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Applied and Environmental Microbiology, September 2000, p. 4119-4123, Vol. 66, No. 9
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Hydrogen Peroxide Sensitivity of Catechol-2,3-Dioxygenase: a Cautionary Note on Use of xylE Reporter Fusions under Aerobic Conditions

Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0524¹; Department of Microbiology and Immunology, University of Colorado Health Sciences, Denver, Colorado 80262²; and Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455-0326³

Received 16 March 2000/Accepted 15 June 2000

	ABSTRACT

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Catechol-2,3-dioxygenase (C23O) of Pseudomonas putida, encoded by the xylE gene, was found to be sensitive to hydrogen peroxide (H₂O₂) when used as a reporter in gene fusion constructs. Exposure of Pseudomonas aeruginosa katA or katA katB mutants harboring katA- or katB-lacZ (encoding -galactosidase) or -xylE fusion plasmids to H₂O₂ stimulated -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 H₂O₂, while a 10,000-fold excess was required for similar inhibition of -galactosidase. Electron paramagnetic resonance spectra of the nitrosyl complexes of C23O showed that H₂O₂ 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.

	TEXT

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The use of gene reporter cassettes, including those encoding -galactosidase (lacZ), luciferase (luxAB), and catechol-2,3-dioxygenase (C23O) (xylE), has been a tremendous benefit to scientists studying gene regulation. The lacZ gene is the most widely used reporter in bacteria, while others that include luxAB, gfp (green fluorescent protein), phoA (alkaline phosphatase), and xylE fusions are used less often. The use of lacZ fusions in bacteria has proven to be the best tool with which to accurately assess promoter activity under conditions of oxidative stress. For example, in Escherichia coli, lacZ fusions were used to show that the transcriptional activity of oxidant-regulated genes including sodA (encoding Mn-superoxide dismutase), katE (encoding catalase/hydroperoxidase II), and katG (encoding catalase/hydroperoxidase I), among others, is markedly increased upon exposure to either O₂- or H₂O₂-generating agents (16, 29, 34).

Unlike the reliable lacZ reporters, other gene fusions have some drawbacks. For example, the use of the Vibrio harveyi luxAB genes (encoding luciferase) in transcriptional gene fusions was cautioned against, because it generates O₂ (12). Fusions linked to phoA are limited to proteins expressed in the periplasm (21), while those linked to gfp are only semiquantitative (24). The use of xylE fusion constructs does not appear to have any of these disadvantages (32).

The product of the xylE gene of Pseudomonas putida, C23O, is an important component in the degradation pathways of toluene and xylenes and catalyzes the dioxygenolytic cleavage of the aromatic ring (8, 11, 18, 25). Interestingly, it has been observed that optimal toluene degradation occurs under reduced oxygen tension with some C23O enzymes (17). This suggests a sensitivity to oxygen or one of its reduced forms, despite the fact that C23O requires oxygen for activity. Oxygen sensitivity may be derived from the fact that each of four C23O subunits contains an essential iron atom which must be in the Fe(II) oxidation state for activity in extradiol-type aromatic dioxygenases (3, 4, 25). Oxidation of this iron would inactivate the enzyme. Indeed, early studies showed that C23O is inactivated by low levels of the oxidizing reagent H₂O₂ in vitro (25). Our later studies of other Fe(II)-containing dioxygenases showed that H₂O₂ treatment resulted in enzyme inactivation and the appearance of electron paramagnetic resonance (EPR) signals attributable to ferric ion in most but not all cases (3, 23, 37). Several other iron-containing enzymes including the antioxidants iron superoxide dismutase and catalase are also sensitive to elevated levels of H₂O₂ (7, 9). In this study, we describe the sensitivity of C23O to H₂O₂ measured both in vivo using isogenic catalase mutants (Table 1) of the aerobic gram-negative bacterium Pseudomonas aeruginosa and in vitro using purified enzyme. We suggest caution in interpreting data obtained using xylE reporter fusions under aerobic conditions.

