Identification of DNA Gyrase Inhibitor (GyrI) in Escherichia coli*

Akira NakanishiDagger §, Tadahiro OshidaDagger , Tadahiro Matsushita, Shinobu Imajoh-Ohmipar , and Tetsuo OhnukiDagger

From the Dagger  Lead Generation Research Laboratory and the  Pharmaceutical Development Research Laboratory, Tanabe Seiyaku Company, Ltd., Saitama 335, and the par  Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108, Japan

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

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.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

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 (1). It has been established that the enzyme is essential for chromosomal replication in vivo (2). Moreover, there have been reports on the involvement of DNA gyrase in transcription from certain operons, DNA repair, and recombination in E. coli (2).

DNA gyrase is composed of two subunits, A (GyrA) and B (GyrB), which are assembled in A2B2 complexes, the active form (3-5). The active complex has been purified from E. coli (6) and reconstituted from the purified GyrA and GyrB (7-9). 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 (10, 11) and quinolones (10, 12). Coumarins bind to GyrB and are competitive inhibitors with respect to ATP (11). 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 in E. coli. LetD (13) encoded on F factor inhibits DNA gyrase activity via the induction of synthesis of heat shock proteins (14). Another regulatory factor, cyclic AMP (cAMP) receptor (15), participates in regulation of the growth phase-dependent transcription of gyrA (15).

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 antisense gyrI gene to decipher importance of GyrI in the regulation of DNA gyrase activity in vivo.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Bacterial Strains and Plasmids-- E. coli strains used in this study were JM109, GI724 (F- lambda - lacIq lacPL8 ampC::Ptrp cI mcrA mcrB INV(rnnD-rnnE)), and KL16 (HfrKL16-P045(thyAright-arrowserA):thi-1 relA1 (lambda )-). A plasmid pLGlacZ7 (16) was used for construction of a reporter plasmid to monitor the strength of the gyrI promoter. pBR322 and pLEX were purchased from Takara and Invitrogen, respectively.

Assay of DNA Supercoiling Activity of DNA Gyrase-- The supercoiling activity was measured by the method of Sato et al. (17). 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 µl) contained 25 mM Tris-HCl (pH 8.0), 67 mM KCl, 5 mM MgCl2, 1.25 mM spermidine hydrochloride, 1.7 mM ATP, 20 µg of E. coli tRNA/ml, and 0.15 µg of relaxed pBR322 DNA. pBR322 was relaxed by using topoisomerase I as described by Takahata and Nishino (19). After the addition of 1 unit (0.23 µg of protein) of the holoenzyme reconstituted from the purified subunits, the reaction mixture was incubated at 37 °C for 2 h. The reaction was stopped by supplementation with 20 µg/ml proteinase K, and the mixture was subjected to 1% agarose gel electrophoresis. The gel was stained with ethidium bromide (0.5 µg/ml) and photographed. The supercoiling activity was calculated from the density of the band of supercoiled DNA, which was quantitated using a densitometer with the negatives.

Purification of GyrA and GyrB Subunits-- Subunits A (GyrA) and B (GyrB) of DNA gyrase were purified from E. coli KL16 (20). Cultivation of the bacteria, preparation of bacterial lysate, and removal of DNA by successive streptomycin and ammonium sulfate precipitation were conducted according to the method of Aoyama and co-workers (17, 20). 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 proteins were eluted stepwise by 0.2 M KCl, 2 M KCl, 5 M urea, and 2 M KCl plus 5 M urea in TED buffer. The holoenzyme and GyrA and GyrB subunits were eluted by TED buffer containing 5 M urea, 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 with TED buffer. The column was washed with TED buffer containing 100 mM KCl, and the activity was eluted with TED buffer containing 2 M KCl. The fractions containing GyrB were purified further by chromatography on novobiocin-Sepharose column again. The sample was applied to the column, washed with TED buffer containing 100 mM KCl, and then eluted with TED buffer containing 5 M urea. When analyzed by SDS-polyacrylamide gel electrophoresis (PAGE),1 the final preparation of GyrA gave a stained band of 105 kDa corresponding to GyrA, whereas that of GyrB produced stained bands of 47 and 43 kDa in addition to a 95-kDa band of GyrB. The contaminating proteins might be degradates of GyrB (21, 22). Each of the purified GyrA and GyrB fractions was pooled separately and dialyzed against 50 mM Tris-HCl (pH 7.5), 50% glycerol, 0.5 mM EDTA, and 1 mM dithiothreitol. Purified GyrA and GyrB were stable for several months when stored at -20 °C.

