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Journal of Bacteriology, April 1999, p. 2307-2313, Vol. 181, No. 7
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Conserved Organization in the cps Gene
Clusters for Expression of Escherichia coli Group
1 K Antigens: Relationship to the Colanic Acid
Biosynthesis Locus and the cps Genes from
Klebsiella pneumoniae
Andrea
Rahn,
Jolyne
Drummelsmith, and
Chris
Whitfield*
Department of Microbiology, The University of
Guelph, Guelph, Ontario, Canada N1G 2W1
Received 29 September 1998/Accepted 16 January 1999
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ABSTRACT |
Group 1 capsules of Escherichia coli are similar to the
capsules produced by strains of Klebsiella spp. in terms of
structure, genetics, and patterns of expression. The striking
similarities between the capsules of these organisms prompted a more
detailed investigation of the cps loci encoding group 1 capsule synthesis. Six strains of K. pneumoniae and 12 strains of E. coli were examined. PCR analysis showed that
the clusters in these strains are conserved in their chromosomal
locations. A highly conserved block of four genes,
orfX-wza-wzb-wzc, was identified in all of the strains. The
wza and wzc genes are required for
translocation and surface assembly of E. coli K30 antigen.
The conservation of these genes points to a common pathway for capsule
translocation. A characteristic JUMPstart sequence was identified
upstream of each cluster which may function in conjunction with RfaH to
inhibit transcriptional termination at a stem-loop structure found
immediately downstream of the "translocation-surface assembly"
region of the cluster. Interestingly, the sequence upstream of the
cps clusters in five E. coli strains and one
Klebsiella strain indicated the presence of IS elements. We
propose that the IS elements were responsible for the transfer of the
cps locus between organisms and that they may continue to
mediate recombination between strains.
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TEXT |
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 (IS
903). 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.
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ACKNOWLEDGMENTS |
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.
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ADDENDUM IN PROOF |
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).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology, The University of Guelph, Guelph, ON, Canada N1G 2W1.
Phone: (519) 824-4120, ext. 3478. Fax: (519) 837-1802. E-mail:
cwhitfie{at}uoguelph.ca.
| |
REFERENCES |
| 1.
|
Allen, P.,
C. A. Hart, and J. R. Saunders.
1987.
Isolation from Klebsiella and characterization of two rcs genes that activate colanic acid capsular biosynthesis in Escherichia coli.
J. Gen. Microbiol.
133:331-340[Abstract/Free Full Text].
|
| 2.
|
Arakawa, Y.,
R. Wacharotayankun,
T. Nagatsuka,
H. Ito,
N. Kato, and M. Ohta.
1995.
Genomic organization of the Klebsiella pneumoniae cps region responsible for serotype K2 capsular polysaccharide synthesis in the virulent strain Chedid.
J. Bacteriol.
177:1788-1796[Abstract/Free Full Text].
|
| 3.
|
Arricau, N.,
D. Hermant,
J. Waxin,
C. Ecobichon,
P. S. Duffey, and M. Y. Popoff.
1998.
The RcsB-RcsC regulatory system of Salmonella typhi differentially modulates the expression of invasion proteins, flagellin and Vi antigen in response to osmolarity.
Mol. Microbiol.
29:835-850[Medline].
|
| 4.
|
Bailey, M. J. A.,
C. Hughes, and V. Koronakis.
1996.
Increased distal gene transcription by the elongation factor RfaH, a specialized homologue of NusG.
Mol. Microbiol.
22:729-737[Medline].
|
| 5.
|
Bailey, M. J. A.,
C. Hughes, and V. Koronakis.
1997.
RfaH and the ops element, components of a novel system controlling bacterial transcription elongation.
Mol. Microbiol.
26:845-851[Medline].
|
| 6.
|
Bereswill, S., and K. Geider.
1997.
Characterization of the rcsB gene from Erwinia amylovora and its influence on exopolysaccharide synthesis and virulence of the fire blight pathogen.
J. Bacteriol.
179:1354-1361[Abstract/Free Full Text].
|
| 7.
|
Bernhard, F.,
K. Poetter,
K. Geider, and D. L. Coplin.
1990.
The rcsA gene from Erwinia amylovora: identification, nucleotide sequence, and regulation of exopolysaccharide biosynthesis.
Mol. Plant-Microbe Interact.
3:429-437[Medline].
|
| 8.
|
Bliss, J. M., and R. P. Silver.
