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Assembly of Cyclic Enterobacterial Common Antigen in Escherichia coli K-12

Department of Biochemistry and Molecular Biology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799

Received 6 May 2005/ Accepted 20 June 2005

We describe here the purification and quantification of a water-soluble cyclic form of enterobacterial common antigen (ECA_CYC) from Escherichia coli K-12 as well as information regarding its subcellular location and the genetic loci involved in its assembly. Structural characterization of purified ECA_CYC molecules obtained from E. coli K-12 revealed that they uniformly contained four trisaccharide repeat units, and they were substituted with from zero to four O-acetyl groups. Cells from overnight cultures contained approximately 2 µg ECA_CYC per milligram (dry weight), and cell fractionation studies revealed that these molecules were localized exclusively in the periplasm. The synthesis and assembly of ECA_CYC were found to require the wzxE and wzyE genes of the wec gene cluster. These genes encode proteins involved in the transmembrane translocation of undecaprenylpyrophosphate-linked ECA trisaccharide repeat units and the polymerization of trisaccharide repeat units, respectively. Surprisingly, synthesis of ECA_CYC was dependent on the wzzE gene, which is required for the modulation of the polysaccharide chain lengths of phosphoglyceride-linked ECA (ECA_PG). The presence of ECA_CYC in extracts of several other gram-negative enteric organisms was also demonstrated; however, it was not detected in cell extracts of Pseudomonas aeruginosa. These data suggest that in addition to ECA_PG, ECA_CYC may be synthesized in many, if not all, members of the Enterobacteriaceae.

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