Mesorhizobium Loti MAFF303099 Research Articles

TonB-Dependent Receptors in Nitrogen-Fixing Nodulating Bacteria

TonB-dependent receptors (TBDRs) allow Gram-negative bacteria to uptake scarce resources from competitive environments with very high affinity. Early reports on TBDRs focused on the uptake of siderophore-iron complexes but recent studies have showed that the spectrum of ligands includes sugars, vitamins, heme, and other non-ferrous cations. To investigate the possible roles of TBDRs in nitrogen-fixing, nodulating bacteria, a bioinformatics approach was adopted to identify their presence in the genome of 13 selected rhizobacteria. The number of TBDR-like genes ranged from 1 (Mesorhizobium loti MAFF303099) to 14 (Azorhizobium caulinodans ORS 571 and Methylobacterium nodulans ORS 2060). These TBDRs can be largely grouped into two clusters, the 'heme' cluster and the 'iron-siderophores' cluster. The only exceptions are a putative nickel-specific TBDR (bll6948) in Bradyrhizobium japonicum USDA110 and two putative sugar-specific TBDRs in B. japonicum USDA110 and A. caulinodans ORS571 genomes, respectively. No TBDR-like sequences lie in the 'Vitamin B12' or 'Non-Fe cations' clusters. A model of the biological roles of TBDRs in free-living and symbiotic states is proposed for B. japonicum.

Crystallization and preliminary X-ray analysis of 4-pyridoxolactonase fromMesorhizobium loti

4-pyridoxolactonase from Mesorhizobium loti MAFF303099 has been overexpressed in Escherichia coli. The recombinant enzyme was purified and was crystallized by the sitting-drop vapour-diffusion method using PEG 4000 and ammonium sulfate as precipitants. Crystals of the free enzyme (form I) and of the 5-pyridoxolactone-bound enzyme (form II) grew under these conditions. Crystals of form I diffracted to 2.0 A resolution and belonged to the monoclinic space group C2, with unit-cell parameters a = 77.93, b = 38.88, c = 81.60 A, beta = 117.33 degrees. Crystals of form II diffracted to 1.9 A resolution and belonged to the monoclinic space group C2, with unit-cell parameters a = 86.24, b = 39.35, c = 82.68 A, beta = 118.02 degrees. The calculated V(M) values suggested that the asymmetric unit contains one molecule in both crystal forms.

Crystallization and preliminary X-ray analysis of AAMS amidohydrolase, the final enzyme in degradation pathway I of pyridoxine

alpha-(N-Acetylaminomethylene)succinic acid (AAMS) amidohydrolase from Mesorhizobium loti MAFF303099, which is involved in a degradation pathway of vitamin B(6) and catalyzes the degradation of AAMS to acetic acid, ammonia, carbon dioxide and succinic semialdehyde, has been overexpressed in Escherichia coli. To elucidate the reaction mechanism based on the tertiary structure, the recombinant enzyme was purified and crystallized by the sitting-drop vapour-diffusion method using PEG 8000 as precipitant. A crystal of the enzyme belonged to the monoclinic space group C2, with unit-cell parameters a = 393.2, b = 58.3, c = 98.9 A, beta = 103.4 degrees , and diffraction data were collected to 2.7 A resolution. The V(M) value and calculation of the self-rotation function suggested that three dimers with a threefold symmetry were possibly present in the asymmetric unit.

Characterization of the Mesorhizobium loti MAFF303099 Type-Three Protein Secretion System

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.

Structure of the PLP Degradative Enzyme 2-Methyl-3-hydroxypyridine-5-carboxylic Acid Oxygenase from Mesorhizobium loti MAFF303099 and Its Mechanistic Implications

A vitamin B(6) degradative pathway has recently been identified and characterized in Mesorhizobium loti MAFF303099. One of the enzymes on this pathway, 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase (MHPCO), is a flavin-dependent enzyme and catalyzes the oxidative ring-opening of 2-methyl-3-hydroxypyridine-5-carboxylic acid to form E-2-(acetamino-methylene)succinate. The gene for this enzyme has been cloned, and the corresponding protein has been overexpressed in Escherichia coli and purified. The crystal structure of MHPCO has been solved to 2.1 A using SAD phasing with and without the substrate MHPC bound. These crystal structures provide insight into the reaction mechanism and suggest roles for active site residues in the catalysis of a novel oxidative ring-opening reaction.

