Sequencing the 500-kb GC-rich symbiotic replicon of Rhizobium sp. NGR234 using dye terminators and a thermostable "sequenase": a beginning.

Abstract

Genomes of the soil-borne nitrogen-fixing symbionts of legumes [Azo(Brady)Rhizobium species] typically have GC contents of 59-65 tool%. As a consequence, compressions (up to 400 per cosmid} are common using automated dye primer shotgun sequencing methods. To overcome this difficulty, we have exclusively applied dye terminators in combination with a thermostable "'sequenase'" for shotgun sequencing GC-rich cosmids from pNGR234a, the S00-kbp symbiotic replicon of ~izobium sp. NGR234. A thermostable sequenase incorporates dye terminators into DNA more efficiently than Taq DNA polymerase, thus reducing the concentrations needed (20-to 250-fold). Unincorporated dye terminators can simply be removed by ethanol precipitation. Here, we present data of pXB296, one of 23 overlapping cosmids representing pNGR234a. We demonstrate that the greatly reduced number of compressions results in a much faster assembly of cosmid sequence data by comparing assembly of the shotgun data from pXB296 and the data from another pNGR234a cosmid (pXBII0) sequenced using dye primer methods. Within the 34,010-bp sequence from pXB2?6, 28 coding regions were predicted. All of them showed significant homologies to known proteins, including oligopeptide permeases, an essential cluster for nitrogen fixation, and the C4-dicarboxylate transporter DctA.Soil bacteria of the genera Azorhizobium, Bradyrhizobium, and Rhizobium establish symbiotic associations with leguminous plants leading to the formation of nitrogen-fixing nodules. Plant exudates (particularly flavonoid compounds) modulate the coordinated expression of many bacterial genes leading to the production of mitogenic Nod factors, which provoke nodule formation (Fellay et al. 1995a). In Azorhizobium and Bradyrhizobium species, symbiotic loci are carried on the chromosome, whereas in Rhizobium species most of them are plasmid-borne (Martinez et al. 1990;Fischer 1994;van Rhijn and Vanderleyden 1995). Complete understanding of legumeRhizobium interactions thus requires a catalog of symbiotic genes and exhaustive analysis of their function.The broad host-range Rhizobium sp. NGR234 carries a plasmid of 500 kbp (pNGR234a), which confers on Agrobacterium tumefaciens recipients the ability to nodulate certain legumes (Broughton et al. 1984). Identification of symbiotic pNGR234a genes has been performed by hostrange extension (complementation) and mutagenesis (Broughton et al. 1986;Lewin et al. 1990), techniques that are not only time-consuming but that fail to identify subtle phenotypes. To facilitate analysis of this replicon, a canonical ordered cosmid library of pNGR234a was constructed (Perret et al. 1991). Flavonoid-inducible loci were mapped to discrete XhoI fragments on these cosmids by competitive RNA hybridization (Fellay et al. 1995b). Other symbiotic loci were identified by subtractive DNA hybridization against the genome of the closely related strain Rhizobium fredii USDA257 (Perret et al. 1994). Nevertheless, a number of symbiotic genes remain to be identified, and for this reason, we wis...

NAD+-dependent Glyceraldehyde-3-phosphate Dehydrogenase from Thermoproteus tenax

“…3), including the predicted protein sequences of NAD ϩ -dependent GAPDH from Thermoproteus tenaxgenes from the genomes of Methanococcus jannaschii (31), Synechocystis sp. (32), and Rhizobium meliloti (33). The analyses resulted in a complex tree topology similar to that found by Habenicht et al (14), which is characterized by several poorly resolved lineages comprising enzymes of different substrate specificities (Fig.…”

Structure and evolution of NGRRS-1, a complex, repeated element in the genome of Rhizobium sp. strain NGR234

“…Unlike those of the two NGRIS-2 copies, which are similar but not identical, the target duplication sites of the three NGRIS-4 elements are identical (5Ј AAGG 3Ј). Genemark analysis predicted four putative genes transcribed in the same direction as in NGRIS-2a and encoding products of 6,105 (Orf1-IS4), 16,836 (Orf2-IS4), 9,703 (Orf3-IS4) and 78,742 (Orf4-IS4) Da ( Fig. 3; …”

“…Although no whole NGRIS-10 element was found, hybridizations and BLAST searches for similar proteins encoded by pNGR234a identified several homologs. Among these, y4UI and y4UH (carried by cosmid pXB296 [16]) en- coded products which are 59% identical and 87% homologous to Orf1-IS10 and Orf2-IS10, respectively. Both putative genes have the same organization as, as well as 60% homology at the nucleotide level to, those of NGRIS-10.…”

“…DNA of cosmids and pBluescript KSϩ clones was prepared by standard alkaline procedures followed by purification on CsCl gradients (45). The complete DNA sequence of cosmid pXB807 was established by using standard protocols for sequencing GC-rich cosmid clones (16). DNA sequences of the NGRRS-1b, -1c, and -1d elements were produced by manual, dideoxy methods (46) using double-stranded templates and Sequenase II (United States Biochemical Corp., Cleveland, Ohio).…”

Transposition of the IS21-related element IS1415 in Rhodococcus erythropolis

“…Z77165 and Z83858) and Rhizobium sp. strain NGR234 (11). The R. erythropolis element was assigned IS1415 by the Plasmid Reference Center (Stanford University School of Medicine, Stanford, Calif.).…”