No abstract

The effect of nitrate on the symbiotic properties of nitrate-reductase-deficient mutants of a strain of cowpea rhizobia (32H1), and of a strain of Rhizobium trifolii (TA1), were examined; the host species were Macroptilium atropurpureum (DC.) Urb. and Trifolium subterraneum L. Nitrate retarded initial nodulation by the mutant strains to an extent similar to that found with the parent strains. It is therefore unlikely that nitrite produced from nitrate by the rhizobia, plays a significant role in the inhibition of nodulation by nitrate. Nitrite is an inhibitor of nitrogenase, and its possible production in the nodule tissue by the action of nitrate reductase could be responsible for the observed inhibition of nitrogen fixation when nodulated plants are exposed to nitrate. However, the results of this investigation show that nitrogen fixation by the plants nodulated by parent or mutant strains was depressed by similar amounts in the presence of nitrate. No nitrite was detected in the nodules. Nodule growth, and to a lesser extent, the nitrogenase specific activity of the nodules (μmol C2H4g(-1) nodule fr. wt. h(-1)), were both affected by the added nitrate.

(1) We constructed Escherichia coli strain JP17 with a deletion in the ATP synthase beta-subunit gene. JP17 is completely deficient in ATP synthase activity and expresses no beta-subunit. Expression of normal beta-subunit from a plasmid restores haploid levels of ATP synthase in membranes. JP17 was shown to be efficacious for studies of beta-subunit mutations. Site-directed mutants were studied directly in JP17. Randomly generated chromosomal mutants were identified by PCR and DNA sequencing, cloned, and expressed in JP17. (2) Eight novel mutations occurring within the putative catalytic nucleotide-binding domain were characterized with respect to their effects on catalysis and structure. The mutations beta C137S, beta G152D, beta G152R, beta E161Q, beta E161R, and beta G251D each impaired catalysis without affecting enzyme assembly or oligomeric structure and are of interest for future studies of catalytic mechanism. The mutations beta D301V and beta D302V, involving strongly conserved carboxyl residues, caused oligomeric instability of F1. However, growth characteristics of these mutants suggested that neither carboxyl side chain is critical for catalysis. (3) The mutations beta R398C and beta R398W rendered ATP synthase resistant to aurovertin, giving strong support to the view that beta R398 is a key residue in the aurovertin-binding site. Neither beta R398C or beta R398W impaired catalysis significantly.