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Journal of Bacteriology, November 2008, p. 7464-7478, Vol. 190, No. 22
0021-9193/08/$08.00+0     doi:10.1128/JB.01075-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Modification of Pseudomonas aeruginosa Pa5196 Type IV Pilins at Multiple Sites with D-Araf by a Novel GT-C Family Arabinosyltransferase, TfpW{triangledown}

Julianne V. Kus,1 John Kelly,2 Luc Tessier,2 Hanjeong Harvey,3 Dennis G. Cvitkovitch,1 and Lori L. Burrows1,3,4*

Faculty of Dentistry, University of Toronto, Toronto, Ontario, Canada,1 Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada K1A 0R6,2 Department of Biochemistry and Biomedical Sciences, McMaster University,3 The Michael G. DeGroote Institute for Infectious Disease Research, Hamilton, Ontario, Canada L8N 3Z54

Received 1 August 2008/ Accepted 5 September 2008

Pseudomonas aeruginosa Pa5196 produces type IV pilins modified with unusual {alpha}1,5-linked D-arabinofuranose ({alpha}1,5-D-Araf) glycans, identical to those in the lipoarabinomannan and arabinogalactan cell wall polymers from Mycobacterium spp. In this work, we identify a second strain of P. aeruginosa, PA7, capable of expressing arabinosylated pilins and use a combination of site-directed mutagenesis, electrospray ionization mass spectrometry (MS), and electron transfer dissociation MS to identify the exact sites and extent of pilin modification in strain Pa5196. Unlike previously characterized type IV pilins that are glycosylated at a single position, those from strain Pa5196 were modified at multiple sites, with modifications of {alpha}β-loop residues Thr64 and Thr66 being important for normal pilus assembly. Trisaccharides of {alpha}1,5-D-Araf were the principal modifications at Thr64 and Thr66, with additional mono- and disaccharides identified on Ser residues within the antiparallel beta sheet region of the pilin. TfpW was hypothesized to encode the pilin glycosyltransferase based on its genetic linkage to the pilin, weak similarity to membrane-bound GT-C family glycosyltransferases (which include the Mycobacterium arabinosyltransferases EmbA/B/C), and the presence of characteristic motifs. Loss of TfpW or mutation of key residues within the signature GT-C glycosyltransferase motif completely abrogated pilin glycosylation, confirming its involvement in this process. A Pa5196 pilA mutant complemented with other Pseudomonas pilins containing potential sites of modification expressed nonglycosylated pilins, showing that TfpW's pilin substrate specificity is restricted. TfpW is the prototype of a new type IV pilin posttranslational modification system and the first reported gram-negative member of the GT-C glycosyltransferase family.

* Corresponding author. Mailing address: Department of Biochemistry and Biomedical Sciences, McMaster University, 4H18 Health Sciences Centre, 1200 Main Street West, Hamilton, Ontario, Canada L8N 3Z5. Phone: (905) 525-9140, ext. 22029. Fax: (905) 522-9033. E-mail: burrowl{at}mcmaster.ca

{triangledown} Published ahead of print on 19 September 2008.

Journal of Bacteriology, November 2008, p. 7464-7478, Vol. 190, No. 22
0021-9193/08/$08.00+0     doi:10.1128/JB.01075-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Harvey, H., Habash, M., Aidoo, F., Burrows, L. L. (2009). Single-Residue Changes in the C-Terminal Disulfide-Bonded Loop of the Pseudomonas aeruginosa Type IV Pilin Influence Pilus Assembly and Twitching Motility. J. Bacteriol. 191: 6513-6524 [Abstract] [Full Text]  


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