Molecular cloning and functional characterization of an ATP-binding cassette transporter OtrC from Streptomyces rimosus

Lan Yu,Long Wang,Yingping Zhuang,Siliang Zhang,Xiangyun Yan,Ju Chu,Meijin Guo

doi:10.1186/1472-6750-12-52

Lan Yu, Long Wang + Show 5 more

Open Access

https://doi.org/10.1186/1472-6750-12-52

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Abstract

BackgroundThe otrC gene of Streptomyces rimosus was previously annotated as an oxytetracycline (OTC) resistance protein. However, the amino acid sequence analysis of OtrC shows that it is a putative ATP-binding cassette (ABC) transporter with multidrug resistance function. To our knowledge, none of the ABC transporters in S. rimosus have yet been characterized. In this study, we aimed to characterize the multidrug exporter function of OtrC and evaluate its relevancy to OTC production.ResultsIn order to investigate OtrC’s function, otrC is cloned and expressed in E. coli The exporter function of OtrC was identified by ATPase activity determination and ethidium bromide efflux assays. Also, the susceptibilities of OtrC-overexpressing cells to several structurally unrelated drugs were compared with those of OtrC-non-expressing cells by minimal inhibitory concentration (MIC) assays, indicating that OtrC functions as a drug exporter with a broad range of drug specificities. The OTC production was enhanced by 1.6-fold in M4018 (P = 0.000877) and 1.4-fold in SR16 (P = 0.00973) duplication mutants, while it decreased to 80% in disruption mutants (P = 0.0182 and 0.0124 in M4018 and SR16, respectively).ConclusionsThe results suggest that OtrC is an ABC transporter with multidrug resistance function, and plays an important role in self-protection by drug efflux mechanisms. This is the first report of such a protein in S. rimosus, and otrC could be a valuable target for genetic manipulation to improve the production of industrial antibiotics.

Highlights

The otrC gene of Streptomyces rimosus was previously annotated as an oxytetracycline (OTC) resistance protein
A NCBI BLAST search of the amino acid sequence of the OtrC protein identified a number of similar proteins, the majority of which were ATP-binding cassette (ABC) components of doxorubicin or multidrug resistance (MDR) ABC transporters from various phyla
The results showed that OtrC-ORF1 exhibits 41% amino acid identity with the doxorubicin resistance protein DrrA from S. peucetius and 43% identity with the multidrug ABC transporter ATP -binding subunit Msr-ORF1 from S. rochei F20

Summary

Introduction

The otrC gene of Streptomyces rimosus was previously annotated as an oxytetracycline (OTC) resistance protein. Numerous resistance proteins have identified, including the ubiquitous ATP-binding cassette (ABC) family of multidrug transporters [1,2,3,4,5,6]. These integral membrane proteins mediate the active extrusion of antibiotics and toxic compounds from cells. The self-protection afforded by the antibiotic producers is dependent on its endogenous drug resistance proteins These resistance proteins may represent targets for antibiotic production improvement strategies [12,13]

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