1 Department of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield S10 2TN, UK
2 School of Biomedical and Molecular Sciences, University of Surrey, Guildford GU2 7XH, UK
Correspondence
Robert K. Poole
r.poole{at}sheffield.ac.uk
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ABSTRACT |
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INTRODUCTION |
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A distinctive feature of C. jejuni is that it is a microaerophile, i.e. oxygen is necessary for growth yet also toxic when at atmospheric concentration. The mechanisms behind microaerophily are not understood but there are many hypotheses (Krieg & Hoffman, 1986). These include an increased susceptibility of microaerophiles to reactive oxygen species (ROS) (Hoffman et al., 1979b
), an increased sensitivity to inhibition by O2 of certain respiratory enzymes (Hoffman et al., 1979a
), and excessive metabolic generation of ROS (Hoffman et al., 1979b
). In light of this, C. jejuni may benefit from the presence of an O2-detoxifying mechanism when exposed to atmospheric O2 conditions. However, in mammalian or avian hosts, C. jejuni is likely to be exposed to lower than optimal O2 concentrations. In such a situation, the presence of high-affinity O2 binding and reduction mechanisms would be useful, such as the cytochrome cbb3-type oxidase predicted by the genome sequence (Parkhill et al., 2000
). The obligately aerobic bacterium Vitreoscilla makes use of a single-domain Hb, Vgb, which is believed to facilitate the transfer of O2 to cytochrome bo' under low-O2 conditions (Park et al., 2002
).
C. jejuni is likely to encounter stresses other than those related to its microaerophilic lifestyle. Among these is nitrosative stress, which can be derived from both macrophages (Stevanin et al., 2002) and, more likely, chemical generation of NO in saliva and the stomach (Duncan et al., 1995
; Li et al., 1997
). It has also been documented that NO synthesis is increased in patients with infective gastroenteritis (Forte et al., 1999
). An established mechanism of protection from NO and related reactive nitrogen intermediates (RNIs) in other bacteria is via the participation of NO-detoxifying haemoglobins (Hbs) (Wu et al., 2003
). C. jejuni possesses two Hbs, representing two of the three classes now well documented in micro-organisms. The flavohaemoglobins (flavoHbs) possess a C-terminal ferredoxin-NADP+ reductase-like domain, whereas the single-domain Hbs are very similar to the N-terminal haem domain of the flavoHbs and to myoglobin. Truncated Hbs (trHbs) are 2040 residues smaller than the single-domain globins, and possess a considerably altered globin fold (Wu et al., 2003
). One of the C. jejuni Hbs, Cgb, is of the single-domain type and functions in the detoxification of NO (Elvers et al., 2004
). A cgb mutant of C. jejuni is hypersensitive to the nitrosating agent S-nitrosoglutathione (GSNO), to S-nitroso-N-acetylpenicillamine (SNAP), and to the NO releaser spermine NONOate. Cgb also protects the respiration of C. jejuni from inhibition by NO (Elvers et al., 2004
).
The second C. jejuni Hb is a trHb, i.e. a member of the most enigmatic group of microbial Hbs. trHbs are found in a number of other microbes (Wittenberg et al., 2002) but little is known concerning their functions. Mycobacterium tuberculosis and Mycobacterium bovis both possess two trHbs, HbO and HbN. HbO is believed to function in the facilitation of O2 transfer (Pathania et al., 2002
) and HbN is thought to detoxify NO (Ouellet et al., 2002
). The C. jejuni trHb (Cj0465c) is the subject of this paper. Attempts were made to elucidate its function, keeping in mind both the functions of other microbial globins and also the needs of C. jejuni as a microaerophilic organism. This study shows that the trHb does not appear to function in the detoxification of NO and is instead involved in the flux of O2 into and within the cell.
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METHODS |
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Construction of a ctb-complemented strain.
