Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK
Correspondence
David J. Kelly
d.kelly{at}sheffield.ac.uk
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ABSTRACT |
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INTRODUCTION |
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C. jejuni is an obligate microaerophile which appears to have a surprisingly complex and highly branched respiratory chain for a relatively small-genome pathogen, allowing the use of a wide variety of electron donors such as formate, hydrogen, D-lactate, succinate, malate and NAD(P)H, and alternative electron acceptors to oxygen, including fumarate, nitrate, nitrite, N- or S- oxides and hydrogen peroxide (Kelly, 2001; Sellars et al., 2002
; Myers & Kelly, 2005
). The addition of sulphite to intact cells of C. jejuni was found by Hoffman & Goodman (1982)
to produce a proton pulse (H+/O ratio=1·58), and sulphite was thus proposed to serve as a potential electron donor. However, the nature of the sulphite oxidation system was not elucidated and no further studies have been carried out. The potential presence of a sulphite respiration system in C. jejuni may be of importance in survival of this pathogenic organism in microaerobic aquatic niches, rich in sulphite, and in some foods, as sulphite is commonly added to processed foods as a preservative. Interestingly, sulphite is also released in the human body by neutrophils in response to stimulation with lipopolysaccharide as part of the host defence, as sulphite is a well-known antimicrobial (Mitsuhashi et al., 1998
).
Sulphite oxidation can support chemolithotrophic and phototrophic growth in a diverse range of bacteria and archaea (Wood, 1988; Sorokin, 1995
; Friedrich, 1998
) and is known to occur either by direct oxidation, usually utilizing a molybdenum-containing sulphite : cytochrome c oxidoreductase (SOR) or by indirect, AMP-dependent oxidation via the intermediate adenylylsulphate (Wood, 1988
; Kappler & Dahl, 2001
). In this study, we demonstrate the ability of the chemoheterotrophic pathogen C. jejuni to utilize sulphite and metabisulphite as respiratory electron donors, identify the proteins constituting the sulphite oxidase and elucidate the pathway for electron transport from sulphite to oxygen.
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METHODS |
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Insertional inactivation of Cj0004c.
PCR primers (forward primer 5'-TGAAGGTATAGGAATGAATG-3', reverse primer 5'-GGAGTTCCTATTTCTAAAGC-3') were designed to amplify a 1·6 kb product containing the Cj0004c gene, which was cloned into pGEM T-easy (Promega) and insertionally inactivated at a unique EcoNI site using the aphAIII (kanamycin resistance) gene containing its own promoter, derived from pJMK30 (van Vliet et al., 1998), producing plasmid pJDM4. As the downstream gene (gyrB) is transcribed in the opposite orientation to Cj0004c, there is no polarity effect of this insertion. Plasmid pJDM4 was then electroporated into C. jejuni 11168, with selection on blood agar plates containing 30 µg kanamycin ml1. The above primers were used in a PCR using genomic DNA from the wild-type 11168 strain or from a kanamycin-resistant transformant, in order to verify the correct mutant construction.
Measurement of respiration rates by oxygen uptake.
Substrate oxidation was determined as described previously (Hughes et al., 1998), by measuring the change in dissolved oxygen concentration of cell suspensions in a Clark-type oxygen electrode linked to a chart recorder and calibrated using air-saturated 25 mM phosphate buffer (pH 7·5) (220 nmol dissolved O2 ml1 at 37 °C). A zero-oxygen baseline was determined by the addition of sodium dithionite. The cell suspension was maintained at 37 °C and stirred at a constant rate. Substrates and inhibitors were added by injection through a fine central pore in the airtight plug. Rates were expressed in nmol O2 utilized min1 (mg cell protein)1.
Analysis of cytochrome spectra, and cytochrome c quantification.
Cytochrome spectra were obtained at room temperature using a Shimadzu UV-2101PC double-beam scanning spectrophotometer. Spectra were scanned from 400 nm to 700 nm. Reduced minus oxidized spectra were obtained by adding a few grains of sodium dithionite or ammonium persulphate as reductant or oxidant, respectively (Jones & Poole, 1985). Reduction of cytochromes by physiological substrates was performed in a sealed cuvette with addition of substrate by injection through a seal in the lid. These spectra were scanned against an ammonium-persulphate-oxidized baseline. The amount of cytochrome c in cell fractions was quantified by the increase in absorbance at 550 nm after reduction of the sample by excess sodium dithionite, using an absorption coefficient of 20 mM1 cm1.
