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Journal of Bacteriology, May 2000, p. 2741-2745, Vol. 182, No. 10
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Regulation of the Escherichia coli K5
Capsule Gene Cluster: Evidence for the Roles of H-NS, BipA, and
Integration Host Factor in Regulation of Group 2 Capsule Gene
Clusters in Pathogenic E. coli
Sonya
Rowe,
Nigel
Hodson,
Gary
Griffiths, and
Ian S.
Roberts*
School of Biological Sciences, University of
Manchester, Manchester M13 9PT, United Kingdom
Received 12 November 1999/Accepted 25 February 2000
 |
ABSTRACT |
The expression of Escherichia coli group 2 capsules (K
antigens) is temperature dependent, with capsules only being expressed at temperatures above 20°C. Thermoregulation is at the level of transcription, with no detectable transcription at 20°C. Using the
E. coli K5 capsule gene cluster as a model system, we have shown that the nucleoid-associated protein H-NS plays a dual role in
regulating transcription of group 2 capsule gene clusters at 37 and
20°C. At 37°C H-NS is required for maximal transcription of group 2 capsule gene clusters, whereas at 20°C H-NS functions to repress
transcription. The BipA protein, previously identified as a
tyrosine-phosphorylated GTPase and essential for virulence in
enteropathogenic E. coli, was shown to play a similar role to H-NS in regulating transcription at 37 and 20°C. The binding of
integration host factor (IHF) to the region 1 promoter was necessary to
potentiate transcription at 37°C and IHF binding demonstrated by
bandshift assays. The IHF binding site was 3' to the site of
transcription initiation, suggesting that sequences in the 5' end of
the first gene (kpsF) in region 1 may play a role in
regulating transcription from this promoter at 37°C. Two additional
cis-acting sequences, conserved in both the region 1 and 3 promoters, were identified, suggesting a role for these sequences in
the coordinate regulation of transcription from these promoters. These
results indicate that a complex regulatory network involving a number
of global regulators exists for the control of expression of group 2 capsules in E. coli.
| |
INTRODUCTION |
Escherichia coli produces
more than 80 chemically and serologically distinct capsules, called K
antigens (18). These capsules have been separated into four
groups on the basis of chemical composition, molecular weight,
intergenic relationships, and regulation of expression (6, 23,
34). The majority of extraintestinal isolates of E. coli associated with invasive disease express group 2 capsules,
with particular capsules being associated with certain diseases
(17). Typical of many virulence factors, expression of group
2 capsules in E. coli is regulated by temperature, with group 2 capsules only being expressed at temperatures above 20°C (18, 34). Environmental regulation of virulence gene
expression is well documented in a broad range of pathogenic bacteria
(19), and it is likely that this offers a mechanism by which
pathogenic bacteria may adapt to particular niches encountered within
the host. The regulation of virulence gene expression by temperature provides a means by which bacteria may selectively express virulence determinants upon entry into the host.
Group 2 capsule gene clusters have a common organization consisting of
three regions (25, 34). A central, capsule-specific region,
region 2, encoding the enzymes for polysaccharide biosynthesis (24), is flanked by regions 1 and 3, which are common to all of the group 2 capsule gene clusters. Regions 1 and 3 encode the eight
Kps proteins, which constitute the common export pathway for the
transport of group 2 polysaccharides out of the cell (5, 22, 25,
26, 34). Region 1 consists of six genes, kpsFEDUCS, organized in a single transcriptional unit with a 
70
promoter which is located 225 bp 5' of kpsF (4, 22,
29). Two integration host factor (IHF) binding site consensus
sequences were identified 110 bp 5' and 130 bp 3' of the transcription
start site (29). Transcription from the promoter 5' of
kpsF generates an 8.0-kb polycistronic transcript that is
then processed to generate a separate, 1.3-kb kpsS
transcript which may enable the differential expression of the KpsS
protein (29).
Region 3 contains two genes, kpsM and kpsT, which
are organized as a single transcriptional unit with a 70
promoter 741 bp 5' of the start of kpsM (30). An
ops sequence, which is conserved in RfaH-regulated genes, is
located 30 bp 5' to the initiation codon of kpsM, and
mutations in rfaH or deletion of the ops sequence
results in a lack of capsule expression (30). By
quantitative reverse transcriptase-PCR, it has been shown that RfaH
acts to allow readthrough transcription originating from the region 3 promoter to proceed into region 2 and that readthrough transcription is
essential for expression of the region 2 genes and group 2 capsule
production (30). As such, the transcriptional organization
of group 2 capsule gene clusters can be regarded as two large
convergent transcripts, one of which originates from the region 1 promoter and covers region 1, and the other originating from the region
3 promoter and spanning regions 2 and 3.
