northern blot analysis with RNA isolated from B. cereus ATCC 14579 and its sigB null mutant. Northern blot analysis also revealed that six other genes were part of sigma(B)-dependent operons. The proteins that are predicted to be encoded by the sigma(B)-dependent genes include an intracellular protease, a Mg2+ transporter, and a thiamine biosynthesis protein (ThiG). Highly conserved promoter sites were found to precede all sigma(B)-dependent genes, with the exception of thiG. By searching the B. cereus genome for this conserved promoter sequence, five more candidate sigma(B)-dependent genes were identified. Northern blot analysis and in vitro transcription experiments with a reconstituted B. cereus sigma(B)-RNA polymerase holoenzyme confirmed the sigma(B) dependency of two of these genes and strongly suggested that two other genes, encoding an oligopeptide-binding OppA-like protein and subunit II of the cytochrome d ubiquinol oxidase, are also sigma(B) dependent. In conclusion, sigma(B) of B. cereus not only regulates genes directly involved in the stress response but may also control specific metabolic rearrangements.
Promoter analysis of the Chilo iridescent virus DNA polymerase and major capsid protein genes
Nalcacioglu, R. ; Marks, H. ; Vlak, J.M. ; Demirbag, Z. ; Oers, M.M. van - \ 2003
Virology 317 (2003). - ISSN 0042-6822 - p. 321 - 329.
swine-fever virus - lymphocystis disease virus - frog virus-3 - macromolecular-synthesis - rna-polymerase - cell-nucleus - genome - replication - sequence - type-6
The DNA polymerase (DNApol) and major capsid protein (MCP) genes were used as models to study promoter activity in Chilo iridescent virus (CIV). Infection of Bombyx mori SPC-BM-36 cells in the presence of inhibitors of DNA or protein synthesis showed that DNApol, as well as helicase, is an immediate-early gene and confirmed that the major capsid protein (MCP) is a late gene. Transcription of DNApol initiated 35 nt upstream and that of MCP 14 nt upstream of the translational start site. In a luciferase reporter gene assay both promoters were active only when cells were infected with CIV. For DNApol sequences between position -27 and -6, relative to the transcriptional start site, were essential for promoter activity. Furthermore, mutation of a G within the sequence TTGTTTT located just upstream of the DNApol transcription initiation site reduced the promoter activity by 25%. Sequences crucial for MCP promoter activity are located between positions -53 and -29. (C) 2003 Elsevier Inc. All rights reserved.
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