Skip to main content
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Eukaryotic Cell
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems
  • Log in
  • My alerts
  • My Cart

Main menu

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • COVID-19 Special Collection
    • Archive
    • Minireviews
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About AAC
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • AAC Podcast
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Eukaryotic Cell
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems

User menu

  • Log in
  • My alerts
  • My Cart

Search

  • Advanced search
Antimicrobial Agents and Chemotherapy
publisher-logosite-logo

Advanced Search

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • COVID-19 Special Collection
    • Archive
    • Minireviews
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About AAC
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • AAC Podcast
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
Mechanisms of Resistance

Purification and Characterization of Recombinant Staphylococcus haemolyticus DNA Gyrase and Topoisomerase IV Expressed in Escherichia coli

Joel C. Bronstein, Stacey L. Olson, Kristin LeVier, Mark Tomilo, Peter C. Weber
Joel C. Bronstein
1Antibacterial Molecular Sciences and Technologies
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • For correspondence: Joel.Bronstein@pfizer.com
Stacey L. Olson
2Manpower Professional, Pfizer Global Research and Development, Ann Arbor, Michigan 48105
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Kristin LeVier
1Antibacterial Molecular Sciences and Technologies
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Mark Tomilo
3Antibacterial Pharmacology
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Peter C. Weber
1Antibacterial Molecular Sciences and Technologies
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
DOI: 10.1128/AAC.48.7.2708-2711.2004
  • Article
  • Figures & Data
  • Info & Metrics
  • PDF
Loading

ABSTRACT

The subunits of DNA gyrase and topoisomerase IV from Staphylococcus haemolyticus were expressed in Escherichia coli, purified to homogeneity, and used to reconstitute active enzymes that were sensitive to known topoisomerase inhibitors. This represents the first description of a method for isolating type II topoisomerases of a coagulase-negative staphylococcal species.

Coagulase-negative staphylococci (CoNS) are ubiquitous human microbes that are the most commonly isolated bacteria in clinical microbiology laboratories. Once thought of as avirulent and often dismissed as culture contaminants, these commensal organisms are becoming more frequently recognized as opportunistic pathogenic agents. In particular, CoNS have been associated with nosocomial infections among immunocompromised individuals, high-risk neonates, and hospitalized patients with imbedded foreign bodies (2, 8, 10, 16, 17, 19, 25). Moreover, treatment of CoNS infections has become increasingly difficult due to the growing prevalence of multiple-antibiotic-resistant phenotypes in clinical isolates (1, 12). One well-characterized species within this group is Staphylococcus haemolyticus, a normal inhabitant of the human skin. While generally considered to be nonpathogenic, there is growing evidence that S. haemolyticus is a causative agent of human disease (4, 9, 20), and as with other CoNS, clinical isolates of S. haemolyticus increasingly display a multiple-antibiotic-resistant phenotype (1, 6, 20, 21, 24).

The escalation of antibiotic resistance observed in clinical isolates of CoNS can be attributed to the widespread and frequently indiscriminant use of antibiotics to treat coagulase-positive Staphylococcus aureus infections in nosocomial patient populations. Since fluoroquinolone antibiotics are commonly used to treat such infections, clinical resistance to these agents in particular has increased dramatically in CoNS in recent years (5, 13, 21). The therapeutic targets of fluoroquinolones are DNA gyrase and topoisomerase IV, two type II topoisomerases that mediate distinct functions within bacterial cells (11). DNA gyrase is responsible for maintaining the topological state of DNA during replication and is the only enzyme known to introduce negative supercoils into DNA. In contrast, topoisomerase IV is a cellular decatenase that separates daughter chromosomes following a round of replication. Both DNA gyrase and topoisomerase IV are heterodimeric enzymes composed of two subunits that form an A2B2 complex and require the free energy of ATP hydrolysis to drive their respective catalytic activities. While these proteins are highly conserved among all bacterial species and have been studied extensively at the biochemical level, the purification and characterization of a DNA gyrase or topoisomerase IV from a CoNS species had not been described to date. Consequently, in this study recombinant subunits of S. haemolyticus topoisomerases were expressed and purified in an attempt to reconstitute active enzymes for use in in vitro inhibition assays and other biochemical work.

