cas3p binding proteins identified by LC-MS/MS analysis

DNA affinity chromatography was performed using the cas3 promoter region (−350 to +50) relative to the translation start site (TSS) was amplified by PCR from A. baumannii ATCC 19606T. The PCR product and pSA508 vector were digested with restriction enzymes and ligated using a T4 DNA ligase. The ligated DNA was transformed into E. coli DH5α and cultured for 16 h at 37℃. To pull down the DNA-binding proteins, a biotinylated DNA fragment, including cas3p, was obtained by PCR using a biotinylated forward primer and a non-biotinylated reverse primer listed in Table S1. To prepare the whole proteins, A. baumannii ATCC 19606T was cultured until an OD600 of 0.5, harvested by centrifugation, and resuspended in diethyl pyrocarbonate water. The bacterial solution was sonicated and centrifuged to obtain a clear lysate. Dynabeads™ M-280 Streptavidin (Thermo Fisher Scientific, USA) and a biotinylated probe DNA were mixed and incubated under rolling at room temperature. Dynabeads were pulled down, and the supernatant was discarded to remove the nonspecifically bound proteins after several washing steps. Bound proteins were eluted using different NaCl concentrations (100, 200, 350, 500, and 1000 mM) at each elution. The eluted protein samples were analyzed by 10% Tricine SDS-PAGE gels and were stained with a silver-staining kit (ELPISBIO, Korea). Bands from the SDS-PAGE gel was cut off for LC-MS/MS analysis (Korea Basic Science Institute, KBSI) to identify the proteins according to their size and elution concentration.