1996.
Coating the surface: a model for expression of capsular polysialic acid in Escherichia coli K1.
Mol. Microbiol.
21:221-231[Medline].
|
| 9.
|
Bugert, P., and K. Geider.
1995.
Molecular analysis of the ams operon required for exopolysaccharide synthesis of Erwinia amylovora.
Mol. Microbiol.
15:917-933[Medline].
|
| 10.
|
Chakraborty, A. K.,
H. Friebolin, and S. Stirm.
1980.
Primary structure of the Escherichia coli serotype K30 capsular polysaccharide.
J. Bacteriol.
141:971-972[Abstract/Free Full Text].
|
| 11.
|
Choy, Y.-M., and G. G. S. Dutton.
1973.
Structure of the capsular polysaccharide of Klebsiella K-type 20.
Can. J. Chem.
51:3015-3020.
|
| 12.
|
Cieslewicz, M., and E. Vimr.
1997.
Reduced polysialic acid capsule expression in Escherichia coli K1 mutants with chromosomal defects in kpsF.
Mol. Microbiol.
26:237-249[Medline].
|
| 13.
|
Clavel, T.,
J. C. Lazzaroni,
A. Vianney, and R. Portalier.
1996.
Expression of the tolQRA genes of Escherichia coli K-12 is controlled by the RcsC sensor protein involved in capsule synthesis.
Mol. Microbiol.
19:19-25[Medline].
|
| 14.
|
Coleman, M.,
R. Pearce,
E. Hitchin,
F. Busfield,
J. W. Mansfield, and I. S. Roberts.
1990.
Molecular cloning, expression and nucleotide sequence of the rcsA gene of Erwinia amylovora, encoding a positive regulator of capsule expression: evidence for a family of related capsule activator proteins.
J. Gen. Microbiol.
136:1799-1806[Abstract/Free Full Text].
|
| 14a.
| Complex Carbohydrate Structure Database Website. 23 January 1999, revision date. http://www.ccrc.uga.edu.
|
| 15.
|
Dodgson, C.,
P. Amor, and C. Whitfield.
1996.
Distribution of the rol gene encoding the regulator of lipopolysaccharide O-chain length in Escherichia coli and its influence on the expression of group I capsular antigens.
J. Bacteriol.
178:1895-1902[Abstract/Free Full Text].
|
| 16.
|
Drummelsmith, J.,
P. A. Amor, and C. Whitfield.
1997.
Polymorphism, duplication and IS1-mediated rearrangement in the chromosomal his-rfb-gnd region of Escherichia coli strains with group IA capsular K antigens.
J. Bacteriol.
179:3232-3238[Abstract/Free Full Text].
|
| 17.
| Drummelsmith, J., and C. Whitfield. Gene products
required for surface expression of the capsular form of the group 1 K
antigen in Escherichia coli (O9a:K30). Mol. Microbiol., in
press.
|
| 18.
|
Ebel, W.,
G. J. Vaughn,
H. K. Peters III, and J. E. Trempey.
1997.
Inactivation of mdoH leads to increased expression of colanic acid capsular polysaccharide in Escherichia coli.
J. Bacteriol.
179:6858-6861[Abstract/Free Full Text].
|
| 19.
|
Gottesman, S.
1995.
Regulation of capsule synthesis: modification of the two-component paradigm by an accessory unstable regulator, p. 253-262.
In
J. A. Hoch, and T. J. Silhavy (ed.), Two-component signal transduction. ASM Press, Washington, D.C.
|
| 20.
|
Grangeasse, C.,
P. Doublet,
C. Vincent,
E. Vaganay,
M. Riberty,
B. Duclos, and A. J. Cozzone.
1998.
Functional characterization of the low-molecular-mass phosphotyrosine-protein phosphatase of Acinetobacter johnsonii.
J. Mol. Biol.
278:339-347[Medline].
|
| 21.
|
Heinrichs, D. E.,
J. A. Yethon, and C. Whitfield.
1998.
Molecular basis for structural diversity in the core regions of the lipopolysaccharides of Escherichia coli and Salmonella enterica.
Mol. Microbiol.
30:221-232[Medline].
|
| 22.
|
Hobbs, M., and P. R. Reeves.
1994.
The JUMPstart sequence: a 39 bp element common to several polysaccharide gene clusters.
Mol. Microbiol.
