γ 32p atp and t4 polynucleotide kinase

The promoters of phoP and pbgP1 were amplified by PCR from the genomic DNA of TT01 with the primers (Supplementary Table 5), and purified with the High Pure PCR Product Purification kit (ROCHE). The 5′ ends of the DNA were labeled with [γ-³²P] ATP and T4 polynucleotide kinase (Promega). The PhoP-His protein was purified and phosphorylated in vitro by incubation with acetyl phosphate56 . Radioactive DNA probe (2000 cpm.ml⁻¹), 200 ng of poly(dI-dC)-poly(dI-dC) (SIGMA) and various amounts of PhoP-His were mixed in binding buffer (50 mM Tris-HCl pH 8, 50 mM KCl, 50 μg.ml⁻¹ BSA), in a total volume of 20 μl, and incubated for 20 minutes at room temperature. The mixture was then loaded onto a native 6% (w/v) polyacrylamide TBE precast gel (Invitrogen) and subjected to electrophoresis in 1% TBE (Tris-borate-EDTA) buffer for 1 h at 100 V. Radioactive species were detected by autoradiography. PhoP-His was activated by in vitro phosphorylation with acetyl phosphate57 (link).

Nuclear extracts were obtained as described before (Romacho et al., 2009 (link)). For electromobility shift assay (EMSA), a commercial oligonucleotide (Promega, Madison, WI, USA) encoding the NF-κB consensus sequence (5′-AGTTGAGGGGACTTTCCCAGGC-3′) was 5′-end labeled using [γ-³²P]ATP and T4 polynucleotide kinase (Promega, Madison, WI, USA). For binding reactions, nuclear extracts (5 μg) were incubated on ice for 15 min in a reaction buffer [40 mM HEPES (pH 7.0), 140 mmol/L NaCl, 5 mM dithiothreitol, 10 μg/mL BSA, 0.01% Nonidet P-40, 4% Ficoll, and 0.05 μg/mL poly(dI-dC).poly(dI-dC)]. After addition of the labeled oligonucleotide (∼50,000 cpm) the reaction mix was further incubated for 20 min at room temperature. For competition experiments a 100-fold excess of unlabeled doubled-stranded oligonucleotide was added to the binding reaction. DNA-protein complexes were resolved on 4% non-denaturing polyacrylamide gels in 0.5x TBE (45 mmol/L Tris-borate, 1 mM EDTA, pH 8.0) at 4°C. Gels were dried and exposed to autoradiography at -80°C. The intensity of the resulting bands was quantified by densitometry, as previously described (Romacho et al., 2009 (link)), using Image J free software. The DNA binding activity of NF-κB was abolished by PDTC (100 μM).

Total RNA was extracted as reported previously (Ferrara et al., 2012 (link)). RNA for RNA/RNA interaction assays was prepared by T7 RNA polymerase transcription of gel-purified DNA fragments. DNA fragments for ErsA RNA and amrZ mRNAs (amrZ, amrZCIS1, amrZΔIS2, amrZCIS1ΔIS2) preparations were amplified from P.aeruginosa PAO1 genomic DNA with oligo pairs 4/5 or 4/6 and 7/8, respectively. The transcription reactions were performed using the Riboprobe^® System-T7 (Promega) with 300 ng of DNA template. DNA probe was 5′-end–labeled with (γ-³²P) ATP and T4 polynucleotide kinase (Promega) according to manufacturer’s instruction. Synthesized RNA was precipitated and resuspended in diethylpyrocarbonate-treated water. Purified RNA was checked by denaturing polyacrylamide gel electrophoresis and quantified using a Qubit Fluorometer.