Cloning, expression, and characterization of a Coxiella burnetii Cu/Zn Superoxide dismutase
© Brennan et al.; licensee BioMed Central. 2015
Received: 11 December 2014
Accepted: 23 April 2015
Published: 12 May 2015
Periplasmically localized copper-zinc co-factored superoxide dismutase (SodC) enzymes have been identified in a wide range of Gram-negative bacteria and are proposed to protect bacteria from exogenously produced toxic oxygen radicals, which indicates the potential significance of a Coxiella burnetii SodC.
Assays for SOD activity demonstrated that the cloned C. burnetii insert codes for a SOD that was active over a wide range of pH and inhibitable with 5 mM H2O2 and 1 mM sodium diethyldithiocarbamate, a characteristic of Cu/ZnSODs that distinguishes them from Fe or Mn SODs. The sodC was expressed by C. burnetii, has a molecular weight of approximately 18 kDa, which is consistent with the predicted molecular weight, and localized towards the periphery of C. burnetii. Over expression of the C. burnetii sodC in an E. coli sodC mutant restored resistance to H2O2 killing to wild type levels.
We have demonstrated that C. burnetii does express a Cu/ZnSOD that is functional at low pH, appears to be excreted, and was able to restore H2O2 resistance in an E. coli sodC mutant. Taken together, these results indicate that the C. burnetii Cu/ZnSOD is a potentially important virulence factor.
Coxiella burnetii, the etiologic agent of Q fever, is an obligate intracellular bacterium that replicates within the phagolysosome of monocytes/macrophages. The ability to survive in the harsh environment of the phagolysosome may require the subversion of macrophage microbicidal mechanisms. Several enzyme systems potentially required to survive in the phagolysosomal compartment, such as catalase, cytoplasmically localized superoxide dismutase (SOD), and acid phosphatase have been partially characterized [1,2]. Catalase and SOD activities were detected in C. burnetii whole cell lysates and were demonstrated to be maximally active at neutral pH suggesting that these enzymes were cytoplasmically localized and may be involved in detoxifying endogenously generated oxygen radicals . Additionally, Heinzen et al. were able to clone a C. burnetii SOD and functionally complement an E. coli sodA sodB double mutant . This C. burnetii SOD was demonstrated to be homologous to known iron-containing SODs. Baca et al. also demonstrated that supernatants from high-speed centrifugation of sonicated C. burnetii contained acid phosphatase activity that was optimally active at low pH, localized to the periplasmic space of C. burnetii, and was capable of inhibiting superoxide anion production by stimulated human neutrophils, suggesting that this enzyme may prevent killing of the bacteria during uptake by inhibiting the respiratory burst [3,4].
The C. burnetii genomic database from TIGR predicted a putative Cu/Zn SOD with a signal sequence (CBU 1822). Recently, Stead et al. reported the presence of a putative SodC protein in supernatants from C. burnetii cultured in acidified citrate cysteine media using a FLAG-tag assay, which indicates the secreted nature of the SOD . Periplasmically localized Cu/Zn SOD enzymes have been identified in a wide range of Gram-negative bacteria and are proposed to protect bacteria from exogenously produced toxic oxygen radicals, which indicate the potential significance of a C. burnetii Cu/Zn SOD. For example, the survival of Mycobacterium tuberculosis and Salmonella typhimurium sodC mutants were reduced by 90% and 5 fold, respectively, compared to wild type when exposed to exogenous superoxide anion [6,7]. Additionally, Strohmeier Gort et al. reported that an Escherichia coli sodC mutant was more sensitive to hydrogen peroxide killing during stationary phase than wild type and were able to restore resistance to hydrogen peroxide killing through complementation . Here we describe the cloning, expression, and characterization of a C. burnetii Cu/Zn SOD.
