Five-gene cluster in Clostridium thermoaceticum consisting of two divergent operons encoding rubredoxin oxidoreductase-rubredoxin and rubrerythrin-type A flavoprotein-high-molecular-weight rubredoxin

A five-gene cluster encoding four nonheme iron proteins and a flavoprotein from the thermophilic anaerobic bacterium Clostridium thermoaceticum (Moore lla thermoacetica) was cloned and sequenced. Based on analysis of deduced a mino acid sequences, the genes were identified as rub (rubredoxin), rbo (ru bredoxin oxidoreductase), rbr (rubrerythrin), fprA (type A flavoprotein), a nd a gene referred to as hrb (high-molecular-weight rubredoxin). Northern b lot analysis demonstrated that the five-gene cluster is organized as two su bclusters, consisting of two divergently transcribed operons, rbr-fprA-hrb and rbo-rub. The rbr, fprA, and rub genes were expressed in Escherichia col i, and their encoded recombinant proteins were purified. The molecular mass es, 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 D esulfovibrio vulgaris Rbr reacted with both native and recombinant Rbr from C. thermoaceticum, indicating that this protein was expressed in the nativ e organism. Since Rbr and Rbo have been recently implicated in oxidative st ress protection in several anaerobic bacteria and archaea, we suggest a sim ilar function of these proteins in oxygen tolerance of C. thermoaceticum.