View this table: [in this window] [in a new window]   TABLE 1.   Strains and plasmids used in this study

Sensitivity of C23O but not -galactosidase to H₂O₂ in vivo using P. aeruginosa catalase gene lacZ and xylE fusions. The katA gene of P. aeruginosa encodes the major, constitutively expressed catalase, KatA (13, 20). In contrast, KatB activity is only detected upon exposure of bacteria to H₂O₂ or the redox-cycling agent paraquat (6). As shown in Fig. 1A, XylE reporter activity in the katA mutant, where catalase activity is virtually undetectable (20), was reduced 93%. The KatA-XylE reporter activity in the katA katB double mutant was reduced ~70%. Since H₂O₂ is required to activate the katB gene (6), it is not surprising that KatB reporter activity was very low or undetectable. In fact, KatB-LacZ reporter activity was reduced ~5,000-fold relative to KatA-LacZ reporter activity (Fig. 1B).

View larger version (22K): [in this window] [in a new window]   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 -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 -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.

Treatment with H₂O₂ stimulates catalase gene lacZ reporter activity but inhibits xylE reporter activity in catalase-deficient strains. To test the hypothesis that H₂O₂ increases katA::lacZ and katB::lacZ but not katA::xylE or katB::xylE activity, aerobic, mid-logarithmic-phase organisms were exposed to a sublethal 1 mM dose of H₂O₂ for 1 h, a condition which markedly increases KatB activity and, to a far lesser extent, KatA activity (6). Figure 2A shows that KatA-LacZ activity was only slightly increased in wild-type bacteria but was increased 1.4-fold following H₂O₂ treatment in the katA katB mutant. KatA-XylE activity was also increased upon exposure to H₂O₂ in wild-type bacteria but was dramatically inhibited in the control and H₂O₂-treated katA katB strains (Fig. 2B). In contrast to KatA activity, KatB activity is stimulated upon exposure to H₂O₂ (6). This is reflected in the KatB-LacZ results shown in Fig. 2B. Wild-type and katA katB mutant KatB-LacZ activity increased 10.7- and 35-fold upon exposure to H₂O₂. In contrast, KatB-XylE activity in the H₂O₂-treated katA katB mutant was reduced ~15-fold and was not detectable in control bacteria.

View larger version (45K): [in this window] [in a new window]   FIG. 2.   H2O2-mediated activation of katA and katB gene transcription: demonstration of increased -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 -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.

Sensitivity of purified C23O but not of -galactosidase to H₂O₂. The in vivo results suggest that H₂O₂ is much more damaging to C23O than to -galactosidase. To compare the sensitivity of C23O and -galactosidase to H₂O₂, aliquots of the purified enzymes were incubated at room temperature with increasing concentrations of H₂O₂. As shown in Fig. 3A, the C23O activity loss was directly correlated with the nearly stoichiometric titration of the active-site Fe(II) in the sample with H₂O₂. Most of the activity was lost after a 1:2 titration, and there was no detectable C23O activity after the enzyme was incubated with a 20-fold excess of H₂O₂. In contrast, 100 mM H₂O₂ (a 10,000-fold excess) was required for ~50% inhibition of -galactosidase activity (Fig. 3B).

View larger version (33K): [in this window] [in a new window]   FIG. 3.   Relative sensitivity of purified P. putida C23O (A) and -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 -galactosidase (10 U; Sigma) were incubated with increasing concentrations of H2O2 at room temperature for 5 min. C23O and -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.

EPR analysis of H₂O₂-treated C23O. The effect of H₂O₂ on the active-site iron of C23O can be directly evaluated by EPR spectroscopy. Our past studies have shown that complexing of the enzyme with NO converts an EPR-silent metal center into an EPR-active species with two of the three resonances symmetrically distributed around g values of 4 (3, 4). This is the characteristic spectrum of a spin (S) 3/2 species that is formed by transferring one electron from the iron to the NO and antiferromagnetic coupling between the resulting species (5). This species is easily distinguished from that resulting from oxidation of the Fe(II) to Fe(III), which yields an S 5/2 EPR spectrum with g values near 4.3. As shown in Fig. 4, untreated C23O yielded an intense S 3/2 EPR spectrum from the active-site Fe(II)-NO complex. Quantitation of the spectra showed that all of the iron in the sample formed a complex. After a 30-min incubation with a twofold excess of H₂O₂ on ice, a dramatic decrease in the S 3/2 spectrum from Fe(II)-NO occurred, accompanied by a 96% (±3%) loss of activity and the appearance of a broad signal from Fe(III) (g = 4.3). Quantitation of the S 3/2 signal showed that 87% (±5%) of the Fe(II) was oxidized. Incubation of an identical sample with a 200-fold excess of H₂O₂ resulted in a complete loss of the S 3/2 signal and activity as well as a further increase in the S 5/2 signal from Fe(III). These results show directly for the first time that the origin of the loss of activity of C23O following H₂O₂ treatment is oxidation of the active-site iron in accord with previous indirect experiments (25). Many studies have indicated that the mechanism of extradiol dioxygenases requires Fe(II), and thus its oxidation would inhibit the enzyme (2, 33). Because the Fe(III) EPR signal of the inactivated C23O is very broad, it is likely either that the iron is lost from the enzyme following oxidation or that the environment of the iron becomes highly disordered.

