Publications (10) View all
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Article: Mycobacterium fluoroquinolone resistance protein B, a novel small GTPase, is involved in the regulation of DNA gyrase and drug resistance.
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ABSTRACT: DNA gyrase plays a vital role in resolving DNA topological problems and is the target of antibiotics such as fluoroquinolones. Mycobacterium fluoroquinolone resistance protein A (MfpA) from Mycobacterium smegmatis is a newly identified DNA gyrase inhibitor that is believed to confer intrinsic resistance to fluoroquinolones. However, MfpA does not prevent drug-induced inhibition of DNA gyrase in vitro, implying the involvement of other as yet unknown factors. Here, we have identified a new factor, named Mycobacterium fluoroquinolone resistance protein B (MfpB), which is involved in the protection of DNA gyrase against drugs both in vivo and in vitro. Genetic results suggest that MfpB is necessary for MfpA protection of DNA gyrase against drugs in vivo; an mfpB knockout mutant showed greater susceptibility to ciprofloxacin than the wild-type, whereas a strain overexpressing MfpA and MfpB showed higher loss of susceptibility. Further biochemical characterization indicated that MfpB is a small GTPase and its GTP bound form interacts directly with MfpA and influences its interaction with DNA gyrase. Mutations in MfpB that decrease its GTPase activity disrupt its protective efficacy. Our studies suggest that MfpB, a small GTPase, is required for MfpA-conferred protection of DNA gyrase.Nucleic Acids Research 12/2012; · 8.03 Impact Factor -
Article: Existence of Separate Domains in Lysin PlyG for Recognizing Bacillus anthracis Spores and Vegetative Cells.
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ABSTRACT: As a potential antimicrobial, the bacteriophage lysin PlyG has been reported to specifically recognize Bacillus anthracis vegetative cells only and to kill B. anthracis vegetative cells and its germinating spores. However, how PlyG interacts with B. anthracis spores remains unclear. Herein, a 60-amino-acid domain in PlyG (residues 106 to 165), located mainly in the previously identified catalytic domain, was found able to specifically recognize B. anthracis spores but not vegetative cells. The exosporium of the spores was found to be the most probable binding target of this domain. This is the first time that a lysin for spore-forming bacteria has been found to have separate domains to recognize spores and vegetative cells, which might help in understanding the coevolution of phages with spore-forming bacteria. Besides providing new biomarkers for developing better assays for identifying B. anthracis spores, the newly found domain may be helpful in developing PlyG as a preventive antibiotic to reduce the threat of anthrax in suspected exposures to B. anthracis spores.Antimicrobial Agents and Chemotherapy 07/2012; 56(10):5031-9. · 4.84 Impact Factor -
Article: Human cytomegalovirus UL94 is a nucleocytoplasmic shuttling protein containing two NLSs and one NES.
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ABSTRACT: The tegument protein UL94 is a human cytomegalovirus (HCMV) late protein and its function has yet to be determined. Using live cell fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) imaging, we found that UL94 is able to shuttle between the nucleus and cytoplasm. Analysis of UL94 mutants fused to EGFP showed that two newly characterized nuclear localization sequences (NLSs) and amino acid 343 play key roles in UL94 nuclear localization. Mutation of these sequences can alter the intracellular distribution of UL94 and disrupt its nucleocytoplasmic shuttling. Amino acid 343 of UL94 was also found to be crucial for its interaction with another HCMV tegument protein pp28. Furthermore, one nuclear export sequence (NES) was identified within UL94. Mutation of the key amino acids in the NES can also alter the intracellular distribution of UL94 and disrupt its shuttling function. Like other proteins containing a leucine-rich export signal, nuclear export of the UL94 was affected by leptomycin B, indicating that it is exported via the Crm1-dependent pathway. Our data provide a basis for further understanding the character and function of HCMV UL94.Virus Research 03/2012; 166(1-2):31-42. · 2.94 Impact Factor -
Article: Purification and characterization of the acetyl-CoA synthetase from Mycobacterium tuberculosis.
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ABSTRACT: Acetyl-CoA (AcCoA) synthetase (Acs) catalyzes the conversion of acetate into AcCoA, which is involved in many catabolic and anabolic pathways. Although this enzyme has been studied for many years in many organisms, the properties of Mycobacterium tuberculosis Acs and the regulation of its activity remain unknown. Here, the putative acs gene of M. tuberculosis H37Rv (Mt-Acs) was expressed as a fusion protein with 6×His-tag on the C-terminus in Escherichia coli. The recombinant Mt-Acs protein was successfully purified and then its enzymatic characteristics were analyzed. The optimal pH and temperature, and the kinetic parameters of Mt-Acs were determined. To investigate whether Mt-Acs is regulated by lysine acetylation as reported for Salmonella enterica Acs, its mutant K617R was also generated. Determination of the enzymatic activity suggests that Lys-617 is critical for its function. We further demonstrated that Mt-Acs underwent auto-acetylation with acetate but not with AcCoA as the acetyl donor, which resulted in the decrease of its activity. CoA, the substrate for AcCoA formation, inhibited the auto-acetylation. Furthermore, the silent information regulator (Sir2) of M. tuberculosis (Mt-Sir2) could catalyze Mt-Acs deacetylation, which resulted in activation of Acs. These results may provide more insights into the physiological roles of Mt-Acs in M. tuberculosis central metabolism.Acta Biochimica et Biophysica Sinica 09/2011; 43(11):891-9. · 1.38 Impact Factor -
Article: Aptamer beacons for visualization of endogenous protein HIV-1 reverse transcriptase in living cells.
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ABSTRACT: Direct visualization of endogenous proteins in living cells remains a challenge. Aptamer beacon is a promising technique to resolve this problem by combining the excellent protein binding specificity of the aptamer with the sensitive signal transduction mechanism of the molecular beacon. In this study, aptamer 93 del against HIV-1 reverse transcriptase (RT) was engineered into aptamer beacons to recognize and image HIV-1 RT. The constructed aptamer beacons could specifically bind to HIV-1 RT and the beacon-RT binding showed effective fluorescence signal transduction in homogeneous solution. In solutions with 1 μM of the aptamer beacon, the effective fluorescence signal increased with increasing concentration of HIV-1 RT from 0.5 μM to 5 μM. When the aptamer beacons were delivered into the living cells that transiently expressed HIV-1 RT, HIV-1 RT could be specifically labeled and imaged. The designed aptamer beacons were further successfully applied for RT imaging in HIV-1 integrated U1 cells. The method developed here may be extended to visualize many other endogenous proteins in living cells using appropriate aptamer beacons.Biosensors & bioelectronics 07/2011; 28(1):270-6. · 5.43 Impact Factor
