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ISSN 1672-9145 Acta Biochim Biophys Sin 2004, 36(8): 519–528 CN 31-1940/Q

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Identification and Characterization of hmr19 Gene Encoding a Multidrug Resistance Efflux Protein from Streptomyces hygroscopicus subsp. yingchengensis Strain 10-22

Lei QIN#, Heng-An WANG¹#, Zhong-Qin WU, Xiao-Feng ZHANG, Mei-Lei JIN, Zi-Xin DENG², and Guo-Ping ZHAO*

Shanghai Research Center of Biotechnology, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, Graduate School of the Chinese Academy of Sciences, Shanghai 200233, China;
¹School of Agriculture and Biology, Shanghai Jiaotong University, Shanghai 201101, China;
²School of Life Science and Biotechnology, Shanghai Jiaotong University, Shanghai 200030, China

Abstract The hmr19 gene was cloned from Streptomyces hygroscopicus subsp. yingchengensis strain 10-22, a bacterium strain producing agricultural antibiotics. Sequence similarity comparison indicates that hmr19 gene may encode a predicted protein with 14 putative transmembrane a-helical spanners, belonging to the drug:H+ antiporter-2 family of the major facilitator superfamily. The expression of hmr19 in the mycelium of strain 10-22 was detected by Western blotting analysis. Gene replacement technology was employed to construct an hmr19 disruption mutant. The growth inhibition test against different antibiotics indicated that the mutant strain was 5-20 fold more susceptible to tetracycline, vancomycin and mitomycin C than the parental wild type strain. The mutant took up tetracycline much faster and accumulated more antibiotics than the wild type strain 10-22. While with the addition of an energy uncoupler, carbonyl cyanide m-chlorophenylhydrazone, the characteristics of the accumulation of [3H]tetracycline in these two strains were almost the same. It was thus concluded that hmr19 encoded a multidrug resistance efflux protein.

Key words multidrug resistance (MDR); gene replacement (knockout); Streptomyces; Streptomyces hygroscopicus subsp. yingchengensis strain 10-22

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Received: March 18, 2004 Accepted: June 18, 2004

This work was supported by the grants from the National Natural Science Foundation of China (No. 30070016) and the National High Technology Research and Development Program of China (No. 2001AA234051)

^#These authors contributed equally to this work

*Corresponding author: Tel, 86-21-54971046; Fax, 86-21-64837495; E-mail, [email protected]