Demonstration of CuZnSOD activity
Expression and subcellular localization of C. burnetii SodC
Complementation of an E. coli sodC mutant with recombinant C. burnetii SodC
There is mounting evidence that demonstrates the importance of Cu/ZnSOD enzymes in bacterial protection against oxidative killing. In fact, inactivation of the sodC gene has been found to cause attenuation of virulence in a wide variety of pathogenic bacteria [8,12-15]. The potential importance of a Cu/ZnSOD for the intracellular survival of C. burnetii is apparent by the obligate intracellular nature of this pathogen. In an effort to begin to characterize the potential role of this enzyme in C. burnetii intracellular survival a 570-bp region containing the signal sequence of the C. burnetii sodC gene was PCR amplified, cloned into pBAD-TOPO, and expressed as a fusion protein in TOP10 E. coli cells. The copper-zinc nature of this SOD was demonstrated by its inhibition by DDC and H2O2 using xanthine oxidase and native PAGE as demonstrated for other bacterial Cu/ZnSOD enzymes [10,16,17]. Several previously characterized Cu/ZnSOD enzymes contain signal sequences and were demonstrated to localize to the periplasmic space [10,14,15,18] leading to the hypothesis that these enzymes aid in the detoxification of superoxide (O2 −) produced by the host. The observations that the C. burnetii SodC is expressed by C. burnetii and localizes towards the periphery supports the hypothesis that this enzyme functions in a low pH environment and may play a role in protecting C. burnetii from exogenously produced reactive oxygen intermediates.
It has been well established that C. burnetii is an acidophile [19-21] and although the cytoplasmic pH of the organism remains near neutral , the periplasmic space is presumably acidic. Therefore, we hypothesized that a periplasmically localized Cu/ZnSOD in C. burnetii would be active at low pH to defend the organism from phagocyte derived O2 −. To test this hypothesis, the ability of purified, recombinant C. burnetii Cu/ZnSOD to inhibit cytochrome C reduction at low pH was determined. The recombinant Cu/ZnSOD did retain activity at a pH of 5.0 suggesting that indeed this enzyme could function in the periplasmic space and protect C. burnetii from host derived O2 −. Interestingly, unlike acid phosphatase and catalase activity previously demonstrated for C. burnetii, we were not able to detect optimal enzymatic activity at any of the three pH values tested for the CuZnSOD. Maximal catalase activity was detected at pH 7.0 with much lower activity detected at pH 4.5, whereas optimum acid phosphatase activity was observed at pH 5.0 and significantly decreased as the pH was raised [1,3]. Bovine Cu/ZnSOD for example has been shown to retain activity in 8.0 M urea or 2% SDS and exhibit essentially constant activity over the pH range 5.0–9.5 [23-25]. However, E. coli Cu/ZnSOD is very thermolabile and sensitive to pH . Thus, whether or not C. burnetii Cu/ZnSOD does have a pH optimum requires further study, but our data clearly demonstrates that this enzyme is active at low pH.
Standard methods such as targeted gene disruption, antibiotic selection and growth on axenic media used to manipulate free living bacteria have not been readily available for C. burnetii. Although the genetic transformation of C. burnetii was first reported in 1996  and later in 2009  along with the ability to grow C. burnetii axenic media , performing targeted mutagenesis remains challenging. This inability to readily manipulate C. burnetii genetically has led to the use of heterologous cloning as a means to study the organism’s genes and regulatory functions. C. burnetii genes such as dnaJ, era, pyrB, sdhCDAB, icd, rnc, and SodA/SodB were used to successfully complement paralogous gene mutations in E. coli [2,29-34]. To test functional expression of the C. burnetii Cu/ZnSOD, pREB102 was transformed into an E. coli sodC mutant (AS454) that was previously demonstrated to be highly susceptible to H2O2 killing in stationary phase . Over expression of the C. burnetii sodC restored resistance of the E. coli sodC mutant to H2O2 killing to wild type levels. Immunoblot analysis confirmed that expression of C. burnetii sodC in AS454 was achieved only upon induction, suggesting that the restored resistance of the complemented mutant was not due to an artifact of pREB102 or some unknown factor (data not shown). This data suggests that this enzyme does possess antioxidant properties and supports the hypothesis that this enzyme may play a role in C. burnetii intracellular survival in an oxidative stress environment.
