The Experts below are selected from a list of 144 Experts worldwide ranked by ideXlab platform
Stephen C. Winans - One of the best experts on this subject based on the ideXlab platform.
-
agrobacterium tumefaciens can obtain sulphur from an opine that is synthesized by Octopine synthase using s methylmethionine as a substrate
Molecular Microbiology, 2012Co-Authors: Ana L Floresmireles, Anatol Eberhard, Stephen C. WinansAbstract:Agrobacterium tumefaciens incites plant tumours that produce nutrients called opines, which are utilized by the bacteria during host colonization. Various opines provide sources of carbon, nitrogen and phosphorous, but virtually nothing was previously known about how A. tumefaciens acquires sulphur during colonization. Some strains encode an operon required for the catabolism of the opine Octopine. This operon contains a gene, msh, that is predicted to direct the conversion of S-methylmethionine (SMM) and homocysteine (HCys) to two equivalents of methionine. Purified Msh carried out this reaction, suggesting that SMM could be an intermediate in opine catabolism. Purified Octopine synthase (Ocs, normally expressed in plant tumours) utilized SMM and pyruvate to produce a novel opine, designated sulfonopine, whose catabolism by the bacteria would regenerate SMM. Sulfonopine was produced by tobacco and Arabidopsis when colonized by A. tumefaciens and was utilized as sole source of sulphur by A. tumefaciens. Purified Ocs also used 13 other proteogenic and non-proteogenic amino acids as substrates, including three that contain sulphur. Sulfonopine and 11 other opines were tested for induction of Octopine catabolic operon and all were able to do so. This is the first study of the acquisition of sulphur, an essential element, by this pathogen.
-
most mutant occr proteins that are defective in positive control hold operator dna in a locked high angle bend
Journal of Bacteriology, 2011Co-Authors: Chingsung Tsai, Chiasui Chen, Stephen C. WinansAbstract:OccR is a LysR-type transcriptional regulator of Agrobacterium tumefaciens that positively regulates the Octopine catabolism operon of the Ti plasmid. Positive control of the occ genes occurs in response to Octopine, a nutrient released from crown gall tumors. OccR also functions as an autorepressor in the presence or absence of Octopine. OccR binds to a site between occQ and occR in the presence or absence of Octopine, although Octopine triggers a conformational change that shortens the DNA footprint and relaxes a DNA bend. In order to determine the roles of this conformational change in transcriptional activation, we isolated 11 OccR mutants that were defective in activation of the occQ promoter but were still capable of autorepression. The mutations in these mutants spanned most of the length of the protein. Two additional positive-control mutants were isolated using site-directed mutagenesis. Twelve mutant proteins displayed a high-angle DNA bend in the presence or absence of Octopine. One mutant, the L26A mutant, showed ligand-responsive DNA binding similar to that of wild-type OccR and therefore must be impaired in a subsequent step in activation.
-
transcriptional regulation and locations of agrobacterium tumefaciens genes required for complete catabolism of Octopine
Journal of Bacteriology, 1997Co-Authors: Kyungyun Cho, Clay Fuqua, Stephen C. WinansAbstract:By screening for Octopine-inducible gene expression, we previously identified all the genes required for utilization of Octopine as a source of carbon, nitrogen, and energy. They are (i) Octopine oxidase, which converts Octopine to arginine and pyruvate and is encoded by the ooxAB operon, (ii) arginase, which converts arginine to ornithine and urea and is encoded by arcA, (iii) ornithine cyclodeaminase, which converts ornithine to proline and ammonia and is encoded by the homologous arcB and ocd genes, and (iv) proline dehydrogenase, which converts proline to glutamate and is encoded by putA. Here we describe the regulation and localization of each of these genes. The ooxA-ooxB-ocd operon was previously shown to reside on the Ti plasmid and to be directly inducible by Octopine. The arcAB operon is directly inducible by arginine, while it is induced by Octopine only in strains that can convert Octopine to arginine. Ornithine may also be a direct inducer of arcAB. putA is directly inducible by proline, while induction by Octopine and by arginine (and probably by ornithine) requires their conversion to proline. Genetic studies indicate that arcAB and putA are localized on a conjugal genetic element. This element can be transferred to other Agrobacterium tumefaciens strains by a mechanism that does not require recA-dependent homologous recombination. Transfer of this genetic element from A. tumefaciens R10 requires at least one tra gene found on its Ti plasmid, indicating that this element is not self-transmissible but is mobilizable by the Ti plasmid. The DNA containing the arcAB and putA genes comigrates with a 243-kb linear molecular weight standard on field inversion electrophoretic gels.
