Department of Microbiology, University of
Sydney, Sydney, New South Wales 2006, Australia
The O antigen is a polymer with a repeated unit. The chain length
in most Escherichia coli strains has a modal value of 10 to
18 O units, but other strains have higher or lower modal values. wzz (cld/rol) mutants have a random chain
length distribution, showing that the modal distribution is determined
by the Wzz protein. Cloned wzz genes from E. coli strains with short (7 to 16), intermediate (10 to 18), and
long (16 to 25) modal chain lengths were transferred to a model system,
and their effects on O111 antigen were studied. The O111 chain length
closely resembled that of the parent strains. We present data based on
the construction of chimeric wzz genes and
site-directed mutagenesis of the wzz gene to show that the modal value of O-antigen chain length of E. coli O1, O2,
O7, and O157 strains can be changed by specific amino acid
substitutions in wzz. It is concluded that
the O-antigen chain length heterogeneity in E. coli strains
is the result of amino acid sequence variation of the Wzz protein.
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INTRODUCTION |
Lipopolysaccharide (LPS), also known
as endotoxin, is a major component of the outer membrane of
gram-negative bacteria. It plays a very important role as a
permeability barrier and protects bacteria from phagocytosis,
detergents, and free fatty acids present in the mammalian colon
(7, 23). LPS consists of three different regions: lipid A,
core oligosaccharide, and O antigen. Lipid A and core oligosaccharide
are synthesized together, while the O antigen is synthesized
independently. The mechanisms for synthesis and polymerization of O
antigens are relatively well known and have been reviewed many times
(26, 38).
The O antigen consists of an oligosaccharide repeat unit of about two
to six sugars, polymerized to give O antigen. The number of O units per
molecule has a characteristic modal distribution which has been shown
in several systems to be determined by the chain length determinant Wzz
(previously Cld or Rol) protein, as wzz mutants have a
random chain length distribution (3, 6, 12, 22). The
wzz gene is located on the chromosome between the
gnd and his genes. The predicted secondary
structure of the Wzz protein has a large, hydrophilic loop with
potential transmembrane helices at each end (3), and Morona
et al. (22) demonstrated that the loop is in the periplasmic
space. The precise mode of action of the Wzz protein is unknown, but it
has been proposed to interact with O-antigen polymerase (Wzy,
previously Rfc) in the periplasm (3) to control Wzy-mediated
polymerization. It should be noted that while most O antigens are
thought to be polymerized by Wzy with chain length determined by Wzz,
there are a few O antigens with other modes of polymerization
(39).
The O-antigen chain length of LPS for a strain is usually determined by
observation of silver-stained gels of LPS, when a ladder of bands is
apparent, each being of LPS with a specific number of O units. When
LPSs with different O antigens are compared, one often finds that the
spacing between bands varies. This is because the size of the O units
varies, which affects the mass difference between LPSs in adjacent
bands. Most of the LPS is in molecules with chain lengths around the
modal value, but there is often a significant amount of short-chain
(one to three O units) LPS. However, there is usually sufficient
material, in at least some gels, in all bands up to the main group of
bands to ascertain by counting bands the number of O units in the LPS
of each band.
The LPS of most Escherichia coli strains has a basic
O-antigen chain length of 10 to 18 O units (9, 19, 24).
However, there is considerable variation, and in some cases, chain
length varies among strains with the same O antigen (1, 10,
14). The LPSs of E. coli can, for convenience, be
subdivided into three groups having short (7 to 16), intermediate (10 to 18), and long (16 to 25) O-antigen chains. Bastin et al.
(3) have shown that wzz genes from different
strains can confer different chain lengths on O111 antigen in a model
system using E. coli K-12 carrying O111 genes. Use of the
one O antigen for the three modes means that all tracks have the same
band spacing. In this study, we used this model system to study the
wzz genes from O1, O2, O7, and O157 strains, and also
modification of chain length after site-directed mutagenesis of
wzz, to show that substitution of one amino acid in the Wzz
protein can change its modal-value specificity.
