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Jos Vanderleyden

Publications and source records attributed to Jos Vanderleyden.

17 recordsLinked to original sources
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

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↗
PubMed2002

Resonance energy transfer in a calcium concentration-dependent cameleon protein.

We report investigations of resonance energy transfer in the green fluorescent protein and calmodulin-based fluorescent indicator constructs for Ca(2+) called cameleons using steady-state and time-resolved spectroscopy of the full construct and of the component green fluorescent protein mutants, namely ECFP (donor) and EYFP (acceptor). EYFP displays a complicated photophysical behavior including protonated and deprotonated species involved in an excited-state proton transfer. When EYFP is excited in the absorption band of the protonated species, a fast nonradiative deactivation occurs involving almost 97% of the excited protonated population and leading to a low efficiency of excited-state proton transfer to the deprotonated species. ECFP displays a multiexponential fluorescence decay with a major contributing component of 3.2 ns. The time-resolved fluorescence data obtained upon excitation at 420 nm of Ca(2+)-free and Ca(2+)-bound YC3.1 cameleon constructs point to the existence of different conformations of calmodulin dependent on Ca(2+) binding. Whereas steady-state data show only an increase in the efficiency of energy transfer upon Ca(2+) binding, the time-resolved data demonstrate the existence of three distinct conformations/populations within the investigated sample. Although the mechanism of the interconversion between the different conformations and the extent of interconversion are still unclear, the time-resolved fluorescence data offer an estimation of the rate constants, of the efficiency of the energy transfer, and of the donor-acceptor distances in the Ca(2+)-free and Ca(2+)-bound YC3.1 samples.

Bacterial Proteins↗
PubMed2002

The functions of Ca(2+) in bacteria: a role for EF-hand proteins?

In bacteria, Ca(2+) is implicated in a wide variety of cellular processes, including the cell cycle and cell division. Dedicated influx and efflux systems tightly control the low cytoplasmic Ca(2+) levels in prokaryotes. Additionally, the growing number of proteins containing various Ca(2+)-binding motifs supports the importance of Ca(2+), which controls various protein functions by affecting protein stability, enzymatic activity or signal transduction. The existence of calmodulin-like proteins (containing EF-hand motifs) in bacteria is a long-standing hypothesis. Analysis of the prokaryotic protein sequences available in the databases has revealed the presence of several calmodulin-like proteins containing two or more authentic EF-hand motifs, suggesting that calmodulin-like proteins could be involved in Ca(2+) regulation in bacteria.

Amino Acid Sequence↗
PubMed2002

Isolation and sequence analysis of the GlnKamtB1amtB2 gene cluster, encoding a PII homologue and two putative ammonium transporters, from Pseudomonas stutzeri A15.

By PCR, using primers based on heterologous amtB genes, an amtB sequence of Pseudomonas stutzeri A15 was amplified. This DNA fragment was used as a probe in Southern hybridisation experiments and resulted in the isolation and sequence analysis of a 6017 bp genomic fragment of P. stutzeri A15 containing glnKamtB1amtB2. GlnK codes for a homologue of the nitrogen regulatory PII protein, amtB1 and amtB2 encode putative ammonium transporters. Whereas a glnKamtB gene cluster is common among bacteria, a tandem repeat of ammonium transporter genes has not been reported before. Apart from the presence of a second amtB gene, the gene organisation on this 6 kbp fragment is very similar to a particular region in the genome of Pseudomonas aeruginosa PAO1, relatively closely related to P. stutzeri. Furthermore, the amtB1 gene shows the highest similarity with P. aeruginosa amtB, whereas the amtB2 gene is more closely related to cyanobacterial amtB genes, which are reported to be monocistronically transcribed and not clustered with glnK homologues. Upstream of glnK, NtrC and RpoN recognition sites can be observed. In the intergenic region of glnKamtB1amtB2 no terminators nor extra promoter sequences were observed, indicating that glnKamtB1amtB2 is possibly transcribed as a nitrogen regulated operon.

Amino Acid Sequence↗
PubMed2002

Flagella-driven chemotaxis towards exudate components is an important trait for tomato root colonization by Pseudomonas fluorescens.

