Department of Microbiology, The University of
Guelph, Guelph, Ontario, Canada N1G 2W1
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Escherichia coli produces
a wide variety of capsular polysaccharides termed K antigens. These
polymers can vary in composition, linkage specificity, and
substitution, allowing for diversity among strains. The major capsule
groups, traditionally designated groups I and II, were defined by
serological properties as well as by the location of the K-antigen
biosynthesis gene cluster, the polymer structure, and the expression
patterns (23). A new and expanded classification system has
recently been proposed (51). This is based on genetic and
biochemical (assembly pathway) data and proposes four capsule groups.
The new group 1 accommodates a subset of K antigens formerly designated
group IA (23). Of the 66 structurally defined K antigens in
E. coli, 16 show characteristics typical of group 1 capsules
(23). Research in this laboratory focuses on the group 1 K
antigens of E. coli and the structurally related capsules
found in Klebsiella spp.
In E. coli, group 1 K antigens are produced in two distinct
forms: a low-molecular-weight form (KLPS), which comprises
K oligosaccharides linked to lipid A-core and resembles a
lipopolysaccharide (LPS)-linked O antigen, and a high-molecular-weight
unlinked form, capsular K antigen, which is associated with the cell
surface. A precise mechanism of attachment for capsular K antigen has
not been described, but it is known that LPS is not involved
(30). In Klebsiella, only the capsular form of K
antigen is produced. The absence of the KLPS form in
Klebsiella may reflect differences in the LPS core structure
and/or the ligase enzyme (21) that attaches polysaccharides to the lipid A-core acceptor.
The genes involved in synthesis and transport of both KLPS
and capsular K antigen in E. coli are encoded at a locus
called cps. A prototype group 1 capsule cluster from
E. coli E69 (O9a:K30) (cpsECK30) has
been analyzed (17). The 16-kb locus contains genes required
for K30 polymerization and translocation. The K30 antigen is
synthesized via the Wzy-dependent polymerization pathway, which has
been described for the biosynthesis of certain O antigens (48). In brief, repeat units are made on the cytoplasmic
face of the plasma membrane by the action of glycosyltransferases, which transfer residues to a lipid (undecaprenol pyrophosphate)-linked biosynthetic intermediate. The lipid-linked repeat units are moved to
the periplasmic face of the membrane by the Wzx protein, where they are
polymerized by the Wzy enzyme. To form KLPS, short
K30-antigenic oligosaccharides are ligated to lipid A-core by the
ligase, WaaL. The capsular form of the K30 antigen is polymerized by
the same pathway; however, surface expression is via an LPS-independent pathway that requires the products of wza and wzc
(see below). These gene products are not required for the assembly of
LPS-linked O antigens, and they provide features that distinguish gene
clusters for biosynthesis of O and K antigens.
Comparative analysis of Klebsiella pneumoniae K2
(cpsKPK2) (2) (GenBank
accession no. D21242) and E. coli K30
(cpsECK30) (17) (GenBank
accession no. AF104912) indicates a shared biosynthetic pathway. The
objective of this study was to investigate the precise relationships
between capsule gene clusters in a number of E. coli and
Klebsiella strains.
A conserved region of genes required for translocation and
polymerization of group 1 K antigens.
Lipid-linked intermediates
are polymerized to form high-molecular-weight K30 polysaccharide via
the Wzy-dependent polymerization pathway and then translocated through
the outer membrane as an unlinked polymer. Surface assembly of capsular
group 1 K antigen minimally requires two additional genes from the
capsule locus, wza and wzc (17). Wza,
Wzb, and Wzc are encoded in the "translocation-surface assembly"
region of the K30 cluster and are thought to be an outer membrane
lipoprotein, a cytoplasmic phosphatase, and an ATP-binding protein,
respectively. Recent work with Acinetobacter johnsonii has
shown that a Wzb homologue, Ptp, is capable of dephosphorylating a Wzc
homologue, Ptk (20). Similar genes have been described in
diverse systems including E. coli K-12 (wza,
wzb, and wzc) (44),
Klebsiella K2 (orf4, orf5, and
orf6) (2), and Erwinia amylovora
(amsH, amsI, and amsA) (9),
where they are believed to be involved in the production of colanic
acid, K2 capsular polysaccharide (CPS), and amylovoran, respectively.
