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.     

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.     

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.     

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.     

Chromosomal nodulation genes: Sym-plasmid containing Agrobacterium strains need chromosomal virulence genes (chvA and chvB) for nodulation

Summary The chromosomal genes chvA and chvB of Agrobacterium tumefaciens, which mediate attachment to plant cells, were found to be essential not only for tumour induction but also for the formation of root nodules on plants.     

Isolation of root hairs from seedlings of Pisum sativum. Identification of root hair specific proteins by in situ labeling

A procedure was developed which allows the large-scale isolation of root hairs from seedlings of Pisum sativum . L. cvs. Kleine Rheinländerin and Rosa Krone. The method may yield up to 50 g fresh weight of root hairs per 3.10⁴ seedlings. In a modified form considerable amounts of root hair material may be harvested, even after incubation of the roots in aqueous solutions. Thus, detailed biochemical studies on the root hair system have become feasible.
The occurrence of specific proteins in membrane fractions of P. sativum root hairs was demonstrated as follows: Incubation of root hairs in situ with 3-azidonaphthalene-2,7-disulfonate – a strongly anionic, photoactivated fluorescent marker – followed by gel electrophoresis of membrane fractions showed the presence of root-hair specific proteins which, since the system was intact, suggests that they are on the outer surface of the cells.     

Rhizobium strain effects on pea: The relation between nitrogen accumulation, phosphoenolpyruvate carboxylase activity in nodules and asparagine in root bleeding sap

Pisum sativum L. cv. Bodil was infected with various strains of Rhizobium leguminosarum (R501, 128c53, B155, 18a or 1044). The Rhizobium genotype influenced the activity of the plant enzyme phosphoenoipyruvate (PEP) carboxylase (EC 4.1.1.31), and the assimilation of fixed N in the root nodules. The specific activity of nodule PEP carboxylase was lowest in the symbioses, which accumulated the least total N (R501 and 128c53). The root bleeding sap of the less effective symbioses contained a lower proportion of asparagine and a higher proportion of glutamine than the more effective symbioses (B155,18a and 1044). The N yield of the symbioses was related neither to the net respiratory CO₂ evolution of the root system nor to the nitrogenase linked nodule respiration. The lower yielding symbioses accumulated a larger proportion of the fixed N in the nodules due to a higher proportion of total dry weight contained in the nodule tissue. However, the concentration of soluble protein in the nodules of the lower-yielding symbioses was lower than that recorded for the higher yileding symbioses. The effect of the Rhizobium strains on N yield was maintained at maturity, and reflected in seed yields.     

Characterization ofR. fredii QB1130, a strain effective on commercial soybean cultivars

Summary Two rhizobial strains (QB1130 and C3A) from northeast China were identified asRhizobium fredii on the basis of growth rate, media acidification and growth on a wide range of carbon substrates. The strains were shown to be distinct from USDA 191 on the basis of plasmid number and size. Bothnif and commonnod genes were located on the 295 kb plasmid of strains QB1130 and USDA 191, while onlynif genes were identified on this plasmid in C3A. When used to inoculate four commercial soybean (Glycine max) cultivars, one of the strains (C3A) was found to be ineffective, while the other (QB1130) was at least as effective as USDA 191, a strain ofR. fredii reported to be widely effective on North American cultivars of soybean. Further, QB1130 was capable of more effective nodulation of cowpea or the uncultivated soybean line, Peking, than either USDA 191 or the slow-growingBradyrhizobium japonicum USDA 16. Strain QB1130 should be useful for studies directed at improving symbiotic performance in soybean, or for studies of the comparative physiology and genetics of FG and SG strains on a single host.