Characterization of rhizobial isolates of Phaseolus vulgaris by staircase electrophoresis of low-molecular-weight RNA.

Low-molecular-weight (LMW) RNA molecules were analyzed to characterize rhizobial isolates that nodulate the common bean growing in Spain. Since LMW RNA profiles, determined by staircase electrophoresis, varied across the rhizobial species nodulating beans, we demonstrated that bean isolates recovered from Spanish soils presumptively could be characterized as Rhizobium etli, Rhizobium gallicum, Rhizobium giardinii, Rhizobium leguminosarum bv. viciae and bv. trifolii, and Sinorhizobium fredii.

Restriction fragment length polymorphism analysis of 16S rDNA and low molecular weight RNA profiling of rhizobial isolates from shrubby legumes endemic to the Canary islands.

Thirty-six strains of slow-growing rhizobia isolated from nodules of four woody legumes endemic to the Canary islands were characterised by 16S rDNA PCR-RFLP analyses (ARDRA) and LMW RNA profiling, and compared with reference strains representing Bradyrhizobium japonicum, B. elkanii, B. liaoningense, and two unclassified Bradyrhizobium sp. (Lupinus) strains. Both techniques showed similar results, indicating the existence of three genotypes among the Canarian isolates. Analysis of the combined RFLP patterns obtained with four endonucleases, showed the existence of predominant genotype comprising 75% of the Canarian isolates (BTA-1 group) and the Bradyrhizobium sp. (Lupinus) strains. A second genotype was shared by nine Canarian isolates (BGA-1 group) and the B. japonicum and B. liaoningense reference strains. The BES-5 strain formed an independent group, as also did the B. elkanii reference strains. LMW RNA profile analysis consistently resolved the same three genotypes detected by 16S ARDRA among the Canarian isolates, and suggested that all these isolates are genotypically more related to B. japonicum than to B. elkanii or B. liaoningense. Cluster analysis of the combined 16S ARDRA and LMW RNA profiles resolved the BTA-1 group with the Bradyrhizobium sp. (Lupinus) strains, and the BES-5 isolate, as a well separated sub-branch of the B. japonicum cluster. Thus, the two types of analyses indicated that the isolates related to BTA-1 conform a group of bradyrhizobial strains that can be clearly distinguishable from representatives of the tree currently described Bradyrhizobium species. No correlation between genotypes, host legumes, and geographic location was found.

Cell-associated pectinolytic and cellulolytic enzymes in Rhizobium leguminosarum biovar trifolii.

The involvement of Rhizobium enzymes that degrade plant cell wall polymers has long been an unresolved question about the infection process in root nodule symbiosis. Here we report the production of enzymes from Rhizobium leguminosarum bv. trifolii that degrade carboxymethyl cellulose and polypectate model substrates with sensitive methods that reliably detect the enzyme activities: a double-layer plate assay, quantitation of reducing sugars with a bicinchoninate reagent, and activity gel electrophoresis-isoelectric focusing. Both enzyme activities were (i) produced commonly by diverse wild-type strains, (ii) cell bound with at least some of the activity associated with the cell envelope, and (iii) not changed appreciably by growth in the presence of the model substrates or a flavone that activates expression of nodulation (nod) genes on the resident symbiotic plasmid (pSym). Equivalent levels of carboxymethyl cellulase activity were found in wild-type strain ANU843 and its pSym-cured derivative, ANU845, consistent with previous results of Morales et al. (V. Morales, E. Martínez-Molina, and D. Hubbell, Plant Soil 80:407-415, 1984). However, polygalacturonase activity was lower in ANU845 and was not restored to wild-type levels in the recombinant derivative of pSym- ANU845 containing the common and host-specific nod genes within a 14-kb HindIII DNA fragment of pSym from ANU843 cloned on plasmid pRt032. Activity gel electrophoresis resolved three carboxymethyl cellulase isozymes of approximately 102, 56, and 33 kDa in cell extracts from ANU843. Isoelectric focusing activity gels revealed one ANU843 polygalacturonase isozyme with a pI of approximately 7.2. These studies show that R. leguminosarum bv. trifolii produces multiple enzymes that cleave glycosidic bonds in plant cell walls and that are cell bound.