Characterizing the cellulose-modifying enzyme BcsG from Escherichia coli

Microbial biofilms are communities of microorganisms that exhibit co-operative behaviour, producing a matrix of exopolysaccharide that enmeshes the community. The well-studied human pathogens Escherichia coli and Salmonella entericaproduce a biofilm matrix comprised chiefly of the biopolymer cellulose, along with amyloid protein fibers termed curli. This biofilm matrix confers surface adherence and acts as a protective barrier to disinfectants, antimicrobials, environmental stressors, and host immune responses. Pertaining to this research, the bcsEFG operon, conserved in the Enterobacteriaceae, encodes an inner membrane-spanning complex responsible for the addition of a phosphoethanolamine (pEtN) modification to microbial cellulose, essential for extracellular matrix assembly and biofilm architecture. Furthermore, E. coli deficient in bcsGproduce a biofilm matrix lacking structural integrity and the self-assembling architecture observed in wild-type E. coli. The presence of a pEtN-substituted cellulose matrix was shown to be important in bladder epithelial colonization by uropathogenic E. coli, further suggesting its role as a virulence factor in etiological agents of urinary tract infections observed in the clinic. The purpose of this research was to characterize theEcBcsG enzyme, a putative pEtN transferase, by resolving its structure, understanding its role in pEtN substitution of the cellulose matrix in E. coli, and by elucidating its catalytic mechanism to enable future efforts in drug discovery. All these research objectives were achieved, shedding light on the biochemical basis of pEtN cellulose in E. coli. The de novo X-ray crystal structure of the C-terminal catalytic domain of EcBcsG was solved using the single-wavelength anomalous diffraction (SAD) technique phased on L-selenomethionine substituted EcBcsG crystals and revealed EcBcsG folds as a zinc-dependent phosphotransferase belonging to the pEtN transferase family. TheEcBcsG active site was mapped using functional complementation, revealing EcBcsG shares a partially conserved active site with other known pEtN transferase family members, including enzymes responsible for resistance to cationic antimicrobial peptides (CAMPs). Using mock cosubstrates para-nitrophenyl phosphoethanolamine (p-NPPE) and cellooligosaccharides, the specific activity of EcBcsG was measured to be 11.45 +/- 0.51 nmol min-1mg-1in the presence of the cellulo-pentasaccharide. The kinetic parameters were measured as Km= 1.673 ±0.382 mM, kcat= 6.876 x 10-3± 4.14 x 10-4s-1, and kcat/Km= 4.110±0.970 M s-1. The enzymatic product of this reaction was identified and confirmed in vitro. Finally, the covalent phospho-enzyme intermediate was isolated, providing evidence for an EcBcsG catalytic mechanism. The structural and functional model of the cellulose modifying complex, resulting from this work, provides new insight into the biochemical basis for the biofilm matrix of E. coli and other Enterobacteriaceae.The research presented here offers opportunities in structure-based drug discovery and other efforts in inhibiting microbial biofilms and including the possibility of limiting the resilience of biofilm-forming pathogens. Additionally, further understanding of bcsEFG-directed phosphoethanolamine cellulose production may enable biosynthetic engineering of new cellulosic materials or confer the advantages of phosphoethanolamine cellulose in new organisms