-
identification of agrobacterium tumefaciens genes that direct the complete catabolism of Octopine
Journal of Bacteriology, 1996Co-Authors: Kyungyun Cho, Clay Fuqua, B S Martin, Stephen C. WinansAbstract:Agrobacterium tumefaciens R10 was mutagenized by using the promoter probe transposon Tn5-gusA7, and a library of approximately 5,000 transcriptional fusions was screened for Octopine-inducible patterns of gene expression. Twenty-one mutants carrying strongly inducible gusA fusions, 20 of which showed defects in the catabolism of Octopine or its metabolites, were obtained. One group of mutants could not use Octopine as a carbon source, while a second group of mutants could not utilize arginine or ornithine and a third group could not utilize Octopine, arginine, ornithine, or proline as a carbon source. Utilization of these compounds as nitrogen sources showed similar but not identical patterns. Fifteen fusions were subcloned together with adjacent DNA. Sequence analysis and further genetic analysis indicated that insertions of the first group are localized in the occ region of the Ti plasmid. Insertions of the second group were localized to a gene encoding ornithine cyclodeaminase. This gene is very similar to, but distinct from, a homolog located on the Ti plasmid. This gene is located immediately downstream from a gene encoding an arginase. Genetic experiments indicated that this arginase gene is essential for Octopine and arginine catabolism. Insertions of the third group was localized to a gene whose product is required for degradation of proline. We therefore have identified all steps required for the catabolism of Octopine to glutamate.
-
a luxr luxi type regulatory system activates agrobacterium ti plasmid conjugal transfer in the presence of a plant tumor metabolite
Journal of Bacteriology, 1994Co-Authors: W C Fuqua, Stephen C. WinansAbstract:Conjugal transfer of Agrobacterium Octopine-type Ti plasmids is activated by Octopine, a metabolite released from plant tumors. Octopine causes conjugal donors to secrete a pheromone, Agrobacterium autoinducer (AAI), and exogenous AAI further stimulates conjugation. The putative AAI synthase and an AAI-responsive transcriptional regulator were found to be encoded by the Ti plasmid traI and traR genes, respectively, and the expression of traR was induced by Octopine. The Octopine-type traR gene product is highly homologous to the TraR protein recently characterized from a nopaline-type Ti plasmid. TraR and TraI are homologous to the LuxR and LuxI regulatory proteins of Vibrio fischeri, and AAI is similar in structure to the diffusable V. fischeri autoinducer, the inducing ligand of LuxR. TraR activated target genes in the presence of AAI and also activated traR and traI themselves, creating two positive-feedback loops. TraR-AAI-mediated activation in wild-type Agrobacterium strains was dramatically enhanced by culturing on solid media, suggesting a possible role in cell density sensing.
Joachim Schroder - One of the best experts on this subject based on the ideXlab platform.
