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Journal of Bacteriology, February 2008, p. 915-925, Vol. 190, No. 3
0021-9193/08/$08.00+0     doi:10.1128/JB.01647-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

The Campylobacter jejuni NADH:Ubiquinone Oxidoreductase (Complex I) Utilizes Flavodoxin Rather than NADH

Dilan R. Weerakoon and Jonathan W. Olson^*

Department of Microbiology, North Carolina State University, Raleigh, North Carolina

Received 11 October 2007/ Accepted 18 November 2007

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Campylobacter jejuni encodes 12 of the 14 subunits that make up the respiratory enzyme NADH:ubiquinone oxidoreductase (also called complex I). The two nuo genes not present in C. jejuni encode the NADH dehydrogenase, and in their place in the operon are the novel genes designated Cj1575c and Cj1574c. A series of mutants was generated in which each of the 12 nuo genes (homologues to known complex I subunits) was disrupted or deleted. Each of the nuo mutants will not grow in amino acid-based medium unless supplemented with an alternative respiratory substrate such as formate. Unlike the nuo genes, Cj1574c is an essential gene and could not be disrupted unless an intact copy of the gene was provided at an unrelated site on the chromosome. A nuo deletion mutant can efficiently respire formate but is deficient in -ketoglutarate respiratory activity compared to the wild type. In C. jejuni, -ketoglutarate respiration is mediated by the enzyme 2-oxoglutarate:acceptor oxidoreductase; mutagenesis of this enzyme abolishes -ketoglutarate-dependent O₂ uptake and fails to reduce the electron transport chain. The electron acceptor for 2-oxoglutarate:acceptor oxidoreductase was determined to be flavodoxin, which was also determined to be an essential protein in C. jejuni. A model is presented in which CJ1574 mediates electron flow into the respiratory transport chain from reduced flavodoxin and through complex I.

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Campylobacter jejuni is the leading cause of human bacterial gastroenteritis, campylobacteriosis, in the world (1, 11, 21). Humans are infected by ingestion of contaminated food, typically poultry, where C. jejuni is a frequent resident (6, 11). The physiology of C. jejuni has adapted to take advantage of life in the lower avian cecum, where C. jejuni predominates (3). Anaerobic fermentation appears to be the dominant lifestyle in the cecum (20), and C. jejuni utilizes fermentation by-products such as small organic acids as both carbon and energy sources (18, 22, 23, 35, 38). C. jejuni itself is nonfermentative and uses oxidative phosphorylation for all its energy demands. The genome sequence of C. jejuni codes for respiratory components that can utilize multiple electron donors and acceptors in a branched electron transport chain (27). One of these components in C. jejuni is a proton pump called complex I, which is widely found in bacteria, archaea, and the mitochondria of eukaryotes (12). This complex (also called NADH:ubiquinone oxidoreductase) is the first enzyme in many respiratory chains and catalyzes the transfer of electrons from NADH to the quinone pool, coupled with the translocation of protons across a membrane (12). The fact that complex I occurs in the C. jejuni genome sequence was somewhat surprising, as it has been shown elsewhere that NADH is a poor respiratory electron donor (14).

The genes that code for complex I of most bacteria are clustered in a conserved order (30) consisting of 14 different genes that are designated either as nuo (NADH:ubiquinone oxidoreductase) (37) or as nqo (NADH:quinone oxidoreductase) (12, 40). Complex I has been well characterized in both Escherichia coli (4, 19) and Paracoccus denitrificans (39), and a model of the subunit structure and function has emerged. Of the 14 subunits, seven subunits (NuoA, NuoH, and NuoJ to NuoN) are integral membrane proteins and the remaining seven (NuoB to NuoG and NuoI) are peripheral subunits. The known redox cofactors are found in the peripheral subunits (12), including those of the NADH dehydrogenase fragment which has been localized to NuoE, NuoF, and NuoG (13). Like these model systems, the complex I genes of C. jejuni are organized in the 14-gene nuo operon; however, nuoE and nuoF are absent from this operon and are replaced with two genes (Cj1575c and Cj1574c) of unknown function (Fig. 1) (27). Since nuoE and nuoF encode the NADH dehydrogenase subunit, it is not surprising that NADH is not the donor to complex I of C. jejuni. The other 12 subunits of complex I in C. jejuni do contain sequence similarities to complex I subunits of other bacteria including E. coli and P. denitrificans (19, 39).

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FIG. 1. Genetic organization of the nuo operon and graphical representation of mutants. (A) Deletions are indicated above the genes; sites of insertions are indicated below the genes. The arrow indicates the chloramphenicol resistance cassette cat; the direction of the arrow indicates the orientation of cat within the gene. (B) Representation of the deleted regions (dotted lines) in the double mutant NuoCDIM+. (C) Representation of the deleted region (dotted line) in NuoMD.

