Copyright C 1992, American Society for Microbiology Cloning, Nucleotide Sequence, and Transcriptional Analyses of the Gene Encoding a Ferredoxin from

A mixed 17-mer oligonucleotide deduced from the N terminus of a ferredoxin isolated from Methanosarcina thermophila was used to probe a Agtll library prepared from M. thermophila genomic DNA; positive clones contained either a 5.7- or 2.1-kbp EcoRI insert. An open reading frame (fdx4) located within the 5.7-kbp insert had a deduced amino acid sequence that was identical to the first 26 N-terminal residues reported for the ferredoxin isolated from M. thermophila, with the exception of the initiator methionine.fdxA had the coding capacity for a 6,230-Da protein which contained eight cysteines with spacings typical of 214Fe-4S] ferredoxins. An open reading frame (ORF1) located within the 2.1-kbp EcoRI fragment also had the potential to encode a 2[4Fe-4S] bacterial-type ferredoxin (5,850 Da).fdxA and ORFI were present as single copies in the genome, and each was transcribed on a monocistronic mRNA. While thefdxA- and ORFl-specific mRNAs were detected in cells grown on methanol and trimethylamine, only thefdx4-specific transcript was present in acetate-grown cells. The apparent transcriptional start sites offdx4 and ORF1, as determined by primer extension analyses, lay 21 to 28 bases downstream of sequences with high identity to the consensus methanogen promoter. Ferredoxins are small, acidic proteins containing nonheme iron and acid-labile sulfur in clusters coordinated by cys-teines. These ubiquitous electron transport proteins partici