Purification of GyrI Protein-- E. coli GI724 (pCA20) was grown on RMG-Amp plates consisting of 6 g of Na2HPO4, 3 g of KH2PO4, 0.5 g of NaCl, 1 g of NH4Cl, 20 g of casamino acids, 5 g of glycerol, 0.2 g of MgCl2, 100 mg of ampicillin, and 15 g of agarose per liter (pH 7.4). Colonies were then inoculated into 500 ml of RM medium consisting of 6 g of Na2HPO4, 3 g of KH2PO4, 0.5 g of NaCl, 1 g of NH4Cl, 20 g of casamino acids, 10 g of glycerol, 0.2 g of MgCl2, and 100 mg of ampicillin per liter (pH 7.4) and grown at 30 °C. When an absorbance at 600 nm (A600) of the culture reached 0.5, tryptophan was added to a final concentration of 100 µg/ml, and the temperature was shifted to 37 °C; the bacterial cells were grown for another 5 h and harvested by centrifugation.

All of the following purification procedures were carried out at 4 °C unless indicated otherwise. The pellet of bacterial cells was suspended in solution (5 ml) consisting of 30 mM Tris-HCl (pH 7.5) and 30 mM NaCl. The cells were lysed with a sonicator and centrifuged at 8,000 rpm for 20 min to remove cell debris. Solid ammonium sulfate (0.39 g/g of supernatant) was added with stirring, and after 20 min, the precipitate was collected by centrifugation and redissolved in 5 ml of TED buffer. The solution thus obtained was loaded onto a Sepharose Q column (Pharmacia, 0.55 cm2 × 16.5 cm) equilibrated with TED buffer. Protein was eluted with a linear gradient of NaCl (0.025-0.7 M) in 140 ml total of TED buffer. An aliquot of each fraction was analyzed using SDS-PAGE, and the fractions containing the 18-kDa protein were pooled. The pooled fractions (4 ml) were applied to a TSKgel TOYOPEARL HW-55 column (TOSO, 0.4 cm2 × 18 cm) and chromatographed with TED buffer. The fractions containing the 18-kDa protein were quickly frozen in small aliquots using liquid nitrogen.

Analysis of Protein-- SDS-PAGE (12%) was carried out according to the method of Laemmli (23). Electrophoresed proteins were transferred onto a polyvinylidene difluoride membrane (Millipore Corp.) using a semidry type electroblotting apparatus at 160 mA for 30 min, with CAPS (24). After blotting, the polyvinylidene difluoride membrane was stained with Coomassie Brilliant Blue, photographed, and destained with 60% methanol followed by extensive rinsing in distilled water. The region of the polyvinylidene difluoride membrane containing the 18-kDa protein band was excised and subjected to Edman degradation. NH2-terminal amino acid sequencing was determined using a peptide sequencer LF3400 (Beckman).

Construction of Plasmids-- The DNA fragment containing the coding and promoter region of gyrI was obtained from the chromosomal DNA of E. coli KL16 by polymerase chain reaction. The primers used were designed from the reported nucleotide sequence of sbcB region (EMBL accession no. U00009) (25, 26) based on an assumption that gyrI might be identical to yeeB (see "Results"). Plasmids pCA20 and pCA19 were constructed for expression of sense and antisense gyrI gene, respectively, as follows. The primers used for cloning gyrI were 5'-CACATATGAACTACGAGATTAAGC-3' containing the 1-19-bp region of gyrI and 5'-CACATATGTTAGTGATGTTTTGGCTGCA-3' containing the 455- 474-bp region of gyrI. Polymerase chain reaction was carried out at 94 °C for 1 min, 40 °C for 1 min, and 72 °C for 3 min for 30 cycles. The amplified 474-bp fragments were digested with NdeI (underlined in the sequence of primers) and ligated into the corresponding cloning site of pLEX vector. Sense or antisense orientation of the inserted gyrI gene was determined by restriction enzyme analysis with EcoRV. The resultant plasmids, pCA20 and pCA19, were introduced into E. coli GI724. In these constructs, expression of the inserted gene depended on the PL promoter, which is tightly regulated by the cI repressor protein encoded on the chromosome; the latter expression was under the control of the trp promoter and repressed by the addition of tryptophan, thus resulting in induction of expression of the inserted gene. Transcription of the sense and antisense gyrI gene was induced by the addition of 100 µg of tryptophan/ml to the culture medium.