Although previously it had been shown that at 20°C there is no
transcription from the region 1 promoter (5, 29), little was
known about the regulation of transcription of group 2 capsule gene
clusters. In this study we demonstrate that the global regulator H-NS
and the tyrosine-phosphorylated GTPase BipA are essential for maximal
transcription from the region 1 and 3 promoters at 37°C.
Paradoxically, both proteins also function in the temperature regulation of these promoters, repressing transcription at 20°C. In
addition, we demonstrate the binding of IHF to the region 1 promoter
and identify the IHF binding site and other cis-acting regulatory sequences important for transcription at 37°C.
| |
MATERIALS AND METHODS |
Bacterial strains, plasmids, and growth conditions.
The
E. coli strains and plasmids used in this study are shown in
Table 1. Bacteria were grown in Luria (L)
broth supplemented with ampicillin (100 µg ml
1),
chloramphenicol (25 µg ml1), kanamycin (50 µg
ml1), or streptomycin (100 µg ml1) where
appropriate at either 37 or 20°C. Tn5 mutagenesis was performed as described before (7). The site of
Tn5 insertion was determined by constructing a plasmid
library of the transposon insertion mutant, followed by screening the
library for clones resistant to kanamycin. The nucleotide sequence
flanking the site of insertion was then determined using primers
specific to the ends of Tn5. Where appropriate, mutations
were introduced into strains by P1vir transduction
(30), and transductants were selected on L-agar supplemented
with kanamycin (50 µg ml1).
DNA procedures.
Plasmid DNA was prepared by alkaline lysis
(27). Deletion of the conA and conB
sites in the region 1 promoter was generated by inverse PCR mutagenesis
(IPCRM) as described before (14) with the following primers;
SR1, 5'-AAATCCTAGGTACCTTGTTCATAATG-3'; SR2,
5'-AAATGGTACCTACTTGACTATTAATAC-3'; SR3,
5'-CAAGTAGGAAACATTTTAACAAATGATA-3'; SR4,
5'-CCAAAAACAATTTATCAATTGATTATTTTC-3'; SR6,
5'-AAATGTTTCCTACTTGACTATTAATAC-3'; and SR8,
5'-CCTAAATTCCTTGTTCATAATGTAGGA-3'. Deletion of the putative IHF binding sites IHF1 and IHF2 was achieved using the primers IHF1D1
(5'-TAGCATAAATAAATTATAGTGGTT-3') and IHF1D2
(5'-TAAACAAAATTTTTAATGAATATAAAACCAT-3') for site IHF1 and
IHF2D1 (5'-AAATTGTTTTTGGTATTAATAGTCAAG-3') and IHF2D2
(5'-TTTTCTTGTAAAAAAAGAACGTATGA-3') for site IHF2.
RNA procedures.
RNA was prepared by hot acid-phenol
extraction (1). To quantitate the specific mRNAs, 50 µg of
total RNA was applied to nylon membranes (Hybond N) using a Bio-Rad
Biodot-SF apparatus and hybridized with DNA probes labeled with
[
-32P]dCTP and random hexanucleotides. The signals due
to bound probe were detected and quantified with a FujiX BAS2000
Phosphoimager with Aida V2.0 software. Hybridization with a
radiolabeled probe containing the 5s RNA gene was used to confirm the
total loading of RNA onto the filter.
Enzyme assays.
Bacterial strains were grown to
mid-logarithmic phase (optical density at 600 nm of 0.5) at either 37 or 20°C, and where appropriate, 
-galactosidase activity was
assayed as described before (20).
Gel retardation experiments.
Two 200-bp
[-32P]dCTP-radiolabeled PCR fragments containing the
putative IHF binding sites were amplified using either primers IHF1F
(5'-CAACCTGTTTATTATGCC-3') and IHF1R
(5'-CACACTCCTACATTATGA-3') for the IHF1 site or primers
IHF2F (5'-GCACCTCCATGAGACATT) and IHF2R
(5'-CCAGGTAAATGTCTTTCAGGAC-3') for the IHF2 site. The
particular radiolabeled PCR product was incubated with appropriate cell
lysates or purified IHF (20 mM), and the binding of IHF to the PCR
fragment was monitored by gel retardation assays as described before
(28).
Measurement of extracellular K5 polysaccharide.
