Genetic and physiological characterization of the Rhodospirillum rubrum carbon monoxide dehydrogenase system.
A 3.7-kb DNA region encoding part of the Rhodospirillum rubrum CO oxidation (coo) system was identified by using oligonucleotide probes. Sequence analysis of the cloned region indicated four complete or partial open reading frames (ORFs) with acceptable codon usage. The complete ORFs, the 573-bp cooF and the 1,920-bp cooS, encode an Fe/S protein and the Ni-containing carbon monoxide dehydrogenase (CODH), respectively. The four 4-cysteine motifs encoded by cooF are typical of a class of proteins associated with other oxidoreductases, including formate dehydrogenase, nitrate reductase, dimethyl sulfoxide reductase, and hydrogenase activities. The R. rubrum CODH is 67% similar to the beta subunit of the Clostridium thermoaceticum CODH and 47% similar to the alpha subunit of the Methanothrix soehngenii CODH; an alignment of these three peptides shows relatively limited overall conservation. Kanamycin cassette insertions into cooF and cooS resulted in R. rubrum strains devoid of CO-dependent H2 production with little (cooF::kan) or no (cooS::kan) methyl viologen-linked CODH activity in vitro, but did not dramatically alter their photoheterotrophic growth on malate in the presence of CO. Upstream of cooF is a 567-bp partial ORF, designated cooH, that we ascribe to the CO-induced hydrogenase, based on sequence similarity with other hydrogenases and the elimination of CO-dependent H2 production upon introduction of a cassette into this region. From mutant characterizations, we posit that cooH and cooFS are not cotranscribed. The second partial ORF starts 67 bp downstream of cooS and would be capable of encoding 35 amino acids with an ATP-binding site motif.
Kerby
R L
RL
Department of Bacteriology, College of Agricultural and Life Sciences, University of Wisconsin-Madison 53706.
Hong
S S
SS
Ensign
S A
SA
Coppoc
L J
LJ
Ludden
P W
PW
Roberts
G P
GP
D000445:Aldehyde Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D002876:Chromosomes, Bacterial; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000302:isolation & purification; D007506:Iron-Sulfur Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes; D016296:Mutagenesis; D016366:Open Reading Frames; D010957:Plasmids; D015183:Restriction Mapping; D012247:Rhodospirillum rubrum / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid
D004269:DNA, Bacterial; D007506:Iron-Sulfur Proteins; D009097:Multienzyme Complexes; D000445:Aldehyde Oxidoreductases; C019823:carbon monoxide dehydrogenase