The electron donor to complex I of C. jejuni has yet to be identified. Finel has suggested that HP1264 and HP1265 (homologues to CJ1575 and CJ1574) in Helicobacter pylori may provide a docking site for a protein that may pass its electrons directly to NuoG (Nqo3) (9), and Myers and Kelly suggest a flavodoxin or ferredoxin as a possible candidate (24). Flavodoxins are small acidic proteins that are involved in electron transfer and contain one molecule of flavin mononucleotide (FMN) that acts as the redox active component (29), while ferredoxins are redox active iron-sulfur proteins classified by the number of Fe-S clusters (5). The genome sequence of C. jejuni codes for several ferredoxins and one flavodoxin (27). The genome also codes for two enzymes that typically use either ferredoxin or flavodoxin as the electron acceptor, pyruvate ferredoxin (flavodoxin) oxidoreductase (PFOR) and 2-oxoglutarate oxidoreductase (OOR). PFORs are iron-sulfur proteins involved in the coenzyme A (CoA)-dependent oxidative decarboxylation of pyruvate to form acetyl-CoA (15, 28). OOR enzymes are involved in the decarboxylation of 2-oxoglutarate in the presence of CoA to form succinyl-CoA and CO₂ (16). In C. jejuni, PFOR is encoded by Cj1476c and codes for a single protein subunit while OOR is composed of four subunits encoded by the genes oorDABC (Cj0535 to Cj0538) (27). PFOR and OOR both play vital roles in central carbon metabolism in C. jejuni, serving the analogous functions of pyruvate dehydrogenase and -ketoglutarate dehydrogenase, enzymes which are not found in the C. jejuni genome (27).

In this article, we demonstrate that complex I functions as a respiratory enzyme that accepts electrons from flavodoxin rather than NADH. We also show that OOR is responsible for -ketoglutarate-dependent respiratory activity and that flavodoxin is the electron acceptor for this enzyme. Furthermore, we show that both Cj1574c and fldA (which encodes flavodoxin) are essential genes in C. jejuni. We suggest that flavodoxin is essential because of its role as a substrate for two important steps in central carbon metabolism and that CJ1575 and CJ1574 are essential in maintaining a pool of oxidized flavodoxin.

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Bacterial strains and growth conditions. C. jejuni strains are listed in Table 1. Cells were grown either on tryptic soy agar plates supplemented with 10% sheep blood (called BA plates) or in Mueller-Hinton (MH) broth. Formate (20 mM for liquid culture and 50 mM for plates), sodium nitrate (10 mM), chloramphenicol (25 µg/ml), and kanamycin (30 µg/ml) were added to the medium as indicated. C. jejuni was routinely cultured microaerobically at 37°C in a trigas incubator (model NU-4950; NuAire, Inc.), in which the gas composition was constantly maintained at 12% O₂ and 5% CO₂ and the balance N₂. E. coli strains are listed in Table 1. Luria-Bertani broth and agar supplemented with ampicillin (at 150 µg/ml), chloramphenicol (at 25 µg/ml), and kanamycin (at 30 µg/ml) were used for growing various E. coli strains, as noted.

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TABLE 1. Strains used in this study

Cloning and construction of nuo mutants. Oligonucleotide primers (Table 2) were designed using the genome sequence of C. jejuni NCTC 11168 (27) using the DNA analysis program MacVector. Unless otherwise stated, PCR amplification was performed with Taq DNA polymerase (Promega) using C. jejuni NCTC 11168 genomic DNA as a template. Genomic DNA was isolated using the MasterPure Complete DNA and RNA purification kit (Epicentre Biotechnologies). The PCR products were cloned into pBluescript II⁺ KS (Stratagene) and pCR2.1 TOPO (Invitrogen) vectors and confirmed by restriction analysis (plasmids are listed in Table 3). The cloned nuo genes were then disrupted by insertion of a chloramphenicol acetyltransferase resistance cassette (cat), removed from pJMA-001 by SmaI digestion (pJMA-001 contains cat from plasmid pRY111 [41]), and cloned into the PvuII site of pGEM-T Easy (Promega) to yield plasmid pJMA-001. Double mutants were obtained by using a kanamycin resistance cassette, aphA-3 (36). Insertion of the cassettes was confirmed by isolation of genomic DNA from the mutants and PCR amplification of the relevant nuo genes, followed by agarose gel electrophoresis to monitor the increase in size of the nuo genes due to the insertion of the cassettes (data not shown).

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TABLE 2. Primers used in this studya

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TABLE 3. Plasmids used in this study

Transformation of C. jejuni. C. jejuni electrocompetent cells were made by harvesting cells by swab from BA plates and washing the cells three times in an ice-cold 9% sucrose and 15% glycerol solution. Cells were used immediately or frozen at –80°C for future use. For transformation, C. jejuni electrocompetent cells were incubated with 1 to 5 µg of plasmid DNA on ice for 10 min. The cells were then placed in a 2-mm electroporation cuvette and given a pulse of 2,500 V in an ECM399 electroporator (BTX, San Diego, CA), and 50 µl of MH broth was added to the cuvette and subsequently incubated on ice for 10 min. The cells were then spotted onto a cold BA plate supplemented with formate and sodium nitrate and incubated for 24 h in an anaerobic jar containing an Anaerobic BBL GasPak Plus with palladium catalyst (anaerobic pack). After 24 h on nonselective medium, the cells were transferred onto BA plates supplemented with chloramphenicol, formate, and sodium nitrate and incubated in an anaerobic jar with an anaerobic pack. Resistant colonies were selected after 3 to 5 days of incubation, and the colonies were screened for recombination of the interrupted version of the gene by PCR of genomic DNA using primers against the targeted gene.

