Deletion and site-directed mutagenesis of the Wolinella succinogenes fumarate reductase operon

Abstract

Wolinella succinogenes is known to grow at the expense of fumarate respiration with formate or sulfide as electron donor. A W. succinogenes mutant (delta frdCAB) lacking the fumarate reductase operon did not grow with fumarate as terminal electron acceptor and either formate or sulfide as electron donor. The delta frdCAB mutant grown with formate and nitrate did not contain fumarate reductase activity and did not catalyze electron transport from sulfide to fumarate, in contrast to the nitrate-grown wild-type strain. A mutant constructed by integration of frdCAB into the delta frdCAB mutant genome showed wild-type properties with respect to growth and enzyme activities. The frdC2 gene located downstream of the fumarate reductase operon frdCAB possibly encodes a diheme cytochrome b that is similar to FrdC (41% identical residues). The corresponding transcript differs from that of frdCAB. A delta frdC2 mutant showed wild-type properties with respect to growth and enzyme activities. Using site-directed mutagenesis, each of the four histidine residues that are predicted to serve as the axial heme ligands in FrdC (His44, His93, His143, and His182) was replaced by alanine or other residues. The resulting mutants did not grow with formate and fumarate and did not contain fumarate reductase activity, FrdA or FrdC when grown with formate and nitrate. In contrast, substitution of two histidine residues that are not considered heme ligands, yielded mutants (H114A and H120A) that grew with fumarate. It is concluded that FrdCAB is an obligatory component of fumarate respiration with formate and with sulfide in W. succinogenes. FrdC2 is not involved in fumarate respiration. Replacement of the putative heme ligands of FrdC prevents formation of a functional fumarate reductase.