Identification of DNA Gyrase Inhibitor (GyrI) inEscherichia coli
1998; Elsevier BV; Volume: 273; Issue: 4 Linguagem: Inglês
10.1074/jbc.273.4.1933
ISSN1083-351X
AutoresAkira Nakanishi, Tadahiro Oshida, Tadahiro Matsushita, Shinobu Imajoh‐Ohmi, Tetsuo Ohnuki,
Tópico(s)DNA and Nucleic Acid Chemistry
ResumoDNA 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 fromE. 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 byyeeB (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. 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 fromE. 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 byyeeB (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. DNA gyrase, a type II topoisomerase in Escherichia coli, has the ability to cut a double-stranded DNA, pass an uncut portion of the duplex between the cut ends, and reseal the cut. It can introduce negative supercoils into covalently closed circular DNA and cause catenation and decatenation of two different DNA duplexes,in vitro (1Menzel R. Gellert M. Adv. Pharmacol. 1994; 29A: 39-69Crossref PubMed Scopus (33) Google Scholar). It has been established that the enzyme is essential for chromosomal replication in vivo (2Wang J.C. Biochim. Biophys. Acta. 1987; 909: 1-9Crossref PubMed Scopus (300) Google Scholar). Moreover, there have been reports on the involvement of DNA gyrase in transcription from certain operons, DNA repair, and recombination inE. coli (2Wang J.C. Biochim. Biophys. Acta. 1987; 909: 1-9Crossref PubMed Scopus (300) Google Scholar).DNA gyrase is composed of two subunits, A (GyrA) and B (GyrB), which are assembled in A2B2 complexes, the active form (3Gellert M. O'Dea M.H. Itoh T. Tomizawa J. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 4474-4478Crossref PubMed Scopus (557) Google Scholar, 4Klevan L. Wang J.C. Biochemistry. 1980; 19: 5229-5234Crossref PubMed Scopus (100) Google Scholar, 5Krueger S. Zaccai G. Wlodawer A. Langowski J. O'Dea M.H. Maxwell A. Gellert M. J. Mol. Biol. 1990; 211: 211-220Crossref PubMed Scopus (38) Google Scholar). The active complex has been purified from E. coli (6Mizuuchi K. O'Dea M.H. Gellert M. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 5960-5963Crossref PubMed Scopus (198) Google Scholar) and reconstituted from the purified GyrA and GyrB (7Higgins N.P. Peebles C.L. Sugino A. Cozzarelli N.R. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 1773-1777Crossref PubMed Scopus (190) Google Scholar, 8Sugino A. Cozzarelli N.R. J. Biol. Chem. 1980; 255: 6299-6306Abstract Full Text PDF PubMed Google Scholar, 9Staudenbauer W.L. Orr E. Nucleic Acids Res. 1981; 9: 3589-3603Crossref PubMed Scopus (127) Google Scholar). GyrA has an active center for the reactions of introducing and resealing the cuts of double-stranded DNA, whereas GyrB powers the reaction by catalyzing ATP hydrolysis.DNA gyrase is a target of two distinct classes of inhibitors, coumarins (10Drlica K. Coughlin S. Pharmacol. Ther. 1989; 44: 107-121Crossref PubMed Scopus (55) Google Scholar, 11Maxwell A. Mol. Microbiol. 1993; 9: 681-686Crossref PubMed Scopus (229) Google Scholar) and quinolones (10Drlica K. Coughlin S. Pharmacol. Ther. 1989; 44: 107-121Crossref PubMed Scopus (55) Google Scholar, 12Maxwell A. J. Antimicrob. Chemother. 1992; 30: 409-414Crossref PubMed Scopus (157) Google Scholar). Coumarins bind to GyrB and are competitive inhibitors with respect to ATP (11Maxwell A. Mol. Microbiol. 1993; 9: 681-686Crossref PubMed Scopus (229) Google Scholar). In contrast, quinolones bind DNA gyrase when the enzyme is complexed with DNA and trap the enzyme in an abortive ternary complex, which, upon treatment with a denaturant, releases cleaved DNA with GyrA covalently attached to the 5′-phosphoryl ends generated at the cut site.There have been several reports on regulating DNA gyrase activity inE. coli. LetD (13Maki S. Takiguchi S. Miki T. Horiuchi T. J. Biol. Chem. 1992; 267: 12244-12251Abstract Full Text PDF PubMed Google Scholar) encoded on F factor inhibits DNA gyrase activity via the induction of synthesis of heat shock proteins (14Kaneko T. Mizushima T. Ohtsuka Y. Kurokawa K. Kataoka K. Miki T. Sekimizu K. Mol. Gen. Genet. 