Screening costramid libraries for chromosomal genes: an alternative interspecific hybridization method

Abstract Broad host-range RK2-based cosmid vectors ('costramids') are increasingly used in molecular genetic studies of Gram-negative soil bacteria such as Rhizobium spp. we describe a simple modification of existing methods, whereby a genomic library constructed in a stringently replicated vector can be screened for genes which are undetectable by colony hybridization due to background cross-hybridization. This method allows the use of 'heterologous' probes (interspecies hybridization) to isolate several presumptive genes of interest from a gene bank of Rhizobium sp. NGR234 made in the costramid pRK7813. These are a gene with homology to the citrate synthase gene ( gltA ) or Escherichia coli , the gene encoding δ-aminol evulinic acid synthase ( hemA ), and a gene or genes regulating dicarboxylate transport.     

Utilization of poly-β-hydroxybutyrate in free-living cultures of Rhizobium ORS571

Abstract The effect of carbon starvation on growth and poly-β-hydroxybutyrate (PHB) utilization in oxygen-limited chemostat cultures of Rhizobium ORS571 was studied. Under oxygen-limited growth conditions PHB was not degraded. When in a nitrogen-fixing oxygen-limited culture, after stopping the medium supply, the dissolved oxygen concentration was maintained at 10 μM, a slow breakdown of PHB was observed. Addition of ammonia and air to a nitrogen-fixing oxygen-limited culture after the medium supply had been stopped, resulted in the simultaneous utilization of PHB and succinate. The possible use of the energy derived from PHB degradation in Rhizobia bacteria and bacteroids is discussed.     

Interspecies homology of nodule development genes in Rhizobium and Bradyrhizobium spp.

Abstract DNA fragments representatives of ndv A and ndv B have been used as probes against genomic DNAs from different Rhizobium and Bradyrhizobium species. ndv A and ndv B homologues were found in all species, indicating extensive conservation of these genes. All Rhizobium species show chromosomal localization of ndv A and ndv B homologues.     

Regulation of urease activity in Rhizobium meliloti

Abstract Using an ureC-lacZ fusion, the expression of urease structural genes of the soil bacterium Rhizobium meliloti strain AK631 was studied in response to different nitrogen sources and nickel contents in the growth medium. The expression of urease genes is repressed by ammonia and is not inducible by urea. Urease activity depends on the nickel concentration of the medium. Nickel uptake is repressed in medium containing ammonia and is not affected by the genes located in the urease operon investigated.     

Pulsed-field gel electrophoresis for genotypic comparison of Rhizobium bacteria that nodulate leguminous trees

Abstract Pulsed-field gel electrophoresis (PFGE) was applied to characterize Rhizobium bacteria isolated from the root nodules of Acacia senegal and Prosopis chilensis trees growing in Sudan and Keya. For the electrophoresis, the total DNA of 42 isolates, embedded in agarose, was digested by a rare-cutting restriction endonuclease, Xba I. The PFGE run resulted in good resolution of the DNA fragments and gave the strains distinctive fingerprint patterns. The patterns were analysed visually and using automated clustering analysis, which divided the strains into groups resembling the results generated by numerical taxonomy. However, several strains had unique banding patterns, which indicates that these strains are genetically very diverse.     

Germintion,nodulation and early growth response of a hard-seeded legume,Lathyrus sylvestris L. (flatpea)

Summary Flatpea (Lathyrus sylvestris L.) requires seed scarification for rapid germination and specificRhizobium leguminosarum strain for nodulation. Methods of inoculation were investigated to determine which would be effective for at least two weeks after seeding. Application of powdered peat to wet seeds was inadequate. Lime pelleting and granular soil implants were effective inoculants, but there appears to be a different advantage to seedling for earch metod. Granular soil implant caused nodulation to a greater depth on the tap root, and lime pelleting caused plants to exhibit higher acetylene reduction activity.     

Introduction of the mercury transposon Tn501 into Rhizobium japonicum strains 31 and 110

Abstract Transposon Tn 501 , which encodes resistance to mercuric ions, was introduced into Rhizobium japonicum 110 and 31 by conjugal transfer. The transposon donor plasmid (pMD100) was able to mobilize into R. japonicum , but could not be maintained. Hg²⁺-resistant colonies were recovered at a frequency of 1.9 × 10⁻⁸/recipient for strain 110, and 1.7 × 10⁻⁷/recipient for strain 31. Presence of Tn 501 in Hg-resistant isolates was verified by Southern analysis and demonstrating transposition of Hg resistance. Transposon mutagenesis has been used to generate auxotrophic mutations at low frequency.     

Rhizoremediation of Dioxin-like Compounds by a Recombinant Rhizobium tropici Strain Expressing Carbazole 1,9a-Dioxygenase Constitutively

A recombinant Rhizobium strain, PBK3-IS, that constitutively expressed the oxygenase component of carbazole 1,9a-dioxygenase from Sphingomonas sp. strain KA1, was constructed. In the water-cultured siratro rhizospheres inoculated with strain PBK3-IS, 48% of the dibenzofuran was removed within 3 days (initial substrate, 25 μg). Similar results were obtained in soil-cultured siratro rhizospheres using sterile vermiculite. When non-sterile field soils were used instead of sterile vermiculite, the inoculated recombinant strain could grow on the siratro root in all soils tested, except for wet paddy field.     

Molecular analysis of an essential gene upstream of rpoN in Rhizobium NGR234

Rhizobium sp. NGR234 is a broad-host range strain. The rpoN gene of this organism encodes a sigma factor which is a primary co-regulator of endosymbiosis. We characterized the locus upstream of rpoN, and identified a contiguous open reading frame, here termed ORF1. DNA sequence analysis of this ORF showed that it encoded a polypeptide highly conserved with a corresponding ORF of Rhizobium meliloti. The gene product contained two ATP/GTP binding pockets. Codon usage in the ORF and the nitrogenase operon nifKDH of NGR234 was similar. Although we used a non-transposable cassette flanked by appropriate sized DNA fragments, we were unable to isolate site-directed mutants in the ORF, whose ATP/GTP binding protein product is thus probably of essential biological function. ORF1 and rpoN exhibited conserved linkage among diverse rhizobia, and in Azotobacter vinelandii. Intragenomic and interspecific homology studies confirmed directly that ORF1 (NGR234) belonged to a large family of ATP-binding protein genes.     

Identification of 3-deoxy-lyxo-2-heptulosaric acid in the core region of lipopolysaccharides from Rhizobiaceae

A 3-deoxy-2-heptulosaric acid (DHA), very probably with the lyxo-configuration, was identified in the R-core region of lipopolysaccharides from nodulating strains of Rhizobium leguminosarum, Rhizobium meliloti and from all three biovars of the phytopathogenic Agrobacterium tumefaciens. Its structure could be deduced from the fragmentation pattern of the corresponding alditol acetates obtained after reduction of the 2-keto and the 1.7-carboxy groups by sodium borohydride or sodium borodeuteride. DHA in lipopolysaccharide was not destroyed by periodate and is therefore not in a terminal position. Two DHA-containing oligosaccharides, namely glucosyl (1----4)-3-deoxy-2-heptulosaric acid and rhamnosyl-rhamnosyl-(1----5)-3-deoxy-2-heptulosaric acid could be tentatively identified by mass spectrometric methods amongst the products of mild acidic hydrolysis of lipopolysaccharides of Rhizobium leguminosarum strain 24. The two types of non-nodulating mutants of Rhizobium leguminosarum included in this study did not contain 3-deoxy-2-heptulosaric acid.