The scarcity of usable nitrogen frequently limits plant growth. A tight metabolic association with rhizobial bacteria allows legumes to obtain nitrogen compounds by bacterial reduction of dinitrogen (N2) to ammonium (NH4+). We present here the annotated DNA sequence of the alpha-proteobacterium Sinorhizobium meliloti, the symbiont of alfalfa. The tripartite 6.7-megabase (Mb) genome comprises a 3.65-Mb chromosome, and 1.35-Mb pSymA and 1.68-Mb pSymB megaplasmids. Genome sequence analysis indicates that all three elements contribute, in varying degrees, to symbiosis and reveals how this genome may have emerged during evolution. The genome sequence will be useful in understanding the dynamics of interkingdom associations and of life in soil environments.

Using physical and genetic data, we have demonstrated that Rhizobium meliloti SU47 has a symbiotic megaplasmid, pRmeSU47b, in addition to the previously described nod-nif megaplasmid pRmeSU47a. This plasm'id includes four loci involved in exopolysaccharide (exo) synthesis as well as two loci involved in thiamine 1'iosynthesis. Mutations at the exo loci have previously been shown to result in the formation of nodules which lack infection threads (Inf)' and fail to fix nitrogen (Fix-). Thus, both megaplasmids contain genes involved in the formation of nitrogen-fixing root nodules. Mutations at two other exo loci were not located on either megaplasmid. To mobilize the megaplasmids, the oriT of plasmid RK2 was inserted into them. On alfalfa, Agrobacterium tumefaciens strains containing pRmeSU47a induced marked root hair curling with no infection threads and Fix-nodules, as reported by others. This plant phenotype was not observed to change with A. tumefaciens strains containing both pRmeSU47a and pRmeSU47b megaplasmids, and strains containing pRmeSU47b alone failed to curl root hairs or form nodules.A great many natural isolates of rhizobia, agrobacteria, and pseudomonads have been shown to carry a variety of large plasmids (for examples, see references 8, 21, and 38). Some of these are megaplasmids, with molecular masses over 450 megadaltons (38). Genes for a few functions have been localized to these large plasmids, notably including pathogenicity gepes for the Ti and Ri plasmids of agrobacteria (6, 21) and symbiotic nodulation (nod) and nitrogen fixation (nif) genes for the Sym megaplasmids of the fast-growing rhizobia (3,8,28,33,37,38). Nevertheless, the significance of this genomic organization remains obscure, although the fact that these are all plant-associated soil bacteria does suggest an underlying evolutionary cause.Rhizobial Sym megaplasmids are being characterized extensively with regard to symbiosis. In addition, Rhizobium meliloti 41 has recently been shown to carry a second megaplasmid, with a molecular weight very nearly that of pSym, on which a region for surface exclusion (although none for symbiotic functions) has tentatively been identified (2). A second megaplasmid has also been identified in R.

Approximately 10% of bacterial genomes are split between two or more large DNA fragments, a genome architecture referred to as a multipartite genome. This multipartite organization is found in many important organisms, including plant symbionts, such as the nitrogen-fixing rhizobia, and plant, animal, and human pathogens, including the genera ,, and . The availability of many complete bacterial genome sequences means that we can now examine on a broad scale the characteristics of the different types of DNA molecules in a genome. Recent work has begun to shed light on the unique properties of each class of replicon, the unique functional role of chromosomal and nonchromosomal DNA molecules, and how the exploitation of novel niches may have driven the evolution of the multipartite genome. The aims of this review are to (i) outline the literature regarding bacterial genomes that are divided into multiple fragments, (ii) provide a meta-analysis of completed bacterial genomes from 1,708 species as a way of reviewing the abundant information present in these genome sequences, and (iii) provide an encompassing model to explain the evolution and function of the multipartite genome structure. This review covers, among other topics, salient genome terminology; mechanisms of multipartite genome formation; the phylogenetic distribution of multipartite genomes; how each part of a genome differs with respect to genomic signatures, genetic variability, and gene functional annotation; how each DNA molecule may interact; as well as the costs and benefits of this genome structure.

