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Jos Vanderleyden

Publications and source records attributed to Jos Vanderleyden.

At least 37 records · Page 2Linked to original sources
PubMed2005

Unraveling the secret lives of bacteria: use of in vivo expression technology and differential fluorescence induction promoter traps as tools for exploring niche-specific gene expression.

A major challenge for microbiologists is to elucidate the strategies deployed by microorganisms to adapt to and thrive in highly complex and dynamic environments. In vitro studies, including those monitoring genomewide changes, have proven their value, but they can, at best, mimic only a subset of the ensemble of abiotic and biotic stimuli that microorganisms experience in their natural habitats. The widely used gene-to-phenotype approach involves the identification of altered niche-related phenotypes on the basis of gene inactivation. However, many traits contributing to ecological performance that, upon inactivation, result in only subtle or difficult to score phenotypic changes are likely to be overlooked by this otherwise powerful approach. Based on the premise that many, if not most, of the corresponding genes will be induced or upregulated in the environment under study, ecologically significant genes can alternatively be traced using the promoter trap techniques differential fluorescence induction and in vivo expression technology (IVET). The potential and limitations are discussed for the different IVET selection strategies and system-specific variants thereof. Based on a compendium of genes that have emerged from these promoter-trapping studies, several functional groups have been distinguished, and their physiological relevance is illustrated with follow-up studies of selected genes. In addition to confirming results from largely complementary approaches such as signature-tagged mutagenesis, some unexpected parallels as well as distinguishing features of microbial phenotypic acclimation in diverse environmental niches have surfaced. On the other hand, by the identification of a large proportion of genes with unknown function, these promoter-trapping studies underscore how little we know about the secret lives of bacteria and other microorganisms.

Adaptation, Physiological↗
PubMed2004

Azospirillum irakense pectate lyase displays a toroidal fold.

The three-dimensional structure of Azospirillum irakense pectate lyase (PelA) has been determined at a resolution of 2.65 A. The crystals are hexagonal, belonging to space group P6(5)22, with unit-cell parameters a = b = 85.37, c = 231.32 angstroms. Phase information was derived from a multiple-wavelength anomalous dispersion (MAD) experiment using a Hg derivative. Refinement of the model converged to Rcryst = 20.08% and Rfree = 25.87%. The overall structure of PelA does not adopt the characteristic parallel beta-helix fold displayed by pectate lyases from polysaccharide lyase (PL) families PL1, PL3 and PL9. Instead, it displays a predominantly alpha-helical structure with irregular coils and short beta-strands, similar to the recently reported structure of the catalytic module of the Cellvibrio japonicus pectate lyase Pel10Acm. The topologies of the two structures have been compared. They show two 'domains' with the interface between them being a wide-open central groove in which the active site is located. The active sites of the crystal structures are also compared and their similarities and differences are discussed.

Amino Acid Sequence↗
PubMed2004

Annotation of the pRhico plasmid of Azospirillum brasilense reveals its role in determining the outer surface composition.

The plant growth-promoting soil bacterium Azospirillum brasilense enhances growth of economically important crops, such as wheat, corn and rice. In order to improve plant growth, a close bacterial association with the plant roots is needed. Genes encoded on a 90-MDa plasmid, denoted pRhico plasmid, present in A. brasilense Sp7, play an important role in plant root interaction. Sequencing, annotation and in silico analysis of this 90-MDa plasmid revealed the presence of a large collection of genes encoding enzymes involved in surface polysaccharide biosynthesis. Analysis of the 90-MDa plasmid genome provided evidence for its essential role in the viability of the bacterial cell.

Azospirillum brasilense↗
PubMed2004

In silico identification and experimental validation of PmrAB targets in Salmonella typhimurium by regulatory motif detection.