Preparation of periplasmic proteins from C. jejuni.
Cells were grown in the microaerobic cabinet at 37 °C in 200 ml BHI-FCS broth overnight. The cell suspension was centrifuged (15 000 g, 20 min at 4 °C) and the resulting pellet was resuspended in 10 ml 20 % (w/v) sucrose, 30 mM Tris/HCl pH 8 at room temperature. EDTA was added to a final concentration of 1 mM and the suspension was poured into a 100 ml conical flask and stirred at 180 r.p.m. in a 25 °C constant-temperature room for 10 min. The suspension was then centrifuged (10 000 g, 10 min at 4 °C) and the pellet was resuspended in ice-cold 10 mM Tris/HCl pH 8 to a volume of 10 ml and stirred at 180 r.p.m. in a 4 °C constant-temperature room for 10 min. The suspension was then centrifuged again (18 000 g, 15 min at 4 °C) and the supernatant collected as the periplasmic fraction. The pellet was also used for further fractionation of cytoplasm and membrane as described below.
Preparation of C. jejuni cell-free extract.
A 200 ml C. jejuni culture in BHI-FCS was grown for 16 h at 37 °C with shaking in a microarobic atmosphere. The cells were harvested by centrifugation (10 000 g, 15 min, 4 °C) and resuspended in 5 ml 10 mM Tris/HCl pH 8. The cell suspension was sonicated (20 kHz, 6 µm amplitude) and centrifuged (20 000 g, 20 min, 4 °C) to remove cell debris.
Localization of SOR activity.
A 1-litre culture of C. jejuni was grown overnight in BHI-FCS and harvested by centrifugation (10 000 g, 15 min, 4 °C). The whole cell pellet was resuspended in 60 ml 20 % (w/v) sucrose, 30 mM Tris/HCl pH 8 and subjected to the osmotic-shock procedure described above, but with a resuspension volume of 60 ml. After decanting the periplasmic fraction, the pellet was resuspended in 5 ml 10 mM Tris/HCl pH 8 and sonicated (20 kHz, 6 µm amplitude) to release the cell contents. This suspension was subjected to low-speed centrifugation (20 000 g, 20 min, 4 °C) to remove cell wall debris and then the supernatant was ultracentrifuged (100 000 g, 80 min, 4 °C) to separate membrane and cytoplasmic fractions. The membrane pellet was resuspended in 3 ml 10 mM Tris/HCl pH 8.
Enzyme assays.
SOR activity was assayed by a method based on that described by Kappler et al. (2000), using a Shimadzu UV-2101PC double-beam scanning spectrophotometer at a wavelength of 550 nm. Rates were obtained by measuring the increase in absorbance of cytochrome c after adding 0·5 mM sodium sulphite to a cuvette containing the following: 900 µl 10 mM Tris/HCl pH 8, 100 µl 10 mg horse heart cytochrome c ml1, 10100 µl cell fraction. An absorption coefficient of 20 mM1 cm1 at 550 nm was used.
Isocitrate dehydrogenase activity was assayed according to Leyland & Kelly (1991) in a Shimadzu UV-2101PC spectrophotometer at a wavelength of 340 nm. Rates were obtained by adding 100 µl 50 mM sodium isocitrate to a cuvette containing the following: 100 µl 50 mM Tris/HCl pH 8; 100 µl 10 mM NADP; 100 µl 10 mM MgCl2; 500590 µl distilled H2O; 10100 µl cell fraction. The specific activity of isocitrate dehydrogenase was calculated using an absorption coefficient of NADPH of 6·22 mM1 cm1 at 340 nm.
Partial purification of SOR activity.
