The Experts below are selected from a list of 2277 Experts worldwide ranked by ideXlab platform
Clive W. Ronson - One of the best experts on this subject based on the ideXlab platform.
-
high quality draft genome sequence of the lotus spp microsymbiont Mesorhizobium loti strain cj3sym
Standards in Genomic Sciences, 2015Co-Authors: Wayne Reeve, T B K Reddy, Christine Munk, Clive W. Ronson, Rekha Seshadri, John T Sullivan, Amrita Pati, Tanja Woyke, Rui Tian, Victor MarkowitzAbstract:Mesorhizobium loti strain CJ3Sym was isolated in 1998 following transfer of the integrative and conjugative element ICEMlSymR7A, also known as the R7A symbiosis island, in a laboratory mating from the donor M. loti strain R7A to a nonsymbiotic recipient Mesorhizobium strain CJ3. Strain CJ3 was originally isolated from a field site in the Rocklands range in New Zealand in 1994. CJ3Sym is an aerobic, Gram-negative, non-spore-forming rod. This report reveals the genome of M. loti strain CJ3Sym currently comprises 70 scaffolds totaling 7,563,725 bp. The high-quality draft genome is arranged in 70 scaffolds of 71 contigs, contains 7,331 protein-coding genes and 70 RNA-only encoding genes, and is part of the GEBA-RNB project proposal.
-
genome sequence of the lotus spp microsymbiont Mesorhizobium loti strain r7a
Standards in Genomic Sciences, 2014Co-Authors: Simon Kelly, Clive W. Ronson, John T Sullivan, Rui Tian, Lambert Brau, Karen W Davenport, Hajnalka E Daligault, Tracy Erkkila, Lynne Goodwin, Wei GuAbstract:Mesorhizobium loti strain NZP2037 was isolated in 1961 in Palmerston North, New Zealand from a Lotus divaricatus root nodule. Compared to most other M. loti strains, it has a broad host range and is one of very few M. loti strains able to form effective nodules on the agriculturally important legume Lotus pedunculatus. NZP2037 is an aerobic, Gram negative, non-spore-forming rod. This report reveals that the genome of M. loti strain NZP2037 does not harbor any plasmids and contains a single scaffold of size 7,462,792 bp which encodes 7,318 protein-coding genes and 70 RNA-only encoding genes. This rhizobial genome is one of 100 sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project.
-
Genome sequence of the Lotus spp. microsymbiont Mesorhizobium loti strain NZP2037
Standards in genomic sciences, 2014Co-Authors: Simon Kelly, Clive W. Ronson, John T Sullivan, Rui Tian, Lambert Brau, Karen W Davenport, Hajnalka E Daligault, Tracy Erkkila, Lynne GoodwinAbstract:Mesorhizobium loti strain NZP2037 was isolated in 1961 in Palmerston North, New Zealand from a Lotus divaricatus root nodule. Compared to most other M. loti strains, it has a broad host range and is one of very few M. loti strains able to form effective nodules on the agriculturally important legume Lotus pedunculatus. NZP2037 is an aerobic, Gram negative, non-spore-forming rod. This report reveals that the genome of M. loti strain NZP2037 does not harbor any plasmids and contains a single scaffold of size 7,462,792 bp which encodes 7,318 protein-coding genes and 70 RNA-only encoding genes. This rhizobial genome is one of 100 sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project.
-
genome sequence of the lotus corniculatus microsymbiont Mesorhizobium loti strain r88b
Standards in Genomic Sciences, 2014Co-Authors: Wayne Reeve, Clive W. Ronson, John T Sullivan, Tanja Woyke, Rui Tian, Lambert Brau, Karen W Davenport, Lynne Goodwin, Patrick S G Chain, Elizabeth A LobosAbstract:Mesorhizobium loti strain R88B was isolated in 1993 in the Rocklands range in Otago, New Zealand from a Lotus corniculatus root nodule. R88B is an aerobic, Gram-negative, non-spore-forming rod. This report reveals the genome of M. loti strain R88B contains a single scaffold of size 7,195,110 bp which encodes 6,950 protein-coding genes and 66 RNA-only encoding genes. This genome does not harbor any plasmids but contains the integrative and conjugative element ICEMl SymR7A, also known as the R7A symbiosis island, acquired by horizontal gene transfer in the field environment from M. loti strain R7A. It also contains a mobilizable genetic element ICEMl adhR88B, that encodes a likely adhesin gene which has integrated downstream of ICEMl SymR7A, and three acquired loci that together allow the utilization of the siderophore ferrichrome. This rhizobial genome is one of 100 sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project.
