Temperature-dependent expression of PA5194 and ptxS in P. aeruginosa. Proteins (A) and RNA (B) were extracted upon induction with 0.1% arabinose, as detailed in Materials and Methods, from cultures of PAO1 carrying either pPA5194-HA (left) or pPtxS-HA (right). Time and temperature of incubation are indicated above the lanes. (A) Proteins were separated by 10% SDS-PAGE, blotted onto an Hybond ECL filter, and hybridized with HA- or S1-specific antibodies, as indicated beside the panels. No bands were detected in the lanes loaded with not induced samples (data not shown). The PA5194-HA (expected MW, 33.2 kDa) and PtxS-HA (expected MW, 39.9 kDa) migrate in this kind of gels slightly slower than the 28 and 36 kDa bands of the PageRuler Plus Prestained Ladder (ThermoScientific), respectively. (B) The RNA was run on a 5% denaturing urea-polyacrylamide gel, blotted onto a nylon Hybond N membrane and hybridized with either the radiolabeled HA oligonucleotide (PA5194) or the PTXS riboprobe. Migration of the 1.0 and 1.5 kb bands of RiboRuler High Range RNA ladder (ThermoScientific) are reported beside the panels. (C) Northern blot analysis of PA5194 and ptxS endogenous mRNAs. Cultures of PAO1 were grown overnight in LD at 37°C and diluted to OD600 = 0.2 in three flasks containing LD. The flasks were incubated at 25°C–37°C–40°C with agitation up to OD600 = 0.8. Ten milliliters of samples were taken for RNA extraction. Twenty micrograms of RNA were run on a 5% denaturing urea-polyacrylamide gel, blotted onto a nylon Hybond N membrane, and hybridized with either the radiolabeled PA5194 or the PTXS riboprobes. Migration of the 1.0- and 0.5-kb band of RiboRuler High Range RNA ladder (ThermoScientific) is reported on the left of the panels. The numbers below the panels represent relative expression (RE) of each mRNA with respect to its expression at 25°C. 5S rRNA signals were used for normalization.
