In vitro transcription analysis of rpoD in Pseudomonas aeruginosa PAO1.

The rpoD gene encoding the principal sigma factor (sigma(70)) of Pseudomonas aeruginosa is transcribed from two promoters, P(C) and P(HS). The sequence of P(C) is similar to the Escherichia coli sigma(70) consensus promoter sequence and that of P(HS) is similar to the E. coli sigma(H) consensus promoter sequence. Synthesis of rpoD mRNA from P(C) is constitutive under both steady-state and heat-shock growth conditions, while that of P(HS) is transiently induced upon heat-shock. To gain a better understanding of the regulation of rpoD expression, we examined in vitro transcription of rpoD using two RNA polymerases (Esigma(70) and Esigma(H), containing sigma(70) and sigma(H), respectively) purified from P. aeruginosa. DNase I footprinting analysis showed specific bindings of Esigma(70) and Esigma(H) to P(C) and P(HS) promoter regions, respectively. In the in vitro runoff transcription assay, Esigma(H) transcribed the template from P(HS) both at 30 degrees C and 42 degrees C but not from P(C). However, Esigma(70) transcribed rpoD not only from P(C) both at 30 degrees C and 42 degrees C but also from P(HS) at 42 degrees C.