Wza and Wzc homologues are found in a variety of bacteria that produce
CPS and extracellular polysaccharide (38), leading to the
conclusion that these represent a common translocation-surface assembly
pathway for cell surface polysaccharides. Although these proteins have been established to function in surface expression of the K30 capsular
antigen (17), their precise role in the process has yet to
be determined.
The region associated with CPS translocation-surface assembly was
examined in detail. Chromosomal DNA was prepared from the strains shown
in Table 1. To reduce the expression of
CPS, bacteria were grown in Luria broth (33) at 42°C. To
isolate chromosomal DNA, bacteria from a 5-ml overnight culture were
collected by centrifugation and resuspended in 1.5 ml of a lysis
solution (50 mM sodium chloride, 2% sodium dodecyl sulfate, 300 mg of
proteinase K per ml). The suspension was incubated at 42°C until
clear. Next, 250 µl of 5 M sodium chloride and 200 µl of 10%
hexadecyltrimethylammonium bromide in 0.7 M sodium chloride were added,
followed by a 30-min incubation at 65°C. The sample was then
subjected to two phenol-chloroform-isoamyl alcohol (25:24:1)
extractions and precipitated with 5% 5 M sodium chloride and 2 volumes
of absolute ethanol. The DNA pellet was washed with 70% ethanol and
resuspended in 100 µl of sterile water. Finally, the DNA was treated
with RNase and subjected to a chloroform-isoamyl alcohol (24:1)
extraction.
The region between wza and wzc was amplified by
PCR. PCRs were performed with Pwo or Expand Long DNA
polymerase (Boehringer Mannheim) in a Perkin-Elmer GeneAmp PCR System
2400 thermocycler. The primers used for DNA amplification are listed in
Table 2 and shown in Fig.
1. PCR products identical in size were
obtained with primers JD89 and JD109 and chromosomal DNA from strains
representing 6 K serotypes of Klebsiella and 12 K serotypes
of E. coli (Fig. 2). The sizes
of the products were consistent with those predicted from the
cpsECK30 and cpsKPK2
sequence data.
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FIG. 1.
Organization of the cpsECK30
locus, which is located between galF and gnd on
the E. coli E69 chromosome (17). Components of
the Wzy-dependent polymerization pathway (wzx and
wzy) are found in the region dedicated to K30 repeat unit
synthesis and polymerization, along with the necessary
glycosyltransferases, wbaP, wcaN,
wcaO, and wbaZ. Genes involved in the
translocation of capsular K30 antigen, wza and
wzc, are near the start of the cluster. The functions of
orfX, orfY, and orfZ are unknown.
Primers used for PCR amplification of selected regions are indicated,
and the sequences are given in Table 2.
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FIG. 2.
PCR analysis of the wza-to-wzc
region from different group 1 K serotypes of E. coli (A) and
Klebsiella (B). For reference, K. pneumoniae K20
strain 889/50 is also shown in panel A and E. coli K30
strain E69 is shown in panel B. PCR products identical in size (1.8 kb)
were obtained for all of the strains tested, indicating a conservation
of gene order between clusters.
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For detailed analysis, selected PCR fragments were purified with Qiagen
nucleotide removal columns and the region was sequenced at the Guelph
Molecular Supercentre (University of Guelph, Guelph, Ontario, Canada).
Analyses revealed that the wza-to-wzc regions in
E. coli A295b (GenBank accession no. AF118245), E75 (GenBank accession no. AF118246), N24c (GenBank accession no. AF118247), 2151 (GenBank accession no. AF118248), and Bi161-42 (GenBank accession no.
AF118249) had 99.5% identity to E. coli E69 at the
nucleotide level and that the equivalent regions in
cpsKPK2 and cpsECK30 were
72% identical at the nucleotide level.