BACKGROUND: The PmrAB (BasSR) two-component regulatory system is required for Salmonella typhimurium virulence. PmrAB-controlled modifications of the lipopolysaccharide (LPS) layer confer resistance to cationic antibiotic polypeptides, which may allow bacteria to survive within macrophages. The PmrAB system also confers resistance to Fe3+-mediated killing. New targets of the system have recently been discovered that seem not to have a role in the well-described functions of PmrAB, suggesting that the PmrAB-dependent regulon might contain additional, unidentified targets. RESULTS: We performed an in silico analysis of possible targets of the PmrAB system. Using a motif model of the PmrA binding site in DNA, genome-wide screening was carried out to detect PmrAB target genes. To increase confidence in the predictions, all putative targets were subjected to a cross-species comparison (phylogenetic footprinting) using a Gibbs sampling-based motif-detection procedure. As well as the known targets, we detected additional targets with unknown functions. Four of these were experimentally validated (yibD, aroQ, mig-13 and sseJ). Site-directed mutagenesis of the PmrA-binding site (PmrA box) in yibD revealed specific sequence requirements. CONCLUSIONS: We demonstrated the efficiency of our procedure by recovering most of the known PmrAB-dependent targets and by identifying unknown targets that we were able to validate experimentally. We also pinpointed directions for further research that could help elucidate the S. typhimurium virulence pathway.

Bacterial Proteins↗
PubMed2004

Quorum sensing and swarming migration in bacteria.

Bacterial cells can produce and sense signal molecules, allowing the whole population to initiate a concerted action once a critical concentration (corresponding to a particular population density) of the signal has been reached, a phenomenon known as quorum sensing. One of the possible quorum sensing-regulated phenotypes is swarming, a flagella-driven movement of differentiated swarmer cells (hyperflagellated, elongated, multinucleated) by which bacteria can spread as a biofilm over a surface. The glycolipid or lipopeptide biosurfactants thereby produced function as wetting agent by reducing the surface tension. Quorum sensing systems are almost always integrated into other regulatory circuits. This effectively expands the range of environmental signals that influence target gene expression beyond population density. In this review, we first discuss the regulation of AHL-mediated surface migration and the involvement of other low-molecular-mass signal molecules (such as the furanosyl borate diester AI-2) in biosurfactant production of different bacteria. In addition, population density-dependent regulation of swarmer cell differentiation is reviewed. Also, several examples of interspecies signalling are reported. Different signal molecules either produced by bacteria (such as other AHLs and diketopiperazines) or excreted by plants (such as furanones, plant signal mimics) might influence the quorum sensing-regulated swarming behaviour in bacteria different from the producer. On the other hand, specific bacteria can reduce the local available concentration of signal molecules produced by others. In the last part, the role and regulation of a surface-associated movement in biofilm formation is discussed. Here we also describe how quorum sensing may disperse existing biofilms and control the interaction between bacteria and higher organisms (such as the Rhizobium-bean symbiosis).

4-Butyrolactone↗
PubMed2004

Genetic and physical map of the pLAFR1 vector.

This paper presents the complete sequencing and annotation of the pLAFR1 vector. pLAFR is a tetracycline-resistant "cosmid" cloning vector, which is derived from the 20 kb plasmid pRK290, a RK2-derivative. Due to its broad host range, the pLAFR1 vector has been widely used in the genetic analysis of a broad number of gram-negative bacterial species. The availability of the complete pLAFR1 sequence will most definitely help in the construction and analysis of clone librares based on pRK290 or pLAFR vectors.

Base Sequence↗
PubMed2004

Regulatory role of Rhizobium etli CNPAF512 fnrN during symbiosis.

The Rhizobium etli CNPAF512 fnrN gene was identified in the fixABCX rpoN(2) region. The corresponding protein contains the hallmark residues characteristic of proteins belonging to the class IB group of Fnr-related proteins. The expression of R. etli fnrN is highly induced under free-living microaerobic conditions and during symbiosis. This microaerobic and symbiotic induction of fnrN is not controlled by the sigma factor RpoN and the symbiotic regulator nifA or fixLJ, but it is due to positive autoregulation. Inoculation of Phaseolus vulgaris with an R. etli fnrN mutant strain resulted in a severe reduction in the bacteroid nitrogen fixation capacity compared to the wild-type capacity, confirming the importance of FnrN during symbiosis. The expression of the R. etli fixN, fixG, and arcA genes is strictly controlled by fnrN under free-living microaerobic conditions and in bacteroids during symbiosis with the host. However, there is an additional level of regulation of fixN and fixG under symbiotic conditions. A phylogenetic analysis of the available rhizobial FnrN and FixK proteins grouped the proteins in three different clusters.