12:855-856[Medline].
|
| 23.
|
Jann, K., and B. Jann.
1997.
Capsules of Escherichia coli, p. 113-143.
In
M. Sussman (ed.), Escherichia coli: mechanisms of virulence. Cambridge University Press, Cambridge, United Kingdom.
|
| 24.
|
Jayaratne, P.,
W. J. Keenleyside,
P. R. MacLachlan,
C. Dodgson, and C. Whitfield.
1993.
Characterization of rcsB and rcsC from Escherichia coli O9:K30:H12 and examination of the role of the rcs regulatory system in expression of group I capsular polysaccharides.
J. Bacteriol.
175:5384-5394[Abstract/Free Full Text].
|
| 25.
|
Keenleyside, W. J.,
P. Jayaratne,
P. R. MacLachlan, and C. Whitfield.
1992.
The rcsA gene of Escherichia coli O9:K30:H12 is involved in the expression of the serotype-specific group I K (capsular) antigen.
J. Bacteriol.
174:8-16[Abstract/Free Full Text].
|
| 26.
|
Kelly, R. F.,
L. L. MacLean,
M. B. Perry, and C. Whitfield.
1995.
Structures of the O-antigens of Klebsiella serotypes O2(2a,2e), O2(2a,2e,2h), and O2(2a,2f,2g), members of a family of related D-galactan O-antigens in Klebsiella spp.
J. Endotoxin Res.
2:131-140.
|
| 27.
|
Kelm, O.,
C. Kiecker,
K. Geider, and F. Bernhard.
1997.
Interaction of the regulator proteins RcsA and RcsB with the promoter of the operon for amylovoran biosynthesis in Erwinia amylovora.
Mol. Gen. Genet.
256:72-83[Medline].
|
| 28.
|
Leeds, J. A., and R. A. Welch.
1997.
Enhancing transcription through the Escherichia coli hemolysin operon, hlyCABD: RfaH and upstream JUMPStart DNA sequences function together via a postinitiation mechanism.
J. Bacteriol.
179:3519-3527[Abstract/Free Full Text].
|
| 29.
|
Leeds, J. A., and R. A. Welch.
1996.
RfaH enhances elongation of Escherichia coli hlyCABD mRNA.
J. Bacteriol.
178:1850-1857[Abstract/Free Full Text].
|
| 30.
|
MacLachlan, P. R.,
W. J. Keenleyside,
C. Dodgson, and C. Whitfield.
1993.
Formation of the K30 (group I) capsule in Escherichia coli O9:K30 does not require attachment to lipopolysaccharide lipid A-core.
J. Bacteriol.
175:7515-7522[Abstract/Free Full Text].
|
| 31.
|
Marolda, C. L., and M. A. Valvano.
1998.
The promoter region of the Escherichia coli O7-specific lipopolysaccharide gene cluster: structural and functional characterization of an upstream untranslated mRNA sequence.
J. Bacteriol.
180:3070-3079[Abstract/Free Full Text].
|
| 32.
|
McCallum, K. L., and C. Whitfield.
1991.
The rcsA gene of Klebsiella pneumoniae O1:K20 is involved in expression of the serotype-specific K (capsular) antigen.
Infect. Immun.
59:494-502[Abstract/Free Full Text].
|
| 33.
|
Miller, J. H.
1992.
A short course in bacterial genetics. A laboratory manual and handbook for Escherichia coli and related bacteria.
Cold Spring Harbor Laboratory Press, Plainview, N.Y.
|
| 34.
|
Nelson, K., and R. K. Selander.
1994.
Intergeneric transfer and recombination of the 6-phosphogluconate dehydrogenase gene (gnd) in enteric bacteria.
Proc. Natl. Acad. Sci. USA
91:10227-10231[Abstract/Free Full Text].
|
| 35.
|
Nieto, J. M.,
M. J. A. Bailey,
C. Hughes, and V. Koronakis.
1996.
Suppression of transcription polarity in the Escherichia coli haemolysin operon by a short upstream element shared by polysaccharide and DNA transfer determinants.
Mol. Microbiol.
19:705-713[Medline].
|
| 36.
|
Ørskov, I., and F. Ørskov.
1984.
Serotyping of Klebsiella.
Methods Microbiol.
14:143-164.
|
| 37.
|
Parker, C. T.,
A. W. Kloser,
C. A. Schnaitman,
M. A. Stein,
S. Gottesman, and B. W. Gibson.
1992.