Construction of the nuo merodiploid strains. pET7574 contains Cj1575c and Cj1574c in frame with a hexahistidine epitope tag. The cat cassette was then inserted into a BlpI restriction site located downstream of Cj1575c and Cj1574c in pET7574 to yield pET7574::CM. The coding region of the two genes and cassette was excised from pET7574::CM by digestion with BglII and StyI and treated with T4 DNA polymerase (Promega) to ensure blunt ends. This fragment was then cloned into the NdeI restriction site of pHydA (Table 3), which contains a 1,193-bp fragment of hydA, to yield pHyd7574::CM. Electrocompetent C. jejuni cells were transformed with pHyd7574::CM to obtain 7574MD. 74MD was constructed as follows: a 2,659-bp fragment of nuoC, nuoD, Cj1575c, Cj1574c, and nuoG was amplified by PCR using C. jejuni wild-type (WT) genomic DNA and primers NuoF and NuoR and cloned into pCR2.1-TOPO to yield pNuo. This plasmid was digested with SspI to create a deletion within Cj1574c, and aphA-3 was inserted into the SspI sites of Cj1574c to yield pNuo::KAN. Restriction analysis revealed that aphA-3 was in the same orientation as Cj1574c. This plasmid was then electroporated with competent 7574MD cells to obtain 74MD. PCR was carried out to confirm that aphA-3 was inserted in the Cj1574c gene of the nuo operon and not in the Cj1574c gene cloned into hydA. NuoMD was constructed as follows: nuoN was digested out of pNuoN (Table 3) via EcoRI, blunt ended with T4 DNA polymerase, and cloned into the BamHI site of pNuoCD (Table 3) in the same orientation as nuoCD to yield pNuoCDN. This plasmid was digested by creating two deletions between the AccB7I site of nuoC and the ClaI site of nuoD and the ClaI sites of nuoD and nuoN. Next, aphA-3 was inserted into the AccB7I/ClaI site to yield pNuoCDN::KAN. Restriction analysis revealed that aphA-3 was in the same orientation as nuoC and nuoN. This plasmid was mobilized via electroporation into competent 7574MD cells to obtain NuoMD.

Construction of the flavodoxin merodiploid strains. A merodiploid flavodoxin strain (FldMD) was constructed as follows: primers designed to engineer an NdeI site to the first codon of the fldA gene (Cj1382c) (FldProF) and a XhoI site in place of the stop codon of fldA (FldProR) and platinum Pfu polymerase (Invitrogen) were used to amplify fldA using C. jejuni WT genomic DNA and cloned into pCR2.1-TOPO to yield pTOPOFld. fldA was excised from pTOPOFld by digestion with NdeI and XhoI and then cloned into the NdeI/XhoI-digested pET-21a(+) in frame with the hexahistidine tag to yield pETFld. aphA-3 was then inserted into the BlpI site of pETFld to yield pET7574::KAN. fldA and aphA-3 were then cut out of pET7574::KAN using BglII and StyI, blunt ended with T4 DNA polymerase, and cloned into the NdeI site of pHydA to yield pHydFld::KAN. This plasmid was used to transform competent C. jejuni to obtain FldMD. FldMD::CM was constructed as follows: the fldA gene (Cj1382c) and two flanking regions of 948 bp upstream of the start codon and 287 bp downstream of the stop codon, respectively, were amplified by PCR using C. jejuni WT genomic DNA and primers FldF and FldR, blunt ended with T4 DNA polymerase, and phosphorylated with T4 polynucleotide kinase (Promega). This product was then cloned into the EcoRV site of pBluescript II KS(+), where the EcoRI restriction site has been destroyed (pKSRI), to yield pRIFld. The cat cassette was then cloned into the EcoRI site of pRIFld. The resulting plasmid, pRIFld::CM, was then electroporated into competent FldMD cells to obtain FldMD::CM. PCR was carried out to confirm that the original fldA gene in the genome contained the cat and not the fldA gene cloned into hydA.

Cloning of oorB and construction of OorB::CM. The oorB (Cj0537) gene was amplified by PCR using the primers OorF and OorR and cloned into pCR2.1-TOPO to yield pOorB. This plasmid was digested with SspI to create a deletion within oorB, and a cat cassette was then inserted into the SspI sites of this plasmid to obtain pOorB::CM. This plasmid was electroporated into competent WT C. jejuni cells to obtain OorB::CM. Restriction analysis revealed that the cat cassette was in the same orientation as the oorB gene.

qRT-PCR. Quantitative reverse transcriptase PCR (qRT-PCR) was performed using the QuantiTect Sybr green RT-PCR kit (Qiagen, Valencia, CA). RNA was extracted from C. jejuni cells grown to mid-log phase by using the MasterPure Complete DNA and RNA purification kit (Epicentre Biotechnologies).

The RT-PCR mixture included 40 ng RNA, 2 µM (each) of the forward and reverse primers, 1x QuantiTect Sybr green RT-PCR Master Mix (Qiagen), and 0.2 µl/reaction mixture QuantiTect reverse transcriptase mix (Qiagen). The reverse transcriptase reaction mixture was held at 50°C for 30 min, followed by a PCR initial activation step for 15 min at 95°C. The mixtures were then amplified for 30 cycles consisting of 94°C for 15 seconds, 60°C for 30 seconds, and 72°C for 30 seconds in an automated thermal cycler (Bio-Rad iCycler; Hercules, CA). The iCycler software was used to determine the threshold cycle for when each transcript could be detected. 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 by standardizing the copy number of that gene to that of an internal control, gyrA (Cj1027c).

