Downloaded from www.microbiologyresearch.org by IP: 202.28.250.96 On: Sat, 28 Nov 2015 04:11:15 Roles of c-type cytochromes in respiration in Neisseria meningitidis Manu Deeudom, 1,2 Michael Koomey 3 and James W. B. Moir 1 Correspondence James W. B. Moir jm46@york.ac.uk 1 Department of Biology (Area 10), University of York, Heslington, York YO10 5YW, UK 2 Department of Microbiology, Faculty of Medicine, Chiangmai University, Chiangmai 50200, Thailand 3 Department of Molecular Biosciences, Centre for Molecular Biology and Neuroscience, University of Oslo, 0316 Oslo, Norway Received 7 May 2008 Revised 6 June 2008 Accepted 16 June 2008 Three c-type cytochromes were identified in Neisseria meningitidis, based on predictions from genome sequences, that were hypothesized to be involved in electron transport to terminal electron acceptor reductases for oxygen (the cytochrome cbb 3 oxidase) and nitrite (the nitrite reductase, AniA). Mutants were generated by allelic exchange with disrupted copies of the genes encoding these cytochromes and the phenotypes of the resultant mutants analysed. It was found that cytochrome c 5 is required for in vivo nitrite reductase activity, whereas cytochromes c x and c 4 are both required for efficient growth using oxygen as an electron acceptor. Mutants in c x , c 4 , and c x +c 4 have a decreased capacity to reduce oxygen, but there is a background oxygen- reduction activity, indicating that there may be other routes for electron transfer from the cytochrome bc 1 complex to the cytochrome cbb 3 oxidase, whereas cytochrome c 5 appears to be the sole route of electrons to the nitrite reductase in N. meningitidis. Interestingly, cytochrome c x is highly similar to a domain of copper nitrite reductases from various proteobacteria, whereas cytochrome c 5 has high identity with a domain of the cytochrome cbb 3 oxidase of Neisseria gonorrhoeae, yet these two proteins function in oxygen respiration and nitrite respiration, respectively. This highlights a limitation of predicting protein function from similarity to known proteins, i.e. very closely related protein domains in different organisms can have different redox partners. INTRODUCTION The b-proteobacterium Neisseria meningitidis is commonly identified as part of the commensal flora of the human nasopharyngeal mucosa. Very occasionally colonization is followed by invasion of the bloodstream by N. meningitidis, leading to life-threatening illness in the form of meningitis or septicaemia (van Deuren et al., 2000). The nasopharyngeal mucosa is habitat to a wide variety of other bacteria, including both aerobes and anaerobes (Brook, 2003). In previous work, we have determined that under aerobic conditions N. meningitidis is able to support respiration by reducing oxygen to water via the enzyme cytochrome cbb 3 oxidase (which is the only oxygen reductase in N. meningitidis), and that when oxygen becomes limiting the bacterium expresses genes encoding a nitrite reductase (aniA) and a nitric oxide reductase (norB) that collectively catalyse the respiratory reduction of nitrite to nitrous oxide (Anjum et al., 2002). N. meningitidis is able to employ this partial denitrification pathway to support growth under microaerobic conditions in the presence of nitrite (Rock et al., 2005). We have proposed an organization for the respiratory chain of N. meningitidis, based on genome sequence analysis and experimental analysis with specific inhibitors (Deeudom et al., 2006). The nitric oxide reductase of N. meningitidis appears more similar to the quinol-oxidizing nitric oxide reductase (qNOR) than the cytochrome c-oxidizing NOR (cNOR) (de Vries & Schro ¨der, 2002), and hence it was proposed that the nitric oxide reductase receives its electrons directly from the ubiquinone pool. This was confirmed experimentally by showing that nitric oxide reduction is insensitive to the cytochrome bc 1 complex inhibitor myxo- thiazol. Contrastingly, the reduction of the other two electron acceptors (oxygen and nitrite) is very sensitive to myxothia- zol. The enzymes responsible for these reductase reactions typically receive their electrons from c-type cytochromes in other micro-organisms, and hence it was proposed that these two enzymes terminate the electron-transport chain down- stream of the cytochrome bc 1 complex and c-type cyto- chromes (Deeudom et al., 2006). The genome of N. meningitidis MC58 reveals the presence of genes encoding three putative c-type cytochromes that might mediate the transfer of electrons between the cytochrome bc 1 complex and the reductases for oxygen and nitrite. These three c-type cytochromes are conserved within the genomes of other N. meningitidis strains and in Neisseria gonorrhoeae. Microbiology (2008), 154, 2857–2864 DOI 10.1099/mic.0.2008/020339-0 2008/020339 G 2008 SGM Printed in Great Britain 2857