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Applied and Environmental Microbiology, March 2008, p. 1367-1375, Vol. 74, No. 5
0099-2240/08/$08.00+0     doi:10.1128/AEM.02261-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Role of Campylobacter jejuni Respiratory Oxidases and Reductases in Host Colonization

Rebecca A. Weingarten,¹ Jesse L. Grimes,² and Jonathan W. Olson^1*

Department of Microbiology, North Carolina State University, Raleigh, North Carolina,¹ Department of Poultry Science, North Carolina State University, Raleigh, North Carolina²

Received 4 October 2007/ Accepted 24 December 2007

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Campylobacter jejuni is the leading cause of human food-borne bacterial gastroenteritis. The C. jejuni genome sequence predicts a branched electron transport chain capable of utilizing multiple electron acceptors. Mutants were constructed by disrupting the coding regions of the respiratory enzymes nitrate reductase (napA::Cm), nitrite reductase (nrfA::Cm), dimethyl sulfoxide, and trimethylamine N-oxide reductase (termed Cj0264::Cm) and the two terminal oxidases, a cyanide-insensitive oxidase (cydA::Cm) and cbb3-type oxidase (ccoN::Cm). Each strain was characterized for the loss of the associated enzymatic function in vitro. The strains were then inoculated into 1-week-old chicks, and the cecal contents were assayed for the presence of C. jejuni 2 weeks postinoculation. cydA::Cm and Cj0264c::Cm strains colonized as well as the wild type; napA::Cm and nrfA::Cm strains colonized at levels significantly lower than the wild type. The ccoN::Cm strain was unable to colonize the chicken; no colonies were recovered at the end of the experiment. While there appears to be a role for anaerobic respiration in host colonization, oxygen is the most important respiratory acceptor for C. jejuni in the chicken cecum.

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Campylobacter jejuni is the primary agent of campylobacteriosis, the most frequent form of human bacterial gastroenteritis worldwide (2, 13). An estimated 2 million cases per year of campylobacteriosis occur in the United States alone (37). C. jejuni's natural hosts appear to be avian species (30), and poultry meat is a major route of transmission into the human population (2, 23). In one recent study, it was found that 71% of packaged chicken taken from 59 retail grocery stores contained Campylobacter spp., and 91% of the stores sold chicken containing Campylobacter spp. (46). C. jejuni is nonpathogenic to poultry and is primarily an enteric bacterium, preferentially localizing in the cecum in experimentally inoculated chicks (7). The chicken cecum is a highly developed organ located at the junction of the large and small intestines that contains more than 10¹¹ bacteria per gram (6, 29). 16S rRNA sequencing has revealed that the cecum tends to be dominated by anaerobic Clostridiaceae-related bacteria (65%), followed by anaerobic bacteria of the genera Fusobacterium, Lactobacillus, and Bacteroides (26), which implies that C. jejuni is exposed to an anaerobic environment in its normal host. A paradox exists, however, in that C. jejuni has long been considered a microaerophile, meaning it is an obligate aerobe that cannot survive fully aerobic conditions (18). C. jejuni is unable to grow fermentatively and must rely on oxidative phosphorylation for all its energy requirements. The genome sequence of C. jejuni strain NCTC 11168 encodes two different terminal oxidases, a cbb3-type cytochrome c oxidoreductase and a bd-type quinol oxidase (33). Recently, it has been demonstrated that cydAB encodes a cyanide-insensitive, low-affinity oxidase rather than the bd-type quinol oxidase that is annotated in the genome (19). In the same report, the cbb3-type cytochrome c oxidoreductase was considered an essential enzyme due to the inability to obtain mutants after repeated transformations (19). Consistent with the anaerobic environment of C. jejuni, the genome sequence also revealed the presence of terminal acceptors alternative to oxygen, allowing for the potential to grow by anaerobic respiration (33). These enzymes include a periplasmic nitrate reductase (Cj0780-0785), nitrite reductase (Cj1357c-1358c), and an enzyme responsible for both dimethyl sulfoxide (DMSO) and trimethylamine N-oxide (TMAO) reduction (Cj0264c-0265c), which we term an SN oxide reductase. Recently the respiration of nitrate and nitrite, DMSO, and TMAO by C. jejuni has been reported (35, 38). However, the importance of these respiratory pathways in the avian host has not been addressed.

We set out to determine the individual roles of the respiratory acceptor enzymes of C. jejuni and their importance in the colonization of chickens. Mutants in all five terminal acceptor enzymes of C. jejuni were constructed and characterized. Insight into the in vivo physiology of Campylobacter will provide a better understanding of how the organism thrives in the intestinal tract of its host. This is especially important for the ultimate goal of removing C. jejuni from poultry flocks and preventing transmission to humans.

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Bacterial strains and growth conditions.
Table 1 lists strains of C. jejuni and Escherichia coli, along with all of the plasmids and primers utilized in this study. The NCTC 11168 strain used in this study is helical in morphology, is fully motile, and is not impaired in poultry colonization. Tryptic soy agar (Difco, Sparks, MD) plates supplemented with 10% defibrinated sheep blood (Gibson Laboratories, Inc., Lexington, KY) (BA plates) were used for growth of C. jejuni. C. jejuni was routinely cultured at 37°C microaerobically in a trigas incubator (model 550D; Fisher Scientific) constantly maintained at 5% CO₂ and 12% O₂, balance N₂ (i.e., the remaining gas volume is N₂). The O₂-sensitive ccoN::Cm mutant was routinely cultured in an atmosphere generated by the Anaerobic BBL Gas Pak Plus system. Mueller-Hinton broth (Difco) with the addition of 50 mM sodium formate (MHF) was used for C. jejuni cultures, and 10 mM sodium nitrate, 5 mM DMSO, or 20 mM TMAO was added as indicated; liquid cultures were incubated at 37°C with shaking. Chloramphenicol (25 µg/ml) was added as indicated. Genetic manipulations were performed with E. coli strain DH5. Luria-Bertani broth and agar were supplemented with ampicillin (100 µg/ml) or chloramphenicol (25 µg/ml), as noted.

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TABLE 1. Strains, plasmids, and primers

Anaerobic growth of C. jejuni.
Anaerobic conditions were achieved by the following protocol. MHF was sparged with a glass sintered gas dispersion tube for 15 min with an anaerobic gas mixture (5% CO₂, 10% H₂, balance N₂). Twenty milliliters of MHF was placed in a 150-ml serum bottle, and the bottle was sealed with a gas-impermeable butyl rubber septum-type stopper (Bellco Glass Inc., Vinland, NJ) secured with an aluminum seal (Wheaton Scientific, Milleville, NJ). The serum bottle was made anaerobic by evacuating the headspace (vacuum gauge reading 102 kPa) for 1 min and was then flushed for 1 min with argon that had passed through a heated copper pellet oxygen scrubber. The process was repeated three times, and the bottle was removed after the last argon flush. Bottles prepared in this way contain argon at atmospheric pressure and are referred to as vacuum sparged.

