TABLE 1.
E. coli K-12 strains and plasmids
| Strain or plasmid | Relevant genotype or characteristics | Reference, source, or construction |
|---|---|---|
| E. coli strains | ||
| AB1157 | F−thr-1 ara-14 leuB6 Δ(gpt-proA)62 lacY1 tsx-33 supE44 galK2 hisG4 rfbD1 mgl-51 rpsL31 kdgK51 xyl-5 mtl-1 argE3 thi-1 | 1 |
| JC11450 | AB1157, spontaneous Su− | A. J. Clark |
| FC40 | ara Δ(lac-proAB)XIIIthi Rifr [F′ proAB+ lacI33ΩlacZ] | 3 |
| CAG12176 | zef-3189::Tn10kan | 33 |
| CAG18604 | zgf-3156::Tn10kan | 33 |
| STL160 | AB1157 Δ(xseA-guaB) zfh-3139::Tn10kan | S. T. Lovett |
| SMR91 | mutL211::Tn5 | Laboratory collection |
| SMR423 | recD1903::Tn10 | Laboratory collection |
| SMR690 | FC40 recJ284::Tn10 | 10 |
| SMR838 | JC11450 ΔxonA300::cat his+ | Laboratory collection |
| SMR839 | JC11450 ΔxonA300::cat | 27 |
| SMR1403 | JC11450 ΔxonA300::cat recJ284::Tn10 | 27 |
| SMR2597 | FC40 Δ(xseA-guaB) zfh-3139::Tn10kan | Laboratory collection |
| SMR3070 | FC40 ΔxonA300::cat | FC40 × P1(SMR839) |
| SMR3404 | FC40 mutL211::Tn5 | FC40 × P1(SMR91) |
| SMR3465 | recD1903::Tn10 ΔxseA18::amp | This studya |
| SMR3472 | FC40 ΔxseA18::amp | SMR2597 × P1(SMR3465)b |
| SMR3481 | FC40 ΔxonA300::cat recJ284::Tn10 ΔxseA18::amp | FC40 × P1(SMR838), P1(SMR690), P1(SMR3472)c |
| SMR3488 | JC11450 ΔxonA300::cat recJ284::Tn10 ΔxseA18::amp | SMR1403 × P1(SMR3472)c |
| SMR3524 | JC11450 mutL211::Tn5 | JC11450 × P1 (SMR3404) |
| SMR4035 | FC40 ΔxonA300::cat zef-3189::Tn10kan | SMR3070 × P1(CAG12176) |
| SMR4036 | FC40 recJ284::Tn10 zgf-3156::Tn10kan | SMR690 × P1(CAG18604) |
| SMR4037 | FC40 ΔxseA18::amp zfh-3139::Tn10kan | SMR3472 × P1(STL160) |
| Plasmids | ||
| pMJ3 | pACYC184 derivative containing xseA and guaBA | This studyd |
| pMJ6 | pMJ3 derivative containing ΔxseA18::amp | This studye |
Constructed by transforming SMR423 with the 5-kb AvaI-BamHI fragment of pMJ6 which contains ΔxseA18::amp and selecting ampicillin-resistant transformants (29).
This transduction confirmed the chromosomal location of ΔxseA18::amp, as all Ampr transductants were Gua+ and the expected linkage to zfh-3139::Tn10kan (4) was observed.
The presence of the null alleles in the triple mutants was confirmed by P1 transduction of each mutation into genetic backgrounds in which the following characteristic phenotypes were observed: recJ284::Tn10 makes recB21 recC22 sbcB15 sbcC201 strains extremely UV sensitive (16); xonA-null mutations decrease transductional recombination via the RecF pathway (2); and xseA mutations enhance sensitivity to low concentrations of nalidixic acid (4). The triple mutants also display UV light sensitivity (discussed in the text), as expected from their reduced-recombination phenotype (27).
A 5-kb BglI-BamHI fragment from Kohara phage λ427 (λ8E3) (11) containing xseA and guaBA was ligated into BglI-BamHI-digested pACYC184 (see, e.g., reference 21). BglI 3′ overhangs were removed with T4 DNA polymerase (exonuclease activity) prior to ligation.
The 690-bp EagI-AflII fragment of xseA (6) (GenBank accession no., J02599) was replaced with an EagI-AflII-digested 1,035-bp PCR fragment containing the bla gene of pBR322 (see, e.g., reference 21). bla was amplified with primers that create EagI and AflII sites (EagI primer: 5′GTACGGCCGAGTAAACTTGGTCTGACA; AflII primer: 5′ATGCTTAAGTAGACGTCAGGTGGCACT).