Isolation of a new hemimethylated DNA binding protein which regulates dnaA gene expression

Abstract

In this report, we show that yccV, a gene of unknown function, encodes a protein having an affinity for a hemimethylated oriC DNA and that the protein negatively controls dnaA gene expression in vivo.

FIG. 1.

yccV sequence and purification of the His-tagged YccV protein. (A) Coding region (122 amino acids) and flanking sequences. The start codon is enclosed in a box. A putative helix-turn-helix motif (according to the hthscan program in GCG sequence analysis software) is underlined with a dotted line. The vertical arrow indicates the location of the mini-Tn10 insertion in the nucleotide sequence. The horizontal arrow indicates the 9 bp duplicated upon transposon insertion. A putative transcription promoter sequence described by Blattner et al. (3) is indicated by italics. A putative transcription terminator (according to the GCG software) is indicated by boldface type. GATC sites are indicated by boldface type. Sequences that exhibit a reasonable match with suggested consensus binding sequences for DnaA protein (22, 25) and for FIS (10) are underlined with thick and thin lines, respectively. (B) Purification. Strain JM109 harboring plasmid pES6ΔHEE1 (Table 1) was grown in LB medium containing ampicillin (100 μg/ml). At an optical density at 600 nm of 0.8, 0.5 mM isopropyl-β-d-thiogalactopyranoside was added to induce synthesis of the fusion protein for 3 h at 37°C. The YccV protein was purified according to the manufacturer's instructions by using 5 ml of chelating Sepharose FF (Pharmacia) coupled with nickel ion. The results of polyacrylamide gel electrophoresis of the eluted fractions followed by Coomassie blue staining are shown. Fractions 7 to 9 containing His-tagged YccV (14.9 kDa) were pooled for further experiments. The protein was dialyzed against buffer containing 50 mM Tris-HCl, 5 mM EDTA, 1 mM dithiothreitol, and 1 M NaCl (pH 7.5) supplemented with 10% glycerol before freezing at −80°C. (C) Western blot analysis of the purified fractions with monoclonal anti-His tag antibodies.

FIG. 2.

Quantitation of DnaA protein. Overnight cultures were diluted 100-fold in LB medium and grown to an optical density at 650 nm of 0.8 at 37°C (C600 derivatives) or at 30°C (KA413 derivatives). Cells were collected by centrifugation and lysed in buffer containing 25 mM Tris-HCl (pH 6.8), 4% sodium dodecyl sulfate, 20% glycerol, and 50 mM β-mercaptoethanol. Total protein contents were estimated by the Bradford assay, and cell extracts were normalized on the basis of their protein concentrations. Extracts were serially diluted into sodium dodecyl sulfate-polyacrylamide gel electrophoresis loading buffer and boiled. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the samples were transferred to a polyvinylidene difluoride membrane for immunoblot analysis. DnaA protein was detected on the membrane by using an anti-DnaA antiserum with the aide of a peroxidase-conjugated secondary antibody in the presence of 1,4-dichloronaphthol. The signal on the membrane was scanned and quantified with the Imagequant program (Molecular Dynamics).

FIG. 3.

Gel shift experiments. A ³²P-labeled double-stranded oligonucleotide (75 bp) corresponding to oriC (nucleotide positions 74 to 148) was used as the probe for gel retardation experiments in the presence of different amounts of YccV protein. Hemimethylated (A and C) or fully methylated (B) probe was generated by annealing equal amounts of the complementary oligonucleotides, including (if necessary) N⁶-methyl deoxyadenosine, at the four GATC sites. After hybridization, the double-stranded oligonucleotide was purified by 12% polyacrylamide gel electrophoresis before labeling with [γ-³²P]ATP (3,000 Ci/mmol; Amersham) for 1 h at 37°C in the presence of T4 polynucleotide kinase (Promega). The labeled oligonucleotide was then purified through a Micro Bio-Spin 30 chromatography column (Bio-Rad). Gel retardation assays were performed as described by Taghbalout et al. (26) by using 20-μl reaction mixtures containing 10 mM Tris-HCl (pH 7.6), 50 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, 10% glycerol, 5 mM MgCl₂, and 0.1% bovine serum albumin, the radioactive probe (5,000 cpm/0.5 fmol), and poly(dI-dC) as a competitor added at a 1,000-fold excess. After 10 min of incubation at room temperature, the reaction mixtures were loaded onto native 5% polyacrylamide gel electrophoresis gels, and electrophoresis was carried out at 4°C with a constant voltage in 0.5× Tris-borate-EDTA buffer. (A and B) YccV prepared as described in the legend to Fig. 1. (C) YccV preparation dialyzed against the final buffer without dithiothreitol was preincubated with 10 mM N-ethylmaleimide (NEM) (+) for 15 min at 37°C. −, no NEM treatment.