Gas analysis.
An OmniStar Gas Analyzer (Pfeiffer Vacuum, Asslar, Germany) was utilized to measure the gas composition of the headspace above the cultures. The gas analyzer used a turbomolecular pump coupled to a quadrapole mass spectrometer to identify gasses based on molecular mass.

Cloning and construction of C. jejuni mutants.
Oligonucleotide primers for cloning genes of interest were designed from the sequenced strain NCTC 11168 (33) and are listed in Table 1. PCR amplification was performed with Taq DNA polymerase (Promega, Madison, WI) using chromosomal DNA isolated from C. jejuni NCTC 11168 as a template. The PCR products (1 kb) were inserted into the appropriate vector (either pKS:XbaI or pCRTOPO2.1) and confirmed by restriction analysis. pKS:XbaI is a version of pBluescript II KS(+) in which the XbaI site has been destroyed by digestion with XbaI, treatment with T4 DNA polymerase (Promega) and deoxynucleoside triphosphates, and religation. The coding region of the gene of interest was disrupted by insertion of an antibiotic resistance cassette into an appropriate restriction endonuclease recognition sequence within the DNA fragment (Table 1). The chloramphenicol resistance gene (cat) was originally isolated from Campylobacter coli (41). pJMA-001 contains the chloramphenicol resistance cassette from plasmid pRY111 (44) cloned into the PvuII site of pGEM-T Easy (Promega). Electrocompetent C. jejuni cells were transformed with each construct to yield the corresponding C. jejuni mutant. Briefly, 1 to 5 µg of plasmid DNA was incubated on ice for 10 min with 50 µl C. jejuni cells that had been previously washed four times with an ice-cold 9% sucrose and 15% glycerol solution. The cells and DNA were then placed in a 2-mm electroporation cuvette and pulsed with 2,500 V in an ECM399 electroporator (BTX, San Diego, CA). Immediately after the pulse, 50 µl of Mueller-Hinton broth (Difco) was added to the cuvette containing the competent cells and the cuvette remained on ice for 10 min. Cells (100 µl) were spotted onto cold nonselective BA plates, and the plates were incubated microaerobically for 24 h. The cells were then transferred to BA plates containing chloramphenicol. In obtaining the ccoN::Cm strain, however, anaerobic jars containing an Anaerobic BBL GasPak Plus with palladium catalyst were utilized, and the BA plates were supplemented with 50 mM formate and 10 mM nitrate. Resistant colonies were passed on selective BA plates, and the correct insertion of the cassette was confirmed by isolation of chromosomal DNA from the mutant strain and PCR amplification of the gene. Agarose gel electrophoresis of the PCR product was used to monitor the size increase of the gene of interest with the antibiotic cassette insertion (data not shown).

qRT-PCR.
The PCR primers used in this study are listed in Table 1 and were designed to amplify 100- to 150-nucleotide fragments of genes of interest. Total RNA was isolated from the parent strain of C. jejuni, as well as the ccoN::Cm, napA::Cm, and nrfA::Cm mutants, using a MasterPure Complete RNA Purification Kit (Epicenter Biotechnologies, Madison, WI). Quantitative reverse transcriptase (qRT) PCR was performed by using the Quantitect Sybr Green RT-PCR kit (Qiagen, Valencia, CA). The PCR mixture (20 µl) contained 40 ng RNA, 10 µl 2x QuantiTect Sybr Green RT-PCR Master Mix, and 0.2 µl QuantiTect RT mixture. The reverse transcriptase cycle was 50°C for 30 min, followed by a PCR initial activation step of 95°C for 15 min. The mixtures were then amplified in 30 cycles of 94°C for 15 seconds, 60°C for 30 seconds, and 72°C for 30 seconds in an automated thermal cycler (iCycler; Bio-Rad, Hercules, CA). The iCycler software was used to determine the threshold cycle at which each transcript could be detected. The threshold cycles were then compared to a standard curve, which was generated independently for each gene, to determine the number of starting RNA molecules. Total RNA in each sample was normalized using the internal control gyrA (Cj1027c).

Nitrite production assay.
In order to determine the nitrite produced in growing cultures, a 1-ml aliquot was removed from the serum bottle, the cells were removed by centrifugation, and 10 µl of the supernatant was added to a reaction mixture containing 500 µl of 1% (wt/vol) sulfanilamide dissolved in 2.5 N HCl and 500 µl of 0.02% (wt/vol) naphthylethylenediamine. Samples were incubated at room temperature for 10 min, the absorbance at 540 nm was measured, and nitrite concentrations were determined by reference to a standard curve.

Sonication.
Cells were disrupted by sonication. The sonication was performed on ice in a W-370 horn cup sonicator (Heat Systems-Ultrasonics, Inc., Farmingdale, NY) for four 45-second pulses at 60% power and 7.0 output control setting.

Protein assay.
The protein concentration was determined using the BCA Protein Assay Kit (Pierce, Rockford, IL) with bovine serum albumin as a standard.

Nitrate reductase activity.
Nitrate reductase activity was determined by the production of nitrite by cell extracts incubated with nitrate as a substrate and reduced benzyl viologen as an electron donor (27, 31). One to 2 µg protein from sonicated vacuum-sparged cell extracts was added to a vacuum-sparged assay mixture containing 0.1 mM benzyl viologen, 20 mM sodium dithionite solution, 54 mM sodium phosphate buffer (pH 6.8), and 10 mM sodium nitrate. The reaction mixture was incubated for 2 min at room temperature, and then the reaction was stopped by removing the stopper and vortexing the mixture until the benzyl viologen was completely oxidized. One percent (wt/vol) sulfanilamide (500 µl) dissolved in 2.5 N HCl and 500 µl of 0.02% (wt/vol) naphthylethylenediamine were added to the reaction mixture, and after 10 min, the absorbance at 540 nm was measured. Nitrite concentrations were then determined by reference to a standard curve. Activity was expressed as nmol nitrite produced min^–1 mg^–1.

Nitrite reductase activity.
Benzyl viologen-linked reductase assays were carried out with sonicated cell extracts in a 1-ml assay volume as described previously (38). Reagents were added by syringe through the stopper, while argon gas was flushed through the cuvette. The reaction mixture (reagents were kept anaerobic during the course of the assay) contained 75 mM sodium phosphate buffer (pH 6.8), 0.2 mM benzyl viologen, and 1 to 5 µg of cell extract in a 1-ml stoppered quartz cuvette. Freshly made 20 mM sodium dithionite was then injected into the cuvette until the absorbance at 585 nm reached 0.9 to 1.3, which represented half-reduced benzyl viologen. An anaerobic solution of sodium nitrite to a final concentration of 2.5 mM was added, and the benzyl viologen oxidation kinetics (benzyl viologen oxidation results in a lower absorbance) was recorded by a spectrophotometer at 585 nm. Nitrite reductase activity was expressed as µmol benzyl viologen oxidized min^–1 mg^–1.

