Insights into phosphoethanolamine cellulose synthesis and secretion across the Gram-negative cell envelope

• Published in: Nature communications Vol. 15; no. 1; pp. 7798 - 17
• Main Authors: Verma, Preeti, Ho, Ruoya, Chambers, Schuyler A., Cegelski, Lynette, Zimmer, Jochen
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 06.09.2024; Nature Publishing Group; Nature Portfolio
• Subjects: 101/28; 631/326/46; 631/45/72/1205; 631/535/1258/1259; 82/16; 82/80; 82/83; BcsC protein; Biofilms; Cell envelopes; Cell Membrane - metabolism; Cell Wall - metabolism; Cellooligosaccharides; Cellulase; Cellulase - genetics; Cellulase - metabolism; Cellulose; Cellulose - biosynthesis; Cellulose - metabolism; Cellulose synthase; Chemical synthesis; E coli; Enterobacteriaceae; Escherichia coli - genetics; Escherichia coli - metabolism; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; Ethanolamines - metabolism; Glucosyltransferases - genetics; Glucosyltransferases - metabolism; Humanities and Social Sciences; Lipids; Membranes; Molecular interactions; multidisciplinary; N-Terminus; Oligosaccharides; Periplasm - metabolism; Polymers; Protein engineering; Protein transport; Proteins; Science; Science (multidisciplinary); Secretion; Translocation
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-024-51838-0

Phosphoethanolamine (pEtN) cellulose is a naturally occurring modified cellulose produced by several Enterobacteriaceae. The minimal components of the E. coli cellulose synthase complex include the catalytically active BcsA enzyme, a hexameric semicircle of the periplasmic BcsB protein, and the outer membrane (OM)-integrated BcsC subunit containing periplasmic tetratricopeptide repeats (TPR). Additional subunits include BcsG, a membrane-anchored periplasmic pEtN transferase associated with BcsA, and BcsZ, a periplasmic cellulase of unknown biological function. While cellulose synthesis and translocation by BcsA are well described, little is known about its pEtN modification and translocation across the cell envelope. We show that the N-terminal cytosolic domain of BcsA positions three BcsG copies near the nascent cellulose polymer. Further, the semicircle’s terminal BcsB subunit tethers the N-terminus of a single BcsC protein in a trans-envelope secretion system. BcsC’s TPR motifs bind a putative cello-oligosaccharide near the entrance to its OM pore. Additionally, we show that only the hydrolytic activity of BcsZ but not the subunit itself is necessary for cellulose secretion, suggesting a secretion mechanism based on enzymatic removal of translocation incompetent cellulose. Lastly, protein engineering introduces cellulose pEtN modification in orthogonal cellulose biosynthetic systems. These findings advance our understanding of pEtN cellulose modification and secretion. Enterobacteriaceae modify cellulose with lipid-derived pEtN groups to promote biofilm cohesion. Here, using structural and biochemical analyses, the authors provide further insights into the molecular interactions of BcsA, BcsG, BcsB, and BcsC facilitating pEtN modification and secretion of cellulose.