Amino Acid Sequence↗
PubMed2003

Single-molecule surface enhanced resonance Raman spectroscopy of the enhanced green fluorescent protein.

In the present contribution, we demonstrated that surface-enhanced resonance Raman scattering spectra from single green fluorescent proteins (GFPs) were obtained. The most important findings are the direct detection of the conversion between a deprotonated and a protonated form of the chromophore at the single-molecule level via the corresponding vibrational fingerprints, and the fact that the enhanced green fluorescent protein (EGFP) also shows a high surface enhanced resonance Raman scattering (SERRS) signal. Our findings show the potential of the technique to study structural dynamics of protein molecules at a single-molecule level.

Green Fluorescent Proteins↗
PubMed2003

Genome-specific higher-order background models to improve motif detection.

Motif detection based on Gibbs sampling is a common procedure used to retrieve regulatory motifs in silico. Using a species-specific background model was previously shown to increase the robustness of the algorithm. Here, we demonstrate that selecting a non-species-adapted background model can have an adverse effect on the results of motif detection. The large differences in the average nucleotide composition of prokaryotic sequences exacerbate the problem of exchanging background models. Therefore, we have developed complex background models for all prokaryotic species with available genome sequences.

Algorithms↗
PubMed2003

Development and application of a dapB-based in vivo expression technology system to study colonization of rice by the endophytic nitrogen-fixing bacterium Pseudomonas stutzeri A15.

Pseudomonas stutzeri A15 is a nitrogen-fixing bacterium isolated from paddy rice. Strain A15 is able to colonize and infect rice roots. This strain may provide rice plants with fixed nitrogen and hence promote plant growth. In this article, we describe the use of dapB-based in vivo expression technology to identify P. stutzeri A15 genes that are specifically induced during colonization and infection (cii). We focused on the identification of P. stutzeri A15 genes that are switched on during rice root colonization and are switched off during free-living growth on synthetic medium. Several transcriptional fusions induced in the rice rhizosphere were isolated. Some of the corresponding genes are involved in the stress response, chemotaxis, metabolism, and global regulation, while others encode putative proteins with unknown functions or without significant homology to known proteins.

Bacterial Proteins↗
PubMed2003

Cloning and heterologous expression of a beta-D-mannosidase (EC 3.2.1.25)-encoding gene from Thermobifida fusca TM51.

Thermobifida fusca TM51, a thermophilic actinomycete isolated from composted horse manure, was found to produce a number of lignocellulose-degrading hydrolases, including endoglucanases, exoglucanases, endoxylanases, beta-xylosidases, endomannanases, and beta-mannosidases, when grown on cellulose or hemicellulose as carbon sources. beta-Mannosidases (EC 3.2.1.25), although contributing to the hydrolysis of hemicellulose fractions, such as galacto-mannans, constitute a lesser-known group of the lytic enzyme systems due to their low representation in the proteins secreted by hemicellulolytic microorganisms. An expression library of T. fusca, prepared in Streptomyces lividans TK24, was screened for beta-mannosidase activity to clone genes coding for mannosidases. One positive clone was identified, and a beta-mannosidase-encoding gene (manB) was isolated. Sequence analysis of the deduced amino acid sequence of the putative ManB protein revealed substantial similarity to known mannosidases in family 2 of the glycosyl hydrolase enzymes. The calculated molecular mass of the predicted protein was 94 kDa, with an estimated pI of 4.87. S. lividans was used as heterologous expression host for the putative beta-mannosidase gene of T. fusca. The purified gene product obtained from the culture filtrate of S. lividans was then subjected to more-detailed biochemical analysis. Temperature and pH optima of the recombinant enzyme were 53 degrees C and 7.17, respectively. Substrate specificity tests revealed that the enzyme exerts only beta-D-mannosidase activity. Its kinetic parameters, determined on para-nitrophenyl beta-D-mannopyranoside (pNP-betaM) substrate were as follows: K(m) = 180 micro M and V(max) = 5.96 micro mol min(-1) mg(-1); the inhibition constant for mannose was K(i) = 5.5 mM. Glucono-lacton had no effect on the enzyme activity. A moderate trans-glycosidase activity was also observed when the enzyme was incubated in the presence of pNP-alphaM and pNP-betaM; under these conditions mannosyl groups were transferred by the enzyme from pNP-betaM to pNP-alphaM resulting in the synthesis of small amounts (1 to 2%) of disaccharides.

