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Journal of Bacteriology, August 2008, p. 5248-5255, Vol. 190, No. 15
0021-9193/08/$08.00+0     doi:10.1128/JB.00028-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Transfer of the First Arabinofuranose Residue to Galactan Is Essential for Mycobacterium smegmatis Viability{triangledown}

Libin Shi,1 Roukun Zhou,2 Zhentong Liu,1 Todd L. Lowary,2 Peter H. Seeberger,3 Bridget L. Stocker,3 Dean C. Crick,1 Kay-Hooi Khoo,4 and Delphi Chatterjee1*

Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado 80523-1682,1 Alberta Ingenuity Centre for Carbohydrate Science and Department of Chemistry, The University of Alberta, Gunning-Lemieux Chemistry Centre, Edmonton, Alberta, T6G 2G2 Canada,2 Laboratorium für Organische Chemie, Swiss Federal Institute of Technology, Wolfgang-Pauli-Strasse 10, CH-8093 Zürich, Switzerland,3 Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan4

Received 7 January 2008/ Accepted 28 May 2008

The mycobacterial arabinan is an elaborate component of the cell wall with multiple glycosyl linkages and no repeating units. In Mycobacterium spp., the Emb proteins (EmbA, EmbB, and EmbC) have been identified as putative mycobacterial arabinosyltransferases implicated in the biogenesis of the cell wall arabinan. Furthermore, it is now evident that the EmbA and EmbB proteins are involved in the assembly of the nonreducing terminal motif of arabinogalactan and EmbC is involved in transferring arabinose, perhaps in the early stage of arabinan synthesis in lipoarabinomannan. It has also been shown that the Emb proteins are a target of the antimycobacterial drug ethambutol (EMB). In the search for additional mycobacterial arabinosyltransferases in addition to the Emb proteins, we disrupted MSMEG_6386 (an orthologue of Rv3792 and a gene upstream of embC) in Mycobacterium smegmatis. Allelic exchange at the chromosomal MSMEG_6386 locus of M. smegmatis could only be achieved in the presence of a rescue plasmid carrying a functional copy of MSMEG_6386 or Rv3792, strongly suggesting that MSMEG_6386 is essential. An in vitro arabinosyltransferase assay using a membrane preparation from M. smegmatis expressing Rv3792 and synthetic β-D-Galf-(1->5)-β-D-Galf-(1->6)-β-D-Galf-octyl and β-D-Galf-(1->6)-β-D-Galf-(1->5)-β-D-Galf-octyl showed that Rv3792 gene product can transfer an arabinose residue to the C-5 position of the internal 6-linked galactose. The reactions were insensitive to EMB, and when {alpha}-D-Manp-(1->6)-{alpha}-D-Manp-(1->6)-{alpha}-D-Manp-octylthiomethyl was used as an acceptor, no product was formed. These observations indicate that transfer of the first arabinofuranose residue to galactan is essential for M. smegmatis viability.

* Corresponding author. Mailing address: Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523-1682. Phone: (970) 491-7495. Fax: (970) 491-1815. E-mail:delphi{at}lamar.colostate.edu

{triangledown} Published ahead of print on 13 June 2008.

Journal of Bacteriology, August 2008, p. 5248-5255, Vol. 190, No. 15
0021-9193/08/$08.00+0     doi:10.1128/JB.00028-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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