1996; 250: 593-600PubMed Google Scholar). Another regulatory factor, cyclic AMP (cAMP) receptor (15Gomez-Gomez J.M. Baquero F. Blazquez J. J. Bacteriol. 1996; 178: 3331-3334Crossref PubMed Google Scholar), participates in regulation of the growth phase-dependent transcription of gyrA (15Gomez-Gomez J.M. Baquero F. Blazquez J. J. Bacteriol. 1996; 178: 3331-3334Crossref PubMed Google Scholar).In this study, 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. We describe here the purification and characterization of GyrI and phenotypic analyses of recombinant strains overproducing GyrI or expressing antisensegyrI gene to decipher importance of GyrI in the regulation of DNA gyrase activity in vivo.DISCUSSIONWe purified from E. coli KL16 the 18-kDa protein that inhibited the supercoiling activity of DNA gyrase and coined it DNA gyrase inhibitory protein (GyrI). The NH2-terminal amino acid sequence and molecular mass of GyrI inferred that the gene encoding GyrI might be identical to the previously reported genes ofyeeB (EMBL accession no. U00009) and sbmC (34Baquero M.R. Bouzon M. Varea J. Moreno F. Mol. Microbiol. 1995; 18: 301-311Crossref PubMed Scopus (42) Google Scholar). The yeeB gene had been identified as an open reading frame in the sbcB region, although its function had not been described. The sbmC gene had been reported to decrease the sensitivity to microcin B17 when overexpressed. Microcin B17, a peptide antibiotic of 43 amino acids, is generated by cleavage of a precursor of 69 amino acids encoded by mcbA on plasmids (35Davagnino J. Herrero M. Furlong D. Moreno F. Kolter R. Proteins. 1986; 1: 230-238Crossref PubMed Scopus (62) Google Scholar) and appeared to trap an abortive cleavable DNA·DNA gyrase complex (36Vizan J.L. Hernandez-Chico C. del Castillo I. Morneo F. EMBO J. 1991; 10: 467-476Crossref PubMed Scopus (154) Google Scholar), a mode of action similar to that of quinolones. The yeeB andsbmC gene was located at 44 min on the E. colichromosome map. However, there have been no reports on the inhibitory activity against DNA gyrase of the gene products of yeeB andsbmC.To investigate the identity of gene encoding GyrI with yeeBand sbmC, we cloned the coding region based on the reported sequence and purified the 18-kDa gene product from the transformant overexpressing it. In vitro assay of DNA gyrase supercoiling activity indicated that the purified 18-kDa protein indeed inhibited the activity. Furthermore, we confirmed that GyrI protein is not intercalated into DNA and does not inhibit the activity of other DNA-processing enzymes (e.g. DNA polymerase) (data not shown). We tentatively named the gene coding for the 18-kDa protein asgyrI to indicate clearly the biological function of the gene product.It was reported that factor LetD regulates the activity of DNA gyrase (13Maki S. Takiguchi S. Miki T. Horiuchi T. J. Biol. Chem. 1992; 267: 12244-12251Abstract Full Text PDF PubMed Google Scholar). LetD encoded by the F factor functions to kill the host E. coli (37Miki T. Yoshioka K. Horiuchi Y. J. Mol. Biol. 1984; 174: 605-625Crossref PubMed Scopus (78) Google Scholar, 38Miki T. Chang Z.-T. Horiuchi Y. J. Mol. Biol. 1984; 174: 627-646Crossref PubMed Scopus (56) Google Scholar, 39Ogura T. Hiraga S. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 4784-4788Crossref PubMed Scopus (399) Google Scholar). The killing effect of LetD is suppressed by a mutation in gyrA or by overexpression of gyrA, suggesting that one target of LetD protein in cells is DNA gyrase (13Maki S. Takiguchi S. Miki T. Horiuchi T. J. Biol. Chem. 1992; 267: 12244-12251Abstract Full Text PDF PubMed Google Scholar). This has been attributed to the following mechanism. Expression of LetD protein leads to synthesis of ς 32, which induces DnaK and GroEL proteins, thus inhibiting DNA gyrase activity (15Gomez-Gomez J.M. Baquero F. Blazquez J. J. Bacteriol. 