A reporter plasmid was constructed for investigation of gene expression of gyrI as follows. A DNA fragment corresponding to the -252 to 970-bp region of gyrI (5774-6995-bp region of the sbcB region) was amplified using the forward and reverse primers 5'-CTGGATCCATCAGCGGGTAGGGGAAATTGA-3' and 5'-GTGGATCCCAGACTAACATCAGCGGTAACG-3', respectively. The putative promoter region of gyrI (285-bp fragment encompassing -252 to +33-bp region of gyrI gene) was obtained by digestion with BamHI and RsaI of the amplified DNA fragment and inserted into a BamHI site of pLGlacZ7 (16) after blunting the insert and the vector with Klenow DNA polymerase. Orientation of the inserted DNA was tested by digestion with EcoRI and BstPI. The resultant reporter plasmid named pCA15 carried a fused gene consisting of 11 amino acid residues of the NH2 terminus of GyrI and beta -galactosidase, a reporter. Another plasmid pCA16 had the insert in the opposite direction compared with pCA15.

Investigation of Expression of gyrI by Reporter System-- The activity of beta -galactosidase in E. coli JM109 transformed with pCA15 was measured as described (27). Overnight culture of the transformant was inoculated into LB medium (10 g of Bacto-tryptone, 5 g of yeast extract, and 10 g of NaCl per liter) containing 50 µg of kanamycin/ml at a inoculum size giving an A600 = 0.1 and cultivated at 37 °C. Aliquots (1 ml) were withdrawn from the culture and analyzed for activity of beta -galactosidase and A600. For measurement of beta -galactosidase activity, the culture aliquots (30 µl) were mixed with 0.97 ml of Z buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4, and 50 mM beta -mercaptoethanol (pH 7.0)), 20 µl of chloroform, and 20 µl of 0.1% SDS, vortexed for 10 s, and then preincubated at 28 °C (5 min). Reaction of beta -galactosidase was started by adding 0.2 ml of 13 mM o-nitrophenyl-beta -D-galactopyranoside and was stopped by adding 0.5 ml of 1 M sodium carbonate. beta -Galactosidase activity was expressed in Miller units (28).

Preparation of Antibody to GyrI-- Antibody to the purified GyrI protein was raised in rabbits by the method of Liu et al. (29). The immunogen (0.1 mg of protein) was injected subcutaneously to rabbits with 0.025 mg of adjuvant (GERBU Biotechnik) (30-32). The second immunization was performed after 3 weeks in the same manner as the first one, and thereafter immunizations were carried out at 7-day intervals. The antibody titer in antiserum was monitored by dot-blot assay. When the antibody titer increased sufficiently, the rabbit was bled from the ear artery (30-50 ml).

Antibody was purified from the antiserum by using E-Z-SEP Polyclonal kit (Pharmacia Biotech Inc.) according to the protocol recommended by the manufacturer, to give an IgG fraction of the antiserum. The purified antibody was aliquoted and stored at -20 °C.

Immunological Detection of GyrI-- Dot-blot assay was carried out as follows. Protein and bovine serum albumin were mixed in phosphate-buffered saline at final concentrations of 0.1 and 1 mg/ml, respectively, to which 0.01 volume of glutaraldehyde was added. The mixture was spotted on a nitrocellulose filter. The filter was washed with TBS (20 mM Tris-HCl (pH 7.5) and 0.15 M NaCl), shaken in TBS containing 5 mM sodium azide and 20 mg of bovine serum albumin per ml for 30 min, and finally washed with TBS. The purified anti-GyrI antibody was diluted sequentially from 100- to 2,000-fold with TBS containing 20 mg of bovine serum albumin/ml, and incubated for 2 h at 37 °C with the antigen-spotted filters. After the incubation, the filter was washed with TBS containing 0.05% (w/v) Tween 20 and incubated with horseradish peroxidase-conjugated secondary antibody (1 µg of anti-rabbit IgG/ml of TBS containing 20 mg of bovine serum albumin/ml). Binding of the antibody was visualized by color development with the peroxidase substrates 3,3'-diaminobenzidine and hydrogen peroxide. Western blotting was performed as described above. The protein band of GyrI was identified by immunological staining of the protein-blotted membrane as in the dot-blot assay.

