Molecular characterisation of ABC transporter type FtsE and FtsX proteins of Mycobacterium tuberculosis
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Elicitation of drug resistance and various survival strategies inside host macrophages have been the hallmarks of Mycobacterium tuberculosis as a successful pathogen. ATP Binding Cassette (ABC) transporter type proteins are known to be involved in the efflux of drugs in bacterial and mammalian systems. FtsE, an ABC transporter type protein, in association with the integral membrane protein FtsX, is involved in the assembly of potassium ion transport proteins and probably of cell division proteins as well, both of which being relevant to tubercle bacillus. In this study, we cloned ftsE gene of M. tuberculosis, overexpressed and purified. The recombinant MtFtsE-6xHis protein and the native MtFtsE protein were found localized on the membrane of E. coli and M. tuberculosis cells, respectively. MtFtsE-6xHis protein showed ATP binding in vitro, for which the K42 residue in the Walker A motif was found essential. While MtFtsE-6xHis protein could partially complement growth defect of E. coli ftsE temperature-sensitive strain MFT1181, co-expression of MtFtsE and MtFtsX efficiently complemented the growth defect, indicating that the MtFtsE and MtFtsX proteins might be performing an associated function. MtFtsE and MtFtsX-6xHis proteins were found to exist as a complex on the membrane of E. coli cells co-expressing the two proteins.
KeywordsFtsE FtsX ABC transporter type protein Walker A motif ATP binding Mycobacterium tuberculosis cell division
3, 3′-dithio-bis(propionic acid N-hydroxysuccinimide ester)
ATP binding Cassette
Signal Recognition Particle
The authors are immensely thankful to Dr. A. Nishimura, National Institute of Genetics, Japan, for providing us E. coli MFT1181 ftsE (Ts) mutant, pSTR-30–SD vector and the wild-type strain E. coli JA200 for the complementation experiments. We thank Ramanujam Srinivasan for critical comments on the work reported here. The work was supported in part by the Genomics Initiative on Microbial Pathogens — Structural Genomics Initiative, funded by the Department of Biotechnology, Government of India, in the Division of Biological Sciences, at Indian Institute of Science. Authors acknowledge the infrastructure and equipment facilities in the Department of Microbiology and Cell Biology supported by the FIST grant from Department of Science and Technology, Government of India, Departmental Special Assistance Programme from University Grants Commission, Government of India, and ICMR Centre for Advanced Study in Molecular Medical Microbiology, at the Department of Microbiology and Cell Biology, Indian Institute of Science. Use of DBT-supported phosphorimager facility is acknowledged. MAM is recipient of the scholarship from Indian Institute of Science and UV acknowledges financial assistance from the Structural Genomics Programme.
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