Cupriavidus metallidurans CH34 resists high concentrations of several metal ions. A recent study reported an ABC-type sugar transporter to be putatively involved in its increased zinc resistance. This transporter has six subunits encoded by the Rmet_2229 to Rmet_2234 genes arranged in an operon and regulated by GlpR encoded by Rmet_2235. However, the role of this transporter in zinc resistance is unknown. The study of how this bacterium resists high zinc concentrations is important as it may offer opportunities in bioremediation and/or bioaugmentation, not leaving out its potential application as an advanced heavy metal whole cell biosensor. This will reduce human exposure to heavy metal-contaminated sites as contamination with heavy metals poses significant threat to human life and the entire ecosystem. Our main aim was to understand the role of this transporter in zinc resistance in Cupriavidus metallidurans CH34. We hypothesized that this transporter is responsible for the increased zinc resistance in a laboratory-evolved C. metallidurans strain. We had as objectives to describe and classify the various subunits by doing a database similarity search with ABCdb, an online database for prokaryotic ABC transporters. To confirm the involvement of this transporter in zinc resistance. To compare the level of glycerol utilization in strain CH34 and its derivatives. To test the function of the individual transporter subunits and to quantify the intracellular levels of zinc in in all the strains. A database similarity search for all the subunits was performed using ABCdb. A plasmid harboring the six genes was used to complement a CH34 derivative in which glpR and the transporter were deleted. Individual knockouts of the six subunits were created in the plasmid by site-directed mutagenesis and used for complementing the CH34 deletion derivative. A dose-response experiment with zinc was performed for all the strains and their growth rates with glycerol as sole carbon source evaluated. A detailed description and classification of the subunits was performed. The involvement of this transporter in zinc resistance was confirmed. The different complementation strains were created, each having a deletion of one of the transporter subunits. All strains could significantly utilize glycerol as sole carbon source. The lower resistance of the CH34 deletion derivative compared to the laboratory-evolved strains confirmed the transporter’s involvement in zinc resistance. The ability of this deletion derivative to utilize glycerol as the sole carbon source suggests that this transporter is not essential for glycerol uptake in this bacterium. The function of the individual subunits could not be tested because the control (complete construct) could not be complemented. Hence, for the six other constructs we do not know if they are able to complement. Evaluation of the complementation strain showed that secondary mutations induced when creating the deletion mutant may have neutralized complementation.
|State||Published - 30 Jun 2017|