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MATERIALS AND METHODS |
Bacterial strains, plasmids, and antisera.
The bacterial
strains and plasmids used in this study are described in Tables
1 and 2.
Bacteria were grown in nutrient broth (10 g of peptone [Amyl Media
Pty., Dandenong, Victoria, Australia]), 5 g of yeast extract
[Amyl Media], and 5 g of NaCl per liter of water adjusted to pH
7.2). This broth was supplemented with ampicillin at a final
concentration of 25 µg ml
1 and kanamycin at a
concentration of 50 µg ml1 when necessary.
E. coli O antisera were used to confirm the serotype of each
strain. Antisera against the O157 antigen was from Denka Seiken Co.
Ltd., Tokyo, Japan. Antisera against the O1, O2, and O7 antigens were
from the Institute of Medical and Veterinary Science, Adelaide, South
Australia, Australia.
LPS analysis.
A modified version of the proteinase K
extraction method (15) was used for LPS preparation.
Cultures (10 ml) were incubated overnight at 37°C with shaking. These
were centrifuged at approximately 10,000 × g for 5 min, and the pellet was washed once with 10 mM Tris (pH 8) and
resuspended in 10 mM Tris (pH 8) to an extinction at 625 nm of 0.6 to
0.7. Subsequently, 1.5 ml of the cell suspension was centrifuged for 5 min in an Eppendorf tube. The pellet was drained, resuspended in 40 µl of electrophoresis buffer (20), boiled for 10 min,
digested with 100 µg of proteinase K (Boehringer Mannheim) at 63 to
65°C for 2 h, and subsequently boiled for 5 min to denature any
traces of proteinase K. The samples were loaded on a sodium dodecyl
sulfate (SDS)-12.5% polyacrylamide gel electrophoresis (PAGE) gel as
described by Lugtenberg et al. (20) and run for 3 h at
50 mA. Silver staining was performed as described by Fomsgaard et al.
(11), except that the developer consisted of 0.28 M sodium carbonate with 0.05% formaldehyde as described by Aucken and Pitt (2).
DNA methods.
Restriction endonucleases and ligase were
obtained from Boehringer Mannheim Biochemicals. Taq
polymerase was obtained from Pharmacia Biotech. The PCR was carried out
as described by Saiki et al. (30), by using a DNA thermal
cycler (Perkin-Elmer Cetus, Norwalk, Conn.). Oligonucleotide primers
were based on the wzz gene sequence of E. coli
HU1124 (5) (GenBank accession no. M89934) and synthesized by
Beckman (Gladesville, New South Wales, Australia) or Auspep (Parkville,
Victoria, Australia). The wzz genes from E. coli
O1, O2, O7, and O157 strains were amplified by primers 465 and 466 (Fig. 1), and PCR products were purified by using the Wizard PCR purification system (Promega), digested with
EcoRI and BamHI, and subsequently cloned into
homologous sites of pUC18. The sequence of bases 16 to 963 of the
978-bp wzz gene was determined for the first nine strains
listed in Table 1, as well as for all of the mutants described in this
paper by using the dye-labelled primer technique and a Perkin-Elmer Catalyst 800 and an automated 377 DNA sequencer (Applied Biosystems) at
the Sydney University and Prince Alfred Macromolecular Analysis Centre,
Sydney, New South Wales, Australia.
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FIG. 1.
Primers used for wzz gene amplification and
site-directed mutagenesis. Primers 466 and 465 were based on the
wzz gene sequence of HU1124 (5), with the
addition of EcoRI and BamHI sites at the 5' end,
respectively. All of the other primers were based on the wzz
gene of E4991/76. Primer 854 contains a PstI site and was
used to create a chimeric wzz gene from C258-94 and
E4991/76. The primers used for site-directed mutagenesis were 704, in
which a glycine residue is inserted between amino acid residues
220 and 221; 705, in which isoleucine replaces valine at residue 224;
881, in which isoleucine replaces methionine at residue 77; 882, in
which serine replaces glutamine at residue 83; 883, in which glutamate
replaces aspartate at residue 90; 945, in which glutamate and
isoleucine replace aspartate and leucine at residues 90 and 91; and
988, in which isoleucine replaces leucine at residue 91. The base
substitutions are in boldface, and the PstI site in primer
854 is indicated by boldface and italics.
