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Cloning of tlrD, 23S rRNA Monomethyltransferase Gene, Overexpression in Eschepichia coli and Its Activity  

Jin, Hyung-Jong (Department of Bioscience and Biotechnology, College of Natural Science, The University of Suwon)
Publication Information
Korean Journal of Microbiology / v.43, no.3, 2007 , pp. 166-172 More about this Journal
Abstract
ERM proteins transfer the methyl group to $A_{2058}$ in 23S rRNA, which reduces the affinity of MLS (macrolide-lincosamide-streptogramin B) antibiotics to 23S rRNA, thereby confer the antibiotic resistance on micro-organisms ranging from antibiotic producers to pathogens and are classified into monomethyltransferase and dimethyltransferase. To investigate the differences between mono- and dimethyltransferase, tirD, a representative monomethylase gene was cloned in Escherichia coli from Streptomyces fradiae which contains ermSF, dimethylase gene as well to overexpress the TlrD for the first time. T7 promoter driven expression system successfully overexpress tlrD as a insoluble aggregate at $37^{\circ}C$ accumulating to around 55% of the total cell protein but unlike ErmSF, culturing at temperature as low as $18^{\circ}C$ did not make insoluble aggregate of protein into soluble protein. Coexpression of Thioredoxin and GroESL, chaperone was not helpful in turning into soluble protein either as in case of ErmSF. These results might suggest that differences between mono- and dimethylase could be investigated on the basis of the characteristics of protein structure. However, a very small amount of soluble protein which could not be detected by SDS-PAGE conferred antibiotic resistance on E. coli as in ErmSF which was expected from the activity exerted by monmethylase in a cell.
Keywords
in vivo activity; minmal inhibitory concentraion (MIC); $MLS_B$ (macrolide-lincosamide-streptogramin B) antibiotic resistance factor protein; overexpression; TlrD;
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1 Blackwell, J.R. and R. Horgan. 1991. A novel strategy for production of a highly expresssed recombinant protein in an active form. FEBS Lett. 295, 10-12   DOI   PUBMED   ScienceOn
2 Jin, H.J. and Y.D. Yang. 2002. Purification and biochemical characterization of the ErmSF macrolide-lincosamide-streptogramin B resistance factor protein expressed as a hexahistidine-tagged protein in Escherichia coli. Protein Expr. Purif. 25, 149-159   DOI   ScienceOn
3 Kovalic, D., R.B. Giannattasio, H.J. Jin, and B. Weisblum. 1994. 23S rRNA domain V, a fragment that can be specifically methylated in vitro by the ErmSF(TlrA) methyltransferase. J Bacteriol. 176, 6992-6998   DOI   PUBMED
4 Roberts, M.C., J. Sutcliffe, P. Courvalin, L.B. Jensen, J. Rood, and H. Seppala. 1999. Nomenclature for macrolide and macrolide-lincomycin-streptogramin B resistance determinants. Antimicrob. Agents Chemother. 43, 2823-2830
5 Roberts, M.C. 2004. Resistance to macrolide, lincosamide, streptogramin, ketolide, and oxazolidinone antibiotics. Mol. Biotechnol. 28,47-62   DOI   ScienceOn
6 Zalacain, M. and E. Cundliffe. 1991. Cloning of tlrD, a fourth resistance gene, from the tylosin producer, Streptomyces fradiae. Gene 97, 137-142   DOI   ScienceOn
7 Liu, M. and S. Douthwaite. 2002. Activity of the ketolide telithromycin is refractory to Erm monomethylation of bacterial rRNA. Antimicrob. Agents Chemother. 46, 1629-1633   DOI
