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Journal of Bacteriology, January 2006, p. 86-95, Vol. 188, No. 1
0021-9193/06/$08.00+0     doi:10.1128/JB.188.1.86-95.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Identification and Characterization of the Genes Involved in Glycosylation Pathways of Mycobacterial Glycopeptidolipid Biosynthesis

Yuji Miyamoto,1 Tetsu Mukai,1 Noboru Nakata,1 Yumi Maeda,1 Masanori Kai,1 Takashi Naka,2 Ikuya Yano,2 and Masahiko Makino1*

Department of Microbiology, Leprosy Research Center, National Institute of Infectious Diseases, 4-2-1 Aobacho, Higashimurayama, Tokyo 189-0002, Japan,1 Japan BCG Central Laboratory, 3-1-5 Matsuyama, Kiyose, Tokyo 204-0022, Japan2

Received 2 August 2005/ Accepted 10 October 2005

Glycopeptidolipids (GPLs) are major components present on the outer layers of the cell walls of several nontuberculous mycobacteria. GPLs are antigenic molecules and have variant oligosaccharides in mycobacteria such as Mycobacterium avium. In this study, we identified four genes (gtf1, gtf2, gtf3, and gtf4) in the genome of Mycobacterium smegmatis. These genes were independently inactivated by homologous recombination in M. smegmatis, and the structures of GPLs from each gene disruptant were analyzed. Thin-layer chromatography, gas chromatography-mass spectrometry, and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analyses revealed that the mutants {Delta}gtf1 and {Delta}gtf2 accumulated the fatty acyl-tetrapeptide core having O-methyl-rhamnose and 6-deoxy-talose as sugar residues, respectively. The mutant {Delta}gtf4 possessed the same GPLs as the wild type, whereas the mutant {Delta}gtf3 lacked two minor GPLs, consisting of 3-O-methyl-rhamnose attached to O-methyl-rhamnose of the fatty acyl-tetrapeptide core. These results indicate that the gtf1 and gtf2 genes are responsible for the early glycosylation steps of GPL biosynthesis and the gtf3 gene is involved in transferring a rhamnose residue not to 6-deoxy-talose but to an O-methyl-rhamnose residue. Moreover, a complementation experiment showed that M. avium gtfA and gtfB, which are deduced glycosyltransferase genes of GPL biosynthesis, restore complete GPL production in the mutants {Delta}gtf1 and {Delta}gtf2, respectively. Our findings propose that both M. smegmatis and M. avium have the common glycosylation pathway in the early steps of GPL biosynthesis but differ at the later stages.

* Corresponding author. Mailing address: Department of Microbiology, Leprosy Research Center, National Institute of Infectious Diseases, 4-2-1 Aobacho, Higashimurayama, Tokyo 189-0002, Japan. Phone: 81-42-391-8059. Fax: 81-42-391-8212. E-mail: mmaki{at}nih.go.jp.

Journal of Bacteriology, January 2006, p. 86-95, Vol. 188, No. 1
0021-9193/06/$08.00+0     doi:10.1128/JB.188.1.86-95.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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