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Biochemical Society Transactions ( 1992) 21
Genetic and biosynthetic aspects of Shigella
flexneri 0-specific lipopolysaccharides
D. Alastair R. Simmons
Department of Bacteriology, University of
Glasgow, Glasgow, G11 6NT, UK.
The 0-specific lipopolysaccharides which
determine the type-specificity and crossreactivity of the different Shigella flexneri
serotypes consist of two distinct regions --a common basal region associated with rough
(R) specificities that can be isolated from
all Sh. flexneri and an 0-specific side-chain
region with a structure unique to each smooth
( S ) serotype [l]. The common basal structure
contains 2-keto-3-deoxy-octonate, L-glyceroD-manno-heptose phosphate, D-glucose, D-galactose and N-acetyl-D-glucosamine incorporated into the growing basal chain in that
order giving a structure identical with the
Escherichia coli R3 core [2]. The 0-specific
side-chains may also be subdivided into two
regions --- a primary unbranched side-chain
with 6 to 8 tetrasaccharide 'repeating-units'
of L-rhamnose and N-acetyl-glucosamine (3:l
respectively) and secondary side-chains of
a-D-glucose and/or Oracetyl residues bound
to the primary side-chain in a linkage that
is specific for each serotype [3]. The
biosynthesis of these lipopolysaccharides,
like that of their analogues from other
Enterobacteriaceae, proceeds in four
distinct stages mediated by specific
synthetases and transferases controlled by
structural and regulatory genes [4].
In the first stage (biosynthesis of basal
region), glycero-manno-heptose phosphate is
bound to the 2-keto-3-deoxy-octonate backbone
of the molecule. Mutation involving deletion
of the genes controlling heptose phosphate
synthetase or transferase produces an antigen
of rough (Re) specificity. Failure to bind
the second sugar, glucose, may be due to the
deletion of a gene controlling UDP-glucose
synthetase or transferase. Two such mutants,
one of each type, have been isolated [5]
both with identical rough (Rd) specificity
as both block the same stage of biosynthesis.
Five mutants of Rc specificity were isolated
which failed to incorporate the third sugar,
galactose, due to loss of the UDP-galactose4-epimerse required for galactose synthesis.
Two mutants of rough (Rb) specificity failed
to synthesise the basal structure for lack of
N-acetylglucosamine transferase but two rough
(Ra) specific mutants which could synthesise
the complete core were unable to synthesise
0-specific chains as they lacked a rhamnose
synthetase [5]. These studies indicate that
smooth-to-rough (S+R) mutation follows the
deletion of one of the genes involved in the
biosynthesis of the basal structure. The
present evidence is that the rfa genes that
control basal structure synthesis, map close
to the mtl locus on the bacterial chromosome
except the gene controlling UDP-galactose4-epimerase which maps near the lac locus.
In the second stage (biosynthesis of the
primary unbranched 0-specific side-chains),
the 'repeating-units' of rhamnose and Nacetylglucosamine are assembled under control
of the rfb genes that map near the his locus.
The 'repeating-units' are then transferred to
a lipid antigen carrier (ACL) and polymerised
under control of the rfc gene. The resulting
unbranched primary chain is structurally and
serologically identical with the Sh. flexneri
variant Y antigen [6'].
In the third stage of biosynthesis, the
secondary side-chains of glucose and/or
0-acetyl residues are incorporated as a postpolymerisation modification of the primary
chain by trans-glucosylases and/or trans-0acetylases under the control of regulating
temperate phages [3,4]. The attachment sites
of these phages which modify the specificity
of the various Sh. flexneri serotypes lie at
the T-locus which maps near the lac locus.
In the fourth stage of lipopolysaccharide
synthesis, the completed 0-specific chains
are attached to the basal structure by the
enzyme translocase under the control of two
genes --- rfaL which recognises the basal
structure and rfbT which recognises the
0-specific side-chain. The gene rfaL maps
with the other rfa genes near the mtl locus
while the gene rfbT maps with the other rfb
genes close to the his locus [7].
Changes in genetic control of the above
biosynthetic pathways explain the different
serotypes and variants of the Sh. flexneri.
The three types of genetic change involved in
these antigenic variations are mutation,
lysogenic conversion and recombination.
Mutations in rfa genes cause enzyme blocks
in the biosynthesis of the basal chain. These
interupt growth of that chain producing an
incomplete rough antigen of serotype Rb, Rc,
Rd or Re depending on the site of the block.
Such mutants usually synthesise 0-specific
side-chains but cannot attached them to the
incomplete basal structure as the terminal
acceptor site is missing. Mutations involving
the loss of rhamnose synthetase or one of the
0-specific side-chain transferases are also
found in stage 2 biosynthesis. These mutants
can synthesise the whole basal structure of
rough type Ra but not 0-specific side-chains.
More recently, it has been shown that the
specificity of the linkage of the a-glucosyl
and 0-acetyl secondary side-chains is phagedependant. The specific serotype can also be
altered by lysogenic conversion when bacteria
are infected by phage from another serotype.
Finally, the antigenic structure of Sh.
flexneri can be modified in hybridisation
experiments with Escherichia coli K12 [6]. In
such recombinants, the Sh. flexneri T-locus
is usually replaced by the analogous region
of E. coli K12 with the concomitant loss of
the phage attachment sites. The incorporation
of the secondary side-chains is therefore
blocked so that the recombinant antigens
display the variant Y specificity associated
with the unsubstituted primary side-chains of
the 0-specific region of the molecule.
1. Johnston, J.H., Johnston, R.J. & Simmons,
D.A.R. (1967) Bi0chem.J. 105, 79-87.
2. Simmons, D.A.R. (1983) Biochem. SOC.
Trans. 11, 104-105.
3. Simmons, D.A.R. (1969) Eur. J. Biochem.
11, 554-575.
4. Simmons, D.A.R. & Romanowska, E. (1987)
J. Med. Microbiol. 23, 289-302.
5. Johnston, J.H., Johnston, R.J. & Simmons,
D.A.R. (1968) Arch. Immunol. Ther. Exp.
16, 252-259.
6. Manson, R., Simmons, D.A.R. & Petrovskaya,
V.G. (1970) Eur. J. Biochem. 17, 472-476.
7. Petrovskaya, V.G. & Licheva, T.A. (1982)
Acta Microbiol. Acad. Sci. Hung. 29, 4153.