In conclusion, we have demonstrated that C. burnetii does express a Cu/ZnSOD that is functional at low pH, which appears to be excreted, and was able to restore H2O2 resistance in an E. coli sodC mutant. These studies provide the framework to evaluate the role that C. burnetii SodC plays in intracellular survival. To address this issue, the potential for regulation of this enzyme during oxidative stress and/or by RpoS is currently under investigation, which should provide insight about the possible role of this enzyme in a developmental life cycle and virulence.
Bacterial strains, plasmids, media, and growth conditions
List of bacterial strains and plasmids
Strain or plasmid
Reference (s) or source
Nine mile, phase I, RSA 493
F−, rpsL, gal
TA cloning vector Apr
pBAD-TOPO with 570 bp C.burnetii sodC insert
Cloning, expression, and purification of rCbSodC
PCR amplification of C. burnetii sodC was performed in a Biometra UNO-Thermoblock (Biometra, Tampa, Fla.). Primers FBB1.2 (5’-GGAAATATTTTGAGGCGCGTC-3’) and RBB1.2 (5’-ACACGCAATTCGCGCACC-3’) (Sigma Genosys, Woodlands, Tx.) were used at final concentrations of 0.2 mM to amplify a 570 bp fragment including the signal sequence of the C. burnetii sodC gene. Amplification consisted of an initial 2 min denaturation step at 94°C followed by 30 cycles of 30 s at 94°C, 30 s at 60°C, and 1 min at 70°C. PCR products were visualized in a 1.2% agarose gel. The PCR product was then directly cloned into pBAD-TOPO (Invitrogen) and transformed into TOP10 cells. Restriction digests were performed on the constructs with BstEII to determine which clones contained the insert in the correct orientation. Plasmid DNA from one of those constructs was sent to the Gene Technologies Laboratory at Texas A & M University for sequencing. This construct was designated pREB102.
Expression of the recombinant SodC peptide was done using the pBAD-TOPO expression system (Invitrogen). Briefly, 10 ml of an overnight culture of TOP10 (pREB102) was inoculated into 1 liter of LB broth containing ampicillin (100 μg/ml) and incubated until an OD600 of 0.5 was reached. Induction was achieved by adding 9 ml of 2% arabinose and incubating the culture for 4 hr at 37°C on a shaker. Cultures were centrifuged at 5,000 rpm for 10 min. Purification of the recombinant SodC protein was then done using a 1 ml HiTrap nickel affinity column (Amersham Biosciences, Piscataway, NJ.). Briefly, the pellet was resuspended in 50 ml of binding buffer (0.02 M NaPO4, 0.5 M NaCl) pH 7.4 containing 50 mg of lysozyme and incubated on ice for 30 min. Cells were lysed using a French press and then centrifuged for 15 min at 5,000 rpm. Twenty milliliters of the French press lysate was filtered through a 0.8/0.45 μm syringe filter and loaded onto the HiTrap nickel column using a parastaltic pump. The column was washed with 8 ml of binding buffer and then washed with 4.5 ml of wash buffer (binding buffer with 100 mM imidazole). His-tagged recombinant protein was eluted using elution buffer (binding buffer with 0.5 M imidazole and collected in 0.1 ml fractions and frozen at-20°C until SDS-PAGE and Western blot analysis.
Superoxide dismutase activity gels
SodC activity was visualized using a method previously described by Beauchamp et al.  incorporating the modification of Steinman . Briefly, after electrophoresis on 12% native PAGE, gels were soaked in a riboflavin solution (0.028 M TEMED, 2.8 x 105 M riboflavin, 0.036 M KPO4 pH 7.8 for 20 min. at 37°C in the dark followed by soaking them in 0.2% nitroblue tetrazolium (NBT) for 10 min. at 37°C in the dark. The gels were then illuminated for approximately 30 min on a transilluminator. SOD activity corresponds to achromatic zones in a uniformly purple background. To identify the copper zinc nature of the cloned C. burnetii SOD, one of the gels was soaked in 5 mM hydrogen peroxide (H2O2) for 1 hr prior to staining.
