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Journal of Bacteriology, December 2008, p. 8075-8085, Vol. 190, No. 24
0021-9193/08/$08.00+0     doi:10.1128/JB.00821-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

A Multicopper Oxidase (Cj1516) and a CopA Homologue (Cj1161) Are Major Components of the Copper Homeostasis System of Campylobacter jejuni{triangledown}

Stephen J. Hall,1 Andrew Hitchcock,1 Clive S. Butler,2 and David J. Kelly1*

Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, United Kingdom,1 School of Biosciences, Centre for Biocatalysis, University of Exeter, Stocker Road, Exeter EX4 4QD, United Kingdom2

Received 11 June 2008/ Accepted 6 October 2008


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ABSTRACT
 
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.


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INTRODUCTION
 
Although Campylobacter jejuni is part of the normal commensal flora of many bird species, it is pathogenic to humans, and ingestion of contaminated poultry is a common route for infection. Consequently, C. jejuni is one of the most important causes of human enteric disease worldwide and continues to be a major public health and economic burden (23). Acute symptoms of C. jejuni infection in humans include diarrhea, fever, and abdominal pain, but complications can include reactive arthritis and neurological sequelae such as the Miller-Fisher and Guillain-Barré syndromes (55). Despite the importance of C. jejuni as a food-borne pathogen and the sequencing of the genomes of a number of strains (19, 27, 40), there are many aspects of the biology of this bacterium, particularly stress responses and homeostatic mechanisms, that remain poorly defined. The molecular mechanisms of pathogenesis of C. jejuni are still not completely understood, although a number of virulence factors have been identified, including motility and chemotaxis, adhesion to and invasion of host cells, and toxin production. Iron acquisition is also an important virulence factor, and in recent years this area has been studied extensively in C. jejuni (36, 47, 63). However, the acquisition, metabolism, and homeostasis of other key metals in C. jejuni, such as copper and zinc, have largely been overlooked.

Metal homeostasis is extremely important in biological systems, and metals such as copper, iron, and zinc are essential for bacterial growth. These metals are usually present in trace amounts in the environment but play important roles in electron transport and redox reactions as cofactors of many enzymes, such as cytochrome c oxidase (44) and superoxide dismutase (41). However, in excess, they can be toxic and thus require specific systems to cope with metal-induced stress. Toxicity occurs via a number of mechanisms, including metal atoms binding to thiol groups and disrupting protein function (38, 46, 56, 61), displacement of metal cofactors in proteins by competition, and the generation of reactive oxygen species through Fenton-like reactions (59).

In Escherichia coli, as many as three distinct systems for copper tolerance have been identified and include the cop/cue and cus systems (38), encoded on the chromosome, and the plasmid-encoded pco system (11). The cus system consists of three proteins (CusCBA) which span the periplasm and outer membrane and CusF, a periplasmic binding protein. This system is involved in the efflux of excess copper in mainly anaerobic situations (22). The plasmid-encoded system pco is present in some strains of E. coli (33) and other organisms, such as Pseudomonas syringae pv. Tomato (5). The system usually consists of seven genes, encoding a multicopper oxidase (MCO), a periplasmic copper binding protein, three other proteins thought to form a membrane transporter, and a two-component regulatory system (5, 11). The cop/cue system consists of CopA, which has been described as the central component of copper homeostasis in E. coli, required for copper resistance under both aerobic and anaerobic conditions (49), and CueO, an MCO operating in the periplasm. Homologues of this system appear to be widespread in bacteria.

MCOs are a diverse family of metalloenzymes which are widely distributed among eukaryotes. They are copper-containing proteins characterized by distinctive structural, spectroscopic, and enzymatic properties (58). The currently well-defined MCOs are Fet3 from Saccharomyces cerevisiae and human ceruloplasmin, both of which have defined roles in iron acquisition (3, 15, 26). Extensive knowledge about the structure and roles of MCOs in eukaryotes contrasts with the situation in prokaryotes, where the widespread existence of MCOs in bacterial genomes (where they are often annotated as laccases) has only recently begun to be recognized (1). Almost all laccases (benzenediol:oxygen oxidoreductases; EC 1.10.3.2) exhibit p-diphenol:O2 oxidoreductase activity, and they are especially common in plants and fungi, but a link between bacterial MCOs and transition metal metabolism is emerging from studies that suggest their involvement in a range of important metal acquisition/homeostasis systems, including those for copper, manganese, and iron. As mentioned above, for E. coli the MCO CueO has been proposed to be involved in the removal of excess copper from the cell as part of a copper efflux system consisting of CueO and CopA, under the control of a MerR-like regulatory element, CueR (24, 25, 38, 39). Manganese oxidation has been suggested as the physiological role for CumA, an MCO present in Pseudomonas putida (10). Compelling evidence has been presented that shows an MCO in Pseudomonas aeruginosa with similarity to Fet3 and CueO to be involved in the acquisition of ferrous iron (29). Mutant strains lacking this protein were unable to grow aerobically with Fe(II) as the sole iron source, and iron uptake analysis showed that the mutant was impaired in Fe(II) uptake but unaffected in Fe(III) uptake (29). Thus, it is clear that the physiological roles of prokaryotic MCOs are diverse and cannot be determined by sequence similarities alone.

In this paper, we identify a periplasmic MCO in C. jejuni that possesses phenoloxidase, ferroxidase, and cuprous oxidase activities. From biochemical and mutant phenotype data, we propose that the major physiological role of this enzyme is the oxidation of copper in the periplasm. However, by acting together with a homologue of the copper(I)-exporting class of P-type ATPases (CopA), this protein can remove and detoxify copper from the cytoplasm, and these two proteins appear to form the major copper homeostasis system in C. jejuni.