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Analysis of the 1,683,333-nt sequence of the pSymB megaplasmid from the symbiotic N2-fixing bacterium Sinorhizobium meliloti revealed that the replicon has a high gene density with a total of 1,570 protein-coding regions, with few insertion elements and regions duplicated elsewhere in the genome. The only copies of an essential arg-tRNA gene and the minCDE genes are located on pSymB. Almost 20% of the pSymB sequence carries genes encoding solute uptake systems, most of which were of the ATP-binding cassette family. Many previously unsuspected genes involved in polysaccharide biosynthesis were identified and these, together with the two known distinct exopolysaccharide synthesis gene clusters, show that 14% of the pSymB sequence is dedicated to polysaccharide synthesis. Other recognizable gene clusters include many involved in catabolic activities such as protocatechuate utilization and phosphonate degradation. The functions of these genes are consistent with the notion that pSymB plays a major role in the saprophytic competence of the bacteria in the soil environment.A mong the bacteria, the ␣-proteobacteria appear unusual because of the presence of multiple replicons within the same bacterial strain (1). In the case of Agrobacterium tumefaciens, the causative agent of crown gall disease, the genome contains both a linear and a circular chromosome (2). Many (but not all) of the bacteria that form N 2 -fixing root nodules on leguminous plants are characterized by the presence of multiple plasmids greater than 400 kb in size. In the case of the N 2 -fixing symbiont Sinorhizobium meliloti, there are three replicons, a 3,654-kb circular chromosome (3, 4) and two megaplasmids 1,354 and 1,683 kb in size (5-7). The smaller of the megaplasmids, variously called pSymA, pNod-Nif, or pRmeSU47a, is known to carry many of the genes involved in root nodule formation (nod) and nitrogen fixation (nif ) (8, 9).The 1,683-kb megaplasmid, referred to as pSymB, pExo, or pRmeSU47b, is known to carry various gene clusters involved in exopolysaccharide (EPS) synthesis, C 4 -dicarboxylate transport, and lactose metabolism (10-12). Early studies focused on mutations that abolished synthesis of the succinoglycan EPS, EPS I, because these mutations resulted in a loss of the ability to form normal N 2 -fixing root nodules. This symbiotic defect was rescued by second-site mutations that increased the synthesis of a second galactoglucan EPS (EPS II), whose biosynthetic genes were also located on the pSymB megaplasmid (13,14). Other genes located on pSymB that are required for the formation of N 2 -fixing root nodules include the C 4 -dicarboxylate (dctA) and phosphate transport (phoCDET) genes and the bacA gene (15-18). The presence of large plasmids in bacteria that form associations with plants was described over 20 years ago (19). However, with the exception of the symbiotic genes in relatively small regions of these plasmids, the broader biological role of the plasmids in the biology of the organism has remained obscure. We constructed a ...

Bacteria react to phosphate starvation by activating genes involved in the transport and assimilation of phosphate as well as other phosphorous compounds. Some soil bacteria have evolved an additional mechanism for saving phosphorus. Under phosphate‐limiting conditions, they replace their membrane phospholipids by lipids not containing phosphorus. Here, we show that the membrane lipid pattern of the free‐living microsymbiotic bacterium Rhizobium (Sinorhizobium) meliloti is altered at low phosphate concentrations. When phosphate is growth limiting, an increase in sulpholipids, ornithine lipids and the de novo synthesis of diacylglyceryl trimethylhomoserine (DGTS) lipids is observed. Rhizobium meliloti phoCDET mutants, deficient in phosphate uptake, synthesize DGTS constitutively at low or high medium phosphate concentrations, suggesting that reduced transport of phosphorus sources to the cytoplasm causes induction of DGTS biosynthesis. Rhizobium meliloti phoU or phoB mutants are unable to form DGTS at low or high phosphate concentrations. However, the functional complementation of phoU or phoB mutants with the phoB gene demonstrates that, of the two genes, only intact phoB is required for the biosynthesis of the membrane lipid DGTS.

The number of solute-binding protein-dependent transporters in rhizobia is dramatically increased compared with the majority of other bacteria so far sequenced. This increase may be due to the high affinity of solute-binding proteins for solutes, permitting the acquisition of a broad range of growth-limiting nutrients from soil and the rhizosphere. The transcriptional induction of these transporters was studied by creating a suite of plasmid and integrated fusions to nearly all ATP-binding cassette (ABC) and tripartite ATP-independent periplasmic (TRAP) transporters of Sinorhizobium meliloti. In total, specific inducers were identified for 76 transport systems, amounting to Ϸ47% of the ABC uptake systems and 53% of the TRAP transporters in S. meliloti. Of these transport systems, 64 are previously uncharacterized in Rhizobia and 24 were induced by solutes not known to be transported by ABC-or TRAP-uptake systems in any organism. This study provides a global expression map of one of the largest transporter families (transportome) and an invaluable tool to both understand their solute specificity and the relationships between members of large paralogous families.ATP-binding cassette ͉ expression ͉ high throughput ͉ transport ͉ tripartite ATP-independent periplasmic

A 5.1 kbp DNA fragment was isolated which complemented C4-dicarboxylate transport mutants (dct) of Rhizobium meliloti. Characterization of this fragment by subcloning, transposon mutagenesis, and complementation analysis revealed three loci, designated dctA, dctB, and dctD. TnphoA-generated alkaline phosphatase fusions to dctA suggested that this gene encodes the structural transport protein and allowed the determination of its direction of transcription. Analysis of the fusions in various mutant backgrounds demonstrated that dctB, dctD, and ntrA products are required for dctA expression. The dctA fusion was constitutively expressed in a dctA mutant background, but was not expressed in dctA dctB or dctA dctD double mutants. This suggests that the constitutive expression in a dctA mutant background is mediated through dctB and dctD. Three independent second-site Dct+ revertant mutations in ntrA mutant strains mapped to the dct locus. Succinate transport in these revertant strains was constitutive, whereas in the wild type, succinate transport was inducible. These results are consistent with the direct requirement of the ntrA gene product for dctA expression. Alfalfa plants inoculated with the dctB and dctD mutants showed reduced nitrogen-fixing activity. Nodules induced by dctA mutants failed to fix nitrogen. These symbiotic phenotypes are consistent with previous suggestions that dctA expression in bacteroids can occur independently of dctB and dctD.