Expression of the A and B subunits of S. haemolyticus DNA gyrase and topoisomerase IV as recombinant proteins in Escherichia coli.

The gyrA and gyrB genes encoding the A and B subunits of DNA gyrase, respectively, and the grlA and grlB genes encoding the A and B subunits of topoisomerase IV, respectively, were identified within S. haemolyticus genomic sequences of the PathoSeq database (version 4.1, September 2001; Elitra Pharmaceuticals, Inc., San Diego, Calif.). The PCR was used to amplify each of these open reading frames from S. haemolyticus genomic DNA, using the following primer pairs: 5′-ATGGCTGACTTACCTCAATCAAG-3′ (forward) and 5′-GGGAAGTCTTGTTTGTTGAAGG-3′ (reverse) for gyrA, 5′-ATGGTGAATACATTGTCAGATGTAAAC-3′ (forward) and 5′-CTACTATTAGAAATCCAAGTTCGCATATAC-3′ (reverse) for gyrB, 5′-ATGAGTGAGATAATTCAAGATTTATC-3′ (forward) and 5′-CTACTATTAGATTGTCATATCTAAGACGTC-3′ (reverse) for grlA, and 5′-ATGCATTACTCAGATGATTCTATTC-3′ (forward) and 5′-TTATTCCTCCTCAGTATATTCTTC-3′ (reverse) for grlB. Genomic DNA was isolated from an S. haemolyticus ATCC 29970 culture grown in Trypticase soy broth at 37°C, using the DNeasy tissue kit according to the manufacturer's instructions (QIAGEN, Valencia, Calif.), except that lysozyme was replaced by lysostaphin (Sigma Chemical Company, St. Louis, Mo.) at a final concentration of 0.1 mg/ml for a 30-min incubation at 37°C. All PCRs were carried out with 100 ng of purified genomic DNA, 25 μM forward and reverse primers, 200 μM deoxynucleoside triphosphate mix, 1 mM MgSO4, and PLATINUM Pfx DNA polymerase and buffer following the manufacturer's instructions (Invitrogen Corporation, Carlsbad, Calif.), except that the gyrA reaction required the addition of Pfx Enhance solution (Invitrogen Corporation) at a concentration of 1×. PCR products were then A-tailed by the addition of recombinant Taq DNA polymerase to the reaction mixture followed by incubation at 72°C for 10 min. Each PCR product was then cloned directly into a pCRT7/CT-TOPO expression vector (Invitrogen Corporation) by immediately mixing 1 μl of PCR product, 1 μl of expression vector, 1 μl of TOPO salt solution, and 3 μl of water. Following a 5-min incubation at room temperature, the resulting product was transformed into chemically competent One Shot E. coli TOP10F′ cells (Invitrogen Corporation). Plasmids containing inserts of the correct size and orientation as determined by restriction site mapping were then confirmed by DNA sequencing (MWG Biotech, Inc., High Point, N.C.). The cloned topoisomerase subunit genes were found to be identical to the corresponding open reading frames present in the PathoSeq database, with the following exceptions: (i) the TTG and AAT start codons of grlA and grlB, respectively, were changed to ATG by using an altered sequence in the forward PCR primers with the intention of maximizing gene expression in the E. coli expression host; (ii) the gyrA construct contained an additional 68 bp of sequence 3′ to the stop codon, which was required for matching the G+C contents of the forward and reverse primers used in the PCR; and (c) the grlB construct was missing the final two amino acids of the PathoSeq open reading frame, which may not be present in all S. haemolyticus isolates as they map to a 17-residue segment that is not conserved in the grlB genes of other gram-positive species.