1996; 178: 3331-3334Crossref PubMed Google Scholar, 40Mizushima T. Ohtsuka Y. Mori H. Miki T. Sekimizu K. Mol. Gen. Genet. 1996; 253: 297-302PubMed Google Scholar). In contrast to LetD, GyrI is the first identified regulatory factor for DNA gyrase which directly inhibits the activity in vitro and is encoded on the chromosome of E. coli.To assess the in vivo importance of the function of GyrI, we examined the morphological phenotype of cells with perturbed expression of gyrI using overexpression of gyrI itself or antisense gyrI. Overexpression of gyrI and expression of antisense gyrI suppressed proliferation of the host cells and decreased the number of the viable cells. Microscopic examination revealed that some population of the cells overexpressing sense or antisense gyrI grew filamentously and had nucleoids with abnormal morphology as described above. The abnormal shapes of cells and nucleoids were similar to those observed in bacterial cells treated with quinolones (41Piddock L.J.V. Wise R. J. Antimicrob. Chemother. 1987; 20: 631-638Crossref PubMed Scopus (141) Google Scholar), suggesting that the abnormality might be caused by perturbation of DNA gyrase activity in the cells expressing sense or antisense gyrI. Thus, it is conceivable that GyrI is involved in regulation of DNA gyrase in vivo.The promoter activity of gyrA, the gene coding for the subunit A of DNA gyrase, increases in the mid-exponential phase to peak in the late exponential growth phase and thereafter decreases to the level of that in the mid-exponential phase (14Kaneko T. Mizushima T. Ohtsuka Y. Kurokawa K. Kataoka K. Miki T. Sekimizu K. Mol. Gen. Genet. 1996; 250: 593-600PubMed Google Scholar). In contrast, transcription of gyrI is expressed mainly from the late growth phase to the stationary phase, as assessed by using the reporter system. By dot-blot assay with the anti-GyrI antibody, it was shown that GyrI was synthesized in a pattern similar to that of transcription of gyrI during cell growth. There was found at the 5′ region (−36 to −31) of gyrI a consensus sequence (TATACT) for recognition by transcription factor ς38, which specifically functions for gene expression in the late growth phase (42Tanaka K. Takayanagi Y. Fujita N. Ishihama A. Takahashi H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3511-3515Crossref PubMed Scopus (190) Google Scholar). To confirm involvement of ς38 in transcription of gyrI, further studies will be needed. The concerted regulation of expression of the genes (gyrA and gyrB) encoding DNA gyrase subunits and the gene encoding the regulatory factor (gyrI) of DNA gyrase might be critical for DNA replication and cell proliferation.This study demonstrated that disturbance (reduction or amplification) of GyrI levels resulted in suppression of bacterial cell proliferation.gyrI/GyrI might be novel and promising targets for development of new antibacterial agents. DNA gyrase, a type II topoisomerase in Escherichia coli, has the ability to cut a double-stranded DNA, pass an uncut portion of the duplex between the cut ends, and reseal the cut. It can introduce negative supercoils into covalently closed circular DNA and cause catenation and decatenation of two different DNA duplexes,in vitro (1Menzel R. Gellert M. Adv. Pharmacol. 1994; 29A: 39-69Crossref PubMed Scopus (33) Google Scholar). It has been established that the enzyme is essential for chromosomal replication in vivo (2Wang J.C. Biochim. Biophys. Acta. 1987; 909: 1-9Crossref PubMed Scopus (300) Google Scholar). Moreover, there have been reports on the involvement of DNA gyrase in transcription from certain operons, DNA repair, and recombination inE. coli (2Wang J.C. Biochim. Biophys. Acta. 1987; 909: 1-9Crossref PubMed Scopus (300) Google Scholar). DNA gyrase is composed of two subunits, A (GyrA) and B (GyrB), which are assembled in A2B2 complexes, the active form (3Gellert M. O'Dea M.H. Itoh T. Tomizawa J. