Metal ion homeostasis mechanisms in the food-borne human pathogen Campylobacter 
jejuni are poorly understood. The Cj1516 gene product is homologous to the 
multicopper oxidase CueO, which is known to contribute to copper tolerance in 
Escherichia coli. Here we show, by optical absorbance and electron paramagnetic 
resonance spectroscopy, that purified recombinant Cj1516 contains both T1 and 
trinuclear copper centers, which are characteristic of multicopper oxidases. 
Inductively coupled plasma mass spectrometry revealed that the protein contained 
approximately six copper atoms per polypeptide. The presence of an N-terminal 
"twin arginine" signal sequence suggested a periplasmic location for Cj1516, 
which was confirmed by the presence of p-phenylenediamine (p-PD) oxidase 
activity in periplasmic fractions of wild-type but not Cj1516 mutant cells. 
Kinetic studies showed that the pure protein exhibited p-PD, ferroxidase, and 
cuprous oxidase activities and was able to oxidize an analogue of the bacterial 
siderophore anthrachelin (3,4-dihydroxybenzoate), although no iron uptake 
impairment was observed in a Cj1516 mutant. However, this mutant was very 
sensitive to increased copper levels in minimal media, suggesting a role in 
copper tolerance. This was supported by increased expression of the Cj1516 gene 
in copper-rich media. A mutation in a second gene, the Cj1161c gene, encoding a 
putative CopA homologue, was also found to result in copper hypersensitivity, 
and a Cj1516 Cj1161c double mutant was found to be more copper sensitive than 
either single mutant. These observations and the apparent lack of alternative 
copper tolerance systems suggest that Cj1516 (CueO) and Cj1161 (CopA) are major 
proteins involved in copper homeostasis in C. jejuni.