Motility is a major trait for competitive tomato root-tip colonization by Pseudomonas fluorescens. To test the hypothesis that this role of motility is based on chemotaxis toward exudate components, cheA mutants that were defective in flagella-driven chemotaxis but retained motility were constructed in four P. fluorescens strains. After inoculation of seedlings with a 1:1 mixture of wild-type and nonmotile mutants all mutants had a strongly reduced competitive root colonizing ability after 7 days of plant growth, both in a gnotobiotic sand system as well as in nonsterile potting soil. The differences were significant on all root parts and increased from root base to root tip. Significant differences at the root tip could already be detected after 2 to 3 days. These experiments show that chemotaxis is an important competitive colonization trait. The best competitive root-tip colonizer, strain WCS365, was tested for chemotaxis toward tomato root exudate and its major identified components. A chemotactic response was detected toward root exudate, some organic acids, and some amino acids from this exudate but not toward its sugars. Comparison of the minimal concentrations required for a chemotactic response with concentrations estimated for exudates suggested that malic acid and citric acid are among major chemo-attractants for P. fluorescens WCS365 cells in the tomato rhizosphere.

Amino Acids↗
PubMed2002

O-antigen structural variation: mechanisms and possible roles in animal/plant-microbe interactions.

Current data from bacterial pathogens of animals and from bacterial symbionts of plants support some of the more general proposed functions for lipopolysaccharides (LPS) and underline the importance of LPS structural versatility and adaptability. Most of the structural heterogeneity of LPS molecules is found in the O-antigen polysaccharide. In this review, the role and mechanisms of this striking flexibility in molecular structure of the O-antigen in bacterial pathogens and symbionts are illustrated by some recent findings. The variation in O-antigen that gives rise to an enormous structural diversity of O-antigens lies in the sugar composition and the linkages between monosaccharides. The chemical composition and structure of the O-antigen is strain-specific (interstrain LPS heterogeneity) but can also vary within one bacterial strain (intrastrain LPS heterogeneity). Both LPS heterogeneities can be achieved through variations at different levels. First of all, O-polysaccharides can be modified non-stoichiometrically with sugar moieties, such as glucosyl and fucosyl residues. The addition of non-carbohydrate substituents, i.e. acetyl or methyl groups, to the O-antigen can also occur with regularity, but in most cases these modifications are again non-stoichiometric. Understanding LPS structural variation in bacterial pathogens is important because several studies have indicated that the composition or size of the O-antigen might be a reliable indicator of virulence potential and that these important features often differ within the same bacterial strain. In general, O-antigen modifications seem to play an important role at several (at least two) stages of the infection process, including the colonization (adherence) step and the ability to bypass or overcome host defense mechanisms. There are many reports of modifications of O-antigen in bacterial pathogens, resulting either from altered gene expression, from lysogenic conversion or from lateral gene transfer followed by recombination. In most cases, the mechanisms underlying these changes have not been resolved. However, in recent studies some progress in understanding has been made. Changes in O-antigen structure mediated by lateral gene transfer, O-antigen conversion and phase variation, including fucosylation, glucosylation, acetylation and changes in O-antigen size, will be discussed. In addition to the observed LPS heterogeneity in bacterial pathogens, the structure of LPS is also altered in bacterial symbionts in response to signals from the plant during symbiosis. It appears to be part of a molecular communication between bacterium and host plant. Experiments ex planta suggest that the bacterium in the rhizosphere prepares its LPS for its roles in symbiosis by refining the LPS structure in response to seed and root compounds and the lower pH at the root surface. Moreover, modifications in LPS induced by conditions associated with infection are another indication that specific structures are important. Also during the differentiation from bacterium to bacteroid, the LPS of Rhizobium undergoes changes in the composition of the O-antigen, presumably in response to the change of environment. Recent findings suggest that, during symbiotic bacteroid development, reduced oxygen tension induces structural modifications in LPS that cause a switch from predominantly hydrophilic to predominantly hydrophobic molecular forms. However, the genetic mechanisms by which the LPS epitope changes are regulated remain unclear. Finally, the possible roles of O-antigen variations in symbiosis will be discussed.

Animal Population Groups↗
PubMed2001

The cin quorum sensing locus of Rhizobium etli CNPAF512 affects growth and symbiotic nitrogen fixation.

