Molecular characterization of Pseudomonas putida KT2440 rpoH gene regulation

摘要

The rpoH gene of Pseudomonas putida KT2440 encoding the heat-shock sigma factor σ32 was cloned and sequenced, and the translated gene product was predicted to be a protein of 32·5?kDa. The unambiguous role of the gene as a sigma factor was confirmed because the cloned P. putida gene complemented the growth defect, at 37 and 42?°C, of an Escherichia coli rpoH mutant strain. Primer extension analysis showed that in P. putida the rpoH gene is expressed from three promoters in cells growing at 30?°C. Two of them, P1 and P3, share homology with the σ70-dependent promoters, while the third one, P2, shows a typical σ24-consensus sequence. The pattern of transcription initiation of the rpoH gene did not change in response to different stresses, i.e. a sudden heat shock or the addition of aromatic compounds. However, the predicted secondary structure of the 5′ region of the mRNA derived from the three different promoters suggests regulation at the level of translation efficiency and/or mRNA half-life. An inverted repeat sequence located 20?bp downstream of the rpoH stop codon was shown to function as a terminator in vivo in P. putida growing at temperatures from 18 to 42?°C.