Article Figures & Data
Figures
- FIG. 1.
Mutagenic and antimutagenic effects of the bacteriophage P1 Hot gene. Shown are the effects of the θ or Hot protein on several dnaQ mutator mutants and on dnaQ + control strains. The strains are derivatives of MG1655 containing the dnaQ49, dnaQ924, dnaQ928, dnaQ930, or dnaQ + allele in either the MG1655 (wt) (light gray), NR13104 (ΔholE) (white), or NR16315 (ΔholE::hot) (dark gray) background. All strains are mismatch repair proficient, except for the dnaQ + mutL::Tn5 set (last entry). Cultures were grown and plated at 30°C (dnaQ49) or 37°C (others). The frequencies of rifampin-resistant mutants were calculated for 12 to 15 independent cultures for each strain, and the data were analyzed using Prism software (GraphPad). The graph shows the median values and interquartile ranges for the frequencies of rifampin-resistant mutants.
- FIG. 2.
Competition of θ and Hot for incorporation into the Pol III core. Shown are the effects of the overproduction of θ or Hot from low-copy-number plasmid pKO3 in a dnaQ + strain containing the ΔholE, holE +, or ΔholE::hot genetic configuration. The mismatch repair-defective strains used were NR17119 (ΔholE), NR17120 (holE +), and NR17121 (ΔholE::hot) (Table 1) containing the indicated pKO3 plasmids (Table 1). Cultures were grown at 30°C in LB plus chloramphenicol, and the plates were likewise incubated at 30°C. The frequencies of rifampin-resistant mutants were determined for 10 to 15 independent cultures for each strain, and the data were analyzed using Prism software (GraphPad). The graph shows the median values and interquartile ranges for the frequencies of rifampin-resistant mutants. The x axis indicates, for each of the three hosts, the three pKO3 plasmids containing ΔholE (white), holE + (light gray), or ΔholE::hot (dark gray).
- FIG. 3.
Amino acid alignment of E. coli θ and P1 Hot along with secondary structure elements determined from NMR spectra (6, 26). Note that the N terminus of Hot contains one extra residue relative to that of θ and that the numbering of the corresponding residues in the two proteins differs by 1. α, α helix, L, loop.
- FIG. 4.
Effects of θ-Hot and Hot-θ chimeric proteins. The strains used were NR17117 (dnaQ49 ΔholE), NR16320 (dnaQ923 ΔholE), and NR16329 (dnaQ930 ΔholE) (Table 1) containing one of five indicated plasmids (Table 1). Cultures were grown overnight at 30°C in LB plus chloramphenicol; plates were incubated at the same temperature as the cultures. Frequencies of rifampin-resistant colonies were determined for 10 independent cultures for each strain, and the data were analyzed using Prism software (GraphPad). The graph shows the median values and interquartile ranges for the frequencies of rifampin-resistant mutants. The x axis indicates, for each dnaQ allele, the five strains containing the following plasmids: pKO3ΔholE, pKO3holE, pKO3ΔholE::hot, pKO3-θ49Hot, and pKO3-Hot50θ. See the text for details.
- FIG. 5.
Effects of the θ11Hot and Hot12θ chimeric proteins. The three panels show the effect of the θ11Hot and Hot12θ chimeric proteins along with the control proteins on the mutability of dnaQ49 (A), dnaQ923 (B), and dnaQ930 (C). The strains used were NR17117 (dnaQ49 ΔholE), NR16320 (dnaQ923 ΔholE), and NR16329 (dnaQ930 ΔholE) containing various plasmids as indicated along the x axis: pKO3-ΔholE, pKO3-holE, pKO3-hot, pKO3-Hot12θ, or pKO3-θ11Hot. The cultures were grown in LB with chloramphenicol at 30°C and 35°C for the dnaQ49 strains (A) and at 30°C for the dnaQ923 and dnaQ930 strains (B and C). Plates were incubated at the same temperature as the cultures. The frequencies of rifampin-resistant colonies were determined for 8 to 12 independent cultures for each strain, and the data were analyzed using Prism software (GraphPad). The graph shows the median values and interquartile ranges for the frequencies of rifampin-resistant mutants.