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For the construction of wzz chimeric genes, DNA was
subjected to restriction enzyme analysis, run on a 1.5%
low-melting-point agarose gel (31), and recovered by using
the Bandpure (Progen Industries Limited) protocol with the following
modifications. The resin-bound DNA was decanted into a Bresaspin column
unit (Bresatec Pty. Ltd., Adelaide, South Australia, Australia) and centrifuged at 10,000 × g for 30 s in a
000-MICR-190 microcentrifuge tube (Elkay, Shrewsbury, Mass.) at room
temperature. The column was subsequently washed three times by
centrifugation with 250 µl of the Bandpure ethanol wash solution.
After the third wash, the column was centrifuged again for 3 min to
remove any residue of the ethanol wash solution and the column was then
transferred into another tube. The silica resin was resuspended three
times with the same 60-µl aliquot of 60°C deionized water and
incubated at 60°C for 5 min in a clean microcentrifuge tube. The
column was centrifuged for 30 s at room temperature. An aliquot (8 to 10 µl) of the eluate was run on another gel to check the
percentage recovered. The construction of chimeric wzz genes
in plasmids pPR1752 and pPR1798 is described in Fig.
2.
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FIG. 2.
Construction of chimeric wzz genes. The
chimeric wzz gene in pPR1752 was constructed by replacing
the 210-bp BglII-EcoRV fragment of the
wzz gene of E4991/76 in pPR1753 with that of F186. The
chimeric wzz gene in pPR1798 was made in two steps: a 424-bp
EcoRI-PstI fragment, which was amplified from the
wzz gene of C258-94 by using primers 466 and 854 and
digested with restriction enzymes, was cloned into pUC18; subsequently,
a 554-bp PstI fragment of the wzz gene of
E4991/76 was cloned into the PstI site of the resulting
plasmid in the right orientation for expression.
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Plasmid pPR1753 carrying the wzz gene of E4991/76 was
subjected to mutagenesis with a U.S.E site-directed mutagenesis kit obtained from Pharmacia Biotech. The conditions and buffers used were
those recommended by the manufacturers; the primers used are listed in
Fig. 1.
Phylogenetic analysis.
DNA and deduced amino acid sequences
were derived and analyzed by using the Australian National Genomic
Information Service (ANGIS) at Sydney University (29). The
MULTICOMP program (28) was used for comparison and alignment
of sequences. A phylogenetic tree was constructed by using the
neighbor-joining method.
Nucleotide sequence accession numbers.
The GenBank
accession numbers for the wzz gene sequences are AF011910
for E4991/76, AF011911 for F186, AF011912 for M70/1-1, AF011913 for
79/311, AF011914 for Bi7509-41, AF011915 for C664-1992, AF011916 for
C258-94, AF011917 for C722-89, and AF011919 for EDL933. The GenBank
accession numbers for the G7 and Bi316-41 wzz genes
sequences are U39305 and U39306, respectively.
| |
RESULTS |
Cloning of wzz genes and effects of Wzz on chain length
specificity.
Strain P4657 (Table 1) is a K-12 derivative which
lacks the chromosomal O-antigen genes but carries those of
E. coli O111 on a plasmid (3). It does not
carry a wzz gene and so has a nonmodal distribution of
O-antigen chain lengths (Fig. 3, track 8). This strain can be used to determine the effects of different wzz genes on chain length distribution in a common genetic
background.
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FIG. 3.
Analysis of short and intermediate chain length Wzz
proteins. LPS was prepared by proteinase K digestion and analyzed by
SDS-12.5% PAGE and silver staining. Tracks 1 to 8 and 9 to 14 are
from two different gels. The strain used for each track is indicated
above the track.