Ion-exchange chromatography was utilized for partial purification of the periplasm. A column packed with 30 ml DEAE-Sepharose (Pharmacia) was connected to a Bio-Rad Biologic HP system. A 50 ml sample of periplasm was loaded onto the anion-exchange column and equilibrated with 50 mM Tris/HCl pH 8·0. Proteins were eluted from the column by a linear gradient of 0 to 0·5 M NaCl. Samples of the protein fractions obtained were assayed for SOR activity using the horse heart cytochrome c assay described above. The SOR-containing fractions were electrophoresed using SDS-PAGE and stained for protein with Coomassie blue or blotted onto PVDF membrane for Western blot analysis as described below.
SDS-PAGE and Western blotting.
SDS-PAGE was carried out according to the method of Laemmli (1970) and gels were stained for protein using Coomassie brilliant blue R. For Western blots, periplasmic extracts were prepared as described above and denatured by heating (100 °C, 5 min) in the presence of SDS sample buffer. The protein was loaded and separated by SDS-PAGE (12 %, w/v, acrylamide), then electroblotted onto a PVDF membrane (Bio-Rad) in a Bio-Rad mini PROTEAN II cell at 11 mA for 16 h. Immunodetection was performed using an ECL detection kit (Amersham Biosciences) according to the instructions of the manufacturer. The primary antiserum for detection of Cj0005 was kindly donated by Christiane Dahl (Institut fur Mikrobiologie und Biotechnologie, Bonn, Germany). The antiserum was prepared in rabbit and was raised against SorA of Starkeya novella, as described by Kappler et al. (2000)
. The working concentration for this serum was a 1 : 10 000 dilution. An anti-rabbit-horseradish peroxidase conjugate (Amersham Biosciences) was used as a secondary antibody (1 : 2000 dilution).
Sequence alignment and phylogenetic analysis.
Multiple protein sequence alignments and phylogenetic analyses were performed using CLUSTAL X (Thompson et al., 1997) with output generated by ESPript (Gouet et al., 1999
). Phyologenetic trees were viewed in TreeView (Page, 1996
).
Determination of protein concentration.
This was done by the Lowry method.
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RESULTS |
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DISCUSSION |
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Sulphite is an extremely widespread inorganic anion in many environments, particularly low-oxygen niches in soil or water, where it is more stable than in aerobic conditions, and is thus available for microbial oxidation or reduction by a variety of pathways (Wood, 1988). The possession of a sulphite respiration system may thus contribute to the survival of C. jejuni in such environments, particularly as this bacterium has a very high-affinity cb-type cytochrome c oxidase which could allow sulphite respiration at extremely low oxygen concentrations. However, for many chemoheterotrophic bacteria sulphite, and particularly metabisulphite (which in aqueous solutions is rapidly converted to the bisulphite anion,
), are well-known growth-inhibitory compounds. Nevertheless, Bolton et al. (1984)
showed enhanced growth and aerotolerance of C. jejuni by addition of a mixture of ferrous sulphate, sodium metabisulphite and sodium pyruvate (FBP) as a growth supplement in laboratory media. FBP is thought to function as a system for reducing oxidative stress by removing free radicals from the medium. Our demonstration that C. jejuni can respire sulphite, and metabisulphite provides an explanation for their relative lack of toxicity for this bacterium and suggests a growth-enhancing role for sulphite/bisulphite as an electron donor. Interestingly, growth of the closely related gastric pathogen Helicobacter pylori was found to be inhibited by the addition of sulphite or metabisulphite to the media (Jiang & Doyle, 2000
) but FP growth supplement, which did not contain metabisulphite, did enhance the growth of H. pylori. Unlike C. jejuni, the genome of H. pylori does not encode a homologue of the SOR system. Thus, sulphite detoxification could be an additional function of this type of sulphite respiration system. This might be important in vivo, where, for example, there is evidence that sulphite can be produced by neutrophils in response to stimulation by lipopolysaccharide (Mitsuhashi et al., 1998
).
