POSTTRANSLATIONAL REGULATION OF NITROGENASE IN RHODOSPIRILLUM-RUBRUM STRAINS OVEREXPRESSING THE REGULATORY ENZYMES DINITROGENASE REDUCTASE ADP-RIBOSYLTRANSFERASE AND DINITROGENASE REDUCTASE ACTIVATING GLYCOHYDROLASE

Rhodospirillum rubrum strains that overexpress the enzymes involved in posttranslational nitrogenase regulation, dinitrogenase reductase ADP -ribosyltransferase (DRAT) and dinitrogenase reductase activating glyc ohydrolase (DRAG), were constructed, and the effect of this overexpres sion on in vivo DRAT and DRAG regulation was investigated, Broad-host- range plasmid constructs containing a fusion of the R. rubrum nifH pro moter and translation initiation sequences to the second codon of draT , the first gene of the dra operon, were constructed, Overexpression p lasmid constructs which overexpressed (i) only functional DRAT, (ii) o nly functional DRAG and presumably the putative downstream open readin g frame (ORF)-encoded protein, or (iii) all three proteins were genera ted and introduced into wild-type R. rubrum, Overexpression of DRAT st ill allowed proper regulation of nitrogenase activity, with ADP-ribosy lation of dinitrogenase reductase by DRAT occurring only upon dark or ammonium stimuli, suggesting that DRAT is still regulated upon overexp ression, However, overexpression of DRAG and the downstream ORF altere d nitrogenase regulation such that dinitrogenase reductase did not acc umulate in the ADP-ribosylated form under inactivation conditions, sug gesting that DRAG was constitutively active and that therefore DRAG re gulation is altered upon overexpression, Proper DRAG regulation was ob served in a strain overexpressing DRAT, DRAG, and the downstream ORF, suggesting that a proper balance of DRAT and DRAG levels is required f or proper DRAG regulation,