The Role of Flavodoxin in The Food-Borne Pathogen Campylobacter jejuni

Abstract

Campylobacter jejuni is the most frequent cause of food-borne gastroenteritis worldwide but remains a poorly understood pathogen. Unusually for an aerobe, it uses pyruvate and 2-oxoglutarate oxidoreductases (POR/OOR) that reduce a flavodoxin (FldA), which acts as the electron donor to Complex I in the respiratory chain, rather than NADH. This study has focussed on additional ways in which FldA is reduced and its physiological roles in the cell, as it is unclear at present why this protein is essential for viability.

We purified and characterised FldA and identified an additional reductase, in addition to POR/OOR. This is called FqrB (Cj0559). The FqrB protein was first characterized in H. pylori and is a member of the NADPH oxidase protein family that contains flavin adenine dinucleotide (FAD) as a cofactor. We were able to delete the fqrB gene in C. jejuni. An fqrB deletion mutant was viable but displayed a significant growth defect, indicating a key role in normal cell growth. FqrB is related to flavoprotein reductases from Gram-positive bacteria that can reduce NrdI, a specialised flavodoxin that is needed for tyrosyl radical formation in NrdF, the beta subunit of Class 1b-type (Mn) ribonucleotide reductase (RNR). However, C. jejuni possesses a single Class 1a-type (Fe) RNR (NrdAB) that would be expected to be ferredoxin dependent. We show that CjFldA is an unusually high potential flavodoxin unrelated to NrdI, yet growth of the fqrB mutant, but not the wild-type or a complemented strain, was stimulated by low deoxyribonucleoside (dRNS) concentrations, suggesting FldA links FqrB and RNR activity. Using purified proteins, we confirmed the NrdB tyrosyl radical could be regenerated in a FldA, FqrB and NADPH dependent manner, as evidenced by both optical and electron paramagnetic (EPR) spectroscopy. Thus, FldA activates ribonucleotide reductase in C. jejuni, explaining its essentiality, which might provide a target for inhibition by anti-microbial agents. Finally, we examined the genomes of sequenced Arcobacter spp, close relatives of Campylobacter which have multiple flavodoxins, FqrB homologues and two types of Complex I and performed some preliminary functional studies on some of these proteins from A. butzleri.