Table 1.
E. coli strains, plasmid DNAs, and synthetic oligonucleotides used in this study.
| E. coli strains: | ||
|---|---|---|
| Strain | Relevant genotype | Source |
| DH5α | endA1 hsdR17(rK- mK+) glnV44 thi-1 recA1 gyrA96 relA1 deoR nupG Δ(lacZYA-argF)U169 (φ80dlacZΔM15) | Invitrogen |
| MS101 | thr-1 araD139 Δ(gpt-proA)62 lacY1 tsx-33 supE44 galK2 hisG4(Oc) rpsL31 xyl-5 mtl-1 argE3(Oc) thi-1 sulA211 dnaN159(Ts) tnaA300::Tn10 | [26] |
| MS146 | MS101 with Δ(araD-polB):: Ω | This work |
| MS125 | MS101 with Δ(dinB-yafN)::kan | [28] |
| MS147 | MS101 with ΔumuDC596::ermGT | This work |
| MS148 | MS101 with Δ(dinB-yafN)::kan ΔmutL::cat | This work |
| Plasmid DNAs: | ||
| Plasmid | Relevant characteristics | Source |
| pWSK29 | AmpR; pSC101 origin; low copy number general cloning vector | [39] |
| pJD100 | AmpR; pWSK29 containing the dnaN+ (β+) gene under the control of its native promoters | [26] |
| pJD109 | AmpR; pJD100 bearing dnaN159 (G66E and G174A substitutions; β159) | [27] |
| pMDS110 | AmpR; pJD100 bearing dnaN lacking residues 362-366 (dnaNC; βC) | This work |
| pMDS111 | AmpR; pJD100 bearing dnaN lacking residues 362-366 and containing a G66E substitution (dnaNC-G66E; βC-G66E) | This work |
| pMDS112 | AmpR; pJD100 bearing dnaN lacking residues 362-366 and containing I272A and L273A substitutions (dnaNC-I272A-L273A; βC-I272A-L273A) | This work |
| pMDS113 | AmpR; pJD100 bearing dnaN containing E93K and L98K substitutions (dnaNE93K-L98K; βE93K-L98K) | This work |
| pMDS114 | AmpR; pJD100 bearing dnaN lacking residues 362-366 and containing E93K and L98K substitutions (dnaNC-E93K-L98K; βC-E93K-L98K) | This work |
| Oligonucleotides: | ||
| Name | Nucleotide sequence (5'→3') | |
| Δ362-366-T | GGCTTATGTTGTCTAATGAATGAGACTG | |
| Δ362-366-B | CAGTCTCATTCATTAGACAACATAAGCC | |
| G66E-T | CAGCCACACGAGCCAGAAGCGACGACCGTTCCGG | |
| G66E-B | CCGGAACGGTCGTCGCTTCTGGCTCGTGTGGCTG | |
| I272A-L273A-T | GTTTGCTCGCGCGGCGGCTGCCTCTAACGAGAAATTCCG | |
| I272A-L273A-B | CGGAATTTCTCGTTAGAGGCAGCCGCCGCGCGAGCAAAC | |
| E93K-L98K-T | CGTGCAGCTGAAAGGTGAACGGATGAAAGTACGCTCCGG | |
| E93K-L98K-B | CCGGAGCGTACTTTCATCCGTTCACCTTTCAGCTGCACG | |