Molecular cloning, expression and biochemical characterization of periplasmic nitrate reductase from Campylobacter jejuni

Campylobacter jejuni, a human gastrointestinal pathogen, uses nitrate for growth under microaerophilic conditions using periplasmic nitrate reductase (Nap). The catalytic subunit, NapA, contains two prosthetic groups, an iron sulfur cluster and a molybdenum cofactor. Here we describe the cloning, expression, purification, and Michaelis-Menten kinetics (kcat of 5.91 ± 0.18 s−1 and a KM (nitrate) of 3.40 ± 0.44 μM) in solution using methyl viologen as an electron donor. The data suggest that the high affinity of NapA for nitrate could support growth of C. jejuni on nitrate in the gastrointestinal tract. Site-directed mutagenesis was used and the codon for the molybdenum coordinating cysteine residue has been exchanged for serine. The resulting variant NapA is 4-fold less active than the native enzyme confirming the importance of this residue. The properties of the C. jejuni enzyme reported here represent the first isolation and characterization of an epsilonproteobacterial NapA. Therefore, the fundamental knowledge of Nap has been expanded.

The Periplasmic Nitrate Reductase NapABC Supports Luminal Growth of Salmonella enterica Serovar Typhimurium during Colitis

The food-borne pathogenSalmonella entericaserovar Typhimurium benefits from acute inflammation in part by using host-derived nitrate to respire anaerobically and compete successfully with the commensal microbes during growth in the intestinal lumen. TheS. Typhimurium genome contains three nitrate reductases, encoded by thenarGHI,narZYV, andnapABCgenes. Work on homologous genes present inEscherichia colisuggests that nitrate reductase A, encoded by thenarGHIgenes, is the main enzyme promoting growth on nitrate as an electron acceptor in anaerobic environments. Using a mouse colitis model, we found, surprisingly, thatS. Typhimurium strains with defects in either nitrate reductase A (narGmutant) or the regulator inducing its transcription in the presence of high concentrations of nitrate (narLmutant) exhibited growth comparable to that of wild-typeS. Typhimurium. In contrast, a strain lacking a functional periplasmic nitrate reductase (napAmutant) exhibited a marked growth defect in the lumen of the colon. InE. coli, thenapABCgenes are transcribed maximally under anaerobic growth conditions in the presence of low nitrate concentrations. Inactivation ofnarP, encoding a response regulator that activatesnapABCtranscription in response to low nitrate concentrations, significantly reduced the growth ofS. Typhimurium in the gut lumen. Cecal nitrate measurements suggested that the murine cecum is a nitrate-limited environment. Collectively, our results suggest thatS. Typhimurium uses the periplasmic nitrate reductase to support its growth on the low nitrate concentrations encountered in the gut, a strategy that may be shared with other enteric pathogens.