flagellar regulon studies. Escherichia coli

This study examined the genes under the control of FlhDC and sigmaF in E. coli. Keywords: wild-type, deletion and overexepression strains Overall design: Under defined steady-state growth condition, we used two different genetic approaches to alter the modulator concentration in cells; (1) moderately expressing FlhDC or sigmaF from anhydrotetracycline (aTc) inducible and Tet repressor-controlled PLtet promoter in a plasmid-borne flhDC or fliA gene; (2) disrupting the expression of FlhDC or sigmaF in flhDC or fliA deletion mutant strains. Samples were taken from culture with wild-type or deletion strains at mid log phase (OD=0.2) or from overexpression strains at mid log phase (OD=0.2) before or 5 minutes after moderate induction. Samples were then RNA-stabilized using Qiagen RNAProtect Bacterial Reagent (Qiagen). Total RNA was then isolated using MasterPure kits (Epicentre Technologies). Purified RNA was reverse-transcribed to cDNA, labeled and hybridized to Affymetrix GeneChip E. coli Antisense Genome Arrays as recommended in the technical manual (www.affymetrix.com).