DMSO reductase assay.
Mueller-Hinton broth-grown cells were harvested by centrifugation and washed once with sterile phosphate-buffered saline (PBS) (pH 7.4). The cell pellet was resuspended in 10 ml PBS and 50 mM formate to a final optical density of approximately 0.2 at 600 nm, and the mixture was vacuum sparged. DMSO was added to a final concentration of 10 mM to start the reaction, and the cultures were shaken at 37°C. An OmniStar gas analyzer (Pfeiffer Vacuum, Asslar, Germany) was utilized to measure the dimethyl sulfide (DMS) evolution into the headspace. A DMS standard curve was generated using >99% pure DMS (Sigma-Aldrich, St. Louis, MO), and the DMS concentrations were determined by reference to the standard curve. DMSO reductase activity was expressed as nmol DMS produced min^–1 mg^–1.

Oxygen uptake assay.
O₂ uptake experiments were performed using a Clarke-type electrode and a YSI (Yellow Springs, OH) model 5300 oxygen monitor. Whole cells were harvested with a swab from BA plates into PBS, washed once with PBS, added to the constantly stirred chamber, and allowed to equilibrate until no change in O₂ consumption was noticed. Formate (final concentration, 5 mM) was added to the chamber through a capillary tube via a Hamilton syringe, the chart recorder measured the oxygen consumption, and the slope of the line determined the rate of oxygen uptake. Rates were expressed as nmol O₂ consumed min^–1 mg¹.

Cytochrome reduction assay.
Cells were harvested with a swab from BA plates into cold PBS (pH 7.4) and washed once. The cells were broken by passage through a French pressure cell (American Instruments Co., Silver Springs, MD) twice at 20,000 lb/in². Broken cells were centrifuged at 12,000 x g for 15 min to remove debris. The supernatant was collected and spun by ultracentrifugation at 100,000 x g for 90 min. Membranes were collected and resuspended in cold PBS (pH 7.4). One-milliliter membrane suspensions were placed into matched quartz cuvettes and left exposed to air to become fully oxidized. A few grains of sodium dithionite were added to one of the cuvettes and capped with a rubber stopper. Spectra (300 to 600 nm) of the reduced samples minus the oxidized samples were collected using a Shimadzu UV-1650PC spectrophotometer.

Chicken colonization.
Lake Wheeler Poultry Facility, operated by the North Carolina State University Poultry Department, supplied Campylobacter-free day-old broiler chicks. For 21 days, the birds were housed in isolation rooms at the Dearstyne Avian Health Center (Department of Poultry Science, North Carolina State University) in isolation brooder batteries (Petersime Incubator Co., Gettysburg, OH) with 10 chicks per battery. The chicks were fed Purina Mills Start & Grow SunFresh Recipe feed (Purina Mills LLC, St. Louis, MO) and water ad libidum. One-week-old chicks were inoculated by oral gavage with 0.1 ml of 10⁸ C. jejuni cells/ml, which had been grown for approximately 16 h on BA plates and cultured at 37°C microaerobically, with the exception of the ccoN::Cm strain, which was grown at 2% O₂, 5% CO₂, balance N₂. Control chicks were inoculated with 0.1 ml sterile PBS (pH 7.4). Two weeks postinoculation, the chickens were humanely sacrificed by CO₂ asphyxiation. Approximately 1 g of cecal contents was collected by necropsy, serially diluted (in PBS), and plated on selective BA media containing 40 µg/ml cefoperazone, 40 µg/ml vancomycin, 10 µg/ml trimethoprim, and 100 µg/ml cycloheximide. Samples from the chickens inoculated with the ccoN::Cm strain and 8 out of 20 wild-type samples were incubated at 2% O₂, 5% CO₂, balance N₂ at 37°C. All other samples were incubated microaerobically at 37°C. After 2 days of incubation, colonies, if any, were counted and the CFU/g cecal content was calculated. The data were analyzed by a one-tailed Mann-Whitney test, using a 95% confidence interval.

This study was conducted under guidelines established by the North Carolina State Animal Care and Use Committee.

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Mutant construction.
Mutants of C. jejuni constructed in this study included napA::Cm (nitrate reductase), nrfA::Cm (nitrite reductase), cydA::Cm (cyanide-insensitive oxidase), ccoN::Cm (cbb3-terminal oxidase), and Cj0264c::Cm (SN oxide reductase) strains (Fig. 1). All mutants were constructed by insertional mutagenesis using a chloramphenicol acetyltransferase (cat) cassette through allelic replacement of a disrupted copy of the genome. The use of the resistance cassette for mutagenesis can interfere with the proper transcription of genes "downstream" of the cassette insertion site (polar effects). To measure the polar effects of downstream genes, qRT-PCR was employed to measure transcript levels between mutant and parent strain genes. The napA::Cm (nitrate reductase) strain contains the cat cassette in the first gene of the nitrate reductase operon, encoded by napAGHBLD (Fig. 1). All six of these genes are involved in the reduction of nitrate; napA is the catalytic subunit, and the other genes function in electron transfer and/or assembly of the enzyme (15). Downstream of the nap operon are two genes, Cj0786 and Cj0787 (which encode a 57-amino-acid hydrophobic protein and a hypothetical protein, respectively). The transcription of these genes was shown not to be affected by the upstream cat insertion, with a transcript abundance of 0.8 ± 0.17 times that of the parent strain. The nrfA::Cm (nitrite reductase) strain also contains the cassette within the catalytic subunit of the enzyme (Fig. 1). Downstream of nrfA is Cj1356c (which encodes an integral membrane protein). The transcript abundance of Cj1356c downstream of nrfA::Cm was 1.1-fold ± 0.27-fold that of the wild type. The ccoN::Cm strain contains the cat cassette, which is inserted into the first gene of the four-gene cco operon (Fig. 1), which together makes up the cbb3-type terminal oxidase (34). Downstream of the ccoNOPQ operon (Cj1490c to Cj1487c) lies Cj1486c, which encodes a putative periplasmic protein. The relative transcript levels of Cj1486c in the ccoN::Cm strain were 0.41-fold ± 0.14-fold changed in comparison to its parent strain, which indicates that this gene is mildly affected by the cat insertion within ccoN. Polar effects are not a concern for the Cj0264c::Cm or cydA::Cm strain. The cat cassette in the cydA::Cm strain is the first gene of the cydAB operon (Cj0081 and Cj0082). The gene downstream of cydAB (Cj0085c, which encodes a putative amino acid racemase) is on the opposite DNA strand. The cat cassette in the Cj0264c::Cm strain is located in the second gene of the two-gene operon. Directly downstream of Cj0264c is zupT (Cj0263), which is also on the opposite DNA strand (Fig. 1).

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FIG. 1. Gene organization of the alternative respiratory pathway operons of C. jejuni. Also shown are the insertion sites of the chloramphenicol cassette (hatched arrows) used for mutagenesis. The direction of the arrow indicates the transcriptional orientation of the gene. Unrelated genes directly downstream of the genes of interest are indicated with black arrows.