8 Schluckebier, G., P. Zhong, K.D. Stewart, TJ. Kavanaugh, and C. Abad-Zapatero. 1999. The 2.2 A structure of the rRNA methyltransferase ErmC' and its complexes with cofactor and cofactor analogs: implications for the reaction mechanism. J Mol. Biol. 289, 277-291   DOI   ScienceOn
9 진형종 2001. MLS (macrolide-lincosamide-streptogramin B) 항생제 내성인자 단백질인ErmSF의 domain발현. 한국미생물학회지 37, 245-252
10 Birmingham, V.A., K.L. Cox, J.L. Larson, S.E. Fishman, C.L. Hershberger, and E.T. Seno. 1986. Cloning and expression of a tylosin resistance gene from a tylosin-producing strain of Streptomyces fradiae. Mol. Gen. Genet. 204, 532-539   DOI
11 Lee, H.J. and H.J. Jin. 2003. Renaturation of recombinant ErmSF and its refolding behavior. Mol. Cells 16, 187-193   PUBMED
12 Kovalic, D., J.H. Kwak, and B. Weisblum. 1991. General method for direct cloning of DNA fragments generated by the polymerase chain reaction. Nucleic Acid Res. 19, 4650
13 'Frontiers in Biotechnology: Antibiotic Resistance' 1994. Science 264,317-476
14 Liu, M. and S. Douthwaite. 2002. Methylation at nucleotide G745 or G748 in 23S rRNA distinguishes gram-negative from grampositive bacteria. Mol. Microbiol. 44, 195-204   DOI   ScienceOn
15 Yasukawa, T., C. Kanei-lshii, T. Maekawa, J. Fujimoto, T. Yamamoto, and S. Ishii. 1995. Increase of solubility of foreign proteins in Escherichia coli by coproduction of the bacterial thioredoxin. J. BioI. Chem. 270, 25328-25331   DOI   ScienceOn
16 Denoya, C. and D. Dubnau. 1989. Mono- and dimethylating activities and kinetic studies of the ermC 23S rRNA methyltransferase. J BioI. Chem. 264,2615-2624
17 Matthews, C.R. 1993. Pathways of protein folding. Annu. Rev. Biochem. 62, 653-683
18 Zalacain, M. and E. Cundliffe. 1989. Methylation of 23S rRNA by tlrA(ermSF), a tylosin resistance determinant from Streptomyces fradiae. J. Bacteriol. 171, 4254-4260   DOI   PUBMED
19 Jin, H.J. 1999. ermSF, a ribosomal RNA adenine N6-methyltransferase gene from Streptomyces fradiae, confers MLS(macrolidelincosamide-streptogramin B) resistance to E. coli when it is expressed. Mol. Cells 9, 252-257   PUBMED
20 Makrides, S.C. 1996. Strategies for achieving high-level expression of genes in Escherichia coli. Microbiol. Rev. 60, 512-538   PUBMED
21 Skerra, A. 1993, Bacterial expression of immunoglobulin fragments. Curr. Opin. Immunol. 5, 256-262   DOI   ScienceOn
22 Skinner, R., E. Cundliffe, and F.J. Schmidt. 1983. Site for Action of a ribosomal RNA methylase responsible for resistance to erythromycin and other antibiotics. J. Biol. Chem. 258, 12702-12706   PUBMED
23 Weisblum, B. 1995. Erythromycin resistance by ribosome modification. Antimicrob. Agents Chemother. 39, 577-585   DOI   ScienceOn
24 Poehlsgaard, J. and S. Douthwaite. 2005. The bacterial ribosome as a target for antibiotics. Nat. Rev. Microbiol. 3,870-881   DOI   ScienceOn
25 Georgiou, G. and P. Valax. 1996. Expression of correctly folded proteins in Escherichia coli. Curr. Opin. Biotechnol. 7, 190-197   DOI   ScienceOn
26 Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T. Nature 227,680-685   DOI   ScienceOn
27 LaVallie, E.R. and J.M. McCoy. 1995. Gene fusion expression systems in Eschrichia coli. Curr. Opin. Biotechnol. 6, 501-506   DOI   ScienceOn
28 Liu, M. and S. Douthwaite. 2002. Resistance to the macrolide antibiotic tylosin is conferred by single methylations at 23S rRNA nucleotides G748 and A2058 acting in synergy. Proc. Natl. Acad. Sci. USA. 99. 14658-14663