The amount
of extracellular K5 polysaccharide produced by MS150 and its
derivatives was quantified using the carbazole assay (3)
with purified K5 polysaccharide as a standard. Since K5 is the only
extracellular uronic acid-containing polysaccharide produced by MS150,
this assay is an accurate measure of the relative amounts of K5 polysaccharide.
| |
RESULTS AND DISCUSSION |
Analysis of the role of IHF on transcription from the region 1 promoter.
Two putative IHF binding sites, termed IHF1 and IHF2,
had previously been identified in the region 1 promoter (Fig.
1), and mutations in the himA
gene were shown to reduce expression of the KpsE protein
(29). To demonstrate the effect of IHF on transcription from
the region 1 promoter, plasmid pDSHcH (Table 1) was introduced into
strains MS150 and MS151, and the level of -galactosidase was assayed
following growth at 37°C. Strain MS150(pDSHcH) expressed 1,398 ± 97 (mean ± standard error of the mean [SEM]) U, whereas strain MS151(pDSHcH) expressed 154 ± 39 U, a sevenfold reduction in the level of -galactosidase. To demonstrate the functional importance of the IHF1 and IHF2 binding sites, IPCRM was
undertaken to generate plasmids pCBIHF-1 and pCBIHF-2 (Table 1), in
which IHF1 or IHF2, respectively, was deleted (Fig. 1). These plasmids were introduced into MS150, and the level of -galactosidase was assayed. Deletion of either IHF1 or IHF2 reduced -galactosidase activity by twofold; strain MS150(pDSHcH) expressed 933 ± 43 U, compared with strains MS150(pCBIHF-1) (519 ± 45 U) and
MS150(pCBIHF-2) (478 ± 33 U).
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FIG. 1.
Analysis of the region 1 promoter. The top line depicts
region 1, with the larger arrow denoting the 8.0-kb polycistronic
transcript and the smaller arrow denoting the processed
kpsS-specific transcript. The stem-loop structure shows the
intragenic Rho-dependent terminator within the kpsF gene. At
the bottom, the promoter region is enlarged. The IHF1, IHF2, conA, and
conB sites are boxed, and the numbers state the distance in base pairs
from the center of the site to the transcriptional start site, depicted
by an arrow.
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The interaction of IHF with the region 1 promoter was shown by gel
shift assays. A radiolabeled PCR fragment spanning IHF1
was amplified
from pDSHcH using primers IHF1F and IHF1R (Fig.
1) and incubated with
extracts from strains HN880, MS150, and
MS151 and with purified IHF
(Fig.
2). In the case of both strains
HN880 and MS150 and with purified IHF, a reproducible band shift
could
be detected which was absent when the fragment was incubated
with
extracts from strain MS151 (Fig.
2). This demonstrates that
IHF is
binding to the region 1 promoter. To localize the site
of interaction
between IHF and the region 1 promoter, the same
primers were used to
amplify the corresponding fragment from plasmid
pCBIHF1, in which the
IHF1 site had been deleted by IPCRM (Table
1). This PCR fragment
lacking IHF1 was then used in gel retardation
assays. Deletion of IHF1
abolished the band shift (Fig.
2), confirming
the binding of IHF at
this site in the region 1 promoter. Primers
IHF2F and IHF2R were then
used to amplify a radiolabeled PCR fragment
containing the IHF2 site
(Fig.
1). However, no IHF-dependent band
shifts were detectable with
this fragment in gel retardation experiments
(data not shown).
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FIG. 2.
Gel retardation experiments. Cell lysates from strains
HN880 (lane b), MS150 (lane c), and MS151 (lane d), along with purified
IHF at a final concentration of 20 nM (lane e), were incubated with 0.5 pmol of [32P]dCTP-labeled PCR product prepared from
either (A) pDSHcH (IHF1+) or (B) pCBIHF1
(IHF1). Following incubation, the samples together with
an aliquot of the PCR product (lane a) were analyzed by electrophoresis
followed by autoradiography.
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|
The identification of an IHF binding site at +130 is a surprising
result, since IHF binding sites are usually 5' to the site
of
transcription initiation (
16). The only other reported
example
of an IHF binding site 3' to the site of transcription
initiation
is in the Pc promoter of bacteriophage Mu (
32).
Generally IHF
acts in concert with other transcriptional factors
(
16) and
is believed to bend the DNA within the promoter
region, to facilitate
the appropriate DNA-protein interactions
necessary for transcriptional
activation (
9,
15,
16). In the
case of the region 1 promoter,
introducing a bend in the DNA centered
on the first residue of
the IHF1 site would suggest that sequences 3'
to IHF1, possibly
in the 5' end of the
kpsF gene, may be
important in the transcriptional
activation of the region 1 promoter at
37°C. The observation that
a
himA mutation had a more
dramatic effect on region 1 transcription
than deletion of the IHF1
site suggests that IHF may also play
an indirect role in the activation
of region 1 transcription,
perhaps by controlling the expression of an
additional
trans-acting
regulator.