Superoxide dismutase assay
The copper-zinc nature of the recombinant C. burnetii SOD was confirmed using a superoxide dismutase assay kit from Cayman Chemical. The assay is based on the utilization of tetrazolium salt for the detection of superoxide anions generated by xanthine oxidase and hypoxanthine. The activity of SODs is determined by their ability to inhibit the reaction. Approximately one unit of each SOD was utilized in the assay. One unit is defined by the amount of SOD that will inhibit the reaction by approximately 50%. The detection of superoxide dismutase activity was done following the manufacturer’s instructions. Specific inhibition of the Cu/Zn SOD activity was achieved by treating the control Cu/Zn SOD and recombinant C. burnetii SOD with 1 mM DDC for 15 min. at room temperature prior to superoxide dismutase activity detection. Manganese (Mn) and Iron (Fe) co-factored SODs from Sigma were included to demonstrate the specificity of DDC for Cu/Zn co-factored SODs. SOD assay kit sample buffer was used to dissolve and dilute all SODs and was included in the no SOD control reactions.
pH effect on the activity of recombinant C. burnetii SodC
The activity of the recombinant SodC was determined at pH 5.0, 7.0, and 9.0 by the method described by Flohè and Ötting  with some modifications. Briefly, 10 μg purified rSodC was added to 50 mM sodium acetate buffer pH 5.0, 50 mM potassium phosphate buffer pH 7.0, or 50 mM Tris–HCl buffer pH 9.0, respectively, each containing 0.01 mM EDTA. Prior to absorbance readings, 20 μM cytochrome c from horse heart (Sigma-Aldrich), 4 μl of 25 mM hypoxanthine (Calbiochem) and xanthine oxidase from bovine milk (Calbiochem). For control reactions, the hypoxanthine and xanthine oxidase were added in the presence of the rSodC elution buffer. The amount of xanthine oxidase added to each buffer system was adjusted to achieve a reduction rate of approximately 0.0125 absorbance/min at 550 nm. The final reaction volume was 100 μl per well in 96 well plates. Plates were incubated at 37°C and reduction was monitored for 10 min by measuring absorbance at 30 sec intervals. Inhibition of cytochrome C reduction was determined by dividing the absorbance values at 10 minutes with rSodC by the absorbance values at 10 minutes without rSodC and multiplying by 100.
sodC expression by C. burnetii
Purified recombinant C. burnetii SodC (rSodC) was combined with the adjuvant Titermax (Sigma) and used to immunize a rabbit for the production of monospecific polyclonal antibodies against rSodC. To determine whether or not a CuZnSOD is expressed by C. burnetii, C. burnetii cells purified from persistently infected L929 murine fibroblasts or E. coli cells overexpressing rSodC were suspended in sample buffer (4% SDS, 10% β-mercaptoethanol, 20% glycerol, and 0.25 M Tris, pH 8.0), boiled for 10 min, separated on 15% SDS-PAGE gels, and transferred to nitrocellulose membranes (Biorad, Hercules, CA). Membranes were blocked with 10% nonfat powdered milk and 0.2% Tween-20 in Tris buffered saline, pH 7.4. Blots were then incubated with rabbit antiserum to rSodC at a 1:2,000 dilution followed by incubation with goat anti-rabbit IgG horseradish peroxidase conjugated secondary antibody at a 1:5,000 dilution (Biorad). The blots were developed using an enhanced chemiluminescence system with luminol substrate (Amersham Biosciences). Images were visualized using Kodak Scientific Imaging film.
