Study of the Expression of Bacterial Multidrug Efflux Pumps in Anaerobic Conditions
Bacterial multidrug efflux pumps belong to a class of membrane transporter proteins that dedicate to the extrusion of a diverse range of substances out of cells including all classes of currently available antibiotics. They constitute an important mechanism of bacterial antibiotic and multidrug resistance. Since many ecological niches of bacteria and the infection foci in animal host display low oxygen tension under which condition bacterial pathogens undergo fundamental changes on their metabolic modes, it is necessary to study the expression profiles of drug efflux pumps under these physiologically and clinically relevant conditions. In this chapter, we first introduce procedures to culture bacteria under anaerobic conditions, which is achieved using screw-capped Pyrex culture tubes without agitation. We then introduce β-galactosidase activity assay using promoter-lacZ (encoding the β-galactosidase enzyme) fusion to measure the expression of efflux pumps at transcriptional level, and Western blot using chromosomal FLAG-tagged construct to examine the expression of these proteins at translational level. Applications of these gene expression studies to reveal the regulatory mechanisms of efflux genes expression as well as their physiological functions are also discussed.
Key wordsMultidrug efflux pump Multidrug resistance Anaerobiosis β-galactosidase activity assay SDS-PAGE Western blot
We are grateful to Prof. Kunihiko Nishino (Institute of Scientific and Industrial Research, Osaka University) for the pNN387 vector and the PgadE-lacZ strain. The studies are supported by the Hong Kong University Grants Council General Research Fund (HKU 17142316) and the Health and Medical Research Fund (HMRF) Hong Kong (No. 13120662) to A.Y.
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