CUMULATIVE TRANSPORTER DELETIONS REVEAL OUR LIMITED UNDERSTANDING OF TRANSPORT IN ESCHERICHIA COLI: INTRODUCING THE D22 STRAIN
Our understanding of how small polyols and related non-glucose sugars cross the cell membrane remains a nebulous, with uncertainty regarding substrate-transporter associations, possible transport bottlenecks and the role of passive diffusion. This situation persists even in model bacteria like Escherichia coli. A strain lacking 22 transporters for small non-glucose sugars was built to shed light on this issue, using recombineering and CRISPR-Cas9 counterselection. All described transporters for the linear polyols glycerol, mannitol and sorbitol, as well as all described pentose importers, were successfully deleted.  A number of promiscuous transporters involved in carbon scavenging responses were also targeted. The resulting D22 strain was evaluated for growth on various polyols as a sole carbon source, and showed no reduction in overall cell fitness after the modifications. Surprisingly, growth rates and lag phase durations on glycerol and ribose were not impacted in D22, suggesting alternative transport routes. However, lag phase durations on mannitol and xylose were significantly increased by 93% and 67% respectively, while growth on mannose was virtually abolished.  The D22 strain may prove a useful tool for screening transporters for mannitol, mannose and xylose with minimal background permeability and represents a step forward in unravelling our understanding of polyol transport.
Our understanding of how small polyols and related non-glucose sugars cross the cell membrane remains a nebulous, with uncertainty regarding substrate-transporter associations, possible transport bottlenecks and the role of passive diffusion. This situation persists even in model bacteria like Escherichia coli. A strain lacking 22 transporters for small non-glucose sugars was built to shed light on this issue, using recombineering and CRISPR-Cas9 counterselection. All described transporters for the linear polyols glycerol, mannitol and sorbitol, as well as all described pentose importers, were successfully deleted.  A number of promiscuous transporters involved in carbon scavenging responses were also targeted. The resulting D22 strain was evaluated for growth on various polyols as a sole carbon source, and showed no reduction in overall cell fitness after the modifications. Surprisingly, growth rates and lag phase durations on glycerol and ribose were not impacted in D22, suggesting alternative transport routes. However, lag phase durations on mannitol and xylose were significantly increased by 93% and 67% respectively, while growth on mannose was virtually abolished.  The D22 strain may prove a useful tool for screening transporters for mannitol, mannose and xylose with minimal background permeability and represents a step forward in unravelling our understanding of polyol transport.
Ghent University