Growth.
Under microaerobic conditions (5% CO₂, 12% O₂, balance N₂), all mutants had generation times similar to that of the wild type (data not shown), with the exception of the ccoN::Cm strain, which is oxygen sensitive (see Table 4). In wild-type cultures, the addition of the respiratory donor formate and/or the acceptor nitrate promoted growth, as shown by a decrease in generation time (Table 2). Despite the presence of oxygen-independent terminal acceptors in the electron transport pathway (22, 33), it has proven difficult to grow C. jejuni anaerobically (38). Under our conditions, we found that the addition of formate to the medium supported anaerobic growth of C. jejuni using the acceptor nitrate, DMSO, or TMAO (Table 2). Either sulfite or hydrogen could replace formate as a potential donor (data not shown).

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TABLE 4. Oxygen effect on growth of ccoN::Cm on BA plates compared to wild type

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TABLE 2. Growth rates of C. jejuni NCTC 11168

O₂ analysis of anaerobic growth conditions.
Three different anaerobic systems were first compared using mass spectrum gas analysis: vacuum-sparged serum bottles, the Coy anaerobic chamber (Coy Laboratories, Grass Lake, MI), and an anaerobic jar containing an Anaerobic BBL GasPak Plus with palladium catalyst. The Coy chamber contained 85% N₂, 5% CO₂, and 10% H₂ and a palladium catalyst that reacts with hydrogen to promote the reduction of any residual oxygen to H₂O. The anaerobic jars had the lowest O₂ partial pressure, followed by our serum bottles, and the Coy chamber had the highest O₂ partial pressure. Overall, it has been shown that the anaerobic chamber has the best efficiency in the recovery of fastidious anaerobes compared to the anaerobic jars and GasPak Plus (9-11). The O₂ partial pressure above anaerobically growing C. jejuni cells was measured. Three samples of the parent strain were grown with or without nitrate in vacuum-sparged bottles, and the O₂ content was measured every 4 hours for 24 h. O₂ concentrations in the headspaces of our anaerobic cultures hovered around zero and never exceeded 0.03%, which was lower than the O₂ measured in the Coy anaerobic chamber. In order to determine what this concentration of O₂ meant physiologically for the cells, the dissolved O₂ in the media was calculated using Henry's law. At the highest O₂ concentration measured (0.03% O₂), the media would contain 0.36 nM O₂.

Nitrate and nitrite reductase activities.
Nitrate reductase activity was readily detectable in both the wild-type strain and the nrfA::Cm strain. Due to an active nitrite reductase in the wild type, however, a specific activity (NO₂ produced min^–1 mg^–1) was reported only for the nrfA::Cm and napA::Cm strains (Table 3). The napA::Cm strain showed negligible activity when grown under microaerobic conditions, but the lack of growth under anaerobic conditions precluded determining a percent O₂ activity. The nrfA::Cm strain showed nitrate reductase activity under both anaerobic and microaerobic conditions, with activity for cells grown under anaerobic conditions approximately threefold higher than that under microaerobic conditions (Table 3).

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TABLE 3. Nitrate and nitrite reductase activities of C. jejuni

Wild-type cells were able to oxidize benzyl viologen using sodium nitrite as the terminal acceptor (Table 3). The nitrite reductase activities of C. jejuni cells grown under anaerobic conditions were threefold higher than those of cells grown microaerobically, and the nrfA::Cm strain was devoid of activity (Table 3).

Nitrate- and nitrite-dependent growth.
Figure 2 shows the nitrate-dependent anaerobic growth of C. jejuni (all growth curves are shown in linear scale in order to visualize the increased absorbance; however, the data were log transformed to determine generation times). The addition of nitrate provided the wild-type strain a generation time of 2.7 ± 0.2 h (Fig. 2A). When nitrate was omitted from the medium, very little growth was detected (Fig. 2A). The napA::Cm strain failed to grow either with or without added nitrate (Fig. 2A), which was predictable, as the strain is unable to reduce nitrate and growth of the parent strain is nitrate dependent. The nrfA::Cm nitrite reductase mutant was able to grow anaerobically under "plus nitrate" conditions (Fig. 2A), although with a longer generation time (3.2 ± 0.07 h) and a lower terminal optical-density reading at 600 nm. In order to track the fate of the added nitrate in anaerobically grown cells, nitrite was measured in the supernatants of cultures of the wild type and the nrfA::Cm strain grown under anaerobic conditions. In both cultures, nitrite accumulated for the first 16 h of growth (Fig. 2B), indicating an active nitrate reductase. At 16 h, the nitrite concentration in the supernatant of the nrfA::Cm strain had accumulated to 10 mM, indicating that the nitrate (initial concentration, 10 mM) had been stoichiometrically converted to nitrite. After the nitrate was exhausted, growth stopped (Fig. 2A), and the nitrite concentration remained 10 mM until the experiment was terminated at 30 h (Fig. 2B). The wild-type culture, however, kept growing after the nitrate was consumed (Fig. 2A), and at 30 h, the accumulated nitrite had also been consumed (Fig. 2B). These data confirm that wild-type C. jejuni can utilize both nitrate and nitrite as terminal electron acceptors and that the nrfA::Cm strain can use nitrate but not nitrite as an electron acceptor.

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FIG. 2. (A) Anaerobic growth of C. jejuni NCTC 11168 (circles) and the napA::Cm (triangles) and nrfA::Cm (squares) strains. Cultures grown in MHF (solid symbols) or MHF plus 10 mM NaNO3 (open symbols) were incubated at 37°C under anaerobic conditions. The growth curve is representative of three independent growth curves. (B) Nitrite concentrations in the supernatants from cultures of the wild type (white bars) and the nrfA::Cm strain (black bars).

The SN oxide reductase mutant.
Cj0264c and Cj0265c are annotated in the genome sequence as genes for a probable molybdopterin-containing oxidoreductase and a probable cytochrome c-type heme-binding periplasmic protein, which together encode an SN oxide reductase (33). DMSO is reduced to DMS, and TMAO is reduced to trimethylamine (TMA). DMSO reduction was quantified by measuring DMS accumulation in the headspaces of serum bottles containing whole cells incubated with sodium formate and DMSO (see Materials and Methods). Wild-type cultures grown under both microaerobic and anaerobic conditions showed the highest DMSO reductase activity during mid-log phase (data not shown). This is in contrast to previous reports that TMA production is highest during the stationary phase of microaerobic and "oxygen-limited" growth of C. jejuni cells (38). The Cj0264c::Cm strain was unable to reduce DMSO (0.61 ± 0.93 nmol DMS min^–1 mg^–1).

DMSO and TMAO can also serve as alternative electron acceptors (28). Wild-type C. jejuni will grow anaerobically with a generation time of 2.9 ± 0.2 h with the addition of DMSO (Fig. 3). Wild-type C. jejuni was also able to respire with TMAO as an electron acceptor, with a generation time of 3.7 ± 0.2 h. The Cj0264c::Cm strain was unable to utilize either DMSO or TMAO as an electron acceptor and therefore was unable to grow under these anaerobic conditions, but it was able to grow anaerobically when supplemented with nitrate and had a generation time of 2.3 ± 0.15 h (Fig. 3).