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 4474-4478Crossref PubMed Scopus (557) Google Scholar, 4Klevan L. Wang J.C. Biochemistry. 1980; 19: 5229-5234Crossref PubMed Scopus (100) Google Scholar, 5Krueger S. Zaccai G. Wlodawer A. Langowski J. O'Dea M.H. Maxwell A. Gellert M. J. Mol. Biol. 1990; 211: 211-220Crossref PubMed Scopus (38) Google Scholar). The active complex has been purified from E. coli (6Mizuuchi K. O'Dea M.H. Gellert M. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 5960-5963Crossref PubMed Scopus (198) Google Scholar) and reconstituted from the purified GyrA and GyrB (7Higgins N.P. Peebles C.L. Sugino A. Cozzarelli N.R. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 1773-1777Crossref PubMed Scopus (190) Google Scholar, 8Sugino A. Cozzarelli N.R. J. Biol. Chem. 1980; 255: 6299-6306Abstract Full Text PDF PubMed Google Scholar, 9Staudenbauer W.L. Orr E. Nucleic Acids Res. 1981; 9: 3589-3603Crossref PubMed Scopus (127) Google Scholar). GyrA has an active center for the reactions of introducing and resealing the cuts of double-stranded DNA, whereas GyrB powers the reaction by catalyzing ATP hydrolysis. DNA gyrase is a target of two distinct classes of inhibitors, coumarins (10Drlica K. Coughlin S. Pharmacol. Ther. 1989; 44: 107-121Crossref PubMed Scopus (55) Google Scholar, 11Maxwell A. Mol. Microbiol. 1993; 9: 681-686Crossref PubMed Scopus (229) Google Scholar) and quinolones (10Drlica K. Coughlin S. Pharmacol. Ther. 1989; 44: 107-121Crossref PubMed Scopus (55) Google Scholar, 12Maxwell A. J. Antimicrob. Chemother. 1992; 30: 409-414Crossref PubMed Scopus (157) Google Scholar). Coumarins bind to GyrB and are competitive inhibitors with respect to ATP (11Maxwell A. Mol. Microbiol. 1993; 9: 681-686Crossref PubMed Scopus (229) Google Scholar). In contrast, quinolones bind DNA gyrase when the enzyme is complexed with DNA and trap the enzyme in an abortive ternary complex, which, upon treatment with a denaturant, releases cleaved DNA with GyrA covalently attached to the 5′-phosphoryl ends generated at the cut site. There have been several reports on regulating DNA gyrase activity inE. coli. LetD (13Maki S. Takiguchi S. Miki T. Horiuchi T. J. Biol. Chem. 1992; 267: 12244-12251Abstract Full Text PDF PubMed Google Scholar) encoded on F factor inhibits DNA gyrase activity via the induction of synthesis of heat shock proteins (14Kaneko T. Mizushima T. Ohtsuka Y. Kurokawa K. Kataoka K. Miki T. Sekimizu K. Mol. Gen. Genet. 1996; 250: 593-600PubMed Google Scholar). Another regulatory factor, cyclic AMP (cAMP) receptor (15Gomez-Gomez J.M. Baquero F. Blazquez J. J. Bacteriol. 1996; 178: 3331-3334Crossref PubMed Google Scholar), participates in regulation of the growth phase-dependent transcription of gyrA (15Gomez-Gomez J.M. Baquero F. Blazquez J. J. Bacteriol. 1996; 178: 3331-3334Crossref PubMed Google Scholar). In this study, 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. We describe here the purification and characterization of GyrI and phenotypic analyses of recombinant strains overproducing GyrI or expressing antisensegyrI gene to decipher importance of GyrI in the regulation of DNA gyrase activity in vivo. DISCUSSIONWe purified from E. coli KL16 the 18-kDa protein that inhibited the supercoiling activity of DNA gyrase and coined it DNA gyrase inhibitory protein (GyrI). The NH2-terminal amino acid sequence and molecular mass of GyrI inferred that the gene encoding GyrI might be identical to the previously reported genes ofyeeB (EMBL accession no. U00009) and sbmC (34Baquero M.R. Bouzon M. Varea J. Moreno F. Mol. Microbiol. 