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  • br Materials and methods Transformation was


    Materials and methods Transformation was performed by electroporation [24]. The EZ-Tn5™ transposon mutants in A. baumannii ATCC 17978 were generated by using the EZ-Tn5™ insertion kit (Epicentre Biotechnologies) as previously described [11]. Surface-associated motility. Motility plates were composed of 0.5% agarose (w/v), 5 g/L of tryptone, and 2.5 g/L of NaCl as previously described [11]. A single colony from a nutrient agar plate (Oxoid) or selective agar plates (supplemented with 50 μg/mL of kanamycin for the A1S_0222 mutant) of either wild-type or A. baumannii ATCC 17978 mutant was taken with the pipette tip and then the surface of a motility plate was touched. Pictures were taken after incubating the plates for 16 h at 37 °C. Construction of protein expression plasmids. The a1s_0222 gene of A. baumannii ATCC 17978 was amplified by PCR using the oligonucleotides 0222-pGEX-6P-3-for: 5′-ATTAGGATCCAATTCAGAGCCTTCGGTATACCAC-3’ (BamHI restriction site underlined) and 0222-pGEX-6P-3-rev: 5′-ATTAGCGGCCGCTTACCAAAGTGCGAGCTGTGTAC-3’ (NotI restriction site underlined). The amplified a1s_0222 gene was inserted into pGEX-6P-3 expression vector (GE Healthcare) after HATU of insert and vector with BamHI and NotI restriction enzymes. The pGEX-6P-3 expression vector carries a glutathione S-transferase (GST-tag) gene, a PreScission protease restriction site, an ampicillin resistance (AmpR) cassette, encodes a tac promotor and is inducible with isopropyl β-d-1-thiogalactopyranoside (IPTG). The plasmid (pGEX-6P-3-A1S_0222) was confirmed by DNA sequencing and transformed into E. coli BL21 (DE3) pLysS expression strain. Protein expression and purification. A 3 mL overnight culture of E. coli BL21 (DE3) pLysS pGEX-6P-3-A1S_0222 was grown in Luria-Bertani (LB)-Medium (10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl, pH 7.4, 100 μg/mL Amp) at 37 °C and 160 rpm. The overnight culture was diluted 1:100 into 200 mL LB (supplemented with 100 μg/mL Amp) and cultured in a 2 L bottle-flask at 20 °C and 160 rpm for 5 h. Expression was induced by addition of 0.05 mM IPTG and cultures were incubated for 16 h at 20 °C and 160 rpm. Cells were then harvested by centrifugation (10,000 × g for 30 min at 4 °C) and the resulting cell pellets were frozen at −80 °C. Cell pellets were solubilized at 4 °C in 20 mL disruption buffer (300 mM NaCl, 1 mM DTT (1,4-dithiothreitol), 5 mM EGTA (ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid), 1 mM EDTA HATU (2,2′,2″,2‴-(ethane-1,2-diyldinitrilo) tetraacetic acid), 2 μL benzonase nuclease (250 U/μL), pH 7.4. The cells were lysed by applying three passages through an EmulsiFlex-C3 homogenizer (Avestin). Lysates were then centrifuged at 20,000 × g for 50 min at 4 °C. The soluble lysate was then added to a GSTPrep™ FF 16/10 column (GE Healthcare Life Sciences). GST affinity chromatography. The GSTPrep™ FF 16/10 column (bed volume 20 mL) was equilibrated with 5 column volumes (CV) of GST binding buffer (300 mM NaCl, 1 mM DTT, 5 mM EGTA, 1 mM EDTA, 5% glycerol (v/v), pH 7.4). The soluble lysate was added to the column and washed with 10 CV of GST binding buffer and eluted with 7 CV of GST elution buffer (300 mM NaCl, 50 mM Tris, 1 mM DTT, 5 mM EGTA, 1 mM EDTA, 5% glycerol (v/v), 10 mM reduced l-glutathione (GSH reduced), pH 7.4). Eluted fractions were analyzed by SDS-PAGE and appropriate fractions were pooled. The GST fusion protein was then incubated for 16 h at 8 °C with 40 units PreScission protease per 14.52 mg recombinant A1S_0222 to cleave off the GST tag (26 kDa). After incubation the pooled fractions were centrifuged at 20,000 g for 20 min at 4 °C and diluted 1:4 in dilution buffer (50 mM Tris, 1 mM EDTA, 1 mM DTT, 5% glycerol (v/v)).