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MATERIALS AND METHODS
 
Bacterial strains, media, and culture conditions. C. jejuni strain NCTC 11168 was routinely cultured at 37°C under microaerobic conditions (10% [vol/vol] O2, 5% [vol/vol] CO2, and 85% [vol/vol] N2 in a MACS growth cabinet [Don Whitley Scientific Ltd., Shipley, United Kingdom]) on Columbia agar containing 5% (vol/vol) lysed horse blood and 10 µg ml–1 each of amphotericin B and vancomycin. Liquid cultures of C. jejuni were routinely grown microaerobically at 200 rpm, either in Mueller-Hinton broth (Oxoid Ltd., United Kingdom) supplemented with 20 mM L-serine (MH-S) or in the defined medium minimal essential medium alpha (MEM-{alpha}) (containing glutamine and deoxyribonucleotides but no phenol red; Invitrogen Ltd.), containing the above antibiotics and 45 µM FeSO4, 20 mM serine, and 20 mM pyruvate. To select for the C. jejuni Cj1516 mutant, kanamycin was added to media at a final concentration of 30 µg ml–1, and to select for the Cj1161c mutant, chloramphenicol was added to media to a final concentration of 30 µg ml–1. E. coli DH5{alpha} was cultured in Luria-Bertani (LB) broth or agar supplemented with appropriate antibiotics at 37°C. For growth experiments, C. jejuni overnight starter cultures were prepared in MH-S and washed three times before inoculation into MEM-{alpha}. Growth was monitored at 600 nm, using an Amersham Pharmacia Biotech Ultrospec 2000 spectrophotometer.

DNA isolation and manipulation. Plasmid DNA was isolated by using a Qiagen miniprep kit (Qiagen Ltd., Crawley, United Kingdom). C. jejuni chromosomal DNA was extracted by using a Wizard genomic DNA purification kit (Promega, Madison, WI). Standard techniques were employed for the cloning, transformation, preparation, and restriction analysis of plasmid DNA from E. coli (52).

Overexpression and purification of Cj1516. For the overexpression of the Cj1516 gene product, primers (forward primer, 5'-ATCAGCTAGCAATAGAAGAAATTTTTTA-3'; and reverse primer, 5'-TAGCGGATCCTTATTCCTTTACTTCTAA-3' [an NheI site is underlined, and a BamHI site is shown in bold italics]) were designed to amplify the complete Cj1516 gene from C. jejuni NCTC 11168 chromosomal DNA by PCR using a proofreading DNA polymerase enzyme (Pwo polymerase; Roche Ltd., United Kingdom). The PCR fragment was then cloned by blunt end ligation into pGEM3ZF (–) (Promega Ltd., United Kingdom) to create pGEM1516. The gene was excised from pGEM1516 by digestion with NheI and BamHI and cloned into similarly digested pET21a(+) (Novagen Ltd., United Kingdom) to give pMCO1516. Automated DNA sequencing (Lark Technologies Inc., Saffron Walden, United Kingdom) showed that the sequence of the Cj1516 gene in pMCO1516 was correct. pMCO1516 was transformed into E. coli BL21(DE3) cells, which were grown aerobically at 25°C in LB medium containing ampicillin (50 µg ml–1) and 1 mM copper sulfate (CuSO4) to an optical density at 600 nm (OD600) of 0.6 before 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added. Induced cells were then grown for a further 16 h before being harvested by centrifugation (30 min, 4°C, 3,500 x g). Cell pellets were resuspended in 10 mM Tris-HCl, pH 8.0, and disrupted by sonication with an MSE Soniprep 150 instrument (Sanyo, United Kingdom), using six 20-s bursts of ultrasound (amplitude, ~15 µm from peak to peak), with 30-s intervals between bursts. Cell debris and soluble matter were separated by centrifugation at 12,000 x g for 20 min at 4°C. The fractions were kept on ice until they were used or stored at –20°C. The supernatant was recovered as a cell extract and fractionated on a DEAE Sepharose fast-flow column (GE Healthcare, United Kingdom) by ion-exchange chromatography. The protein was eluted from the resin by a gradient of 0 to 500 mM NaCl in 10 mM Tris-HCl, pH 7.5. Fractions were pooled, adjusted to 1 M ammonium sulfate, and further fractionated by hydrophobic interaction chromatography using a 10-ml phenyl Sepharose (Sigma) column. A salt gradient of 1 M to 0 M (NH4)2SO4 was used to elute the proteins. Recombinant Cj1516 elution from the chromatography columns was detected by monitoring the A607 due to type 1 (blue) copper content. The final purification step utilized the apparent thermal stability of MCOs and involved heat treatment of the samples as previously described (30). Briefly, pooled fractions collected from the hydrophobic interaction step were incubated at 70°C for 5 min before being centrifuged to remove denatured proteins. Phenoloxidase activity was monitored before and after heat treatment to ensure that the activity of the recombinant protein was not lost.

Construction of mutants. A feoB mutant was constructed by Mariner transposon insertion into the Cj1398 gene and was kindly provided by A. Grant, Cambridge Veterinary School, University of Cambridge, United Kingdom. Cj1516 and Cj1161c mutants were constructed by insertion of kanamycin and chloramphenicol resistance cassettes, respectively, into each gene in the same transcriptional orientation. The Cj1516 gene was amplified using the following specific primers: Cj1516For, 5'-CAAAGTCCGCTACAAGTACAAC-3'; and Cj1516rev, 5'-CCGATCTTGAAACACGACATAGA-3'. The resulting 1.59-kb fragment containing the coding region of the gene was cloned into the pGEM 3Zf (–) vector (Promega, United Kingdom). Transformants were recovered by selection on plates containing ampicillin (50 µg ml–1). The kanamycin resistance cassette derived by PCR from plasmid pJMK30 was cloned into the unique SwaI restriction site in the center of the Cj1516 gene to produce plasmid p1516kan. For construction of a Cj1161c mutant strain, primers Cj1161cF (5'-ATGCATGGAAGAATTGCGTAT-3') and Cj1161cR (5'-ATGCTCTTAAAGAATTAAGCACTACA-3') were used to amplify a 2.085-kb fragment containing the entire coding region of the Cj1161c gene, and this fragment was cloned into the pGEM-T Easy vector to produce plasmid pGEM1161c. The chloramphenicol resistance cassette derived from pAV35 (64) was cloned into the unique SwaI restriction site in the Cj1161c gene in pGEM1161c to produce p1161cCat.

The p1516kan and p1161cCat plasmids were transformed by electroporation into C. jejuni NCTC 11168, and transformants were selected using Columbia blood agar plates supplemented with either kanamycin (final concentration, 30 µg ml–1) or chloramphenicol (final concentration, 30 µg ml–1). Correct insertion of the antibiotic resistance cassettes into the target genes was confirmed by PCR. Specific primers used to amplify the Cj1516 and Cj1161c genes (see above) were used to confirm the allelic exchange by double crossover in each mutant. This was demonstrated by increases in PCR product size of 0.8 kb and 1.4 kb for the chloramphenicol and kanamycin resistance cassette insertions, respectively. The Cj1516 mutant strain was designated SJH400, and the Cj1161c mutant strain was designated AH100. A double mutant was created by electroporation of AH100 with the p1516kan plasmid and selection on Columbia agar blood plates containing both kanamycin and chloramphenicol.