A possible role of class 1 plant hemoglobin at the early stage of legume-rhizobium symbiosis

Leguminous plants form root nodules, in which symbiotic rhizobia fix atmospheric nitrogen and supply the fixation products to their host plants as a nitrogen source. On the process of establishing the symbiosis, rhizobia induce genes involved in the defense system of their host plants. However, the host defense responses will be cancelled by unknown mechanism. We focused on nitric oxide (NO) as a key molecule of plant defense system and class 1 plant hemoglobin (Hb) as a scavenger of NO. The inoculation of a symbiotic rhizobium, Mesorhizobium loti MAFF303099, induced transiently NO production and expression of a class 1 Hb gene LjHb1 in the roots of a model legume Lotus japonicus. In this addendum, we show that the lipopolysaccharide of M. loti induces NO production and expression of LjHb1 in L. japonicus, and we propose the role of NO and Hb at the early stage of symbiosis.

Functional differences of two distinct catalases in Mesorhizobium loti MAFF303099 under free-living and symbiotic conditions.

Protection against reactive oxygen species (ROS) is important for legume-nodulating rhizobia during the establishment and maintenance of symbiosis, as well as under free-living conditions, because legume hosts might assail incoming microbes with ROS and because nitrogenase is extremely sensitive to ROS. We generated mutants of two potential catalase genes in Mesorhizobium loti MAFF303099 to investigate their physiological significance. Biochemical results indicated that genes with the locus tags mlr2101 and mlr6940 encoded a monofunctional catalase and a bifunctional catalase-peroxidase, respectively, that were named katE and katG. Under free-living conditions, the katG mutant demonstrated an extended generation time and elevated sensitivity to exogenous H(2)O(2), whereas the katE mutant exhibited no generation time extension and only a slight increase in sensitivity to exogenous H(2)O(2). However, the katE mutant showed a marked decrease in its survival rate during the stationary phase. With regard to symbiotic capacities with Lotus japonicus, the katG mutant was indistinguishable from the wild type; nevertheless, the mutants with disrupted katE formed nodules with decreased nitrogen fixation capacities (about 50 to 60%) compared to those formed by the wild type. These mutant phenotypes agreed with the expression profiles showing that transcription of katG, but not katE, was high during the exponential growth phase and that transcription levels of katE versus sigA were elevated during stationary phase and were approximately fourfold higher in bacteroids than mid-exponential-phase cells. Our results revealed functional separation of the two catalases, as well as the importance of KatE under conditions of strong growth limitation.

Legume–rhizobial symbiosis: an anorexic model?

Legume–rhizobial symbiosis: an anorexic model?

PLP Catabolism: Identification of the 2-(Acetamidomethylene)succinate Hydrolase Gene in Mesorhizobium loti MAFF303099

The function of the mlr6787 gene from Mesorhizobium loti MAFF303099 has been identified. This gene encodes 2-(acetamidomethylene)succinate hydrolase, an enzyme involved in the catabolism of pyridoxal 5'-phosphate (vitamin B 6). This enzyme was overexpressed in Escherichia coli, purified to homogeneity, and characterized. 2-(Acetamidomethylene)succinate hydrolase catalyzes the hydrolysis of 2-(acetamidomethylene)succinate to yield succinic semialdehyde, acetic acid, carbon dioxide, and ammonia. The k cat and K M for this reaction were 0.6 s (-1) and 143 microM, respectively. The enzyme was shown to utilize the E isomer of 2-(acetamidomethylene)succinate.

