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Journal of Bacteriology, May 2008, p. 3670-3680, Vol. 190, No. 10
0021-9193/08/$08.00+0     doi:10.1128/JB.01920-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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

Yoshikane Itoh,1,§ John D. Rice,2 Carlos Goller,1 Archana Pannuri,1 Jeannette Taylor,3 Jeffrey Meisner,1 Terry J. Beveridge,4,{dagger} James F. Preston III,2 and Tony Romeo1*

Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, Georgia 30322,1 Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida 32611,2 Integrated Microscopy and Microanalytical Facility, Emory University, Atlanta, Georgia 30322,3 Department of Molecular and Cellular Biology and AFMnet-NCE, College of Biological Science, University of Guelph, Guelph, Ontario, Canada N1G 2W14

Received 10 December 2007/ Accepted 8 March 2008

The linear homopolymer poly-β-1,6-N-acetyl-D-glucosamine (β-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 β-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 β-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 β-1,6-GlcNAc into the growth medium. Thus, covalent modification of β-1,6-GlcNAc by N-deacetylation serves distinct biological functions in gram-negative and gram-positive species, dictated by cell envelope differences.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, Emory University School of Medicine, 3105 Rollins Research Center, 1510 Clifton Rd. N.E., Atlanta, GA 30322. Phone: (404) 727-3734. Fax: (404) 727-3659. E-mail: romeo{at}microbio.emory.edu

{triangledown} Published ahead of print on 21 March 2008.

§ Present address: Faculty of Environmental Earth Science, Hokkaido University, Kita 10 Nishi 5, Kita-ku, Sapporo 060-0810, Japan.

{dagger} Deceased.

Journal of Bacteriology, May 2008, p. 3670-3680, Vol. 190, No. 10
0021-9193/08/$08.00+0     doi:10.1128/JB.01920-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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