-
Genome Sequence and Gene Functions in Mesorhizobium loti and Relatives
Compendium of Plant Genomes, 2014Co-Authors: Kazuhiko Saeki, Clive W. RonsonAbstract:Mesorhizobium loti is a collective name for mesorhizobial species that establish nitrogen-fixing symbiosis with Lotus species. Accumulating genetic and genomic data indicate that diverse strains of M. loti have been generated through lateral integration of symbiosis islands into core chromosomes of a range of bacteria. The M. loti symbiosis islands probably derived from a common ancestral island and are evolving by acquiring accessory genetic elements while maintaining gene sets essential for nodulation and nitrogen fixation together with genes for some supportive processes. This view was supported by preliminary mappings of next-generation sequencing data of three strains, R7A, NZP2037, and NZP2213, on the whole-genome sequence of the strain MAFF303099. Common properties of M. loti genes involved in symbiosis and their regulation are also described along with genetic resources to study M. loti.
John T Sullivan - One of the best experts on this subject based on the ideXlab platform.
-
high quality draft genome sequence of the lotus spp microsymbiont Mesorhizobium loti strain cj3sym
Standards in Genomic Sciences, 2015Co-Authors: Wayne Reeve, T B K Reddy, Christine Munk, Clive W. Ronson, Rekha Seshadri, John T Sullivan, Amrita Pati, Tanja Woyke, Rui Tian, Victor MarkowitzAbstract:Mesorhizobium loti strain CJ3Sym was isolated in 1998 following transfer of the integrative and conjugative element ICEMlSymR7A, also known as the R7A symbiosis island, in a laboratory mating from the donor M. loti strain R7A to a nonsymbiotic recipient Mesorhizobium strain CJ3. Strain CJ3 was originally isolated from a field site in the Rocklands range in New Zealand in 1994. CJ3Sym is an aerobic, Gram-negative, non-spore-forming rod. This report reveals the genome of M. loti strain CJ3Sym currently comprises 70 scaffolds totaling 7,563,725 bp. The high-quality draft genome is arranged in 70 scaffolds of 71 contigs, contains 7,331 protein-coding genes and 70 RNA-only encoding genes, and is part of the GEBA-RNB project proposal.
-
genome sequence of the lotus spp microsymbiont Mesorhizobium loti strain r7a
Standards in Genomic Sciences, 2014Co-Authors: Simon Kelly, Clive W. Ronson, John T Sullivan, Rui Tian, Lambert Brau, Karen W Davenport, Hajnalka E Daligault, Tracy Erkkila, Lynne Goodwin, Wei GuAbstract:Mesorhizobium loti strain NZP2037 was isolated in 1961 in Palmerston North, New Zealand from a Lotus divaricatus root nodule. Compared to most other M. loti strains, it has a broad host range and is one of very few M. loti strains able to form effective nodules on the agriculturally important legume Lotus pedunculatus. NZP2037 is an aerobic, Gram negative, non-spore-forming rod. This report reveals that the genome of M. loti strain NZP2037 does not harbor any plasmids and contains a single scaffold of size 7,462,792 bp which encodes 7,318 protein-coding genes and 70 RNA-only encoding genes. This rhizobial genome is one of 100 sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project.
-
Genome sequence of the Lotus spp. microsymbiont Mesorhizobium loti strain NZP2037
Standards in genomic sciences, 2014Co-Authors: Simon Kelly, Clive W. Ronson, John T Sullivan, Rui Tian, Lambert Brau, Karen W Davenport, Hajnalka E Daligault, Tracy Erkkila, Lynne GoodwinAbstract:Mesorhizobium loti strain NZP2037 was isolated in 1961 in Palmerston North, New Zealand from a Lotus divaricatus root nodule. Compared to most other M. loti strains, it has a broad host range and is one of very few M. loti strains able to form effective nodules on the agriculturally important legume Lotus pedunculatus. NZP2037 is an aerobic, Gram negative, non-spore-forming rod. This report reveals that the genome of M. loti strain NZP2037 does not harbor any plasmids and contains a single scaffold of size 7,462,792 bp which encodes 7,318 protein-coding genes and 70 RNA-only encoding genes. This rhizobial genome is one of 100 sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project.