-
Octopine and nopaline oxidases from ti plasmids of agrobacterium tumefaciens molecular analysis relationship and functional characterization
Journal of Bacteriology, 1994Co-Authors: H Zanker, G Lurz, U Langridge, D Kreusch, Peter Langridge, Joachim SchroderAbstract:The occ and noc regions of pTiAch5 (Octopine) and pTiC58 (nopaline) Ti plasmids are responsible for the catabolic utilization of Octopine and nopaline in Agrobacterium spp. The first enzymatic step is the oxidative cleavage into L-arginine and pyruvate or 2-ketoglutarate, respectively, by membrane-bound opine oxidases requiring two polypeptides (subunits B and A) for function. The DNA sequences showed that the subunits of pTiAch5 and pTiC58 are related, but none of the proteins revealed significant similarities to the biosynthetic enzymes expressed in transformed plant cells. The four proteins had no extensive overall similarity to other proteins, but the 35 N-terminal amino acids contained motifs found in many enzymes utilizing flavin adenine dinucleotide, flavin mononucleotide, or NAD(P)+ as cofactors. However, the activities were completely independent of added cofactors, and the nature of the electron acceptor remained unclear. Membrane solubilization led to complete loss of enzyme activity. The nopaline oxidase accepted nopaline and Octopine (Vmax ratio, 5:1) with similar Km values (1.1 mM). The Octopine oxidase had high activity with Octopine (Km = 1 mM) and barely detectable activity with nopaline. The subunits from the occ and the noc regions were exchangeable. The combinations ooxB-noxA and noxB-ooxA both produced active enzymes which oxidized Octopine and nopaline at similar rates, suggesting that both subunits contributed to the substrate specificity. These experiments also showed that the formation of functional enzyme required close proximity of the subunit genes on the same plasmid and that even a reversal of the gene order (A-B instead of B-A) led to reduced activity. Images
-
Octopine and nopaline oxidases from ti plasmids of agrobacterium tumefaciens molecular analysis relationship and functional characterization
Journal of Bacteriology, 1994Co-Authors: H Zanker, G Lurz, U Langridge, D Kreusch, Peter Langridge, Joachim SchroderAbstract:The occ and noc regions of pTiAch5 (Octopine) and pTiC58 (nopaline) Ti plasmids are responsible for the catabolic utilization of Octopine and nopaline in Agrobacterium spp. The first enzymatic step is the oxidative cleavage into L-arginine and pyruvate or 2-ketoglutarate, respectively, by membrane-bound opine oxidases requiring two polypeptides (subunits B and A) for function. The DNA sequences showed that the subunits of pTiAch5 and pTiC58 are related, but none of the proteins revealed significant similarities to the biosynthetic enzymes expressed in transformed plant cells. The four proteins had no extensive overall similarity to other proteins, but the 35 N-terminal amino acids contained motifs found in many enzymes utilizing flavin adenine dinucleotide, flavin mononucleotide, or NAD(P)+ as cofactors. However, the activities were completely independent of added cofactors, and the nature of the electron acceptor remained unclear. Membrane solubilization led to complete loss of enzyme activity. The nopaline oxidase accepted nopaline and Octopine (Vmax ratio, 5:1) with similar Km values (1.1 mM). The Octopine oxidase had high activity with Octopine (Km = 1 mM) and barely detectable activity with nopaline. The subunits from the occ and the noc regions were exchangeable. The combinations ooxB-noxA and noxB-ooxA both produced active enzymes which oxidized Octopine and nopaline at similar rates, suggesting that both subunits contributed to the substrate specificity. These experiments also showed that the formation of functional enzyme required close proximity of the subunit genes on the same plasmid and that even a reversal of the gene order (A-B instead of B-A) led to reduced activity.
-
opine regulated promoters and lysr type regulators in the nopaline noc and Octopine occ catabolic regions of ti plasmids of agrobacterium tumefaciens
Journal of Bacteriology, 1994Co-Authors: Johannes Von Lintig, D Kreusch, Joachim SchroderAbstract:Essential steps in the uptake and catabolism of the plant tumor metabolites nopaline and Octopine in Agrobacterium spp. are performed by proteins encoded in the nopaline catabolic (noc) and Octopine catabolic (occ) regions of Ti plasmids. We investigated the opine activation of the genes by using (i) promoter studies of Agrobacterium spp. and (ii) analysis of the promoter interaction with the regulatory proteins NocR (noc) and OccR (occ). The noc region contained two nopaline-induced promoters (Pi1[noc] and Pi2[noc]) and one autogenously regulated promoter (Pr [control of NocR expression]). Pi2 and Pr overlapped and were divergently oriented (Pi2 [noc]). DNA binding studies and DNase I footprints indicated that