Real-time PCR. MEM-{alpha} (containing serine and pyruvate as C sources, plus ferrous sulfate, sodium metabisulfite, and sodium pyruvate [FBP] as the iron source) broth cultures (50 ml) of wild-type (WT) and mutant strains were harvested directly into a mix of 187.5 µl of prechilled phenol made up in 3.56 ml of 100% ethanol to stabilize the RNA. Samples were then centrifuged at 8,000 x g for 4 min (4°C). Total RNA was purified from cell pellets by use of an RNeasy Mini kit (Qiagen, United Kingdom) as recommended by the supplier. The RNA concentration and purity were determined using an Eppendorf biophotometer. Gene-specific primers were designed to amplify 50- to 150-nucleotide fragments of the gyrA (internal control), Cj1516, Cj1161c, moaD, and Cj1160c genes, using Primer 3 software (http://primer3.sourceforge.net/). One-step real-time reverse transcription-PCR (RT-PCR) was performed on total RNA samples, using a Brilliant SYBR green QRT-PCR kit (Stratagene). Reaction mixtures contained 50 ng of RNA, 12.5 µl 2x SYBR green QRT-PCR master mix (containing an optimized PCR buffer, 2.5 mM MgCl2, GAUC nucleotides, SureStart Taq DNA polymerase, SYBR green, and stabilizers), 200 nM specific primer mix, and 1 µl StrataScript RT/RNase block and were made up to 25 µl with superpure RNase-free water. Each reaction was carried out in a total volume of 25 µl in a 96-well optical reaction plate (Stratagene). Experiments were performed in a Stratagene mx3005p thermocycler with the following thermal cycling conditions: 50°C for 30 min for cDNA synthesis; 95°C for 10 min; and 40 cycles of 95°C for 30 s, 50°C for 1 min, and 72°C for 30 s. The data were analyzed using MxPro QPCR software (Stratagene) and further processed in Microsoft Excel. A standard curve was established for each gene studied, using genomic DNA, to confirm that the primers amplified at the same rate and to validate the experiment. The relative levels of expression of moaD in the Cj1516 mutant and of the Cj1160c gene in the Cj1161c mutant compared with those in the WT were calculated following the protocol for the standard curve method in Applied Biosystems user bulletin 2 (relative quantification of gene expression; ABI Prism 7700 sequence detection system). No-template reactions were included as negative controls.

Phenoloxidase assays and kinetics. Phenoloxidase assays were carried out on purified recombinant Cj1516 protein or cellular periplasmic fractions prepared by the osmotic shock method as described previously (35). The 1-ml assay volume consisted of 50 mM sodium acetate buffer, pH 5.7, containing 0.8 µM of pure Cj1516 protein or 50 µg periplasmic protein. The assay was started by the addition of p-phenylenediamine (p-PD) to final concentrations of 0 to 8 mM for assays containing excess copper and 0 to 60 mM for those without excess copper, and rates were recorded at 487 nm using a Shimadzu UV-2401 dual-wavelength scanning spectrophotometer (Shimadzu Ltd.). All assays were performed at 37°C. Specific activities were calculated using an extinction coefficient for p-PD of 14.7 mM–1 cm–1 at 487 nm. Sigmaplot 8.0 (SPSS Inc.) was used for calculation of Vmax and Km values, data were averaged from at least three separate assays, and the hyperbolic curve-fitting algorithms of Sigmaplot were used to analyze the data.

Ferroxidase assays and kinetics. Ferroxidase assays were performed on purified recombinant Cj1516 protein. The 1-ml assay volume contained 50 mM sodium acetate buffer, pH 5.7, and 0.8 µM Cj1516 protein. The assay was started by the addition of ammonium ferrous sulfate to final concentrations of 0.01 to 0.3 mM, and rates were recorded at 315 nm as described above. All assays were performed at 37°C in matching quartz cuvettes. Specific activities were calculated using an extinction coefficient for Fe(III) of 2.2 mM–1 cm–1 at 315 nm. Sigmaplot 8.0 (SPSS Inc.) was used to calculate Vmax and Km values as described above.

Siderophore oxidase assays and kinetics. Oxidation assays of an analogue of the bacterial siderophore anthrachelin were performed on pure recombinant Cj1516 protein. Each assay mixture contained 50 mM sodium acetate buffer, pH 5.7, and 0.8 µM of pure Cj1516 protein. The assay was started by the addition of 3,4-dihydroxybenzoate (3,4-DHB) to a concentration range of 0 to 8 mM for assays containing excess copper and 0 to 60 mM for those without excess copper, and rates were recorded at 487 nm using a Shimadzu UV-2401 dual-wavelength scanning spectrophotometer as described above. All assays were performed at 37°C. Specific activities were calculated using an extinction coefficient for 3,4-DHB of 2.3 mM–1 cm–1 at 400 nm. Sigmaplot 8.0 (SPSS Inc.) was used for the calculation of Vmax and Km values as described above.

Measurement of metal ion oxidation-linked oxygen consumption. Metal ion oxidation by pure Cj1516 protein was determined by measuring the change in dissolved oxygen concentration in a Clark-type polargraphic oxygen electrode (Rank Brothers Ltd., Bottisham, Cambridge, United Kingdom) comprising a water-jacketed Perspex chamber that was stirred magnetically, linked to a chart recorder, and calibrated using air-saturated water. One hundred percent saturation was assumed to be 220 µM O2. A zero-oxygen baseline was determined by the addition of sodium dithionite. The chamber was maintained at 37°C and stirred at a constant rate. Substrates were added by injection through a fine central pore in the airtight plug. Substrates used were manganese chloride, ammonium ferrous sulfate, and a caged form of copper(I), which consisted of the compound tetrakis (acetonitrile) copper(I) hexafluorophosphate (Sigma-Aldrich, United Kingdom) dissolved in argon-sparged 5% acetonitrile [referred to hereafter as caged copper(I)]. Cuprous oxidase assays were carried out as described previously (54). Pure Cj1516 (1.3 µM) was used in each cuprous oxidase assay, and 0.8 µM Cj1516 was used for manganese and ferrous iron assays. All assays were performed in 100 mM Tris-acetate buffer, pH 5.7, and rates were expressed in µmol O2 utilized min–1 mg protein–1. For analysis of cuprous oxidase kinetics, the means of three assay measurements at various substrate concentrations were used. The hyperbolic curve-fitting algorithms of GraphPad Prism 5.0 for Mac (GraphPad Software, San Diego, CA) were used to analyze the data and to calculate Km and Vmax values based on the Michaelis-Menten equation.

