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.

Figure 1. Presence or absence of cytochrome c′ from gonococcal strains F62 and JCGC214

Total proteins from samples of aerobically grown bacteria were harvested in the middle of exponential growth, lysed in sample buffer and separated by SDS/PAGE. The gel was then stained for peroxidase activity associated with covalently bound haem. Lane 1, the cytochrome c′ mutant strain JCGC214; lane 2, the parental strain F62. The densitometer trace on the left shows the prominent top band of CcoO (a component of the only cytochrome oxidase in the gonococcus) and cytochrome c₄; the less intense lowest band in track 2, absent from the mutant, is the 16 kDa cytochrome c′.

Figure 2. Growth of the gonococcal strains F62 and its cycP mutant, JCGC214, in oxygen-limited cultures in the presence and absence of nitrite

Triplicate cultures of N. gonorrhoeae strains F62 and JCGC214 were grown under oxygen-limited conditions and the attenuance of the cultures was measured at regular intervals. (a) Attenuance of the cultures of F62 and JCGC214 grown in the presence of 3 mM nitrite. The graph is plotted on a semi-exponential scale and shows the means±S.D. of absorbance readings for three experiments. The inset shows the same data plotted on a linear scale (OD=attenuance). (b) Strains F62 and JCGC214 were also grown without nitrite added to the growth medium. The graph shows the means±S.D. for samples from triplicate cultures.

Figure 3. Effect of globomycin on the production of cytochrome c′

Whole cell proteins (300 μg) from BL21(λDE3) pST2 pST205 were separated by SDS/PAGE and stained for haem-dependent peroxidase activity. Transformed bacteria were grown aerobically in Lennox broth to an A₆₅₀ 0.5 and expression of cytochrome c′ was induced with 0.5 mM IPTG for 2 h. Globomycin at a final concentration of 20 μg/ml was added at the time of induction. Lanes 1 and 2, induced bacteria, without globomycin, after 2 h; lane 3, uninduced bacteria; lanes 4 and 5, induced bacteria in the presence of globomycin after 2 h. The approx. 14 kDa band (band 1) corresponds to mature cytochrome c′ from which the signal peptide had been cleaved; the approx. 16 kDa band 2 corresponds to cytochrome c′ with its signal peptide still attached.

Figure 4. The location of gonococcal cytochrome c′ in E. coli

Cellular fractions (300 μg of protein) from BL21(DE3) pST2 pST205 were separated by SDS/PAGE and stained for haem-dependent peroxidase activity. Transformed bacteria were grown to stationary phase, fractionated in a French press and soluble and membrane fractions were prepared by centrifugation. Lane 1, whole cells; lanes 2 and 3, soluble fraction; lanes 4 and 5, membrane fraction.

Figure 5. The separation of E. coli membranes containing gonococcal cytochrome c′ by sucrose density-gradient centrifugation

E. coli membranes containing gonococcal cytochrome c′ were loaded on to a sucrose step gradient and centrifuged at 100000 g for 48 h. Fractions were extracted from the top of the gradient. Proteins were separated by SDS/PAGE and the gel was stained first for haem-dependent peroxidase activity to locate cytochrome c′ (results not shown) and then restained for total protein. Cytochrome c′ was detected in fractions 8–14 but was most abundant in fractions 10–14 (illustrated above). Note the significant changes in the protein profile down the gradient with the outer membrane marker OmpF accumulating in lower fractions.

Figure 6. Spectral analysis of oxidized and reduced E. coli membranes containing gonococcal cytochrome c′

Membranes extracted from aerobically grown BL21(λDE3) pST205 pST2 and BL21(λDE3) pST2 were diluted to a protein concentration of 1.8 mg/ml and analysed by spectroscopy. (a) Membranes from bacteria expressing cytochrome c′ were oxidized with ammonium persulphate and the absorbance spectrum between 350 and 700 nm was recorded. The membranes were then reduced with dithionite and the spectrum was repeated. The wavelengths of absorbance maxima are shown for the oxidized and reduced spectra. (b) The ammonium persulphate oxidized spectrum was subtracted from the dithionite reduced spectrum to obtain the difference spectrum and compared with a difference spectrum for non-expressing cells. I, the Soret region; and II, the α and β regions for the membranes extracted from non-expressing and cytochrome c′-expressing bacteria.

Figure 7. Spectral analysis of E. coli membranes containing gonococcal cytochrome c′ in the presence of nitric oxide

The absorbance of membranes containing cytochrome c′ oxidized with ammonium persulphate (ferric) was recorded between 350 and 700 nm. The sample was sealed with a septum and sparged with N₂ gas to generate anaerobic conditions. The sample was then sparged with NO for 5 min and the new spectrum was recorded. The spectrum of membranes containing gonococcal cytochrome c′ in the absence of NO was subtracted from the spectrum obtained after exposure to NO to determine the difference spectrum.