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. 2010 Jan;192(2):381-90.
doi: 10.1128/JB.00980-09. Epub 2009 Nov 20.

The Major Catalase Gene (katA) of Pseudomonas Aeruginosa PA14 Is Under Both Positive and Negative Control of the Global Transactivator OxyR in Response to Hydrogen Peroxide

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The Major Catalase Gene (katA) of Pseudomonas Aeruginosa PA14 Is Under Both Positive and Negative Control of the Global Transactivator OxyR in Response to Hydrogen Peroxide

Yun-Jeong Heo et al. J Bacteriol. .
Free PMC article

Abstract

The adaptive response to hydrogen peroxide (H(2)O(2)) in Pseudomonas aeruginosa involves the major catalase, KatA, and OxyR. However, neither the molecular basis nor the relationship between the aforementioned proteins has been established. Here, we demonstrate that the transcriptional activation of the katA promoter (katAp) in response to H(2)O(2) was abrogated in the P. aeruginosa PA14 oxyR null mutant. Promoter deletion analyses revealed that H(2)O(2)-mediated induction was dependent on a region of DNA -76 to -36 upstream of the H(2)O(2)-responsive transcriptional start site. This region harbored the potential operator sites (OxyR-responsive element [ORE]) of the Escherichia coli OxyR binding consensus. Deletion of the entire ORE not only abolished H(2)O(2)-mediated induction but also elevated the basal transcription, suggesting the involvement of OxyR and the ORE in both transcriptional activation and repression. OxyR bound to the ORE both in vivo and in vitro, demonstrating that OxyR directly regulates the katAp. Three distinct mobility species of oxidized OxyR were observed in response to 1 mM H(2)O(2), as assessed by free thiol trapping using 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid. These oxidized species were not observed for the double mutants with mutations in the conserved cysteine (Cys) residues (C199 and C208). The uninduced transcription of katAp was elevated in an oxyR mutant with a mutation of Cys to serine at 199 (C199S) and even higher in the oxyR mutant with a mutation of Cys to alanine at 199 (C199A) but not in oxyR mutants with mutations in C208 (C208S and C208A). In both the C199S and the C208S mutant, however, katAp transcription was still induced by H(2)O(2) treatment, unlike in the oxyR null mutant and the C199A mutant. The double mutants with mutations in both Cys residues (C199S C208S and C199A C208S) did not differ from the C199A mutant. Taken together, our results suggest that P. aeruginosa OxyR is a bona fide transcriptional regulator of the katA gene, sensing H(2)O(2) based on the conserved Cys residues, involving more than one oxidation as well as activation state in vivo.