Spectroscopy. UV-visible electronic absorbance spectra were collected by using a Shimadzu UV-2401 spectrophotometer (Shimadzu Ltd., Japan). Pure Cj1516 protein in 10 mM Tris-HCl, pH 7.5, was scanned as isolated or with the addition of 5 mM EDTA or sodium dithionite. Electron paramagnetic resonance (EPR) spectra were recorded with a Bruker (Billerica, MA) EMX spectrometer (X band, 9.38 GHz) equipped with an ER4112HV liquid-helium-flow cryostat system. Spectra were recorded at a temperature of 30 K, a modulation amplitude of 6 mT, and a microwave power of 0.2 mW. The protein for analysis was used as isolated.

Protein and copper content determinations. The concentration of protein was determined by the Bradford method (9), using bovine serum albumin as the standard. Copper content was determined by inductively coupled plasma mass spectrometry, using an Agilent 4500 spectrometer (Agilent Systems) operated by the University of Sheffield Centre for Chemical Instrumentation and Analytical Services.

Copper tolerance growth experiments. Triplicate 10-ml cultures of each strain (WT NCTC 11168 and the Cj1516, Cj1161c, and Cj1516 Cj1161c mutants) were grown in MEM-{alpha} containing copper(II) sulfate in the concentration range of 0 to 1 mM. Cultures were incubated microaerobically from a starting OD600 of 0.1 to stationary phase at 37°C with shaking. The final OD600 was recorded using an Amersham Pharmacia Ultrospec 2000 spectrophotometer.

Iron-limited growth experiments. Ferrous iron-restricted and -replete experiments were performed with the WT and the Cj1516 and feoB mutants. Starter cultures were grown microaerobically in MH-S medium at 37°C to late exponential phase and then harvested and washed in MEM-{alpha} to remove excess iron. The washed cells were then used to inoculate 200 ml of MEM-{alpha} (iron deficient). Iron-replete cultures in MEM-{alpha} were additionally supplemented with FBP (14). FBP was added as both a ferrous iron source and an oxidative stress protectant. The final concentration of iron in the cultures was 45 µM. Cultures were incubated to stationary phase microaerobically at 37°C with shaking. Growth was monitored by measuring the OD600 every hour, using an Amersham Pharmacia Ultrospec 2000 spectrophotometer. Experiments were repeated three times with independent cultures.


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RESULTS
 
Identification of a C. jejuni periplasmic MCO. The Cj1516 gene in the genome sequence of strain NCTC 11168 is described as encoding a periplasmic oxidoreductase with an unassigned function. The full-length deduced protein is ~60 kDa and has 38% identity with the MCO CueO of E. coli. MCOs are characterized by three different Cu(II) centers which couple four one-electron oxidation reactions to the four-electron reduction of oxygen to water. These three classes reflect the geometric and electronic structures of the active site (58). They are type 1 (T1) or blue copper, comprising a single Cu atom complexed to two histidines; type 2 (T2) sites, containing a single Cu atom; and type 3 (T3) sites, containing two Cu atoms in a binuclear center. The T2 and T3 sites together form a trinuclear center complexed with a total of six histidines (58). These copper centers have been observed in the crystal structure of CueO of E. coli (50). Figure 1 shows the protein sequence alignment of Cj1516 with sequences of known MCOs, some of which have defined or suggested roles in iron acquisition and copper tolerance. Clearly, Cj1516 possesses all of the amino acid residues critical for the formation of the T1 and trinuclear centers. Further sequence analysis using the TatP (7) and SignalP (6) web servers suggests that the protein is secreted into the periplasm via the Tat system (8) due to the presence of a typical Tat signal motif (Fig. 1), and cleavage is predicted to remove 20 amino acids during export. The mature protein is expected to be ~56 kDa. The presence of the Cj1516 protein in the periplasm of C. jejuni was shown by assaying the characteristic phenoloxidase activity with the chromogenic substrate p-PD. Rates of ~800 nmol p-PD oxidized min–1 mg protein–1 were found in periplasmic fractions of WT cells, whereas a Cj1516 mutant (see below) completely lacked this activity.


Figure 1
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  		FIG. 1. (A) Sequence alignment of Cj1516 with related MCOs. The twin arginine signal motif (TAT) and the four pairs of histidine residues involved in copper ligand formation are shown in shaded boxes. The residues ligating the T1 copper (cupredoxin domain) are also indicated. Alignments were performed using CLC Workbench and ClustalX. (B) Gene context and mutagenesis strategy for the Cj1516 gene. Note the presence of Moco biosynthesis genes (moaDEA2) downstream of the Cj1516 gene. (C) Gene context and mutagenesis strategy for the Cj1161c gene. The genes upstream of the Cj1161c gene may also have a role in copper homeostasis, as discussed in the text. Arrows above the kanamycin (kan) and chloramphenicol (cat) cassettes used for mutagenesis indicate the directions of transcription of the resistance gene promoters.

Overexpression and purification of Cj1516. The Cj1516 gene in the NCTC 11168 strain of C. jejuni was PCR amplified and cloned into the expression vector pET21a(+), such that the recombinant protein would be expressed from the T7 promoter with the original C. jejuni signal sequence and without any tags. Induction of E. coli BL21(DE3)(pMCO1516) with IPTG at 37°C resulted in only insoluble protein. However, induction at 25°C resulted in the overproduction of a soluble protein that represented about 30% of the total protein at 16 h (Fig. 2A). It was purified to homogeneity from cell extracts by a combination of ion-exchange and hydrophobic interaction chromatography and heat treatment. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that the protein migrated as a single band with a molecular mass of ~56 kDa (Fig. 2A, right panel). The eluted recombinant protein was monitored at each step by phenoloxidase activity with p-PD and by identifying fractions that were blue due to the presence of fully oxidized T1 copper centers, a phenomenon observed previously (30). Optical spectroscopy of these blue fractions confirmed the presence of the protein (Fig. 2B), which was obtained at an approximately 80% yield based on phenoloxidase activity.


