Transcription analysis of rpoH in Pseudomonas putida

We previously determined the complete DNA sequence of the rpoH gene encoding the heat-shock sigma factor (sigmaH) of Pseudomonas putida. In the present study, the transcriptional start sites of rpoH were determined to be 41 nucleotides (T1), 153 nucleotides (T2) and 157 nucleotides (T3) upstream from the translational start codon (AUG) of rpoH by rapid amplification of cDNA 5'-ends. Based on the locations of T2 and T3, a sigma70-type promoter (P2) was determined to be located in the open reading frame region of upstream ftsX in addition to the sigmaE-type promoter (P1; DNA Res. 6 (1999) 241). In the in vitro transcription assay with reconstituted RNA polymerases (Esigma70, EsigmaE, EsigmaH and EsigmaS) of Pseudomonas aeruginosa, EsigmaE transcribed rpoH from T1 and Esigma(70) transcribed it from T2 and T3. In both cases, the level of transcription was higher at 42 degrees C than at 30 degrees C. No transcript was detected when EsigmaH or EsigmaS was used. These results indicate that EsigmaE and Esigma70 recognize P1 promoter and P2 promoter, respectively, and also prove that the synthesis of rpoH mRNA is inducible upon heat shock.