Hydrogen peroxide sensitivity of catechol-2,3-dioxygenase: a cautionary note on use of xylE reporter fusions under aerobic conditions

Abstract

Catechol-2,3-dioxygenase (C23O) of Pseudomonas putida, encoded by the xylE gene, was found to be sensitive to hydrogen peroxide (H(2)O(2)) when used as a reporter in gene fusion constructs. Exposure of Pseudomonas aeruginosa katA or katA katB mutants harboring katA- or katB-lacZ (encoding beta-galactosidase) or -xylE fusion plasmids to H(2)O(2) stimulated beta-galactosidase activity, while there was little or no detectable C23O activity in these strains. More than 95% of C23O activity was lost after a 5-min exposure to equimolar H(2)O(2), while a 10,000-fold excess was required for similar inhibition of beta-galactosidase. Electron paramagnetic resonance spectra of the nitrosyl complexes of C23O showed that H(2)O(2) nearly stoichiometrically oxidized the essential active-site ferrous ion, thus accounting for the loss of activity. Our results suggest using caution in interpreting data derived from xylE reporter fusions under aerobic conditions, especially where oxidative stress is present or when catalase-deficient strains are used.

FIG. 1

Quantification of catalase gene reporter activity in wild-type and catalase mutant bacteria. P. aeruginosa PAO1 katA, katB, and katA katB strains (13) harboring plasmids containing katA- or katB-lacZ or -xylE transcriptional fusions were grown aerobically for 24 h in L broth containing carbenicillin (0.4 mg/ml) for plasmid maintenance. Bacteria were washed twice in either ice-cold 50 mM potassium phosphate buffer (pH 7.5) (for C23O assays) or Z buffer (for β-galactosidase assays) (22) containing 39 mM 2-mercaptoethanol and were sonicated in an ice water bath for 10 s with a Heat-Systems, Inc. (Farmington, N.Y.), model W-225 sonicator at setting 5. Cell extracts were assayed for C23O and β-galactosidase activities as previously described (19, 28). The results are expressed as the means ± standard errors of the means of three replicates. (A) Lane 1, PAO1/pkatA::xylE; lane 2, PAO1/pkatB::xylE; lane 3, katA mutant/pkatA::xylE; lane 4, katA mutant/pkatB::xylE; lane 5, katB mutant/pkatA::xylE; lane 6, katB mutant/pkatB::xylE; lane 7, katA katB mutant/pkatA::xylE; lane 8, katA katB mutant/pkatB::xylE. (B) Lanes are identical to those in panel A except that the fusions are to lacZ. The values in panel A are all statistically significantly different from one another (P < 0.01, Student's t test). The differences between lanes 1, 3, 5, and 7 of panel B are not statistically significant.

FIG. 2

H₂O₂-mediated activation of katA and katB gene transcription: demonstration of increased β-galactosidase activity but not C23O activity in catalase-deficient bacteria. Bacteria were grown aerobically to mid-exponential phase (optical density at 600 nm = 0.6) in L broth and were allowed to grow an additional hour in the presence (lanes 2 and 4) or absence (lane 1 and 3) of 1 mM H₂O₂. Cell extracts were then assayed for β-galactosidase (A and C) and C23O (B and D) activity. The results are expressed as the percentage of reporter activity in uninduced wild-type bacteria (n = 3). Lane 1, PAO1; lane 2, PAO1 + H₂O₂; lane 3, katA katB; lane 4, katA katB + H₂O₂. In panel A, values in lanes 1, 2, and 4 are not statistically significant, yet that in lane 3 is significantly reduced (P < 0.05). All remaining values are statistically significantly different at P values of < 0.01.

FIG. 3

Relative sensitivity of purified P. putida C23O (A) and β-galactosidase (B) to H₂O₂. C23O was isolated from P. putida mt-2 cells maintained on m-toluate and grown in 10-liter batch cultures on benzoate as the sole carbon source from 1-liter liquid starter cultures containing 1:2 m-toluate-benzoate as carbon sources. The total culture time on benzoate was 12 h, and the extradiol catechol oxidation activity was approximately 10⁵ U/100 g (wet weight) of cells. The purification was as previously described (4), except that 50 mM morpholinepropanesulfonic acid (MOPS) buffer (pH 7.0) was used in place of potassium phosphate buffer in all steps. Samples with a specific activity greater than 150 U/mg were pooled, concentrated, and stored at −80°C in small aliquots until use. Purified P. putida C23O (10 U) and E. coli β-galactosidase (10 U; Sigma) were incubated with increasing concentrations of H₂O₂ at room temperature for 5 min. C23O and β-galactosidase activities were then assayed as previously described (19, 28) and expressed as the percentage of the control without H₂O₂. The bars indicate the means + standard errors of the means of three replicates. Concentrations of H₂O₂ added follow. (A) Lane 1, control; lane 2, 1 μM; lane 3, 10 μM; lane 4, 100 μM. (B) Lane 1, control; lane 2, 1 mM; lane 3, 10 mM; lane 4, 100 mM; lane 5, 200 mM; lane 6, 300 mM; lane 7, 400 mM; lane 8, 500 mM.

FIG. 4

EPR spectra of the Fe-nitrosyl complex of purified P. putida C23O show that loss of activity correlates with oxidation of the active-site Fe(II). Purified P. putida C23O (160 μM) was incubated on ice with 0 μM (——), 300 μM (....), or 30 mM (– – –) H₂O₂ for 30 min at pH 7. Then 7.5 U of bovine liver catalase was added to destroy any residual H₂O₂, and the enzyme activity was determined (this concentration of catalase cannot be detected by EPR). The samples were then transferred by a gastight syringe to an EPR tube under argon. Nitric oxide (NO) was added by slowly bubbling the gas through the sample under argon flow. Trace oxygen was removed from the argon gas by passage over a BASF copper catalyst at 160°C. Samples were flushed with argon after NO addition to remove excess NO from the headspace. The samples were frozen by slow immersion in liquid N₂, and the EPR spectra were recorded. The enzyme activity of the EPR samples after the measurement was determined by first thawing them under argon. The samples were transferred by a gastight syringe to a serum-stoppered vial under argon. Subsequently, the NO was removed by cycles of evacuation and flushing with Ar. Activity measurements of the samples were approximately unchanged from those determined before exposure to NO. The loss of signal in the 4-g region is proportional to the oxidation of Fe(II) to Fe(III) in the sample. At least three slightly different S 3/2 species are present with resonance pairs at g values of 4.18 and 3.82, 4.11 and 3.91, and 4.02 and 3.98. Multiple species are usually seen for nitrosyl complexes of Fe(II) dioxygenases. EPR measurement conditions using a Bruker E-500 spectrometer equipped with an Oxford ESR-910 liquid helium cryostat follow: temperature, 2 K; modulation amplitude, 10 G; modulation frequency, 100 kHz; microwave power, 200 μW; and microwave frequency, 9.63 GHz. Data were digitally recorded and analyzed as previously reported (10). Spin quantitations were performed by single or double integration of the first derivative spectra (1) using an Fe(II)-NO-EDTA complex as a standard. EPR spectra of S 3/2 and S 5/2 complexes were analyzed as previously reported (23, 37). Iron content was quantified by atomic absorption.