Genetic relationship of Sinorhizobium meliloti and Sinorhizobium medicae strains isolated from Caucasian region

Sinorhizobium meliloti and Sinorhizobium medicae are two closely related species of the genus Sinorhizobium showing a similar host range, nodulating leguminous species of the genera Medicago, Melilotus and Trigonella, but their phylogenic relationship has not been elucidated yet. In this paper we report the application of three different molecular markers, (i) RFLP of nodD genes, (ii) 16S-23S rDNA intergenic gene spacer fingerprinting and (iii) amplification fragment length polymorphism to S. meliloti and S. medicae strains isolated from the Caucasian area, which is the region of origin of the host plant Medicago. The analysis of data could suggest the origin of S. medicae strains from an ancestral S. meliloti population.     

Characterization of an indican-hydrolyzing enzyme from Sinorhizobium meliloti

A novel β-glucosidase capable of hydrolyzing indican to indigo was mined and isolated from Sinorhizobium meliloti using a systematic approach. The corresponding gene was amplified by PCR and overexpressed in the soluble fraction as an MBP fusion protein. The resulting enzyme easily purified to apparent homogeneity via a consecutive step in the affinity column. The recombinant enzyme was determined to be a monomer with a calculated molecular mass of 52 kDa and showed the maximum activity for indican at pH 7.0 and 45 °C. The kinetic parameters for indican, K_M and V_max, were determined to be 0.97 mM and 355.6 μM/min/mg protein, respectively, at pH 7.0 and 35 °C. Additionally, this enzyme hydrolyzed both the β-(1-4)- and β-(1-6)-glucosidic bonds and revealed a minor activity against α-d-glucosides. Furthermore, the enzyme was severely inhibited by DTT, indicating a possibility that the oxidation of amino acids could play a crucial role in the activity of the enzyme.     

Functional and topological analysis of phosphatidylcholine synthase from Sinorhizobium meliloti

Phosphatidylcholine (PC) is the major membrane-forming phospholipid in eukaryotes and is estimated to be present in about 15% of eubacteria. It can be synthesized in bacteria by either of two pathways, the phospholipid N-methylation pathway or the phosphatidylcholine synthase (Pcs) pathway. Pcs belongs to the CDP-alcohol phosphotransferase superfamily and synthesizes PC and CMP in one step from CDP-diacylglycerol and choline. In this study, we aligned sequences of characterized Pcs enzymes to identify conserved amino acid residues. Alanine scanning mutagenesis was performed on 55 of these conserved residues. The mutation of nine residues caused a drastic to complete loss (<20% of wild type activity) of Pcs activity. Six of these essential residues were subjected to further mutagenesis studies replacing them by amino acids with similar properties or size. A topological analysis of sinorhizobial Pcs showed the presence of eight transmembrane helices, with the C- and N-terminus located in the cytoplasm. The majority of the conserved residues is predicted to be either located within the cytoplasmic loops or on the cytoplasmic side of the membrane which can be expected for an enzyme using one membrane-associated and one soluble substrate.     

Crystal structure of the flavoprotein ArsH from Sinorhizobium meliloti

Purified ArsH from Sinorhizobium meliloti exhibits NADPH:FMN-dependent reduction of molecular O2 to hydrogen peroxide and catalyzes reduction of azo dyes. The structure of ArsH was determined at 1.8A resolution. ArsH crystallizes with eight molecules in the asymmetric unit forming two tetramers. Each monomer has a core domain with a central five-stranded parallel beta-sheet and two monomers interact to form a classical flavodoxin-like dimer. The N- and C-terminal extensions of ArsH are involved in interactions between subunits and tetramer formation. The structure may provide insight in how ArsH participates in arsenic detoxification.     

Characterization of cis-4-hydroxy-D-proline dehydrogenase from Sinorhizobium meliloti

The hypO gene from Sinorhizobium meliloti, located within the trans-4-hydroxy-L-proline metabolic gene cluster, was first successfully expressed in the host Pseudomonas putida. Purified HypO protein functioned as a FAD-containing cis-4-hydroxy-D-proline dehydrogenase with a homomeric structure. In contrast to other known enzymes, significant activity for D-proline was found, confirming a previously proposed potential involvement in D-proline metabolism.     