-
genome sequence of the lotus corniculatus microsymbiont Mesorhizobium loti strain r88b
Standards in Genomic Sciences, 2014Co-Authors: Wayne Reeve, Clive W. Ronson, John T Sullivan, Tanja Woyke, Rui Tian, Lambert Brau, Karen W Davenport, Lynne Goodwin, Patrick S G Chain, Elizabeth A LobosAbstract:Mesorhizobium loti strain R88B was isolated in 1993 in the Rocklands range in Otago, New Zealand from a Lotus corniculatus root nodule. R88B is an aerobic, Gram-negative, non-spore-forming rod. This report reveals the genome of M. loti strain R88B contains a single scaffold of size 7,195,110 bp which encodes 6,950 protein-coding genes and 66 RNA-only encoding genes. This genome does not harbor any plasmids but contains the integrative and conjugative element ICEMl SymR7A, also known as the R7A symbiosis island, acquired by horizontal gene transfer in the field environment from M. loti strain R7A. It also contains a mobilizable genetic element ICEMl adhR88B, that encodes a likely adhesin gene which has integrated downstream of ICEMl SymR7A, and three acquired loci that together allow the utilization of the siderophore ferrichrome. This rhizobial genome is one of 100 sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project.
-
Nodulation gene mutants of Mesorhizobium loti R7A-nodZ and nolL mutants have host-specific phenotypes on Lotus spp.
Molecular plant-microbe interactions : MPMI, 2009Co-Authors: P Rodpothong, John T Sullivan, Simona Radutoiu, Kriangsak Songsrirote, David Sumpton, Kenneth W. J.-t. Cheung, Jane Thomas-oates, Jens Stougaard, Clive W. RonsonAbstract:Rhizobial Nod factors induce plant responses and facilitate bacterial infection, leading to the development of nitrogen-fixing root nodules on host legumes. Nodule initiation is highly dependent on Nod-factor structure and, hence, on at least some of the nodulation genes that encode Nod-factor production. Here, we report the effects of mutations in Mesorhizobium loti R7A nodulation genes on nodulation of four Lotus spp. and on Nod-factor structure. Most mutants, including a ΔnodSΔnolO double mutant that produced Nod factors lacking the carbamoyl and possibly N-methyl groups on the nonreducing terminal residue, were unaffected for nodulation. R7AΔnodZ and R7AΔnolL mutants that produced Nod factors without the (acetyl)fucose on the reducing terminal residue had a host-specific phenotype, forming mainly uninfected nodule primordia on Lotus filicaulis and L. corniculatus and effective nodules with a delay on L. japonicus. The mutants also showed significantly reduced infection thread formation and Nin gene in...
Toshiharu Yagi - One of the best experts on this subject based on the ideXlab platform.