Complementation of the ctb mutation in RKP1386 was achieved by insertion of the wild-type gene into an isolated pseudogene, a technique which has been successful with other mutations (Elvers et al., 2005). PCR was used to amplify ctb and its promoter from C. jejuni NCTC 11168 genomic DNA using oligonucleotide primers HB3 (5'-GGATCCTTTAGCCTCTTCGCCTTCAT-3') and HB4 (5'-GGTACCGTGGAGATTTGCTCGATGGT-3') containing a BamHI and a KpnI restriction site respectively. The resulting fragment was digested with BamHI and KpnI and then ligated into the corresponding sites of pGEMCWH01 (C. Holmes & P. A. Lund, University of Birmingham, UK, unpublished results), forming plasmid pKE112. Plasmid pGEMCWH01 contains the pseudogene Cj0752 into which a multiple cloning site has been inserted. The chloramphenicol-resistance cassette from pAV103 (kindly donated by J. Ketley, University of Leicester) was amplified by PCR using oligonucleotide primers CAT3 (5'-GGTACCTGCTCGGCGGTGTTCCTTTCCAAG-3') and CAT4 (5'-GGTACCTGCGCCCTTTAGTTCCTAAAGGGT-3') with unique restriction sites for KpnI. This cassette was then inserted into the KpnI site of pKE112 to create construct pKE115. The suicide plasmid pKE115 therefore contains a wild-type copy of ctb and, downstream, a selectable chloramphenicol-resistance marker flanked by Cj0752. The plasmid pKE115 was confirmed using PCR and restriction digestion. This plasmid was introduced into competent cells of RKP1386 by electroporation and transformants were selected on MuellerHinton agar with chloramphenicol (10 µg ml1). The homologous recombination that resulted in a double crossover event was verified by PCR using HB3, HB4 and primers for Cj0752. The resulting strain was designated RKP5404.
Growth curves and viable counts.
For growth curves, C. jejuni was grown as above. Samples (1 ml) were removed at 2, 4, 7, 10, 12, 24 and 48 h and the apparent absorbance (OD600) measured on a Jenway 6100 spectrophotometer. Viable counts were performed using 96-well plates and multiwell pipettes, in triplicate, and each dilution series was plated three times using 10 µl drops. To measure survival in air, C. jejuni were grown for 14 h microaerobically and then transferred to an air incubator, where they were shaken at the same temperature and speed as before for 12 h. Samples (10 µl) were taken in triplicate every 90 min. C. jejuni cells were exposed to NOC-5 and -7, paraquat and H2O2 in microtitre plates. Cells were grown for 8 h before exposure. NOC compounds were used at 5, 20, 50 and 100 µM and the exposure time was 1 h. H2O2 was used at 0·01, 0·1, 0·2 and 0·4 mM, and paraquat at 1, 5, 10, and 100 µM; the exposure time for both was 30 min. Serial dilutions were performed immediately thereafter.
Disk diffusion assays.
The sensitivity of C. jejuni NCTC 11168, RKP1386 (ctb), RKP1336 (cgb) and RKP1389 (cgb ctb) to a range of stress-inducing agents, tert-butyl hydroperoxide (39 mM), paraquat (117 mM), cumene hydroperoxide (158 mM), hydrogen peroxide (265 mM) and S-nitrosoglutathione (100 mM) was assayed in a plate diffusion assay using the method described by Baillon et al. (1999).
Determination of culture oxygen transfer rate constants.
Baffled flasks (250 ml) were loaded with 100, 150 and 200 ml distilled H2O and deoxygenated in the microaerobic cabinet using a few grains of dithionite. The flasks were then shaken at 115 r.p.m. and their reoxygenation within the cabinet followed using a Hanna portable waterproof dissolved oxygen probe (model HI 9142). A plot was made of the percentage deoxygenation against time on a semilog scale, and the rate constant K was determined by dividing ln 2 (0·693) by the time at which the water was 50 % deoxygenated (Poole, 1976). The oxygen electrode was calibrated by placing in stirred water for the 100 % air saturation value; dithionite was added to determine the zero point.
