TABLE 4.
Ability to construct triple-exonuclease mutants without direct selectiona
| Genetic background | Relevant genotype of:
|
No. of col.c | Frequency of cotransducing exo mutation with linked Tn10kan (mean ± SD)b | |
|---|---|---|---|---|
| Donor | Recipient | |||
| JC11450 | ΔxseA18::amp zfh-3139::Tn10kan | xonA+ xseA+ recJ+ | 148 | 0.19 ± 0.03 |
| ΔxonA300::cat recJ284::Tn10 | 573 | 0.19 ± 0.03 | ||
| ΔxonA300::cat zef-3189::Tn10kan | xonA+ xseA+ recJ+ | 161 | 0.24 ± 0.05 | |
| recJ284::Tn10 ΔxseA18::amp | 170 | 0.27 ± 0.05 | ||
| recJ284::Tn10 zgf-3156::Tn10kan | xonA+ xseA+ recJ+ | 123 | 0.13 ± 0.04 | |
| ΔxonA300::cat ΔxseA18::amp | 244 | 0.20 ± 0.05 | ||
| FC40 | ΔxseA18::amp zfh-3139::Tn10kan | xonA+ xseA+ recJ+ | 886 | 0.19 ± 0.01 |
| ΔxonA300::cat recJ284::Tn10 | 1,420 | 0.18 ± 0.01 | ||
| ΔxonA300::cat zef-3189::Tn10kan | xonA+ xseA+ recJ+ | 1,010 | 0.27 ± 0.01 | |
| recJ284::Tn10 ΔxseA18::amp | 902 | 0.21 ± 0.01 | ||
| recJ284::Tn10 zgf-3156::Tn10kan | xonA+ xseA+ recJ+ | 540 | 0.17 ± 0.01 | |
| ΔxonA300::cat ΔxseA18::amp | 576 | 0.15 ± 0.02 | ||
If secondary suppressor mutations are necessarily selected when constructing triple-exonuclease null mutants, then most transductants of a double-exonuclease mutant with the final nuclease allele would be absent from the transduction progeny—only the rare double-nuclease mutant that already carries a secondary suppressor mutation would form a detectable transductant colony. The frequency of cotransduction of the third nuclease allele with a nearby selectable marker would then appear to be much lower when transducing into double-nuclease mutant recipient strains than into exonuclease-proficient recipients. These frequencies were compared for all of the possible orders of construction of triple-nuclease mutants in both strain backgrounds used in this study. In each case, a nearby selectable kanamycin resistance-encoding transposon, Tn10kan, was selected, and the number of colonies also carrying the linked nuclease allele was determined by assaying the (other) drug resistance encoded by the cassette disrupting each nuclease gene.
Three separate P1 transductional crosses were performed. Standard transduction methods were used (21).
The total number of transductant colonies screened.