-
Crystal structure of pyridoxine 4-oxidase from Mesorhizobium loti
Acta Crystallographica Section A Foundations and Advances, 2014Co-Authors: Andrew Njagi Mugo, Yu Yoshikane, Toshiharu Yagi, Jun Kobayashi, Bunzo Mikami, Taiji Yamasaki, Kouhei OhnishiAbstract:Mesorhizobium loti MAFF303099, a nitrogen-fixing symbiotic bacterium, harbors degradation pathway I for pyridoxine (PN); a free form of vitamin B6. Pyridoxine 4-oxidase (PNOX), a monomeric glucose-methanol-choline (GMC) oxidoreductase family enzyme, is the first enzyme in the pathway. It catalyzes FAD-dependent oxidation of pyridoxine (PN) into pyridoxal. PNOX with a C-terminal His6 tag was overexpressed in E.coli JM109 cells and purified with a Ni-NTA agarose column and a QA52 column. The tertiary structures of PNOX and a complex of PNOX with pyridoxamine (PM), which is a substrate analog, were determined at 2.2 Å and at 2.1 Å resolutions, respectively. The overall structure consisted of FAD-binding and substrate-binding domains. The FAD interacts with the PNOX protein through a network of hydrogen bonds, which are mainly found in the ribose and pyrophosphate moieties of the FAD molecule. The surface structure of PNOX molecule showed that it had an opening socket for access of substrates. The opening was followed by a tunnel that was linked to the active site cavity. In the active site, His460, His462, and Pro504 were located on the re-face of the isoalloxazine ring of FAD. PM binds to the active site through several hydrogen bonds. The side chains of His462 and His460 are located at 2.7 and 3.1 Å from the N4′ atom of PM. The activities of H460A and H462A mutant PNOXs were very low, and H460A/H462A double mutant PNOX exhibited no activity. His462 may act as a general base for abstraction of a proton from the 4′-hydroxyl of PN. His460 may play a role in the binding and positioning of PN. The C4′ atom in PM is located at 3.2 Å, and the hydride ion from the C4′ atom may be transferred to the N5 atom of the isoalloxazine ring. The comparison of active site residues in GMC oxidoreductase family shows that Pro504 in PNOX corresponds to Asn or His of the conserved His-Asn or His-His pair in other GMC oxidoreductases.
-
Structure of 4-pyridoxolactonase from Mesorhizobium loti.
Acta crystallographica. Section F Structural biology communications, 2014Co-Authors: Jun Kobayashi, Yu Yoshikane, Seiki Baba, Kimihiko Mizutani, Toshiharu Yagi, Nobuyuki Takahashi, Bunzo MikamiAbstract:4-Pyridoxolactonase from Mesorhizobium loti catalyzes the zinc-dependent lactone-ring hydrolysis of 4-pyridoxolactone (4PAL) to 4-pyridoxic acid (4PA) in vitamin B6 degradation pathway I. The crystal structures of 4-pyridoxolactonase and its complex with 5-pyridoxolactone (5PAL; the competitive inhibitor) were determined. The overall structure was an αβ/βα sandwich fold, and two zinc ions were coordinated. This strongly suggested that the enzyme belongs to subclass B3 of the class B β-lactamases. In the complex structure, the carbonyl group of 5PAL pointed away from the active site, revealing why it acts as a competitive inhibitor. Based on docking simulation with 4PAL, 4PA and a reaction intermediate, 4-pyridoxolactonase probably catalyzes the reaction through a subclass B2-like mechanism, not the subclass B3 mechanism.
-
Crystal structure of pyridoxine 4-oxidase from Mesorhizobium loti.
Biochimica et biophysica acta, 2013Co-Authors: Andrew Njagi Mugo, Yu Yoshikane, Jun Kobayashi, Bunzo Mikami, Kouhei Ohnishi, Taiji Yamasaki, Toshiharu YagiAbstract:Abstract Pyridoxine 4-oxidase (PNOX) from Mesorhizobium loti is a monomeric glucose–methanol–choline (GMC) oxidoreductase family enzyme, catalyzes FAD-dependent oxidation of pyridoxine (PN) into pyridoxal, and is the first enzyme in pathway I for the degradation of PN. The tertiary structures of PNOX with a C-terminal His 6 -tag and PNOX–pyridoxamine (PM) complex were determined at 2.2 A and at 2.1 A resolutions, respectively. The overall structure consisted of FAD-binding and substrate-binding domains. In the active site, His460, His462, and Pro504 were located on the re-face of the isoalloxazine ring of FAD. PM binds to the active site through several hydrogen bonds. The side chains of His462 and His460 are located at 2.7 and 3.1 A from the N4′ atom of PM. The activities of His460Ala and His462Ala mutant PNOXs were very low, and 460Ala/His462Ala double mutant PNOX exhibited no activity. His462 may act as a general base for the abstraction of a proton from the 4′-hydroxyl of PN. His460 may play a role in the binding and positioning of PN. The C4′ atom in PM is located at 3.2 A, and the hydride ion from the C4′ atom may be transferred to the N5 atom of the isoalloxazine ring. The comparison of active site residues in GMC oxidoreductase shows that Pro504 in PNOX corresponds to Asn or His of the conserved His–Asn or His–His pair in other GMC oxidoreductases. The function of the novel proline residue was discussed.
