IN-VIVO NICKEL INSERTION INTO THE CARBON-MONOXIDE DEHYDROGENASE OF RHODOSPIRILLUM-RUBRUM - MOLECULAR AND PHYSIOLOGICAL CHARACTERIZATION OF COOCTJ

The products of cooCTJ are involved in normal in vivo Ni insertion int o the carbon monoxide dehydrogenase (CODH) of Rhodospirillum rubrum. L ocated on a 1.5-kb DNA segment immediately downstream of the CODH stru ctural gene (cooS), two of the genes encode proteins that bear moths r eminiscent of other (urease and hydrogenase) Ni-insertion systems: a n ucleoside triphosphate-binding moth near the N terminus of CooC and a run of 15 histidine residues regularly spaced over the last 30 amino a cids of the C terminus of CooJ. A Gm(r) Omega-linker cassette was deve loped to create both polar and nonpolar (60 bp) insertions in the cooC TJ region, and these, along with several deletions, were introduced in to R. rubrum by homologous recombination, Analysis of the exogenous Ni levels required to sustain CO-dependent growth of the R. rubrum mutan ts demonstrated different phenotypes: whereas the mild-type strain and a mutant bearing a partial cooJ deletion (of the region encoding the histidine-rich segment) grew at 0.5 mu M Ni supplementation, strains b earing Gm(r) Omega-linker cassettes in cooT and cooJ required approxim ately 50-fold-higher Ni levels and all cooC insertion strains, bearing polar or nonpolar insertions, grew optimally at 550 mu M Ni.