Immunogold transmission electron microscopy
L929 murine fibroblast cells persistently infected with C. burnetii, Nine Mile, (RSA493), were fixed and processed as described previously . Cells were fixed with 0.2% picric acid, 1% glutaraldehyde, 4% paraformaldehyde, 0.5 mM CaCl2 in phosphate buffered saline (PBS, 140 mM NaCl2, 3 mM KCl2, 2 mM KPO4, 10 mM NaPO4), pH 7.4 for 3 hr at room temperature while turning end over end. Cells were spun at 10,000 x g for 10 min and the pellet was incubated for 1 hr at 4°C after resuspension in 50 mM NH4Cl, 250 mM sucrose, PBS. Cells were then centrifuged at 10,0000 x g for 10 min. Ammonium chloride was removed by resuspending the pellet in 3.5% sucrose, 0.5 mM CaCl2 in PBS pH 7.4 overnight at 4°C. Phosphate buffers were removed by washing 4 x 15 min with 0.1 M maleate buffer, with 3.5% sucrose, pH 6.5. Post fixation staining was carried out with 2% uranyl acetate in sucrose/maleate buffer, pH 6.0 for 2 hr at 0°C protected from light. Dehydration and infiltration into LR White was carried out a room temperature in 45 min. steps of: 50% acetone, 70% acetone, 90% acetone, 1:1 100% ethanol/LR White, 3:7 100% ethanol/LR White, 100% LR White, then fresh LR White overnight, second change of fresh LR White before samples were enclosed in gelatin capsules (Electron Microscopy Scences, Hatfield, PA). Polymerization was carried out at 50°C for 24 hr. Silver to gold sections were collected on 300 mesh nickel grids (Electron Microscopy Sciences). All staining was carried out in the BioWave microwave (Ted Pella, Inc., Redding, CA.) at 30°C. Blocking (2% nomal goat sea), primary (rabbit anti-sodC, 1:25 dilution in 2% normal goat sera), and secondary antibody incubations (goat anti-mouse conjugate 12 nm gold particles, (Jackson ImmunoResearch laboratories, West Grove, PA.), diluted 1:40 in 2% normal goat sera were done in three cycles of 1 min on, 1 min off at full power. Primary antibody was washed three times in Tris buffered Saline (TBS, 5 mM Tris, 15 mM NaCl2), pH 7.4, for one min per wash. Secondary antibody was washed in TBS pH 8.4, 3 x 1 min. Secondary antibody was fixed in 1% glutaraldehyde for 1 min with gentle agitation at room temperature. Fixed grids were briefly washed with water then stained with 2% uranyl acetate in the microwave for six seconds at full power. Grids were briefly washed in water with gentle agitation at room temperature. Grids were viewed with a JEOL operated at 100 kV.
Complementation of E. coli sodC null mutant
Complementation studies were carried out using an E. coli sodC mutant (AS454) previously demonstrated to be more sensitive to killing by exogenous H2O2 than the wild type parental strain (AN387) during early stationary phase . To genetically complement the sodC mutant, plasmid pREB102 was electroporated into AS454. Transformants were selected on LB agar plates containing ampicillin (150 μg/ml). AN387, AS454, and AS454 (pREB102) were grown in LB broth or LB broth containing ampicillin (100 μg/ml) overnight at 37°C on a shaker, subcultured into fresh media at a starting OD600 of ≈ 0.01. In order achieve expression of sodC, AS454 and AS454 (pREB102) were either induced or not induced with 2% arabinose 4 hr prior to H2O2 challenge. At 45 min intervals aliquots for H2O2 challenges were removed, diluted 1:10,000 in PBS, and challenged with 2 mM H2O2 for 30 min while shaking at 37°C as previously described . Survival was determined as the percentage of colony counts (cfu/mL) from surviving bacteria after H2O2 treatment and from untreated bacteria by plating on LB plates with or without ampicillin (150 μg/ml).
This work was supported by the National Institutes of Health grant A1037744 (James E Samuel) and 3M Non-tenured Faculty Grant (Robert E Brennan). We especially thank Dr. James Imlay, University of Illinois at Urbana-Champaign for generously providing the E. coli sodC mutant strain (AS454) and wild type parental strain (AN387).
