Two new enzymes that liberate undecaprenyl-phosphate to replenish the carrier lipid pool during envelope stress

The 55-carbon isoprenoid, undecaprenyl-phosphate (UndP), is a universal carrier lipid that ferries most glycans and glycopolymers across the cytoplasmic membrane in bacteria. In addition to peptidoglycan precursors, UndP transports O-antigen, capsule, wall teichoic acids, and sugar modifications. How this shared but limited lipid is distributed among competing pathways is just beginning to be elucidated. We recently reported that in the bacterium <i>Bacillus subtilis</i>, the stress-response sigma factor SigM and its cognate anti-sigma factor complex respond to changes in the free UndP pool. When levels are low, SigM activates genes that increase flux through the essential cell wall synthesis pathway, promote the recycling of the lipid carrier, and liberate the carrier from other polymer pathways. Here, we report that two additional enzymes under SigM control help maintain the free pool of UndP. One, UshA (YqjL), resembles alpha-beta hydrolases and liberates UndP from undecaprenyl-monophosphate-linked sugars. The other, UpsH (YpbG), resembles metallophosphoesterases and releases UndP from undecaprenyl-diphosphate-linked wall teichoic acids polymers but not lipid-linked peptidoglycan precursors. UshA becomes critical for growth when UndP-linked sugars are sequestered, and the carrier lipid pool is depleted. Similarly, UpsH becomes essential for viability when UndPP-linked intermediates accumulate. Mutations in the predicted catalytic residues of both putative hydrolases abrogate their function arguing that they act directly to release UndP. These findings define two new enzymes that liberate the carrier lipid from UndP- and UndPP-linked intermediates and bolster the model that the SigM stress-response pathway maintains the UndP pool and prioritizes its use for peptidoglycan synthesis.IMPORTANCEMotivated by the success of naturally occurring glycopeptide antibiotics like vancomycin, one arm of recent antibiotic discovery efforts has focused on compounds that bind lipid-linked precursors used to build extracytoplasmic polymers. Trapping these precursors depletes the universal carrier lipid undecaprenyl-phosphate, which is required for the synthesis of virtually all surface polymers, including peptidoglycan. Understanding how cells respond to this stress to restore the carrier lipid pool is critical to identifying effective drugs. Here, we report the identification of two new enzymes that are produced in response to the depletion of the carrier lipid pool. These enzymes recover the carrier lipid but cleave distinct lipid-linked precursors to do so.