Identification of cis-acting elements in the region 1 promoter.
Comparison of the region 1 and 3 promoters identified
two conserved sequences, termed conA and conB, which are separated by 17 bp and are located 5' to the 
35 region in both promoters
(29). It has been suggested that these sequences could
represent cis-acting regulatory sequences that are important
in coordinating transcription from the region 1 and 3 promoters
(25, 29). To elucidate the possible role of these sequences,
IPCRM was used to generate plasmids pSR4A, pSR4B, and pSR4AB, from
which conA, conB, and both conA and conB have been deleted,
respectively (Fig. 1, Table 1). These plasmids were introduced into
strain MS150, and the level of -galactosidase at 37 and 20°C was
assayed. Deletion of either or both conA and conB reduced
-galactosidase activity by greater than twofold but had no effect on
stimulating transcription at 20°C (Table 2). One interpretation of these data is
that the deletions are having a generic effect on disrupting promoter
function. However, this is unlikely to be the case for two reasons.
First, it has been shown that sequences 5' to 50 are not important
for the binding of RNA polymerase and the initiation of transcription from constitutive 70 promoters (8). Second,
in regulated 70 promoters, such 5' sequences define
cis-acting sites for the binding of regulatory proteins
(8, 10). As such, the most likely interpretation is that
conA and conB define a cis-acting region necessary for
maximum transcription from the region 1 promoter. The proximity of IHF2
to conA and conB (Fig. 1), deletion of which also reduces transcription
by twofold, would be in keeping with this notion. The conservation of
conA and conB in the region 3 promoter could represent a mechanism to
coordinate transcription from the region 1 and 3 promoters at 37°C.
Role of H-NS and BipA in transcription from the region 1 and 3 promoters.
The global regulator H-NS has been shown to play a key
role in the temperature regulation of a number of virulence genes
(2). To investigate the role of H-NS in the temperature
regulation of the region 1 promoter, plasmid pDSHcH was introduced into
strain MS152, which contains the
hns::kan null mutation from strain
MC4100 (Table 1), and the level of -galactosidase activity was
assayed following growth at 37°C. Strain MS150(pDSHcH) expressed
1,030 ± 21 U, whereas strain MS152(pDSHcH) expressed 404 ± 36 U. These results were confirmed by RNA dot blot analysis with a
kpsE-specific probe to RNA extracted from MS152 grown at 37 and 20°C (Table 3). The hns
mutation resulted in comparable levels of transcription at both 20 and
37°C (Table 3), indicating that H-NS was required for maximal
transcription at 37°C as well as acting to repress transcription
at 20°C. To determine the effect of the hns mutation on
capsule production, the levels of extracellular K5 polysaccharide produced by MS152 were determined. The hns mutation reduced
the extracellular K5 polysaccharide level by 50% at 37°C but
increased K5 polysaccharide production at 20°C, with comparable
levels of K5 polysaccharide being made at both temperatures (Table
4).
To identify other regulatory genes, plasmid pDSHcH was introduced into
a pool of Tn
5 mutants of MS150, and colonies expressing
reduced
-galactosidase activity were identified on medium
supplemented
with X-Gal
(5-bromo-4-chloro-3-indolyl-
-
D-galactopyranoside).
Two
colonies were identified, and in both cases the Tn
5 had
inserted
at different sites into the
E. coli bipA gene. One
of these strains,
termed MS153 (Table
1), was used for further study,
and the level
of
-galactosidase was assayed. The
bipA
mutation reduced
-galactosidase
activity by fivefold in cells grown
at 37°C, 1,030 ± 21 U for
strain MS150(pDSHcH) versus 182 ± 43 U for strain MS153 (pDSHcH).
The effects of the
bipA mutation were confirmed by RNA dot blot
analysis with a
kpsE-specific probe to RNA extracted from MS150
and MS153
grown at 37 and 20°C. At 37°C the
bipA mutation caused
a
threefold reduction in transcription from the region 1 promoter
(Table
3), while at 20°C the
bipA mutation increased
transcription
to levels comparable to those seen in an
hns
mutant (Table
3).
The effects of the
bipA mutation on region
1 transcription were
mirrored in the levels of extracellular K5
polysaccharide produced
at 37 and 20°C (Table
4).