Figure 2
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  		FIG. 2. Purification and optical properties of Cj1516. (A) (Left) SDS-PAGE demonstrating expression of an ~56-kDa protein, indicated by a black arrow, in the BL21({lambda}DE3)/pET1516 strain after induction with IPTG (lanes 4 to 7 [1, 3, 5, and 16 h postinduction]). Expression appeared to be maximal after 16 h of growth (lane 7). This protein was absent in the same strain without induction (lane 3) and from the control strain BL21({lambda}DE3)/pET21a after overnight growth and addition of IPTG (lane 2). Lane 1 contains prestained molecular size markers (Bio-Rad, United Kingdom). Cells were grown at 25°C with shaking, and 1 mM IPTG was used for induction. (Right) SDS-PAGE analysis of Cj1516 purification steps. CE, crude extract; DEAE, ion-exchange column fraction; HIC, hydrophobic interaction column fraction; HT, heat treatment step. (B) Optical absorbance spectroscopy of Cj1516. The spectrum for the protein, as purified (solid line) (23 µM protein in 10 mM Tris-HCl, pH 7.5), shows the T1 copper site signal at 607 nm. The addition of 5 mM EDTA reduced the T1 signal (dashed line), while reduction with sodium dithionite abolished it (dotted line). (Inset) The T3 copper site signal at 330 nm is apparent as a weak feature in this concentrated protein preparation (470 µM protein). The y axis scales have the same units (absorbance and extinction coefficient) as those in the main figure.

N-terminal amino acid sequencing revealed a sequence of YANPMH, which is identical to residues 21 to 26 of the deduced complete sequence and consistent with correct cleavage at the AYA signal peptidase recognition site, predicted using the signal sequence web servers SignalP 3.0 (6) and TatP 1.0 (7). The molecular mass of the mature processed protein was calculated to be 56.6 kDa using the tools at http://expasy.org.

Protein copper content. Copper content in the protein was determined using inductively coupled plasma mass spectrometry. The copper content was found to be 6.4 atoms per polypeptide chain. No significant amounts of any other transition metals were present.

Optical absorbance spectroscopy and EPR spectroscopy show that Cj1516 is an MCO. Optical absorbance spectroscopy performed on the 56-kDa blue protein, as purified, revealed a characteristic peak ({lambda}max = 607 nm, {varepsilon} = 2,300 M–1 cm–1) indicative of a T1 center (Fig. 2B). Treatment with the copper chelator EDTA considerably reduced the intensity of the T1 absorbance, and reduction of the protein with sodium dithionite abolished it (Fig. 2B). T3 binuclear copper centers produce a characteristic but weak absorbance at around 330 nm, which is evident in the inset in Fig. 2B, where a scan of this region with concentrated protein was performed. A weak 420-nm absorbance was also observed. This has not been reported for other MCOs, so it could represent a minor contaminant. None of the spectral features were significantly changed in the presence of 1 mM copper(II) sulfate.

T2 (or normal) copper centers do not produce intense features within the visible absorption spectrum, but along with T1 sites, they exhibit characteristic features that can be observed by EPR spectroscopy, owing to the open shell configuration of electrons in oxidized (cupric) atoms providing an unpaired electron in the outer shell. Conversely, while being visible at 330 nm by optical spectroscopy, T3 centers are EPR "silent" due to the coupling of the two copper atoms via a bridging ligand and thus the loss of unpaired ferromagnetically active electrons (58). Figure 3A shows the results of EPR spectroscopy performed on Cj1516 as purified. The protein exhibited EPR features typical of multicopper proteins, displaying a spectrum with narrow hyperfine splitting (g[perp], ~2.05; g||, ~2.209; and A||, ~76.79 x 10–4 cm–1) for the T1 center. Underlying features characteristic of a T2 center are also evident in the lower field and display an approximate hyperfine splitting A|| value of 156.11 x 10–4 cm–1. Figure 3B shows the effects of the addition of ferrous iron to the sample. Rapid reduction of the centers to Cu(I), with the concomitant oxidation of ferrous [Fe(II)] to ferric [Fe(III)] iron, resulted in the obvious loss of the T1 signal. These observations are consistent with the ferroxidase activity of the protein described below.


Figure 3
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  		FIG. 3. EPR spectra of Cj1516 T1 and T2 copper centers. A Bruker EMX spectrometer (X band, 9.38 GHz) was used to analyze the copper center active sites of the MCO Cj1516. (A) Spectrum recorded for Cj1516 as isolated, with type 1 copper center hyperfine splitting displayed. Type 2 copper center hyperfine splitting is shown in the 8x amplified signal. (B) Spectrum for Cj1516 after addition of Fe(II) in the form of 1 mM ammonium ferrous sulfate. The protein was in 50 mM sodium acetate, pH 5.0, for both spectra.

Spectrophotometric analysis of substrate specificity and kinetics of Cj1516 and the effect of excess copper. Cj1516 oxidized phenolic compounds such as p-PD, N,N,N,N-tetramethyl-p-phenylenediamine (data not shown), and 3,4-DHB (anthrachelin). The Vmax and Km values for p-PD after the addition of 1 mM CuSO4 to the assay were markedly different from those in the absence of excess copper, suggesting an enhancement in activity, as seen with other MCOs (30, 51) (Table 1). The higher affinity and high Vmax values for 3,4-DHB imply that this is a better substrate than p-PD. Ferroxidase activity measured using the optical method, as previously described (30), was also observed and greatly enhanced by the addition of excess copper: a fivefold increase in Vmax and a twofold decrease in the Km value were observed (Table 1). Oxidation of phenolic compounds did not take place in the absence of enzyme, even with excess copper present (data not shown), suggesting that free copper does not take part in a redox cycle, in agreement with the work of others (30).


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  		TABLE 1. Kinetic parameters for Cj1516 enzyme activitiesa

Cj1516 exhibits iron- and copper-dependent oxygen uptake. MCOs are oxygen-dependent enzymes and, as such, can be assayed by measuring the substrate-linked uptake of oxygen by use of a Clark-type oxygen electrode. Manganese was tested as a possible substrate, but no uptake of oxygen was observed (Fig. 4A). However, consistent with the data in Table 1, Fig. 4B shows that significant oxygen consumption occurred when Fe(II) in the form of ammonium ferrous sulfate was used as a substrate. Cuprous oxidase activity was also measured with the oxygen electrode, using the previously described caged copper(I) substrate (51) to minimize interference from chemical oxidation of the unstable copper(I). High rates of oxygen uptake upon addition of the compound demonstrated that Cj1516 is capable of oxidizing cuprous copper (Fig. 4C). In the absence of enzyme, negligible background rates were observed at the caged copper concentrations used (data not shown). The concentration dependence of the cuprous oxidase activity followed Michaelis-Menten kinetics, as shown in Fig. 4D. A Km of 180 µM was calculated for cuprous copper, similar to that previously measured for CueO (54) and in the same region as that of Fe(II) for Cj1516 (Table 1). The Vmax was the highest of any for the substrates tested (Table 1).