View larger version (22K): [in this window] [in a new window]   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.

Conclusions. In this study, we demonstrate that C23O is very sensitive to approximately stoichiometric levels of H₂O₂ both when it is present as a gene fusion product in vivo and as the purified enzyme in vitro. Arguably, the attractiveness of the xylE reporter system in monitoring gene-promoter activity is based upon its rapid spectrophotometric assay that is zero-order with respect to its substrate catechol. However, this study raises concern over the interpretation of data obtained under highly aerobic conditions. Recently we have cloned, overexpressed, and characterized another extradiol dioxygenase similar to C23O that catalyzes the cleavage of 3,4-dihydroxyphenyl acetic acid (homoprotocatechuate) between the 2- and 3-ring carbons to yield a yellow product analogous to the product of the C23O-catalyzed reaction (23, 35). Homoprotocatechuate-2,3-dioxygenase (H23O, encoded by hpcd) is unique among Fe(II)-containing dioxygenases in that it exhibits very low sensitivity to H₂O₂. Moreover, a homologous H23O containing Mn(II) rather than Fe(II) has also been cloned and characterized and was also found to be insensitive to H₂O₂ (27, 36). Thus, gene fusions to either of these enzymes could offer an alternative to C23O when experiments require highly aerobic and/or oxidative stress conditions. Such experiments are in progress.

	ACKNOWLEDGMENTS

This work was supported by Public Health Service grant AI-45041 and Cystic Fibrosis grant HASSET97PO to D.J.H. and NIGMS grant 24689 to J.D.L.

	FOOTNOTES

^* Corresponding author. Mailing address: Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, 231 Bethesda Ave., Cincinnati, OH 45267-0524. Phone: (513) 558-1154. E-mail: Daniel.Hassett{at}UC.Edu.