1995; 18: 301-311Crossref PubMed Scopus (42) Google Scholar). The yeeB gene had been identified as an open reading frame in the sbcB region, although its function had not been described. The sbmC gene had been reported to decrease the sensitivity to microcin B17 when overexpressed. Microcin B17, a peptide antibiotic of 43 amino acids, is generated by cleavage of a precursor of 69 amino acids encoded by mcbA on plasmids (35Davagnino J. Herrero M. Furlong D. Moreno F. Kolter R. Proteins. 1986; 1: 230-238Crossref PubMed Scopus (62) Google Scholar) and appeared to trap an abortive cleavable DNA·DNA gyrase complex (36Vizan J.L. Hernandez-Chico C. del Castillo I. Morneo F. EMBO J. 1991; 10: 467-476Crossref PubMed Scopus (154) Google Scholar), a mode of action similar to that of quinolones. The yeeB andsbmC gene was located at 44 min on the E. colichromosome map. However, there have been no reports on the inhibitory activity against DNA gyrase of the gene products of yeeB andsbmC.To investigate the identity of gene encoding GyrI with yeeBand sbmC, we cloned the coding region based on the reported sequence and purified the 18-kDa gene product from the transformant overexpressing it. In vitro assay of DNA gyrase supercoiling activity indicated that the purified 18-kDa protein indeed inhibited the activity. Furthermore, we confirmed that GyrI protein is not intercalated into DNA and does not inhibit the activity of other DNA-processing enzymes (e.g. DNA polymerase) (data not shown). We tentatively named the gene coding for the 18-kDa protein asgyrI to indicate clearly the biological function of the gene product.It was reported that factor LetD regulates the activity of DNA gyrase (13Maki S. Takiguchi S. Miki T. Horiuchi T. J. Biol. Chem. 1992; 267: 12244-12251Abstract Full Text PDF PubMed Google Scholar). LetD encoded by the F factor functions to kill the host E. coli (37Miki T. Yoshioka K. Horiuchi Y. J. Mol. Biol. 1984; 174: 605-625Crossref PubMed Scopus (78) Google Scholar, 38Miki T. Chang Z.-T. Horiuchi Y. J. Mol. Biol. 1984; 174: 627-646Crossref PubMed Scopus (56) Google Scholar, 39Ogura T. Hiraga S. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 4784-4788Crossref PubMed Scopus (399) Google Scholar). The killing effect of LetD is suppressed by a mutation in gyrA or by overexpression of gyrA, suggesting that one target of LetD protein in cells is DNA gyrase (13Maki S. Takiguchi S. Miki T. Horiuchi T. J. Biol. Chem. 1992; 267: 12244-12251Abstract Full Text PDF PubMed Google Scholar). This has been attributed to the following mechanism. Expression of LetD protein leads to synthesis of ς 32, which induces DnaK and GroEL proteins, thus inhibiting DNA gyrase activity (15Gomez-Gomez J.M. Baquero F. Blazquez J. J. Bacteriol. 1996; 178: 3331-3334Crossref PubMed Google Scholar, 40Mizushima T. Ohtsuka Y. Mori H. Miki T. Sekimizu K. Mol. Gen. Genet. 1996; 253: 297-302PubMed Google Scholar). In contrast to LetD, GyrI is the first identified regulatory factor for DNA gyrase which directly inhibits the activity in vitro and is encoded on the chromosome of E. coli.To assess the in vivo importance of the function of GyrI, we examined the morphological phenotype of cells with perturbed expression of gyrI using overexpression of gyrI itself or antisense gyrI. Overexpression of gyrI and expression of antisense gyrI suppressed proliferation of the host cells and decreased the number of the viable cells. Microscopic examination revealed that some population of the cells overexpressing sense or antisense gyrI grew filamentously and had nucleoids with abnormal morphology as described above. The abnormal shapes of cells and nucleoids were similar to those observed in bacterial cells treated with quinolones (41Piddock L.J.V. Wise R. J. Antimicrob. Chemother. 1987; 20: 631-638Crossref PubMed Scopus (141) Google Scholar), suggesting that the abnormality might be caused by perturbation of DNA gyrase activity in the cells expressing sense or antisense gyrI. Thus, it is conceivable that GyrI is involved in regulation of DNA gyrase in vivo.The promoter activity of gyrA, the gene coding for the subunit A of DNA gyrase, increases in the mid-exponential phase to peak in the late exponential growth phase and thereafter decreases to the level of that in the mid-exponential phase (14Kaneko T. Mizushima T. Ohtsuka Y. Kurokawa K. Kataoka K. Miki T. Sekimizu K. Mol. Gen. Genet. 