Expression constructs containing native, full-length, untagged sequences of the gyrA, gyrB, and grlA genes were transformed into BL21-Gold (DE3) pLysS-competent cells (Stratagene, La Jolla, Calif.), whereas the expression construct containing the native, full-length, untagged sequence of the grlB gene was transformed into E. coli BL21-Gold (DE3)-competent cells (Stratagene). Transformants were grown overnight at 37°C on Luria-Bertani (LB) agar plates containing 0.15-mg/ml ampicillin. The resulting colonies, which were used to inoculate LB broth supplemented with 0.2-mg/ml carbenicillin and 50-μg/ml chloramphenicol (omitted for grlB transformants), were then grown to log phase at 30°C and stored overnight at 4°C. This culture was centrifuged at 10,000 × g for 5 min, and the cell pellet was resuspended in fresh LB medium supplemented with 0.2-mg/ml carbenicillin. A portion of this cell suspension was then transferred to a larger culture of LB medium supplemented with 0.2-mg/ml carbenicillin and grown to log phase at 30°C. Expression of the protein subunits was induced by the addition of 0.2 mM isopropyl-β-d-thiogalactopyranoside (IPTG) to the culture followed by an additional 2 h of incubation at 30°C. The induced cells were then recovered by centrifugation, washed with an ice-cold solution of 50 mM Tris-HCl (pH 8.0) and 2 mM EDTA, and stored as a pellet at −80°C.

Purification of recombinant A and B subunits of S. haemolyticus DNA gyrase and topoisomerase IV.

Frozen cell pellets were resuspended in TED buffer (50 mM Tris-HCl [pH 7.6], 1 mM EDTA, 5 mM dithiothreitol [DTT]) containing 0.5 mM phenylmethylsulfonyl fluoride and 1-mg/ml lysozyme. Following a 30-min incubation on ice, the cells were lysed by Dounce homogenization. The resulting lysate was probe sonicated to reduce viscosity, centrifuged to remove insoluble material, dialyzed into TGED buffer (TED buffer containing 10% glycerol), and then subjected to streptomycin sulfate precipitation (1% final concentration). Precipitated protein was recovered by centrifugation and dissolved in TGED buffer containing 1 M NaCl. Insoluble material was again removed by centrifugation, and the clarified supernatant was subjected to ammonium sulfate precipitation (60% saturation). This precipitated protein was recovered by centrifugation, dissolved in TGED buffer, and dialyzed overnight into TGED buffer. The clarified lysate was applied to a HiPrep 16/10 heparin FF column (Amersham Biosciences Corporation, Piscataway, N.J.) that was preequilibrated in TGED buffer. Bound protein was eluted with a gradient of 0 to 0.6 M NaCl in TGED buffer. Fractions that contained the subunit of interest as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were pooled, dialyzed, and applied to a Mono Q HR 10/10 column (Amersham Biosciences Corporation, Piscataway, N.J.) that was preequilibrated in TGED buffer. The bound subunit resolved into a single protein band following elution with a gradient of 0 to 0.45 M NaCl in TGED buffer. Fractions containing the purified protein were pooled; diluted with 1 volume of 50 mM Tris-HCl (pH 7.6), 1 mM EDTA, 200 mM KCl, and 20% glycerol (to give concentrations of 50 mM Tris-HCl [pH 7.6], 1 mM EDTA, 2.5 mM DTT, 100 mM KCl, and 10% glycerol in the final protein preparation); and then stored at −80°C. Unless noted otherwise, all steps of the purification process were performed at 4°C.

The purity and molecular mass of each subunit in the final protein preparations were evaluated by SDS-PAGE. Each of the four subunits was determined to be greater than 95% pure, and their apparent molecular masses were consistent with the predicted molecular masses of the polypeptides encoded by gyrA (101 kDa), gyrB (73 kDa), grlA (90 kDa), and grlB (74 kDa) (Fig. 1). The yields ranged from 6 to 19 mg of purified protein per liter of culture, and none of the purified subunits exhibited detectable DNA-independent ATPase activity (J. C. Bronstein and S. L. Olson, unpublished data).

FIG. 1.
  • Open in new tab
  • Download powerpoint
FIG. 1.

SDS-PAGE analysis of recombinant A and B subunits of S. haemolyticus DNA gyrase and topoisomerase IV. A total of 0.5 μg of the final protein preparations of each topoisomerase subunit was electrophoresed on 8% SDS-PAGE gels and stained with SimplyBlue SafeStain (Invitrogen Corporation). The mobility of molecular mass markers (in kilodaltons) is shown to the left.

Characterization of the enzymatic properties of recombinant S. haemolyticus DNA gyrase and topoisomerase IV.