	REFERENCES

Top Abstract Text References

1.	Aasa, R., and T. Vänngård. 1975. EPR signal intensity and powder shapes: a re-examination. J. Magn. Reson. 19:308-315.
2.	Arciero, D. M., and J. D. Lipscomb. 1986. Binding of 17O-labeled substrate and inhibitors to protocatechuate 4,5-dioxygenase-nitrosyl complex. Evidence for direct substrate binding to the active site Fe2+ of extradiol dioxygenases. J. Biol. Chem. 261:2170-2178[Abstract/Free Full Text].
3.	Arciero, D. M., J. D. Lipscomb, B. H. Huynh, T. A. Kent, and E. Munck. 1983. EPR and Mossbauer studies of protocatechuate 4,5-dioxygenase. Characterization of a new Fe2+ environment. J. Biol. Chem. 258:14981-14991[Abstract/Free Full Text].
4.	Arciero, D. M., A. M. Orville, and J. D. Lipscomb. 1985. [17O]water and nitric oxide binding by protocatechuate 4,5-dioxygenase and catechol 2,3-dioxygenase. Evidence for binding of exogenous ligands to the active site Fe2+ of extradiol dioxygenases. J. Biol. Chem. 260:14035-14044[Abstract/Free Full Text].
5.	Brown, C. A., M. A. Pavlosky, T. E. Westre, Y. Zhang, B. Hedman, K. O. Hodgson, and E. I. Solomon. 1995. Spectroscopic and theoretical description of the electronic structure of S=3/2 iron-nitrosyl complexes and their relation to O2 activation by non-heme iron enzyme active sites. J. Am. Chem. Soc. 117:715-732[CrossRef].
6.	Brown, S. M., M. L. Howell, M. L. Vasil, A. J. Anderson, and D. J. Hassett. 1995. Cloning and characterization of the katB gene of Pseudomonas aeruginosa encoding a hydrogen peroxide-inducible catalase: purification of KatB, cellular localization, and demonstration that it is essential for optimal resistance to hydrogen peroxide. J. Bacteriol. 177:6536-6544[Abstract/Free Full Text].
7.	Clare, D. A., J. Blum, and I. Fridovich. 1984. A hybrid superoxide dismutase containing both functional iron and manganese. J. Biol. Chem. 259:5932-5936[Abstract/Free Full Text].
8.	Dagley, S. 1978. Microbial catabolism, the carbon cycle and environmental pollution. Naturwissenschaften 65:85-95[CrossRef][Medline].
9.	Darr, D., and I. Fridovich. 1985. Inhibition of catalase by 3,3'-diaminobenzidine. Biochem. J. 226:781-787[Medline].
10.	Fox, B. G., W. A. Froland, J. E. Dege, and J. D. Lipscomb. 1989. Methane monooxygenase from Methylosinus trichosporium OB3b. Purification and properties of a three-component system with high specific activity from a type II methanotroph. J. Biol. Chem. 264:10023-10033[Abstract/Free Full Text].
11.	Franklin, F. C., M. Bagdasarian, M. M. Bagdasarian, and K. N. Timmis. 1981. Molecular and functional analysis of the TOL plasmid pWWO from Pseudomonas putida and cloning of genes for the entire regulated aromatic ring meta cleavage pathway. Proc. Natl. Acad. Sci. USA 78:7458-7462[Abstract/Free Full Text].
12.	González-Flecha, B., and B. Demple. 1994. Intracellular generation of superoxide as a by-product of Vibrio harveyi luciferase expressed in Escherichia coli. J. Bacteriol. 176:2293-2299[Abstract/Free Full Text].
13.	Hassett, D. J., J.-F. Ma, J. G. Elkins, T. R. McDermott, U. A. Ochsner, S. E. H. West, C.-T. Huang, J. Fredericks, S. Burnett, P. S. Stewart, G. McPheters, L. Passador, and B. H. Iglewski. 1999. Quorum sensing in Pseudomonas aeruginosa controls expression of catalase and superoxide dismutase genes and mediates biofilm susceptibility to hydrogen peroxide. Mol. Microbiol. 34:1082-1093[CrossRef][Medline].
14.	Holloway, B. W. 1969. Genetics of Pseudomonas. Bacteriol. Rev. 33:419-443[Free Full Text].
15.	Holloway, B. W., V. Krishnapillai, and A. F. Morgan. 1979. Chromosomal genetics of Pseudomonas. Microbiol. Rev. 43:73-102[Free Full Text].
16.	Ivanova, A., C. Miller, G. Glinsky, and A. Eisenstark. 1994. Role of rpoS (katF) in OxyR-independent regulation of hydroperoxidase I in Escherichia coli. Mol. Microbiol. 12:571-578[Medline].
17.	Kukor, J. J., and R. H. Olsen. 1996. Catechol 2,3-dioxygenases functional in oxygen-limited (hypoxic) environments. Appl. Environ. Microbiol. 62:1728-1740[Abstract].
18.	Lipscomb, J. D., and A. M. Orville. 1992. Mechanistic aspects of dihydroxybenzoate dioxygenases, p. 243-298. In H. Sigel, and I. Sigel (ed.), Metal ions in biological systems. Marcel Dekker, Inc., New York, N.Y.
