Abstract
DNA gyrase is an essential enzyme in DNA replication in Escherichia coli. It mediates the introduction of negative supercoils near oriC, removal of positive supercoils ahead of the growing DNA fork, and separation of the two daughter duplexes. In the course of purifying DNA gyrase from E. coli KL16, we found an 18-kDa protein that inhibited the supercoiling activity of DNA gyrase, and we coined it DNA gyrase inhibitory protein (GyrI). Its NH2-terminal amino acid sequence of 16 residues was determined to be identical to that of a putative gene product (a polypeptide of 157 amino acids) encoded by yeeB (EMBL accession no. U00009) and sbmC (Baquero, M. R., Bouzon, M., Varea, J., and Moreno, F. (1995) Mol. Microbiol. 18, 301-311) of E. coli. Assuming the identity of the gene (gyrI) encoding GyrI with the previously reported genes yeeB and sbmC, we cloned the gene after amplification by polymerase chain reaction and purified the 18-kDa protein from an E. coli strain overexpressing it. The purified 18-kDa protein was confirmed to inhibit the supercoiling activity of DNA gyrase in vitro. In vivo, both overexpression and antisense expression of the gyrI gene induced filamentous growth of cells and suppressed cell proliferation. GyrI protein is the first identified chromosomally nucleoid-encoded regulatory factor of DNA gyrase in E. coli.
Highlights
DNA gyrase is an essential enzyme in DNA replication in Escherichia coli
In the course of purifying DNA gyrase from E. coli KL16, we found an 18-kDa protein that inhibited the supercoiling activity of DNA gyrase, and we coined it DNA gyrase inhibitory protein (GyrI)
We discovered an 18-kDa protein, termed DNA gyrase inhibitory protein (GyrI), which could inhibit the supercoiling activity of DNA gyrase in E. coli KL16
Summary
Bacterial Strains and Plasmids—E. coli strains used in this study were JM109, GI724 (FϪ Ϫ lacIq lacPL8 ampC::Ptrp cI mcrA mcrB INV(rnnD-rnnE)), and KL16 (HfrKL16-P045(thyA3serA):thi-1 relA1 ()Ϫ). One unit of enzyme activity was defined as the amount that brought 50% of relaxed pBR322 to the supercoiled position in agarose gel electrophoresis as described by Gellert et al (18). The reaction mixture (10 l) contained 25 mM Tris-HCl (pH 8.0), 67 mM KCl, 5 mM MgCl2, 1.25 mM spermidine hydrochloride, 1.7 mM ATP, 20 g of E. coli tRNA/ml, and 0.15 g of relaxed pBR322 DNA. The solution obtained after ammonium sulfate precipitation was loaded onto a novobiocin-Sepharose column previously equilibrated with TED buffer (10 mM Tris-HCl (pH 7.5), 1 mM EDTA, and 1 mM dithiothreitol). The holoenzyme and GyrA and GyrB subunits were eluted by TED buffer containing 5 M urea, 2 M KCl, and 2 M KCl plus 5 M urea, respectively. The fractions containing GyrA were loaded onto a column of heparin-Sepharose CL-6B equilibrated
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