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FIG. 3. Anaerobic growth of C. jejuni NCTC 11168 (open symbols) and the Cj0264c::Cm strain (closed symbols). Cultures grown in MHF (circles), MHF plus 10 mM NaNO3 (triangles), or MHF plus 5 mM DMSO (squares) were incubated at 37°C under anaerobic conditions. The growth curve is representative of three independent growth curves.

Terminal oxidase mutants.
Mutants were constructed in both terminal oxidases: a cyanide-insensitive oxidase (cydA::Cm) and a cbb3-type cytochrome c oxidoreductase (ccoN::Cm). The formate-dependent respiration rates of C. jejuni whole cells were measured. Wild-type whole cells consumed oxygen at a rate of 123 ± 4 nmol O₂ min^–1 mg^–1, and the cydA::Cm strain had a lower rate of 102 ± 2 nmol O₂ min^–1 mg^–1. The ccoN::Cm strain exhibited a negligible rate of oxygen consumption at 5.2 ± 3.6 nmol O₂ min^–1 mg^–1. Dithionite-reduced minus air-oxidized spectra on isolated membrane particles of all three strains revealed no gross changes in the cytochrome composition; however, the soret peak (421 nm) was 75% reduced in the ccoN::Cm strain compared to both the wild type and the cydA::Cm strain (data not shown). The intensity of the soret peak is correlated with the total cytochrome content, as each type of cytochrome contributes to this value; thus, the ccoN::Cm strain contained fewer total cytochromes on a per-milligram basis. No cytochrome d peaks were detected, consistent with a recent work that concluded that C. jejuni does not contain the d-type cytochrome (19).

The cydA::Cm strain grew anaerobically when supplemented with formate and nitrate (Fig. 4) and had generation times similar to those of its parent strain under microaerobic conditions (data not shown). The ccoN::Cm strain failed to grow at oxygen tensions above 8% O₂ on BA plates (Table 4). Under anaerobic conditions, the ccoN::Cm strain grew similarly to the wild type in the anaerobic jar with an Anaerobic BBL GasPak Plus with palladium catalyst, with single, isolated colonies of the tertiary streak (Table 4). In O₂ tensions from 2 to 6%, isolated colonies were still present; however, these colonies were smaller than for the wild type. At 7% O₂, no isolated colonies were formed, but some growth was seen on the primary streak. No growth was observed at 8% O₂ or above. Generation times for the ccoN::Cm strain were similar to those of the wild type in anaerobic cultures (2.35 ± 0.22 h), but the terminal optical density was lower than for the wild type (Fig. 5).

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FIG. 4. Anaerobic growth of C. jejuni NCTC 11168 (closed squares) and the cydA::Cm (open circles) and ccoN::Cm (open triangles) strains. Cultures grown in MHF plus 10 mM NaNO3 were incubated at 37°C under anaerobic conditions.

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FIG. 5. Chicken colonization abilities of various strains. (A) CFU/gram cecal contents of C. jejuni wild type (square) and the napA::Cm (triangle) and nrfA::Cm (inverted triangle) strains. (B) CFU/gram cecal contents of the wild type (squares) and the cydA::Cm (inverted triangles) and Cj0264c::Cm (diamonds) strains. The horizontal bars represent the median value for each group. *, P < 0.05 compared to the wild type.

Chicken colonization.
The wild type and each strain (10⁷ CFU) were inoculated into 8 to 20 birds by oral gavage. Two weeks postinoculation, the birds were sacrificed by CO₂ asphyxiation and the cecal contents were removed and assayed for viable C. jejuni. The cydA::Cm and Cj0264c::Cm strains colonized as well as the wild type (Fig. 5B). The napA::Cm strain colonized at significantly lower rates than the wild type (P = 0.022). The nrfA::Cm strain colonized at levels significantly lower than wild type (P = 0.015) (Fig. 5A). No ccoN::Cm bacteria were recovered from the chicken ceca. Care was taken to make sure the cecal samples were not exposed to O₂ during the recovery of colonies. PBS was sparged with N₂ before the dilution of cells, and the cells were incubated in a 2% O₂ incubator. Wild-type C. jejuni (ranging from 8.7 x 10⁷ to 4.9 x 10⁸ CFU/gram cecal content) was recovered from the eight wild-type samples incubated at 2% O₂. No C. jejuni was found in the control birds that were given PBS as the inoculum (data not shown).

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The respiratory chain of C. jejuni is surprisingly flexible. The branched pathways allow C. jejuni to thrive in its natural environment, utilizing multiple respiratory donors and acceptors. Besides encoding two separate terminal oxidases, the genome sequence predicts a nitrate reductase, a nitrite reductase, and an SN oxide reductase (33). Anaerobic growth of C. jejuni is reasonable in light of its normal ecological niche. C. jejuni preferentially colonizes the ceca of experimentally inoculated chickens (7, 30). The avian cecum is a specialized organ used in the anaerobic fermentation of undigested polysaccharides, especially cellulose (8). Not surprisingly, molecular 16S rRNA analysis of the chicken cecum revealed that it is dominated by obligate anaerobic bacteria (3, 26), suggesting that C. jejuni often encounters anaerobic conditions despite its microaerophilic designation (18). We set out to determine the importance of the various respiratory acceptors in C. jejuni host colonization using strains with mutations in each of the respiratory acceptor enzymes.

Under specific in vitro conditions, nitrate was able to support anaerobic growth of C. jejuni (Fig. 2A). For optimal growth under anaerobic conditions, both the nitrate and nitrite reductases must be intact (Fig. 2B). Mutants deficient in either reductase were unable to reduce their respective substrates (Table 3). Denitrification seems to play an important role in the chicken cecum; cultured chicken cecal isolates have been shown to reduce nitrate (36). Our chicken colonization experiments showed the importance of the nitrate and nitrite reductases of C. jejuni in the ceca of chickens. The napA::Cm and nrfA::Cm strains both colonized the ceca of chickens at significantly lower levels than the wild type (Fig. 5A). Recently, a nrfA mutant in C. jejuni strain 81-176 was shown not to be impaired in host colonization compared to the parent strain (35). This discrepancy may arise from differences in the colonization assay protocol, in which the authors used an inoculating dose (1 x 10⁹ CFU) 100-fold higher than we used (1 x 10⁷ CFU). C. jejuni chick colonization has been shown to be dose dependent (1, 45), and thus, subtle differences in colonization potential may be observed only at the lower inoculating dose. C. jejuni can also utilize DMSO and TMAO as respiratory acceptors. Both these activities can be attributed to a single heterodimeric enzyme (encoded by Cj0264c and Cj0265c), a property that has been shown in other SN oxide reductases (25). Growth with DMSO or TMAO was slightly slower than with nitrate (Table 2), and anaerobic cultures had a lower terminal optical density than nitrate-grown cells (Fig. 3). This could be because DMSO and TMAO are poor electron acceptors compared to nitrate. The standard redox potentials for the DMS/DMSO and TMA/TMAO couples are +160 mV and +130 mV, respectively (14, 42), compared to +420 mV for nitrate/nitrite. The Cj0264c::Cm strain colonized the chicken at levels similar to those for the wild type (Fig. 5B). There have been very few published data on in vivo S- or N-oxide concentrations. The DMSO reductase of Actinobacillus pleuropneumoniae has been identified as a virulence factor in the porcine pathogen (which causes pleuroneumonia), as mutants are attenuated for pyrogenicity and have lower endoscopy scores for inflammation. Colonization and persistence, however, were not affected in this strain (5).