-
The mll6786 gene encodes a repressor protein controlling the degradation pathway for vitamin B6 in Mesorhizobium loti
FEMS microbiology letters, 2012Co-Authors: Takayuki Nagase, Yu Yoshikane, Huy Nhat Chu, Kouhei Ohnishi, Andrew Njagi Mugo, Toshiharu YagiAbstract:Pyridoxine is converted to succinic semialdehyde, acetate, ammonia and CO2 through the actions of eight enzymes. The genes encoding the enzymes occur as a cluster on the chromosomal DNA of Mesorhizobium loti , a symbiotic nitrogen-fixing bacterium. Here, it was found that disruption of the mll6786 gene, which is located between the genes encoding the first and eighth enzymes of the pathway, caused constitutive expression of the eight enzymes. The protein encoded by the mll6786 gene is a member of the GntR family and is designated as PyrR. PyrR comprises 223 amino acid residues and is a dimeric protein with a subunit molecular mass of 25; kDa. The purified PyrR with a C-terminal His6-tag could bind to an intergenic 67-bp DNA region, which contains a palindrome sequence and a deduced promoter sequence, between the mll6786 and mlr6787 genes, encoding PyrR and AAMS amidohydrolase, respectively.
-
Crystallization and preliminary X-ray analysis of SDR-type pyridoxal dehydrogenase from Mesorhizobium loti.
Acta Crystallographica Section F Structural Biology and Crystallization Communications, 2010Co-Authors: Huy Nhat Chu, Yu Yoshikane, Jun Kobayashi, Bunzo Mikami, Toshiharu YagiAbstract:Pyridoxal 4-dehydrogenase from Mesorhizobium loti MAFF303099 was overexpressed in Escherichia coli. The recombinant selenomethionine-substituted enzyme was purified and crystallized by the sitting-drop vapour-diffusion method using PEG 4000 as precipitant. Crystals grew in the presence of 0.45 mM NAD+. The crystals diffracted to 2.9 A resolution and belonged to the monoclinic space group P21, with unit-cell parameters a = 86.20, b = 51.11, c = 91.73 A, β = 89.36°. The calculated V M values suggested that the asymmetric unit contained four molecules.
Viviana C Lepek - One of the best experts on this subject based on the ideXlab platform.
-
The transcriptional factor TtsI is involved in a negative regulation of swimming motility in Mesorhizobium loti MAFF303099.
FEMS microbiology letters, 2016Co-Authors: Cecilia M. Duarte, Laura A. Basile, Andrés Zalguizuri, Viviana C LepekAbstract:Mesorhizobium loti MAFF303099 has a functional Type III secretion system (T3SS) that is involved in the determination of competitiveness for legume nodulation. Here we demonstrate that the transcriptional factor TtsI, which positively regulates T3SS genes expression, is involved in a negative regulation of M. loti swimming motility in soft-agar. Conditions that induce T3SS expression affect flagella production. The same conditions also affect promoter activity of M. loti visN gene, a homolog to the positive regulator of flagellar genes that has been described in other rhizobia. Defects in T3SS complex assembly at membranes limited the negative regulation of motility by the expression of TtsI.
-
the absence of protein y4ys affects negatively the abundance of t3ss Mesorhizobium loti secretin rhcc2 in bacterial membranes
Frontiers in Plant Science, 2015Co-Authors: Virginia Mercante, Cintia Sanchez, Cecilia M. Duarte, Andrés Zalguizuri, Gustavo Caetanoanolles, Viviana C LepekAbstract:Mesorhizobium loti MAFF303099 has a functional type III secretion system (T3SS) that is involved in the determination of nodulation competitiveness on Lotus. The M. loti T3SS cluster contains gene y4yS (mlr8765) that codes for a protein of unknown function (Y4yS). A mutation in the y4yS gene favors the M. loti symbiotic competitive ability on Lotus tenuis cv. Esmeralda and affects negatively the secretion of proteins through T3SS. Here we localize Y4yS in the bacterial membrane using a translational reporter peptide fusion. In silico analysis indicated that this protein presents a tetratricopeptide repeat (TPR) domain, a signal peptide and a canonical lipobox LGCC in the N-terminal sequence. These features that are shared with proteins required for the formation of the secretin complex in type IV secretion systems and in the Tad system, together with its localization, suggest that the y4yS-encoded protein is required for the formation of the M. loti T3SS secretin (RhcC2) complex. Remarkably, analysis of RhcC2 in the wild-type and M. loti y4yS mutant strains indicated that the absence of Y4yS affects negatively the accumulation of normal levels of RhcC2 in the membrane.
