TABLE 2.
λ red gam lytic crosses in recD mutants lacking activities of various exonucleases
| Strain | Description | Total yield(× 107) | Relative J+R+ recombinants |
|---|---|---|---|
| DE100 | RecD− | 94.0 | 1.0a |
| AB1157 | Wild type | 9.2 | 0.28 ± 0.138 |
| DE1061 | RecD− RecJ− | 16.2 | 0.79 ± 0.099 |
| DE302 | RecJ− | 5.3 | 0.34 ± 0.230 |
| DE1063 | RecD− RecJ− ExoVII− | 0.25 | 0.0064 ± 0.0039 |
| DE303 | RecJ− ExoVII− | 2.5 | 0.23 ± 0.092 |
| DE1062 | RecD− RecJ− ExoI− | 8.5 | 0.13 ± 0.081 |
| DE1050 | RecD− ExoI− | 53.0 | 0.51 ± 0.198 |
| DE1052 | RecD− ExoVII− | 97.5 | 1.03 ± 0.225 |
| DE1055 | RecD− ExoI− ExoX− | 83.0 | 0.55 ± 0.101 |
| DE1054 | RecD− ExoI− ExoVII− | 14.1 | 1.22 ± 0.295 |
| DE1059 | RecD− ExoI− ExoVII− ExoX− | 4.67 | 1.10 ± 0.302 |
| DE1060 | RecD− ExoI− ExoVII− SbcD− | 1.85 | 1.34 ± 0.354 |
| DE101 | RecB− | 5.1 | 0.022 ± 0.005 |
The λ-lytic crosses were performed essentially as described (Ðermić et al. 2006). Bacteria were grown in tryptone broth with 0.3% maltose at 34° to a density of 108 cells/ml (an OD600 of 0.25). A total of 0.3 ml of each culture was mixed with 0.1 ml of phage mixture. The multiplicity of infection was 10 for MMS555 (Jam) and 0.2 for MMS754 (Rts). After 15 min of incubation at 34°, unadsorbed phages (on average, ∼1% of total infecting phages) were removed by centrifugation. Infected cells were resuspended in 1 ml of tryptone broth with Nozu supplements (Arber et al. 1983) and gently aerated for 90 min at 34°. The remaining cells were then lysed with 0.2 ml of chloroform. The phage titers were determined on V371 host strain at 34° for total Am+ phage and at 42° for Am+Ts+ recombinants. Plaques were counted after 18–24 hr of growth on tryptone plates. The frequency of recombinants was calculated as the recombinant titer divided by the total (Am+) titer.
Recombination frequency of 1.0 corresponds to 13 J+R+ recombinants per 100 J+ phages. All values are averages of at least three independent experiments ± standard deviations.