Role of the rfaG and rfaP genes in determining the lipopolysaccharide core structure and cell surface properties of Escherichia coli K-12.
J. Bacteriol.
174:2525-2538[Abstract/Free Full Text].
|
| 38.
|
Paulsen, I. T.,
A. M. Beness, and M. J. J. Saier.
1997.
Computer-based analyses of the protein constituents of transport systems catalysing export of complex carbohydrates in bacteria.
Microbiology
142:2685-2699.
|
| 39.
|
Russo, T. A.,
G. Sharma,
J. Weiss, and C. Brown.
1995.
The construction and characterization of colanic acid deficient mutants in an extraintestinal isolate of Escherichia coli (O4/K54/H5).
Microb. Pathog.
18:269-278[Medline].
|
| 40.
|
Simpson, D. A.,
T. C. Hammarton, and I. S. Roberts.
1996.
Transcriptional organization and regulation of expression of region 1 of the Escherichia coli K5 capsule gene cluster.
J. Bacteriol.
178:6466-6474[Abstract/Free Full Text].
|
| 41.
|
Sledjeski, D. D., and S. Gottesman.
1996.
Osmotic shock induction of capsule synthesis in Escherichia coli K-12.
J. Bacteriol.
178:1204-1206[Abstract/Free Full Text].
|
| 42.
|
Stevens, M. P.,
B. R. Clarke, and I. S. Roberts.
1997.
Regulation of the Escherichia coli K5 capsule gene cluster by transcription antitermination.
Mol. Microbiol.
24:1001-1012[Medline].
|
| 43.
|
Stevens, M. P.,
P. Hänfling,
B. Jann,
K. Jann, and I. S. Roberts.
1994.
Regulation of Escherichia coli K5 capsular polysaccharide expression: evidence for involvement of RfaH in the expression of group II capsules.
FEMS Microbiol. Lett.
124:93-98[Medline].
|
| 44.
|
Stevenson, G.,
K. Andrianopoulos,
M. Hobbs, and P. R. Reeves.
1996.
Organization of the Escherichia coli K-12 gene cluster responsible for production of the extracellular polysaccharide colanic acid.
J. Bacteriol.
178:4885-4893[Abstract/Free Full Text].
|
| 45.
|
Stout, V.
1996.
Identification of the promoter region for the colanic acid polysaccharide biosynthetic genes in Escherichia coli K-12.
J. Bacteriol.
178:4273-4280[Abstract/Free Full Text].
|
| 46.
|
Sugiyama, T.,
N. Kido,
Y. Kato,
N. Koide,
T. Yoshida, and T. Yokochi.
1998.
Generation of Escherichia coli O9a serotype, a subtype of E. coli O9, by transfer of the wb* gene cluster of Klebsiella O3 into E. coli via recombination.
J. Bacteriol.
180:2775-2778[Abstract/Free Full Text].
|
| 47.
|
Wacharotayankun, R.,
Y. Arakawa,
M. Ohta,
T. Hasegawa,
M. Mori,
T. Horii, and N. Kato.
1992.
Involvement of rcsB in Klebsiella K2 capsule synthesis in Escherichia coli K-12.
J. Bacteriol.
174:1063-1067[Abstract/Free Full Text].
|
| 48.
|
Whitfield, C.
1995.
Biosynthesis of lipopolysaccharide O-antigens.
Trends Microbiol.
3:178-185[Medline].
|
| 49.
|
Whitfield, C.,
W. J. Keenleyside, and B. R. Clarke.
1994.
Structure, function and synthesis of cell surface polysaccharides in Escherichia coli, p. 437-494.
In
C. L. Gyles (ed.), Escherichia coli in domestic animals and man. CAB International, Wallingford, Oxon, United Kingdom.
|
| 50.
|
Whitfield, C.,
W. J. Keenleyside,
P. R. MacLachlan,
P. Jayaratne, and A. J. Clarke.
1995.
Identification of rcs genes in Escherichia coli O9:K30:H12 and involvement in regulation of expression of group IA K30 capsular polysaccharide.
Methods Mol. Genet.
6:301-321.
|
| 51.
| Whitfield, C., and I. S. Roberts. Structure,
assembly, and regulation of expression of capsules in Escherichia
coli. Mol. Microbiol., in press.
|
Journal of Bacteriology, April 1999, p. 2307-2313, Vol. 181, No. 7
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