title: In vivo nickel insertion into the carbon monoxide dehydrogenase of Rhodospirillum rubrum: molecular and physiological characterization of cooCTJ.
abstract: 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)omega-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 microM Ni supplementation, strains bearing Gm(r)omega-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 microM Ni.
authors.affiliation: Department of Bacteriology, College of Agricultural and Life Sciences, University of Wisconsin-Madison, 53706, USA.
authors.forename: R L
authors.initials: RL
authors.lastname: Kerby
authors.forename: P W
authors.initials: PW
authors.lastname: Ludden
authors.forename: G P
authors.initials: GP
authors.lastname: Roberts
mesh_terms: D000445:Aldehyde Oxidoreductases / Q000737:chemistry* / Q000502:physiology; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics / Q000502:physiology*; D015180:Electrophoresis, Gel, Two-Dimensional; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000502:physiology; D009532:Nickel / Q000378:metabolism*; D009876:Operon; D011994:Recombinant Proteins; D012247:Rhodospirillum rubrum / Q000201:enzymology* / Q000235:genetics; D013329:Structure-Activity Relationship; D056511:Metallochaperones
chemical_list: D000445:Aldehyde Oxidoreductases; D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D009097:Multienzyme Complexes; D009532:Nickel; D011994:Recombinant Proteins; C088906:CooC protein, E coli; C105283:CooJ protein, Rhodospirillum rubrum; D056511:Metallochaperones; C105282:CooT protein, Rhodospirillum rubrum; C019823:carbon monoxide dehydrogenase