Flavodoxin expression, purification, and characterization. E. coli strain BL21 Rosetta (Novagen) was transformed with pETFld. A 5-ml overnight culture of pETFld Rosetta grown at 37°C in Luria-Bertani broth supplemented with ampicillin was used to inoculate a fresh 500-ml Luria-Bertani broth. This inoculated culture was shaken at 37°C. When the optical density of the culture at 600 nm (OD₆₀₀) was between 0.5 and 0.7, the culture was induced with a final concentration of 0.5 mM IPTG (isopropyl-β-D-thiogalactopyranoside). Two hours postinduction the cells were harvested by centrifugation, washed two times with lysis buffer (50 mM NaH₂PO₄, 300 mM NaCl, 10 mM imidazole), and resuspended with 40 ml lysis buffer. The cells were then broken by passage through a French pressure cell (Thermo Spectronic) three times at 20,000 lb/in². The crude extract was cleared by centrifugation at 12,000 x g for 5 min. The supernatant was collected and passed over a Ni-nitrilotriacetic acid agarose (5-ml bed volume) column (Qiagen) preequilibrated with lysis buffer. The column was then washed with 5 column volumes of wash buffer (50 mM NaH₂PO₄, 300 mM NaCl, 20 mM imidazole). Protein was eluted with elution buffer (50 mM NaH₂PO₄, 300 mM NaCl, 250 mM imidazole), and 2-ml fractions were collected. Elution fractions were assayed for protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as described by Laemmli (17). Protein-containing fractions were dialyzed overnight in a 1-liter solution of 10 mM Tris, 10 mM NaCl, 1 mM dithiothreitol, and 10% glycerol at 4°C. Spectra were obtained using this purified flavodoxin in a Shimadzu UV-1650PC spectrophotometer. Spectra from commercially purchased FMN (MP Biomedicals, Inc.) and flavin adenine dinucleotide (FAD; Alexis Biochemicals) were used to compare the flavodoxin spectrum.

Cytochrome reduction assays. Cells were harvested with a swab into N₂-sparged phosphate-buffered saline (PBS), washed once, and sonicated in sealed tubes under N₂ to obtain cell extracts (CE). One milliliter each of CE was put into two quartz cuvettes, which were subsequently sealed with rubber stoppers, flushed with N₂ gas for 3 min, and then placed in a Shimadzu UV-1650PC spectrophotometer. A baseline spectrum was produced first without the addition of any substrates and second with the simultaneous addition of a 5 mM final concentration of -ketoglutarate and a 0.5 mM final concentration of CoA. Protein concentrations were determined with the bicinchoninic acid protein assay kit (Pierce).

Reduction of flavodoxin. Purified flavodoxin and C. jejuni CE were mixed in a 4:1 ratio in a capped quartz cuvette to obtain a total volume of 1 ml; a blank cuvette contained only C. jejuni CE and buffer. The cuvettes were flushed with N₂ for 3 min and placed in a Shimadzu UV-1650PC spectrophotometer. A baseline spectrum was produced without the addition of substrate to verify the characteristic flavodoxin spectra. Next, a mixture of -ketoglutarate and CoA was added to both quartz cuvettes simultaneously to give a final concentration of 5 mM and 0.5 mM, respectively. The cuvettes were inverted several times, and a spectral analysis was conducted immediately, 1 min, and 5 min after the addition of these substrates. To determine the flavodoxin reduction kinetics, the absorbance at 460 nm was monitored following addition of -ketoglutarate and CoA.

Oxygen uptake experiments. O₂ was quantified using a YSI Model 5300 biological oxygen monitor (Yellow Springs Instrument Co., Yellow Springs, OH) and a Clark-type electrode. The electrode was inserted into a 5-ml-capacity glass chamber that was continuously stirred. Five milliliters of whole cells or CE was added to the chamber and allowed to equilibrate until no change in dissolved O₂ was observed for several minutes. Upon equilibration, substrate was added through a capillary tube via a Hamilton syringe into the chamber and the dissolved O₂ was recorded by chart recorder. After each experiment the chamber was calibrated using known concentrations of dissolved O₂. Substrate concentrations used were as follows: -ketoglutarate and CoA, 5 mM and 0.5 mM, respectively; formate, 5 mM; NADH, 5 mM; NADPH, 5 mM.

