Carbon monoxide-induced activation of gene expression in Rhodospirillum rubrum requires the product of cooA, a member of the cyclic AMP receptor protein family of transcriptional regulators

Abstract

Induction of the CO-oxidizing system of the photosynthetic bacterium Rhodospirillum rubrum is regulated at the level of gene expression by the presence of CO. In this paper, we describe the identification of a gene that is required for CO-induced gene expression. An 11-kb deletion of the region adjacent to the previously characterized cooFSCTJ region resulted in a mutant unable to synthesize CO dehydrogenase in response to CO and unable to grow utilizing CO as an energy source. A 2.5-kb region that corresponded to a portion of the deleted region complemented this mutant for its CO regulation defect, restoring its ability to grow utilizing CO as an energy source. When the 2.5-kb region was sequenced, one open reading frame, designated cooA, predicted a product showing similarity to members of the cyclic AMP receptor protein (CRP) family of transcriptional regulators. The product, CooA, is 28% identical (51% similar) to CRP and 18% identical (45% similar) to FNR from Escherichia coli. The insertion of a drug resistance cassette into cooA resulted in a mutant that could not grow utilizing CO as an energy source. CooA contains a number of cysteine residues substituted at, or adjacent to, positions that correspond to residues that contact cyclic AMP in the crystal structure of CRP. A model based on this observation is proposed for the recognition of CO by CooA. Adjacent to cooA are two genes, nadB and nadC, with predicted products similar to proteins in other bacteria that catalyze reactions in the de novo synthesis of NAD. A mutant with the cooA-nadBC region deleted displayed an auxotrophy for nicotinic acid, while the cooA insertion mutant did not.The purple, nonsulfur, phototrophic bacterium Rhodospirillum rubrum synthesizes an enzyme system for carbon monoxide (CO) oxidation. Anaerobically in the dark, R. rubrum can utilize the CO-oxidizing system to generate energy (28). The system consists of at least three proteins that together couple CO oxidation to H 2 evolution: CooS (CO dehydrogenase), which oxidizes CO; CooF, a CooS-associated Fe-S protein; and CooH, a CO-tolerant hydrogenase (4,5,15). CooS and CooF have been purified to homogeneity and biochemically characterized (5, 6, 13-15). The cooH, cooF, and cooS genes are adjacent to one another on the chromosome and have been cloned, sequenced, and mutationally characterized. Mutations in any of these genes eliminate the ability of R. rubrum to evolve H 2 from CO and to utilize CO as an energy source (27,28).Unlike the case for many other bacteria capable of oxidizing CO anaerobically (11), the presence of the CO-oxidizing system in R. rubrum is dependent upon exogenous CO. Induction of this system by CO occurs in cells grown either photoheterotrophically (4, 5) or anaerobically in the dark (28). For cells growing photoheterotrophically in malate-ammonium medium, CooS activity is induced at least 1,000-fold by CO; under these conditions, CooS can constitute 2 to 5% of cellular protein (5). CooS accumulation following exposure to CO requires protein synthesis, in...

The Identification, Purification, and Characterization of CooJ

“…Cultures of wild type R. rubrum (strain UR2) or mutant strain UR294 were cultured in 0.5 liter of Ormerod's medium (28) with NH 4 Cl as the nitrogen source in the presence or absence of CO in the head-space. Cultures were harvested by centrifugation at 6000 ϫ g. The cell paste was frozen in liquid nitrogen until cells were used.…”

“…In the presence of CO, the CooA regulatory protein recognizes CO and binds to the promoter regions of the cooFSCTJ and cooMKLXUH operons, initiating the expression of the CO oxidation system (2)(3)(4). CO dehydrogenase (CODH) 1 is encoded by the cooS gene (5) and is a redox enzyme that catalyzes the oxidation of CO to CO 2 .…”

Heme Environmental Structure of CooA Is Modulated by the Target DNA Binding

“…A gene region designated as cooA was demonstrated to be responsible for the regulation, whose product protein, CooA, shows high homology with the known transcriptional regulators, such as cyclic AMP receptor protein (CRP) 1 (28% identical, 51% similar) (4). Like the mechanism of CRP activation by cyclic AMP, CO is assumed to bind with CooA (3,(5)(6)(7)(8)(9)(10), which causes the protein-CO complex to bind specifically to the target DNA, resulting in the expression of CO oxidizing enzymes (7,10).…”

“…The rebinding kinetics of CO after photodissociation from CO-bound hemes reflect the environment around the hemes. The characterization of CO-bound CooA is of special interest, since it is the CO-bound form of CooA that plays crucial and physiological role in its gene activation (1)(2)(3)(4)(5)(6)(7)(8)(9)(10), and provides a clue to elucidate the activation mechanism of CooA by CO. We have, therefore, undertaken the resonance Raman investigation on the CO-bound and reduced forms of CooA. CO rebinding kinetics of CooA were also determined by a laser photolysis method.…”

“…CooA is a homodimer containing 222 amino acid residues in each subunit (2,7). Recent success of the high level expression of CooA in Escherichia coli has enabled us and other researchers to obtain enough homogeneous protein for spectroscopic investigations (1,2).…”