Actinomycetales↗
PubMed2003

Identification of a third sulfate activation system in Sinorhizobium sp. strain BR816: the CysDN sulfate activation complex.

Sinorhizobium sp. strain BR816 possesses two nodPQ copies, providing activated sulfate (3'-phosphoadenosine-5'-phosphosulfate [PAPS]) needed for the biosynthesis of sulfated Nod factors. It was previously shown that the Nod factors synthesized by a nodPQ double mutant are not structurally different from those of the wild-type strain. In this study, we describe the characterization of a third sulfate activation locus. Two open reading frames were fully characterized and displayed the highest similarity with the Sinorhizobium meliloti housekeeping ATP sulfurylase subunits, encoded by the cysDN genes. The growth characteristics as well as the levels of Nod factor sulfation of a cysD mutant (FAJ1600) and a nodP1 nodQ2 cysD triple mutant (FAJ1604) were determined. FAJ1600 shows a prolonged lag phase only with inorganic sulfate as the sole sulfur source, compared to the wild-type parent. On the other hand, FAJ1604 requires cysteine for growth and produces sulfate-free Nod factors. Apigenin-induced nod gene expression for Nod factor synthesis does not influence the growth characteristics of any of the strains studied in the presence of different sulfur sources. In this way, it could be demonstrated that the "household" CysDN sulfate activation complex of Sinorhizobium sp. strain BR816 can additionally ensure Nod factor sulfation, whereas the symbiotic PAPS pool, generated by the nodPQ sulfate activation loci, can be engaged for sulfation of amino acids. Finally, our results show that rhizobial growth defects are likely the reason for a decreased nitrogen fixation capacity of bean plants inoculated with cysD mutant strains, which can be restored by adding methionine to the plant nutrient solution.

Bacterial Proteins↗
PubMed2002

Prediction and overview of the RpoN-regulon in closely related species of the Rhizobiales.

BACKGROUND: In the rhizobia, a group of symbiotic Gram-negative soil bacteria, RpoN (sigma54, sigmaN, NtrA) is best known as the sigma factor enabling transcription of the nitrogen fixation genes. Recent reports, however, demonstrate the involvement of RpoN in other symbiotic functions, although no large-scale effort has yet been undertaken to unravel the RpoN-regulon in rhizobia. We screened two complete rhizobial genomes (Mesorhizobium loti, Sinorhizobium meliloti) and four symbiotic regions (Rhizobium etli, Rhizobium sp. NGR234, Bradyrhizobium japonicum, M. loti) for the presence of the highly conserved RpoN-binding sites. A comparison was also made with two closely related non-symbiotic members of the Rhizobiales (Agrobacterium tumefaciens, Brucella melitensis). RESULTS: A highly specific weight-matrix-based screening method was applied to predict members of the RpoN-regulon, which were stored in a highly annotated and manually curated dataset. Possible enhancer-binding proteins (EBPs) controlling the expression of RpoN-dependent genes were predicted with a profile hidden Markov model. CONCLUSIONS: The methodology used to predict RpoN-binding sites proved highly effective as nearly all known RpoN-controlled genes were identified. In addition, many new RpoN-dependent functions were found. The dependency of several of these diverse functions on RpoN seems species-specific. Around 30% of the identified genes are hypothetical. Rhizobia appear to have recruited RpoN for symbiotic processes, whereas the role of RpoN in A. tumefaciens and B. melitensis remains largely to be elucidated. All species screened possess at least one uncharacterized EBP as well as the usual ones. Lastly, RpoN could significantly broaden its working range by direct interfering with the binding of regulatory proteins to the promoter DNA.

