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Journal of Bacteriology, March 2001, p. 1560-1567, Vol. 183, No. 5
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.5.1560-1567.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Five-Gene Cluster in Clostridium thermoaceticum Consisting of Two Divergent Operons Encoding Rubredoxin Oxidoreductase- Rubredoxin and Rubrerythrin-Type A Flavoprotein- High-Molecular-Weight Rubredoxin

Amaresh Das,1 Eric D. Coulter,2 Donald M. Kurtz Jr.,2 and Lars G. Ljungdahl1,*

Center for Biological Resource Recovery and Department of Biochemistry and Molecular Biology,1 and Department of Chemistry and Center for Metalloenzyme Studies,2 University of Georgia, Athens, Georgia 30602

Received 18 August 2000/Accepted 6 December 2000

A five-gene cluster encoding four nonheme iron proteins and a flavoprotein from the thermophilic anaerobic bacterium Clostridium thermoaceticum (Moorella thermoacetica) was cloned and sequenced. Based on analysis of deduced amino acid sequences, the genes were identified as rub (rubredoxin), rbo (rubredoxin oxidoreductase), rbr (rubrerythrin), fprA (type A flavoprotein), and a gene referred to as hrb (high-molecular-weight rubredoxin). Northern blot analysis demonstrated that the five-gene cluster is organized as two subclusters, consisting of two divergently transcribed operons, rbr-fprA-hrb and rbo-rub. The rbr, fprA, and rub genes were expressed in Escherichia coli, and their encoded recombinant proteins were purified. The molecular masses, UV-visible absorption spectra, and cofactor contents of the recombinant rubrerythrin, rubredoxin, and type A flavoprotein were similar to those of respective homologs from other microorganisms. Antibodies raised against Desulfovibrio vulgaris Rbr reacted with both native and recombinant Rbr from C. thermoaceticum, indicating that this protein was expressed in the native organism. Since Rbr and Rbo have been recently implicated in oxidative stress protection in several anaerobic bacteria and archaea, we suggest a similar function of these proteins in oxygen tolerance of C. thermoaceticum.

* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, A214 Life Sciences Building, The University of Georgia, Athens, GA 30602-7229. Phone: (706) 542-7640. Fax: (706) 542-2222. E-mail: larsljd{at}arches.uga.edu.

Journal of Bacteriology, March 2001, p. 1560-1567, Vol. 183, No. 5
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.5.1560-1567.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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