Quantitative RNA dot blots with the
kpsT gene as a
radiolabeled probe to RNA extracted from MS150, MS152, and MS153 grown
at 37 and 20°C confirmed that H-NS and BipA regulated transcription
from the region 3 promoter. At 37°C the
hns mutation
reduced region
3 transcription twofold, while the
bipA
mutation had a greater
effect, reducing transcription more than
threefold (Table
3).
At 20°C both the
hns and
bipA mutations increased transcription
twofold from the
region 3 promoter (Table
3).
H-NS has been implicated in the temperature regulation of a number of
genes, with H-NS repressing gene expression at low temperature
(
2,
11,
31,
33). Generally,
hns mutations increase
transcription
at both the permissive and nonpermissive temperatures to
comparable
levels. In the case of the region 1 and 3 promoters, the
situation
is more complex, since the
hns mutation has a more
modest effect
on increasing transcription at 20°C and does not
completely abolish
temperature regulation (Table
3). In addition to its
effect on
transcription at 20°C, the
hns mutation reduced
transcription
from both the region 1 and 3 promoters at 37°C by
twofold, as
assayed by reporter gene activity and quantitative RNA dot
blot
analysis. Taken together, these data provide compelling evidence
that H-NS is required for maximal transcription from both the
region 1 and 3 promoters at 37°C. The effect of the
hns mutation
was mirrored in reduced levels of extracellular K5 polysaccharide
at
37°C (Table
4). The observation that H-NS has a dual role,
being
involved in the activation of group 2 capsule gene expression
at 37°C
while also playing a role in repression at 20°C, is unique
for
H-NS-mediated thermoregulation. At this stage, the mechanism
by which
H-NS functions in this dual role is
unclear.
The isolation of mutants that have transposon insertions in the
bipA gene and are altered in transcription of both the
region
1 and 3 promoters at 37 and 20°C is intriguing. The BipA
protein
was identified as a protein that was phosphorylated on a
tyrosine
residue in enteropathogenic
E. coli (EPEC) strains
(
12,
13).
In addition, EPEC
bipA mutants do not
trigger cytoskeletal rearrangements
in host cells, are hypersensitive
to the host defense protein
BPI, and demonstrate increased mobility and
flagellum expression
(
12). The observation that the
E. coli BipA protein has GTPase
activity and is homologous to both
elongation factor G and the
TetO resistance protein, both of which
interact with the ribosome,
leads to the suggestion that the BipA
protein may represent a
new class of regulators which function via
interactions with the
ribosome (
12). The effect of the
bipA mutation on transcription
from the region 1 and 3 promoters is unlikely to be via a direct
interaction between the BipA
protein and the respective promoter,
and the most likely explanation is
that BipA regulates the activity
of proteins that are required for the
regulation of transcription
of the region 1 and 3
promoters.
In summary, we can begin to develop a model to explain the regulation
of group 2 capsule gene expression in pathogenic
E. coli. At
37°C the region 1 and 3 promoters are active, and both
H-NS and BipA
are required for maximal transcription. The identification
of
cis-acting sequences that are conserved within the region 1
and 3 promoters would suggest that additional regulatory proteins
interact to stimulate transcription at 37°C. In the case of the
region 1 promoter, there is an additional requirement for IHF,
which is
likely to be providing an architectural role in bending
the DNA to
allow the assembly of the nucleoprotein complex. In
the case of region
3, RfaH will be interacting with the RNA polymerase
complex to permit
transcription elongation to proceed from region
3 into region 2. Paradoxically, at 20°C, both H-NS and BipA also
play roles in
repressing transcription, suggesting key roles for
these proteins in
the regulation of group 2 capsule expression.
Studies are ongoing to
further elucidate this complex regulatory
circuit and establish roles
for additional regulatory
proteins.
| |
ACKNOWLEDGMENTS |
We kindly thank Howard Nash for strain HN880, Steve Goodman for
purified IHF, and Jay Hinton for helpful discussions.
Work in the laboratory of I.S.R. is supported by the BBSRC of the UK,
the Wellcome Trust, and the Lister Institute of Preventive Medicine.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: School of
Biological Sciences, 1.800 Stopford Building, University of Manchester,
Oxford Rd., Manchester M13 9PT, United Kingdom. Phone: 00 44 161 275 5601. Fax: 00 44 161 275 5656. E-mail:
ISRobert{at}fs1.scg.man.ac.uk.
| |
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Journal of Bacteriology, May 2000, p. 2741-2745, Vol. 182, No. 10
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
