Organization of the electron transfer chain to oxygen in the obligate human pathogen Neisseria gonorrhoeae: roles for cytochromes c4 and c5, but not cytochrome c2, in oxygen reduction

Abstract

Although Neisseria gonorrhoeae is a prolific source of eight c-type cytochromes, little is known about how its electron transfer pathways to oxygen are organized. In this study, the roles in the respiratory chain to oxygen of cytochromes c(2), c(4), and c(5), encoded by the genes cccA, cycA, and cycB, respectively, have been investigated. Single mutations in genes for either cytochrome c(4) or c(5) resulted in an increased sensitivity to growth inhibition by excess oxygen and small decreases in the respiratory capacity of the parent, which were complemented by the chromosomal integration of an ectopic, isopropyl-beta-d-thiogalactopyranoside (IPTG)-inducible copy of the cycA or cycB gene. In contrast, a cccA mutant reduced oxygen slightly more rapidly than the parent, suggesting that cccA is expressed but cytochrome c(2) is not involved in electron transfer to cytochrome oxidase. The deletion of cccA increased the sensitivity of the cycB mutant to excess oxygen but decreased the sensitivity of the cycA mutant. Despite many attempts, a double mutant defective in both cytochromes c(4) and c(5) could not be isolated. However, a strain with the ectopically encoded, IPTG-inducible cycB gene with deletions in both cycA and cycB was constructed: the growth and survival of this strain were dependent upon the addition of IPTG, so gonococcal survival is dependent upon the synthesis of either cytochrome c(4) or c(5). These results define the gonococcal electron transfer chain to oxygen in which cytochromes c(4) and c(5), but not cytochrome c(2), provide alternative pathways for electron transfer from the cytochrome bc(1) complex to the terminal oxidase cytochrome cbb(3).

FIG. 1.

(A) SDS-PAGE gel of the cccA, cycA, and cycB mutants stained for covalently bound heme. N. gonorrhoeae strains F62 (lane 1), JCGC851 (cccA) (lane 2), JCGC800 (cycA) (lane 3), JCGC850 (cycB) (lane 4), JCGC853 (cccA cycA) (lane 5), and JCGC852 (cccA cycB) (lane 6) were grown on plates at 37°C in a candle jar overnight. Samples were collected, lysed, and separated by SDS-PAGE. The gel was then heme stained to detect c-type cytochromes. (B) Western analysis of the accumulation of AniA as cultures became oxygen limited. Equivalent amounts of bacterial biomass were loaded onto each track of all the gels shown. (Top) Comparison of the cccA mutant that lacks the cytochrome c₂ gene with parental strain F62. Tracks on either side of the vertical black bar were from independent gels. (Middle) Comparison of strain F62 with the cycA mutant that lacks cytochrome c₄. (Bottom) Comparison of F62 with the cycB mutant that lacks cytochrome c₅.

FIG. 2.

Effects of different levels of aeration on the growth of cccA, cycA, and cycB mutants compared with those of the parental strain together with double mutants defective in both cytochromes c₂ and c₄ or c₂ and c₅. Shown is a comparison of the effect of limiting or excess aeration on the growth of gonococcal strains F62 (parental strain) with single mutants defective in cytochrome c₄ (strain JCGC800), cytochrome c₅ (JCGC850), or cytochrome c₂ (JCGC851) grown in either 60 ml (limiting aeration) (A) or 10 ml (oxygen sufficient) (B) of GC broth in 100-ml conical flasks. Cultures were shaken at 100 rpm and incubated at 37°C. The amount of the inoculum was adjusted so that each culture started with the same concentration of cells. (C and D) Growth of the cccA cycA double mutant defective in cytochromes c₂ and c₄ (strain JCGC 853) with the parent and the single mutant defective in only cytochrome c₄ under oxygen-limited (C) and oxygen-sufficient (D) conditions. (E and F) Oxygen-limited (E) and oxygen-sufficient (F) growth of the cccA cycB double mutant defective in cytochromes c₂ and c₅ (strain JCGC852) compared with those of the parental strain and the cycB single mutant. An OD₆₅₀ of 1.0 corresponds to a biomass of 0.4 mg ml⁻¹.

FIG. 3.

(A) Cultures of parent strain F62 containing a second, IPTG-inducible, ectopic copy of cycA (labeled c₄I) (triangles), the cycA mutant (labeled Δc₄) (circles), and the cycA mutant complemented with the IPTG-inducible copy of cycA (labeled c₄I Δc₄) (squares). (B) Cultures of parent strain F62 containing the IPTG-inducible copy of cycB (labeled c₅I) (triangles), the cycB mutant (labeled Δc₅) (circles), and the cycB mutant complemented with the IPTG-inducible copy of cycB (labeled c₅I Δc₅) (squares). For A and B, cells were grown at 37°C in 30 ml of GCB in 100-ml conical flasks with shaking at 100 rpm. Continuous lines indicate that no IPTG was added to cultures, whereas dotted lines indicate cultures where 1 mM IPTG was added at time zero. (C) Cultures of parental strain F62 (triangles) and the cycA cycB mutant containing the IPTG-inducible copy of cycB (circles). Growth conditions were the same as those described above (A and B). Continuous lines indicate that no IPTG was added to cultures, whereas dotted lines indicate cultures where 0.1 mM IPTG was added at time zero.

FIG. 4.

Scheme for electron transfer from NADH to oxygen in the gonococcus. Cyt, cytochrome; Nuo, NADH dehydrogenase; UQ, ubiquinone.