Rhizobium sp. strain NGR234 is a unique alphaproteobacterium (order Rhizobiales) that forms nitrogenfixing nodules with more legumes than any other microsymbiont. We report here that the 3.93-Mbp chromosome (cNGR234) encodes most functions required for cellular growth. Few essential functions are encoded on the 2.43-Mbp megaplasmid (pNGR234b), and none are present on the second 0.54-Mbp symbiotic plasmid (pNGR234a). Among many striking features, the 6.9-Mbp genome encodes more different secretion systems than any other known rhizobia and probably most known bacteria. Altogether, 132 genes and proteins are linked to secretory processes. Secretion systems identified include general and export pathways, a twin arginine translocase secretion system, six type I transporter genes, one functional and one putative type III system, three type IV attachment systems, and two putative type IV conjugation pili. Type V and VI transporters were not identified, however. NGR234 also carries genes and regulatory networks linked to the metabolism of a wide range of aromatic and nonaromatic compounds. In this way, NGR234 can quickly adapt to changing environmental stimuli in soils, rhizospheres, and plants. Finally, NGR234 carries at least six loci linked to the quenching of quorum-sensing signals, as well as one gene (ngrI) that possibly encodes a novel type of autoinducer I molecule.Diverse soil bacteria interact with plants in ways that range from symbiotic to pathogenic. Symbiotic Eubacteria (both alpha-and betaproteobacteria, collectively called rhizobia) form nitrogen-fixing associations of tremendous environmental importance (41, 66). Although some rhizobia are able to reduce atmospheric nitrogen to ammonia under saprophytic, free-living conditions, the reduced oxygen tensions found within the intracellular environment of specialized organs called nodules, maximizes this process (16). As legume roots penetrate the soil, they come in contact with rhizobia. Symbiotic interactions are initiated by the exchange of diverse molecules between the partners. Among them, plants liberate flavonoids into the rhizosphere that upregulate rhizobial genes. As a result, lipo-chito-oligo-saccharidic Nod factors are produced that trigger the nodulation pathway in susceptible legumes. Then, in many hosts, rhizobia enter the roots through root hairs, make their way to the cortex, multiply and fill the intracellular spaces of mature nodules. Centripetal progression of rhizobia into the plant and their maturation into nitrogen-fixing symbiosomes depends on the continued exchange of diverse signals.Many, but not all of these signals have been identified; one sure way to take stock of what is necessary for effective symbiosis is to sequence the partners. We began this work by assembling overlapping sets of cosmids (contigs) of the microsymbiont Rhizobium sp. strain NGR234 (hereafter NGR234) (63), which enabled us to elucidate the nucleotide sequence of the symbiotic (pNGR243a) plasmid (29). Similar techniques permitted the assembly of sections of th...

Rhizobium leguminosarum bv. phaseoli CFN42 contains six plasmids (pa to pf), and pd has been shown to be the symbiotic plasmid. To determine the participation of the other plasmids in cellular functions, we used a positive selection scheme to isolate derivatives cured of each plasmid. These were obtained for all except one (pe), of which only deleted derivatives were recovered. In regard to symbiosis, we found that in addition to pd, pb is also indispensable for nodulation, partly owing to the presence of genes involved in lipopolysaccharide synthesis. The positive contribution of pb, pc, pe, and pf to the symbiotic capacity of the strain was revealed in competition experiments. The strains that were cured (or deleted for pe) were significantly less competitive than the wild type. Analysis of the growth capacity of the cured strains showed the participation of the plasmids in free-living conditions: the pf- strain was unable to grow on minimal medium, while strains cured of any other plasmid had significantly reduced growth capacity in this medium. Even on rich medium, strains lacking pb or pc or deleted for pe had a diminished growth rate compared with the wild type. Complementation of the cured strains with the corresponding wild-type plasmid restored their original phenotypes, thus confirming that the effects seen were due only to loss of plasmids. The results indicate global participation of the Rhizobium genome in symbiotic and free-living functions.