Microscopic Observation of Nucleoids-- Nucleoids of E. coli cells were stained with ethidium bromide (33). The bacterial cells were washed with M9 medium (42 mM Na2HPO4, 22 mM KH2PO4, 8.5 mM NaCl, and 18 mM NH4Cl (pH 7.4)), suspended in a solution containing 10 mM Tris-HCl (pH 7.4), 5 mM EDTA, 0.05% 2-mercaptoethanol, 0.05% chloramphenicol, and 0.8% ethanol, and then mixed with an equal volume of 0.01% ethidium bromide solution. The nucleoids were observed and photographed with an incident fluorescence microscope equipped with phase-contrast optics (Nikon HFX).

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Purification of 18-kDa Protein-- DNA gyrase was partially purified by using affinity chromatography on a novobiocin-Sepharose column (Fig. 1). The holoenzyme of DNA gyrase consisting of both GyrA and GyrB subunits was eluted by 5 M urea. The fractions containing GyrA were identified by activity to enhance the supercoiling activity of the dilute holoenzyme at a concentration at which its activity was hardly detected (20). The activity of the GyrA subunit was found in the fractions eluted by 2 M KCl. GyrB subunit was found in the fractions of 2 M KCl plus 5 M urea when tested by crossing with the GyrA fraction. DNA supercoiling activity was not detected in the other fractions even when assayed in the presence of subunit GyrA or GyrB.


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Fig. 1.   Partially purified 18-kDa protein inhibits DNA gyrase. Partially purified extract of E. coli KL16 was separated by stepwise elution from a novobiocin-Sepharose column. Solutions used for elution are indicated under the abscissa. The ordinate represents absorbance at 280 nm of the fractions. The fractions containing the holoenzyme consisting of GyrA and GyrB subunits (black-triangle) were identified by supercoiling activity. The fractions containing GyrA (bullet ) or GyrB (×) were detected by DNA supercoiling activity in the presence of the holoenzyme (0.5 unit) or GyrA (0.5 unit), respectively. As assayed by supercoiling activity, the other fractions (open circle ) did not appear to contain GyrA and/or GyrB. SDS-PAGE analysis of fractions 25 and 32 is shown in the inset. The stained bands of intact GyrA (105 kDa) and GyrB (95 kDa) subunits and the 18-kDa protein are marked, respectively. Addition of fraction 32 (8 µl) to the reaction mixture (20 µl) containing 1 unit of DNA gyrase inhibited supercoiling of relaxed pBR322, whereas addition of fraction 25 did not (inset).

All of the fractions eluted from the affinity column were analyzed by SDS-PAGE. It was found that fraction 32, which exhibited no supercoiling activity, showed a protein banding pattern similar to that of the fraction containing both GyrA and GyrB, except that fraction 32 contained an additional protein band of 18 kDa (Fig. 1). Only in this fraction was the 18-kDa protein detected. When added to the reaction mixture for DNA supercoiling, fraction 32 inhibited the activity (Fig. 1). This result suggested that the 18-kDa protein might affect the DNA supercoiling activity of DNA gyrase. The 18-kDa protein was purified by gel filtration from fraction 32 for more detailed studies. The purified 18-kDa protein was examined for its ability to inhibit DNA gyrase supercoiling activity. Supercoiling activity was inhibited 91 and 97% by fraction 32 (22 µg/ml) and purified 18-kDa protein (6.8 µg/ml), respectively. This result suggested that the 18-kDa protein might be responsible for the inhibition.

