The Mesorhizobium loti strain R7A symbiosis island is a 502-kb chromosomally integrated element which transfers to nonsymbiotic mesorhizobia in the environment, converting them to Lotus symbionts. It integrates into a phenylalanine tRNA gene in a process mediated by a P4-type integrase encoded at the left end of the element. We have determined the nucleotide sequence of the island and compared its deduced genetic complement with that reported for the 611-kb putative symbiosis island of M. loti strain MAFF303099. The two islands share 248 kb of DNA, with multiple deletions and insertions of up to 168 kb interrupting highly conserved colinear DNA regions in the two strains. The shared DNA regions contain all the genes likely to be required for Nod factor synthesis, nitrogen fixation, and island transfer. Transfer genes include a trb operon and a cluster of potential tra genes which are also present on the strain MAFF303099 plasmid pMLb. The island lacks plasmid replication genes, suggesting that it is a site-specific conjugative transposon. The R7A island encodes a type IV secretion system with strong similarity to the vir pilus from Agrobacterium tumefaciens that is deleted from MAFF303099, which in turn encodes a type III secretion system not found on the R7A island. The 414 genes on the R7A island also include putative regulatory genes, transport genes, and an array of metabolic genes. Most of the unique hypothetical genes on the R7A island are strain-specific and clustered, suggesting that they may represent other acquired genetic elements rather than symbiotically relevant DNA.The symbiosis between legumes and the root nodule bacteria collectively known as rhizobia is of critical agronomic and environmental importance, accounting for the majority of the nitrogen fixed through biological processes. Rhizobia are phylogenetically diverse, falling into five genera of ␣-proteobacteria (Rhizobium, Bradyrhizobium, Sinorhizobium, Azorhizobium, and Mesorhizobium) (64, 70) and at least two genera of ␤-proteobacteria (Burkholderia and Ralstonia) (35). It is thought that the rhizobial lineages diverged well before the evolution of legumes and that the genes required for the formation of the symbiosis were subsequently acquired by lateral transfer from undefined sources (8,33). Reflecting the accessory nature of the traits, several species of rhizobia contain the genes required for nodulation and nitrogen fixation on large plasmids that can be cured under laboratory conditions without affecting the survival of the bacteria (31). Exceptions in which the symbiosis genes are encoded on the chromosome include Bradyrhizobium species, in which the symbiosis genes are clustered but not known to be mobile (18), and at least one strain of Mesorhizobium loti, strain ICMP3153, in which the genes are located on a mobile symbiosis island (57).M. loti is the microsymbiont of several Lotus species, including Lotus corniculatus and L. japonicus. The symbiosis island of M. loti strain ICMP3153 was discovered through its ability to transfe...

We report the purification and biochemical characterization of the cobalamin biosynthetic enzyme nicotinate-mononucleotide:5,6-dimethylbenzimidazole phosphoribosyltransferase (CobT) from Salmonella typhimurium. cobT was overexpressed and the protein purified to approximately 97% homogeneity. NH 2 -terminal sequence analysis confirmed that the protein encoded by cobT was purified. Homogeneous CobT catalyzed the synthesis of N 1 -(5-phospho-␣-D-ribosyl)-5,6-dimethylbenzimidazole. The identity of high performance liquid chromatography-purified product was confirmed by fast atom bombardment mass spectrometry. CobT activity was optimal at 45°C and pH 10.0. The apparent K m for nicotinate mononucleotide was 680 M; the apparent K m for 5,6-dimethylbenzimidazole was less than 10 M. CobT used nicotinamide mononucleotide as a ribose phosphate donor. CobT phosphoribosylated alternative base substrates including benzimidazole, 4,5-dimethyl-1,2-phenylenediamine, imidazole, histidine, adenine, and guanine in vitro. The resulting ribotides were incorporated into cobamides that were differentially utilized by methionine synthase (EC 2.1.1.13), ethanolamine ammonia-lyase (EC 4.3.1.7), and 1,2-propanediol dehydratase (EC 4.2.1.28) in vivo. The lack of base substrate specificity by CobT may explain the inability to isolate mutants blocked in the synthesis of 5,6-dimethylbenzimidazole in this bacterium.

We present in vitro evidence which demonstrates that CobT is the nicotinate nucleotide:5,6-dimethylbenzimidazole (DMB) Most of the cobalamin biosynthetic (cob) genes of Salmonella typhimurium make up a large 17-kb operon located at 41 min of the chromosome (9, 16). The results of nutritional analyses performed with cob mutants with lesions in the 41-min region yielded three phenotypically distinct classes of strains. The mutants were classified as being blocked in the synthesis of adenosyl-cobinamide, auxotrophic for 5,6-dimethylbenzimidazole (DMB), or unable to synthesize adenosyl-cobalamin from its precursors adenosyl-cobinamide and DMB; i.e., these mutants cannot assemble the nucleotide loop of cobalamin which joins DMB to the corrin ring (6, 9,14). Previous reports refer to these mutant classes as cobI, cobII, and cobIII, respectively (9).Analysis of the DNA sequence of the region encoding the functions necessary for nucleotide loop assembly suggested that it was made up of three open reading frames designated cobU, cobS, and cobT (16). These genes were shown to direct the synthesis of three polypeptides with molecular masses that were in good agreement with those predicted by the DNA sequence (14). On the basis of homology to Pseudomonas denitrificans cob genes with assigned biochemical activities, the following functions for the CobU, CobT, and CobS proteins were proposed: (i) CobU, a bifunctional ATP:adenosyl-cobinamide kinase, GTP:adenosyl-cobinamide-phosphate guanylyltransferase whose final product is adenosyl-cobinamide-GDP; (ii) CobT, the nicotinate nucleotide (NaMN):DMB phospho-* Corresponding author. Mailing address:

No abstract

Eleven Sinorhizobium meliloti 1021 loci whose expression was induced under low oxygen concentrations were identified in a collection of 5,000 strains carrying Tn5-1063 (luxAB) transcriptional reporter gene fusions. The 11 Tn5-1063-tagged loci were cloned and characterized. The dependence of the expression of the tagged loci on the FixL/FixJ oxygen-sensing two-component regulatory system was examined. Three of the loci were found to be dependent upon fixL and fixJ for their expression, while one locus showed a partial dependence. The remaining seven loci showed fixL-and fixJ-independent induction of expression in response to oxygen limitation. This suggests that in S. meliloti, additional regulatory system(s) exist that respond either directly or indirectly to oxygen limitation conditions.