Figure 4
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  		FIG. 4. Substrate-linked oxygen consumption of purified Cj1516. Pure Cj1516 protein was assayed for oxidase activities in 100 mM Tris-acetate buffer, pH 5.7, using a Clark-type oxygen electrode as described in Materials and Methods. The substrates used were manganese(II) chloride (A), ferrous ammonium sulfate (B), and caged copper(I) (C). (D) Dependence of the rate of oxygen consumption on the caged copper(I) concentration. The data from three independent titrations were fitted to the Michaelis-Menten equation (black line). The kinetic parameters from this titration are given in Table 1.

Ferrous iron acquisition is not affected in a Cj1516 mutant. A mutant in Cj1516 was constructed by the insertion of a kanamycin resistance cassette into a unique SwaI site within the cloned gene (Fig. 1B). After electroporation into WT cells, several antibiotic-resistant colonies were selected, and a PCR with gene-specific primers showed that the mutant construction had been successful (data not shown). Intact cells and periplasmic protein fractions of this mutant completely lacked p-PD oxidase activity. The kanamycin resistance cassette used was inserted with the same polarity as the Cj1516 gene and therefore should not interfere with downstream transcription. However, the genes downstream of the Cj1516 gene are predicted to encode the proteins MoaD, MoaE, and MoeA2 (Fig. 1B), all of which are essential for the synthesis of the molybdopterin cofactor (Moco) of molybdoenzymes. Since it is now known that copper is needed for the correct biosynthesis of this cofactor (31), we wanted to ensure that mutation of Cj1516 did not interfere with Moco synthesis. C. jejuni expresses a number of Moco-containing proteins that function as part of the electron transport pathway (35, 45, 53), including trimethylamine-N-oxide reductase (Cj0264). We found that trimethylamine-N-oxide reductase activity in the previously described methyl viologen (MV) assay (53) was comparable in the Cj1516 mutant to that of the WT parent strain, with both giving high rates of ~2.5 µmol MV oxidized min–1 mg–1 protein in intact cells, indicating that molybdenum cofactor synthesis was not affected in the mutant. In addition, RT-PCR analysis using primers for moaD showed that its expression was not altered in the mutant compared to that in the WT parent strain (1.33- ± 0.82-fold change in the mutant versus the WT).

In order to determine any effects on iron acquisition in a Cj1516 mutant, iron-limited growth experiments were carried out. However, since C. jejuni NCTC 11168 possesses the well-known FeoB ferrous iron transporting protein (36), a feoB mutant strain was also used for comparison in these experiments. The feoB mutant was created by a chloramphenicol resistance Mariner transposon insertion into the FeoB-encoding Cj1398 gene (kindly provided by A. Grant, Cambridge, United Kingdom). The WT and Cj1516 and feoB mutant strains were grown in liquid culture in the presence and absence of a ferrous iron source. None of these strains grew significantly in the absence of ferrous iron (Fig. 5), but after 16 h of microaerobic growth in the presence of ferrous iron, both the WT and Cj1516 strains had grown to an OD600 of ~1.2. The Cj1516 mutant clearly showed no iron acquisition-related phenotype in this growth assay. However, the feoB mutant strain was completely unable to grow with ferrous iron (Fig. 5). Thus, the data suggest that growth of C. jejuni on ferrous iron does not involve Cj1516 and can be explained entirely by the activity of the FeoB transporter, confirming the importance of FeoB as an iron acquisition protein, as described in a previous study (36).


Figure 5
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  		FIG. 5. Growth of WT and mutant strains under iron-limited and iron-replete conditions. Cultures were grown in MEM-{alpha} in the absence of added iron (white bars) and the presence of 45 µM ferrous iron (black bars) as described in Materials and Methods. Data are the means and standard deviations of the final ODs reached after 16 h of growth of three biological replicate cultures.

Bioinformatic evidence suggests that the Cj1161c gene encodes a copper-exporting P1B-type ATPase. The genome sequence of C. jejuni contains other genes encoding proteins with homology to well-known copper management proteins (40). In addition to the putative MCO Cj1516, two genes (Cj1161c and Cj1155c) encode Cop-like proteins. Cop proteins are members of the large P-type ATPase family, coupling the hydrolysis of ATP to the transport of a substrate (2, 57). More specifically, they belong to the heavy metal transporter subgroup P1B. P1B-type ATPases have a distinct structure compared to those of other P-type ATPases, characterized by a reduced number of transmembrane (TM) helices, typically having 8 instead of the 10 or more in P2- or P3-ATPases (34, 62). Within this subgroup, the presence of conserved amino acid residues in TM helices 6, 7, and 8 further classifies the proteins into groups based on the type of metal ion transported (2, 12, 57). Analysis of the protein sequence of Cj1155c of C. jejuni revealed that this protein contains a modified version of the highly conserved signature phosphorylation site motif DKTGT found in all P-type ATPases (2). However, it does contain the CPC motif as well as an N-terminal CXXC motif found in copper-transporting ATPases. The annotation and location of the gene within an apparent operon encoding homologues of the cytochrome c oxidase maturation protein cluster CcoGHIS found in many bacteria (31) suggest that it is involved in the assembly of the copper-containing terminal oxidase encoded by the Cj1487c to Cj1490c genes in the C. jejuni NCTC 11168 genome (40). Analysis of the amino acid sequence of Cj1161 showed this protein to be a more likely candidate as a Cop-like P-ATPase copper exporter. The TMHMM v2.0 prediction program suggested a total of eight TM helices with two cytoplasmic loops, which probably accommodate the phosphorylation site (data not shown). The protein also contains the DKTGT signature, the CPC motif, and the N-terminal metal binding domain motif CXXC. In addition, the protein also contains amino acids in TM helices 6, 7, and 8 proposed to participate in determining metal selectivity. All of these are defining features of proteins in the P1B-1-ATPase group, which are involved in the export of Cu(I) from cytoplasm to periplasm (2).