Effects of rhizobacterial ACC deaminase activity on Arabidopsis indicate that ethylene mediates local root responses to plant growth-promoting rhizobacteria

1-Amino cyclopropane-1-carboxylic acid deaminase (AcdS) is an enzyme that degrades the precursor of plant hormone ethylene. AcdS activity has been identified in many soil bacteria. It has been proposed to play an important role in plant-growth promotion by rhizobacteria. It would lower ethylene level via uncharacterized signaling pathways in the host plant. To further investigate the role of AcdS and the involvement of ethylene signaling pathway in plant development responses to rhizobacteria, we used the model plant Arabidopsis thaliana. We compared the changes in root architecture and root hair length induced by four rhizobacteria ( Phyllobacterium brassicacearum STM196, Pseudomonas putida UW4, Rhizobium leguminosarum bv. viciae 128C53K, Mesorhizobium loti MAFF303099) and by their respective acdS-deficient mutants. All the mutant strains induced similar changes in lateral root development as their WT counterparts. By contrast, root hairs of seedlings inoculated with the acdS mutant strains were significantly longer than those of the plants inoculated with the WT strains. Overall, our results would suggest that rhizobacterial AcdS activity affects local regulatory mechanisms in plant roots, and not lateral root development that is under systemic regulation involving shoot–root dialog.

Gene Identification and Characterization of the Pyridoxine Degradative Enzyme 4-Pyridoxic Acid Dehydrogenase from the Nitrogen-fixing Symbiotic Bacterium Mesorhizobium loti MAFF303099

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.

Enhanced symbiotic nitrogen fixation by Lotus japonicus containing an antisense .BETA.-1,3-glucanase gene

We hypothesized that root nodule formation would be promoted by lower expression of a b-1,3-glucanase gene (designated as LjGlu1), because expression of this gene is increased in transgenic Lotus japonicus that shows reduced nodulation. In order to suppress the expression of this gene, we introduced the antisense LjGlu1 gene into L. japonicus via Agrobacterium rhizogenes. Although there was no significant difference in shoot and root growth between L. japonicus possessing the antisense construct and plants containing an empty vector, the number of root nodules 28 days after inoculation with Mesorhizobium loti MAFF303099 increased in the antisense plant compared with the control plant. Moreover, the nitrogen fixation activity of the antisense plant was drastically enhanced. The mechanism of enhanced nitrogen fixation by suppression of the expression of LjGlu1 is unknown, but this phenomenon should be an important breeding target for leguminous plants. This is the first report of strong enhancement of nitrogen fixation activity by manipulation of a gene of the leguminous host plant.

Gene Identification and Structural Characterization of the Pyridoxal 5‘-Phosphate Degradative Protein 3-Hydroxy-2-methylpyridine-4,5-dicarboxylate Decarboxylase from Mesorhizobium loti MAFF303099,

The function of the mlr6791 gene from Mesorhizobium loti MAFF303099 has been identified. This gene encodes 3-hydroxy-2-methylpyridine-4,5-dicarboxylate decarboxylase (HMPDdc), an enzyme involved in the catabolism of pyridoxal 5'-phosphate (Vitamin B6). This enzyme was overexpressed in Escherichia coli and characterized. HMPDdc is a 26 kDa protein that catalyzes the decarboxylation of 3-hydroxy-2-methylpyridine-4,5-dicarboxylate to 3-hydroxy-2-methylpyridine-5-carboxylate. The KM and kcat were found to be 366 microM and 0.6 s-1, respectively. The structure of this enzyme was determined at 1.9 A resolution using SAD phasing and belongs to the class II aldolase/adducin superfamily. While the decarboxylation of hydroxy-substituted benzene rings is a common motif in biosynthesis, the mechanism of this reaction is still poorly characterized. The structural studies described here suggest that catalysis of such decarboxylations proceeds by an aldolase-like mechanism.

