The PcfaB mutant promoters were generated by overlap extension PCR mutagenesis as described previously (Gallegos et al., 1996). The internal oligonucleotides used for mutagenesis exhibited one mismatch with respect to the wild-type sequence (primer sequences will be made available upon request); the external primers were EcoRIcfaB2 and PstIcfaB2. The PCR fragments were cut with EcoRI and PstI and cloned into pMP220 (Spaink et click here al., 1987), previously cut with the same enzymes, to construct the plasmid
pMPcfaBKT2440. This plasmid was electroporated into P. putida KT2440 and into P. putida C1R1, a P. putida KT2440 RpoS mutant (Ramos-González & Molin, 1998). Cultures were grown overnight at 30 °C in LB medium plus tetracycline, and the following morning, were diluted to an OD660 nm of 0.1. β-Galactosidase activity was measured along the growth curve. Phenylacetate (20 mM) was added when the cultures reached the early stationary phase (OD660 nm 2) and β-galactosidase activity was measured 1 h after the addition of this stressor. Pseudomonas putida KT2440 was grown in LB medium and samples were taken at different
points along the growth curve. RNA isolation from www.selleckchem.com/PD-1-PD-L1.html the pellets was performed by TRIzol reagent (Invitrogen). The RNA samples were treated with DNase I (1 U/5 μg RNA) (Roche) at 37 °C for 1 h. Agarose gel electrophoresis and quantification at 260 and 280 nm were performed to assess the integrity and purity of the RNA. The different RNA samples were diluted to a final concentration of 1 μg μL−1 2-hydroxyphytanoyl-CoA lyase and used to synthesize cDNA using 200 U of Superscript IIa reverse transcriptase (Invitrogen) in a mixture containing 25 ng of random primers, 10 mM of dNTP Mix (Roche) and 40 U of RNase OUT (Roche), following the manufacturer’s instructions. Serial dilutions (1/5; 1/25; 1/125) of the cDNA samples were carried
out. Three microliters of the 16S cDNA dilutions and 5 μL of the cti and cfa cDNA dilutions were used to perform real-time PCR using 12.5 μL of IQ™ SYBR® Green Supermix (BioRad) in a 25 μL reaction containing 600 nM of the appropriate primer. Amplification and detection of specific products was performed using the BioRad-IQ5 system with the following profile: one cycle at 95 °C for 5 min plus 40 amplification cycles (95 °C for 10 s, 57 °C for 30 s, 72 °C for 30 s). Amplification was carried out in triplicate for each cDNA preparation. Controls without a template or with the sample before the reverse transcription were included for each reaction on the same plate. The critical threshold cycle (CT) is defined as the cycle at which the fluorescence becomes detectable above background. All values were compared using the CT method, where the fluorescence of each gene () was normalized to the housekeeping gene 16S (ΔCT).