Wild-type cultures fail to grow in anaerobic serum bottles without the addition of an alternative respiratory acceptor (Table 2). This clearly indicates these cultures are not using O₂ as a respiratory electron acceptor. Furthermore, our O₂ analysis of anaerobically grown cells confirmed that the subnanomolar dissolved O₂ levels are far below the recently calculated K_m values for either of the terminal oxidases of C. jejuni (19). It has been postulated that C. jejuni requires O₂ for production of deoxynucleotides via the enzyme ribonucleotide reductase (RNR) (38). C. jejuni encodes a single I-type RNR (33). I-type RNR requires molecular oxygen for generation of the tyrosyl radical for catalysis; however, other facultative anaerobes also encode only I-type RNRs (16, 40). It is not known if RNR could function at the O₂ levels detected in our anaerobic cultures, as the O₂ affinity of this enzyme has not been determined.

C. jejuni encodes two terminal oxidases: a cbb3-type cytochrome c oxidoreductase (encoded by ccoNOQP) and an enzyme annotated in the genome sequence as a bd-type quinol oxidase, encoded by cydAB (33). It has been shown that no d-type cytochromes can be detected in C. jejuni using multiple spectrometric techniques, which leaves the original annotation of cydAB in doubt (19). The same group used a cydAB mutant to assign this oxidase to the low-O₂-affinity, cyanide-insensitive component of the respiratory chain (19). In our hands, cydA::Cm grew as well as the wild type in both microaerobic and anaerobic growth (Fig. 4) and retained over 80% of the wild-type respiratory activity. The cydA::Cm strain also colonized the chicken as well as the wild type did (Fig. 5B). In both the laboratory setting and the host, the cbb3-type terminal oxidase appeared to predominate. In vitro, the ccoN::Cm strain exhibited less than 5% of the wild-type formate-dependent O₂ uptake and was severely affected in microaerobic growth: no colonies could be recovered above 7% O₂ (Table 4). There are two possible explanations for the observed oxygen toxicity in the ccoN::Cm strain. (i) It is possible that the cells are susceptible to reactive oxygen species. Cyanide-insensitive oxidases, such as the one encoded by cydAB, produce H₂O₂ (18, 19, 24). If large amounts of H₂O₂ are produced when the cyanide-insensitive enzyme is the sole oxidase, the oxidative-protection enzymes (specifically, catalase and alkyl-hydroperoxide reductase) could be overwhelmed. (ii) The cbb3-type oxidase keeps cytoplasmic O₂ tensions low enough to keep O₂-sensitive, metabolically important enzymes functioning. This phenomenon (termed respiratory protection) was first described in Azotobacter vinelandii as a way of using respiration to protect the O₂-sensitive nitrogenase from inactivation (20). While either or both of these explanations could contribute to the O₂-sensitive phenotype of the ccoN::Cm strain, we think the second (respiratory protection) is more likely to be the cause. O₂-sensitive enzymes have long been considered to be the cause of C. jejuni's microaerophilic requirement (24). Of particular interest is the enzyme pyruvate-ferredoxin oxidoreductase, which has been implicated in conferring oxygen sensitivity on C. jejuni, as well as other species (32, 39). No colonies of the ccoN::Cm strain were recovered from the ceca of inoculated chickens. While we can conclude that an active cbb3-type oxidase is important for colonization of the host, we cannot confirm that the ccoN::Cm strain ever reached the cecum. It is entirely possible that cells of the ccoN::Cm strain did not survive passage through the more oxygen-rich areas of the upper gastrointestinal tract.

There is clearly a role for both anaerobic and aerobic respiration in C. jejuni host colonization. A high-affinity terminal oxidase mutant was unable to colonize, and nitrate and nitrite reductase mutants were impaired in this ability, using a chicken model. These studies agree with transcriptional profiling of C. jejuni during cecal colonization, in which the genes for the three enzymes (ccoNOPQ, napAGB, and nrfAH) were all shown to be up-regulated in vivo (43). This work also parallels recent studies of E. coli's ability to colonize the mouse intestine. It was shown that while the anaerobic-respiration pathways are important in colonization, it is the high-affinity oxidase that is crucial in E. coli's ability to be maintained in the mouse intestine (21). While it might seem surprising that O₂ plays such an important role in what is typically characterized as an anaerobic niche (4), it now appears that there may be more O₂ in the animal intestine than was previously thought. New imaging techniques have made it possible to measure oxygen concentrations in the gastrointestinal tracts of living mice (17). When the dissolved O₂ levels were converted to percent oxygen tension, it was shown that O₂ levels in the gut fell from a high of 7% in the stomach to 4.2% in the midduodenum and 1.4% in the mid-small intestine and midcolon to a low of 0.4% in the distal sigmoid colon-rectal junction (17). In experimentally inoculated chickens, C. jejuni localized to the cecum, which projects from the proximal colon at the junction with the small intestine (7, 30). Although the cecum would likely be at the low range of O₂ concentration, C. jejuni preferentially localizes to the mucosal crypts in the cecum, into which oxygen could diffuse from adjacent epithelial cells (7, 24). The bacteria do not physically attach to the microvilli, however, and could easily be displaced to more anaerobic regions via cecal mixing (24). The cecal wall is constantly contracting, which causes the mixing of the contents required for the filling and evacuation of the organ (8, 12). The shifting environment of the cecum is likely the impetus behind the redundant respiratory pathways. The ability of the nonfermentative C. jejuni to use alternative respiratory acceptors gives the bacterium an advantage when exposed to anaerobic conditions, even transiently. The single disruption of the nitrate or nitrite reductase creates a significant decrease in colonization, but colonization persists (albeit at lower levels). The ability to use multiple alternative acceptors (nitrate, nitrite, DMSO, and TMAO) for anaerobic growth may compensate for the loss of any one enzyme. The role of the cyanide-insensitive oxidase encoded by cydAB remains elusive. The low affinity of this enzyme for O₂ (19) seems to rule out a role in the host, and our colonization data bear this out. There is a clear role for the cbb3-type terminal oxidase both in vitro and in vivo. The disruption of this oxidase (in the ccoN::Cm strain) abolished C. jejuni's ability to colonize the chicken cecum, and mutants were unable to grow at elevated O₂ levels in vitro. This stresses the importance of the high-affinity terminal oxidase of C. jejuni, implying that oxygen is a key factor in chicken colonization despite the anaerobic conditions to which C. jejuni is consistently exposed.

