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Making the Enterobacterial Common Antigen Glycan and Measuring Its Substrate Sequestration.

Colleen R Eade1, Timothy W Wallen1, Claire E Gates1,2

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Disrupting enterobacterial common antigen (ECA) synthesis in bacteria depletes essential polyisoprenoid phosphate (BP) carriers. This study provides biochemical evidence of BP sequestration and its impact on bacterial glycan production.

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Area of Science:

  • Microbiology
  • Biochemistry
  • Glycobiology

Background:

  • Enterobacterial common antigen (ECA) is a crucial polysaccharide in Enterobacteriaceae, influencing outer membrane permeability.
  • ECA biosynthesis competes for shared substrates, notably polyisoprenoid phosphate (BP), a carrier for multiple essential glycans.
  • Understanding substrate competition is vital for deciphering bacterial glycan synthesis regulation.

Purpose of the Study:

  • To provide biochemical evidence for the impact of glycan pathway disruption on endogenous polyisoprenoid pools.
  • To investigate the effect of ECA biosynthesis disruption on polyisoprenoid phosphate (BP) levels.
  • To establish methods for assessing ECA glycan and understanding polysaccharide interdependence.

Main Methods:

  • In vitro enzymatic synthesis of ECA repeat units and LC-MS characterization of standards.
  • Cloning and expression of genes involved in ECA biosynthesis.
  • Generation of ECA-deficient mutants and analysis of accumulated intermediates and BP levels via LC-MS.

Main Results:

  • Successfully reconstituted the ECA biosynthesis pathway in vitro, generating characterized standards.
  • ECA-deficient mutants accumulated intermediates indistinguishable from synthesized standards.
  • A significant decrease in cellular BP levels was observed in ECA-deficient mutants, correlating with pathway disruption.

Conclusions:

  • This study provides the first direct biochemical evidence for polyisoprenoid phosphate (BP) sequestration upon disruption of bacterial glycan biosynthesis pathways.
  • Genetic disruption of ECA synthesis leads to the accumulation of specific glycan intermediates and depletion of BP.
  • Reveals a dynamic interdependence within the bacterial polysaccharide repertoire, highlighting substrate competition as a regulatory mechanism.