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

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 g 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.

Pivotal Roles of the Outer Membrane Polysaccharide Export and Polysaccharide Copolymerase Protein Families in Export of Extracellular Polysaccharides in Gram-Negative Bacteria

“…ECA is found in many genera of the Enterobacteriaceae (53). Wzz E participates in the biosynthesis of both ECA PG (4), a phosphoglyceride-linked form, and ECA CYC (45), an unusual cyclic molecule containing four to six ECA repeat units. In the absence of any detailed characterization of the PCP proteins associated with group E OPX proteins, we have retained the PCP-1 designation.…”

O Acetylation of the Enterobacterial Common Antigen Polysaccharide Is Catalyzed by the Product of the yiaH Gene of Escherichia coli K-12

“…A water-soluble cyclic form of ECA (ECA CYC ) has also been demonstrated to be present in many gram-negative enteric bacteria (7,14,28,48), and the available data suggest that ECA CYC may indeed occur in all members of the Enterobacteriaceae (19). Recent studies have demonstrated that ECA CYC is located exclusively in the periplasm of Escherichia coli K-12, and cells from cultures grown overnight were found to contain approximately 2 g of ECA CYC (dry weight) per milligram (19).…”

“…Recent studies have demonstrated that ECA CYC is located exclusively in the periplasm of Escherichia coli K-12, and cells from cultures grown overnight were found to contain approximately 2 g of ECA CYC (dry weight) per milligram (19). Structural characterization of the ECA CYC molecules isolated from E. coli K-12 revealed that they uniformly consist of four trisaccharide repeat units, and each molecule of ECA CYC contains from zero to four O-acetyl groups (15,19). Similar to ECA PG , the O-acetyl groups of ECA CYC are also linked to the 6 position of GlcNAc residues (14).…”

Enterobacterial common antigen and O-specific polysaccharide coexist in the lipopolysaccharide of Yersinia enterocolitica serotype O : 3

“…It was particularly dramatic in wzzE and the downstream wecB-wecA genes. These genes encode for ECA chain length regulatory protein and for enzymes required for the biosynthesis of ECA glycosyl precursors, respectively (Barr et al, 1999;Kajimura et al, 2005). The immunoblotting analyses of the present work suggested coexistence of OPS and ECA in the Y. enterocolitica O : 3 LPS.…”

“…This polymer can either covalently anchor to the outer membrane via its own L-glycerophospholipid (ECA PG ) or utilize in some bacteria the core oligosaccharide as an attachment point (ECA LPS ). The third form of ECA, found in the periplasmic space, is a cyclic form and has no lipid anchor (ECA CYC ) (Kajimura et al, 2005;Kuhn et al, 1988;Lugowski et al, 1983). Of these three ECA types, only ECA LPS is naturally immunogenic and it has been detected from Escherichia coli, Proteus mirabilis and Yersinia enterocolitica Kasperkiewicz et al, 2004;Kuhn et al, 1988;Rabsztyn et al, 2011;Radziejewska-Lebrecht et al, 2003).…”