Enzymatic activities of topoisomerases reconstituted from their purified subunits were monitored by either the introduction of supercoils into relaxed closed circular pBR322 plasmid DNA (the DNA gyrase assay) or the decatenation of interlinked kinetoplast DNA minicircles (the topoisomerase IV assay). The DNA gyrase assay contained 35 mM Tris-HCl (pH 7.5), 250 mM potassium glutamate, 5 mM MgCl2, 2 mM DTT, 50-μg/ml bovine serum albumin, 1 mM ATP, 250 ng of relaxed pBR322 DNA (TopoGEN, Inc., Columbus, Ohio), and 50 ng of each gyrase subunit in a 30-μl reaction mixture that was incubated at 37°C for 45 min. The reaction was terminated by the addition of 7 μl of a stop solution containing 1.5 μl of 10% SDS, 1.5 μl of 10-mg/ml proteinase K, and 4 μl of 10× BlueJuice gel loading buffer (Invitrogen Corporation). The topoisomerase IV assay was identical to the DNA gyrase assay, except that it employed 120 ng of kinetoplast DNA (TopoGEN, Inc.) as a substrate, 20 ng of each topoisomerase IV subunit, and a 15-min incubation time. The products of either assay were resolved on 0.8% agarose gels, stained with ethidium bromide, and quantitated on a Gel Dock 2000 gel documentation system (Bio-Rad Laboratories, Hercules, Calif.). In experiments that included topoisomerase inhibitors, the percent supercoiled pBR322 or percent decatenated kinetoplast DNA minicircles relative to drug-free control reactions was calculated for each inhibitor concentration; these were then plotted against the log of the respective inhibitor concentrations, and 50% inhibitory concentrations (IC50s) were calculated from the resulting curves.

Both of the recombinant S. haemolyticus topoisomerases were found to be enzymatically active, as evidenced by the ability of reconstituted DNA gyrase and topoisomerase IV to supercoil relaxed pBR322 DNA and decatenate kinetoplast DNA, respectively (Fig. 2). The specific activities for DNA gyrase and topoisomerase IV were calculated to be 22 and 180 U/μg, respectively, where a unit of topoisomerase activity is defined as the amount of enzyme needed to supercoil or decatenate 50% of substrate in 1 h at 37°C under the assay conditions described above (3, 18). These topoisomerases were also shown to exhibit sensitivity to a panel of fluoroquinolone and coumarin antibiotics (Table 1 and Fig. 2), further confirming the authenticity of their observed enzymatic properties. The IC50s of these inhibitors against S. haemolyticus DNA gyrase and topoisomerase IV were found to be comparable to those reported for the corresponding enzymes from S. aureus (3, 7, 18, 22, 23). Additionally, in the presence of ciprofloxacin and clinafloxacin, a linear DNA product indicative of cleavage complex formation could be detected within reaction mixtures following agarose gel electrophoresis (J. C. Bronstein and M. Tomilo, unpublished data). Whole-cell inhibitory activities against S. haemolyticus (Table 1), as determined by susceptibility testing following National Committee for Clinical Laboratory Standards guidelines (14, 15), were also found to be comparable to those reported from S. aureus (3, 18, 23). These results suggest that S. haemolyticus topoisomerases may someday prove to be a useful surrogate for their S. aureus counterparts in routine biochemical assays.

FIG. 2.
  • Open in new tab
  • Download powerpoint
FIG. 2.

Agarose gel electrophoresis of DNA gyrase and topoisomerase IV assays containing recombinant S. haemolyticus enzymes. (A) DNA gyrase assays. All reactions contained relaxed closed circular pBR322 plasmid as the substrate, and the reaction mixture in lane 3 contained 0.7 μM coumermycin A1 as an inhibitor. The mobility of the relaxed DNA substrate (R) and the supercoiled DNA product (S) are indicated at the right. (B) Topoisomerase IV assays. All reactions contained interlinked kinetoplast DNA minicircles as the substrate, and the reaction mixture in lane 3 contained 6.2 μM coumermycin A1 as an inhibitor. The mobilities of the linked DNA substrate (L) and the free DNA product (F) are indicated to the right.

View this table:
  • View inline
  • View popup
TABLE 1.

