Extension of host range of Rhizobium leguminosarum bv. trifolii caused by point mutations in nodD that result in alterations in regulatory function and recognition of inducer molecules.

The positive activation of several nodulation genes in strain ANU843 of Rhizobium leguminosarum biovar trifolii is mediated by the product of the nodD gene and by the interaction of NodD with plant-secreted inducer and anti-inducer compounds. We have mutagenized the nodD gene of strain ANU843 with nitrosoguanidine and have found that the ability of the mutated nodD products to interact with inducer and anti-inducer compounds is affected by the amino acid sequence in at least two key regions, including a novel area between amino acids 77 and 123. Several novel classes of mutants were recognized by phenotypic and molecular analysis of the mutant nodD genes. Classes 1 and 4 mutants were able to induce nodA expression independently of the addition of inducer and anti-inducer compounds and were unable to mediate autoregulation of the nodD gene. Classes 2 and 3 mutants retained several properties of the wild-type nodD, including the ability to interact with inducer and anti-inducer compounds and the capacity to autoregulate nodD expression. In addition, class 2 mutants showed an inducer-independent ability to mediate nodA expression to 10-fold higher levels over control strains. The class 3 mutant showed reactivity to compounds that had little or no inducing ability with the wild-type nodD. An alteration in NodD function was demonstrated with classes 2 and 3 mutants, which showed greatly enhanced ability to complement a Tn5-induced mutation in the nodD1 gene of strain NGR234 and to restore nodulation ability on the tropical legume siratro. Mutants of nodD possessing inducer-independent ability to activate nod gene expression (classes 1, 2, and 4) were capable of extending the host range of R. l. bv. trifolii to the nonlegume Parasponia. DNA sequence analysis showed that single base changes were responsible for the altered phenotypic properties of five of six mutants examined. Four of the six mutations affected amino acid residues in a putative receiver domain in the N-terminal end of the nodD protein.

Asymbiotic acetylene reduction by a fast-growing cowpea Rhizobium strain with nitrogenase structural genes located on a symbiotic plasmid.

A procedure was designed which enabled the detection of ex planta nitrogenase activity in the fast-growing cowpea Rhizobium strain IHP100. Nitrogenase activity in agar culture under air occurred at a rate similar to that found for Bradyrhizobium strain CB756 but lower than that for Rhizobium strain ORS571. Hybridization studies showed that both nod and nif genes were located on a 410-kilobase Sym plasmid in strain IHP100.

In vitro expression of nitrogenase activity in Parasponia-Rhizobium strain ANU 289.

Rhizobium strain ANU 289 derepressed nitrogenase activity under defined in vitro conditions. Acetylene reduction was detected both in agar and liquid stationary culture. The strain is capable of nitrogen-fixing nodulation of legumes [such as siratro (Macroptilium atropurpureum Urb] as well as the non-legumes Parasponia andersonii and P. rugosa. Nitrogenase activity as high as 40-70 nmol C2H4 per mg protein after 7 days of incubation was detected. Strain ANU 289 was similar to Rhizobium strains 32 H1 and CB 756 with regard to oxygen requirement in the gas phase for development of nitrogenase activity between 0 and 10% O2, but showed increased sensitivity to oxygen repression at 20% O2. Strain ANU 289 also showed pronounced sensitivity to exogenous glutamine compared to strains 32 H1 and CB 756.

Transposon mutagenesis in rhizobia which can nodulate both legumes and the nonlegume parasponia.

The successful use of transposon mutagenesis in a slow-growing Rhizobium permitted the isolation of auxotrophic and symbiotically defective mutants. In these mutants, it was shown that different single-copy Tn5 insertions had occurred. Mutant phenotypes indicated that the control of nitrogenase activity in vitro and in planta is different.