Determination of Km and Vmax values of whole-cell respiration.
These measurements were made using the deoxygenation of oxyleghaemoglobin (oxyLHb) and oxymyoglobin (oxyMb) as sensitive reporters of oxygen concentration, according to the protocol and calculations detailed by Appleby & Bergerson (1980). LHb was kindly donated by Dr F. J. Bergersen, CSIRO Division of Plant Industry, Canberra, Australia. The LHb was treated with a fivefold excess of sodium dithionite and oxygenated by passage down a 50x5·0 cm column of Sephadex G25 (Amersham) in aerated 50 mM potassium phosphate pH 7·4, 1 mM EDTA. Horse skeletal muscle Mb (Sigma) was oxygenated similarly, using a PD-10 column (Amersham). The oxyLHb or oxyMb was diluted to a concentration of 1015 µM in a cuvette with 25 mM potassium phosphate, pH 7·0, plus 1 mM EDTA. The cuvette was sealed and additions of C. jejuni cells and formate as respiratory electron donor, 5 mM final concentration, were made. These studies were carried out on a custom-built SDB3.5 dual-wavelength scanning spectrophotometer (University of Pennsylvania School of Medicine Biomedical Instrumentation Group and Current Designs, Philadelphia, PA, USA) (Kalnenieks et al., 1998
) in time-scanning mode. The
A was measured between 576 and 565 nm for oxyLHb, and 582 and 565 nm for oxyMb. The data were transferred to Excel and calculations were performed according to Appleby & Bergersen (1980)
.
Cloning and expression of ctb in E. coli.
Genomic DNA was isolated from NCTC 11168 using guanidium thiocyanate (Pitcher et al., 1989). The forward (RP268, 5'-AAAATTAACATTTAACCATGGCTTATATGAAATTTGAAAC-3') and reverse (RP267, 5'-GAAAAGGTAAAAAAAGCTTTGGCAAAAAAATTG-3') primers to amplify ctb contained an NcoI or HindIII site respectively. The 0·38 kb fragment (corresponding to ctb) was recovered with a Qiaquick gel extraction kit (Qiagen), cloned between the NcoI and HindIII sites of pBAD/His (Invitrogen) to make pLW1 and transformed into E. coli TOP10 using the method of Inoue et al. (1990)
to make strain RKP4979. The construct pLW1 was checked by sequencing from just upstream of the promoter through to the end of the insert. For Ctb overexpression, starter cultures in LB were grown overnight. A 1 % inoculum was used in expression cultures supplemented with ampicillin, 200 µM
-aminolaevulinic acid and 12 µM FeCl3. The cultures were shaken at 200 r.p.m. until an OD600 of
0·5 had been reached; they were then induced with 0·02 % arabinose and grown for a further 4 h. Concentrations of
-aminolaevulinic acid, FeCl3 and arabinose were selected after preliminary optimization studies.
Ctb purification and expression studies.
Polyclonal antiserum against Ctb was raised by immunizing rabbits with pure Ctb. Cells from 4 litres of culture were harvested by centrifugation at 5000 g for 10 min at 4 °C and resuspended in 80 ml 50 mM Tris/HCl (pH 7·0). Cells were disrupted by sonication and centrifuged at 21 000 g for 15 min at 4 °C to remove cell debris. The reddish-brown supernatant was loaded onto a 30 ml DEAE Sepharose Fast Flow (Pharmacia Biotech) column equilibrated with 50 mM Tris/HCl (pH 7·0). The column was washed with 40 ml of the same buffer and Ctb eluted with a NaCl gradient (from 0 to 0·5 M) in 50 mM Tris/HCl (pH 7·0). Fractions to be carried forward for the next step were chosen on the basis of coincidence of the haem and protein peaks in the UV absorption profile. The eluate was concentrated to 1·4 ml using a Vivaspin 20 concentrator (Vivascience) with a molecular mass cut-off of 5 kDa. This fraction was further purified by gel filtration. A Superdex-200 column (16x60 cm, Amersham Biosciences) was equilibrated with 50 mM Tris/HCl (pH 7·0), 0·2 M NaCl. A 1 ml portion of the previous fraction was applied and eluted in the equilibration buffer at a flow rate of 1 ml min1. The haem and protein elution profiles were monitored at 412 and 280 nm respectively. Pure Ctb was stored at 4 °C.
