Index >> Rhizobium and Legume Root Nodulation >> Antibiotic Genetics

	Genetics Genetic studies on Rhizobium began by obtaining records of simple mutations, the action of phage on Rhizobium and transformation attempts with different strains of nodule bacteria. Markers generally employed are resistance to antibiotics and biochemical requirements of an amino acid or a simple sugar. X-rays have been used to obtain a range of biochemical mutants.
Rhizobium has been successfully used as an acceptor bacterium for transformation of tumour-inducing capacity from Agrobacterium tumefaciens, an instance of inter-generic transformation. Transformation of nodulating capacity between Rhizobium species has been reported. In this way, R. japonicum has been made to nodulate lucerne and R. meliloti to nodulate lupin. In R. trifolii, avirulent strains have been made virulent by transformation experiments. Mutations to ineffectiveness are common in soil. It has been shown that phage resistant mutants of R. trifolii tend to be ineffective. When wild-type ineffective strains are treated with phage, effective variants do not arise. On the other hand, ineffective mutants occasionally revert to effectiveness when treated with phage. A-Rhizobial Colonies on YEMA medium having Congo red shoowing glistening colonies B-Electron Photomicrographs of Rhizobia showing polar, Sub-Polar and Peritrichous Flagella
Loss of effectiveness is found to be closely associated with mutation to viomycin and neomycin resistance in strains of R. leguminosarum, R. trifolii and R. meliloti. Both viomycin resistance and ineffectiveness remain unchanged in clones re-isolated from nodules. Ability to from nodules (ineffectiveness) on the homologous host is, however, retained in all antibiotic resistant strains. Genetic circular linkage maps have been constructed for R. meliloti, R. leguminosarum and R. lupini and studies are in progress to locate more genes on the nuclear material.
Conjugation of R. phaseoli with R. trifolii as donor has been accomplished which extended the progeny to nodulate T. repens. This acq11ir d ability was got rid of by treatment of bacteria with acridine orange. Other experiments with R. meliloti have also shown the loss of ability to nodulate upon treatment with acridine orange. These results suggest that infectivity is a plasmid controlled property, thereby indicating the possibility of obtaining strains with a wide host range. Over the past ten years, the availability of methods to analyze DNA, more particularly from plasmids, has made it possible to identify high molecular weight plasmids (megaplasmids with mol. wt. more than 100 x 106) in fast-growing rhizobia. These plasmids carry heritable factors for nodulation and nitrogen-fixation.
	The use of transposon-induced mutation technology coupled with other novel methods of in vivostrain constructions using R plasmids has improved our knowledge concerning the genes controlling symbiotic functions. Mutants of Bradyrhizobium japonicum requiring tryptophan or unable to nodulate (Nod-), fix nitrogen (fix-) or nodulate but not fix nitrogen (Nod+, fix-) have been obtained by physical and chemical mutagenesis.

Similarly, mutants with more hydrogenase (hup⁺) or ones lacking in this enzyme (hup^-) and those unable to curl root hairs (Hac^-) have also been obtained. These studies have helped in understanding the genetic control of symbiosis.

The question whether all or part of the genes responsible for legume symbiosis are located on the rhizobial chromosome or on extrachromosomal elements is being increasingly examined with the discovery of extrachromosomal megaplasmids controlling the functioning and expression of the nitrogen-fixation process (nod and nif functions) in legumes.

However, it is not easy to say how many are partitioned to the chromosome even though genetic mapping data have shown that fix-mutants of R. meliloti can be traced to the bacterial chromosome. Plasmids are known to carry nod and nif function in fast-growing Rhizobium species (R. meliloti, R. leguminosarum, R. trifolii) whereas evidence for the location of these genes in the slow-growing Bradyrhizobium japonicum is gradually emerging.

The current knowledge on the genetics of legume root nodulation is complicated owing to the capacity of rhizobia to enter plant roots, induce nitrogen-fixing nodules and regulate the translocation of ammonium ion through a series of events which are controlled by genes located in large megaplasmids, as stated earlier. These genes include the nitrogen fixation genes (nif), the common nodulation genes (nod ABC) and the host range genes (host specific nod H and Q).

Several nod genes (nod ABC), nod FE and nod H have been identified in R. meliloti on a 'Sym' plasmid, by means of genetic analysis and DNA sequencing. The common nod ABC seem to be required in all Rhizobium systems for eliciting root hair deformation and root cell division. The root hair deformation and cell division processes appear to be caused by the products of (or) signals from Rhizobium nod ABC genes. The nod H and Q genes which are Rhizobium species dependent are necessary for deciding host specificity.

If nod ABC genes are mutated, then live Rhizobium cells cannot deform root hairs whereas if nod H is mutated, specificity may be lost but not root hair deformation. The root hair deformation activity is known as 'Had' activity. The signal product of nod ABC genes from R. meliloti causing Had activity in alfalfa root hairs has been enriched, purified and named as Nod Rm1 factor which is a sulfated and acylated glucosamine oligosaccharide.