Chromosomal location of the hdfR gene. The hdfR gene was previously divided into two ORFs, pssR and yifA, by E. coli genome sequencing for strain MG1655. Numbers indicate nucleotide positions, in kilobases. Arrows indicate directions of the ORFs. The nucleotide and deduced amino acid sequences of the hdfR gene have been submitted to GenBank (accession no. AF25103). ΔG indicates the location of the missing guanine nucleotide in the reported sequence (accession no. AE000453). The insertion site of TnphoA132 (Tn) is also indicated.

Effects of hns and hdfR mutations on the transcription of the flhDC operon. β-Galactosidase activities expressed from the strains containing the flhDC-lacZ transcriptional fusion in λMS205 were measured in cells grown in TB medium (1% tryptone, 0.25% NaCl) at 35°C to an optical density at 600 nm of 0.4 to 0.5. Strains used were MS358 (λMS205 hns⁺ hdfR⁺), MS359 (MS358 hns), MS372 (MS358 hdfR), MS373 (MS358 hns hdfR), MS372/pMS272ΔH (hdfR/pHdfR), and MS373/pMS272ΔH (hns hdfR/pHdfR). The activities of β-galactosidase are presented in Miller units (14), with standard deviations (error bars) estimated from three independent samples.

Gel mobility shift of the DNA fragment containing the flhDC promoter by purified HdfR-His₆. The probe DNA corresponding to the region from −626 to +185 bases from the transcription start was obtained by PCR and labeled with [γ-³²P]ATP using polynucleotide kinase (Boehringer Mannheim). For a 20-μl reaction volume, 1 ng of DNA probe (about 3,000 cpm), 2 μg of sheared salmon sperm DNA, and 0 to 100 ng of purified proteins were used. The buffer contained 50 mM KCl, 20 mM Tris-Cl (pH 7.9), 1 mM dithiothreitol, 10% glycerol, and 125 μg of bovine serum albumin/ml. The whole mixture was stored on ice for 30 min and loaded onto a 5% low-ionic-strength polyacrylamide gel (2). The DNA species are appropriately marked. The unlabeled upper band appears to be a denatured form of the probe generated during the purification step. Amounts of purified HdfR-His₆ proteins used are none (lane 1), 10 ng (lane 2), 20 ng (lane 3), 50 ng (lane 4), and 100 ng (lane 5).