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In an attempt to understand the genetic basis of O-antigen chain length
heterogeneity, we transferred cloned wzz genes from E. coli O1, O2, O7, and O157 strains into strain P4657. In Fig. 3, we
show short and intermediate chain length LPSs from both the parent
strain and clones in the O111 model system. Tracks 1 and 2 contain LPSs
from intermediate- and short-mode O2 strains, respectively. Track 3 contains the LPS of a short-mode O1 strain which has the same modal
chain length as that in track 2 but different mobility due to the
O-antigen difference. Tracks 4 to 6 show the LPSs of the model host
strain carrying wzz genes from the same three strains.
Tracks 9 and 11 show the LPSs of intermediate- and short-mode O7
strains, respectively, and tracks 10 and 12 show the LPSs of the model
host strain with wzz genes from the same two strains. The
LPSs of two O157 long-mode strains are shown in tracks 1 and 3 of Fig.
4, and the LPSs of the model host strain carrying their wzz genes are shown in tracks 2 and 4, respectively. We also cloned the wzz gene of
intermediate-mode O157:H7 strain C664-1992 to give pPR1756, which gave
results comparable to those of other intermediate-mode strains (data
not shown). In summary, when the wzz genes from O1, O2, O7,
and O157 strains were expressed in our model system, the length of the
O111 chain, in general, corresponded well to the chain lengths of the
donor strains (Fig. 3 and 4).
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FIG. 4.
Analysis of long and intermediate chain length Wzz
proteins. LPS was prepared by proteinase K digestion and analyzed by
SDS-12.5% PAGE and silver staining. Tracks 1 to 4 and 5 to 12 are
from two different gels. The strain used for each track is indicated
above the track.
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Sequence alignment and amino acid variation in Wzz proteins.
We sequenced the cloned wzz genes. Alignment of the deduced
amino acid sequences reveals that they are highly conserved but there
are minor variations which must be involved in the functional specificity of the protein (Fig. 5). The
most consistent amino acid variations between intermediate- and
short-mode Wzz proteins are the presence of glycine at position 221 and
the change from valine to isoleucine at position 224 in the C'-terminal
half of the short-mode proteins (Fig. 5). In the long-mode Wzz
proteins, there is substantial difference from intermediate-mode
proteins in the N'-terminal half (Fig. 5).
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FIG. 5.
Comparison of amino acid sequences of wild-type and
mutant Wzz proteins. Only residues which vary are shown, the consensus
residues are shown at top, and for specific proteins only the amino
acids which vary from the consensus are shown. Restriction enzyme names
with arrows indicate the sites used for wzz chimeric gene
construction. The numbers above the consensus residues indicate the
positions of the amino acid residues. S, short chain; I, intermediate
length chain; I-L, intermediate-to-long chain; L, long chain. Boldface
letters represent the amino acids changed in pPR1753 (E4991/76)
derivatives. The E. coli HU1124 wzz gene sequence
is that of Batchelor et al. (5) (GenBank accession no.
Z17241). The E. coli Flexneri 2a wzz gene
sequence (Sfl1) is that of Morona et al. (22) (GenBank
accession no. X71970). The wzz genes of E. coli
G7 and Bi316-42 confer a long-chain modal distribution (data not
shown). The E. coli Dysenteriae (W30864) and K-12
wzz gene sequences are from Klee et al. (18)
(GenBank accession no. Y07560 and Y07559, respectively).
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The new wzz sequences were combined with other E. coli sequences to construct a tree (Fig.
6), which shows that the wzz
genes for the intermediate and long modes form two major clusters and the short-mode wzz genes form a third cluster derived from
the intermediate-mode genes. The M92 (O111) wzz gene was
used as the outgroup because of its relatively low sequence similarity
with the other E. coli wzz genes (5, 12).
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FIG. 6.
Phylogenetic tree generated by the neighbor-joining
method on the basis of wzz gene DNA sequences. The
wzz genes are represented by the strain names. The
wzz genes of E. coli G7 and Bi316-42 confer a
long-chain modal distribution (data not shown). The E. coli
M92 wzz gene sequence was used as the outgroup
(3). Abbreviations: I, intermediate-length chain mode; S,
short-chain mode; L, long-chain mode.