 
This work was supported by USDA-NRI grant no. 2004-04553.

We are grateful to Jason Andrus and Debbie Threadgill of the University of North Carolina, Chapel Hill, for the kind gift of pJMA-001 and for helpful discussions.

 
* Corresponding author. Mailing address: Department of Microbiology, North Carolina State University, Campus Box 7615, Raleigh, NC 27695. Phone: (919) 515-7860. Fax: (919) 515-7867. E-mail: jwolson{at}ncsu.edu

Published ahead of print on 11 January 2008.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1
1. Ahmed, I. H., G. Manning, T. M. Wassenaar, S. Cawthraw, and D. G. Newell. 2002. Identification of genetic differences between two Campylobacter jejuni strains with different colonization potentials. Microbiology 148:1203-1212.[Abstract/Free Full Text]
2
2. Altekruse, S. F., N. J. Stern, P. I. Fields, and D. L. Swerdlow. 1999. Campylobacter jejuni—an emerging foodborne pathogen. Emerg. Infect. Dis. 5:28-35.[Medline]
3
3. Amit-Romach, E., D. Sklan, and Z. Uni. 2004. Microflora ecology of the chicken intestine using 16S ribosomal DNA primers. Poult. Sci. 83:1093-1098.[Abstract/Free Full Text]
4
4. Backhed, F., R. E. Ley, J. L. Sonnenburg, D. A. Peterson, and J. I. Gordon. 2005. Host-bacterial mutualism in the human intestine. Science 307:1915-1920.[Abstract/Free Full Text]
5
5. Baltes, N., I. Hennig-Pauka, I. Jacobsen, A. D. Gruber, and G. F. Gerlach. 2003. Identification of dimethyl sulfoxide reductase in Actinobacillus pleuropneumoniae and its role in infection. Infect. Immun. 71:6784-6792.[Abstract/Free Full Text]
6
6. Barnes, E. M., G. C. Mead, D. A. Barnum, and E. G. Harry. 1972. The intestinal flora of the chicken in the period 2 to 6 weeks of age, with particular reference to the anaerobic bacteria. Br. Poult. Sci. 13:311-326.[Medline]
7
7. Beery, J. T., M. B. Hugdahl, and M. P. Doyle. 1988. Colonization of gastrointestinal tracts of chicks by Campylobacter jejuni. Appl. Environ. Microbiol. 54:2365-2370.[Abstract/Free Full Text]
8
8. Clench, M. H., and J. R. Mathias. 1995. The avian cecum: a review. Wilson Bull. 107:93-121.
9
9. Cox, M. E., R. J. Kohr, and C. K. Samia. 1997. Comparison of quality control results with use of anaerobic chambers versus anaerobic jars. Clin. Infect. Dis. 25(Suppl. 2):S137-S138.[Medline]
10
10. Doan, N., A. Contreras, J. Flynn, J. Morrison, and J. Slots. 1999. Proficiencies of three anaerobic culture systems for recovering periodontal pathogenic bacteria. J. Clin. Microbiol. 37:171-174.[Abstract/Free Full Text]
11
11. Downes, J., J. I. Mangels, J. Holden, M. J. Ferraro, and E. J. Baron. 1990. Evaluation of two single-plate incubation systems and the anaerobic chamber for the cultivation of anaerobic bacteria. J. Clin. Microbiol. 28:246-248.[Abstract/Free Full Text]
12
12. Duke, G. E. 1986. Alimentary canal: secretion and digestion. Springer-Verlag, New York, NY.
13
13. Friedman, C. R., J. Niemann, H. C. Wegener, and R. V. Tauxe. 2000. Epidemiology of Campylobacter jejuni infections in the United States and other industrialized nations, p. 121-138. In I. Nachamkin and M. J. Blaser (ed.), Campylobacter. ASM Press, Washington, DC.
14
14. Gon, S., M. T. Giudici-Orticoni, V. Mejean, and C. Iobbi-Nivol. 2001. Electron transfer and binding of the c-type cytochrome TorC to the trimethylamine N-oxide reductase in Escherichia coli. J. Biol. Chem. 276:11545-11551.[Abstract/Free Full Text]
15
15. Gonzalez, P. J., C. Correia, I. Moura, C. D. Brondino, and J. J. Moura. 2006. Bacterial nitrate reductases: molecular and biological aspects of nitrate reduction. J. Inorg. Biochem. 100:1015-1023.[CrossRef][Medline]
16
16. Hartig, E., A. Hartmann, M. Schatzle, A. M. Albertini, and D. Jahn. 2006. The Bacillus subtilis nrdEF genes, encoding a class Ib ribonucleotide reductase, are essential for aerobic and anaerobic growth. Appl. Environ. Microbiol. 72:5260-5265.[Abstract/Free Full Text]
17
17. He, G., R. A. Shankar, M. Chzhan, A. Samouilov, P. Kuppusamy, and J. L. Zweier. 1999. Noninvasive measurement of anatomic structure and intraluminal oxygenation in the gastrointestinal tract of living mice with spatial and spectral EPR imaging. Proc. Natl. Acad. Sci. USA 96:4586-4591.[Abstract/Free Full Text]
18
18. Hoffman, P. S., and T. G. Goodman. 1982. Respiratory physiology and energy conservation efficiency of Campylobacter jejuni. J. Bacteriol. 150:319-326.[Abstract/Free Full Text]
19
19. Jackson, R. J., K. T. Elvers, L. J. Lee, M. D. Gidley, L. M. Wainwright, J. Lightfoot, S. F. Park, and R. K. Poole. 2007. Oxygen reactivity of both respiratory oxidases in Campylobacter jejuni: the cydAB genes encode a cyanide-resistant, low-affinity oxidase that is not of the cytochrome bd type. J. Bacteriol. 189:1604-1615.[Abstract/Free Full Text]
20
20. Jones, C. W., J. M. Brice, V. Wright, and B. A. Ackrell. 1973. Respiratory protection of nitrogenase in Azotobacter vinelandii. FEBS Lett. 29:77-81.[CrossRef][Medline]
21
21. Jones, S. A., F. Z. Chowdhury, A. J. Fabich, A. Anderson, D. M. Schreiner, A. L. House, S. M. Autieri, M. P. Leatham, J. J. Lins, M. Jorgensen, P. S. Cohen, and T. Conway. 2007. Respiration of Escherichia coli in the mouse intestine. Infect. Immun. 75:4891-4899.[Abstract/Free Full Text]
22
22. Kelly, D. J. 2001. The physiology and metabolism of Campylobacter jejuni and Helicobacter pylori. Symp. Ser. Soc. Appl. Microbiol. 30:16S-24S.[Medline]
23
23. Ketley, J. M. 1997. Pathogenesis of enteric infection by Campylobacter. Microbiology 143:5-21.[Free Full Text]
24
