The products of cooCTJ are involved in normal in vivo Ni insertion into the carbon monoxide dehydrogenase (CODH) of Rhodospirillum rubrum. Located on a 1.5-kb DNA segment immediately downstream of the CODH structural gene (cooS), two of the genes encode proteins that bear motifs reminiscent of other (urease and hydrogenase) Ni-insertion systems: a nucleoside triphosphate-binding motif near the N terminus of CooC and a run of 15 histidine residues regularly spaced over the last 30 amino acids of the C terminus of CooJ. A Gm r ⍀-linker cassette was developed to create both polar and nonpolar (60 bp) insertions in the cooCTJ region, and these, along with several deletions, were introduced into R. rubrum by homologous recombination. Analysis of the exogenous Ni levels required to sustain CO-dependent growth of the R. rubrum mutants demonstrated different phenotypes: whereas the wild-type strain and a mutant bearing a partial cooJ deletion (of the region encoding the histidine-rich segment) grew at 0.5 M Ni supplementation, strains bearing Gm r ⍀-linker cassettes in cooT and cooJ required approximately 50-fold-higher Ni levels and all cooC insertion strains, bearing polar or nonpolar insertions, grew optimally at 550 M Ni.The phototrophic bacterium Rhodospirillum rubrum induces synthesis of an Ni-and Fe-containing carbon monoxide dehydrogenase (CODH) upon anaerobic exposure to CO (3, 4) and couples CO oxidation to H 2 evolution as a source of energy (31). Limited evidence suggests that accessory functions are necessary for posttranslational Ni insertion and CODH activity in R. rubrum: an Ni-deficient apo-CODH is found in induced cultures deprived of Ni; substantially higher Ni concentrations are required for activation of apo-CODH in vitro than in vivo; and there is an elevated Ni requirement for CO-dependent growth of a mutant bearing an insertion in cooC, a gene immediately downstream of the CODH structural gene (5,13,31).Aside from the physiology of Ni insertion, the R. rubrum CODH has been well characterized biochemically (4, 14), genes encoding its synthesis and several additional proteins likely involved in H 2 production have been cloned and sequenced (16,17,30), active CODH has been heterologously expressed (24), and the CO-responsive transcriptional regulator is under investigation (24, 53). The hydrogenase (CooH) and CODH (CooS) genes occur in two operons, cooMKLXUH and cooFSCTJ, whose coding regions are separated by a 450-bp interval, with the CO-responsive transcriptional activator (CooA) encoded 137 nucleotides downstream of cooJ.Formation of the Ni-containing center(s) of other Ni-CODH enzymes has not been characterized, although the activities are prevalent in diverse anaerobic archaea and bacteria (15). Both the spectroscopic similarities of purified enzymes (26) and evident conservation of a limited number of potential metal ligands in an alignment of bacterial and archaeal CODH sequences (e.g., see reference 30) are consistent with conservation of structure and function of the Ni-containing C center...