The Escherichia coli cellulose synthase subunit G (BcsG) is a Zn2+-dependent phosphoethanolamine transferase

Abstract

Bacterial biofilms are cellular communities that produce an adherent matrix. Exopolysaccharides are key structural components of this matrix and are required for the assembly and architecture of biofilms produced by a wide variety of microorganisms. The human bacterial pathogens Escherichia coli and Salmonella enterica produce a biofilm matrix composed primarily of the exopolysaccharide phosphoethanolamine (pEtN) cellulose. Once thought to be composed of only underivatized cellulose, the pEtN modification present in these matrices has been implicated in the overall architecture and integrity of the biofilm. However, an understanding of the mechanism underlying pEtN derivatization of the cellulose exopolysaccharide remains elusive. The bacterial cellulose synthase subunit G (BcsG) is a predicted inner membrane–localized metalloenzyme that has been proposed to catalyze the transfer of the pEtN group from membrane phospholipids to cellulose. Here we present evidence that the C-terminal domain of BcsG from E. coli (EcBcsGΔN) functions as a phosphoethanolamine transferase in vitro with substrate preference for cellulosic materials. Structural characterization of EcBcsGΔN revealed that it belongs to the alkaline phosphatase superfamily, contains a Zn2+ ion at its active center, and is structurally similar to characterized enzymes that confer colistin resistance in Gram-negative bacteria. Informed by our structural studies, we present a functional complementation experiment in E. coli AR3110, indicating that the activity of the BcsG C-terminal domain is essential for integrity of the pellicular biofilm. Furthermore, our results established a similar but distinct active-site architecture and catalytic mechanism shared between BcsG and the colistin resistance enzymes.

Identification of the Clostridial cellulose synthase and characterization of the cognate glycosyl hydrolase, CcsZ

“…At present, an understanding of the synthase responsible for bacterial cellulose biosynthesis has been largely limited to the study of Gram-negative model organisms. The Gram-negative bacterial cellulose synthesis (Bcs) complex is encoded by the bcsABCZ locus and represents the essential biosynthetic machinery required for production of the β-(1-4)-glucan polymer [16] that is subsequently derivatized by at least three distinct accessory cellulose modification systems described in Gram-negative bacteria [11,[17][18][19]. The proposed functions of the bcsABCZ gene products were largely inferred from structural and functional studies of other Gram-negative exopolysaccharide systems (e.g., the poly-β-(1-6)-N-acetyl-D-gluccoasmine (PNAG) biosynthetic system from Escherichia coli [20] and Staphylococcal species [21][22][23] or the alginate biosynthesis pathway from Pseudomonas aeruginosa [24]).…”

Inactivation Efficacy of 405 nm LED Against Cronobacter sakazakii Biofilm

“…Cellulose is a major component of biofilm matrix and may also act as an adhesion factor (El Hag et al, 2017). bcsA and bcsG genes are necessary to produce cellulose, and are involved in cell-cell aggregation and biofilm formation (Hu et al, 2015;Anderson et al, 2020). Our study suggested that LED may control the synthesis of cellulose and adhesion ability of C. Sakazakii cells by regulating the function of bcsA and bcsG, thus influencing the biofilm forming process.…”

Variation in the ratio of curli and phosphoethanolamine cellulose associated with biofilm architecture and properties

“…[53,83,84] In addition, increased curli content in UTI89 pellicles yields a mechanically more robust film with increased elasticity. [73,85] The cellulosic component contributes to cohesive integrity of UTI89 pellicles, [12] and one recent study of BcsG indicated that pEtN modification does contribute to pellicle integrity in AR3110, although in different growth conditions at 30 C. [33] Thus, we sought to address the question of how pellicle formation in UTI89 and AR3110 might differ, knowing that the composition of the isolated ECM above revealed variation in the ratio of pEtN cellulose and curli. Then, we explored the influence of the pEtN modification in AR3110 and UTI89 pellicle formation and evaluated bcsA mutants.…”

“…Recently, two groups reported crystal structures of the periplasmic C‐terminal domain of BcsG and confirmed its catalytic ability to remove pEtN from phospholipid head groups [ 18 ] and to add pEtN to a carbohydrate substrate. [ 33 ]…”

“…Recently, two groups reported crystal structures of the periplasmic C-terminal domain of BcsG and confirmed its catalytic ability to remove pEtN from phospholipid head groups [18] and to add pEtN to a carbohydrate substrate. [33] F I G U R E 1 A, Chemical structure of cellulose. B, Representation of chemical structure of phosphoethanolamine (pEtN) cellulose, here shown with one modified glucose unit adjacent to an unmodified glucose.…”