Production of antibody was performed by S. Smith in The University of Sheffield Antibody Resource Centre. Protein (250500 µg) in a maximum volume of 250 µl PBS (137 mM NaCl, 2·7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4) was mixed with an equal volume of Freund's adjuvant. This was passed through a 23G needle until an emulsion formed. Two rabbits were each injected subcutaneously with a maximum of 500 µl. After 22 days, the injections were repeated but with incomplete Freund's adjuvant; this was performed again another 20 days later. Sample bleeds took place after a further 10 days, followed by Western blotting to check the antisera. After a final booster injection, the rabbits were bled out 10 days later. The antibody was aliquoted and stored at 70 °C; the aliquot in use was stored at 4 °C.
For Western blotting, crude extracts were resolved on SDS-PAGE using 520 % Tris/HCl Ready Gels (Bio-Rad). The Bio-Rad protein assay was used with BSA standards to ensure that 20 µg protein was loaded per lane. Proteins were transferred onto a nitrocellulose membrane in a mini-transblot apparatus (Bio-Rad) at 75 mA overnight. Immobilized proteins were probed with primary anti-Ctb antibody and secondary antibody (horseradish-peroxidase-conjugated anti-rabbit IgG, Sigma) and developed using ECL detection reagents (Amersham).
Polarographic measurements of O2 consumption.
A digital Clark-type electrode system (model 10: Rank Brothers) was used. C. jejuni cells were grown to early exponential phase and resuspended in 50 mM potassium phosphate (pH 7·3). Supernatant subcellular fractions were prepared by sonicating whole cells and centrifuging out cell debris at 21 000 g for 15 min. Membranes were spun out at 142 000 g for 1 h. The electrode chamber was stirred and used at a working volume of 1·5 ml at 42 °C. The electrode was calibrated with air-saturated 50 mM potassium phosphate (pH 7·3), assuming an oxygen concentration of 220 µM. A few grains of dithionite were used to zero the electrode. The same buffer was used to perform experiments. Intact cells or supernatant subcellular fractions were added in sufficient amount to give measurable O2 uptake rates. Formate (5 mM) was used as a reductant for cells and NADH (0·5 mM) for supernatant fractions (all expressed as final concentrations).
RNA extraction.
RNA was extracted from C. jejuni NCTC 11168 and RKP1386 (ctb mutant) using RNeasy and RNAprotect purification and stabilization kits (Qiagen). Both strains were grown in 50 ml MuellerHinton medium microaerobically at 37 °C to an OD600 of 0·2 and then 5 ml of each strain was directly pipetted into 10 ml RNAprotect (Qiagen). The cells were pelleted by centrifugation and lysed with TE buffer (100 µl) containing lysozyme (1 mg ml1; Sigma). The RNA was extracted and contaminating DNA was removed for 1 µg total RNA by treating with a DNA-free kit (Ambion) according to the manufacturer's instructions.
Reverse transcriptase PCR.
First-strand cDNA synthesis was performed using 100 ng total RNA with random hexamers (250 ng, Invitrogen) and Superscript III RT (Invitrogen). PCR was carried out under the conditions described previously (Elvers et al., 2004) with the following oligonucleotides: to amplify recG (Cj0464), forward primer 5'-GTGCGGCTTTGATGGTTTAT-3' and reverse primer 5'-TCTTGGAATAGGCGTAGCTGA-3' were used, and for Cj0466, forward primer 5'-ATGAAGGCGAAGAGGCTAAA-3' and reverse primer 5'-TCGAGATAAAGATTGCGCTCTA-3' were used. DNA gyrase subunit A gene (Cj1027c) was used as a housekeeping control gene with forward primer 5'-AGGCACACGCTTAATATAACCA-3' and reverse primer gyrA2 5'-GGTGTGATTATGTTGGCAATTCAT-3'. The PCR products were analysed by gel electrophoresis on 1·5 % agarose.