-
Dual effect of Mesorhizobium loti T3SS functionality on the symbiotic process
FEMS microbiology letters, 2012Co-Authors: Cintia Sanchez, Virginia Mercante, María F. Babuin, Viviana C LepekAbstract:Mesorhizobium loti MAFF303099 has a functional type III secretory system (T3SS) involved in the nodulation process on Lotus tenuis and Lotus japonicus. Four putative M. loti T3SS effectors (Mlr6358, Mlr6331, Mlr6361, and Mlr6316) have been previously described, and it has been demonstrated that the N-terminal regions of Mlr6361 and Mlr6358 mediate the secretion via a T3SS. Here, we demonstrate the capacity of Mlr6316 and Mlr6331 N-terminal regions to direct the secretion of a translational fusion to a reporter peptide through T3SS. By using single, double, and triple mutants, we demonstrated the positive and negative participation of some of these proteins in the determination of competitiveness on Lotus spp. Low competitiveness values correlated with low nodulation efficiency for a mutant deficient in three of the putative M. loti effectors. Our data suggest that the net effect of M. loti T3SS function on symbiotic process with Lotus results from a balance between positive and negative effects.
-
characterization of the Mesorhizobium loti maff303099 type three protein secretion system
Molecular Plant-microbe Interactions, 2009Co-Authors: Cintia Sanchez, Florencia Iannino, William J Deakin, Rodolfo A Ugalde, Viviana C LepekAbstract:Type III secretion systems (T3SS) have been found in several species of rhizobia. Proteins (termed effectors) secreted by this system are involved in host-range determination and influence nodulation efficiency. Mesorhizobium loti MAFF303099 possesses a functional T3SS in its symbiotic island whose expression is induced by flavonoids. As in other rhizobia, conserved cis-elements (tts box) were found in the promoter regions of genes or operons encoding T3SS components. Using a bioinformatics approach, we searched for other tts-box-controlled genes, and confirmed this transcriptional regulation for some of them using lacZ fusions to the predicted promoter regions. Translational fusions to a reporter peptide were created to demonstrate T3SS-mediated secretion of two new MAFF303099 effectors. Finally, we showed that mutation of the M. loti MAFF303099 T3SS affects its competitiveness on Lotus glaber and investigated, at the molecular level, responses of the model legume L. japonicus to the T3SS.
-
nodule development induced by Mesorhizobium loti mutant strains affected in polysaccharide synthesis
Molecular Plant-microbe Interactions, 2005Co-Authors: Alejandra Dantuono, Rodolfo A Ugalde, Adriana C Casabuono, Alicia S Couto, Viviana C LepekAbstract:The role of Mesorhizobium loti surface polysaccharides on the nodulation process is not yet fully understood. In this article, we describe the nodulation phenotype of mutants affected in the synthesis of lipopolysaccharide (LPS) and β(1,2) cyclic glucan. M. loti lps β2 mutant produces LPS with reduced amount of O-antigen, whereas M. loti lps β1 mutant produces LPS totally devoid of O-antigen. Both genes are clustered in the chromosome. Based on amino acid sequence homology, LPS sugar composition, and enzymatic activity, we concluded that lps β2 codes for an enzyme involved in the transformation of dTDP-glucose into dTDP-rhamnose, the sugar donor of rhamnose for the synthesis of O-antigen. On the other hand, lps β1 codes for a glucosyl transferase involved in the biosynthesis of the O-antigen. Although LPS mutants elicited normal nodules, both show reduced competitiveness compared with the wild type. M. loti β(1-2) cyclic glucan synthase (cgs) mutant induces white, empty, ineffective pseudonodules in Lotus...
Kouhei Ohnishi - One of the best experts on this subject based on the ideXlab platform.