DNA adenine methylation changes dramatically during establishment of symbiosis

The DNA adenine methylation status on specific 5'-GANTC-3' sites and its change during the establishment of plant-microbe interactions was demonstrated in several species of alpha-proteobacteria. Restriction landmark genome scanning (RLGS), which is a high-resolution two dimensional DNA electrophoresis method, was used to monitor the genomewide change in methylation. In the case of Mesorhizobium loti MAFF303099, real RLGS images obtained with the restriction enzyme MboI, which digests at GATC sites, almost perfectly matched the virtual RLGS images generated based on genome sequences. However, only a few spots were observed when the restriction enzyme HinfI was used, suggesting that most GANTC (HinfI) sites were tightly methylated and specific sites were unmethylated. DNA gel blot analysis with the cloned specifically unmethylated regions (SUMs) showed that some SUMs were methylated differentially in bacteroids compared to free-living bacteria. SUMs have also been identified in other symbiotic and parasitic bacteria. These results suggest that DNA adenine methylation may contribute to the establishment and/or maintenance of symbiotic and parasitic relationships.

Molecular cloning, identification and characterization of 2-methyl-3-hydroxypyridine-5-carboxylic-acid-dioxygenase-coding gene from the nitrogen-fixing symbiotic bacterium Mesorhizobium loti

The gene (mlr6788) of a nitrogen-fixing symbiotic bacterium Mesorhizobium loti MAFF303099 has been identified as a gene coding for 2-methyl-3-hydroxypyridine-5-carboxylic acid dioxygenase (MHPCO), the seventh enzyme in degradation pathway I for pyridoxine, a free form of vitamin B(6). The gene was cloned and overexpressed in Escherichia coli cells co-transformed with chaperonin genes. The homogeneous recombinant enzyme showed similar enzymatic properties to the enzyme from Pseudomonas sp. MA-1. MHPCO was essential for the assimilation of pyridoxine in M. loti, but not for its growth in a nutrient-rich medium. From the infection experiment of a symbiotic plant Lotus japonicus with an M. loti mlr6788 gene disruptant, MHPCO was demonstrated to be dispensable for at least nodule formation on roots of seedlings in symbiosis.

4-Pyridoxolactonase from a symbiotic nitrogen-fixing bacterium Mesorhizobium loti: Cloning, expression, and characterization

4-Pyridoxolactonase is involved in the degradation pathway for pyridoxine, a free form of vitamin B 6. The gene (mlr6805) encoding the putative 4-pyridoxolactonase of nitrogen fixing symbiotic microorganism Mesorhizobium loti MAFF303099 has been identified based on the genome database. The gene was cloned and overexpressed in a cotransformant Escherichia coli cell. The recombinant enzyme was dimeric protein and contained one mole of Zn 2+ per mole of subunit. The enzyme showed about 30% identity with various N-acylhomoserine lactone lactonases and metallo-β-lactamases. The phylogram made with ClustalW shows that 4-pyridoxolactonase makes a cluster with Agrobacterium tumefaciens acyl-homoserine lactone lactonase. The alignment of amino acid sequences suggests that 4-pyridoxolactonase has three histidine residues probably involved in binding of Zn 2+.

Two Rhizobial Strains,Mesorhizobium lotiMAFF303099 andBradyrhizobium japonicumUSDA110, Encode Haloalkane Dehalogenases with Novel Structures and Substrate Specificities

Haloalkane dehalogenases are key enzymes for the degradation of halogenated aliphatic pollutants. Two rhizobial strains, Mesorhizobium loti MAFF303099 and Bradyrhizobium japonicum USDA110, have open reading frames (ORFs), mlr5434 and blr1087, respectively, that encode putative haloalkane dehalogenase homologues. The crude extracts of Escherichia coli strains expressing mlr5434 and blr1087 showed the ability to dehalogenate 18 halogenated compounds, indicating that these ORFs indeed encode haloalkane dehalogenases. Therefore, these ORFs were referred to as dmlA (dehalogenase from Mesorhizobium loti) and dbjA (dehalogenase from Bradyrhizobium japonicum), respectively. The principal component analysis of the substrate specificities of various haloalkane dehalogenases clearly showed that DbjA and DmlA constitute a novel substrate specificity class with extraordinarily high activity towards beta-methylated compounds. Comparison of the circular dichroism spectra of DbjA and other dehalogenases strongly suggested that DbjA contains more alpha-helices than the other dehalogenases. The dehalogenase activity of resting cells and Northern blot analyses both revealed that the dmlA and dbjA genes were expressed under normal culture conditions in MAFF303099 and USDA110 strain cells, respectively.

