STRAUB, PETER F. Biology Program,The Richard Stockton College, Pomona, N.J. 08240-0195. - Construction of a putA -GUS reporter gene for insertion in the nitrogen fixing symbiont Bradyrhizobium japonicum.
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