FIG 4.

(A) Nadi assay of the WT and various mutant strains. This method is commonly used to specifically detect cytochrome c oxidase activity (26), which is based on the rapid formation of indophenol blue from colorless α-naphthol catalyzed by cytochrome c oxidases with N,N-dimethyl-p-phenylenediamine monohydrochloride as an exogenous electron donor. Five microliters of cultures grown anaerobically for 24 h, which were adjusted to about 10⁷ CFU/ml, were dropped onto an agar plate in the presence of nitrate. Strains were incubated at 30°C for 4 days under anaerobic conditions and photographed after a 5-min Nadi reaction. (B) Nadi assay of anaerobically grown complementation strains. Plasmid pLYJ138 contains a WT cbb₃ allele, while pLYJ139 and pLYJ140 harbor WT aa₃ and bd alleles, respectively. (C) TEM images of Δcbb₃ and Δaa₃ Δcbb₃ strains complemented with plasmid pLY138, harboring the WT cbb₃ allele, grown in microaerobic nitrate medium. Bars, 500 nm (whole cells) and 100 nm (magnetosomes).