Tables
- TABLE 1.
E. coli strains and plasmids used in this study
Strain or plasmid Relevant genotype Reference, source, or construction E. coli strains CAG18486 eda-51::Tn10 30 CAG12068 zeb-3190::Tn10 30 CC101 to CC106 F′CC101 to F′CC106 4 KA796 ara thi Δ(pro lac) 29 MG1655 Wild type 30 NR9464 mutL::Tn5 27 NR9695 dnaQ49 zae-502::Tn10 28 NR11572 dnaQ923 zae-502::Tn10 33 NR11573 dnaQ924 zae-502::Tn10 33 NR11641 dnaQ928 zae-502::Tn10 33 NR11642 dnaQ930 zae-502::Tn10 33 NR13104 ΔholE203 3 NR16315 ΔholE204::hot 3 NR16319 dnaQ923 MG1655 × P1/NR11572 NR16320 ΔholE203 dnaQ923 NR13104 × P1/NR11572 NR16321 ΔholE204::hot dnaQ923 NR16315 × P1/NR11572 NR16322 dnaQ924 MG1655 × P1/NR11573 NR16323 ΔholE203 dnaQ924 NR13104 × P1/NR11573 NR16324 ΔholE204::hot dnaQ924 NR16315 × P1/NR11573 NR16325 dnaQ928 MG1655 × P1/NR11641 NR16326 ΔholE203 dnaQ928 NR13104 × P1/NR11641 NR16327 ΔholE204::hot dnaQ928 NR16315 × P1/NR11641 NR16328 dnaQ930, zae-502::Tn10 MG1655 × P1/NR11642 NR16329 ΔholE203 dnaQ930 NR13104 × P1/NR11642 NR16330 ΔholE204::hot dnaQ930 NR16315 × P1/NR11642 NR16785 ΔholE204::hot eda-51::Tn10 NR16315 × P1/CAG18486 NR16786 ΔholE204::hot zeb-3190::Tn10 NR16315 × P1/CAG12068 NR16787 ara, thi, Δprolac, ΔholE202::cat KA796 × P1/RM4193 NR17012 ΔholE204::hot eda-51::Tn10 KA796 × P1/NR16785 NR17013 ΔholE204::hot zeb-3190::Tn10 KA796 × P1/NR16786 NR17116 dnaQ49 MG1655 × P1/NR9695 NR17117 ΔholE203 dnaQ49 NR13104 × P1/NR9695 NR17118 ΔholE204::hot dnaQ49 NR16315 × P1/NR9695 NR17119 mutL::Tn5 MG1655 × P1/NR9464 NR17120 ΔholE203 mutL::Tn5 NR13104 × P1/NR9464 NR17121 ΔholE204::hot mutL::Tn5 NR16315 × P1/NR9464 RM4193 ΔholE202::cat 31 Plasmids pKO3 21 pKO3-ΔholE 3 pKO3-holE 3 pKO3-hot 3 pKO3-θ49Hot This work pKO3-Hot50θ This work pKO3-θ11Hot This work PKO3-Hot12θ This work - TABLE 2.
Oligonucleotide primers
Primer Sequencea Pr1 5′-GAGAAATGCGGCCGCTGTAGTGTCCTTTCGTTTTATGCCC-3′ Pr6 5′-CAAATCAGTCGACGCCAGCAGGTCGGGTTCTCC-3′ hotN-lowb 5′-CAGCTGCGCAGATTCTCTGGTTG-3′ holEC-upb 5′-CAGAGAATCTGCGCAGCTGGTTTCG-3′ holEN-lowc 5′-ATGAAATAGGTGCGCAAATGTTCAGGCTG-3′ hotC-upc 5′-TGAACATTTGCGCACCTATTTCATGGAAC-3′ HotNLowd 5′-CACTTTATCCATTTCTTCCTGACTTTTAGCTGC-3′ HolECUpd 5′-GTCAGGAAGAAATGGATAAAGTGAATGTCG-3′ HolENLowe 5′-CTTATCCCGTTCTGTTTGATCCAGTTTAGC-3′ HotCUpe 5′-GATCAAACAGAACGGGATAAGGTTAACG-3′ SeqHotUp 5′-GGAATATTGCAGCTAAAAGTC-3′ SeqHotLow 5′-CTATTTCTTTACGGCATCATC-3′ SeqholE Up 5′-CTGAAGAATCTGGCTAAACTG-3′ SeqholE Low 5′-GTTTTATTTAAGTTTGGGCTC-3′ ↵ a Sequences in boldface type correspond to holE or hot coding sequences.
