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

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Abstract
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 .