TABLE 4.
The flaA promoter is transcribed by ς54-holoenzyme and the flaE, flaD, and flaB promoters are transcribed by ς28-holoenzyme in S. typhimuriuma
Genotypeb | β-Galactosidase activity in strain with mutation:
|
|||||
---|---|---|---|---|---|---|
flaAp-′lacZ | flaCp-′lacZ | flaEp-′lacZ | flaDp-′lacZ | flaBp-′lacZ | glnAp-′lacZc | |
Wild type | 23 | 651 | 2,125 | 6,649 | 9,049 | 508 |
Wild type + FlrAd | 1,808 | 493 | 1,239 | 5,165 | 5,133 | 2,047 |
ntrA | 27 | 241 | 1,285 | 2,260 | 2,374 | 503 |
ntrA + FlrAd | 33 | 564 | 1,372 | 5,931 | 6,014 | 288 |
fliA | 28 | 206 | 21 | 173 | 40 | |
fliA + FlrAd | 2,311 | 164 | 26 | 174 | 42 |
Assays were performed as described in Materials and Methods. Strains were grown in LB supplemented with 2 mM glutamine and 0.05% arabinose; cultures were assayed in triplicate at an optical density at 600 nm of ∼0.2 to 0.4. Results are the average of three samples expressed in Miller units (38). Strain KK140 (putPA::′lacZ) grown in this medium has 8 Miller units of activity, which can be considered background activity.
The actual strains used (Table 1) were KK164, KK173, KK167, KK156, and KK159 (wild type with flaAp-, flaCp-, flaEp-, flaDp-, and flaBp-′lacZ fusions, respectively), KK165, KK174, KK168, KK157, and KK160 (ntrA209::Tn10 with flaAp-, flaCp-, flaEp-, flaDp-, and flaBp-′lacZ fusions, respectively), and KK166, KK175, KK169, KK158, and KK161 (fliA5059::Tn10dTc with flaAp-, flaCp-, flaEp-, flaDp-, and flaBp-′lacZ fusions, respectively).
The genetic background in these reporter strains is Δ(ntrB-C); NtrC is the activator of ς54-dependent transcription of the glnA promoter, so we wished to measure activation by the heterologous activator FlrA in the absence of NtrC. Residual transcription in a ntrA Δ(ntrB-C) strain originates from a second ς70-dependent glnA promoter (32). The actual strains used (Table 1) were KK188 and KK189 [Δ(ntrB-C) and ntrA Δ(ntrB-C) with glnAp-′lacZ, respectively].
These strains harbor plasmid pKEK94 (see Materials and Methods), which carries the gene encoding the ς54-activator FlrA from V. cholerae (25) under the control of the arabinose-inducible promoter PBAD. ς54 activators in high concentrations can activate ς54-dependent transcription from solution (45); in the present study, this protein serves to identify any ς54-dependent promoter.