To investigate whether this inhibition was caused by nuclease activity, the 18-kDa protein (3 µg/ml) was incubated in the presence of relaxed or supercoiled pBR322 DNAs for 2 h at 37 °C and subsequently analyzed by agarose gel electrophoresis. Neither of the plasmid DNAs was degradated by the 18-kDa protein (data not shown). Furthermore, we examined the protease activity of the purified 18-kDa protein using subunits A and B of DNA gyrase as substrates. Their size and the amount of DNA gyrase subunits A and B were not influenced by the addition of the 18-kDa protein when examined by SDS-PAGE (data not shown). The above results suggested that the 18-kDa protein had neither nuclease nor protease activity.

Taken together, the results strongly suggested that the 18-kDa protein was an inhibitor of the supercoiling activity of DNA gyrase. We tentatively termed it DNA gyrase inhibitory protein (GyrI).

Analysis of NH2-terminal Amino Acid Sequence of GyrI Protein-- The NH2-terminal amino acid sequence of the GyrI protein was determined to be MNYEIKQEEKRTVAGF. Homology searching of protein data bases revealed that a putative gene product (157 amino acid residues, 18,095 Da) encoded by yeeB of E. coli had the same NH2-terminal sequence (EMBL U00009). The function of this gene product has not been characterized to date. yeeB was mapped at 44 min of the E. coli genetic map, near the sbcB gene. Another synonymous gene (sbmC) encoding the same NH2-terminal amino acids as GyrI, had been characterized as a gene that confers resistance to microcin B17 on E. coli when overexpressed (34). Microcin B17, a peptide antibiotic of 43 amino acids, induced breakage of double-stranded DNA in a DNA gyrase-dependent manner.

Exogenous Expression and Purification of 18-kDa Protein-- We assumed that the gene encoding GyrI might be identical to yeeB and sbmC, and we amplified the gene based on the DNA sequence of the sbcB region by polymerase chain reaction. The amplified DNA was inserted into the pLEX vector, resulting in construction of pCA20.

E. coli GI724 was transformed with pCA20 and grown in RM medium containing 1% (w/v) glycerol and 2% (w/v) casamino acids. Induction of the expression of inserted gene was achieved by the addition of 100 µg of tryptophan/ml to the growth medium, and cultivation was continued for 5 h. SDS-PAGE analysis identified a Coomassie Blue-stained band of 18 kDa in cell lysate from tryptophan-induced E. coli containing pCA20. In contrast, the protein band was absent from the lysate of uninduced cells. The 18-kDa protein was purified by the procedures of ammonium sulfate fractionation, Sepharose Q column chromatography, and TSKgel filtration (Fig. 2A). A yield of 0.55 mg/liter of culture of the purified 18-kDa protein was obtained.


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Fig. 2.   Purification of 18-kDa protein and inhibitory activity of purified 18-kDa protein against DNA gyrase. Panel A, purification of 18-kDa protein. Protein samples from each step of the purification were subjected to SDS-PAGE (12%). Lane 1, crude extract; lane 2, a dialyzed sample after ammonium sulfate precipitation; lane 3, pooled fractions after Q-Sepharose Fast Flow chromatography; lane 4, pooled fractions after TSKgel TOYOPEARL HW-55 chromatography; M, molecular mass markers. Panel B, inhibition of supercoiling activity of DNA gyrase by the purified 18-kDa protein. Lane 1, control without 18-kDa protein; lanes 2, 3, and 4 are 18-kDa protein at 2, 4, and 8 µg/ml, respectively. Lane 5, control relaxed and lane 6, supercoiled pBR322 DNA without 18-kDa protein and DNA gyrase are presented as shown. The positions of intracellularly negatively supercoiled and relaxed pBR322 DNAs are indicated by arrows. The pure 18-kDa protein was obtained from the E. coli strain overproducing GyrI. For details, see "Experimental Procedures."

Inhibition of DNA Supercoiling Activity by 18-kDa Protein-- The inhibitory effects on DNA gyrase by the purified 18-kDa protein are shown in Fig. 2B. The 18-kDa protein-free control (Fig. 2B, lane 1) contained relaxed pBR322 DNA and 1 unit of the DNA gyrase holoenzyme. The 18-kDa protein dose-dependently inhibited DNA supercoiling at doses of 2-8 µg/ml with complete inhibition at 8 µg/ml (Fig. 2B). The purified 18-kDa protein from the yeeB-overexpressing transformant inhibited DNA gyrase with a potency similar to that of the protein purified from E. coli KL16. Thus, we concluded that the gene encoding GyrI was identical to yeeB and sbmC. We tentatively named this gene gyrI to correlate the names of the gene and the gene product.

