The Late-Stage Steps ofBurkholderia cenocepaciaProtein O-Linked Glycan Biosynthesis Are Conditionally Essential

Abstract

PeriplasmicO-linked protein glycosylation is a highly conserved process observed across theBurkholderiagenus. Within Burkholderia, protein glycosylation requires the five gene cluster known as theO-glycosylationcluster (OGC,ogcXABEI) which facilitates the construction of theO-linked trisaccharide attached to periplasmic proteins. Previous studies have reported conflicting results regarding the essentiality ofogcAandogcX, predicted to be involved in the last steps inO-linked glycan biosynthesis. Within this work, we aimed to dissect the impact of the loss ofogcAandogcXonBurkholderia cenocepaciaviability. We demonstrate that the loss ofogcAorogcXappears detrimental if glycosylation is initiated leading to marked phenotypic effects. Proteomic analysis supports that the loss ofogcA/ogcXboth blocks glycosylation and drives pleotropic effects in the membrane proteome, resulting in the loss of membrane integrity. Consistent with this, strains lackingogcAandogcXexhibit increased sensitivity to membrane stressors including antibiotics and demonstrate marked changes in membrane permeability. These effects are consistent with fouling of the undecaprenyl pool due to dead-endO-linked glycan intermediates, and consistent with this, we show that modulation of the undecaprenyl pool through the overexpression of undecaprenyl pyrophosphate synthase (UppS) as well as early-stage OGC biosynthesis genes (ogcIandogcB) impactsB. cenocepaciaviability. These findings demonstrate disruptingO-linked glycan biosynthesis or transport appears to dramatically impactB. cenocepaciaviability, supporting the assignment ofogcAandogcXas conditionally essential.ImportanceProtein glycosylation, a conserved process in Burkholderia species, utilizes glycans generated by the O-glycosylation cluster (OGC), which is composed of five genes (ogcX, ogcA,ogcB,ogcE, andogcI). In this study, we demonstrate that the loss of ogcA or ogcX significantly affects the physiology ofBurkholderia cenocepacia. Using complementary genetic approaches and proteomic techniques, we show that the loss of ogcA or ogcX blocks glycosylation, alters the cell membrane, and sensitizes cells to stressors such as antibiotics. This increased sensitivity to membrane stress is caused by the accumulation of dead-end O-linked glycan intermediates, which sequester the limited and essential undecaprenyl pool withinB. cenocepacia. These findings highlight thatogcAandogcXare conditionally essential forB. cenocepaciasurvival and provides mechanistic insight into how protein glycosylation fidelity—the use of specific glycans for protein glycosylation—is controlled inBurkholderiaspecies.