19.	Ma, J.-F., P. W. Hager, M. L. Howell, P. V. Phibbs, and D. J. Hassett. 1998. Cloning and characterization of the Pseudomonas aeruginosa zwf gene encoding glucose-6-phosphate dehydrogenase, an enzyme important in resistance to methyl viologen (paraquat). J. Bacteriol. 180:1741-1749[Abstract/Free Full Text].
20.	Ma, J.-F., U. A. Ochsner, M. G. Klotz, V. K. Nanayakkara, M. L. Howell, Z. Johnson, J. E. Posey, M. L. Vasil, J. J. Monaco, and D. J. Hassett. 1999. Bacterioferritin A modulates catalase A (KatA) activity and resistance to hydrogen peroxide in Pseudomonas aeruginosa. J. Bacteriol. 181:3730-3742[Abstract/Free Full Text].
21.	Michaelis, S., L. Guarente, and J. Beckwith. 1983. In vitro construction and characterization of phoA-lacZ gene fusions in Escherichia coli. J. Bacteriol. 154:356-365[Abstract/Free Full Text].
22.	Miller, J. H. 1992. A short course in bacterial genetics, p. 72-74. Cold Spring Harbor Laboratory Press, Plainview, N.Y.
23.	Miller, M. A., and J. D. Lipscomb. 1996. Homoprotocatechuate 2,3-dioxygenase from Brevibacterium fuscum. A dioxygenase with catalase activity. J. Biol. Chem. 271:5524-5535[Abstract/Free Full Text].
24.	Miller, W. G., and S. E. Lindow. 1997. An improved GFP cloning cassette designed for prokaryotic transcriptional fusions. Gene 191:149-153[CrossRef][Medline].
25.	Nozaki, M., K. Ono, T. Nakazawa, S. Kotani, and O. Hayaishi. 1968. Metapyrocatechase. II. The role of iron and sulfhydryl groups. J. Biol. Chem. 243:2682-2690[Abstract/Free Full Text].
26.	Ochsner, U. A., M. L. Vasil, E. Alsabbagh, K. Parvatiyar, and D. J. Hassett. 2000. Role of the Pseudomonas aeruginosa oxyR-recG operon in oxidative stress defense and DNA repair: OxyR-dependent regulation of katB-ankB, ahpB, and ahpC-ahpF. J. Bacteriol. 182:4533-4544[Abstract/Free Full Text].
27.	Que, L., Jr., J. Widom, and R. L. Crawford. 1981. 3,4-Dihydroxyphenylacetate 2,3-dioxygenase. A manganese(II) dioxygenase from Bacillus brevis. J. Biol. Chem. 256:10941-10944[Abstract/Free Full Text].
28.	Rothmel, R. K., A. M. Charkabarty, A. M. Berry, and A. Darzins. 1991. Genetic systems in Pseudomonas. Methods Enzymol. 204:485-514[Medline].
29.	Schellhorn, H. E., and H. M. Hassan. 1988. Transcriptional regulation of katE in Escherichia coli K-12. J. Bacteriol. 170:4286-4292[Abstract/Free Full Text].
30.	Schweizer, H. P. 1991. Improved broad-host-range lac-based plasmid vectors for the isolation and characterization of protein fusions in Pseudomonas aeruginosa. Gene 103:87-92[CrossRef][Medline].
31.	Schweizer, H. P. 1993. Small broad-host-range gentamicin resistance gene cassettes for site-specific insertion and deletion mutagenesis. BioTechniques 15:831-833[Medline].
32.	Schweizer, H. P. 1993. Two plasmids, X1918 and Z1918, for easy recovery of the xylE and lacZ reporter genes. Gene 134:89-91[CrossRef][Medline].
33.	Shu, L., Y. M. Chiou, A. M. Orville, M. A. Miller, J. D. Lipscomb, and L. Que, Jr. 1995. X-ray absorption spectroscopic studies of the Fe(II) active site of catechol 2,3-dioxygenase. Implications for the extradiol cleavage mechanism. Biochemistry 34:6649-6659[CrossRef][Medline].
34.	Touati, D. 1988. Transcriptional and posttranscriptional regulation of manganese superoxide dismutase biosynthesis in Escherichia coli, studied with operon and protein fusions. J. Bacteriol. 170:2511-2520[Abstract/Free Full Text].
35.	Wang, Y. Z., and J. D. Lipscomb. 1997. Cloning, overexpression, and mutagenesis of the gene for homoprotocatechuate 2,3-dioxygenase from Brevibacterium fuscum. Protein Expr. Purif. 10:1-9[CrossRef][Medline].
36.	Whiting, A. K., Y. R. Boldt, M. P. Hendrich, L. P. Wackett, and L. Que, Jr. 1996. Manganese(II)-dependent extradiol-cleaving catechol dioxygenase from Arthrobacter globiformis CM-2. Biochemistry 35:160-170[CrossRef][Medline].
37.	Wolgel, S. A., J. E. Dege, P. E. Perkins-Olson, C. H. Juarez-Garcia, R. L. Crawford, E. Münck, and J. D. Lipscomb. 1993. Purification and characterization of protocatechuate 2,3-dioxygenase from Bacillus macerans: a new extradiol catecholic dioxygenase. J. Bacteriol. 175:4414-4426[Abstract/Free Full Text].

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