The complete coding sequence of the nitro-

Several lines of evidence suggest that reiterated sequences in the human genome are targets for nonallelic homologous recombination (NAHR), which facilitates genomic rearrangements. We have used a PCR-based approach to identify breakpoint regions of rearranged structures in the human genome. In particular, we have identified intrachromosomal identical repeats that are located in reverse orientation, which may lead to chromosomal inversions. A bioinformatic workflow pathway to select appropriate regions for analysis was developed. Three such regions overlapping with known human genes, located on chromosomes 3, 15, and 19, were analyzed. The relative proportion of wild-type to rearranged structures was determined in DNA samples from blood obtained from different, unrelated individuals. The results obtained indicate that recurrent genomic rearrangements occur at relatively high frequency in somatic cells. Interestingly, the rearrangements studied were significantly more abundant in adults than in newborn individuals, suggesting that such DNA rearrangements might start to appear during embryogenesis or fetal life and continue to accumulate after birth. The relevance of our results in regard to human genomic variation is discussed.

Repeated DNA sequences are a general characteristic of eucaryotic genomes. Although several examples of DNA reiteration have been found in procaryotic organisms, only in the case of the archaebacteria Halobacterium halobium and Halobacterium volcanii [C. Sapienza and W. F. Doolittle, Nature (London) 295: [384][385][386][387][388][389] 1982], has DNA reiteration been reported as a common genomic feature. (1,13,14,17,23,24,30,39,49), but it has been demonstrated as a common genomic feature only in a few cases. For instance, Sapienza and Doolittle (42) showed that DNA reiteration is a common feature in the genome of the archaebacteria Halobacterium halobium and Halobacterium volcanii.We have previously reported that nitrogen fixation genes (nif genes) are reiterated in Rhizobium phaseoli (33,34) MATERIALS AND METHODSBacterial strains and plasmids. The bacterial strains and plasmids used are listed in Table 1.Molecular cloning. To prepare bacterial gene libraries, total DNA from each strain was digested with EcoRI restriction endonuclease and the fragments were cloned in the EcoRI site of pBR329 by using Escherichia coli MC1061 as a recipient for transformation.Filter blot hybridization. Total DNA digested with EcoRI restriction endonuclease was subjected to electrophoresis in a 1% agarose gel and blotted onto nitrocellulose by the method of Southern (48). Recombinant plasmids were labeled with 32P by nick translation (36) at high specific activity (108 cpm/,Lg of DNA) and used as probes for hybridization.For each lane, 2 x 106 cpm of probe DNA was used. The blots were prehybridized for 2 h at 65TC in 5 x SSC (1 x SSC is 0.15 M NaCl plus 0.015 M sodium citrate) containing 0.2% Ficoll (Pharmacia Fine Chemicals, Piscataway, N.J.), 0.2% bovine serum albumin, 0.2% polyvinylpirrolidone, 0.1 M phosphates (pH 6.7), and 0.05 mg of salmon sperm DNA per ml. The blots were then hybridized for 16 h at 65°C in the same solution with the labeled probe. The filters were washed once with 2 x SSC containing 0.01% sodium dodecyl sulfate for 10 min at room temperature and three times with 0.1 x SSC containing 0.1% sodium dodecyl sulfate for 30 min at 50°C. Autoradiography in the presence of intensifying screens was carried out at -70°C for 24 h.Transfer of R. phaseoli plasmids to A. tumefaciens. R. phaseoli CFN-42 and CFN-285 were randomly mutagenized with TnS-mob (45). Different Kmr derivatives of each strain were mated with the plasmidless A. tumefaciens GM19023 in triparental matings by using pJB3JI as a helper plasmid for mobilization. Agrobacterium transconjugants were analyzed for plasmid profiles by the Eckhardt procedure (12), and strains carrying each different plasmid were selected. A