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RESULTS |
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To elucidate the role of Ctb, a deficient mutant, RKP1386, was constructed as described in Methods, with the antibiotic-resistance marker orientated with the same transcriptional polarity as ctb. To eliminate the possibility that the phenotype of the ctb mutant would be masked by Cgb, the second globin, a mutant deficient in both C. jejuni globins, RKP1389, was also constructed (see Methods). The kanamycin-resistance cassette used in the construction of the ctb mutant contains its own promoter but lacks a transcriptional terminator and, when the resistance cassette is inserted with the same transcriptional polarity as the mutated gene, mutations have been shown to be non-polar on downstream genes (Hickey et al., 2000). Furthermore, the insertion of the kanamycin-resistance cassette did not affect expression of the genes immediately proximal to ctb as determined by reverse transcriptase PCR (data not shown) and polar effects are minimal. To test unequivocally that the ctb gene is directly linked to the observed phenotypes, we also constructed a strain (RKP5404) in which the ctb mutation was complemented by transforming RKP1386 with a plasmid containing ctb under the control of its own promoter that then recombined onto the C. jejuni chromosome at a second location.
Growth of C. jejuni at various aeration rates
The presence of the single-domain globin Vgb in microaerobically growing cells offers a growth advantage to E. coli (Khosla & Bailey, 1988) and the implication is that Vgb functions to this end in Vitreoscilla. For this reason, growth curves were performed on C. jejuni grown at different aeration rates in order to determine if the presence of Ctb conferred a positive effect under conditions of either low or high oxygen provision. The oxygen transfer rate to cultures was varied by using 100, 150 or 200 ml medium in 250 ml baffled flasks since, in a rotary shaker, the O2 solution rate falls rapidly as the liquid volume is increased (Pirt, 1975
). Values of K (the O2 transfer constant) were calculated for each volume in flasks shaken at 115 r.p.m. An O2 transfer constant of 0·06 min1 was found for 200 ml H2O in a 250 ml flask, 0·16 min1 for 150 ml and 0·43 min1 for 100 ml. Thus, the volume of medium determines the rate of oxygen transfer into the culture.
For each strain, the growth curves were performed in triplicate on at least two separate occasions. Wild-type C. jejuni cells (Fig. 1a) grown in 100 ml medium reached a significantly higher OD600 (1·1) than the same strain grown in a higher volume of medium (data not shown). However, when inoculated into 100 ml medium, the ctb mutant grew significantly less well than the wild-type, achieving only 0·7 OD units (P=6·77x105, assuming an equal variance (two tailed, Fig. 1a
). There was no difference in growth rate of the two strains for the majority of the exponential phase (110 h), but the mutant slowed in growth as it approached stationary phase. No significant difference was observed between the ctb mutant and wild-type cells when grown at lower oxygen transfer rates (i.e. in 150 and 200 ml MuellerHinton broth) but yields attained by both strains decreased as K decreased (data not shown). The cgb mutant and the wild-type strains grew similarly in all three volumes of medium (Fig. 1b
for 100 ml MuellerHinton; data not shown for 150 and 200 ml). The strain mutated in both globins consistently reached the same final OD600 as the wild-type (Fig. 1b
for 100 ml MuellerHinton; data not shown for 150 and 200 ml). The complemented ctb mutant strain did not display the attenuated growth of the mutant strain when grown in 100 ml medium (Fig. 1a
). Hence complementation with the ctb gene reversed the effect of the mutation.