NocR bound specifically to single binding sites in Pi1[noc] and Pi2/Pr[noc] and that Pi2 and Pr were regulated from the same binding site. The binding was independent of the inducer nopaline, and nopaline caused small changes in the footprint. The promoters in the noc and occ regions shared sequence motif and contained the sequence T-N11-A, which is characteristic for LysR-type-regulated promoters. The occ region contained one Octopine-induced and one autogenously regulated promoter (Pi/Pr[occ]) in the same arrangement as Pi2/Pr[noc] in the noc region. Promoter deletions indicated that sequences flanking the OccR binding site determined the extent of induction, although they did not bind OccR. The promoter bound OccR in the absence and presence of Octopine. The opine caused a change in the mobility of the DNA-protein complex with the complete promoter. The resected fragments did not reveal this opine-induced shift, and it was also not detectable with the DNA-NocR complexes with the two promoters of the noc region. Images
-
opine transport genes in the Octopine occ and nopaline noc catabolic regions in ti plasmids of agrobacterium tumefaciens
Journal of Bacteriology, 1992Co-Authors: H Zanker, Johannes Von Lintig, Joachim SchroderAbstract:The occ and noc regions of Octopine and nopaline Ti plasmids in Agrobacterium tumefaciens are responsible for the catabolic utilization of Octopine and nopaline, respectively. Opine-inducible promoters, genes for regulatory proteins and for catabolic enzymes, had been identified in previous work. However, both regions contained additional DNA stretches which were under the control of opine-inducible promoters, but the functions were unknown. We investigated these stretches by DNA sequence and functional analyses. The sequences showed that both of the catabolic regions contain a set of four genes which are transcribed in the same direction. The occ and noc region genes are related, but the arrangement of the genes is different. The deduced polypeptides are related to those of binding protein-dependent transport systems of basic amino acids in other bacteria. The comparison suggested that three of the polypeptides are located in the membrane and that one is a periplasmic protein. We constructed cassettes which contained either the putative transport genes only or the complete occ or noc region; all constructs, however, included the elements necessary for opine-induced expression of the genes (the regulatory gene and the inducible promoters). Uptake studies with 3H-labelled Octopine showed that the putative transport genes in the occ region code for Octopine uptake proteins. The corresponding studies with 3H-labelled nopaline and the noc region cassettes indicated that the uptake of nopaline requires the putative transport genes and additional functions from the left part of the noc region.
H Zanker - One of the best experts on this subject based on the ideXlab platform.
-
Octopine and nopaline oxidases from ti plasmids of agrobacterium tumefaciens molecular analysis relationship and functional characterization
Journal of Bacteriology, 1994Co-Authors: H Zanker, G Lurz, U Langridge, D Kreusch, Peter Langridge, Joachim SchroderAbstract:The occ and noc regions of pTiAch5 (Octopine) and pTiC58 (nopaline) Ti plasmids are responsible for the catabolic utilization of Octopine and nopaline in Agrobacterium spp. The first enzymatic step is the oxidative cleavage into L-arginine and pyruvate or 2-ketoglutarate, respectively, by membrane-bound opine oxidases requiring two polypeptides (subunits B and A) for function. The DNA sequences showed that the subunits of pTiAch5 and pTiC58 are related, but none of the proteins revealed significant similarities to the biosynthetic enzymes expressed in transformed plant cells. The four proteins had no extensive overall similarity to other proteins, but the 35 N-terminal amino acids contained motifs found in many enzymes utilizing flavin adenine dinucleotide, flavin mononucleotide, or NAD(P)+ as cofactors. However, the activities were completely independent of added cofactors, and the nature of the electron acceptor remained unclear. Membrane solubilization led to complete loss of enzyme activity. The nopaline oxidase accepted nopaline and Octopine (Vmax ratio, 5:1) with similar Km values (1.1 mM). The Octopine oxidase had high activity with Octopine (Km = 1 mM) and barely detectable activity with nopaline. The subunits from the occ and the noc regions were exchangeable. The combinations ooxB-noxA and noxB-ooxA both produced active enzymes which oxidized Octopine and nopaline at similar rates, suggesting that both subunits contributed to the substrate specificity. These experiments also showed that the formation of functional enzyme required close proximity of the subunit genes on the same plasmid and that even a reversal of the gene order (A-B instead of B-A) led to reduced activity. Images
