A collection of symbiotically defective mutants of Rhizobium meliloti JJ1c10 was derived by Tn5 mutagenesis using the suicide vector pGS9. They include two Nod− and about 250 Fix− mutants. The mutants were found to be heterogenous in acetylene reduction activity and in the morphology and ultrastructure of the nodules which they induced. Over 90% were found to contain bona fide Tn5 insertions in a variety of DNA restriction fragments. When Tn5-carrying DNA segments cloned from 24 of the mutants were introduced into the equivalent location in the genome of the wild-type strain by recombination-mediated replacement, only eight produced a symbiotically defective phenotype similar to that of the original mutant. This result indicated that many of the symbiosis mutations were not directly caused by Tn5 insertion. DNA segments apparently containing mutated fix genes but not containing Tn5 were found in eight mutants by identifying cosmids carrying wild-type DNA which complemented their symbiosis defects. Probing of the DNA of these mutants with their complementing cosmids revealed no detectable physical alteration of the homologous DNA. A segment of DNA including the hsn and nifHDK genes was favoured for these non-Tn5 mutations. Three regions of the genome in which Tn5 caused fix mutations were identified. One of these was the known megaplasmid nod-nif region. The other two regions, designated fix-e5 and fix-h21, were found to be chromosomal. Mutants in one of these chromosomal regions fluoresced more intensely on calcofluor plates than the wild type.
DNA hybridization with the cloned nodulation region of Rhizobium melioti as a probe revealed DNA homology with four Hindm fragments, 12.5, 6.8, 5.2, and 0.3 kilobases (kb) in size, of the symbiotic plasmid pRjaUSDA193. Both hybridization and complementation studies suggest that the common nodulation genes nodABC and nodD of R. fredi USDA 193 are present on the 5.2-kb HindIII and 2.8-kb EcoRI fragments, respectively, of the Sym plasmid. Both fragments together could confer nodulation ability on soybeans when present in Sym plasmid-cured (Sym-) and wild-type (Sym+) Rhizobium strains or in a Ti plasmid-cured Agrobacterium tumefaciens strain. Furthermore, the 2.8-kb EcoRI fragment alone was able to form nodulelike structures on Glycine mar L. cv. "Peking" (soybean). Microscopic examination of these nodules revealed bacterial invasion of the cells, probably via root hair penetration. Bacterial strains harboring plasmids carrying the 5.2-and 2.8-kb nod fragments elicited root-hair-curling responses on infection. These data suggest that the genes responsible for host range determination and some of the early events of nodulation may be coded for by the 5.2-kb Hindm and 2.8-kb EcoRI fragments.Bacteria of the genus Rhizobium form symbiotic associations with leguminous plants and fix nitrogen in specialized structures called root nodules. This is a complex, multistage process involving induction of root hair deformation (curling, branching, or both), infection of root hairs, development and differentiation of root nodules, proliferation of bacteria within the host cells, and finally, the reduction of molecular nitrogen into ammonia (3, 37, 38). Recently, a new group of fast-growing rhizobia that nodulate soybeans was isolated in mainland China (20). These fast-growing soybean strains, previously known as Rhizobium japonicum, have been redesignated Rhizobiumfredii (35). Studies of the nodulation process in fast-growing Rhizobium strains have demonstrated the presence of a series of genetic loci coding for nodulation (nod) and nitrogenase (nif) genes, on a very large symbiotic (pSym) plasmid (2,7,22,23,30,33). The nod and nif genes of R. fredii strains have also been shown to be present on a large plasmid (26,28). Strain USDA 194 is an exception, in which the symbiotic genes are present either on a megaplasmid or on the chromosome (25). R. fredii USDA 193, used in this study, nodulates a highly inbred soybean (Glycine max L. cv. "Peking") effectively and the North American cultivars of soybean ineffectively (20).Long et al. (23) cloned a region of the symbiotic plasmid of Rhizobium meliloti 1021(pRmSL26), which complements two nodulation-deficient mutants of R. meliloti. Transposon mutagenesis and DNA sequence analysis (8, 19) revealed a cluster of four nod genes, nodABC and nodD, located on this fragment, with nodD read divergently from nodABC. Others have found that mutations in nodA, -B, and -C genes fail to induce root-hair-curling (Hac-) as well as nodule formation
In Rhizobiumjaponicum strain Nitragin 61A76, morphologically distinct types of bacteria were found to occur in yeast extract-mannitol broth cultures, at both mid-log and stationary phases. Of these only the capsular form, characterized by a smooth cell envelope, storage granules (glycogen and poly-,8-hydroxybutyric acid), and an amorphous extracellular capsule, bound soybean lectin. The binding site was localized in the capsular material. Less than 1% of the bacterial population differentiated into these capsular forms, which were also able to attach to the soybean root hair surface.
Treatment of soybean root nodule tissue with a nonionic detergent. Nonidet P-40, after aldehyde fixation, results in a selective solubilization of membranes. The cell wall membrane of bacteroids and of free-living Rhizobium is resistant to this treatment. Fragments of "extra" membrane present inside the membrane envelope enclosing the bacteroids are also resistant to the detergent and are morphologically similar to the outer membrane of the cell wall of Rhizobium grown in broth culture. These observations, along with electrophoretic profiles of detergent resistant membranes from nodules, free-living Rhizobium, and isolated nodule bacteroids, suggest that the Rhizobium cell wall membrane undergoes significant changes during establishment of the root nodule symbiosis.
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