Effect of Overexpression of gyrI on Growth of E. coli-- To investigate whether the expression of gyrI has an effect on cell growth, we used the expression plasmid pCA20, which contains the former. Colony-forming units (CFU) of the transformants carrying the plasmid (pCA20) were normal when the cells were grown in the absence of induction of gyrI expression (absence of tryptophan) (Fig. 3). Tryptophan was added to the culture at A600 = 0.02 to induce gyrI. Under these conditions the CFU was reduced from 107 to 106 CFU/ml 5 h after the addition of tryptophan, i.e. induction of gyrI overexpression (Fig. 3). The CFU of transformants carrying the vector pLEX were not influenced by the addition of tryptophan (data not shown). The morphological phenotypes of the transformants were examined using ethidium bromide staining of the nucleoids (Fig. 4). The morphology of cells carrying the plasmid pCA20 were normal when cultured in the absence of tryptophan (Fig. 4A), whereas in the presence of tryptophan, some of the cells exhibited filamentous morphology and contained abnormal shapes of nucleoids (Fig. 4B); some of the host cells lost their nucleoids, and some of those contained small condensed, fragmented, or elongated nucleoids. The phenotype of this strain carrying the control vector pLEX was normal irrespective of the presence or absence of tryptophan.


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Fig. 3.   Effect of overproduction of GyrI on bacterial growth. E. coli GI724 transformed with plasmid pCA20 that contained gyrI was grown in the presence of ampicillin (100 µg/ml) at 30 °C overnight. The bacterial cells were transferred into fresh RM medium with (induction of gyrI) and without tryptophan (100 µg/ml) to give A600 = 0.02, and incubated at 37 °C. A600 and CFU were measured at 1-h intervals; CFU were determined by diluting and plating the culture on LB plates containing ampicillin (100 µg/ml). Open (open circle ) and closed (bullet ) circles represent data of A600 for the culture without and with tryptophan, respectively. Open (square ) and closed (black-square) squares represent data of CFU for the culture without and with tryptophan, respectively.


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Fig. 4.   Microscopic observation of nucleoids of the bacterial cells overexpressing gyrI. Growing cells of E. coli GI724 (pCA20) were inoculated in fresh RM medium with and without tryptophan (100 µg/ml) at an inoculum size to give A600 = 0.02 and incubated at 37 °C. After 1 h, the bacterial cells were stained with ethidium bromide to observe the morphology of nucleoids. Arrows indicate the locations of nucleoids. Panel A, cultured without tryptophan; panel B, cultured with tryptophan (induction of gyrI). Bar represents 2.5 µm.

Effect of Expression of Antisense gyrI Gene on Growth of E. coli-- We next assessed the physiological importance of GyrI by the means of expression of antisense gyrI gene (Fig. 5). To confirm that synthesis of GyrI was actually inhibited by the antisense gyrI we carried out Western blotting analysis with anti-GyrI antibody. Antibody-reactive proteins were not detected in the cells when expression of antisense gyrI was induced (data not shown). In the absence of tryptophan, the CFU of transformants carrying the plasmid pCA19, which contained antisense gyrI gene, increased from 2 × 107 to 1.4 × 109 CFU/ml. In the presence of tryptophan, the CFU did not change or tended to decrease. The bacterial cells carrying pCA19 exhibited filamentous growth in the presence of tryptophan as in the case of overexpression of gyrI (data not shown).


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Fig. 5.   Effect of expression of antisense gyrI on bacterial growth. E. coli GI724 transformed with plasmid pCA19 containing antisense gyrI was grown in the presence of ampicillin (100 µg/ml) at 30 °C overnight. The bacterial cells were inoculated into fresh RM medium with and without tryptophan (100 µg/ml) to give A600 = 0.02, and incubated at 37 °C. A600 and CFU were measured at 1-h intervals; CFU were determined by diluting and plating the culture on LB plates containing ampicillin (100 µg/ml). Open (open circle ) and closed (bullet ) circles represent data of A600 for the culture without and with tryptophan (induced), respectively. Open (square ) and closed (black-square) squares represent data of CFU for the culture without and with tryptophan (induced), respectively.