Analysis of the Cj0004c/Cj0005c operon has demonstrated that C. jejuni encodes a SOR enzyme for sulphite oxidation, which is very similar to that of St. novella. The latter has been studied in detail and shown to be heterodimeric, and clearly distinct from the eukaryotic SOR enzymes, which are homodimeric proteins containing haem b and with a molybdopterin cofactor residing in each subunit (Kappler et al., 2000). The MPT cofactor is unusual for a prokaryote, with the majority of known bacterial molybdoenzymes containing the dinucleotide form of the pterin cofactor (Hille, 1996
; Kisker et al., 1997
, 1999
). The St. novella SOR Km value for sulphite was 27 µM and a periplasmically located cytochrome c550 has been identified as the natural electron acceptor, with a low Km value of 2·5 µM (Kappler et al., 2000
). While the corresponding SorA molybdoproteins of C. jejuni and St. novella are clearly homologous, the SorB monohaem cytochrome subunits of the two bacteria are not closely related, and it seems that SorA homologues in other bacteria may also be associated with distinct cytochrome subunits serving the same function. Although we were unable to mutate Cj0005c, several lines of evidence showed that the cytochrome encoded by Cj0004c was essential for sulphite respiration. The Cj0004c mutant possessed no detectable rate of oxygen respiration after the addition of sulphite or metabisulphite as an electron donor. Additionally, no cytochrome c reduction was detected upon addition of sulphite to periplasmic extracts. The mutation did not affect the expression of the large molybdopterin-containing subunit, Cj0005, which is thought to contain the active site responsible for the oxidation of sulphite. A more complete analysis involving attempts to remutagenize Cj0005c and complement both genes should be carried out, although genetic tools for C. jejuni are still somewhat limited.
SOR activity is located mainly in the periplasm of C. jejuni. Cell fractionation of C. jejuni is not as reliable as with model bacteria like Escherichia coli, and the smaller percentages of activity detected in other cell compartments are probably due to an unavoidable lysis of some cells during the osmotic shock procedure and the enzyme non-specifically binding to the membrane fraction. A periplasmic location would be in agreement with the predicted topology of the subunits and the signal sequences contained on the N-terminus of each subunit. The TAT system has been shown to export folded or partially folded proteins with the cofactors previously inserted in the cytoplasm (Sargent et al., 1998; Berks et al., 2003
). The smaller cytochrome c subunit would be exported via the Sec pathway as an unfolded polypeptide with the haem group inserted in the periplasm. SOR activity was linked to the Cj0005 protein by the partial purification of the enzyme using anion-exchange chromotography. The fractions containing the highest SOR activity correlated with the presence of Cj0005, which was detected using the St. novella SorA antibody. However, the correlation was not exact, with the fraction of highest activity not being the same as the fraction with the highest concentration of Cj0005. A possible explanation would be that a functioning Cj0004 protein must be present for the detection of the SOR activity using the horse heart cytochrome c reduction method. The Cj0004 protein could be eluted at a different time to the Cj0005 protein and would result in a fraction which has the highest concentration of Cj0005 but does not have the full SOR activity. Unfortunately, the Cj0004 protein could not be positively identified on haem stains of column fractions.
This study provides more evidence for the highly diverse and branched structure of the respiratory chains of this pathogen, and it is now clear that many of the alternative electron transport pathways employ periplasmic molybdoenzyme dehydrogenases or reductases. These include formate dehydrogenase, nitrate reductase and DMSO/TMAO reductase, in addition to the sulphite oxidase (Kelly 2001; Myers & Kelly, 2005
; Sellars et al., 2002
). The one remaining uncharacterized periplasmic molybdoenzyme encoded by Cj0379 is related to YedY in E. coli (Loschi et al., 2004
) and is distantly related to Cj0005, but is not a sulphite oxidase (J.D. Myers & D. J. Kelly, unpublished). Fig. 7(a)
shows how the SOR system connects with these other electron-transport chains in C. jejuni, and Fig. 7(b)
shows how electrons from sulphite are transferred to oxygen via the periplasmic SOR and the membrane bound cb-type cytochrome c oxidase. It is clear that the ability to use such a wide range of electron donors and electron acceptors allows considerable respiratory chain flexibility, particularly under conditions of severe oxygen limitation, and this could be a key factor in the organism being able to survive and grow in a number of different niches, possibly including human and animal hosts.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 19 August 2004;
revised 7 October 2004;
accepted 11 September 2004.
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