The observation that group 1 capsule clusters from E. coli
and Klebsiella are organized with a conserved block of
translocation-surface assembly genes is reminiscent of the modular
structure of gene clusters required for the manufacture of group 2 capsules in E. coli. Group 2 (kps) clusters
comprise three regions (51). Region 1 encodes proteins
necessary for translocation of polysaccharide through the periplasm and
across the outer membrane, as appears to be the case with the initial
genes (wza to wzc) of the group 1 cps
clusters. The serotype-specific central region (region 2) is
responsible for biosynthesis and polymerization of oligosaccharide repeat units. This is equivalent to the genes downstream of
wzc in the group 1 capsule clusters. Region 3 contains genes
encoding the subunits for an ATP-binding cassette transporter that is
required for capsule export across the cytoplasmic membrane
(8). The region 3 components are not required in
Wzy-dependent polysaccharides, such as the group 1 capsules (48,
51). In the group 2 clusters, regions 1 and 3 are conserved in
strains that produce structurally distinct capsules and the gene
products are functionally interchangeable between these strains
(51). Although functional identity has not been formally
shown for the translocation-surface assembly region of group 1 capsules, the high degree of conservation seems to indicate that this
would be the case. Some conserved feature of the group 1 K antigens or
a component in their assembly must therefore be recognized for the
translocation-surface assembly processes to be completed.
orfX is a unique component of group 1 K antigen
cps gene clusters.
Although homologues of
wza, wzb, and wzc are found in other
systems, including the cpsK-12 cluster for
colanic acid biosynthesis, orfX homologues have been
found only in cpsECK30 and
cpsKPK2 (where it is designated orf3)
(2, 17). In both cases, orfX represents the first
gene in the cluster. PCRs were performed with primers designed to
amplify the region between orfX (JD95 for
Klebsiella and JD99 for E. coli) and a gene
upstream of the K-antigen cluster, galF (GALF1) (Table 2;
Fig. 1). The sizes of the fragments obtained varied considerably due to
polymorphism upstream of cps (see below). However, sequence
data showed orfX to be conserved in position and virtually
identical at the nucleotide level in all of the E. coli
strains (E56b, Bi161-42, A295b, N24c, and E75) and
Klebsiella strains (A5054, 889/50, 6613, and 708)
investigated (data not shown). In E. coli K30,
orfX mutants do not appear to be impaired in
translocation-surface assembly of the capsular K antigen
(17) and the precise role of orfX remains
unclear. However, the high degree of conservation in orfX
homologues would indicate that it is important in the production of
group 1 capsules.
Identical cps gene clusters in E. coli K30
and K. pneumoniae K20.
There are notable structural
similarities between group 1 K antigens in E. coli and
K. pneumoniae (structures are available online in the
Complex Carbohydrate Structure Database [14a]). In the
example of Klebsiella K20 (11) and E. coli K30 (10), the capsular antigen structures are
identical. The relationship between these two gene clusters was also
examined by PCR and sequencing. Similar to the E. coli
strains analyzed above, the wza-to-wzc region in
these strains was shown to be 99.5% identical (Klebsiella K20 GenBank accession no. AF118250).
Genes downstream of wza, wzb, and wzc
are involved in repeat-unit biosynthesis and polymerization. Genes in
this region, in particular, the glycosyltransferases, will vary with
the structure of the polymer being produced by any given strain. Among
group 1 capsules, this region has been described in detail for E. coli K30 (17) and Klebsiella K2
(2), and although both encode the same classes of gene
products, they exhibit only low levels of similarity. PCR amplification
of the region from wcaO to wzx with primers JD90
and JD53 (Table 2; Fig. 1) yields products identical in size (3.6 kb)
for both E. coli K30 and K. pneumoniae K20 (data
not shown). At the nucleotide level, the products are 99% identical
over the 550 bp sequenced from each end. The high degree of
conservation at the nucleotide level for both the wza-wzc and the wcaO-to-wzx regions may indicate the
transfer of cps gene clusters between K. pneumoniae and E. coli (see below).
