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

Genotypebβ-Galactosidase activity in strain with mutation:
Wild type236512,1256,6499,049508
Wild type + FlrAd1,8084931,2395,1655,1332,047
ntrA + FlrAd335641,3725,9316,014288
fliA + FlrAd2,3111642617442
  • a 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.

  • b The actual strains used (Table 1) were KK164, KK173, KK167, KK156, and KK159 (wild type with flaAp-,flaCp-, flaEp-, flaDp-, andflaBp-′lacZ fusions, respectively), KK165, KK174, KK168, KK157, and KK160 (ntrA209::Tn10with flaAp-, flaCp-, flaEp-,flaDp-, and flaBp-′lacZ fusions, respectively), and KK166, KK175, KK169, KK158, and KK161 (fliA5059::Tn10dTc withflaAp-, flaCp-, flaEp-,flaDp-, and flaBp-′lacZ fusions, respectively).

  • c The genetic background in these reporter strains is Δ(ntrB-C); NtrC is the activator of ς54-dependent transcription of the glnApromoter, so we wished to measure activation by the heterologous activator FlrA in the absence of NtrC. Residual transcription in antrA 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].

  • d 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.