Whole-cell and in vitro activities of fluoroquinolone and coumarin antibiotics against S. haemolyticus and its topoisomerases

In summary, this report describes a simple and reliable procedure for the expression and purification of the subunits that comprise the DNA gyrase and topoisomerase IV of S. haemolyticus and their reconstitution into fully active enzymes. This represents the first characterization of topoisomerases from a species of CoNS, which are now recognized as a growing source of opportunistic infection in hospital settings. The ability to purify topoisomerases from S. haemolyticus and other CoNS, including fluoroquinolone-resistant strains, should provide researchers with new biochemical tools with which to assess the efficacy of current and future antibiotics targeting this class of enzymes.

Nucleotide sequence accession number.

Sequences for the S. haemolyticus gyrA, gyrB, grlA, and grlB genes are available in GenBank under accession no. AY341071 , AY341072 , AY341073 , and AY341074 , respectively.

ACKNOWLEDGMENTS

We thank T. McQuade for assistance in topoisomerase assay optimization, H. Johnson and R. Brandon for topoisomerase assay data, M. Huband for MIC data, and R. Collins for SDS-PAGE of purified proteins.

FOOTNOTES

    • Received 3 September 2003.
    • Returned for modification 22 November 2003.
    • Accepted 31 March 2004.
  • Copyright © 2004 American Society for Microbiology

