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. 2001 Aug;183(15):4405-12.
doi: 10.1128/JB.183.15.4405-4412.2001.

Complex regulation of the organic hydroperoxide resistance gene (ohr) from Xanthomonas involves OhrR, a novel organic peroxide-inducible negative regulator, and posttranscriptional modifications

R Sukchawalit  1 , S Loprasert, S Atichartpongkul, S Mongkolsuk
Affiliations

Complex regulation of the organic hydroperoxide resistance gene (ohr) from Xanthomonas involves OhrR, a novel organic peroxide-inducible negative regulator, and posttranscriptional modifications

R Sukchawalit et al. J Bacteriol. 2001 Aug.

Abstract

Analysis of the sequence immediate upstream of ohr revealed an open reading frame, designated ohrR, with the potential to encode a 17-kDa peptide with moderate amino acid sequence homology to the MarR family of negative regulators of gene expression. ohrR was transcribed as bicistronic mRNA with ohr, while ohr mRNA was found to be 95% monocistronic and 5% bicistronic with ohrR. Expression of both genes was induced by tert-butyl hydroperoxide (tBOOH) treatment. High-level expression of ohrR negatively regulated ohr expression. This repression could be overcome by tBOOH treatment. In vivo promoter analysis showed that the ohrR promoter (P1) has organic peroxide-inducible, strong activity, while the ohr promoter (P2) has constitutive, weak activity. Only P1 is autoregulated by OhrR. ohr primer extension results revealed three major primer extension products corresponding to the 5' ends of ohr mRNA, and their levels were strongly induced by tBOOH treatment. Sequence analysis of regions upstream of these sites showed no typical Xanthomonas promoter. Instead, the regions can form a stem-loop secondary structure with the 5' ends of ohr mRNA located in the loop section. The secondary structure resembles the structure recognized and processed by RNase III enzyme. These findings suggest that the P1 promoter is responsible for tBOOH-induced expression of the ohrR-ohr operon. The bicistronic mRNA is then processed by RNase III-like enzymes to give high levels of ohr mRNA, while ohrR mRNA is rapidly degraded.

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Figures

FIG. 1
FIG. 1
OxyR-independent tBOOH induction of ohr. Northern analysis of ohr and ahpC expression in uninduced (U) or tBOOH-induced (T) cultures of X. campestris pv. phaseoli (Xp) and an oxyR mutant (Xp oxyR) is shown. The arrowheads to the left and right indicate the positions of ohr and ahpC mRNAs, respectively.
FIG. 2
FIG. 2
Phylogenetic tree for OhrR and other members of the MarR family. Analysis and construction of the tree were performed as described in Materials and Methods. Proteins, GenBank accession numbers (in parentheses), and organisms are as follows: BadR (U75363), Rhodopseudomonas palustris; HpcR (S56952), E. coli; MarR (P27245), E. coli; MexR (U23763), P. aeruginosa; MoaI (D63524), Klebsiella aerogenes; OhrR (AF036166), X. campestris pv. phaseoli (this study); OhrR-Ac (Y09102), Acinetobacter sp.; OhrR1-Pa (D83290) and OhrR2-Pa (G83292), P. aeruginosa; ORF145 (Y13052), Staphylococcus sciuri; OhrR-Vc (B82389), V. cholerae; OhrR-Sc (AL163672), S. coelicolor; PecS (P42195), Erwinia chrysanthemi; SlyA (P40676), Salmonella enterica serovar Typhimurium; YdgJ (D69783), YhbI (Z99108), and YkmA (E69857), B. subtilis. The bar indicates genetic distance.
FIG. 3
FIG. 3
Gene order and transcriptional organization of ohrR-ohr. (A) The bars above the map of ohrR-ohr indicate the locations and sizes of the fragments used in the construction of ohrR mutants (Xp designations). The sizes and directions of the arrows represent the amounts and directions of transcription, respectively. Hc, HincII; K, KpnI; N, NotI; P, PstI; ScI, SacI; ScII, SacII. (B) Northern analysis of ohrR and ohr. Ten micrograms of RNA samples from tBOOH-induced cultures were separated in formaldehyde-agarose gels, and the RNA was transferred to nylon membranes. The membranes were hybridized separately to radioactively labeled ohrR or ohr probes. The arrowheads indicate monocistronic ohr mRNA and bicistronic ohrR-ohr mRNA. (C) RT-PCR of RNA samples from tBOOH-induced X. campestris pv. phaseoli cultures. RNA extraction and DNase I treatment were done as described in Materials and Methods. The conditions for PCR and the primers used are described in Materials and Methods. Lane M, molecular weight markers; lane 1, PCR of a positive control DNA sample; lane 2, RT-PCR of an RNA sample from tBOOH-induced X. campestris pv. phaseoli cultures; lane 3, the same RNA sample and PCR conditions as in lane 2 except that the RT step was omitted; lane 4, PCR of reagents (negative control).
FIG. 4
FIG. 4
Northern analysis of the effects of ohrR on ohr expression. Northern blotting of various X. campestris pv. phaseoli cells (Xp designations) was performed as described in Materials and Methods. The Northern blot was probed with radioactively labeled ohr. U, uninduced; T, tBOOH induced; C, CuOOH induced.
FIG. 5
FIG. 5
In vivo ohrR and ohr promoter analysis. Cat levels were determined by Western immunoblotting performed as described in Materials and Methods. Forty micrograms of total protein was loaded in each lane. U and I, lysates prepared from uninduced and tBOOH-induced cultures, respectively. (A) Analysis of in vivo promoter activities of ohrR (pP1) and ohr (pP2). (B) Effects of OhrR on pP1 and pP2. Western analysis of Cat levels in various strains (X. campestris pv. phaseoli [Xp] and ohrR mutant [Xp ohrR1] harboring pP1 or pP2 and with or without pBBRohrR) is shown.
FIG. 6
FIG. 6
Primer extension analysis of ohr mRNA and proposed processing sites of bicistronic ohrR-ohr mRNA.(A) Primer extension was performed with 10 μg of RNA isolated from uninduced (U) or tBOOH-induced (O) X. campestris pv. phaseoli cultures. G, A, T, and C are the sequence ladder. (B) Stem-loop secondary structure of the region around the putative RNA processing sites of the ohrR-ohr bicistronic mRNA. RBS, ribosome binding site. The arrows mark the locations of primer extension products in panel A and the locations of putative RNA processing sites in panel B.
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