1996; 250: 593-600PubMed Google Scholar). In contrast, transcription of gyrI is expressed mainly from the late growth phase to the stationary phase, as assessed by using the reporter system. By dot-blot assay with the anti-GyrI antibody, it was shown that GyrI was synthesized in a pattern similar to that of transcription of gyrI during cell growth. There was found at the 5′ region (−36 to −31) of gyrI a consensus sequence (TATACT) for recognition by transcription factor ς38, which specifically functions for gene expression in the late growth phase (42Tanaka K. Takayanagi Y. Fujita N. Ishihama A. Takahashi H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3511-3515Crossref PubMed Scopus (190) Google Scholar). To confirm involvement of ς38 in transcription of gyrI, further studies will be needed. The concerted regulation of expression of the genes (gyrA and gyrB) encoding DNA gyrase subunits and the gene encoding the regulatory factor (gyrI) of DNA gyrase might be critical for DNA replication and cell proliferation.This study demonstrated that disturbance (reduction or amplification) of GyrI levels resulted in suppression of bacterial cell proliferation.gyrI/GyrI might be novel and promising targets for development of new antibacterial agents. We purified from E. coli KL16 the 18-kDa protein that inhibited the supercoiling activity of DNA gyrase and coined it DNA gyrase inhibitory protein (GyrI). The NH2-terminal amino acid sequence and molecular mass of GyrI inferred that the gene encoding GyrI might be identical to the previously reported genes ofyeeB (EMBL accession no. U00009) and sbmC (34Baquero M.R. Bouzon M. Varea J. Moreno F. Mol. Microbiol. 1995; 18: 301-311Crossref PubMed Scopus (42) Google Scholar). The yeeB gene had been identified as an open reading frame in the sbcB region, although its function had not been described. The sbmC gene had been reported to decrease the sensitivity to microcin B17 when overexpressed. Microcin B17, a peptide antibiotic of 43 amino acids, is generated by cleavage of a precursor of 69 amino acids encoded by mcbA on plasmids (35Davagnino J. Herrero M. Furlong D. Moreno F. Kolter R. Proteins. 1986; 1: 230-238Crossref PubMed Scopus (62) Google Scholar) and appeared to trap an abortive cleavable DNA·DNA gyrase complex (36Vizan J.L. Hernandez-Chico C. del Castillo I. Morneo F. EMBO J. 1991; 10: 467-476Crossref PubMed Scopus (154) Google Scholar), a mode of action similar to that of quinolones. The yeeB andsbmC gene was located at 44 min on the E. colichromosome map. However, there have been no reports on the inhibitory activity against DNA gyrase of the gene products of yeeB andsbmC. To investigate the identity of gene encoding GyrI with yeeBand sbmC, we cloned the coding region based on the reported sequence and purified the 18-kDa gene product from the transformant overexpressing it. In vitro assay of DNA gyrase supercoiling activity indicated that the purified 18-kDa protein indeed inhibited the activity. Furthermore, we confirmed that GyrI protein is not intercalated into DNA and does not inhibit the activity of other DNA-processing enzymes (e.g. DNA polymerase) (data not shown). We tentatively named the gene coding for the 18-kDa protein asgyrI to indicate clearly the biological function of the gene product. It was reported that factor LetD regulates the activity of DNA gyrase (13Maki S. Takiguchi S. Miki T. Horiuchi T. J. Biol. Chem. 1992; 267: 12244-12251Abstract Full Text PDF PubMed Google Scholar). LetD encoded by the F factor functions to kill the host E. coli (37Miki T. Yoshioka K. Horiuchi Y. J. Mol. Biol. 1984; 174: 605-625Crossref PubMed Scopus (78) Google Scholar, 38Miki T. Chang Z.