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Determination of Vmax and Km of C. jejuni whole cells for O2
Since we had obtained evidence for a role for Ctb in promoting aerobic growth and oxygen consumption, we examined the oxygen consumption of cells at low oxygen tensions to determine whether Ctb affected the Km or Vmax of the cell for O2. In the past this technique has been used to determine the Km values of several high-affinity oxidases for O2 (Contreras et al., 1999; D'Mello et al., 1994
, 1995
, 1996
; Smith et al., 1990
). The technique relies on the spectrophotometric differences between an oxygenated (exogenous) reporter Hb and its deoxy form. Oxygenated globin (LHb or Mb) was placed with buffer in a cuvette to which further O2 entry was prevented. Additions of reductant and cells were made: initially cells respired using oxygen present in the buffer; when this had been exhausted they started to deoxygenate the Hb. This deoxygenation was followed spectrally; a typical kinetic trace showing oxygen consumption by the cells is represented in Fig. 3
. Both oxyLHb and oxyMb were used. LHb has the higher oxygen affinity and is appropriate for determining respiration by the high-affinity (R. Jackson & R. K. Poole, unpublished) cytochrome cbb3-type oxidase. Use of oxyMb is suited to observing respiration by the low-affinity oxidase. The experiments were carried out using wild-type cells, and the ctb, cgb and cgb ctb mutants. Only data that were within the working range of the two globins were examined (D'Mello et al., 1994
). Fig. 4
displays, as examples, plots of total oxygen (that bound to LHb plus that present in solution in the cuvette) against time and EadieHofstee plots for the ctb and cgb mutants using oxyLHb as a source of O2. Both EadieHofstee and LineweaverBurk plots were obtained for all strains. However, EadieHofstee plots were taken to be the more reliable linear transformation as the LineweaverBurk plot has a tendency to give overly large estimates of Vmax and Km when applied to unweighted data (Dowd & Riggs, 1965
). The t test, assuming an equal variance (two tailed), was used to analyse the significance of the data. No significant difference in Km values was seen between the wild-type and the globin mutants (Table 2
) using either oxyLHb or oxyMb as reporters. However, the Vmax of the ctb mutant was 2·5-fold greater than the wild-type when assayed using oxyLHb (P=0·0027). The difference was not so pronounced with oxyMb, the ctb mutant value being 1·6-fold greater than the wild-type (P=0·00015). The cgb and double mutant values were not significantly different from the wild-type. All scans were repeated at least four times for each combination of strain and oxygenated globin.
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DISCUSSION |
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The ctb mutant was not markedly sensitive to the majority of the agents of oxidative stress tested, showing only marginal sensitivity to paraquat. This diminishes the likelihood that the attenuated growth and oxygen consumption of this strain under conditions of high oxygen are due to a heightened sensitivity to oxidative stress. Interestingly, although the ctb mutant survived in air considerably less well than wild-type cells, both the cgb mutant and the double mutant mimicked the drop in its viability. The latter two mutants did not, however, display the reduced growth and oxygen consumption of the ctb mutant. A mutant of E. coli defective in the flavoHb Hmp has increased sensitivity to paraquat (Membrillo-Hernández et al., 1999) but the basis of this effect is not understood. No such sensitivity has been found for other microbes mutated in trHbs or indeed any other microbial globins (Wu et al., 2003
).