- Akporiaye ET, Baca OG. Superoxide anion production and superoxide dismutase and catalase activities in Coxiella burnetii. J Bacterol. 1983;154:520–3.Google Scholar
- Heinzen RA, Frazier ME, Mallavia LP. Coxiella burnetii superoxide dismutase gene: cloning, sequencing, and expression in Escherichia coli. Infect Immun. 1992;60:3814–23.PubMed CentralPubMedGoogle Scholar
- Baca OG, Roman MJ, Glew RH, Christner RF, Buhler JE, Aragon AS. Acid phosphatase activity in Coxiella burnetii: a possible virulence factor. Infect Immun. 1993;61:4232–9.PubMed CentralPubMedGoogle Scholar
- Li YP, Curley G, Lopez M, Chavez M, Glew R, Aragon A, et al. Protein-tyrosine phosphatase activity of Coxiella burnetii that inhibits human neutrophils. Acta Virol. 1996;40:163–272.Google Scholar
- Stead C, Anders O, Beare PA, Sandoz KM, Heinzen RA. Sec-mediated secretion by Coxiella burnetii. BMC Microbiol. 2013;13:222–32.View ArticlePubMed CentralPubMedGoogle Scholar
- De Groote MA, Ochsner UA, Shiloh MU, Nathan C, McCord JM, Dinauer MC, et al. Periplasmic superoxide dismutase protects Salmonella from products of phagocyte NAPDH-oxidase and nitric oxide synthase. Proc Nat Acad Sci. 1997;94:13997–4001.View ArticlePubMed CentralPubMedGoogle Scholar
- Piddington DL, Fang FC, Leassig T, Cooper AM, Orme IM, Buchmeier NA. Cu, Zn superoxide dismutase of mycobacteria tuberculosis contributes to survival in activated macrophages that are generating an oxidative burst. Infect Immun. 2001;69:4980–7.View ArticlePubMed CentralPubMedGoogle Scholar
- Strohmeier Gort AS, Ferber DM, Imlay JA. The regulation and role of the periplasmic copper, zinc superoxide dismutase of Escherichia coli. Mol Microbiol. 1999;32:179–91.View ArticleGoogle Scholar
- Crapo JD, McCord JM, Fridovich I. Preparation and assay of superoxide dismutases. Methods Enzym. 1978;53:382–93.View ArticleGoogle Scholar
- Benov LT, Fridovich I. Escherichia coli expresses a copper- and zinc-containing superoxide dismutase. J Biol Chem. 1994;269:25310–4.PubMedGoogle Scholar
- Sharma SK, Evans DB, Vosters AF, Chattopadhyay D, Hoogerheide JG, Campbell CM. Immobilized metal affinity chromatography of bacterially expressed proteins engineered to contain an alternating-histidine domain. Methods. 1992;4:57–67.View ArticleGoogle Scholar
- Latimer E, Simmers J, Sriranganathan N, Roop II RM, Schurig GG, Boyle BSM. Brucella abortus deficeint in copper/zinc superoxide dismutase is virulent in BALB/c mice. Microbial Path. 1992;12:105–13.View ArticleGoogle Scholar
- St John G, Steinman HM. Periplasmic copper-zinc superoxide dismutase of Legionella pneumophila: Role in stationary phase survival. J Bacteriol. 1996;178:1578–84.PubMed CentralPubMedGoogle Scholar
- Fang FC, DeGroote MA, Foster JW, Baumler AJ, Ochsner U, Testerman T, et al. Virulent Salmonella typhimurium has two periplasmic Cu, Zn-superoxide dismutases. Proc Nat Acad Sci. 1999;96:7502–7.View ArticlePubMed CentralPubMedGoogle Scholar
- Sansone A, Watson PR, Wallis TS, Langford PR, Kroll JS. The role of two periplasmic copper- and zinc-cofactored superoxide dismutases in the virulence of Sallmonella choleraesuis. Microbiol. 2002;148:719–26.Google Scholar
- Beauchamp CO, Fridovich I. Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Annals of Biochem. 1971;72:276–87.View ArticleGoogle Scholar
- Steinman HM. Bacteriocuprein: superoxide dismutases in Pseudomonads. J Bacteriol. 1985;162:1255–60.PubMed CentralPubMedGoogle Scholar
