Characterization of the Respiratory Donor Complex I in Campylobacter jejuni

Abstract

Campylobacter jejuni is an Epsilon proteobacterium that is one of the leading causes of human bacterial gastroenteritis, campylobacteriosis, in the world. C. jejuni does not utilize carbohydrates, does not have an intact glycolytic pathway, nor can it grow fermentatively. It uses oxidative phosphorylation and the electron transport chain to fulfill its energy requirements. Inspection of its electron transport chain reveals a highly branched electron transport chain that comprises of a number of electron donor and acceptor enzymes. One of these electron donor enzymes in C. jejuni is the protein pump Complex I also known as NADH:Ubiquinone Oxidoreductase. This enzyme is encoded by the 14 gene nuo operon nuoA-N. However, two of the nuo genes that typically encode for the NADH dehydrogenase module, nuoE and nuoF, are missing and are replaced by two novel genes Cj1575c and Cj1574c respectively. These genes share no homology at all to the nuoE and nuoF genes of other bacteria and thereby provide evidence that NADH is not the electron donor to the complex I in C. jejuni. This study was conducted to characterize the complex I of C. jejuni and identify the electron donor to this enzyme. Twelve of the 14 nuo genes can be disrupted either via an insertion or deletion mutation. However, Cj1575c and Cj1574c are essential genes and could not be disrupted unless extra copies of these genes were provided at a separate location in the chromosome. Disruption of all 12 nuo genes results in strains with similar phenotypes: they will not grow in Mueller Hinton broth unless supplemented with an alternative respiratory substrate such as hydrogen or formate. We show here that flavodoxin, another essential protein in C. jejuni, is the electron donor to complex I, and that flavodoxin accepts electrons from the enzyme 2-oxoglutarate:acceptor oxidoreductase (OOR). From this data it appears that the machinery required for hydrogen and formate respiration operates separately from complex I. In order to determine the relative importance of these three respiratory pathways in host colonization, mutants in hydrogenase, formate dehydrogenase and a double mutant in these enzymes were constructed. These mutants as well as an OOR mutant (OorB::CM) and several complex I (nuo) mutants were used in chicken colonization assays. Individual mutations in hydrogenase or formate dehydrogenase do not display any defectiveness in colonizing chickens. However, a double mutant in these enzymes, OorB::CM and nuo mutants are heavily deficient in chicken colonization indicating that these enzymes may play an important role in conjunction with in vivo colonization.