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.

Alcanivorax borkumensis is a cosmopolitan marine bacterium that uses oil hydrocarbons as its exclusive source of carbon and energy. Although barely detectable in unpolluted environments, A. borkumensis becomes the dominant microbe in oil-polluted waters. A. borkumensis SK2 has a streamlined genome with a paucity of mobile genetic elements and energy generation–related genes, but with a plethora of genes accounting for its wide hydrocarbon substrate range and efficient oil-degradation capabilities. The genome further specifies systems for scavenging of nutrients, particularly organic and inorganic nitrogen and oligo-elements, biofilm formation at the oil-water interface, biosurfactant production and niche-specific stress responses. The unique combination of these features provides A. borkumensis SK2 with a competitive edge in oil-polluted environments. This genome sequence provides the basis for the future design of strategies to mitigate the ecological damage caused by oil spills. Supplementary information The online version of this article (doi:10.1038/nbt1232) contains supplementary material, which is available to authorized users.

Sinorhizobium meliloti is an ␣-proteobacterium that forms agronomically important N 2-fixing root nodules in legumes. We report here the complete sequence of the largest constituent of its genome, a 62.7% GC-rich 3,654,135-bp circular chromosome. Annotation allowed assignment of a function to 59% of the 3,341 predicted protein-coding ORFs, the rest exhibiting partial, weak, or no similarity with any known sequence. Unexpectedly, the level of reiteration within this replicon is low, with only two genes duplicated with more than 90% nucleotide sequence identity, transposon elements accounting for 2.2% of the sequence, and a few hundred short repeated palindromic motifs (RIME1, RIME2, and C) widespread over the chromosome. Three regions with a significantly lower GC content are most likely of external origin. Detailed annotation revealed that this replicon contains all housekeeping genes except two essential genes that are located on pSymB. Amino acid͞peptide transport and degradation and sugar metabolism appear as two major features of the S. meliloti chromosome. The presence in this replicon of a large number of nucleotide cyclases with a peculiar structure, as well as of genes homologous to virulence determinants of animal and plant pathogens, opens perspectives in the study of this bacterium both as a free-living soil microorganism and as a plant symbiont.

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 ...

Sinorhizobium meliloti is an alpha-proteobacterium that alternates between a free-living phase in bulk soil or in the rhizosphere of plants and a symbiotic phase within the host plant cells, where the bacteria ultimately differentiate into nitrogen-fixing organelle-like cells, called bacteroids. As a step toward understanding the physiology of S. meliloti in its free-living and symbiotic forms and the transition between the two, gene expression profiles were determined under two sets of biological conditions: growth under oxic versus microoxic conditions, and in free-living versus symbiotic state. Data acquisition was based on both macro- and microarrays. Transcriptome profiles highlighted a profound modification of gene expression during bacteroid differentiation, with 16% of genes being altered. The data are consistent with an overall slow down of bacteroid metabolism during adaptation to symbiotic life and acquisition of nitrogen fixation capability. A large number of genes of unknown function, including potential regulators, that may play a role in symbiosis were identified. Transcriptome profiling in response to oxygen limitation indicated that up to 5% of the genes were oxygen regulated. However, the microoxic and bacteroid transcriptomes only partially overlap, implying that oxygen contributes to a limited extent to the control of symbiotic gene expression.

Azoarcus sp. strain BH72, a mutualistic endophyte of rice and other grasses, is of agrobiotechnological interest because it supplies biologically fixed nitrogen to its host and colonizes plants in remarkably high numbers without eliciting disease symptoms. The complete genome sequence is 4,376,040-bp long and contains 3,992 predicted protein-coding sequences. Genome comparison with the Azoarcus-related soil bacterium strain EbN1 revealed a surprisingly low degree of synteny. Coding sequences involved in the synthesis of surface components potentially important for plant-microbe interactions were more closely related to those of plant-associated bacteria. Strain BH72 appears to be 'disarmed' compared to plant pathogens, having only a few enzymes that degrade plant cell walls; it lacks type III and IV secretion systems, related toxins and an N-acyl homoserine lactones-based communication system. The genome contains remarkably few mobile elements, indicating a low rate of recent gene transfer that is presumably due to adaptation to a stable, low-stress microenvironment.Endophytic bacteria reside within the living tissue of plants without substantively harming them. They are of high interest for agrobiotechnological applications, such as the improvement of plant growth and health, phytoremediation 1 or even as biofertilizer 2 . Supply of nitrogen derived from fixation of atmospheric N 2 by grass endophytes, such as Gluconacetobacter diazotrophicus and Azoarcus sp. strain BH72, which has been shown to occur in sugarcane 3 and Kallar grass 2 , is a process of potential agronomical and ecological importance.Although the lifestyle of these endophytes is relatively well documented, the molecular mechanisms by which they interact beneficially with plants have only been poorly elucidated. A combination of features makes Azoarcus sp. strain BH72 an excellent model grassendophyte 4 . (i) It supplies nitrogen derived from N 2 fixation to its host, Kallar grass (Leptochloa fusca (L.) Kunth); in planta it is usually not culturable, but can be detected by culture-independent methods based on nifH-encoding nitrogenase reductase, the key enzyme for N 2 fixation 2 . (ii) It colonizes nondiseased plants in remarkably high numbers: estimates range from 10 8 cells (culturable cells per gram root dry weight (RDW) of field-grown Kallar grass 5 ) to 10 10 cells (estimated on the basis of abundance of bacterial nifH-mRNA in roots) 2 . (iii) It is the only cultured grass endophyte shown by molecular methods to be the most actively N 2 -fixing bacterium of the natural population in roots 2 . (iv) It also colonizes the roots of rice, a cereal of global importance, in high numbers (10 9 cells per g RDW) in the laboratory, and spreads systemically into shoots 6 . Plant stress response is only very limited in a compatible, that is, well-colonized rice cultivar 7 . Notably, Azoarcus sp. strain BH72 is capable of endophytic N 2 -fixation inside the roots of rice 8 .For a wider application in agriculture, more knowledge is required on mechanisms o...

A regulatory network of Sinorhizobium meliloti genes involved in adaptation to iron-limiting conditions and the involvement of the rhizobial iron regulator gene (rirA) were analyzed by mutation and microarray analyses. A constructed S. meliloti rirA mutant exhibited growth defects and enhanced H 2 O 2 sensitivity in the presence of iron, but symbiotic nitrogen fixation was not affected. To identify iron-responsive and RirA-regulated S. meliloti genes, a transcriptome approach using whole-genome microarrays was used. Altogether, 45 genes were found to be jointly derepressed by mutation of rirA and under different iron-limited conditions. As expected, a number of genes involved in iron transport (e.g., hmuPSTU, shmR, rhbABCDEF, rhtX, and rhtA) and also genes with predicted functions in energy metabolism (e.g., fixN3, fixP3, and qxtAB) and exopolysaccharide production (e.g., exoY and exoN) were found in this group of genes. In addition, the iron deficiency response of S. meliloti also involved rirA-independent expression changes, including repression of the S. meliloti flagellar regulon. Finally, the RirA modulon also includes genes that are not iron responsive, including a gene cluster putatively involved in Fe-S cluster formation (sufA, sufS, sufD, sufC, and sufB).

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