Mutations in either Cj1516 or Cj1161c lead to a copper-sensitive phenotype. A mutant in Cj1161c was constructed by the insertion of a chloramphenicol resistance cassette into a unique SwaI site within the cloned gene (Fig. 1C). After electroporation into WT cells, several chloramphenicol-resistant colonies were selected, and a PCR with gene-specific primers showed that the mutant construction had been successful (data not shown). RT-PCR analysis showed that expression of the small downstream gene Cj1160c was unaffected in this mutant (1.12- ± 0.64-fold change in the mutant versus the WT parent strain), indicating no polar effects of the insertion in Cj1161c. To fully explore the hypothesis that both the proteins encoded by the Cj1516 and Cj1161c genes are involved in copper homeostasis, a double mutant strain was created by transforming the p1516kan plasmid into the Cj1161c mutant AH100. Several colonies resistant to both kanamycin and chloramphenicol were selected, and a PCR of the genomic DNA of these colonies with gene-specific primers showed that mutant construction was successful.

Copper sensitivity of growth was determined in MEM-{alpha} containing 0 to 1 mM copper(II) sulfate (Fig. 6). The Cj1516, Cj1161c, and double mutants were all more sensitive than the parent strain NCTC 11168 to low or intermediate copper levels. However, the Cj1516 mutant was more sensitive to lower copper concentrations (0.1 to 0.2 mM) than the Cj1161c strain, which showed a clear difference from the WT only at 0.4 to 0.6 mM copper(II) sulfate. The double mutant was more sensitive than either single mutant (particularly apparent at 0.1 to 0.2 mM), indicating an additive effect of the mutations. The data show that both Cj1516 and Cj1161 have a role in mediating resistance to copper in C. jejuni.


Figure 6
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  		FIG. 6. Effect of copper on the growth of C. jejuni WT and mutant strains. WT and mutant cultures were grown microaerobically to stationary phase in MEM-{alpha} containing increasing concentrations of copper(II) sulfate as described in Materials and Methods. Black bars, WT; white bars, Cj1516 mutant; gray bars, Cj1161c mutant; hatched bars, Cj1516 Cj1161c double mutant. Data are the means and standard deviations of the final ODs reached after 16 h of growth of three biological replicate cultures. Note that complete inhibition of growth is represented by an OD of 0.1, as this was the initial inoculation OD.

Regulation of Cj1516 and Cj1161c gene expression by copper. Given the evidence obtained above suggesting that both Cj1516 and Cj1161 are part of the copper homeostasis system in C. jejuni, we used RT-PCR to determine the effect of copper ions during growth on the expression levels of the cognate genes. WT cells were grown to mid-exponential phase (OD600 of 0.4) in MEM-{alpha} without added copper (the medium contains a trace amount of copper, but this was not quantified) or with the addition of 50 µM copper(II) sulfate, a concentration that did not affect the growth rate of the cells. RT-PCR analysis showed that expression of the Cj1516 gene was 33- ± 7-fold upregulated in the excess copper cultures compared to those with no additional copper, while expression of the Cj1161c gene was not appreciably altered (0.95- ± 0.19-fold difference with and without copper).


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DISCUSSION
 
The acquisition, utilization, and management of transition metals are crucially important in pathogenic bacteria and contribute to their survival in host and external environments. For C. jejuni, these processes have been studied in detail only for iron. However, the importance of other metals, including copper, which is required as a cofactor for proteins such as the major electron transport terminal oxidase complex, the cytochrome c oxidase, is obvious. Copper is also now known to be required in the biosynthesis of Moco (32), yet it is also extremely toxic in excess and requires strict management. In this study, we have demonstrated that C. jejuni possesses mechanisms for dealing with excess copper and that the removal of these mechanisms renders the organism susceptible to the toxic effects of this transition metal. We have determined the function of an unknown protein, Cj1516, by biochemical characterization, mutagenesis, and phenotypic analysis and have shown by mutation that a second gene encoding a probable CopA-like protein is also involved in copper management.

The data presented here demonstrate that Cj1516 is a protein which binds copper atoms in the specific copper center formations characteristic of MCOs (58). These have been demonstrated in a number of bacterial proteins, for instance, the related protein CueO in E. coli (24). Bioinformatic analysis (Fig. 1) revealed the presence of the critical copper ion binding residues present in other MCOs. There are also at least two MXXM motifs (Fig. 1) that could also act as copper binding sites. Inductively coupled plasma mass spectrometry analysis showed Cj1516 to contain approximately six copper atoms per molecule, similar to E. coli CueO, whose copper content has been quoted as four, with two more atoms present, one of which is a labile "regulatory" copper atom and one of which is a surface copper atom (50, 51). In spectroscopic studies, the protein displayed optical and EPR spectra consistent with the presence of T1, T2, and T3 copper centers. A strong electronic absorption maximum at 607 nm indicated a T1 copper center, and a shoulder at around 330 nm indicated a T3 copper center (Fig. 2). The narrow hyperfine splitting observed in the EPR spectra was indicative of a T2 center and further proof of a T1 center. Upon the addition of ferrous iron to the pure protein sample, T1 signals were lost, presumably due to reduction of the center, thus providing initial evidence of ferroxidase capabilities.