-T. Horiuchi Y. J. Mol. Biol. 1984; 174: 627-646Crossref PubMed Scopus (56) Google Scholar, 39Ogura T. Hiraga S. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 4784-4788Crossref PubMed Scopus (399) Google Scholar). The killing effect of LetD is suppressed by a mutation in gyrA or by overexpression of gyrA, suggesting that one target of LetD protein in cells is DNA gyrase (13Maki S. Takiguchi S. Miki T. Horiuchi T. J. Biol. Chem. 1992; 267: 12244-12251Abstract Full Text PDF PubMed Google Scholar). This has been attributed to the following mechanism. Expression of LetD protein leads to synthesis of ς 32, which induces DnaK and GroEL proteins, thus inhibiting DNA gyrase activity (15Gomez-Gomez J.M. Baquero F. Blazquez J. J. Bacteriol. 1996; 178: 3331-3334Crossref PubMed Google Scholar, 40Mizushima T. Ohtsuka Y. Mori H. Miki T. Sekimizu K. Mol. Gen. Genet. 1996; 253: 297-302PubMed Google Scholar). In contrast to LetD, GyrI is the first identified regulatory factor for DNA gyrase which directly inhibits the activity in vitro and is encoded on the chromosome of E. coli. To assess the in vivo importance of the function of GyrI, we examined the morphological phenotype of cells with perturbed expression of gyrI using overexpression of gyrI itself or antisense gyrI. Overexpression of gyrI and expression of antisense gyrI suppressed proliferation of the host cells and decreased the number of the viable cells. Microscopic examination revealed that some population of the cells overexpressing sense or antisense gyrI grew filamentously and had nucleoids with abnormal morphology as described above. The abnormal shapes of cells and nucleoids were similar to those observed in bacterial cells treated with quinolones (41Piddock L.J.V. Wise R. J. Antimicrob. Chemother. 1987; 20: 631-638Crossref PubMed Scopus (141) Google Scholar), suggesting that the abnormality might be caused by perturbation of DNA gyrase activity in the cells expressing sense or antisense gyrI. Thus, it is conceivable that GyrI is involved in regulation of DNA gyrase in vivo. The promoter activity of gyrA, the gene coding for the subunit A of DNA gyrase, increases in the mid-exponential phase to peak in the late exponential growth phase and thereafter decreases to the level of that in the mid-exponential phase (14Kaneko T. Mizushima T. Ohtsuka Y. Kurokawa K. Kataoka K. Miki T. Sekimizu K. Mol. Gen. Genet. 1996; 250: 593-600PubMed Google Scholar). In contrast, transcription of gyrI is expressed mainly from the late growth phase to the stationary phase, as assessed by using the reporter system. By dot-blot assay with the anti-GyrI antibody, it was shown that GyrI was synthesized in a pattern similar to that of transcription of gyrI during cell growth. There was found at the 5′ region (−36 to −31) of gyrI a consensus sequence (TATACT) for recognition by transcription factor ς38, which specifically functions for gene expression in the late growth phase (42Tanaka K. Takayanagi Y. Fujita N. Ishihama A. Takahashi H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3511-3515Crossref PubMed Scopus (190) Google Scholar). To confirm involvement of ς38 in transcription of gyrI, further studies will be needed. The concerted regulation of expression of the genes (gyrA and gyrB) encoding DNA gyrase subunits and the gene encoding the regulatory factor (gyrI) of DNA gyrase might be critical for DNA replication and cell proliferation. This study demonstrated that disturbance (reduction or amplification) of GyrI levels resulted in suppression of bacterial cell proliferation.gyrI/GyrI might be novel and promising targets for development of new antibacterial agents. We thank Dr. Saburo Komatsubara, Dr. Motoaki Ohashi, Dr. Keisuke Kawashima, Dr. Tetsuya Tosa, Mr. Yoshiyasu Ohta, and Dr. Shiro Kanegasaki for support and encouragement through this study and Dr. Charles W. Mahoney for critical reading of the manuscript.
Referência(s)