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. 2015 Jul 31;197(17):2792–2809. doi: 10.1128/JB.00101-15

FIG 1.

FIG 1

In vivo activity and expression of fluorescence-tagged Pol IV. (A) NQO sensitivities of strains carrying fluorescence-tagged Pol IV. For each strain, 10-μl aliquots of 10−1 to 10−6 dilutions of saturated cultures were spotted on an LB agar plate containing NQO; the 10−6 dilutions spotted on a plate containing no NQO are shown at the right. WT (wild type), FC40; ΔdinB, PFB243; ΔdinB/pDinB-12L-EYFP, PFB243/pPFB913; ΔdinB/pDinBE104A-12L-EYFP, PFB243/pPFB1173; ΔdinB/pmCh-DinB, PFB243/pPFV407; WT/VC (vector control), FC40/pPFB1179; ΔdinB/VC, PFB243/pPFB1179; ΔdinB/pDinB+, PFB243/pPYG768; ΔdinB/pDinB-20L-EYFP, PFB243/pPFB1188. (B) Growth-dependent mutation rates of strains carrying fluorescence-tagged Pol IV. Shown are mutation rates to Tcr ± 95% confidence intervals as determined by fluctuation tests in two experiments with 40 cultures per strain (see Materials and Methods). WT, FC1373; ΔdinB, PFB1284; ΔdinB/pDinB+, PFB1284/pPYG768; ΔdinB/pDinB-20L-EYFP, PFB1284/pPFB1188; ΔdinB/pDinBE104A-12L-EYFP, PFB1284/pPFB1173; ΔdinB/pmCh-10L-DinB, PFB1284/pPFV407. (C) Adaptive-mutation activity of DinB-20L-EYFP+. Shown are the mean cumulative numbers of Lac+ colonies appearing on days 3 to 5 on lactose minimal medium during a small-scale adaptive-mutation assay (see Materials and Methods). Each bar is the mean of four cultures; the error bars are SEM. WT/VC, FC40/pPFB1179; ΔdinB/VC, B243/pPFB1179; ΔdinB/pDinB+, PFB243/pPYG768; ΔdinB/pDinB-20L-EYFP, PFB243/pPFB1188. (D) Expression of fluorescence-tagged Pol IV proteins compared to wild-type Pol IV. Shown are immunoblots of DinB+, DinB-20L-EYFP, and DinBE104A-12L-EYFP before and after induction with Nal. The blots were probed with anti-Pol IV antibody, stripped, and then reprobed with anti-GroEL antibody to show GroEL as a loading control. The band intensities were quantified and normalized to the GroEL bands. The numbers below the lanes give the induced levels of DinB+ and DinB fusion proteins relative to the uninduced levels. The strains used were as follows: DinB+, PFB243/pPYG768; VC, pFB243/pPFB1172; DinB-20L-EYFP, PFB243/pPFB1188; DinBE104A-12L-EYFP, PFB243/pPFB1173.