-
Crystal structure of pyridoxine 4-oxidase from Mesorhizobium loti
Acta Crystallographica Section A Foundations and Advances, 2014Co-Authors: Andrew Njagi Mugo, Yu Yoshikane, Toshiharu Yagi, Jun Kobayashi, Bunzo Mikami, Taiji Yamasaki, Kouhei OhnishiAbstract:Mesorhizobium loti MAFF303099, a nitrogen-fixing symbiotic bacterium, harbors degradation pathway I for pyridoxine (PN); a free form of vitamin B6. Pyridoxine 4-oxidase (PNOX), a monomeric glucose-methanol-choline (GMC) oxidoreductase family enzyme, is the first enzyme in the pathway. It catalyzes FAD-dependent oxidation of pyridoxine (PN) into pyridoxal. PNOX with a C-terminal His6 tag was overexpressed in E.coli JM109 cells and purified with a Ni-NTA agarose column and a QA52 column. The tertiary structures of PNOX and a complex of PNOX with pyridoxamine (PM), which is a substrate analog, were determined at 2.2 Å and at 2.1 Å resolutions, respectively. The overall structure consisted of FAD-binding and substrate-binding domains. The FAD interacts with the PNOX protein through a network of hydrogen bonds, which are mainly found in the ribose and pyrophosphate moieties of the FAD molecule. The surface structure of PNOX molecule showed that it had an opening socket for access of substrates. The opening was followed by a tunnel that was linked to the active site cavity. In the active site, His460, His462, and Pro504 were located on the re-face of the isoalloxazine ring of FAD. PM binds to the active site through several hydrogen bonds. The side chains of His462 and His460 are located at 2.7 and 3.1 Å from the N4′ atom of PM. The activities of H460A and H462A mutant PNOXs were very low, and H460A/H462A double mutant PNOX exhibited no activity. His462 may act as a general base for abstraction of a proton from the 4′-hydroxyl of PN. His460 may play a role in the binding and positioning of PN. The C4′ atom in PM is located at 3.2 Å, and the hydride ion from the C4′ atom may be transferred to the N5 atom of the isoalloxazine ring. The comparison of active site residues in GMC oxidoreductase family shows that Pro504 in PNOX corresponds to Asn or His of the conserved His-Asn or His-His pair in other GMC oxidoreductases.
-
Crystal structure of pyridoxine 4-oxidase from Mesorhizobium loti.
Biochimica et biophysica acta, 2013Co-Authors: Andrew Njagi Mugo, Yu Yoshikane, Jun Kobayashi, Bunzo Mikami, Kouhei Ohnishi, Taiji Yamasaki, Toshiharu YagiAbstract:Abstract Pyridoxine 4-oxidase (PNOX) from Mesorhizobium loti is a monomeric glucose–methanol–choline (GMC) oxidoreductase family enzyme, catalyzes FAD-dependent oxidation of pyridoxine (PN) into pyridoxal, and is the first enzyme in pathway I for the degradation of PN. The tertiary structures of PNOX with a C-terminal His 6 -tag and PNOX–pyridoxamine (PM) complex were determined at 2.2 A and at 2.1 A resolutions, respectively. The overall structure consisted of FAD-binding and substrate-binding domains. In the active site, His460, His462, and Pro504 were located on the re-face of the isoalloxazine ring of FAD. PM binds to the active site through several hydrogen bonds. The side chains of His462 and His460 are located at 2.7 and 3.1 A from the N4′ atom of PM. The activities of His460Ala and His462Ala mutant PNOXs were very low, and 460Ala/His462Ala double mutant PNOX exhibited no activity. His462 may act as a general base for the abstraction of a proton from the 4′-hydroxyl of PN. His460 may play a role in the binding and positioning of PN. The C4′ atom in PM is located at 3.2 A, and the hydride ion from the C4′ atom may be transferred to the N5 atom of the isoalloxazine ring. The comparison of active site residues in GMC oxidoreductase shows that Pro504 in PNOX corresponds to Asn or His of the conserved His–Asn or His–His pair in other GMC oxidoreductases. The function of the novel proline residue was discussed.