-
Octopine and nopaline oxidases from ti plasmids of agrobacterium tumefaciens molecular analysis relationship and functional characterization
Journal of Bacteriology, 1994Co-Authors: H Zanker, G Lurz, U Langridge, D Kreusch, Peter Langridge, Joachim SchroderAbstract:The occ and noc regions of pTiAch5 (Octopine) and pTiC58 (nopaline) Ti plasmids are responsible for the catabolic utilization of Octopine and nopaline in Agrobacterium spp. The first enzymatic step is the oxidative cleavage into L-arginine and pyruvate or 2-ketoglutarate, respectively, by membrane-bound opine oxidases requiring two polypeptides (subunits B and A) for function. The DNA sequences showed that the subunits of pTiAch5 and pTiC58 are related, but none of the proteins revealed significant similarities to the biosynthetic enzymes expressed in transformed plant cells. The four proteins had no extensive overall similarity to other proteins, but the 35 N-terminal amino acids contained motifs found in many enzymes utilizing flavin adenine dinucleotide, flavin mononucleotide, or NAD(P)+ as cofactors. However, the activities were completely independent of added cofactors, and the nature of the electron acceptor remained unclear. Membrane solubilization led to complete loss of enzyme activity. The nopaline oxidase accepted nopaline and Octopine (Vmax ratio, 5:1) with similar Km values (1.1 mM). The Octopine oxidase had high activity with Octopine (Km = 1 mM) and barely detectable activity with nopaline. The subunits from the occ and the noc regions were exchangeable. The combinations ooxB-noxA and noxB-ooxA both produced active enzymes which oxidized Octopine and nopaline at similar rates, suggesting that both subunits contributed to the substrate specificity. These experiments also showed that the formation of functional enzyme required close proximity of the subunit genes on the same plasmid and that even a reversal of the gene order (A-B instead of B-A) led to reduced activity.
-
opine transport genes in the Octopine occ and nopaline noc catabolic regions in ti plasmids of agrobacterium tumefaciens
Journal of Bacteriology, 1992Co-Authors: H Zanker, Johannes Von Lintig, Joachim SchroderAbstract:The occ and noc regions of Octopine and nopaline Ti plasmids in Agrobacterium tumefaciens are responsible for the catabolic utilization of Octopine and nopaline, respectively. Opine-inducible promoters, genes for regulatory proteins and for catabolic enzymes, had been identified in previous work. However, both regions contained additional DNA stretches which were under the control of opine-inducible promoters, but the functions were unknown. We investigated these stretches by DNA sequence and functional analyses. The sequences showed that both of the catabolic regions contain a set of four genes which are transcribed in the same direction. The occ and noc region genes are related, but the arrangement of the genes is different. The deduced polypeptides are related to those of binding protein-dependent transport systems of basic amino acids in other bacteria. The comparison suggested that three of the polypeptides are located in the membrane and that one is a periplasmic protein. We constructed cassettes which contained either the putative transport genes only or the complete occ or noc region; all constructs, however, included the elements necessary for opine-induced expression of the genes (the regulatory gene and the inducible promoters). Uptake studies with 3H-labelled Octopine showed that the putative transport genes in the occ region code for Octopine uptake proteins. The corresponding studies with 3H-labelled nopaline and the noc region cassettes indicated that the uptake of nopaline requires the putative transport genes and additional functions from the left part of the noc region.
William Scott Chilton - One of the best experts on this subject based on the ideXlab platform.
-
Structure and characterization of the crown gall opines heliopine, vitopine and ridéopine
Phytochemistry, 2001Co-Authors: William Scott Chilton, Marydell Chilton, Annik Petit, Yves DessauxAbstract:Abstract The crown gall opines heliopine from tumors induced by Octopine type Agrobacterium tumefaciens strains A6, A136(pTiB6-806), E9, A652 and 1590-1 and vitopine from tumor induced by grapevine strains S4 and T2 are identical to synthetic N 2 -(1′ R -carboxyethyl)- l -glutamine. Tumors produced by strains S4 and T2 do not contain Octopine or lysopine, but they do contain heliopine and the new opine rideopine identified as N -(4′-aminobutyl)- d -glutamic acid. Grapevine strains S4 and T2 grow normally on tumor heliopine or synthetic heliopine and on tumor and synthetic rideopine as well as on rideopine lactam as sole carbon source. While Octopine strains A6 and A136(pTiB6-806) do not grow on heliopine, mutant colonies do appear after a few weeks. Heliopine catabolism by Octopine strains is not induced by Octopine.