Conservation of regulatory regions upstream of cps.
Gene
clusters for bacterial polysaccharides are characteristically preceded
by a 39-bp JUMPstart (for "just upstream of many polysaccharide
starts") element (22). The JUMPstart element has also been
identified upstream of gene clusters involved in F conjugation pilus
assembly and hemolysin toxin secretion (reviewed in reference
5). A second element, ops (for "operon
polarity suppressor") is an 8-bp motif located within the JUMPstart
sequence (35). Both elements are believed to play a role in
transcriptional antitermination of the gene clusters (5, 31,
35). Their role has been described in regulation of E. coli group 2 K-antigen (kps [42, 43])
and LPS O-antigen (rfb [31]) biosynthesis.
The role of the JUMPstart element has been best characterized for the
hemolysin operon, where it has been shown to control operon polarity
but not transcript stability (5, 35), in concert with a NusG
homologue, RfaH (4, 28, 29). The element can function over
long distances (2 kb); however, it does so only when present on the
nascent transcript (35). In one possible model, the
JUMPstart sequence functions by facilitating the formation of a number
of stem-loop structures on the mRNA during transcript elongation
(31). This region recruits RfaH and potentially other proteins. Binding of RfaH to stem-loop III may inhibit the formation of
the other stem-loops, which are thought to induce premature termination
when present.
To investigate the conservation of these regulatory elements, regions
immediately upstream of the group 1 K-antigen clusters were amplified
and sequenced. A conserved JUMPstart element was present in both
E. coli strains (E56b, Bi161-42, A295b, N24c, and E75) and
Klebsiella strains (A5054, 889/50, 6613, and 708) (Fig.
3A). This finding suggests that group 1 capsule clusters are subject to transcriptional control via
antitermination. Interestingly, the cps gene clusters share
a feature noted upstream of the O-antigen biosynthesis region of
E. coli O7 (wb*) (31), i.e., the
presence of two ops elements.
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FIG. 3.
(A) JUMPstart sequences from polysaccharide gene
clusters directing the synthesis of amylovoran (ams)
(27), colanic acid (cps) (45), O7
antigen (wb*) (31), and the E. coli
and Klebsiella group 1 capsule clusters examined in this
study. Variations from the E. coli K30 sequence are
highlighted in boldface. The two ops elements found in the
E. coli O7 JUMPstart sequence are underlined. (B) Stem-loop
structures which have been identified in
cpsECK30 and cpsKPK2.
They are located immediately downstream of wzc and may
function as transcription terminators.
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In the hemolysin system, antitermination is required to avoid extreme
operon polarity in a situation where the structural gene for the toxin
is separated from those required for toxin maturation and export by a
stem-loop structure (5). Analysis of the available
cpsECK30 and cpsKPK2
sequences identified a stem-loop structure in the intergenic region
between wzc and the initial gene of the repeat-unit
synthesis region (Fig. 3B). This potentially provides a strong
transcriptional terminator, thereby allowing differential expression of
structural components for capsule translocation and the highly active
enzymes involved in polymer synthesis. Equivalent stem-loop structures
are also predicted from the nucleotide sequence downstream of the
wzc homologue in the gene clusters for amylovoran (9) and colanic acid (44) (data not shown).
Differential expression of genes required for translocation-surface
assembly and synthesis is known to occur in the E. coli
group 2 kps clusters. In these systems, regions 2 and 3 are
organized into one transcriptional unit under the control of the region
3 promoter. Region 2 genes rely on transcriptional antitermination by
RfaH to avoid operon polarity problems (42). The
kps region 1 is not regulated via antitermination but has
been shown to be thermoregulated (12, 40). In addition, the
first gene of region 1, kpsF, plays a poorly understood role
in regulation (12). It remains to be established whether the
product of orfX plays a similar role in expression of group
1 K antigens.
Group 1 K antigens are known to be regulated by the Rcs (for
"regulator of capsule synthesis") system in both E. coli
(24, 25) and Klebsiella (1, 32, 47).
