Abstract
The betaproteobacterium Castellaniella defragrans 65Phen grows on monoterpenes at concentrations toxic to many bacteria. Tolerance mechanisms include modifications of the membrane fatty acid composition and the mineralization of monoterpenes. In this study, we characterized an efflux transporter associated to the monoterpene metabolism. The inner-membrane transporter AmeD (apolar monoterpene efflux) affiliated to the HAE3 (hydrophobe/amphiphile efflux) family within the Resistance-Nodulation-Division (RND) superfamily. RND pumps of the HAE3 family are known for transporting substrates into the periplasm. AmeD is co-expressed with the outer membrane protein AmeA and the periplasmic proteins AmeB and AmeC, suggesting an export channel into the environment similar to HAE1-type RND exporters. Proteins AmeABCD are encoded within a genetic island involved in the metabolism of acyclic and cyclic monoterpenes. The deletion of ameABCD translated into a decrease in tolerance to monoterpenes in liquid cultures. The addition of acetate as cosubstrate in limonene-containing cultures partially alleviated monoterpene toxicity in the deletion mutant. Accumulation of Nile Red in cells of C. defragrans required dissipation of the proton motive force with carbonyl cyanide m-chlorophenylhydrazone (CCCP). Cells lacking AmeABCD accumulated more Nile Red, suggesting an export function of the proteins. Our observations suggest that the tetrapartite RND transporter AmeABCD acts as an exporter during monoterpene detoxification in C. defragrans.
Highlights
Monoterpenes are a diverse group of volatile biogenic hydrocarbons found mainly in the essential oils of plants
Our observations suggest that the tetrapartite RND transporter AmeABCD acts as an exporter during monoterpene detoxification in C. defragrans
To assess the role of the putative RND transporter during monoterpene utilization, the wild-type strain and the deletion mutant 65Phen DameABCD were compared in growth experiments (Fig. 2)
Summary
Monoterpenes are a diverse group of volatile biogenic hydrocarbons found mainly in the essential oils of plants. These compounds exhibit a myriad of biological functions such as pollinator attraction, plant–plant communication and as antimicrobials (Mahmoud and Croteau 2002) Due to their hydrophobic nature, monoterpenes tend to accumulate in cellular membranes altering the proton gradient, the electron transport and the stability of membrane proteins (Abrahim et al 2003; Brennan et al 2012; Griffin et al 1999). RND efflux transporters active on volatile hydrocarbons belong to the hydrophobe/amphiphile efflux-1 (HAE1) family (Eswaran et al 2004; Garcia et al 2010; Nikaido 2011; Tseng et al 1999) Members of this family are mostly tripartite consisting of an inner membrane substrate/ proton antiporter, an outer membrane pore and a periplasmic membrane fusion protein (MFP). Substrate specificity is determined by the inner membrane RND pump which recruits substrates from the periplasm or from the outer leaflet of the inner membrane (Daury et al 2016; Nikaido 2011)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
