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The O antigen is highly variable and typically consists of repeat units of 2-5
sugars in a polymer that can be more than 100 sugars long. The range of repeat
unit numbers is specific for a particular strain and is controlled by different strate-
gies, depending on which of the two biosynthetic pathways is used for O-antigen
biosynthesis: the Wzy-dependent pathway or the ABC transporter-dependent
pathway (
Figure 17.3
) (
Raetz and Whitfield, 2002
). In all cases, biosynthesis of
O antigens begins at the cytoplasmic face of the inner membrane using the lipid
carrier undecaprenyl phosphate. In
E. coli,
the enzyme WecA initiates O-PS
biosynthesis by transferring GlcNAc-1-phosphate from UDP-GlcNAc to unde-
caprenyl phosphate to form undecaprenyl pyrophosphoryl-GlcNAc. In some
cases, this is epimerized to undecaprenyl pyrophosphoryl-GalNAc (
Rush et al.,
2010
). The GlcNAc/GalNAc residues occur in each repeat unit in O-PSs formed
by the Wzy-dependent pathway, or just once at the reducing terminus in the
ABC transporter-dependent process.
In the Wzy-dependent pathway, individual repeat units are synthesized on
undecaprenyl pyrophosphoryl-GlcNAc/GalNAc using nucleoside phosphate
sugar donors by enzymes encoded in the locus. These repeat units are then
flipped to the periplasmic side of the inner membrane by the flippase Wzx
and polymerized into the complete O antigen by the polymerase Wzy. Wzy
transfers the growing glycan from one undecaprenyl pyrophosphate carrier to
the incoming lipid-linked repeat unit. Insight into this pathway has benefitted
from model systems, including the O7 and O86 antigens (
Table 17.1
), studied
in the laboratories of Miguel Valvano and George Peng Wang, respectively. In
the ABC transporter-dependent pathway, the O antigen is synthesized entirely
at the cytoplasmic face of the inner membrane through the sequential action of
glycosyltransferases that add sugars to undecaprenyl pyrophosphoryl-GlcNAc,
before the completed chain is exported via an ABC transporter. This process
is less common than the Wzy-dependent pathway and the resulting repeat-unit
structures tend to be less elaborate. The serotype O8/O9/O9a antigens provide
influential prototypes for this type of assembly process and many of the steps
have been resolved by the research group of Klaus Jann and the Whitfield lab
(
Table 17.1
). The
E. coli
WaaL ligase can operate effectively with nascent
undecaprenyl-linked O-PSs from either pathway.
The LPS species extracted from a bacterial cell shows heterogeneity best
illustrated by profiles separated by SDS-PAGE (
Hitchcock and Brown, 1983
).
The most obvious variations occur in the chain-lengths of O-PS, evident as a
ladder of high-molecular-weight molecules. The length of the O-PS has impor-
tant functional consequences (see below) and is established by different mecha-
nisms, depending on the assembly pathway. In the Wzy-dependent pathway,
the length of the O antigen is controlled by the protein Wzz, a transmembrane
protein with a large periplasmic domain that belongs to the polysaccharide co-
polymerase (PCP) family (reviewed in
Cuthbertson et al., 2009
;
Morona et al.,
2009
). In the ABC transporter-dependent pathway, chain length of some O-PS
molecules is controlled by the addition of novel residues (ones not found in
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