A Mechanistic Basis for Phosphoethanolamine Modification of the Cellulose Biofilm Matrix in Escherichia coli

Abstract

Biofilms are communities of self-enmeshed bacteria in a matrix of exopolysaccharides. The widely distributed human pathogen and commensal Escherichia coli produces a biofilm matrix composed of phosphoethanolamine (pEtN)-modified cellulose and amyloid protein fibers, termed curli. The addition of pEtN to the cellulose exopolysaccharide is accomplished by the action of the pEtN transferase, BcsG, and is essential for the overall integrity of the biofilm. Here, using the synthetic co-substrates p-nitrophenyl phosphoethanolamine and β-D-cellopentaose, we demonstrate using an in vitro pEtN transferase assay that full activity of the pEtN transferase domain of BcsG from E. coli (EcBcsG ΔN ) requires Zn 2+ binding, a catalytic nucleophile/acid-base arrangement (Ser 278 /Cys 243 /His 396 ), disulfide bond formation, and other newly uncovered essential residues. We further confirm that EcBcsG ΔN catalysis proceeds by a ping-pong bisubstrate−biproduct reaction mechanism and displays inefficient kinetic behavior (k cat /K M = 1.81 × 10 −4 ± 2.81 × 10 −5 M −1 s −1 ), which is typical of exopolysaccharide-modifying enzymes in bacteria. Thus, the results presented, especially with respect to donor binding (as reflected by K M ), have importantly broadened our understanding of the substrate profile and catalytic mechanism of this class of enzymes, which may aid in the development of inhibitors targeting BcsG or other characterized members of the pEtN transferase family, including the intrinsic and mobile colistin resistance factors.

Molecular insights into phosphoethanolamine cellulose formation and secretion

“…5a. Cells expressing the IMC only, however, reveal background staining, similar to cells producing unmodified cellulose due to the Ser278 to Ala substitution in BcsG 25 , Fig. 5a.…”

“…The conserved Ser residue (Ser278) is assumed to serve as the nucleophile to attack the electrophilic phosphorous of a phosphatidylethanolamine (PE) lipid to form a covalent reaction intermediate with pEtN and releasing diacylglycerol, Fig. S1b 25 . The pEtN group is then subject to attack by the glucose C6 hydroxyl oxygen, resulting in transfer of the pEtN group to cellulose and release of the phospho-enzyme intermediate.…”

“…BcsG is a membrane-bound pEtN transferase containing five N-terminal TM helices, followed by a periplasmic catalytic domain, Fig. 1c and S1a [23][24][25] . The homologous enzymes EptA and EptC catalyze pEtN modification of lipid A and N-glycans, respectively 26,27 .…”

WssI from the Gram-negative bacterial cellulose synthase is an O-acetyltransferase that acts on cello-oligomers with several acetyl donor substrates

“…, in tomato, wheat, and tobacco) ( 41 , 42 ). While these novel findings represent an important area of study to understand robust biofilm formation by these model organisms, they also extend to the growing list of organisms that possess homologs of the wss gene cluster and offer a juxtaposition to bacteria that instead use the recently identified phosphoethanolamine modification of cellulose ( 15 , 20 , 21 , 22 ). Finally, this work not only expands our knowledge of the O-acetylation of polysaccharides in bacterial biofilms by demonstrating both parallels to the prototypical alginate system but also areas that are finely tuned to these unique polymers that are likely to have broad implications for the colonization and persistence of particular niches, like the lungs of cystic fibrosis patients that are colonized by both Pseudomonas and Achromobacter species ( 31 , 37 ).…”

“…Of the organisms on this list, E. coli and Salmonella enterica are the most extensively studied with respect to bacterial cellulose biosynthesis and have served as model organisms for understanding the synthesis and export of the polymer ( 3 , 16 , 17 , 19 ). In these two model organisms, microbial cellulose is decorated by the transferase BcsG with phosphoethanolamine, which is predicted to aid in resistance to cellulases because of altered phenotypic effects when the decorated cellulose fibrils are part of the biofilm along with amyloid curli fibers ( 15 , 20 , 21 , 22 ).…”