Two Classes of Rhizobiuk Geies Required for the Nodulation of Legumes

Abstract

Two different classes of genes in the bacterium Rhizobium leguminosarum biovar viciae, both of which are essential for the nodulation of its host legumes were identified. One group of genes is on a large sym plasmid and is involved in the early stages of the infection process and in the determination of host-range specificity. Several nod genes on the sym plasmid are not transcribed in cells growing in normal growth media but, when exposed to root exudate of legumes, are expressed at high levels. The inducer molecules in the exudate are certain flavones and flavanones and it was shown that their induction required the regulatory gene nodD. In R. I. bv. viciae, which nodulates peas, nodD is also autoregulatory, being capable of repressing its own expression. Mutant forms of nodD altered in their regulatory properties were isolated by in vitro mutagenesis of nodD with hydroxylamine. One class of mutation abolished both autoregulation and the induction of other nod genes in the presence of inducer flavonoids. Other mutations specifically inhibited the ability of nodD to autoregulate and a third mutant type was unaffected in autoregulation but was defective in activation of the other nod genes. A fourth group of nodD mutations, which activated transcription of other nod genes in the absence of inducer flavonoids was isolated; these “constitutive” forms of nodD caused a reduction in the number of nodules on peas and the nodules that formed failed to fix nitrogen. Using the gel retardation assay, it was shown that the nodD gene product bound to a DNA fragment lieing upstream of nodD and that this binding is probably responsible for the autoregulatory properties of nodD. In addition to genes on the sym plasmid, other genes, involved in the synthesis of the high molecular weight acidic exopolysaccharide [EPS], are needed for nodule formation on peas. Mutations which abolished EPS production resulted in strains which made non-mucoid colonies and which failed to nodulate. Vhen peas were co-inoculated with two different types of Nod⁻ mutants [ie. one strain lacked its sym plasmid and the other was defective in EPS synthesis] normal numbers of nodules were formed and, in all cases, the nodules were occupied by the strain lacking its sym plasmid. Thus, a strain that does not contain a sym plasmid was “helped” into the nodule by a strain that contains such a plasmid but which is Nod⁻ because of its failure to make EPS.