Characterization of cytochromes c₅₅₀ and c₅₅₅ from Bradyrhizobium japonicum: Cloning, mutagenesis, and sequencing of the c₅₅₅ gene (cycC)

The major soluble c-type cytochromes in cultured cells of Bradyrhizobium japonicum USDA 110 comprised a CO-reactive c₅₅₅ (M(r), ~15,500) and a non-CO-reactive c₅₅₀ (M(r), ~12,500). Levels of cytochrome per gram of soluble protein in aerobic, anaerobic, and symbiotic cells were 32, 21, and 30 nmol, respectively, for c₅₅₅ and 31, 44, and 65 nmol, respectively, for c₅₅₀. The midpoint redox potentials (E(m,7)) of the purified cytochromes were +236 mV for c₅₅₅ and +277 mV for c₅₅₀. The CO reactivity of c₅₅₅ was pH dependent, with maximal reactivity at pH 10 or greater. Rabbit antiserum was produced against purified c₅₅₅ and used to screen a B. japonicum USDA 110 genomic DNA expression library in λgt11 for a downstream portion of the c₅₅₅ gene (cycC). This sequence was then used to probe a cosmid library for the entire c₅₅₅ locus. The nucleotide sequence shows an open reading frame of 149 amino acids, with an apparent signal sequence at the N terminus and a heme-binding site near the C terminus. The deduced amino acid sequence is similar to those of the cytochromes c₅₅₆ of Rhodopseudomonas palustris and Agrobacterium tumefaciens. The cycC gene was mutagenized by insertion of a kanamycin resistance cassette and homologously recombined into the B. japonicum genome. The resulting mutant made no c₅₅₅ but made normal amounts of c₅₅₀. The levels of membrane cytochromes were unaffected. The mutant and wild type exhibited identical phenotypes when used to modulate plants of soybean (Glycine max L. Merr.), with no significant differences in nodule number, nodule mass, or total amount of N₂ fixed.