24. Krieg, N. R., and P. S. Hoffman. 1986. Microaerophily and oxygen toxicity. Annu. Rev. Microbiol. 40:107-130.[CrossRef][Medline]
25
25. Kurihara, F. N., and T. Satoh. 1988. A single enzyme is responsible for both dimethylsulfoxide and trimethylamine-N-oxide respirations as the terminal reductase in a photodenitrifier, Rhodobacter sphaeroides f.s. denitrificans. Plant Cell Physiol. 29:377-379.[Abstract/Free Full Text]
26
26. Lu, J., U. Idris, B. Harmon, C. Hofacre, J. J. Maurer, and M. D. Lee. 2003. Diversity and succession of the intestinal bacterial community of the maturing broiler chicken. Appl. Environ. Microbiol. 69:6816-6824.[Abstract/Free Full Text]
27
27. MacGregor, C. H., C. A. Schnaitman, and D. E. Normansell. 1974. Purification and properties of nitrate reductase from Escherichia coli K12. J. Biol. Chem. 249:5321-5327.[Abstract/Free Full Text]
28
28. McCrindle, S. L., U. Kappler, and A. G. McEwan. 2005. Microbial dimethylsulfoxide and trimethylamine-N-oxide respiration. Adv. Microb. Physiol. 50:147-198.[CrossRef][Medline]
29
29. Mead, G. C. 1989. Microbes of the avian cecum: types present and substrates utilized. J. Exp. Zool. Suppl. 3:48-54.[Medline]
30
30. Newell, D. G., and C. Fearnley. 2003. Sources of Campylobacter colonization in broiler chickens. Appl. Environ. Microbiol. 69:4343-4351.[Free Full Text]
31
31. Nicholas, D. J. D., and A. Nason. 1957. Determination of nitrate and nitrite. Methods Enzymol. 3:981-984.[CrossRef]
32
32. Pan, N., and J. A. Imlay. 2001. How does oxygen inhibit central metabolism in the obligate anaerobe Bacteroides thetaiotaomicron. Mol. Microbiol. 39:1562-1571.[CrossRef][Medline]
33
33. Parkhill, J., B. W. Wren, K. Mungall, J. M. Ketley, C. Churcher, D. Basham, T. Chillingworth, R. M. Davies, T. Feltwell, S. Holroyd, K. Jagels, A. V. Karlyshev, S. Moule, M. J. Pallen, C. W. Penn, M. A. Quail, M. A. Rajandream, K. M. Rutherford, A. H. van Vliet, S. Whitehead, and B. G. Barrell. 2000. The genome sequence of the food-borne pathogen Campylobacter jejuni reveals hypervariable sequences. Nature 403:665-668.[CrossRef][Medline]
34
34. Pitcher, R. S., and N. J. Watmough. 2004. The bacterial cytochrome cbb₃ oxidases. Biochim. Biophys. Acta 1655:388-399.[Medline]
35
35. Pittman, M. S., K. T. Elvers, L. Lee, M. A. Jones, R. K. Poole, S. F. Park, and D. J. Kelly. 2007. Growth of Campylobacter jejuni on nitrate and nitrite: electron transport to NapA and NrfA via NrfH and distinct roles for NrfA and the globin Cgb in protection against nitrosative stress. Mol. Microbiol. 63:575-590.[CrossRef][Medline]
36
36. Salanitro, J. P., I. G. Fairchilds, and Y. D. Zgornicki. 1974. Isolation, culture characteristics, and identification of anaerobic bacteria from the chicken cecum. Appl. Microbiol. 27:678-687.[Medline]
37
37. Samuel, M. C., D. J. Vugia, S. Shallow, R. Marcus, S. Segler, T. McGivern, H. Kassenborg, K. Reilly, M. Kennedy, F. Angulo, and R. V. Tauxe. 2004. Epidemiology of sporadic Campylobacter infection in the United States and declining trend in incidence, FoodNet 1996-1999. Clin. Infect. Dis. 38(Suppl. 3):S165-S174.[CrossRef][Medline]
38
38. Sellars, M. J., S. J. Hall, and D. J. Kelly. 2002. Growth of Campylobacter jejuni supported by respiration of fumarate, nitrate, nitrite, trimethylamine-N-oxide, or dimethyl sulfoxide requires oxygen. J. Bacteriol. 184:4187-4196.[Abstract/Free Full Text]
39
39. St Maurice, M., N. Cremades, M. A. Croxen, G. Sisson, J. Sancho, and P. S. Hoffman. 2007. Flavodoxin:quinone reductase (FqrB): a redox partner of pyruvate:ferredoxin oxidoreductase that reversibly couples pyruvate oxidation to NADPH production in Helicobacter pylori and Campylobacter jejuni. J. Bacteriol. 189:4764-4773.[Abstract/Free Full Text]
40
40. Torrents, E., I. Roca, and I. Gibert. 2003. Corynebacterium ammoniagenes class Ib ribonucleotide reductase: transcriptional regulation of an atypical genomic organization in the nrd cluster. Microbiology 149:1011-1020.[Abstract/Free Full Text]
41
41. Wang, Y., and D. E. Taylor. 1990. Chloramphenicol resistance in Campylobacter coli: nucleotide sequence, expression, and cloning vector construction. Gene 94:23-28.[CrossRef][Medline]
42
42. Wood, P. M. 1981. The redox potential of dimethylsulfoxide reduction to dimethylsulfide—evaluation and biochemical implications. FEBS Lett. 124:11-14.[CrossRef][Medline]
43
43. Woodall, C. A., M. A. Jones, P. A. Barrow, J. Hinds, G. L. Marsden, D. J. Kelly, N. Dorrell, B. W. Wren, and D. J. Maskell. 2005. Campylobacter jejuni gene expression in the chick cecum: evidence for adaptation to a low-oxygen environment. Infect. Immun. 73:5278-5285.[Abstract/Free Full Text]
44
44. Yao, R., R. A. Alm, T. J. Trust, and P. Guerry. 1993. Construction of new Campylobacter cloning vectors and a new mutational cat cassette. Gene 130:127-130.[CrossRef][Medline]
45
45. Young, C. R., R. L. Ziprin, M. E. Hume, and L. H. Stanker. 1998. Dose response and organ invasion of day-of-hatch leghorn chicks by different isolates of Campylobacter jejuni. Avian Dis. 43:763-767.[CrossRef]
46
46. Zhao, C., B. Ge, J. De Villena, R. Sudler, E. Yeh, S. Zhao, D. G. White, D. Wagner, and J. Meng. 2001. Prevalence of Campylobacter spp., Escherichia coli, and Salmonella serovars in retail chicken, turkey, pork, and beef from the Greater Washington, D.C., area. Appl. Environ. Microbiol. 67:5431-5436.[Abstract/Free Full Text]

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Applied and Environmental Microbiology, March 2008, p. 1367-1375, Vol. 74, No. 5
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