Figure 1. Identification and analysis of YgaQ and RpmG genes and proteins as mitomycin C resistance factors during an ASKA library screen.

(A) Viability spot test to illustrate MMC sensitivity of the E. coli ΔruvAB strain used for screening the ASKA library for MMC^R. (B) The screening procedure. Plasmid DNA isolated from combining typically 96 colonies from an individual ASKA library agar plate was transformed into E. coli ΔruvAB. Growth of colonies after plating out on LB agar containing MMC was used to assess MMC^R when compared with that given by plasmid expression of Hjc resolvase as a positive control, as shown in the panel. Further experimental details, including how ruvAB induced false positives were avoided, are given in the methods section. (C) Example of a MMC^R clone arising from the ASKA screen. The panels show details of agar plates after gridding individual colonies in the presence or absence of MMC as indicated. (D) Analysis of MMC^R provided by expression of YgaQ or RpmG, dependent on addition of IPTG to growth media. The graph compares viable colony counts from spot tests in triplicate using ΔruvAB cells transformed by either pHjc (a positive control that restores MMC^R (17)) and its corresponding empty vector (empty 1, pT7-7), or by ASKA plasmids (Supplementary Table S2) harbouring rpmG (SA2) or ygaQ (VM6) and its empty plasmid control (empty 2). A photograph of an example viability spot test for these clones is presented in the panels below. (E) Western blot of total cell protein extracted from cultures used to make the viability spot tests shown in (D). YgaQ and RpmG proteins were detected using antibody against their hexa-histidine tag.