Genetic regulation of nitrogen fixation in rhizobia.

Using physical and genetic data, we have demonstrated that Rhizobium meliloti SU47 has a symbiotic megaplasmid, pRmeSU47b, in addition to the previously described nod-nif megaplasmid pRmeSU47a This plasmid includes four loci involved in exopolysaccharide (exo) synthesis as well as two loci involved in thiamine biosynthesis Mutations at the exo loci have previously been shown to result in the formation of nodules which lack infection threads (Inf-) and fail to fix nitrogen (Fix-) Thus, both megaplasmids contain genes involved in the formation of nitrogen-fixing root nodules Mutations at two other exo loci were not located on either megaplasmid To mobilize the megaplasmids, the oriT of plasmid RK2 was inserted into them On alfalfa, Agrobacterium tumefaciens strains containing pRmeSU47a induced marked root hair curling with no infection threads and Fix- nodules, as reported by others This plant phenotype was not observed to change with A tumefaciens strains containing both pRmeSU47a and pRmeSU47b megaplasmids, and strains containing pRmeSU47b alone failed to curl root hairs or form nodules

Second symbiotic megaplasmid in Rhizobium meliloti carrying exopolysaccharide and thiamine synthesis genes.

Organization of the bacterial chromosome.

Populations of fluorescent pseudomonads isolated from an uncultivated soil and from the roots of two plant species were previously shown to differ (P. Lemanceau, T. Corberand, L. Gardan, X. Latour, G. Laguerre, J.-M. Boeufgras, and C. Alabouvette, Appl. Environ. Microbiol. 61:1004-1012, 1995). The diversities of fluorescent pseudomonads, from two uncultivated soils and from the roots of two plant species cultivated in these two soils, were compared. The phenotypic diversity of the bacterial isolates was characterized on the basis of biochemical and physiological tests and on the basis of their ability to utilize 147 different organic compounds. The genotypic diversity of the isolates was characterized on the basis of the types of 16S genes coding for rRNA (rDNA), their repetitive extragenic palindromic patterns by PCR, and plasmid profiles. Taxonomic identification of the isolates was achieved with both biochemical and physiological tests and by comparing their 16S rDNA types to those of reference and type strains of fluorescent Pseudomonas spp. Numerical analysis of phenotypic characteristics allowed the clustering of isolates that showed high levels of similarity. This analysis indicated that both soil type and host plant had an effect on the diversity of fluorescent pseudomonads. However, of the two factors studied, the soil was clearly the dominating one. Indeed, the populations associated with the roots of each plant species varied from one soil to the other. This variation could possibly be ascribed to the differences recorded between the phenotypically diverse populations of fluorescent pseudomonads from the two uncultivated soils. The plant selection was, at least partly, plant specific. It was not related to bacterial species and biovars or to the presence of plasmid DNA. The phenotypic clustering of isolates was well correlated with genotypic characterization by repetitive extragenic palindrome-PCR fingerprinting.

The Composition of Fluorescent Pseudomonad Populations Associated with Roots Is Influenced by Plant and Soil Type

Summary

A system which allows direct selection for curing of plasmids in Gram-negative bacteria was used to generate derivatives of Rhizobium leguminosarum VF39 cured of each of six plasmids present in this strain. Phenotypes could be correlated with the absence of five of the six plasmids. The smallest plasmid, pRIeVF39a, carries genes for the production of a melanin-like pigment as has been previously reported. Plasmid pRIeVF39d carries nodulation and nitrogen fixation genes. Curing of the plasmids pRleVF39c and pRleVF39e gave rise to strains which formed Fix− nodules on peas, lentils, and faba beans. The nodules formed by the strains cured of pRIeVF39c contained few, if any, bacteria. Analysis of washed cells by SDS-PAGE showed that this strain is defective in lipopolysaccharide (LPS) production; the defect could be complemented by introducing plasmids from several other R. leguminosarum strains, and by the R. leguminosarum biovar phaseoli LPS gene clones pCos126 and pDel27. The nodules formed by the strain cured of pRleVF39e had a reduced symbiotic zone, an enlarged senescence zone, and an abundance of starch granules. This strain grew at a much slower rate than the wild type, was unable to grow on minimal medium, and no longer produced melanin. These defects could be complemented by at least one other Rhizobium plasmid, pRle336e, a plasmid of strain 336 which is distinct from the nodulation plasmid (pRle336c) and the plasmid (pRle336d) which could complement the LPS defect associated with the loss of pRleVF39c. This demonstrates that genes necessary for symbiosis can be carried on at least three different plasmids in R. leguminosarum.

Beate Burkardt

Papers

Visualization and exact molecular weight determination of a Rhizobium meliloti megaplasmid.

Physical characterization of Rhizobium meliloti megaplasmids.