Repeat unit polysaccharides of bacteria: a model for polymerization resembling that of ribosomes and fatty acid synthetase, with a novel mechanism for determining chain length

Abstract

We report the identification and sequence from Escherichia coli and Salmonella enterica strains of the cld gene, encoding the chain-length determinant (CLD) which confers a modal distribution of chain length on the O-antigen component of lipopolysaccharide (LPS). The distribution of chain lengths in the absence of this gene fits a model in which as the chain is extended there is a constant probability of 0.165 of transfer of growing chain to LPS core, with termination of chain extension. The data for E. coli O111 fit a model in which the CLD reduces this probability for short chains and increases it to 0.4 for longer chains, leading to a reduced number of short chain molecules but an increase in numbers of longer molecules and transfer of essentially all molecules by chain length 21. We put forward a model for O-antigen polymerase which resembles the ribosome and fatty acid synthetase in having two sites, with the growing chain being transferred from a D site onto the new unit at the R site to extend the chain and then back to the D site to repeat the process. It is proposed that the CLD protein and polymerase form a complex which has two states: 'E' facilitating extension and 'T' facilitating transfer to core. The complex is postulated to enter the E state as O-antigen polymerization starts, and to shift to the T state after a predetermined time, the CLD acting as a molecular clock. The CLD is not O-antigen or species-specific but the modal value does depend on the source of the cld gene.

Sequence-structure relationships in polysaccharide co-polymerase (PCP) proteins

“…During LPS Oag biosynthesis, Wzz proteins control Oag modal length distribution through two proposed mechanisms: as a molecular timer of Oag polymerization [9] or in an organizational manner to determine the crucial ratio of the polymerase Wzy to the Oag ligase WaaL [10]. Much of the work aimed at understanding the mechanism behind Wzz-mediated chain length regulation has come from mutagenesis and genetic studies, which revealed that chain modal length regulation is a property of the entire protein [10][11][12][13][14][15][16][17].…”

“…Much of the work aimed at understanding the mechanism behind Wzz-mediated chain length regulation has come from mutagenesis and genetic studies, which revealed that chain modal length regulation is a property of the entire protein [10][11][12][13][14][15][16][17]. In agreement with secondary structure predictions, genetic and biochemical analyses have shown that Wzz is largely a-helical in structure and is likely to function as an oligomer [9,12,18,19]. These approaches have been limited in giving insight into the molecular mechanism involved in polysaccharide chain modal length control.…”

Coiled-coil regions play a role in the function of the Shigella flexneri O-antigen chain length regulator WzzpHS2

“…They have two transmembrane (TM) domains flanking a periplasmic domain (Morona et al, 1995) and a conserved proline-rich region, PX 2 PX 4 SPKX 1 X 10 GGMXGAG, overlapping the second TM region (Becker & Pühler, 1998;Becker et al, 1995;Daniels & Morona, 1999;Morona et al, 2000b). The periplasmic domain of Wzz proteins is predicted to be mostly a-helical in structure (Bastin et al, 1993) and has significant coiled-coil (CC) potential (Morona et al, 2000b). The features described above are illustrated for Wzz pHS2 in Fig.…”

The Edwardsiella ictaluri O polysaccharide biosynthesis gene cluster and the role of O polysaccharide in resistance to normal catfish serum and catfish neutrophils

“…Ugd converts UDP-Glc into UDP-glucuronic acid. In the Enterobacteriaceae, ugd is often located within the OPS biosynthesis gene cluster adjacent to a gene encoding an OPS chain length determinant (wzz) (Amor & Whitfield, 1997;Bastin et al, 1993).…”