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PMID: 7536735 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Expression of the O9 polysaccharide of Escherichia coli: sequencing of the E. coli O9 rfb gene cluster, characterization of mannosyl transferases, and evidence for an ATP-binding cassette transport system.

Journal of bacteriology ·Vol. 177 ·No. 8 ·1995-04-00 ·Pages 2178-87

Kido N, Torgov VI, Sugiyama T, Uchiya K, Sugihara H, Komatsu T, Kato N, Jann K

Abstract

The rfb gene cluster of Escherichia coli O9 directs the synthesis of the O9-specific polysaccharide which has the structure -->2-alpha-Man-(1-->2)-alpha-Man-(1-->2)-alpha-Man-(1-->3)-alpha- Man-(1-->. The E. coli O9 rfb cluster has been sequenced, and six genes, in addition to the previously described rfbK and rfbM, were identified. They correspond to six open reading frames (ORFs) encoding polypeptides of 261, 431, 708, 815, 381, and 274 amino acids. They are all transcribed in the counter direction to those of the his operon. No gene was found between rfb and his. A higher G+C content indicated that E. coli O9 rfb evolved independently of the rfb clusters from other E. coli strains and from Shigella and Salmonella spp. Deletion mutagenesis, in combination with analysis of the in vitro synthesis of the O9 mannan in membranes isolated from the mutants, showed that three genes (termed mtfA, -B, and -C, encoding polypeptides of 815, 381, and 274 amino acids, respectively) directed alpha-mannosyl transferases. MtfC (from ORF274), the first mannosyl transferase, transfers a mannose to the endogenous acceptor. It critically depended on a functional rfe gene (which directs the synthesis of the endogenous acceptor) and initiates the growth of the polysaccharide chain. MtfB (from ORF381) then transfers two mannoses into the 3 position of the previous mannose, and MtfA (from ORF815) transfers three mannoses into the 2 position. Further chain growth needs only the two transferases MtfA and MtfB. Thus, there are fewer transferases needed than the number of sugars in the repeating unit. Analysis of the predicted amino acid sequence of the ORF261 and ORF431 proteins indicated that they function as components of an ATP-binding cassette transport system. A possible correlation between the mechanism of polymerization and mode of membrane translocation of the products is discussed.

Related Genes
rfb
MeSH Terms
ATP-Binding Cassette Transporters/genetics,metabolism Carbohydrate Sequence Chromosome Mapping DNA, Bacterial/genetics Escherichia coli/genetics,metabolism Escherichia coli Proteins Genes, Bacterial Mannosyltransferases/genetics,metabolism Molecular Sequence Data Multigene Family O Antigens Open Reading Frames Polysaccharides, Bacterial/biosynthesis,chemistry,genetics Substrate Specificity Transferases (Other Substituted Phosphate Groups)/genetics,metabolism
Chemicals
ATP-Binding Cassette Transporters DNA, Bacterial Escherichia coli Proteins O Antigens Polysaccharides, Bacterial Mannosyltransferases Transferases (Other Substituted Phosphate Groups) wecA protein, E coli
Authors & Affiliations
8 authors, click to expand affiliations / ORCID
Kido N
Max-Planck-Institute für Immunobiologie, Freiburg, Germany.
Torgov V I
Sugiyama T
Uchiya K
Sugihara H
Komatsu T
Kato N
Jann K
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Article Info
Journal
Journal of bacteriology
Abbr.
J Bacteriol
ISSN
0021-9193
DOI
10.1128/jb.177.8.2178-2187.1995
Published
1995-04-00
Pages
2178-87
Language
English
Region
United States
NLM ID
2985120R
PMCID
PMC176863
Subset
IM
Databases
GENBANK
D43637
External Links
PubMed source DOI: 10.1128/jb.177.8.2178-2187.1995 PMC Full Text View journal details
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