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Journal of Bacteriology, May 2000, p. 2567-2573, Vol. 182, No. 9
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

A Single Amino Acid Substitution in a Mannosyltransferase, WbdA, Converts the Escherichia coli O9 Polysaccharide into O9a: Generation of a New O-Serotype Group

Nobuo Kido1,* and Hidemitsu Kobayashi2

Unit of Biosystems, School of Informatics and Sciences, Nagoya University, Nagoya, Aichi 464-8601,1 and Department of Food Hygienic Chemistry, Faculty of Home Economics, Kyushu Women's University, Kitakyushu City, Fukuoka 807-8586,2 Japan

Received 13 September 1999/Accepted 14 February 2000

wbdA is a mannosyltransferase gene that is involved in synthesis of the Escherichia coli O9a polysaccharide, a mannose homopolymer with a repeating unit of 2-alpha Man-1,2-alpha Man-1,3-alpha Man-1,3-alpha Man-1. The equivalent structural O polysaccharide in the E. coli O9 and Klebsiella O3 strains is 2-alpha Man-1,2-alpha Man-1,2-alpha Man-1,3-alpha Man-1,3-alpha Man-1, with an excess of one mannose in the 1,2 linkage. We have cloned wbdA genes from these O9 and O3 strains and shown by genetic and functional studies that wbdA is the only gene determining the O-polysaccharide structure of O9 or O9a. Based on functional analysis of chimeric genes and site-directed mutagenesis, we showed that a single amino acid substitution, C55R, in WbdA of E. coli O9 converts the O9 polysaccharide into O9a. DNA sequencing revealed the substitution to be conserved in other E. coli O9a strains. The reverse substitution, R55C, in WbdA of E. coli O9a resulted in lipopolysaccharide synthesis showing no ladder profile instead of the conversion of O9a to O9. This suggests that more than one amino acid substitution in WbdA is required for conversion from O9a to O9.

* Corresponding author. Mailing address: Unit of Biosystems, School of Informatics and Sciences, Nagoya University, Nagoya, Aichi 464-8601, Japan. Phone: 81 52 7894816. Fax: 81 52 7894818. E-mail: j45811a{at}nucc.cc.nagoya-u.ac.jp.

Journal of Bacteriology, May 2000, p. 2567-2573, Vol. 182, No. 9
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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