The data derived from the deoxygenation of oxygenated globins also signal an O2-related role for Ctb. These measurements of O2 uptake are different from those obtained in the O2 electrode as, in this experiment, C. jejuni is consuming O2 at very low buffered O2 tensions. The absence of Ctb, or indeed Cgb, did not affect the affinity of either oxidase for O2. The values for Km remained close to 40 nM and 0·9 µM, which have recently been assigned to the cco- and cyd-encoded oxidases, respectively (R. Jackson & R. K. Poole, unpublished). However, the Vmax of cells for O2 was markedly altered by the absence of Ctb. The deoxygenation of oxyLHb by the ctb mutant gave a Vmax value 2·5 times greater than that given by wild-type cells. When oxyMb was used as a source of O2, the Vmax displayed by cells deficient in Ctb was 1·6-fold greater than that displayed by wild-type cells. Fig. 5 shows that the combined use of oxyLHb, oxyMb and the Clark-type oxygen electrode provides an observable range of free O2 covering more than four orders of magnitude. Bergersen & Turner (1979)
have suggested that, at high levels of oxygenation of LHb, respiration rates (of Rhizobium spp.) are usually diminished, perhaps because there is an insufficient concentration gradient of oxyLHb for facilitated diffusion of oxygen to permit maximal respiration rates. Indeed, in Fig. 5
, the data points obtained for both strains with oxyLHb at free O2 concentration above 0·1 µM are lower than those obtained with oxyMb; the brackets (Fig. 5
) indicate the preferred ranges of O2 concentration for each globin. These ranges may be combined with the ability of the O2 electrode to measure O2 between air saturation (approx. 200 µM) and (at best) the oxygen concentrations appropriate for oxidases with a Km of approximately 1 µM (Lundsgaard et al., 1978
). The combined data (Fig. 5
) show that the presence of Ctb reduces oxygen consumption below about 1 µM but increases it between 1 µM and air saturation levels. Thus the physiological function of Ctb in vivo may be to moderate oxygen consumption. The optimal oxygen concentration for growth of campylobacters in the laboratory lies in the range 315 % (by volume in the gas phase) but to our knowledge the optimal oxygen concentrations in solution under physiological conditions (i.e. in natural environments avian and mammalian hosts and foods) have not been defined. However, at 25 °C and assuming a Henry's law constant of 1·2 M atm1 (see http://www.henrys-law.org) the oxygen solubilities in water equate to about 40200 µM (315 % in the gas phase). These are close to the electrode-determined oxygen concentrations in Fig. 5
, suggesting that, at high external concentrations, Ctb may function to increase oxygen consumption and decrease oxygen concentrations. This function might be regarded as analogous to respiratory protection in Azotobacter vinelandii, in which the oxygen-labile nitrogenase is protected by rapid O2 consumption catalysed by cytochrome bd (Kelly et al., 1990
). Conversely, at low external oxygen concentrations, Ctb may serve to spare oxygen consumption in this obligately microaerobic bacterium.
Our results relate only to external oxygen concentrations, and the intracellular oxygen levels in campylobacters (or any other bacteria) are not known precisely. However, Unden et al. (1995) have predicted, on the basis of oxygen diffusion constants and bacterial cellular dimensions that, under both aerobic (about 220 µM) and microaerobic (20·2 µM) conditions, the bacterial intracellular oxygen tension approximates to extracellular levels. Fig. 8
outlines a hypothetical situation in vivo. In the wild-type cell (Fig. 8a
), Ctb is proposed to act as a chaperone for oxygen, delivering it to a/both terminal oxidase(s) in a manner similar to that proposed for Vgb expressed in E. coli (Park et al., 2002
) or LHb-facilitated O2 flux in soybean nodule cells, where the diffusive flux of free O2 through the cytoplasm is augmented by a flux of oxyLHb (Bergerson, 1996
). In a ctb mutant cell (Fig. 8b
), oxygen accesses the oxidase without globin-mediated facilitation; however, when O2 is bound by neither the oxidase nor by Ctb, some oxygen may be utilized by Cgb (or a diaphorase) and reduced, perhaps to superoxide. Indeed, in the absence of nitric oxide, Hmp, the E. coli flavoHb, will convert oxygen to superoxide (Membrillo-Hernández et al., 1996
; Wu et al., 2004
). Because of the strong homology between Cgb and the haem domain of Hmp (33 % identity; Elvers et al., 2004
) it is possible that Cgb could function in the same way in the absence of NO. A partner reductase (at present not identified) would be necessary to transfer electrons to the Cgb haem for O2 reduction. There seems no reason to suppose that the respiratory flux of electrons would be different in a globin mutant; hence it is possible that the value of Vmax may be higher in ctb mutant cells because fewer electrons are needed to reduce oxygen to superoxide (equation 1) than are needed to reduce it to H2O (equation 2).
