Article Figures & Data
Figures
- Fig. 1.
OxyR-independent tBOOH induction of ohr. Northern analysis of ohr and ahpCexpression in uninduced (U) or tBOOH-induced (T) cultures of X. campestris pv. phaseoli (Xp) and anoxyR mutant (Xp oxyR) is shown. The arrowheads to the left and right indicate the positions ofohr and ahpC mRNAs, respectively.
- 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 ), Acinetobactersp.; 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 entericaserovar Typhimurium; YdgJ (D69783 ), YhbI (Z99108 ), and YkmA (E69857 ), B. subtilis. The bar indicates genetic distance.
- Fig. 3.
Gene order and transcriptional organization ofohrR-ohr. (A) The bars above the map ofohrR-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.
Northern analysis of the effects of ohrRon ohr expression. Northern blotting of variousX. campestris pv. phaseoli cells (Xpdesignations) 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.
In vivo ohrR and ohrpromoter 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) andohr (pP2). (B) Effects of OhrR on pP1 and pP2. Western analysis of Cat levels in various strains (X. campestrispv. phaseoli [Xp] and ohrR mutant [Xp ohrR1] harboring pP1 or pP2 and with or without pBBRohrR) is shown.
- Fig. 6.
Primer extension analysis of ohr mRNA and proposed processing sites of bicistronic ohrR-ohrmRNA.(A) Primer extension was performed with 10 μg of RNA isolated from uninduced (U) or tBOOH-induced (O) X. campestrispv. 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.