Despite the similarities between eukaryotic and prokaryotic MCOs, only a few bacterial proteins, such as CueO (30) and an MCO from P. aeruginosa (29), have been shown to exhibit the same phenoloxidase and ferroxidase activities as those seen for eukaryotic enzymes such as Fet3p and human ceruloplasmin. Biochemical characterization clearly showed that Cj1516 exhibits both of these activities. For P. aeruginosa, a mutation in the MCO-encoding gene led to the loss of ferrous iron acquisition in the organism under aerobic conditions (29). A model similar to that for S. cerevisiae, in which an MCO (Fet3) oxidizes iron for uptake by an integral membrane permease (Ftr1) (3), was proposed for P. aeruginosa (29) and also for the magnetotactic bacterium MV-1 (16). In this organism, it is anticipated that an additional gene product with homology to p19, a periplasmic Fur-regulated protein in C. jejuni, is involved, along with an MCO and a permease-like protein, in iron acquisition (16). In C. jejuni, the Fur-regulated periplasmic protein p19 (Cj1659) is encoded as part of a large gene cluster also containing an iron permease gene (Cj1658). A similar gene arrangement is also found in an iron uptake pathogenicity island in the gammaproteobacterium Yersinia pestis (13). These observations led us to the possibility that Cj1516 was likely to be involved in iron metabolism, and the data presented here show that the enzyme is clearly able to oxidize Fe(II) with reasonable kinetics. In addition, a previous global transcriptomic study showed that Cj1516 gene expression is induced threefold under conditions of iron limitation (28). However, we did not observe a phenotype relating to ferrous iron acquisition in growth experiments involving a Cj1516 null mutant. These data suggest that the ferroxidase activity of the protein may not be physiologically relevant, at least under the growth conditions used. Manganese oxidation is also a feature of some bacterial MCOs (20, 21), yet the purified Cj1516 protein did not exhibit manganese-linked oxygen uptake. However, Cj1516 exhibited high rates of cuprous oxidase activity, and a Cj1516 mutant was much more copper sensitive than the WT parent strain. Interestingly, we also found by RT-PCR analysis that the expression of Cj1516 in WT cells was significantly increased after growth in the presence of copper. All of these observations are consistent with the conclusion that the physiological role of Cj1516 is in periplasmic copper detoxification.

A second gene, the Cj1161c gene, has also been shown to encode a protein with striking similarity to a specific group of Cu(I)-exporting proteins belonging to the P-type ATPase family. It is likely that this gene encodes a P1B-1-ATPase copper transporting protein similar to CopA, which has a central role in copper homeostasis in E. coli (42, 48). A mutation in the Cj1161c gene resulted in increased sensitivity to excess copper in growth studies compared to that of the WT strain, although this mutant was less sensitive than the Cj1516 mutant (Fig. 6). The role of CopA has been well documented in recent years for a number of bacteria (4, 37, 48), and it has been found to export Cu(I) ions from the cytoplasm to the periplasm. It has also been proposed that an MCO protein is then involved in further detoxification of the Cu(I) ions by oxidation to Cu(II), a less toxic and less membrane-permeative form of copper in the periplasmic compartment (24). Thus, in C. jejuni, Cj1161 is likely to provide the bacterium with an efficient copper(I) export system, with the MCO Cj1516 providing periplasmic protection by oxidation to Cu(II). These distinct roles may explain the differential copper(II) sensitivity results for the respective mutant strains, and the additive effects of the mutations are consistent with Cj1516 and Cj1161 operating together as part of a general copper homeostasis system. Both proteins are also likely to be important for copper tolerance when oxygen is limiting, since C. jejuni lacks the Cus copper efflux system found in other bacteria, which operates under anaerobic conditions (25, 38).

The exact mechanism by which bacterial MCOs confer copper resistance has yet to be established. However, the most widely held view is that the MCOs oxidize toxic Cu(I) to the much less toxic Cu(II). E. coli CueO is also capable of oxidizing catecholate siderophores, and the resulting pigments may then sequester copper. Campylobacter jejuni does not synthesize its own siderophores (18, 43), but it does utilize siderophores produced by other organisms (17). Consistent with this, Cj1516 is able to oxidize both Cu(I) and the catecholate siderophore analogue 3,4-DHB, which may also have a role in copper tolerance.

The Cue/Cop system described for E. coli (24, 42, 48) is regulated by CueR in the cytoplasm, although none of the genes are in the same operon. CueR is a MerR-like transcriptional regulator with a helix-turn-helix motif and is induced by copper (60). Interestingly, as noted above, the Cj1516 gene is copper regulated, but we could not find an obvious homologue of CueR in the genome of C. jejuni 11168, although there is an example of a MerR-like protein, encoded by the Cj1563c gene, and it is possible that this protein could fulfill the regulatory role. Recently, it was proposed that genes involved in molybdenum cofactor biosynthesis in E. coli are regulated by excess copper via CueR (32, 65), and several genes involved in molybdenum cofactor biosynthesis, moaD, moaE, and moeA2, are located downstream of the Cj1516 gene in C. jejuni NCTC 11168 (Fig. 1B). It should also be noted that the Cj1516 gene is conserved in other sequenced C. jejuni strains (e.g., 81-176, RM1221, and 81116) and that Moco biosynthesis genes are also located in similar positions in these strains. Although we did not find evidence for copper regulation of the Cj1161c gene, the corresponding mutant is relatively less copper sensitive than the Cj1516 mutant, so perhaps much higher concentrations of copper than those used here would be needed to observe any regulation.

In addition to the Cj1161c gene, in the same region a number of unusual and functionally undefined genes are present which may also be involved in the putative Cop system and, indeed, may form an operon which includes the Cj1161c gene (Fig. 1B). The Cj1162 to Cj1164c genes encode proteins which are all predicted to possess at least one CXXC motif each. The Cj1163c gene contains a histidine-rich N-terminal domain. It is also predicted to possess six TM helices and, as such, is similar in structure to the P-type ATPases. However, it lacks the highly conserved and essential ATP binding motif DKTGT. Both the Cj1162c and Cj1164c genes encode small hypothetical proteins (64 amino acids and 87 amino acids, respectively), with each containing a CXXC motif. In the other sequenced strains of C. jejuni, a very similar gene arrangement to that found in the reference strain NCTC 11168 exists. No data exist with respect to these three genes, but given their location, they merit further investigation for a copper-related role.

In conclusion, we have identified and characterized two gene products involved in the homeostasis of copper in C. jejuni. The Cj1516 and Cj1161c genes encode an MCO and a copper-transporting P-type ATPase, respectively, and should be redesignated CueO and CopA based on their counterparts in other bacteria. Our studies have shown that the removal of these genes renders the organism more sensitive to copper, and we were unable to identify any other genes associated with the known alternative systems of copper management in the organism. The function and regulation of these copper homeostasis genes in C. jejuni require further investigation.


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ACKNOWLEDGMENTS
 
This work was supported by UK Biotechnology and Biological Sciences Research Council grant BB/D008395/1 to D.J.K.

We thank Wendy Trotter for help with RT-PCR experiments.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, United Kingdom. Phone: 44 114 222 4414. Fax: 44 114 272 8697. E-mail: d.kelly{at}sheffield.ac.uk Back

{triangledown} Published ahead of print on 17 October 2008. Back


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Journal of Bacteriology, December 2008, p. 8075-8085, Vol. 190, No. 24
0021-9193/08/$08.00+0     doi:10.1128/JB.00821-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.




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