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Analysis of the genetic basis of short-mode chain length
distribution.
There are a few substitutions in the region around
residue 220 which distinguish short- and intermediate-mode Wzz proteins (see above). We therefore made a chimeric wzz gene with the
BglII-EcoRV segment of short-mode strain F186
replacing that of intermediate-mode strain E4991/76 (Fig. 2). The
resulting gene in pPR1752 conferred the short-mode O-antigen chain
length distribution of 7 to 16 O units (Fig. 3, track 7), compared with
10 to 18 O units (Fig. 3, track 4) for the E4991/76 gene in pPR1753.
The additional glycine at position 221 and the isoleucine at position
224 were present only in short-mode Wzz proteins (Fig. 5). We used
site-directed mutagenesis of pPR1753 to make clones containing the
additional glycine residue or the V224I substitution, and
complementation studies were performed with P4657. Plasmid pPR1776
(V224I) conferred a chain length of the lower value of 7 to 16 O units
(Fig. 3, track 14), whereas the presence of the additional glycine
residue alone in pPR1775 (Fig. 3, track 13) did not cause any shift in O-antigen chain length from that conferred by pPR1753 (Fig. 3, track
4). Note that pPR1793 (Fig. 3, track 10) confers the same pattern as
pPR1753 when run on the same gel (data not shown) and can replace
pPR1753 for comparison with pPR1775 and pPR1776. These results suggest
that the isoleucine residue alone is sufficient to cause the shift to
short-mode chain length.
Analysis of the genetic basis of long-mode chain length
distribution.
The long-mode Wzz proteins differ
substantially from those of intermediate-mode strains in the
amino-terminal half (Fig. 5). The C258-89 and E4991/76 genes differ at
103 sites in the 138-to-573 segment, of which 74 are synonymous
substitutions. The flanking segments 1 to 137 and 574 to 978 contain only four and seven substitutions, respectively, of which four
and six, respectively, are synonymous.
As for the analysis of short-mode chain length distribution, we started
by making a chimeric gene. An EcoRI-PstI 424-bp
C258-94 fragment encoding the first 140 amino acids of Wzz was cloned into pUC18, followed by insertion of an E4991/76 554-bp PstI
fragment encoding the remaining 186 amino acids, to give pPR1798 (Fig. 2). The chimeric wzz gene confers a typical long-mode chain
length (Fig. 4, track 7), indicating that the substitutions needed to confer a long O-antigen chain length are between residues 1 and 140. LPS from the model system with the intermediate-mode gene from EDL933
is shown for comparison. Note that it gives essentially the same LPS
profile as the parent wzz gene from E4991/76.
Salmonella enterica strains generally have a long-mode chain
length distribution: Wzz of S. enterica LT2 has, in the
first 140 residues, only residues 77, 83, 90, and 91 in common with the
long-mode E. coli strains. These residues seem most likely to be involved in determination of the long mode, and point mutations were made in pPR1753 to give substitutions M77I in pPR1801, Q83S in
pPR1802, D90E in pPR1803, and L91I in pPR1815. The first two failed to
change the O-antigen chain length (Fig. 4, tracks 8 and 9), but the
D90E mutation in pPR1803 shifted the chain length up to approximately
13 to 21 O units (Fig. 4, track 10), midway between those of the long-
and intermediate-mode parents. On the other hand, the L91I mutation
shifted the chain length down to 7 to 16 O units, typical of a
short-mode strain (Fig. 4, track 11). These results indicate that
glutamate 90 plays an important role in determination of long-mode
chain length but that the long-mode chain length of 16 to 25 O units
can only be restored if one or more additional amino acid substitutions
are present. We then made the D90E-L91I double mutation in pPR1804,
which gave an O-antigen chain length of 7 to 16 O units, similar to
that of a wild-type short-mode strain (Fig. 4, track 12). It appears
that the effect of isoleucine 91 in the double mutation is dominant
over that of glutamate 90.
