PNAS Crosson et al. 10.1073/pnas.0503022102. |
Fig. 6.
(A) Expression of the fixLJ promoter under hypoxic conditions in wild-type and D fixK backgrounds. (B) Expression of fixK promoter under high oxygen conditions in wild-type and D fixT backgrounds. Transcriptional promoter fusions were generated by amplifying 500 bases upstream of the predicted start codons of fixLJ and fixK and cloning into vector pRKlac290 (see Table 7). Strains carrying the fusion vectors were grown in quadruplicate to OD = 0.3 in PYE medium supplemented with 1 m g/ml oxyteteracycline. All strains were grown at 30°C in a shaker at 250 rpm. A 200-m l aliquot was removed and quickly permeablized with 50 m l of chloroform. 550 m l of Z-buffer (60 mM Na2HPO4/40 mM NaH2PO4/10 mM KCl/1 mM MgSO4/50 mM 2-mercaptoethanol, pH 7.0) and 200 m l of o-nitrophenyl-b -D-galactopyranoside (ONPG) was added to the permeablized cells to determine activity. Hypoxic conditions were created by purging the cultures with oxygen-free N2 for 10 min and removing a culture aliquot. Activity in Miller Units was determined by measuring OD420.