Figures

FIG. 1.
FIG. 1.
Promoter region of the katA gene. (A) The transcriptional start site of the katA gene was determined by high-resolution S1 nuclease mapping. Total RNA (50 μg) was prepared from cells grown to the mid-logarithmic (Log; OD600 = 0.3) and the stationary (Stat; OD600 = 3.0) growth phases, with or without 1 mM H2O2 treatment for 10 min. The sequencing ladder (C, T, A, and G) was generated as described in Materials and Methods. The transcriptional start site is indicated by the arrow, and the putative −10 box is designated. (B) The alignment of the katA promoter region with the P. aeruginosa OxyR-regulated promoters (ahpB, ahpC, and katB) and the E. coli OxyR-regulated promoters (ahpC and oxyS) is shown, with the OxyR binding consensus indicated (O1 to O4; ATAG-t-N5-a-CTAT-N7-ATAG-t-N5-a-CTAT). The strongly conserved nucleotides are designated by underlining, and the weakly conserved nucleotides are indicated by lowercase letters within the consensus. The −35 and −10 boxes and the experimentally determined +1 site are indicated. The heavy line above the katAp sequence indicates the PhoB binding site (60). N21(−76), N22(−56), and N23(−35) are the truncated promoter series, with the 5′ ends (see Fig. 2) indicated by the bent arrows. The numbers represent the nucleotide positions relative to the transcriptional start site.
FIG. 2.
FIG. 2.
Effects of deletions on the katA promoter activity. (A) Schematic representations of promoter deletions, with the OxyR consensus (ORE) and PhoB box (Pho-box) shown. (B) β-Galactosidase (β-gal) assay. The β-galactosidase activities were determined for the wild-type cells harboring one of the pQF50-derived lacZ fusions with the full-length promoter construct (N10) containing a potential inverted repeat (Ω) and for deletion mutants encompassing the intact ORE (N21) or the O1- and O2-truncated ORE (N22) or missing the entire ORE (N23). Cells were treated with (+) or without (−) 1 mM H2O2 for 20 min at the mid-logarithmic growth phase. β-Galactosidase activities from the mutant promoters are expressed as Miller units, with standard deviations from the three independent experiments. The statistical significance based on Student's t test is indicated as follows: *, P < 0.005; **, P < 0.001.
FIG. 3.
FIG. 3.
H2O2-induced transcription of katAp requires OxyR. (A) β-Galactosidase (β-gal) activities driven from the katA N10 promoter (Fig. 2) were determined for PA14 (WT) and various mutants (oxyR, rpoS, phoB, lasR rhlR, mvfR, and oxyR mutants complemented with mTn7-oxyR) treated with (+) or without (−) 1 mM H2O2 for 20 min. The cells were grown as described in the legend for Fig. 2. The β-galactosidase activities are represented in Miller units, with standard deviations from the three independent experiments. The statistical significance based on Student's t test is indicated as follows: *, P < 0.005; **, P < 0.001. (B) The KatA protein level in the oxyR mutant was determined by Western blot analysis. Both PA14 (WT) and oxyR mutant cells were grown to the mid-logarithmic growth phase (OD600 = 0.3) and treated with 1 mM H2O2. Total protein (50 μg) was prepared from the cells harvested before treatment and at 10, 20, and 30 min after the H2O2 treatment, followed by Western blotting with anti-KatA and anti-RpoA antisera.
FIG. 4.
FIG. 4.
OxyR binds the katA promoter region in vitro and in vivo. (A, B, and C) The indicated amounts of the purified OxyR proteins were treated with 1 mM dithiothreitol and then incubated with 10 fmol of the radiolabeled katA promoter fragments N21 (340 bp) and N23 (199 bp) and the synthetic ORE consensus oligonucleotide (50 bp). The radiolabeled pelA promoter fragment (164 bp) was included as the negative control in the binding reaction. The open arrowheads indicate the free probes, and the filled arrowheads indicate the OxyR-bound complexes. The numbers indicate the amounts of OxyR (pmol) in 20 μl of binding buffer, with 9 pmol corresponding to 450 nM. (D) DNase I footprinting analysis of OxyR, tested under the same conditions described above. The DNA probes (339 bp) radiolabeled at the 5′ end of the bottom strand were incubated with increasing amounts of OxyR as indicated, followed by DNase I treatment. The samples were run on a 6% polyacrylamide sequencing gel with the corresponding sequencing ladder. The region protected by oxidized OxyR is indicated by a thick solid line, with the slightly protected region overlapping with the −35 box indicated by a dashed line. The ORE (O1 to O4) and the promoter elements (−35 and −10 boxes and +1 site) are designated. (E) The wild-type and the oxyR DNA-binding domain (S33N) mutant bacteria were grown to the late logarithmic growth phase (OD600 = 0.7), treated with (+) or without (−) 10 mM H2O2 for 1 min, and then subjected to the chromatin immunoprecipitation assay as described in Materials and Methods. The samples were precipitated with either anti-OxyR antiserum (O) or preimmune serum (S) (as the negative template control). Then, the nonprecipitated, input samples (I) and the precipitated samples were analyzed by PCR targeting the katA, ahpC (positive control), and pelA (negative control) promoter regions.
FIG. 5.
FIG. 5.
H2O2-induced modification of OxyR in vivo. Total protein (50 μg) from the oxyR null mutants containing either the FLAG-tagged OxyR protein (WT) or one of the FLAG-tagged OxyR muteins with mutations in the conserved Cys residues (C199S, C208S, and C199S C208S) that were harvested before (0 min) or after (1, 5, and 10 min) treatment with 1 mM H2O2 and alkylated using 10 mM 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid (AMS) was used to monitor the OxyR status, based on Western blotting using the anti-FLAG antiserum. The single band for the WT protein in the uninduced condition (0 min) (∼36 kDa, species R), the two faster-migrating bands (species I and II), and the slower-migrating band (∼50 kDa, species III) under the H2O2-induced conditions are designated.
FIG. 6.
FIG. 6.
katA transcription in oxyR cysteine mutants. (A) Total proteins from the wild type (PA14) and the oxyR mutants (oxyR null, C199A, C199S, C208S, C199A C208S, and C199S C208S mutants) grown to the mid-logarithmic growth phase (OD600 = 0.3) with (+) or without (−)1 mM H2O2 treatment for 20 min were analyzed by Western blotting. Fifty micrograms of total proteins was used for Western blot analysis using anti-KatA, anti-RpoA, or anti-OxyR antiserum. (B) Fifty micrograms of total RNA from the wild type (PA14) and the oxyR mutants (oxyR null, C199A, C199S, C208S, C199A C208S, and C199S C208S mutants) that had been grown to the mid-logarithmic growth phase (OD600 = 0.3) with 1 mM H2O2 and harvested before treatment and at 10 and 20 min after H2O2 treatment was used for the katA, katB, and rpoA transcript analysis by S1 nuclease protection. The probes were prepared as described in Materials and Methods.

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