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DISCUSSION |
A single amino acid substitution can change the chain length from
intermediate to short.
In the present study, we looked at the
genetic basis of O-antigen chain length variation in E. coli. Most strains have an intermediate chain length of 10 to 18 units, but others have short- or long-mode chain length distributions.
The wzz sequences covered 948 bp, comprising all but the
initial and final 15 bp. However, clones in which the 948-bp segments
from long- or short-mode strains replace that segment of an
intermediate-mode strain confer long- or short-mode chain length,
respectively, indicating that this region is sufficient to determine
the chain length mode and justifies our use of this segment for
analysis. We made chimeric genes and used site-directed mutagenesis to
show that substitution of one or two amino acids in these regions of
wzz could determine the difference between modal values. A
single amino acid change (V224I) was sufficient to change the chain
length of the wzz gene of E4991/76 from the typical
intermediate mode to the typical short-mode distribution. An
intermediate-mode wzz gene was converted to a long-mode gene by replacement of 140 amino acids at the 5' end, but in this case, an
individual amino acid substitution (D90E) conferred only a partial
shift toward the long mode.
Wide distribution of amino acids affecting chain length.
Our
first experiments suggested that a specific residue was
responsible for each modal value, but subsequent experiments
showed the situation to be more complex.
Also, while we were completing this report, Klee et al. (18)
showed that the wzz genes of E. coli K-12 and
Dysenteriae type 1 confer what we call intermediate-mode distribution
while E. coli Flexneri 2a (22) confers a
short-mode distribution (shorter than usual at about 5 to 13 O units).
We use the terms E. coli Dysenteriae and Flexneri instead of
Shigella dysenteriae and S. flexneri because
it is clear that E. coli and all Shigella spp. are sufficiently similar to be placed in the same species (8, 25). Klee et al. observed that only lysine 267 and phenylalanine 270 are present in strain Flexneri but not in either of K-12 or Dysenteriae and, on this basis, suggested that this region may be
responsible for the different biological activities. However, E. coli Flexneri differs at six other residues from either
Dysenteriae or K-12 and our data show that lysine 267 and phenylalanine
270 are not present in other intermediate-mode genes and, indeed, are
unique among the sequences in Fig. 5. The wzz gene of
Flexneri 2a has valine at residue 224, whereas isoleucine is
characteristic of our short-mode strains and a mutation at this site
can change an intermediate-mode gene to a short-mode gene. These cases
all involve the same general region of the protein. Even this rule is
broken by mutation L91I, which we had expected to increase the modal
value of intermediate-mode strain E4991/76 but, surprisingly, shifted
the O-antigen chain length to the short-mode range of 7 to 16 O units.
This does not exhaust the possibilities, as the difference between the
intermediate and short modes of strains K-12 and Flexneri must involve
one or more of the substitutions at residues 116, 217, 267, 270, and
285, none of which were picked up in our study.
The E. coli K-12 intermediate chain length wzz
gene sequenced by Klee et al. (18) closely resembles our
short-mode chain length genes, including the presence of isoleucine
224, which is characteristic of our short-mode genes. Comparison of the
K-12 wzz gene with those of our short-mode strains reveals
two differences: the presence of arginine 116 and methionine 285, which
must, in some way, override the effect of isoleucine 224. These sites
are also away from those we subjected to mutagenesis.
We conclude that while chain length distribution can be changed
dramatically by substitution of a single key amino acid, it is not
possible to define a specific area or amino acids for each specific
mode. It appears that rather than a few specific sites determining the
modal value, modal-value determination may be an overall property of
the protein. All of the substitutions found to affect chain length are
between the transmembrane segments in the region shown to be in the
periplasm by Morona et al. (22). This indicates that the
effect of Wzz on chain length occurs in the periplasm, which is perhaps
to be expected, as that is where O-antigen polymerization occurs.