-
diversity of opines and opine catabolizing bacteria isolated from naturally occurring crown gall tumors
Applied and Environmental Microbiology, 1997Co-Authors: Larry W Moore, William Scott Chilton, M L CanfieldAbstract:The diversity of opines from 43 naturally occurring crown gall tumors on several plant species was analyzed for the presence of agropine, chrysopine, iminodiacid, an unidentified leucinopine-like iminodiacid (IDA-B), mannopine, Octopine, nopaline, DL- and LL-succinamopine, leucinopine and heliopine. Opine utilization patterns of agrobacteria and fluorescent pseudomonads resident in a tumor were then analyzed and compared for agreement with the opine isolated from that tumor. Nopaline was the most common opine found and was detected in tumors from cherry, blackberry, grape, and plum. Octopine was not found, although Octopine-catabolizing bacteria were isolated from several tumors. A new, previously undescribed iminodiacid of the succinamopine-leucinopine type (provisionally designated IDA-B) was isolated from tumors of wild blackberry. Field tumors from apple, blueberry and grape yielded no detectable opines, even though opine-utilizing bacteria were present. Bacterial isolates from plum and cherry showed the best correspondence between the opine in tumors (nopaline) and the presence of bacteria that catabolized that opine. However, several unusual opine catabolic combinations were identified, including isolates that catabolized a variety of opines but were nonpathogenic. More variability was observed among isolates from field tumors on the remaining plant species. We isolated novel mannopine-nopaline type agrobacteria from field tumors of cherry, plum and blackberry that induced tumors containing either mannopine (plus agropine) or nopaline, but not both. Epidemiologically, the galled plants from an area were not of clonal origin (same Ti plasmid), indicating that the field tumors from a small area were incited by more than one type of Ti plasmid.
Yves Dessaux - One of the best experts on this subject based on the ideXlab platform.
-
Structural basis for high specificity of Octopine binding in the plant pathogen Agrobacterium tumefaciens
Scientific Reports, 2017Co-Authors: Armelle Vigouroux, Yves Dessaux, Denis Faure, Abbas El Sahili, Julien Lang, Magali Aumont-nicaise, Solange MoréraAbstract:Agrobacterium pathogens of Octopine- and nopaline-types force host plants to produce either Octopine or nopaline compounds, which they use as nutrients. Two Agrobacterium ABC-transporters and their cognate periplasmic binding proteins (PBPs) OccJ and NocT import Octopine and nopaline/Octopine, respectively. Here, we show that both Octopine transport and degradation confer a selective advantage to Octopine-type A. tumefaciens when it colonizes plants. We report the X-ray structures of the unliganded PBP OccJ and its complex with Octopine as well as a structural comparison with NocT and the related PBP LAO from Salmonella enterica, which binds amino acids (lysine, arginine and ornithine). We investigated the specificity of OccJ, NocT and LAO using several ligands such as amino acids, Octopine, nopaline and Octopine analogues. OccJ displays a high selectivity and nanomolar range affinity for Octopine. Altogether, the structural and affinity data allowed to define an Octopine binding signature in PBPs and to construct a OccJ mutant impaired in Octopine binding, a selective Octopine-binding NocT and a non-selective Octopine-binding LAO by changing one single residue in these PBPs. We proposed the PBP OccJ as a major trait in the ecological specialization of Octopine-type Agrobacterium pathogens when they colonize and exploit the plant host.
-
Structure and characterization of the crown gall opines heliopine, vitopine and ridéopine
Phytochemistry, 2001Co-Authors: William Scott Chilton, Marydell Chilton, Annik Petit, Yves DessauxAbstract:Abstract The crown gall opines heliopine from tumors induced by Octopine type Agrobacterium tumefaciens strains A6, A136(pTiB6-806), E9, A652 and 1590-1 and vitopine from tumor induced by grapevine strains S4 and T2 are identical to synthetic N 2 -(1′ R -carboxyethyl)- l -glutamine. Tumors produced by strains S4 and T2 do not contain Octopine or lysopine, but they do contain heliopine and the new opine rideopine identified as N -(4′-aminobutyl)- d -glutamic acid. Grapevine strains S4 and T2 grow normally on tumor heliopine or synthetic heliopine and on tumor and synthetic rideopine as well as on rideopine lactam as sole carbon source. While Octopine strains A6 and A136(pTiB6-806) do not grow on heliopine, mutant colonies do appear after a few weeks. Heliopine catabolism by Octopine strains is not induced by Octopine.
