EstG is a novel esterase required for cell envelope integrity in Caulobacter

Proper regulation of the bacterial cell envelope is critical for cell survival. Identification and characterization of enzymes that maintain cell envelope homeostasis is crucial, as they can be targets for effective antibiotics. In this study, we have identified a novel enzyme, called EstG, whose activity protects cells from a variety of lethal assaults in the ⍺-proteobacterium Caulobacter crescentus. Despite homology to transpeptidase family cell wall enzymes and an ability to protect against cell-wall-targeting antibiotics, EstG does not demonstrate biochemical activity toward cell wall substrates. Instead, EstG is genetically connected to the periplasmic enzymes OpgH and BglX, responsible for synthesis and hydrolysis of osmoregulated periplasmic glucans (OPGs), respectively. The crystal structure of EstG revealed similarities to esterases and transesterases, and we demonstrated esterase activity of EstG in vitro. Using biochemical fractionation, we identified a cyclic hexamer of glucose as a likely substrate of EstG. This molecule is the first OPG described in Caulobacter and establishes a novel class of OPGs, the regulation and modification of which are important for stress survival and adaptation to fluctuating environments. Our data indicate that EstG, BglX, and OpgH comprise a previously unknown OPG pathway in Caulobacter. Ultimately, we propose that EstG is a novel enzyme that instead of acting on the cell wall, acts on cyclic OPGs to provide resistance to a variety of cellular stresses. •Cells lacking EstG are sensitive to an array of antibiotic and other stresses•EstG has esterase activity and acts on a cyclic osmoregulated periplasmic glucan•Mutations in OpgH rescue antibiotic sensitivity of the ΔestG mutant•BglX is involved in the OPG pathway and impacts envelope homeostasis Daitch et al. establish a novel osmoregulated periplasmic glucan (OPG) pathway in Caulobacter. EstG is a periplasmic esterase that acts on a cyclic OPG to promote cell envelope homeostasis and protect against cell stress. The enzymes OpgH and BglX also contribute to envelope integrity through synthesis and hydrolysis of Caulobacter OPGs.