SDS-PAGE and immunoblotting. To prepare membrane particles, 10 plates of each strain were harvested with a sterile swab into cold PBS, washed twice with PBS, and resuspended to 10 ml in cold PBS. The cells were broken by passage three times through a French pressure cell at 20,000 lb/in², and the lysate was cleared of unbroken cells by centrifugation at 12,000 x g for 5 min. The cleared supernatant was then subjected to fractionation by ultracentrifugation (150,000 x g for 90 min) in a Beckman L8-55 ultracentrifuge to isolate the membranes. The supernatant (soluble fraction) was saved for immunoblotting. The membrane fraction was washed once by resuspension in 10 ml cold PBS by Dounce homogenization, followed by centrifugation (150,000 x g for 90 min), and finally resuspended with a Dounce homogenizer. Ten micrograms of the soluble and membrane fractions was subjected to SDS-PAGE and transferred electrophoretically onto 0.2-µm nitrocellulose membranes (Bio-Rad Laboratories). The membranes were incubated in blocking solution (5% nonfat dry milk powder in Tris-buffered saline) for 30 min at room temperature. The primary antibody (purified mouse monoclonal tetra-His antibody [Qiagen]) was diluted 1:2,000 in blocking solution and incubated overnight at 4°C. The membranes were washed three times for 5 min each time with Tris-buffered saline and then incubated with a 1:3,000 dilution of purified goat anti-mouse-alkaline phosphatase conjugate antibody (Bio-Rad Laboratories) for 2 hours at room temperature. The membranes were washed twice with Tris-buffered saline, and a final wash was conducted with alkaline Tris buffer (100 mM Tris [pH 9.5], 100 mM NaCl, 50 mM MgCl₂). After washing, the membranes were developed with the alkaline phosphatase color development reagents 5-bromo-4-chloro-3-indoyl phosphate p-toluidine salt and p-nitroblue tetrazolium chloride purchased from Bio-Rad Laboratories.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Twelve nuo genes of complex I are dispensable. Twelve of the 14 genes in the complex I operon were disrupted via either an insertion or a deletion mutation (Fig. 1A). NuoAB contains a deletion comprising 34% of nuoA and 53% of nuoB; the deleted DNA was replaced with cat (41). nuoC was disrupted both by an insertion with cat within the coding region (NuoC::CM) and also as part of a deletion comprising nuoC (15% deletion) and nuoD (99% deletion) to obtain NuoCD. Two nuoD mutants were isolated with cat insertions near the middle (NuoD::CM) and near the end (Nuo::CM) of the gene. nuoG was disrupted by insertion of cat (in both orientations) within the coding region, and the resulting mutants were designated NuoG::CM⁺ and NuoG::CM^– (for all mutants, a superscript plus sign indicates that the cassette is transcribed in the same orientation as the gene and a superscript minus sign indicates cassette transcription opposite the gene). nuoG was also disrupted as part of a deletion comprising nuoG (56% deletion), nuoH, and nuoI (23% deletion) to obtain NuoGI⁺ and NuoGI^–. nuoI was disrupted by insertion of cat (in both orientations) within the coding region (NuoI::CM⁺ and NuoI::CM^–) and also as part of a deletion comprising 77% of nuoI; all of nuoJ, nuoK, and nuoL; and 89% of nuoM to obtain NuoIM⁺ and NuoIM^–. nuoN was disrupted by insertion of cat (in both orientations) to obtain NuoN::CM⁺ and NuoN::CM^–. Two versions of a double mutant were obtained (Fig. 1B), using NuoCD as the parent strain and insertion of aphA-3 (in both orientations) into a deleted region consisting of nuoI, nuoJ, nuoK, nuoL, and nuoM, to yield NuoCDIM⁺ and NuoCDIM^–. Each of these mutants was isolated on BA plates supplemented with formate (50 mM) and nitrate (10 mM) under an anaerobic atmosphere created by a GasPak Plus anaerobic pouch. All nuo mutants display similar growth phenotypes: they will not grow in liquid culture (cultures fail to double twice in 30 h) or on plates (no isolated colonies) unless the medium is supplemented with an alternative respiratory substrate such as formate. When formate is provided, these mutants displayed growth rates similar to that of the WT (Table 4; Fig. 2). Growth is entirely dependent on formate in these strains, which is shown graphically for strain NuoMD in Fig. 2. Final culture OD₆₀₀ is proportional to the initial concentration of formate (10 mM-supplemented cultures grow to a final OD₆₀₀ of 0.18; 20 mM-supplemented cultures grow to a value of 0.36), and after the formate has been consumed growth ceases.

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TABLE 4. Generation times of various strains in MH broth and MH broth plus 20 mM formate

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FIG. 2. Growth curve of NuoMD in MH broth in a microaerobic atmosphere (5% CO2, 12% O2, balance N2). Conditions: unsupplemented MH broth (squares), MH broth plus 10 mM formate (triangles), and MH broth plus 20 mM formate (circles). The y axis is in a linear scale to show the difference in the terminal optical density.

Expression of Cj1574c is required for viability. Despite many attempts, we were unsuccessful at mutating Cj1574c via either an insertion or a deletion mutation. We also noticed that nuo genes upstream of Cj1574c could be mutated only with cat that was in the same orientation as the nuo genes, while genes downstream of Cj1574c could be mutated with cat in either orientation (Fig. 1). qRT-PCR reveals that expression of the genes downstream of cat is affected differentially depending on the cat orientation. To show this effect quantitatively, we measured the transcription of nuoH in NuoG::CM⁺ and NuoG::CM^– strains that are identical except for the orientation of cat. When cat is in the same orientation as the operon (NuoG::CM⁺), transcription is reduced eightfold in comparison to WT, but when cat is transcribed opposite (NuoG::CM^–) from the nuo genes, transcription is down 50-fold (Table 5). That no strains with opposite-orientation cat cassettes upstream of Cj1574c could be isolated gives support to the conclusion that expression of Cj1574c is required for viability in C. jejuni.

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TABLE 5. Transcription of nuoH and Cj1574c in various strains as measured by qRT-PCR

The Cj1574c merodiploid strains. A merodiploid Cj1575c and Cj1574c C. jejuni strain was constructed by cloning the coding regions of both into the coding region of hydA (Cj1267c). Insertions into hydA have no phenotype relevant to these studies, and transcription of hydA is constitutive (data not shown), making this a useful locus for unrelated protein expression. The resulting strain (designated 7574MD) contains two copies of Cj1575c and Cj1574c. When 7574MD was used as the parent strain, it was possible to delete a portion of Cj1574c found within the nuo operon, yielding 74MD. Furthermore, a large deletion of the nuo operon could be made, in which nuoC to nuoN (including Cj1574c and Cj1575c) were deleted and replaced with the aphA-3 cassette (designated NuoMD [Fig. 1C]). qRT-PCR of WT and NuoMD strains indicates that Cj1574c expression levels are threefold higher in NuoMD than in the WT (Table 5). 74MD is unable to grow in MH broth without formate supplementation (Table 4). This result was unexpected, as this strain contains intact copies of all 12 nuo genes and is also complemented with Cj1575c and Cj1574c. qRT-PCR assays of nuoH in WT and 74MD, however, reveal that the aphA-3 cassette within Cj1574c reduced the expression of downstream genes 80-fold (Table 5). The requirement for formate in this strain can be attributed to the loss of transcription of nuoG to nuoN.

