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Journal of Bacteriology, November 2009, p. 7050-7062, Vol. 191, No. 22
0021-9193/09/$08.00+0     doi:10.1128/JB.00861-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Motility and Flagellar Glycosylation in Clostridium difficile{triangledown} ,{dagger}

Susan M. Twine,1 Christopher W. Reid,1 Annie Aubry,1 David R. McMullin,1 Kelly M. Fulton,1 John Austin,2 and Susan M. Logan1*

Institute for Biological Sciences, National Research Council, Ottawa, ON K1A 0R6,1 Bureau of Microbial Hazards, HFPB, Health Canada, Sir Frederick G. Banting Research Centre, Ottawa, ON K1A 0K9, Canada2

Received 30 June 2009/ Accepted 7 September 2009

In this study, intact flagellin proteins were purified from strains of Clostridium difficile and analyzed using quadrupole time of flight and linear ion trap mass spectrometers. Top-down studies showed the flagellin proteins to have a mass greater than that predicted from the corresponding gene sequence. These top-down studies revealed marker ions characteristic of glycan modifications. Additionally, diversity in the observed masses of glycan modifications was seen between strains. Electron transfer dissociation mass spectrometry was used to demonstrate that the glycan was attached to the flagellin protein backbone in O linkage via a HexNAc residue in all strains examined. Bioinformatic analysis of C. difficile genomes revealed diversity with respect to glycan biosynthesis gene content within the flagellar biosynthesis locus, likely reflected by the observed flagellar glycan diversity. In C. difficile strain 630, insertional inactivation of a glycosyltransferase gene (CD0240) present in all sequenced genomes resulted in an inability to produce flagellar filaments at the cell surface and only minor amounts of unmodified flagellin protein.

* Corresponding author. Mailing address: Institute for Biological Sciences, National Research Council, 100 Sussex Drive, Ottawa, ON K1A 0R6, Canada. Phone: (613) 990-0839. Fax: (613) 952-9092. E-mail: susan.logan{at}nrc-cnrc.gc.ca

{triangledown} Published ahead of print on 11 September 2009.

{dagger} Supplemental material for this article may be found at http://jb.asm.org/.

Journal of Bacteriology, November 2009, p. 7050-7062, Vol. 191, No. 22
0021-9193/09/$08.00+0     doi:10.1128/JB.00861-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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