Symbiotic induction of pyruvate dehydrogenase genes from Sinorhizobium meliloti

Genes coding for components of the pyruvate dehydrogenase (PDH) multienzyme complex (PDHc) from Sinorhizobium meliloti, the alfalfa symbiont, have been isolated on the basis of their high expression in symbiotic bacteria. The Elp component, PDH, is encoded by two genes, pdhAalpha (1,047 bp) and pdhAbeta (1,383 bp), a situation encountered in the alpha-proteobacteria Rickettsia prowazekii and Zymomonas mobilis as well as in some gram-positive bacteria and in mitochondria. pdhAalpha and pdhAbeta precede pdhB (1,344 bp), which encodes the E2p component, dihydrolipoamide acetyltransferase, of the PDHc. No gene encoding the E3 component, lipoamide dehydrogenase, was found in the immediate vicinity of pdhA and pdhB genes. pdhAalpha, pdhAbeta and pdhB likely constitute an operon. Here, we provide evidence that pdhA expression is induced in the symbiotic stage, compared with free-living conditions. We demonstrate that symbiotic expression of pdhA genes does not depend on the fix LJ regulatory cascade that regulates nitrogen fixation and respiration gene expression in symbiotic S. meliloti cells. Induction of pdhA expression could be obtained under free-living conditions upon the addition of pyruvate to the culture medium. Induction by pyruvate and symbiotic activation of pdh gene expression take place at the same promoter.     

Characterization of an Atypical Superoxide Dismutase from Sinorhizobium meliloti

Sinorhizobium meliloti Rm5000 is an aerobic bacterium that can live free in the soil or in symbiosis with the roots of leguminous plants. A single detectable superoxide dismutase (SOD) was found in free-living growth conditions. The corresponding gene was isolated from a genomic library by using a sod fragment amplified by PCR from degenerate primers as a probe. The sodA gene was located in the chromosome. It is transcribed monocistronically and encodes a 200-amino-acid protein with a theoretical M(r) of 22,430 and pI of 5. 8. S. meliloti SOD complemented a deficient E. coli mutant, restoring aerobic growth of a sodA sodB recA strain, when the gene was expressed from the synthetic tac promoter but not from its own promoter. Amino acid sequence alignment showed great similarity with Fe-containing SODs (FeSODs), but the enzyme was not inactivated by H(2)O(2). The native enzyme was purified and found to be a dimeric protein, with a specific activity of 4,000 U/mg. Despite its Fe-type sequence, atomic absorption spectroscopy showed manganese to be the cofactor (0.75 mol of manganese and 0.24 mol of iron per mol of monomer). The apoenzyme was prepared from crude extracts of S. meliloti. Activity was restored by dialysis against either MnCl(2) or Fe(NH(4))(2)(SO(4))(2), demonstrating the cambialistic nature of the S. meliloti SOD. The recovered activity with manganese was sevenfold higher than with iron. Both reconstituted enzymes were resistant to H(2)O(2). Sequence comparison with 70 FeSODs and MnSODs indicates that S. meliloti SOD contains several atypical residues at specific sites that might account for the activation by manganese and resistance to H(2)O(2) of this unusual Fe-type SOD.     

苜蓿中华根瘤菌042BM noeAB基因的转录调控

对苜蓿中华根瘤菌(Sinorhizobiummeliloti) 0 4 2BMnoeAB基因的表达调控进行研究。结果发现,葫芦巴碱不能使noeAB的表达水平提高,证明它们的转录不受nodD2的调控。当nodD3和syrM同时存在时,noeAB的表达水平没有明显的变化,表明它们也不受nodD3 syrM系统的调控。在FY基本培养基上,毛地黄黄酮的诱导使noeAB基因的表达水平提高16倍,而在不添加该诱导物的TY培养基上,noeAB基因的表达水平也能够提高30倍以上,说明noeAB是受nodD1控制的,但除受毛地黄黄酮诱导外,noeAB还可能受到其他因子的调节     

Control of Gluconate Utilization in Sinorhizobium meliloti

The Sinorhizobium meliloti megaplasmid pSymA has previously been implicated in gluconate utilization. We report a locus on pSymA encoding a putative tripartite ATP-independent periplasmic (TRAP) transporter that is required for gluconate utilization. The expression of this locus is negatively regulated by a GntR family regulator encoded adjacent to the transporter operon.