Mutational and biochemical analysis of cytochrome c′, a nitric oxide-binding lipoprotein important for adaptation of Neisseria gonorrhoeae to oxygen-limited growth

Abstract

Neisseria gonorrhoeae is a prolific source of c-type cytochromes. Five of the constitutively expressed cytochromes are predicted, based on in silico analysis of the N. gonorrhoeae genome, to be components of the cytochrome bc1 complex, cytochrome c oxidase cbb3 or periplasmic cytochromes involved in electron transfer reactions typical of a bacterium with a microaerobic physiology. Cytochrome c peroxidase was previously shown to be a lipoprotein expressed only during oxygen-limited growth. The final c-type cytochrome, cytochrome c', similar to cytochrome c peroxidase, includes a lipobox required for targeting to the outer membrane. Maturation of cytochrome c' was partially inhibited by globomycin, an antibiotic that specifically inhibits signal peptidase II, resulting in the accumulation of the prolipoprotein in the cytoplasmic membrane. Disruption of the gonococcal cycP gene resulted in an extended lag phase during microaerobic growth in the presence but not in the absence of nitrite, suggesting that cytochrome c' protects the bacteria from NO generated by nitrite reduction during adaptation to oxygen-limited growth. The cytochrome c' gene was overexpressed in Escherichia coli and recombinant cytochrome c' was shown to be targeted to the outer membrane. Spectroscopic evidence is presented showing that gonococcal cytochrome c' is similar to previously characterized cytochrome c' proteins and that it binds NO in vitro. The demonstration that two of the seven gonococcal c-type cytochromes fulfil specialized functions and are outer membrane lipoproteins suggests that the localization of these lipoproteins close to the bacterial surface provides effective protection against external assaults from reactive oxygen and reactive nitrogen species.

Defenses against Oxidative Stress inNeisseria gonorrhoeae: a System Tailored for a Challenging Environment

“…A cycP mutant strain of N. gonorrhoeae has an extended lag phase during microaerobic growth in the presence of nitrite. It is suggested that the constitutive synthesis of cytochrome cЈ provides an instant defense by binding nitric oxide until NorB has accumulated to levels sufficient to reduce nitric oxide and by preventing the generation of peroxynitrite from free nitric oxide and superoxide (238). Cytochrome cЈ has also been shown to bind nitric oxide and to act as a buffer against this toxic radical species in N. meningitidis (6) and P. denitrificans (249).…”

“…Cytochrome cЈ (CycP; NG108), an outer membrane lipoprotein that binds nitric oxide in vitro, is believed to protect N. gonorrhoeae from endogenously and/or exogenously generated nitrosative stress ( Fig. 2B) (238). A cycP mutant strain of N. gonorrhoeae has an extended lag phase during microaerobic growth in the presence of nitrite.…”

Legless pathogens: how bacterial physiology provides the key to understanding pathogenicity

“…Gonococci defend themselves against attack from outside the cell by synthesizing a range of outer membrane redox proteins. These include an inducible cytochrome c peroxidase CCP and a constitutive c-type cytochrome, cytochrome c9 or CycP, which binds nitric oxide (Turner et al, 2003(Turner et al, , 2005. CycP is believed to act as a buffer system that limits damage by NO while the norB expression is induced (Anjum et al, 2002).…”

A Critical Role for the cccA Gene Product, Cytochrome c 2 , in Diverting Electrons from Aerobic Respiration to Denitrification in Neisseria gonorrhoeae

“…The sixth c-type cytochrome readily detected during aerobic growth is an NO-binding protein, CycP or cytochrome c=, which provides some protection against nitrosative stress (14)(15)(16). During oxygen-limited growth, a seventh c-type cytochrome, the cytochrome c peroxidase (Ccp), is synthesized as part of the FNR (fumarate and nitrate reductase) regulon but is absent from N. meningitidis.…”

“…During oxygen-limited growth, a seventh c-type cytochrome, the cytochrome c peroxidase (Ccp), is synthesized as part of the FNR (fumarate and nitrate reductase) regulon but is absent from N. meningitidis. Both cytochrome c peroxidase and cytochrome c= are outer membrane lipoproteins that provide protection against reactive oxygen and reactive nitrogen species, respectively (16,17). The nitrite reductase, AniA, and a lipid-associated azurin, Laz, are also outer membrane redox proteins (18)(19)(20)(21).…”