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.

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 ...

BlnI or AvrII (5'-CCTAGG) sites are very rare in the Salmonella typhimurium LT2 genome. BlnI was used to construct a physical map which was correlated with the genetic map by using three methods. First, TnlO carries BlnI sites, and the extra restriction sites produced by 34 genetically mapped TnlO insertions were physically mapped by using pulsed-field gel electrophoresis. Second, six genetically mapped Mud-P22 prophage insertions were used to assign BlnI fragments. Integration of Mud-P22 introduces 30 kb of DNA that can easily be detected by a "shift up" in all but the largest BlnI fragments. Finally, induced Mud-P22 insertions package more than 100 kb of genomic DNA adjacent to one side of the insertion. Some of the smaller BlnI fragments were localized by hybridization to a dot blot array of 52 lysates from induced Mud-P22 insertions. Of the 10 BlnI sites mapped, 6 probably occur in or near the 16S rRNA genes at about 55, 71, 83, 86, 88.5, and 89.5 A number of restriction maps of bacterial genomes have been constructed by using endonucleases that cleave infrequently (22) and then separating the resulting fragments by pulsed-field gel electrophoresis (PFGE) (2,5,7,8,10,17,19,20,(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(36)(37)(38)(39). The physical order of these fragments has usually been determined by using Southern blotting with genetically mapped probes such as cloned genes (34) or transposons (38) or by hybridization of fragments from one restriction digest to fragments from a different digest (1, 4). An alternative strategy employs a genetically mapped transposon integration that carries a rare restriction site that can be used to cleave and thus locate a restriction fragment in the genetic map (22,35). In this paper, we took advantage of the serendipitous occurrence of two BlnI sites in the transposon TnlO. BinI sites are very rare in enterobacterial genomes (22). We correlated genetically mapped TnlO insertions with the BlnI physical map of Salmonella typhimurium LT2. In addition, Mud-P22 prophage insertions (42), which lack BlnI sites, introduce 30 kb and were used to monitor changes in the length of BlnI fragments. This correlated genetically mapped Mud-P22 insertions with the BlnI physical map.While the methods mentioned above can quickly locate the largest restriction fragments, smaller fragments are less likely to contain a transposon integration or to be detected by probing with cloned genes. The position of a small fragment is likely to be only crudely mapped by blotting to pulsed-field digests. We had available a panel of 52 genetically mapped Mud-P22 integrations. These can be induced to amplify and package 100 kb or more in one direction from the site of integration (42). We used these induced lysates to produce a dot blot array which could then be probed with the smaller BlnI restriction fragments. MATERUILS AND METHODSPhage and bacterial strains. Most of the strains used are listed in Tables 2 and 3. Strain MS1017 (12) lacks the * Corresponding author. mitomycin C-inducible Fels-2 prophage...

Microbial genomes are thought to be mosaic, making it difficult to decipher how these genomes have evolved. Whole-genome nearest-neighbor analysis was applied to the Sinorhizobium meliloti pSymB replicon to determine its origin, the degree of horizontal transfer, and the conservation of gene order. Prediction of the nearest neighbor based on contextual information, i.e., the nearest phylogenetic neighbor of adjacent genes, provided useful information for genes for which phylogenetic relationships could not be established. A large portion of pSymB genes are most closely related to genes in the Agrobacterium tumefaciens linear chromosome, including the rep and min genes. This suggests a common origin for these replicons. Genes with the nearest neighbor from the same species tend to be grouped in "patches". Gene order within these patches is conserved, but the content of the patches is not limited to operons. These data show that 13% of pSymB genes have nearest neighbors in species that are not members of the Rhizobiaceae family (including two archaea), and that these likely represent genes that have been involved in horizontal transfer.