The physical and genetic map of the Bradyrhizobium japonicum chromosome revealed that nitrogen fixation and nodulation genes are clustered. Because of the complex interactions between the bacterium and the plant, we expected this chromosomal sector to contain additional genes that are involved in the maintenance of an efficient symbiosis. Therefore, we determined the nucleotide sequence of a 410-kb region. The overall G؉C nucleotide content was 59.1%. Using a minimum gene length of 150 nucleotides, 388 open reading frames (ORFs) were selected as coding regions. Thirty-five percent of the predicted proteins showed similarity to proteins of rhizobia. Sixteen percent were similar only to proteins of other bacteria. No database match was found for 29%. Repetitive DNA sequence-derived ORFs accounted for the rest. The sequenced region contained all nitrogen fixation genes and, apart from nodM, all nodulation genes that were known to exist in B. japonicum. We found several genes that seem to encode transport systems for ferric citrate, molybdate, or carbon sources. Some of them are preceded by ؊24/؊12 promoter elements. A number of putative outer membrane proteins and cell wall-modifying enzymes as well as a type III secretion system might be involved in the interaction with the host.Nodulation (nod) genes and nitrogen fixation (nif) genes are the key determinants in the interaction between rhizobia and their host plants (14, 47). However, other loci influence the efficiency of the interaction or change the host range. Sequencing of the symbiotic plasmid of Rhizobium sp. strain NGR234 revealed a gene cluster that encodes a type III secretion system (22). Secreted proteins are encoded within the same cluster (95). The closely related Sinorhizobium fredii carries a type III secretion system as well (51, 61). Mutations within the secretion systems of the two strains influence symbiosis in a hostdependent manner. Plant and animal pathogens use related systems to target proteins to host cells (35), but such proteins have not been identified in rhizobia.During symbiosis, rhizobia exclusively rely on the carbon supply from the plant. Although bacteroids can utilize a wide range of carbon compounds, dicarboxylic acids are most likely the main carbon and energy source for bacteroids (45,83). The main argument is that several strains that have a defect in the dicarboxylic acid transport system show a Fix Ϫ phenotype (7,17,19,79,94) or are at least strongly impaired in nitrogen fixation (37).In our earlier work, we established a correlated physical and genetic map of the Bradyrhizobium japonicum genome (28,53) and discovered that all known nod and nif genes were clustered within a chromosomal region of about 400 kb. Furthermore, we found that the GϩC content of these genes was 58 mol% (76), considerably lower than the 61 to 65 mol% reported for the whole genome (43). Therefore, we concluded that the symbiotic genes have integrated into the chromosome after horizontal gene transfer from a different strain. In the absence of genomi...

Functional cloning led to the isolation of a novel methotrexate (MTX) resistance gene in the protozoan parasite Leishmania. The gene corresponds to orfG, an open reading frame (ORF) of the LD1/CD1 genomic locus that is frequently amplified in several Leishmania stocks. A functional ORF G-green fluorescence protein fusion was localized to the plasma membrane. Transport studies indicated that ORF G is a high affinity biopterin transporter. ORF G also transports folic acid, with a lower affinity, but does not transport the drug analog MTX. Disruption of both alleles of orfG led to a mutant strain that became hypersensitive to MTX and had no measurable biopterin transport. Leishmania tarentolae MTX-resistant cells without their high affinity folate transporters have a rearranged orfG gene and increased orfG RNA levels. Overexpression of orfG leads to increased biopterin uptake and, in folate-rich medium, to increased folate uptake. MTX-resistant cells compensate for mutations in their high affinity folate/MTX transporter by overexpressing ORF G, which increases the uptake of pterins and selectively increases the uptake of folic acid, but not MTX.

We describe a compilation of 79 known genes of Bradyrhizobiumjaponicum 110, 63 of which were placed on a correlated physical and genetic map of the chromosome. Genomic DNA was restricted with enzymes PacI, PmeI, and Swal, which yielded two, five, and nine fragments, respectively. Linkage of some of the fragments was established by performing Southern blot hybridization experiments. For probes we used isolated, labelled fragments that were produced either by Pmel or by Swal. Genes were mapped on individual restriction fragments by performing gene-directed mutagenesis. The principle of this method was to introduce recognition sites for all three restriction enzymes mentioned above into or very near the desired gene loci. Pulsed-field gel electrophoresis of restricted mutant DNA then resulted in an altered fragment pattern compared with wild-type DNA. This allowed us to identify overlapping fragments and to determine the exact position of any selected gene locus. The technique was limited only by the accuracy of the fragment size estimates. After linkage of all of the restriction fragments we concluded that the B.japonicum genome consists of a single, circular chromosome that is approximately 8,700 kb long. Genes directly concerned with nodulation and symbiotic nitrogen fixation are clustered in a chromosomal section that is about 380 kb long.The incentive for doing genetic work with bacterial species belonging to the genera Rhizobium, Bradyrhizobium, and Azorhizobium clearly stems from an interest in the characterization of genes essential for root nodule formation, endosymbiotic bacteroid (symbiosome) development, and symbiotic nitrogen fixation. Many genes involved in nodulation (nod) and nitrogen fixation (nif, fix), as well as genes concerned with bacteroid proliferation and physiology, have now been investigated in great detail (for reviews see references 8, 45, 61, and 64). By contrast, analysis of the chromosomes of rhizobial species has not progressed with the same speed. Chromosome mapping by conjugative gene transfer, a favorite research subject of Rhizobium geneticists in the late 1970s (9,53,54,69,70,91), has received the attention of workers in only a few laboratories in the last few years (48,51,74). Certainly one reason for this attention was the discovery that the nif and nod genes in Rhizobium species are for the most part located closely linked on symbiotic plasmids, which greatly facilitates molecular analysis of these genes (21,75,76). With the advent of pulsedfield gel electrophoresis (PFGE), which is ideal for the separation of very large DNA restriction fragments on agarose gels (14,57,93,95), the mapping of bacterial genomes became much easier; however, this powerful technique has been exploited to only a limited extent for investigation of rhizobial genomes (20,(97)(98)(99).Symbiotic plasmids have been found in Rhizobium species but not in species belonging to the genus Bradyrhizobium (65, 76); hence, it is generally believed that the Bradyrhizobium nif, fix, and nod genes are located on t...

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