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 PHS is transiently induced upon heat-shock. To gain a better understanding of the regulation of r(POD) expression, we examined in vitro transcription of rpoD using two RNA polymerases (E sigma(70) and E sigma(H), containing sigma(70) and sigma(H), respectively) purified from P. aeruginosa. DNase I footprinting analysis showed specific bindings of E sigma(70) and E sigma(H) to P-C and P-HS promoter regions, respectively. In the in vitro runoff transcription assay, E sigma(H) transcribed the template from P-HS both at 30 degrees C and 42 degrees C but not from P-C. However, E sigma(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. (C) 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.