Structural and genetic characterization of the Escherichia coli O180 O antigen and identification of a UDP-GlcNAc 6-dehydrogenase.
The O antigen is an essential component of the lipopolysaccharides on the surface of Gram-negative bacteria and its variation provides a major basis for serotyping schemes. The Escherichia coli O-antigen form O180 was first designated in 2004, and O180 strains were found to contain virulence factors and cause diarrhea. Different O-antigen forms are almost entirely due to genetic variations in the O-antigen gene clusters. In this study, the chemical structure and gene cluster of E. coli O180 O antigen were investigated. A tetrasaccharide repeating unit with the following structure: →4)-β-D-ManpNAc3NAcA-(1 → 2)-α-L-Rhap(I)-(1 → 3)-β-L-Rhap(II)-(1 → 4)-α-D-GlcpNAc-(1→was identified in the E. coli O180 O antigen, including the residue D-ManpNAc3NAcA (2,3-diacetamido-2,3-dideoxy-D-mannopyranuronic acid) that had not been hitherto identified in E. coli. Genes in the O-antigen gene cluster were assigned functions based on their similarities with those from available databases, and five genes involved in the synthesis of UDP-D-ManpNAc3NAcA (the nucleotide-activated form of D-ManpNAc3NAcA) were identified. The gnaA gene, encoding the enzyme involved in the initial step of the UDP-D-ManpNAc3NAcA biosynthetic pathway, was cloned and the enzyme product was expressed, purified and assayed for its activity. GnaA was characterized using capillary electrophoresis and electrospray ionization mass spectrometry and identified as a UDP-GlcNAc 6-dehydrogenase. The kinetic and physicochemical parameters of GnaA also were determined.
TEDA School of Biological Sciences and Biotechnology, Nankai University, TEDA, People's Republic of China.
Quan
Q
Wang
Andrei V
AV
Perepelov
Lothar
L
Beutin
Sof'ya N
SN
Senchenkova
Yanli
Y
Xu
Alexander S
AS
Shashkov
Peng
P
Ding
Yuriy A
YA
Knirel
Lu
L
Feng
D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli / Q000737:chemistry*; D006836:Hydro-Lyases / Q000737:chemistry* / Q000378:metabolism*; D019081:O Antigens / Q000737:chemistry* / Q000235:genetics*; D014574:Uronic Acids / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D001426:Bacterial Proteins; D019081:O Antigens; C506191:UDP-2,3-diacetamido-2,3-dideoxy-D-mannuronic acid; D014574:Uronic Acids; D006836:Hydro-Lyases; C441972:wbpM protein, Pseudomonas aeruginosa