- Stabel TJ, Sha Z, Mayfield JE. Periplasmic location of Brucella abortus Cu/Zn superoxide dismutase. Vet Microb. 1994;38:307–14.View ArticleGoogle Scholar
- Hackstadt T, Williams JC. Biochemical stratagem for obligate parasitism of eukaryotic cells by Coxiella burnetii. Proc Nat Acad Sci. 1981;78:3240–4.View ArticlePubMed CentralPubMedGoogle Scholar
- Zuerner RL, Thompson HA. Protein synthesis in by intact Coxiella burnetii cells. J Bacteriol. 1983;156:186–91.PubMed CentralPubMedGoogle Scholar
- Chen S, Vodkin M, Thompson HA, Williams JC. Isolated Coxiella burnetii synthesizes DNA during acid activation in the absence of host cells. J Gen Microbiol. 1990;136:89–96.View ArticlePubMedGoogle Scholar
- Hackstadt T. Estimation of the cytoplasmic pH of Coxiella burnetii and effect of substrate oxidation on proton motive force. J Bacteriol. 1983;154:591–7.PubMed CentralPubMedGoogle Scholar
- Klug D, Rabini J, Fridovich I. A direct demonstration of the catalytic action of superoxide dismutase through the use of pulse radiolysis. J Biol Chem. 1972;247:4839–42.PubMedGoogle Scholar
- Foreman HJ, Fridovich I. On the stability of bovine superoxide dismutase. The effects of metals. J Biol Chem. 1973;248:2645–9.Google Scholar
- Malinowski DP, Fridovich I. Subunit association and side-chain reactivities of bovine erythrocyte superoxide dismutase in denaturing solvents. Biochem. 1979;18:5055–60.View ArticleGoogle Scholar
- Suhan ML, Chen SY, Thompson HA. Transformation of Coxiella burnetii to ampicillin resistance. J Bacteriol. 1996;178:2701–8.PubMed CentralPubMedGoogle Scholar
- Beare PA, Howe D, Cockrell DC, Omlsand A, Hansen B, Heinzen RA. Characterization of a Coxiella burnetii ftsZ mutant generated by Himar1 transposon mutagenesis. J Bacteriol. 2009;191:1369–81.View ArticlePubMed CentralPubMedGoogle Scholar
- Omsland A, Cockrell DC, Howe D, Fischer ER, Virtaneva K, Sturdevant DE, et al. Host cell-free growth of the Q fever bacterium Coxiella burnetii. Proc Natl Acad Sci U S A. 2009;106:4430–4.View ArticlePubMed CentralPubMedGoogle Scholar
- Hoover TA, Williams JC. Characterization of Coxiella burnetii pyrB. Ann NY Acad Sci. 1990;590:485–90.View ArticlePubMedGoogle Scholar
- Zuber M, Hoover TA, Powell BS, Court DL. Analysis of the rnc locus of Coxiella burnetii cells. J Bacteriol. 1994;156:186–91.Google Scholar
- Zuber M, Hoover TA, Court DL. Cloning, sequencing and expression of the dnaJ gene of Coxiella burnetii. Gene. 1995;152:99–102.View ArticlePubMedGoogle Scholar
- Heinzen RA, Mo YY, Robertson SJ, Mallavia LP. Characterization of the succinate dehydrogenase-encoding gene cluster (sdh) from the rickettsia Coxiella burnetii. Gene. 1995;155:27–34.View ArticlePubMedGoogle Scholar
- Thompson HA, Suhan ML. Genetics of Coxiella burnetii. FEMS Microbiol Let. 1996;145:130–46.View ArticleGoogle Scholar
- Nguyen SV, To H, Yamaguchi T, Fukushi H, Hirai K. Molecular cloning of an immunogenic and acid-induced isocitrate dehydrogenase gene from Coxiella burnetii. FEMMS Microbiol Let. 1999;175:101–6.View ArticleGoogle Scholar
- Flohe L, Otting F. Superoxide dismutase assays. Methods Enzym. 1984;105:93–104.View ArticleGoogle Scholar
- Berryman MA, Rodewald RD. An enhanced method for post-embedding immunocytochemical staining which preserves cell membrane. J Histochem Cytochem. 1990;38:159–70.View ArticlePubMedGoogle Scholar
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.