To study the effects of drought induced proline accumulation in soybean nodules on the resident nitrogen fixing bacteria (B. japonicum), a reporter gene was constructed by fusing ca.1 kb of upstream region of a cloned B. japonicum putA gene with a b-glucuronidase (GUS) reporter gene (with the technical assistance of Dr. T. Sutliff). The GUS gene was cloned into a new XbaI site that was made by site directed mutagenesis of the putA start codon. A spectinomycin resistance gene was cloned in to complete the expression cassette. The expression cassette, in pBluescript, was tested in E. coli by incubation of cells with and without 250 mM proline followed by assay with the chromogenic substrate X-Gluc. GUS activity was consistently higher in the proline treated samples indicating the reporter construction was expressing. The expression cassette was then isolated by restriction digest, blunted and linkered with Not1 linkers and cloned into the B. japonicum insertion plasmid pAY19, prepared by HindIII digestion and linkered with Not1. The plasmid pAY19 directs chromosomal insertion into a non-coding region of the nifDK locus. This work will compliment previous putA insertional knockout mutants in the symbiont that have been shown to affect seed yield under drought in the whole plant by allowing the putA gene activity to be monitored in vivo without disrupting bacteroid proline concentrations as in the case of the knockout mutants.

Key words: Bradyrhizobium japonicum, drought, proline, putA