This two-component regulatory system is best characterized for colanic
acid production in E. coli K-12 (19); however,
essentially identical systems operate in E. amylovora
(6, 7, 14, 27). It is believed that the RcsC protein senses
an environmental signal and, along with a second protein, RcsF,
modulates the activity of RcsB through phosphorylation. RcsB can
interact with a Lon protease-sensitive protein, RcsA, and upregulate
cps transcription. Both RcsA and RcsB have helix-turn-helix
DNA-binding motifs and bind to the promoter region of the
ams cluster for amylovoran production in E. amylovora (27). A potential binding site has also been
identified upstream of the colanic acid cluster based on the titration
of regulatory proteins by using promoter DNA and sequence homology to
the E. amylovora binding site (27, 45). The
environmental signal sensed by the Rcs system is uncharacterized but
may involve membrane perturbations (13, 18, 37) or osmotic
stress (3, 18, 41).
Although group 1 capsules, colanic acid, and amylovoran are all Rcs
regulated, there are some important differences in their expression
patterns. Most notable is the observation that colanic acid and
amylovoran are optimally produced at 20°C and are not manufactured at
37°C. This is not surprising for colanic acid since this polymer is
not a virulence determinant (39) and its function may be
more important in environments outside the host (18, 41).
E. amylovora is a plant pathogen associated with infections
at environmental temperatures. However, group 1 capsules are virulence
determinants and are produced at 37°C (49). The ability to
express cps gene products at 37°C may be due to altered interactions of an RcsA-RcsB dimer with the cps promoter or
to the involvement of additional (as yet uncharacterized) regulatory proteins. The Rcs proteins themselves are highly conserved in E. coli K30 and E. coli K-12 (24, 25),
suggesting that they are unlikely to determine the different patterns
of expression. The regions upstream of cps in E. coli and K. pneumoniae strains with group 1 K antigens
lack the published RcsA-RcsB-binding sequences (27).
Therefore, although the Rcs system may function to regulate group 1 K
antigens, details of this interaction may differ considerably from
those for the colanic acid and amylovoran systems.
Analysis of the Klebsiella K2 cps upstream region
resulted in the identification of a partial sequence of a putative
54 promoter (2). This is conserved in all the
E. coli and K. pneumoniae strains examined here.
However, this putative promoter lies downstream of the JUMPstart
element and thus could not operate in situations where antitermination
by RfaH is required. The precise physiological significance of this
"promoter" is therefore unclear.
Possible lateral transfer of group 1 cps genes.
The data presented above demonstrates that the K-antigen gene clusters
of E. coli and Klebsiella are highly conserved in
organization and in cps nucleotide sequence. This is
consistent with the possibility of lateral transfer of group 1 capsule
gene clusters between these organisms. Further evidence in support of
this contention was obtained by the identification of IS elements
upstream of the cps gene clusters. In E. coli
K30, a partial IS1 element (250 bp) truncates an adjacent
gene, orf2. orf2 is also found immediately upstream of the
cpsKPK2 cluster (2) but is absent in
E. coli K-12. orf2 is not essential for K-antigen
production in K. pneumoniae (2). A survey of
other strains showed that many of the E. coli cps clusters
are flanked by IS sequences (Fig. 4). Of
the six E. coli strains examined, five contained IS
elements. The type of IS element present is highly variable, with only
IS1 being identified in more than one strain (E. coli E69 and E56b). In contrast, only one of the five K. pneumoniae strains had an IS element. The location of the IS
sequences seems to be quite highly conserved, with only two of six
strains (E. coli A295b and K. pneumoniae 889/50)
showing slight variation.
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FIG. 4.
Diagrammatic representation of the region upstream of
cps in the E. coli and K. pneumoniae
strains investigated. The regions for E. coli E69
(17) and K. pneumoniae Chedid (2) are
based on published data. The location and type of IS element
identified, if any, is indicated. The putative 54
promoters and conserved JUMPstart elements are located between the IS
elements (or orf2) and orfX, the first gene of
each cluster.
