The sulfate-reducing strain AK-01 activates alkanes via addition of the subterminal carbon to the double bond of fumarate. This reaction is similar to the action of the glycyl radical enzyme benzylsuccinate synthase (Bss). It was hypothesized that strain AK-01 possesses a similar enzyme. Degenerate bssA primers and inverse PCR were used to amplify two unlinked genes (assA1 and assA2), which encode catalytic subunits of glycyl radical type enzymes. Subsequent genome sequencing of AK-01 revealed two ass operons. SDS-PAGE analysis of AK-01 grown on n-hexadecane revealed a 95-kDa protein which is absent in hexadecanoate-grown cells. LC-MS/MS data obtained from a tryptic digest of this protein match the deduced amino acid sequence encoded by assA1, thus confirming AssA1's involvement in alkane metabolism. This report is the first description of a gene involved in anaerobic n-alkane metabolism in a sulfate-reducer and provides evidence for a novel glycyl radical enzyme. (C) 2007 Elsevier Inc. All rights reserved.