Apai restriction enzyme

Genotyping and the clonal relationship between A. baumannii strains were determined by pulsed-field gel electrophoresis (PFGE) using ApaI restriction enzyme (Thermo Fisher Scientific, USA) for digestion. The Lambda Ladder 48.5–727.5 kb PFG Marker (New England Biolabs, US) was used as DNA size marker. DNA fragments were separated in 1% ultra-pure agarose gels (Invitrogen) using the CHEF Mapper apparatus (Bio-Rad, Munich, Germany) under the following conditions: voltage 6 V/cm; temperature at 14 °C; with switch times ranging from 5 s to 30s; at an angle of 120°, for 19 h. After that, the gels were stained by ethidium bromide, and the DNA bands were visualized and photographed under UV transilluminator. DNA banding pattern images were analyzed using Bionumeric 7.6.3 software (Applied Maths NV, St-Martens-Latem Belgium) and the dendrogram was constructed by UPGMA method (Unweighted Pair Group Method with Arithmetic Mean), based on Dice’s similarity coefficient at a 1.5% tolerance [6 (link)]. The similarity of 80% or higher was defined as the same PFGE genotype, whereas the similarity of < 80% indicated various PFGE genotypes.

The mCherry cDNA was cloned in a human proinsulin vector (OriGene) between the insulin B and A chains using ApaI restriction enzyme (ThermoScientific), similar to the Emerald tagging strategy by Watkins (Watkins et al., 2002 (link)). After subcloning RINS1_mCherryonly using NheI/BamHI, the superfolder sfGFP was fused to the C-terminus of the insulin A chain using BamHI and NotI. RINS1mut was generated by mutation of amino acids 55 and 94 by mutagenesis PCR using the primers listed (Key Resources Table). EGFP-phogrin (Torii et al., 2004 (link)) was kindly provided by Seiji Torii (Gunma University) and CMV-B-GECO1 was a gift from Robert Campbell (Addgene plasmid #32448) (Zhao et al., 2011 (link)).

Genetic similarities among clinical and environmental isolates of A. baumannii were investigated by PFGE as previously described [37 (link)]. Briefly, an overnight culture of bacteria was suspended in 100 μl of cell suspension buffer and was mixed with an identical volume of 2% low melting agarose and distributed in a plug mold. Genomic DNA in agarose plugs was lysed in the cell lysis solutions I and II, washed and digested with ApaI restriction enzyme (Thermo Scientific, USA). The Lambda PFG Ladder (New England, Biolabs) was used as a DNA size marker. Electrophoresis of digested DNA was performed in a pulsed-field electrophoresis system (Chef Mapper; Bio-Rad Laboratories, USA) by programming two states with the following conditions: temperature 14 °C; voltage 6 V/cm; switch angle, 120°; switch ramp 2.2–35 s for 19 h.