-
The mll6786 gene encodes a repressor protein controlling the degradation pathway for vitamin B6 in Mesorhizobium loti
FEMS microbiology letters, 2012Co-Authors: Takayuki Nagase, Yu Yoshikane, Huy Nhat Chu, Kouhei Ohnishi, Andrew Njagi Mugo, Toshiharu YagiAbstract:Pyridoxine is converted to succinic semialdehyde, acetate, ammonia and CO2 through the actions of eight enzymes. The genes encoding the enzymes occur as a cluster on the chromosomal DNA of Mesorhizobium loti , a symbiotic nitrogen-fixing bacterium. Here, it was found that disruption of the mll6786 gene, which is located between the genes encoding the first and eighth enzymes of the pathway, caused constitutive expression of the eight enzymes. The protein encoded by the mll6786 gene is a member of the GntR family and is designated as PyrR. PyrR comprises 223 amino acid residues and is a dimeric protein with a subunit molecular mass of 25; kDa. The purified PyrR with a C-terminal His6-tag could bind to an intergenic 67-bp DNA region, which contains a palindrome sequence and a deduced promoter sequence, between the mll6786 and mlr6787 genes, encoding PyrR and AAMS amidohydrolase, respectively.
-
Gene identification and characterization of the pyridoxine degradative enzyme 4-pyridoxic acid dehydrogenase from the nitrogen-fixing symbiotic bacterium Mesorhizobium loti MAFF303099.
Journal of biochemistry, 2008Co-Authors: Nana Yokochi, Yu Yoshikane, Kouhei Ohnishi, Toshiharu YagiAbstract:The gene encoding 4-pyridoxic acid dehydrogenase was identified as mlr6792 in a chromosome of a nitrogen-fixing symbiotic bacterium Mesorhizobium loti MAFF303099. The enzyme is the fourth enzyme in the vitamin B(6) (pyridoxine)-degradation pathway I. The recombinant enzyme with a his-tag over-expressed in Escherichia coli cells was a membrane-bound protein, and purified to homogeneity. The enzyme was a monomeric protein with a molecular weight of 59,000, and a flavoprotein containing one mole of FAD per mole of subunit. The optimum pH and temperature, and K(m) for 4-pyridoxic acid were pH 8.5 and 30 degrees C, and 29 muM, respectively. The enzyme was a glucose-methanol-choline (GMC) family protein with two signature patterns, FAD-binding residues, a putative active site histidine residue and a probable transmembrane segment.
-
crystal structure of pyridoxamine pyruvate aminotransferase from Mesorhizobium loti maff303099
Journal of Biological Chemistry, 2008Co-Authors: Yu Yoshikane, Kimihiko Mizutani, Bunzo Mikami, Nana Yokochi, Kouhei Ohnishi, Masayuki Yamasaki, Hideyuki Hayashi, Toshiharu YagiAbstract:Abstract Pyridoxamine-pyruvate aminotransferase (PPAT; EC 2.6.1.30) is a pyridoxal 5′-phosphate-independent aminotransferase and catalyzes reversible transamination between pyridoxamine and pyruvate to form pyridoxal and l-alanine. The crystal structure of PPAT from Mesorhizobium loti has been solved in space group P43212 and was refined to an R factor of 15.6% (Rfree = 20.6%) at 2.0A resolution. In addition, the structures of PPAT in complexes with pyridoxamine, pyridoxal, and pyridoxyl-l-alanine have been refined to R factors of 15.6, 15.4, and 14.5% (Rfree = 18.6, 18.1, and 18.4%) at 1.7, 1.7, and 2.0A resolution, respectively. PPAT is a homotetramer and each subunit is composed of a large N-terminal domain, consisting of seven β-sheets and eight α-helices, and a smaller C-terminal domain, consisting of three β-sheets and four α-helices. The substrate pyridoxal is bound through an aldimine linkage to Lys-197 in the active site. The α-carboxylate group of the substrate amino/keto acid is hydrogen-bonded to Arg-336 and Arg-345. The structures revealed that the bulky side chain of Glu-68 interfered with the binding of the phosphate moiety of pyridoxal 5′-phosphate and made PPAT specific to pyridoxal. The reaction mechanism of the enzyme is discussed based on the structures and kinetics results.