Controlled expression of branch-forming mannosyltransferase is critical for mycobacterial lipoarabinomannan biosynthesis.
Lipomannan (LM) and lipoarabinomannan (LAM) are phosphatidylinositol-anchored glycans present in the mycobacterial cell wall. In Mycobacterium smegmatis, the mannan core of LM/LAM constitutes a linear chain of 20-25 alpha1,6-mannoses elaborated by 8-9 alpha1,2-monomannose side branches. At least two alpha1,6-mannosyltransferases mediate the linear mannose chain elongation, and one branching alpha1,2-mannosyltransferase (encoded by MSMEG_4247) transfers monomannose branches. An MSMEG_4247 deletion mutant accumulates branchless LAM and interestingly fails to accumulate LM, suggesting an unexpected role of mannose branching for LM synthesis or maintenance. To understand the roles of MSMEG_4247-mediated branching more clearly, we analyzed the MSMEG_4247 deletion mutant in detail. Our study showed that the deletion mutant restored the synthesis of wild-type LM and LAM upon the expression of MSMEG_4247 at wild-type levels. In striking contrast, overexpression of MSMEG_4247 resulted in the accumulation of dwarfed LM/LAM, although monomannose branching was restored. The dwarfed LAM carried a mannan chain less than half the length of wild-type LAM and was elaborated by an arabinan that was about 4 times smaller. Induced overexpression of an elongating alpha1,6-mannosyltransferase competed with the overexpressed branching enzyme, alleviating the dwarfing effect of the branching enzyme. In wild-type cells, LM and LAM decreased in quantity in the stationary phase, and the expression levels of branching and elongating mannosyltransferases were reduced in concert, presumably to avoid producing abnormal LM/LAM. These data suggest that the coordinated expressions of branching and elongating mannosyltransferases are critical for mannan backbone elongation.
Sena
Chubert B C
CB
Research Institute for Microbial Diseases and WPI-Immunology Frontier Research Center, Osaka University, Osaka 565-0871, Japan.
Fukuda
Takeshi
T
Miyanagi
Kana
K
Matsumoto
Sohkichi
S
Kobayashi
Kazuo
K
Murakami
Yoshiko
Y
Maeda
Yusuke
Y
Kinoshita
Taroh
T
Morita
Yasu S
YS
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002473:Cell Wall / Q000201:enzymology* / Q000235:genetics; D017353:Gene Deletion; D008070:Lipopolysaccharides / Q000096:biosynthesis* / Q000235:genetics; D008358:Mannose / Q000378:metabolism*; D008364:Mannosyltransferases / Q000235:genetics / Q000378:metabolism*; D020102:Mycobacterium smegmatis / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D008070:Lipopolysaccharides; C050016:lipoarabinomannan; D008364:Mannosyltransferases; D008358:Mannose