Localization of CJ1574. Purified CJ1575 and CJ1574 expressed from E. coli were not immune reactive when inoculated into two separate rabbits and did not provide antisera able to identify these proteins (data not shown). We therefore used an epitope-tagged (hexahistidine) version of CJ1574 to create the merodiploid strains. Since this tag has been cloned in frame with Cj1574c, the resulting protein product would contain a hexahistidine epitope tag on the C terminus. Anti-His antibodies recognize an appropriately sized protein expressed in all three strains expressing CJ1574 from the hyd operon (Fig. 3). To determine CJ1574 localization, membrane and soluble fractions of each strain were prepared and blotted (Fig. 3). The results indicate that CJ1574 was present in both the membrane and the soluble fractions in all three strains; however, the ratio of membrane-associated protein was highest in strain NuoMD.

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FIG. 3. Immunoblot assay of C. jejuni extracts using anti-His primary antibody. Lane 1, prestained low-range standards composed of phosphorylase b (113 kDa), bovine serum albumin (92 kDa), ovalbumin (52.3 kDa), carbonic anhydrase (35.3 kDa), soybean trypsin inhibitor (28.7 kDa), and lysozyme (21.3 kDa); lane 2, 7574MD membrane fraction; lane 3, 7475MD soluble fraction; lane 4, 74MD membrane fraction; lane 5, 74MD soluble fraction; lane 6, NuoMD membrane fraction; lane 7, NuoMD soluble fraction.

Respiratory activities of WT and NuoMD. Respiration rates on various substrates were determined by O₂ uptake using a Clark-type O₂ electrode and CE from WT and NuoMD (Table 6). In agreement with previous studies (8, 14), formate was the preferred substrate for both WT and NuoMD (Table 6). The only substrate tested that was significantly affected by the nuo deletion was -ketoglutarate, a known respiratory substrate in C. jejuni (23, 38). WT CE respired -ketoglutarate at a rate of 16.4 nmol of O₂ consumed minute^–1 mg of protein^–1 versus 3.7 nmol of O₂ consumed minute^–1 mg of protein^–1 for NuoMD (Table 6). Significantly, C. jejuni CE had low rates of respiratory activity with either NADH or NADPH, and these rates were unaffected by the nuo deletion (Table 6).

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TABLE 6. Respiration rates in strains (CE) with various substrates

OOR is responsible for -ketoglutarate respiration in C. jejuni. The decreased ability of NuoMD to respire -ketoglutarate prompted us to look at this substrate as a possible donor to complex I. Previous studies have shown that -ketoglutarate supports respiration in C. jejuni strain 11168 (23) and C. jejuni strain ACC 29428 (38); however, the enzymes responsible have not been characterized. We targeted the OorB subunit of the enzyme OOR for mutagenesis by insertion of cat into the oorB (Cj0537) coding region to yield OorB::CM. CE of OorB::CM displayed negligible -ketoglutarate and CoA-dependent O₂ uptake (Table 6), indicating that OOR is the sole enzyme responsible for -ketoglutarate respiration. Furthermore, we were able to show that -ketoglutarate (plus CoA) initiated the reduction of the electron transport chain, as evidenced by the emergence of peaks at 421 nm, 524 nm, and 553 nm, characteristic of reduced cytochromes. Addition of -ketoglutarate and CoA to CE of OorB::CM results in no cytochrome reduction, even after a 30-min incubation (data not shown). These data indicate that -ketoglutarate is oxidized by OOR and that electrons liberated are transferred to O₂ via the respiratory electron transport chain.

Flavodoxin is the electron acceptor of OOR and an essential protein. Attempts to mutate flavodoxin (fldA, Cj1382c) in C. jejuni were unsuccessful despite many attempts. We used the same strategy to make an fldA merodiploid strain as we used with 7574MD, and fldA was cloned in frame with a His tag coding sequence into the hyd operon, disrupting hydA to obtain FldMD. When FldMD was used as the parent strain, we were able to interrupt the original fldA gene to obtain FldMD::CM, indicating that flavodoxin is required for viability in C. jejuni. A His-tagged version of C. jejuni flavodoxin was expressed at high levels in E. coli and purified to near homogeneity in one step using nickel-chelate affinity chromatography (Fig. 4A). The recombinant flavodoxin contains a flavin cofactor as determined by visible spectra; however, the nature of the cofactor remains ambiguous. Comparison of purified flavodoxin to a commercially purchased FMN and FAD (Fig. 4B) reveals that although the gross spectra are similar, the absorption maximum for flavodoxin (460 nm) is shifted in relation to both that of FMN (445 nm) and that of FAD (450 nm). Addition of dithionite to the protein leads to reduction of the flavin, which can be monitored by a decrease in absorbance at 460 nm (data not shown). We used the redox state of the flavodoxin to determine if -ketoglutarate (via OOR) was the electron donor. Purified flavodoxin was incubated with CE of WT and OorB::CM along with -ketoglutarate and CoA in a stoppered quartz cuvette. Flavodoxin reduction was monitored by a decrease in absorbance of the peak at 460 nm (Fig. 5A). WT CE reduce recombinant flavodoxin at a rate of 213 ± 19 nmol min^–1 mg of protein^–1 (Fig. 5A), and OorB::CM CE are unable to reduce flavodoxin (Fig. 5B). These data indicate that in C. jejuni (unlike in H. pylori) flavodoxin is an efficient electron acceptor of OOR (16). Previous studies have shown that the flavodoxin of H. pylori is essential for the survival for this organism (10).

