Gel shift kit

The open reading frame of oqxR was cloned into a pET28 expression vector, followed by induction of expression in E. coli strain BL21. The expressed protein was purified by using a nickel-chelated NTA column and performing size exclusion chromatography in an ÄKTA protein purification system. The promoter region of oqxA was amplified from K. pneumoniae MGH78578 by PCR using primers listed in Table S2. EMSA was performed using a Gel Shift Kit, 2nd generation (Roche). Briefly, amplified DNA was labeled with digoxigenin and incubated with OqxR at room temperature for 30 min, followed by electrophoresis in a precast 6% acrylamide non-denaturing gel with ice-cold Tris-borate-EDTA buffer. The contents were transferred to a nylon membrane by a semi-dry electrotransfer system, followed by UV cross-linking. The membrane was probed against anti-dig antibody prior to addition of the substrate for chemiluminescence detection.

For the electrophoretic mobility shift assay (EMSA), a 50 bp nif promoter fragment (from −47 to +3 relative to the transcription start site of nifB in P. sabinae T27) was synthesized by Sangon Biotech Co., Ltd (Shanghai). To do this, two DNA fragments corresponding to the sequences of the first strand (5′- GGAGAAGTGAATTGACTGTATTTGTCCCTGTCTCTAAGA-TGTAATTATAT-3′) and the complementary DNA strand (5′- ATATAATTACATCTTAGAGAC-AGGGACAAATACAGTCAATTCACTTCTCC-3′) were synthesized. The two strands were annealed and then labeled with digoxin using the DIG gel shift kit (Roche). The binding shift experiment of E. coli σ⁷⁰-RNAP (RNA polymerase) (Epicentre) or σ⁷⁰ of P. sabinae T27 to the nif promoter was carried out using a gel shift kit (Roche). At the same time, a scrambled 39 bp DNA fragment formed by annealing the following complementary oligonucleotides (5′- GTACGGAGTATCCAGCTCCGTAGCATGCAAATCCTCTGG-3′) and (5′-CCAGAGGATTTGCATGCTACGGAGCTGGATACTCCGTAC -3′) was used to assay non-specific binding.