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. 1998 Jan;180(2):303–316. doi: 10.1128/jb.180.2.303-316.1998

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
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-, 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). 

c

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]. 

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. 

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