Bacterial Proteins↗
PubMed2002

Sensitivity function-based model reduction: A bacterial gene expression case study.

Mathematical models used to predict the behavior of genetically modified organisms require 1). a (rather) large number of state variables, and 2). complicated kinetic expressions containing a large number of parameters. Since these models are hardly identifiable and of limited use in model-based optimization and control strategies, a generic methodology based on sensitivity function analysis is presented to reduce the model complexity at the level of the kinetics, while maintaining high prediction power. As a case study to illustrate the method and results obtained, the influence of the dissolved oxygen concentration on the cytN gene expression in the bacterium Azospirillum brasilense Sp7 is modeled. As a first modeling approach, available mechanistic knowledge is incorporated into a mass balance equation model with 3 states and 14 parameters. The large differences in order of magnitude of the model parameters identified on the available experimental data indicate 1). possible structural problems in the kinetic model and, associated with this, 2). a possibly too high number of model parameters. A careful sensitivity function analysis reveals that a reduced model with only seven parameters is almost as accurate as the original model.

Azospirillum brasilense↗
PubMed2002

Expression of the NH(+)(4)-transporter gene LEAMT1;2 is induced in tomato roots upon association with N(2)-fixing bacteria.

Plants growing in close association with N(2)-fixing bacteria are able to overcome growth limitations in N-depleted soils. The molecular mechanism by which free-living, N(2)-fixing bacteria promote plant growth is still a matter of debate. By inoculating N-depleted tomato (Lycopersicon esculentum Mill.) plants with Azospirillum brasilense or Azoarcus sp. we could demonstrate the induction of the root NH(+)(4)-transporter gene, LEAMT1;2 (L. esculentum ammonium transporter 1;2), indicating that bacterial NH(+)(4) might be used as an N source under these conditions. Azospirillum brasilense (nif(-)) mutants, which lack the structural nifDK genes, failed to induce LEAMT1;2 expression. This suggests that root-associated N(2)-fixing bacteria do excrete NH(+)(4) to levels that can be sensed by tomato roots and is in agreement with the induction of expression of LEAMT1;2 with as low as > or = 1 microM external NH(+)(4). While peak expression was obtained with 2-5 microM NH(+)(4), a further increase in NH(+)(4) reduced LEAMT1;2-mRNA levels in a concentration-dependent manner. The inhibition of LEAMT1;2 expression by glutamine and the glutamine synthetase blocker L-methionine sulfoximine (MSX) provided evidence for the control of LEAMT1;2 expression by cytoplasmic NH(+)(4) concentration or the plant N status. Since micromolar concentrations of NH(+)(4) strongly increased the LEAMT1;2-mRNA levels, the transported NH(+)(4) ion itself could represent a key signal in the associative interaction between higher plants and N(2)-fixing micro-organisms.

Azospirillum↗
PubMed2002

A major chemotaxis gene cluster in Azospirillum brasilense and relationships between chemotaxis operons in alpha-proteobacteria.

Azospirillum brasilense shows chemotaxis to a variety of nutrients and oxygen. Genes encoding the central signal transduction pathway in chemotaxis were identified by phenotypic complementation of generally non-chemotactic mutants. Sequencing of a DNA fragment, which complemented two different mutants, revealed a region of five open reading frames translated in one direction and encoding homologs of known genes comprising excitation and adaptation pathways for chemotaxis in other bacterial species. The major chemotaxis gene cluster appears to be essential for all known behavioral responses that direct swimming motility in A. brasilense. Phylogenetic and genomic analysis revealed three groups of chemotaxis operons in alpha-proteobacterial species and assigned the A. brasilense operon to one of them. Interestingly, operons that are shown to be major regulators of behavior in several alpha-proteobacterial species are not orthologous.

Alphaproteobacteria↗