↵ b Primers with complementary 5′ ends used to create pKO3-Hot50θ.
↵ c Primers with complementary 5′ ends used to create pKO3-θ49Hot.
↵ d Primers with complementary 5′ ends used to create pKO3-Hot12θ.
↵ e Primers with complementary 5′ ends used in creation of pKO3-θ11Hot.
- TABLE 3.
Differential mutator effects of ΔholE and ΔholE::hot: Lac+ or Rifr mutant frequencies in mismatch repair-defective mutL strainsa
lac allele from strain Mutation scored No. of mutants (per 106 cells) Mutator effect (fold) holE+ ΔholE ΔholE::hot ΔholE/ holE+ hot+/ holE+ Allb Rifr 0.44 0.71 0.7 1.6 1.6 CC101 A · T→C · G 0.0036 0.0027 0.0046 0.75 1.3 CC102 G · C→A · T 0.44 0.45 0.79 1 1.8 CC103 G · C→C · G 0 0 0 CC104 G · C→T · A 0.009 0.038 0.017 4.2 1.9 CC105 A · T→T · A 0.0037 0.015 0.004 4 1 CC106 A · T→G · C 0.17 0.55 0.36 3.2 2.1 FC40c lac (−1) FSd 0.096 0.093 0.27 0.97 2.8 ↵ a Strains are mutL::Tn5 derivatives of KA796 (holE+), NR16787 (ΔholE), and NR17012 (ΔholE::hot eda-51::Tn10) or NR17013 (ΔholE::hot zeb-3190::Tn10) (Table 1) and contain the F′(pro lac) episome from strains CC101 through CC106 (Table 1), permitting measurement of the indicated, specific lac reversion frequencies. The eda or zeb transposon insertions did not affect the hot mutator effect (results not shown). For each strain with each F′(pro lac), 22 independent LB cultures were grown overnight at 37°C. The 22 cultures were derived from multiple isolates (transductants) for each strain. The frequencies of lac + revertants and of Rifr mutants were determined as described in Materials and Methods. The mutant frequencies were analyzed by Prism software, and statistically significant differences between hot + and ΔholE or hot + and ΔholE::hot strains (mutator effects) were assessed using nonparametric tests. Differences with P values of <0.05 are shown in boldface type.
↵ b The Rifr data reflect the median value of eight independent experiments, each comprised of 22 cultures.
↵ c This strain was tested in the mismatch repair-proficient background.
↵ d FS, frameshift mutation.
- TABLE 4.
Effect of point mutants in Hot12θ and θ11Hot chimeric proteins on the mutability of dnaQ49, dnaQ923, and dnaQ930 mutantsa
Proteinb No. of Rifr mutants per 106 cells dnaQ49 dnaQ923 dnaQ930 32°C 35°C None (ΔholE) 65 55 27 0.33 θ 0.69 13 0.33 0.29 Hot 0.4 2.0 0.30 13 Hot12θ 0.72 12 2.4 44 Hot12θ (Y2Η) 0.5 34 37 50 Hot12θ (Α7Τ) 12 55 0.42 0.59 Hot12θ (N17H) 0.66 20 38 40 Hot12θ (W51C) 71 27 28 3.2 θ11Hot 0.52 1.7 0.11 1.4 θ11Hot (D9Y) 0.78 8.8 0.17 0.96 θ11Hot (V17I) 0.39 0.12 0.64 θ11Hot (F53S) 63 92 7.5 0.87