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FIG. 4. Purification of flavodoxin. (A) SDS-PAGE. Lane 1, low-range standards (Mr of each standard is to the left of the gel); lane 2, crude extract; lane 3, supernatant of crude extract; lane 4, purified flavodoxin. (B) Absorption spectra of purified flavodoxin (solid line), commercially purchased FMN (dashed line), and commercially purchased FAD (dotted line).

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FIG. 5. Reduction of flavodoxin with CE and -ketoglutarate and CoA. (A) WT CE. Spectrum with no addition (solid line), immediately after the addition of substrates (dashed line), 1 min after the addition of substrates (dashed line with single dots), and 5 min after the addition of substrates (dashed line with double dots). (B) OorB::CM CE. Spectra with no addition (solid line), immediately after the addition of substrates (dashed line), 1 min after the addition of substrates (dashed line with single dots), and 5 min after the addition of substrates (dashed line with double dots).

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In order to fulfill its energy demands, C. jejuni relies solely on the respiratory chain and oxidative phosphorylation. Known respiratory substrates for C. jejuni include hydrogen, formate, succinate, malate, lactate, sulfite, and -ketoglutarate (14, 23, 25, 38). Curiously, despite the fact that NADH is a poor respiratory substrate in C. jejuni (14), the genome sequence predicts the presence of 12 nuo (for NADH ubiquinone oxidoreductase) genes, also called complex I (27). This apparent paradox is explained by the fact that the nuo genes that encode the NADH dehydrogenase module subunits (nuoE and nuoF) are absent from the operon. In the place of nuoE and nuoF (between nuoD and nuoG [Fig. 1A]) are two novel genes, Cj1574c and Cj1575c. We present a model (Fig. 6) whereby these novel proteins act as electron acceptors from a flavodoxin rather than NADH. In this model flavodoxin acts (like NADH) as an intermediate between central carbon metabolism and the electron transport chain.

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FIG. 6. Model of the C. jejuni respiratory pathway through complex I. Arrows indicate the flow of electrons.

Despite the lack of NADH dehydrogenase activity, C. jejuni complex I is a major point of entry of electrons into the respiratory chain. nuo mutants fail to grow in MH broth, but growth can be restored when provided with an alternative respiratory substrate such as formate (Table 4). In contrast to the 12 nuo genes, Cj1574c (the sixth gene of the nuo operon [Fig. 1A]) is essential for viability of C. jejuni. We draw this conclusion based on three criteria. First, repeated attempts to interrupt or delete the gene via allelic replacement were unsuccessful, despite the ability to interrupt or delete the other 12 genes of the operon. Second, genes "upstream" of Cj1574c can be interrupted only when the drug cassette is in an orientation that allows transcription of downstream genes. Third, when a second copy of Cj1574c was provided on a second location in the genome (a Cj1574c merodiploid), the entire nuo operon was dispensable.

Strain NuoMD contains a large (10.5-kb) deletion from the middle of nuoC to the beginning of nuoN (Fig. 1C). Importantly, Cj1574c is expressed in this strain from the inserted copy both transcriptionally (Table 5) and translationally (as measured by immunoblotting [Fig. 3]). We used this strain to identify the physiological donor to complex I. Respiratory activities (as measured by O₂ uptake) were similar to those of the parent strain for all respiratory substrates tested except for -ketoglutarate (Table 6). -Ketoglutarate is a tricarboxylic acid (TCA) cycle intermediate and the entry point for many amino acids into central carbon metabolism, making it especially important in an asaccharolytic organism such as C. jejuni that garners most of its carbon and energy from amino acids (18, 22, 35). OOR (encoded by oorDABC [Cj0535 to Cj0538]) is a functional equivalent to -ketoglutarate dehydrogenase in that it catalyzes the decarboxylation of -ketoglutarate to form succinyl-CoA (16). The OOR mutant strain supports neither -ketoglutarate-dependent cytochrome reduction nor -ketoglutarate-dependent O₂ uptake (Table 6). Although the oxidative TCA cycle is interrupted in OorB::CM, the mutation is not lethal due to the presence of reductive TCA enzymes (e.g., fumarate reductase) which allow for the synthesis of important biosynthetic precursors. One major difference between OOR and -ketoglutarate dehydrogenase is that the reducing equivalents from OOR are transferred to a low-potential protein electron acceptor rather than NADH. In search of the physiological electron acceptor to OOR, we mutated two ferredoxin genes (Cj0333c and Cj0369c) and found no effect on -ketoglutarate respiratory activity (data not shown). Attempts to mutate the C. jejuni flavodoxin gene (fldA) proved unsuccessful unless a second copy of fldA was provided on the chromosome. We conclude that fldA (like Cj1574c) is an essential gene in C. jejuni. Flavodoxin has also been shown to be essential in H. pylori (10). C. jejuni flavodoxin expressed heterologously in E. coli and purified by nickel chelate affinity chromatography (Fig. 4A) contains a flavin cofactor that can be monitored spectrophotometrically (Fig. 4B). Extracts of WT C. jejuni reduce flavodoxin when provided with -ketoglutarate and CoA (Fig. 5A); extracts of OorB::CM do not (Fig. 5B). These data indicate that the electron acceptor for OOR in C. jejuni is flavodoxin, in contrast to H. pylori, where the electron acceptor was determined not to be flavodoxin (16).