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Thus, in ctb mutant cells, more oxygen can potentially be consumed per electron than in wild-type cells, since the O2-buffering/chaperone role of Ctb is lacking.
This hypothesis can be extended to the cgb mutant, which would lack the superoxide-yielding reaction envisaged in Fig. 8(b). The data shown in Table 2
indicate that the cgb mutant is not significantly different from wild-type in the measured Vmax value. However, the double (cgb ctb) mutant did not display the markedly elevated Vmax of the ctb mutant (Table 2
). We suggest that this may be the result of the lack of a Cgb-catalysed incomplete O2 reduction and an adequate O2 flux to the oxidase in the absence of the O2-binding Ctb. Fumarate reductase (Sellars et al., 2002
) may also reduce oxygen to superoxide, as documented in E. coli (Imlay, 1995
). However, the rates of oxygen binding to, and dissociation from, the two globins have not been measured. Inspection of Fig. 5
shows that this effect of mutation in ctb is evident only at extracellular O2 concentrations below about 1 µM. Even if the intracellular concentration of oxygen equates to that outside (see above), it seems plausible that intracellular Ctb concentration might be sufficient for such a role since immunoblotting (Fig. 7
) shows significant levels of synthesis under all conditions tested. That bacterial globins can significantly facilitate oxygen transport to an oxidase has been suggested by the modelling by Khosla & Bailey (1989)
of the effects of periplasmic Vgb when expressed in E. coli.
A different explanation must be proposed for the effects of ctb mutation at much higher extracellular concentrations (Fig. 5). Here, we argue that Ctb may play an important oxygen-protective physiological role by increasing oxygen uptake rates (see above), perhaps by facilitated diffusion or direct interaction of the oxygen-bound Ctb with a terminal oxidase. Such a mechanism has recently been demonstrated for the interaction of Vgb with subunit I (where oxygen is reduced at the haem-Cu binuclear centre) of the cytochrome bo'-type oxidase in Vitreoscilla (Park et al., 2002
). Such interaction is proposed to result in enhancement of microaerobic growth. Within the trHb family, only HbO of M. tuberculosis has been previously shown to facilitate oxygen transfer. In E. coli membrane vesicles, HbO was able to enhance oxygen consumption activity approximately twofold and Liu et al. (2004)
demonstrated a dynamic and weak interaction between E. coli cytochrome bo subunit I and HbO.
Ctb is present in C. jejuni at a readily detectable basal level. Although exposure to RNIs resulted in upregulation of ctb (Fig. 7), the ctb mutant was not hypersensitive to NOC compounds or GSNO. This is in contrast to the results obtained for the cgb mutant, which displayed increased sensitivity to all the agents of nitrosative stress tested. However, while the double globin mutant was sensitive to GSNO, it was not hypersensitive to the NOC compounds (Fig. 6
). This is most likely due to the fact that exposure to GSNO was over a period of 24 h, considerably longer than the 1 h to which cells were exposed to NOC compounds. Furthermore, NO-donating compounds (NOCs) and nitrosating agents (GSNO) have different chemistries and hence differing targets and effects within the cell (Cooper, 1999
; Li et al., 2001
; Nikov et al., 2003
). The hypothesis outlined in Fig. 8
is also relevant to conditions of nitrosative stress. If detoxification of NO by Cgb requires O2, its availability may limit oxidase-catalysed respiration. Under such conditions, the facilitation of O2 transfer to the oxidases by Ctb would be beneficial and would be promoted by the increased expression of the trHb. Thus, since the functions of the two C. jejuni globins appear to be intimately related they might be expected to be co-regulated. This is indeed the case, since both cgb and ctb have recently been shown to be positively regulated by NssR, a member of the Crp-Fnr superfamily, in response to nitrosative stress (Elvers et al., 2005
).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 14 June 2005;
revised 17 August 2005;
accepted 22 August 2005.
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