Rhizobium etli CNPAF512 produces an autoinducer that inhibits growth of Rhizobium leguminosarum bv. viciae 248 and activates the Agrobacterium tumefaciens tra reporter system. Production of this compound in R. etli is dependent on two genes, named cinR and cinI, postulated to code for a transcriptional regulator and an autoinducer synthase, respectively. NMR analysis of the purified molecule indicates that the R. etli autoinducer produced by CinI is a saturated long chain 3-hydroxy-acyl-homoserine lactone, abbreviated as 3OH-(slc)-HSL. Using cin-gusA fusions, expression of cinI and cinR was shown to be growth phase-dependent. Deletion analysis of the cinI promoter region indicates that a regulatory element negatively controls cinI expression. Mutational analysis revealed that expression of the cinI gene is positively regulated by the CinR/3OH-(slc)-HSL complex. Besides 3OH-(slc)-HSL, R. etli produces at least six other autoinducer molecules, for which the structures have not yet been revealed, and of which the synthesis requires the previously identified raiI and raiR genes. At least three different autoinducers, including a compound co-migrating with 3OH-(slc)-HSL, are produced in R. etli bacteroids isolated from bean nodules. This is further substantiated by the observation that cinI and cinR are both expressed under symbiotic conditions. Acetylene reduction activity of nodules induced by the cin mutants was reduced with 60-70% compared with wild-type nodules, indicating that the R. etli 3OH-(slc)-HSL is involved in the symbiotic process. This was further confirmed by transmission electron microscopy of nodules induced by the wild type and the cinI mutant. Symbiosomes carrying cinI mutant bacteroids did not fully differentiate compared with wild-type symbiosomes. Finally, it was observed that the cinR gene and raiR control growth of R. etli.

4-Butyrolactone↗
PubMed1998

Bacterial chemotactic motility is important for the initiation of wheat root colonization by Azospirillum brasilense.

Bacteria of the genus Azospirillum are able to colonize plant roots. Using the beta-glucuronidase (GUS) reporter system, various Azospirillum mutants, including mutants affected in chemotactic motility or extracellular polysaccharide biosynthesis, were investigated for their capacity to initiate wheat root colonization at the root hair zones. Only non-flagellated mutants and a generally non-chemotactic mutant exhibited a strongly reduced colonization ability as compared to the wild-type. No role of the Azospirillum calcofluor-binding polysaccharide in primary wheat root colonization could be observed. This is the first report demonstrating directly, by using different motility mutants, the requirement of bacterial motility in the establishment of the Azospirillum-plant root association.

Azospirillum brasilense↗
PubMed1997

Structural analysis of the 6 kb cryptic plasmid pFAJ2600 from Rhodococcus erythropolis NI86/21 and construction of Escherichia coli-Rhodococcus shuttle vectors.

The complete nucleotide sequence of the 5936 bp cryptic plasmid pFAJ2600 from Rhodococcus erythropolis NI86/21 was determined. Based on the characteristics of its putative replication genes, repA and repB, pFAJ2600 was assigned to the family of pAL5000-related small replicons identified in Mycobacterium (pAL5000), Corynebacterium (pXZ10142), Brevibacterium (pRBL1), Bifidobacterium (pMB1) and Neisseria (pJD1). The replication systems of these plasmids show striking similarities to the ones used by the ColE2 family of plasmids from Enterobacteria with respect to both trans-acting factors and ori sequences. Two possible plasmid stabilization systems are encoded on pFAJ2600: a site-specific recombinase (PmrA) related to the Escherichia coli Xer proteins for plasmid multimer resolution and an ATPase (ParA) related to the A-type of proteins in sop/par partitioning systems. The proposed replication termination region of pFAJ2600 has features in common with the Ter loci of Bacillus subtilis. Chimeras composed of a pUC18-Cmr derivative inserted in the parA-repA intergenic region of vector pFAJ2600 produced vectors that could be shuttled between Escherichia coli and several Rhodococcus species (R. erythropolis, R. fascians, R. rhodochrous, R. ruber). The pFAJ2600-based shuttle vector pFAJ2574 was stably maintained in R. erythropolis and R. fascians growing under non-selective conditions.

Amino Acid Sequence↗