It is interesting that the two amino acid substitutions (V224I and
L91I) directly shown to convert from the intermediate to the short mode
are conservative changes in hydrophobic amino acids. It is more likely
that such substitutions change the packing of residues in a protein
than that they affect residues involved in some active site. We have
proposed that Wzz exists in two allosteric states (3) and
the substitutions may affect the ease with which Wzz moves between
them, perhaps in this way influencing the modal chain length determined
by Wzz. However, analysis of the tertiary structure of Wzz may be
necessary to determine the conformational domains involved in the
determination of O-antigen chain length specificity and the role of
these residues.
Phylogeny of the wzz gene.
The short-,
intermediate-, and long-mode wzz genes we sequenced form
three clusters in a neighbor-joining tree (Fig. 6). The single
substitutions made by mutagenesis had litte or no effect on this tree
(data not shown). The most likely explanation is that the majority of
nonsynonymous substitutions are not related to chain length specificity
but, like the synonymous substitutions, indicate phylogenetic
relationships.
The long-mode genes show substantial deviation from typical
intermediate-mode genes. There are 17% synonymous substitutions in the
138- to 573-bp segment of the E. coli wzz gene, which cannot be due to selection on the protein, indicating that this segment was
transferred to E. coli after a long period of divergence. For comparison, E. coli and S. enterica genes
characteristically differ at 15 to 20% of the bases (32).
Within that 138 to 573-bp segment of Wzz are clusters of nonsynonymous
substitutions, for example, from position 231 amino acid residue 77 and
position 306 amino acid residue 102. These nonsynonymous substitutions confer the difference in modal chain length, as the segment to base 420 (amino acid residue 140) in an otherwise intermediate-mode gene gives a
long O-antigen chain length mode (Fig. 3 and 4). The level of
synonymous substitutions is such that the segment from approximately
position 138 to position 573 is most likely derived from a related
species which has a long-mode wzz gene, and the lateral
transfer was presumably selected because it conferred a long-mode chain
length distribution.
The role of Wzz.
Wzz both confers a modal distribution and
determines the modal value. There is very little information on the
selective advantage of either a modal chain length or a specific
average chain length, but it has been shown (36) that lack
of a functional wzz gene in E. coli Flexneri 2a
leads to changes in the distribution of IcsA and reduced
virulence. It has been shown that the plasmid-encoded Wzz protein in
E. coli Flexneri is required for serum resistance and full inflammation, while the chromosome-encoded Wzz protein is
required for normal invasiveness and intercellular spread
(16). There are also reports of an association of O-antigen
chain length with serum resistance (13, 17, 23, 24, 33).
The effects of specific O-antigen chain lengths on serum resistance do
not seem to have been studied (27), but there seems to be
some association between O-antigen chain length and the pathogenicity
of E. coli. Most E. coli strains have
intermediate-mode chain lengths, and the few short- and long-mode
strains do not seem to be randomly distributed. For example, E. coli O1:K1 strains, mostly found in urinary tract infections and
newborn meningitis, all display a typical short mode (19,
21). Also, half of the E. coli O2:K1 strains found in
urinary tract infections have short O-antigen chains (1).
Furthermore, E. coli O83:K1 strains, which cause newborn
meningitis and urinary tract infections, also show the short O-antigen
chain length mode (34, 35). These facts lead us to believe
that the specific chain length range of the O antigen may be an
important virulence factor. However, the way in which O-antigen chain
length exerts an effect is still unknown.
A.V.F. was supported by a University of Sydney F. H. Loxton
Postgraduate Scholarship. This work was supported by an Australian Research Council grant.
We are grateful to Michael Heuzenroeder (IMVS), Adelaide, South
Australia, Australia, and Flemming Scheutz (Statens Seruminstitut, Copenhagen, Denmark) for providing strains and to Chris Murray (IMVS)
for providing antisera.
| 1.
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Achtman, M.,
M. Heuzenroeder,
B. Kusecek,
H. Ochman,
D. Caugant,
R. K. Selander,
V. Väisanen-Rhen,
T. K. Korhonen,
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Abstract
Introduction