Taken together, these data indicate that complex I in C. jejuni is an entry point for electrons into the respiratory electron transport chain from a reduced flavodoxin. We also conclude that CJ1574 (and possibly CJ1575, but we will speculate only on CJ1574 here) is required for viability of C. jejuni and involved in both electron transport and flavodoxin redox cycling. In our model (Fig. 6), we believe that in WT cells CJ1574 is part of complex I and facilitates the transfer of electrons into the complex. Furthermore, in the absence of a functional complex I (such is the case in all the nuo mutants), CJ1574 retains its role in facilitating the oxidation of flavodoxin. This model explains why CJ1574 is essential, as without this activity the flavodoxin pool would remain reduced. Oxidized flavodoxin is required for the activity of both OOR and PFOR (15, 31), both of which are important enzymes of central carbon metabolism. The importance of a pool of oxidized flavodoxin is indicated by the essential nature of both flavodoxin and CJ1574.

O₂ is the final electron acceptor of flavodoxin regardless of whether complex I is intact. Although greatly reduced, the nuo deletion strain retains partial -ketoglutarate-dependent O₂ uptake activity (Table 6), and CJ1574 is still membrane associated (Fig. 3). We conclude that this low level of CJ1574-mediated electron flow in the nuo mutants is sufficient to provide a pool of oxidized flavodoxin but does not provide enough energy to support growth of the cultures in the absence of formate (Table 4). A recent study has shown that in H. pylori flavodoxin interacts with a flavin quinone reductase that the authors termed FqrB (31). FqrB was shown to mediate the transfer of electrons from flavodoxin to NADP to form NADPH. FqrB is also present in C. jejuni and can accept electrons from reduced flavodoxin (31). Although we feel it most likely that CJ1574 and FqrB accept electrons from flavodoxin independently, we cannot rule out the possibility that FqrB is an intermediate in electron flow from flavodoxin to the respiratory chain. We do not believe, however, that NADPH is involved in the electron transfer to the respiratory chain. The level of NADPH-dependent respiratory activity observed (Table 6) is too low to account for the rate of -ketoglutarate-dependent O₂ uptake.

C. jejuni belongs to the epsilon class of the proteobacteria, the diversity of which has only recently been recognized. In addition to the well-studied pathogens of the genera Campylobacter and Helicobacter, the Epsilonproteobacteria also include many marine and terrestrial aquatic species (7) and may be the dominant bacterial species in deep-sea hydrothermal vent systems (33). Abandoning the NADH dehydrogenase module form appears to be an early event in the evolution of the Epsilonproteobacteria, as nuoE and nuoF are absent from all epsilonproteobacterial genome sequences, despite the presence of nuo operons (Fig. 7). Three different strategies have been employed by these bacteria to cope with the loss of nuoE and nuoF: replacement with Cj1575c and Cj1574c homologues (as in Campylobacter and Helicobacter species), deletion (along with nuoG) without replacement (as in Caminibacter mediatlanticus TB-2 and one of the Sulfurovum strain NBC37-1 operons), and duplication of nuoG and recruitment of a Cj1575c homologue and either gltD or fdhB (as in Wolinella succinogenes, Nitratiruptor strain SB155-2, Thiomicrospira denitrificans ATCC 33889, and one of the Sulfurovum strain NBC37-1 operons) (Fig. 7). The different strategies employed by the Epsilonproteobacteria likely reflect the different needs of each bacterium adapted to its specific environment. The -proteobacters of the hydrothermal vent systems grow autotrophically using the reductive TCA cycle to fix CO₂ and relying heavily on inorganics for energy (7, 32). The presence of the reversible enzymes PFOR and OOR (both of which are required for the reductive TCA cycle) in the Epsilonproteobacteria is likely the legacy of this autotrophic lifestyle (32). C. jejuni, on the other hand, thrives in the resource-rich environment of the animal intestinal tract and employs an oxidative TCA cycle to garner energy from organic compounds found in its ecological niche. In this evolutionary context, the adaptation of complex I to accept electrons from flavodoxin is a logical response to inheriting a TCA cycle that employs PFOR and OOR. A better understanding of this unusual respiratory pathway (at least two of the components of which are essential for C. jejuni viability) could prove vital in devising strategies to eliminate this important human pathogen from the food supply.

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FIG. 7. Genetic organization of the nuo operons from various Epsilonproteobacteria. Only the regions between nuoD and nuoH are shown; each bacterium also encodes nuoA to nuoC and nuoI to nuoN. References for each genome sequence are in parentheses: C. jejuni (27), H. pylori (34), Wolinella succinogenes (2), Caminibacter mediatlanticus TB-2 (C. Vetriani, S. Ferriera, J. Johnson, S. Kravitz, K. Beeson, G. Sutton, Y.-H. Rogers, R. Friedman, M. Frazier, and J. C. Venter, direct submission to EMBL/GenBank/DDBJ, 2007), Nitratiruptor strain SB155-2 and the two operons from Sulfurovum strain NBC37-1 (26), and Thiomicrospira denitrificans ATCC 33889 (A. Copeland, S. Lucas, A. Lapidus, K. Barry, J. C. Detter, T. Glavina, N. Hammon, S. Israni, S. Pitluck, P. Chain, S. Malfatti, M. Shin, L. Vergez, J. Schmutz, F. Larimer, M. Land, N. Kyrpides, A. Lykidis, and P. Richardson, unpublished data).

 
This work was supported by USDA-NRI grant 2004-04553.

We thank Debbie Threadgill and Jay Andrus for the kind gift of pJMA-001.

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

Published ahead of print on 7 December 2007.

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Journal of Bacteriology, February 2008, p. 915-925, Vol. 190, No. 3
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