Expression Profile of gyrI Gene during Cell Growth-- To investigate the expression of gyrI during cell growth, we constructed a plasmid, pCA15, which carried a gyrI-lacZ fused gene. The fused gene consisted of a putative promoter and NH2-terminal region of gyrI (-251 to 33-bp region), which contained a palindrome structure of sigma -factor-dependent terminator for yeeC 5' adjacent to gyrI, a typical consensus sequence of -35 and -10 region for gyrI, and the structural gene of lacZ fused in a proper reading frame. E. coli JM109 transformed by pCA15 was grown in LB medium at 37 °C, and aliquots of the culture were taken periodically during the growth transition from the exponential growth phase to stationary phase. The level of expression of beta -galactosidase, i.e. strength of a promoter of gyrI, started to increase at the late exponential phase and reached the maximum level in the stationary phase (Fig. 6A).


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Fig. 6.   Profiles of transcription of gyrI and expression of GyrI during growth phase of bacteria. Panel A, reporter assay for assessing transcriptional activity of the promoter of gyrI. E. coli JM109 harboring the reporter plasmid (pCA15) or the plasmid (pCA16) carrying the promoter region inserted in the opposite direction compared with pCA15 was grown in LB medium with kanamycin (50 µg/ml) at 37 °C overnight. The precultures were inoculated into the same medium to give 0.1 absorbance at 600 nm (A600) and cultivated at 37 °C. At 1-h intervals, aliquots were removed and their A600 and beta -galactosidase activity were measured. Open (open circle ) and closed (bullet ) circles represent data of A600 for the bacterial strain harboring pCA15 (sense promoter) and pCA16 (inverted promoter), respectively. Open (square ) and closed (black-square) squares represent data of beta -galactosidase activity for bacterial strains harboring pCA15 and pCA16, respectively. Panel B, dot-blot assay for measuring the amount of GyrI synthesized during the growth phase. E. coli KL16 was grown in LB medium, and the GyrI protein was detected by dot-blot analysis. The bacterial cells were harvested at the indicated times of cultivation and used for dot-blot analysis using anti-GyrI antibody. Open (open circle ) and closed (bullet ) circles represent the growth of bacteria (A600) and the amount of GyrI expressed in arbitrary units, respectively.

Next, we examined synthesis of GyrI in E. coli KL16 during cell growth by a quantitative dot-blot assay using polyclonal antibodies against GyrI (Fig. 6B). The relative amounts of GyrI protein were determined by scanning the blots with a densitometer. The content of the protein increased about 2-fold/cell mass when the comparison was made between the cells of 0.75 and 1.3 A600.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

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 of yeeB (EMBL accession no. U00009) and sbmC (34). 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 (35) and appeared to trap an abortive cleavable DNA·DNA gyrase complex (36), a mode of action similar to that of quinolones. The yeeB and sbmC gene was located at 44 min on the E. coli chromosome map. However, there have been no reports on the inhibitory activity against DNA gyrase of the gene products of yeeB and sbmC.

To investigate the identity of gene encoding GyrI with yeeB and 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 as gyrI to indicate clearly the biological function of the gene product.

It was reported that factor LetD regulates the activity of DNA gyrase (13). LetD encoded by the F factor functions to kill the host E. coli (37-39). 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 (13). This has been attributed to the following mechanism. Expression of LetD protein leads to synthesis of sigma  32, which induces DnaK and GroEL proteins, thus inhibiting DNA gyrase activity (15, 40). 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 (41), 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 (14). 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 sigma 38, which specifically functions for gene expression in the late growth phase (42). To confirm involvement of sigma 38 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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Lead Generation Research Laboratory, Tanabe Seiyaku Co., Ltd., 2-50 Kawagishi-2-chome, Toda-shi, Saitama 335, Japan. Tel.: 81-48-433-2545; Fax: 81-48-433-2734; E-mail: anaka{at}tanabe.co.jp.

1 The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; CAPS, 3-(cyclohexylamino)propanesulfonic acid; bp, base pair; CFU, colony-forming units.

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Top
Abstract
Introduction
Procedures
Results
Discussion
References

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