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The presence of IS elements adjacent to most of the tested E. coli cps clusters and their absence in all but one of the
cps regions examined in Klebsiella strains
suggest that IS elements may have mediated an initial transfer of the
cluster to E. coli from Klebsiella and could
continue to be important for the exchange of genes encoding different
capsule types between strains. Support for this hypothesis can be found
in an analysis of gnd (6-phosphogluconate dehydrogenase)
alleles from a variety of E. coli and Klebsiella isolates. The cps genes map near his and
gnd in E. coli, and Nelson and Selander
(34) have suggested that diversity in gnd
represents a surprisingly high degree of recombination. This was
attributed to cotransfer with adjacent loci (primarily rfb)
whose activities are subject to diversifying selection because of the
host immune response. Nelson and Selander also argued for lateral
transfer of gnd genes from Klebsiella to E. coli. E. coli strains with the 16 known group 1 K antigens have a
limited array of LPS O antigens (23); of the >170 O
serotypes, only O9, O9a, O8, O20, and O101 are represented.
Klebsiella strains have 77 different K antigens
(36) but fewer than 10 structurally distinct O antigens (26, 46). Notably, some O-antigen structures are shared, and lateral transfer of the O3 gene cluster to E. coli has been
proposed (46). Collectively, these data suggest that
transfer of a large region of DNA including the rfb and
cps loci may have occurred. In such a scenario, the extended
region between galF and his
(orf2-cps-ugd-rfb-gnd) from Klebsiella would
replace the "typical" E. coli region (cps [colanic acid]-rfb-gnd-ugd-wzz). This is consistent with
our previous analysis of the regions surrounding gnd that
confirmed the lack of wzz in E. coli strains with
group 1 capsules (15, 16). Such organization also explains
why expression of colanic acid and expression of a group 1 capsule are
mutually exclusive. In previous work, we proposed that the
cpsK-12 and cpsECK30
systems are allelic (25, 50). From the differences in
organization (the presence of orfX), altered upstream
sequences, and the IS/orf2 region missing in E. coli K-12, these can no longer be considered alleles. Colanic acid
is therefore not simply a widespread serotype of group 1 K antigen and
should not be included as such.
The simple conclusion that all E. coli group 1 capsules have
arisen by lateral transfer of DNA from Klebsiella is
complicated by several observations. First, not all E. coli
group 1 capsules have structurally identical counterparts in
Klebsiella, although this could reflect further gene
transfer and recombination events within cps after the
initial transfer, thereby resulting in the production of novel
structures. However, of the strains examined here, one E. coli isolate (E75) lacks an IS element immediately upstream of
cps and only one Klebsiella strain (889/50) has
an IS element (IS903). These exceptions suggest that the
events that resulted in group 1 capsule diversity in E. coli
and Klebsiella are more complicated. As noted above, the
E. coli K30 and K. pneumoniae K20 (889/50)
cps gene clusters appear to be identical, and there are two
possible explanations for this. One is that the cluster was mobilized
to these strains from a common (unknown) source by a process involving
different IS elements. The other is that the gene exchanges between
Klebsiella and E. coli are possibly not
restricted to unidirectional lateral transfer events. There is
currently no data to resolve these possibilities.
This work was supported by funding to C.W. from the Medical
Research Council of Canada (MT-9623). A.R. and J.D. are recipients of
NSERC PGS-B scholarships.
While this paper was under review, further analysis of the
RcsA-RcsB binding site in Escherichia coli K-12 was reported
(W. Ebel and J. E. Trempy, J. Bacteriol. 181:577-584,
1999). A conserved motif (the RcsA box) was identified upstream of both cps and rcsA in E. coli K-12. This
motif is not present in the regions upstream of the group 1 capsule
gene clusters reported here. The binding site for RcsA-RcsB in
Erwinia and related bacteria has also been further
elucidated (M. Wehland, C. Kiecker, D. L. Coplin, O. Kelm, W. Saenger,
and F. Bernhard, J. Biol. Chem. 274:3300-3307, 1999).
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