Symbiotic Rhizobium and Nitric Oxide Induce Gene Expression of Non-symbiotic Hemoglobin in Lotus japonicus

We characterized the expression profiles of LjHb1 and LjHb2, non-symbiotic hemoglobin (non-sym-Hb) genes of Lotus japonicus. Although LjHb1 and LjHb2 showed 77% homology in their cDNA sequences, LjHb2 is located in a unique position in the phylogenetic tree of plant Hbs. The 5'-upstream regions of both genes contain the motif AAAGGG at a position similar to that in promoters of other non-sym-Hb genes. Expression profiles obtained by using quantitative RT-PCR showed that LjHb1 and LjHb2 were expressed in all tissues of mature plants, and expression was enhanced in mature root nodules. LjHb1 was strongly induced under both hypoxic and cold conditions, and by the application of nitric oxide (NO) donor, whereas LjHb2 was induced only by the application of sucrose. LjHb1 was also induced transiently by the inoculation with the symbiotic rhizobium Mesorhizobium loti MAFF303099. Observations using fluorescence microscopy revealed the induction of LjHb1 expression corresponded to the generation of NO. These results suggest that non-sym-Hb and NO have important roles in stress adaptation and in the early stage of legume-rhizobium symbiosis.

The nitrogen-fixing symbiotic bacteriumMesorhizobium lotihas and expresses the gene encoding pyridoxine 4-oxidase involved in the degradation of vitamin B6

The gene product of mll6785 of a nitrogen-fixing symbiotic bacterium Mesorhizobium loti MAFF303099 was identified as pyridoxine 4-oxidase, the first enzyme in the vitamin B6-degradation pathway. The gene was cloned and ligated into pET-21a(+). Escherichia coli BL21(DE3) was co-transformed with the constructed plasmid plus pKY206 containing groESL genes encoding chaperonins. The overexpressed protein was purified to homogeneity by the ammonium sulfate fractionation and three chromatography steps. The enzymatic properties of the purified protein, such as Km values for pyridoxine (213 ± 19 μM) and oxygen (78 ± 10 μM), were compared to those of pyridoxine 4-oxidase from two bacteria with known vitamin B6-degradation pathway. M. loti grown in a Rhizobium medium showed the enzyme activity. The results suggest that M. loti also contains the degradation pathway of vitamin B6.

The nitrogen-fixing symbiotic bacterium Mesorhizobium loti has and expresses the gene encoding pyridoxine 4-oxidase involved in the degradation of vitamin B 6

The gene product of mll6785 of a nitrogen-fixing symbiotic bacterium Mesorhizobium loti MAFF303099 was identified as pyridoxine 4-oxidase, the first enzyme in the vitamin B 6-degradation pathway. The gene was cloned and ligated into pET-21a(+). Escherichia coli BL21(DE3) was co-transformed with the constructed plasmid plus pKY206 containing groESL genes encoding chaperonins. The overexpressed protein was purified to homogeneity by the ammonium sulfate fractionation and three chromatography steps. The enzymatic properties of the purified protein, such as K m values for pyridoxine (213 ± 19 μM) and oxygen (78 ± 10 μM), were compared to those of pyridoxine 4-oxidase from two bacteria with known vitamin B 6-degradation pathway. M. loti grown in a Rhizobium medium showed the enzyme activity. The results suggest that M. loti also contains the degradation pathway of vitamin B 6.