Table 2.
Measurements of TnpR‐mediated resolution activated by the citH promoter under various conditions in culture.*
| Growth conditions† | Strains | ||
|---|---|---|---|
| XcvResPcitH | XcvResPcitHrev | XcvRes | |
| NB | No‡ | No | N.A. |
| XVM2 | Complete§ | 90% | No |
| M9 + citrate | Complete | No | No |
| M9 +l‐malate | No | No | N.A. |
| M9 +l‐succinate | No | No | N.A. |
| M9 + sucrose | No | No | No |
| M9 + fructose | No | No | No |
| M9 + glucose | No | No | N.A. |
| M9 + glucose + citrate | Complete | No | N.A. |
| M9 + isocitrate | No | No | N.A. |
| M9 + sucrose + fructose | 90% | 65% | No |
| M9 + sucrose + fructose + citrate | Complete | No | No |
| M9 + sucrose pH 5.7, 6.2 or 6.7 | No | No | No |
| M9 + sucrose pH 5 or 8 | No | No | N.A. |
| M9 + citrate pH 5 or 8 | Complete | No | N.A. |
The TnpR resolution assay is based on the fact that tnpR expression, activated by a tested promoter, leads to the excision of a KmR gene. Strains were grown in liquid medium under different conditions, and serial dilutions were plated on both nutrient agar (NA) (total cells) and NA/kanamycin (Km) (unresolved cells). The percentage resolution is determined from [(average total cfu − average unresolved cfu)/average total cfu] × 100. Tested strains were as follows: XcvResPcitH, tnpR under the control of the citH promoter; XcvResPcitHrev, the citH promoter in the opposite orientation relative to tnpR; and XcvRes, carrying the res cassette but no tnpR.
Where not indicated, the pH of the medium was 7.
‘No’ indicates no resolution and means that the differences in colony‐forming unit (cfu) counts between Km‐lacking versus Km‐containing plates were smaller than 5%.
‘Complete’ indicates complete resolution (differences between counts in Km‐lacking versus Km‐containing plates were greater than 95%). Resolution was assessed for each strain in at least two independent experiments with similar results for each medium, with the exception of N.A. (not assessed).