Roles of pgaABCD Genes in Synthesis, Modification, and Export of the Escherichia coli Biofilm Adhesin, Poly--1,6-N-acetyl-D-glucosamine (PGA)

Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, Georgia 30322, Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida 32611, Integrated Microscopy & Microanalytical Facility, Emory University, Atlanta, Georgia 30322, and Department of Molecular and Cellular Biology and AFMnet-NCE, College of Biological Science, University of Guelph, Guelph, Ontario, Canada N1G 2W1

^* To whom correspondence should be addressed. Email: romeo{at}microbio.emory.edu.

	Abstract

The linear homopolymer poly--1,6-N-acetyl-D-glucosamine (-1,6-GlcNAc, PGA) serves as an adhesin for 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. Deletion of genes for the predicted outer membrane proteins PgaA or PgaB did not prevent PGA synthesis, but blocked its export, as shown by 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. Overexpression of pgaB increased 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. Previous studies indicated that N-deacetylation of -1,6-GlcNAc in Staphylococcus epidermidis by the PgaB homolog, IcaB, anchors it to the cell surface. Deletion of icaB resulted in -1,6-GlcNAc release into the growth medium. Thus, covalent modification of -1,6-GlcNAc by N-deacetylation serves distinct biological functions in gram-negative vs. gram-positive species, dictated by cell envelope differences.