REFERENCES

  1. 1.↵
    Archer, G. L., and M. W. Climo. 1994. Antimicrobial susceptibility of coagulase-negative staphylococci. Antimicrob. Agents Chemother.38:2231-2237.
    OpenUrlFREE Full Text
  2. 2.↵
    Bjorkqvist, M., B. Soderquist, E. Tornqvist, L. Sjorberg, H. Fredlund, I. Kuhn, P. Colque-Navarro, and J. Schollin. 2002. Phenotypic and genotypic characterization of blood isolates of coagulase-negative staphylococci in the newborn. APMIS110:332-339.
    OpenUrlCrossRefPubMedWeb of Science
  3. 3.↵
    Blanche, F., B. Cameron, F.-X. Bernard, L. Maton, B. Manse, L. Ferrero, N. Ratet, C. Lecoq, A. Goniot, D. Bisch, and J. Crouzet. 1996. Differential behaviors of Staphylococcus aureus and Escherichia coli type II DNA topoisomerases. Antimicrob. Agents Chemother.40:2714-2720.
    OpenUrlAbstract/FREE Full Text
  4. 4.↵
    Degener, J. E., M. E. Heck, W. J. van Leeuwen, C. Heemskerk, A. Crielaard, P. Joosten, and P. Caesar. 1994. Nosocomial infection by Staphylococcus haemolyticus and typing methods for epidemiological study. J. Clin. Microbiol.32:2260-2265.
    OpenUrlAbstract/FREE Full Text
  5. 5.↵
    Dubin, D. T., J. E. Fitzgibbon, M. D. Nahvi, and J. E. John. 1999. Topoisomerase sequences of coagulase-negative staphylococcal isolates resistant to ciprofloxacin or trovafloxacin. Antimicrob. Agents Chemother.43:1631-1637.
    OpenUrlAbstract/FREE Full Text
  6. 6.↵
    Froggatt, J. W., J. L. Johnston, D. W. Galetto, and G. L. Archer. 1989. Antimicrobial resistance in nosocomial isolates of Staphylococcus haemolyticus. Antimicrob. Agents Chemother.33:460-466.
    OpenUrlAbstract/FREE Full Text
  7. 7.↵
    Gootz, T. D., R. P. Zaniewski, S. L. Haskell, F. S. Kaczmarek, and A. E. Maurice. 1999. Activities of trovafloxacin compared with those of other fluoroquinolones against purified topoisomerases and gyrA and grlA mutants of Staphylococcus aureus. Antimicrob. Agents Chemother.43:1845-1855.
    OpenUrlAbstract/FREE Full Text
  8. 8.↵
    Huebner, J., and D. A. Goldmann. 1999. Coagulase-negative staphylococci: role as pathogens. Annu. Rev. Med.50:223-236.
    OpenUrlCrossRefPubMedWeb of Science
  9. 9.↵
    Isaac, D. W., T. A. Pearson, C. A. Hurwitz, and C. C. Patrick. 1993. Clinical and microbiological aspects of Staphylococcus haemolyticus infections. Pediatr. Infect. Dis. J.12:1018-1021.
    OpenUrlPubMed
  10. 10.↵
    Kloos, W. E., and T. L. Bannerman. 1994. Update on clinical significance of coagulase-negative staphylococci. Clin. Microbiol. Rev.7:117-140.
    OpenUrlAbstract/FREE Full Text
  11. 11.↵
    Levine, C., H. Hiasa, and K. J. Marians. 1998. DNA gyrase and topoisomerase IV: biochemical activities, physiological roles during chromosome replication, and drug sensitivities. Biochim. Biophys. Acta1400:29-43.
    OpenUrlCrossRefPubMedWeb of Science
  12. 12.↵
    Livermore, D. M. 2000. Antibiotic resistance in staphylococci. Int. J. Antimicrob. Agents16(Suppl. 1):3-10.
    OpenUrlCrossRef
  13. 13.↵
    Mulder, J. G., J. G. Kosterink, and J. E. Degener. 1997. The relationship between the use of flucloxacillin, vancomycin, aminoglycosides and ciprofloxacin and the susceptibility patterns of coagulase-negative staphylococci recovered from blood cultures. J. Antimicrob. Chemother.40:701-706.
    OpenUrlCrossRefPubMedWeb of Science
  14. 14.↵
    National Committee for Clinical Laboratory Standards. 2003. Methods for dilution antimicrobial tests for bacteria that grow aerobically. Approved standard M7-A6, 6th ed. National Committee for Clinical Laboratory Standards, Wayne, Pa.
  15. 15.↵
    National Committee for Clinical Laboratory Standards. 2003. MIC testing supplemental tables. M100-S13 (for use with M7-A6), National Committee for Clinical Laboratory Standards, Wayne, Pa.
  16. 16.↵
    Patrick, C. C. 1990. Coagulase-negative staphylococci: pathogens with increasing clinical significance. J. Pediatr.116:497-507.
    OpenUrlCrossRefPubMedWeb of Science
  17. 17.↵
    Rupp, M. E., and G. L. Archer. 1994. Coagulase-negative staphylococci: pathogens associated with medical progress. Clin. Infect. Dis.19:231-243.
    OpenUrlCrossRefPubMedWeb of Science
  18. 18.↵
    Sakei, A. Y. C., L. L. Shen, C.-M. Chen, J. Baranowski, and C. G. Lerner. 1999. DNA cleavage activities of Staphylococcus aureus gyrase and topoisomerase IV stimulated by quinolones and 2-pyridones. Antimicrob. Agents Chemother.43:1574-1577.
    OpenUrlAbstract/FREE Full Text
  19. 19.↵
    Schulin, T., and A. Voss. 2001. Coagulase-negative staphylococci as a cause of infections related to intravascular prosthetic devices: limitations of present therapy. Eur. Soc. Clin. Microbiol. Infect. Dis.7:1-7.
    OpenUrl
  20. 20.↵
    Tabe, Y., A. Nakamura, T. Oguri, and J. Igari. 1998. Molecular characterization of epidemic multiresistant Staphylococcal haemolyticus isolates. Diagn. Microbiol. Infect. Dis.32:177-183.
    OpenUrlCrossRefPubMedWeb of Science
  21. 21.↵
    Takahata, M., M. Yonezawa, N. Matsubara, Y. Watanabe, H. Narita, T. Matsunaga, H. Igarashi, M. Kawahara, S. Onodera, and Y. Oishi. 1997. Antibacterial activity of quinolones against coagulase-negative staphylococci and the quinolone resistance-determining region of the gyrA genes from six species. J. Antimicrob. Chemother.40:383-386.
    OpenUrlCrossRefPubMed
  22. 22.↵
    Tanaka, M., K. Sato, Y. Kimura, I. Hayakawa, Y. Osada, and T. Nishino. 1991. Inhibition by quinolones of DNA gyrase from Staphylococcus aureus. Antimicrob. Agents Chemother.35:1489-1491.
    OpenUrlAbstract/FREE Full Text
  23. 23.↵
    Tanaka, M., Y. Onodera, Y. Uchida, K. Sato, and I. Hayakawa. 1997. Inhibitory activities of quinolones against DNA gyrase and topoisomerase IV purified from Staphylococcus aureus. Antimicrob. Agents Chemother.41:2362-2366.
    OpenUrlAbstract/FREE Full Text
  24. 24.↵
    Veach, L. A., M. P. Pfaller, M. Barrett, F. P. Koontz, and R. P. Wenzel. 1990. Vancomycin resistance in Staphylococcus haemolyticus causing colonization and bloodstream infection. J. Clin. Microbiol.28:2064-2068.
    OpenUrlAbstract/FREE Full Text
  25. 25.↵
    von Eiff, C., R. A. Proctor, and G. Peters. 2001. Coagulase-negative staphylococci: pathogens have a major role in nosocomial infections. Postgrad. Med.110:63-76.
    OpenUrl
PreviousNext
Back to top
Download PDF
Citation Tools
Purification and Characterization of Recombinant Staphylococcus haemolyticus DNA Gyrase and Topoisomerase IV Expressed in Escherichia coli
Joel C. Bronstein, Stacey L. Olson, Kristin LeVier, Mark Tomilo, Peter C. Weber
Antimicrobial Agents and Chemotherapy Jun 2004, 48 (7) 2708-2711; DOI: 10.1128/AAC.48.7.2708-2711.2004

