Roles of pgaABCD Genes in Synthesis, Modification, and Export of the Escherichia coli Biofilm Adhesin Poly-β-1,6- N -Acetyl- d -Glucosamine

Abstract

The linear homopolymer poly-␤-1,6-N-acetyl-D-glucosamine (␤-1,6-GlcNAc; PGA) serves as an adhesin for the maintenance of biofilm structural stability in diverse eubacteria. Its function in Escherichia coli K-12 requires the gene products of the pgaABCD operon, all of which are necessary for biofilm formation. PgaC is an apparent glycosyltransferase that is required for PGA synthesis. Using a monoclonal antibody directed against E. coli PGA, we now demonstrate that PgaD is also needed for PGA formation. The deletion of genes for the predicted outer membrane proteins PgaA and PgaB did not prevent PGA synthesis but did block its export, as shown by the results of immunoelectron microscopy (IEM) and antibody adsorption assays. IEM also revealed a conditional localization of PGA at the cell poles, the initial attachment site for biofilm formation. PgaA contains a predicted ␤-barrel porin and a superhelical domain containing tetratricopeptide repeats, which may mediate protein-protein interactions, implying that it forms the outer membrane secretin for PGA. PgaB contains predicted carbohydrate binding and polysaccharide N-deacetylase domains. The overexpression of pgaB increased the primary amine content (glucosamine) of PGA. Site-directed mutations targeting the N-deacetylase catalytic activity of PgaB blocked PGA export and biofilm formation, implying that N-deacetylation promotes PGA export through the PgaA porin. The results of previous studies indicated that N-deacetylation of ␤-1,6-GlcNAc in Staphylococcus epidermidis by the PgaB homolog, IcaB, anchors it to the cell surface. The deletion of icaB resulted in release of ␤-1,6-GlcNAc into the growth medium. Thus, covalent modification of ␤-1,6-GlcNAc by N-deacetylation serves distinct biological functions in gram-negative and gram-positive species, dictated by cell envelope differences.

Comprehensive overexpression analysis of cyclic-di-GMP signalling proteins in the phytopathogen Pectobacterium atrosepticum reveals diverse effects on motility and virulence phenotypes

“…atrosepticum could be induced to flocculate through c-di-GMP-dependent production of the exopolysaccharide adhesin poly-b-1,6-N-acetyl-D-glucosamine (PNAG) (Itoh et al, 2008;Pérez-Mendoza et al, 2011b;Tagliabue et al, 2010). PNAG (and the associated flocculation phenotype) was expressed upon induction of a heterologous, constitutively active PleD, revealing that Pe.…”

Structural Basis for Translocation of a Biofilm-supporting Exopolysaccharide across the Bacterial Outer Membrane

“…In E. coli, four genes of the pgaABCD operon encode proteins required for the synthesis and translocation of dPNAG across the bacterial double-layer membrane (14,17). PgaA, a tetratricopeptide repeat (TPR)-containing outer membrane protein, forms the secretion pore in the outer membrane and translocates the dPNAG polymer from the periplasm to the cell surface (14,17).…”

The diguanylate cyclase YddV controls production of the exopolysaccharide poly-N-acetylglucosamine (PNAG) through regulation of the PNAG biosynthetic pgaABCD operon

“…LG26, a DbcsA : : kan mutant impaired in cellulose production; AM73, a DcsgA/DbcsA double mutant; and AM56, a DpgaA : : cat mutant unable to export PNAG and to expose it on the cell surface (Itoh et al, 2008). We expected that phenotypes depending on an increase in production of cell surface-associated structures caused by DGC overexpression would be abolished by inactivation of the corresponding target genes.…”