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Print

Alerts
Sign In to Email Alerts with your Email Address
Email

Thank you for sharing this Antimicrobial Agents and Chemotherapy article.

NOTE: We request your email address only to inform the recipient that it was you who recommended this article, and that it is not junk mail. We do not retain these email addresses.

Enter multiple addresses on separate lines or separate them with commas.
Purification and Characterization of Recombinant Staphylococcus haemolyticus DNA Gyrase and Topoisomerase IV Expressed in Escherichia coli
(Your Name) has forwarded a page to you from Antimicrobial Agents and Chemotherapy
(Your Name) thought you would be interested in this article in Antimicrobial Agents and Chemotherapy.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Share
Purification and Characterization of Recombinant Staphylococcus haemolyticus DNA Gyrase and Topoisomerase IV Expressed in Escherichia coli
Joel C. Bronstein, Stacey L. Olson, Kristin LeVier, Mark Tomilo, Peter C. Weber
Antimicrobial Agents and Chemotherapy Jun 2004, 48 (7) 2708-2711; DOI: 10.1128/AAC.48.7.2708-2711.2004
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
  • Top
  • Article
    • ABSTRACT
    • Expression of the A and B subunits of S. haemolyticus DNA gyrase and topoisomerase IV as recombinant proteins in Escherichia coli.
    • Purification of recombinant A and B subunits of S. haemolyticus DNA gyrase and topoisomerase IV.
    • Characterization of the enzymatic properties of recombinant S. haemolyticus DNA gyrase and topoisomerase IV.
    • Nucleotide sequence accession number.
    • ACKNOWLEDGMENTS
    • FOOTNOTES
    • REFERENCES
  • Figures & Data
  • Info & Metrics
  • PDF

KEYWORDS

DNA gyrase
DNA Topoisomerase IV
Escherichia coli
Staphylococcus haemolyticus

Related Articles

Cited By...

About

  • About AAC
  • Editor in Chief
  • Editorial Board
  • Policies
  • For Reviewers
  • For the Media
  • For Librarians
  • For Advertisers
  • Alerts
  • AAC Podcast
  • RSS
  • FAQ
  • Permissions
  • Journal Announcements

Authors

  • ASM Author Center
  • Submit a Manuscript
  • Article Types
  • Ethics
  • Contact Us

Follow #AACJournal

@ASMicrobiology

       

ASM Journals

ASM journals are the most prominent publications in the field, delivering up-to-date and authoritative coverage of both basic and clinical microbiology.

About ASM | Contact Us | Press Room

 

ASM is a member of

Scientific Society Publisher Alliance

 

American Society for Microbiology
1752 N St. NW
Washington, DC 20036
Phone: (202) 737-3600

Copyright © 2021 American Society for Microbiology | Privacy Policy | Website feedback

Print ISSN: 0066-4804; Online ISSN: 1098-6596