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J Bacteriol, January 1998, p. 338-349, Vol. 180, No. 2
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Identification, Genomic Organization, and Analysis of the Group
III Capsular Polysaccharide Genes kpsD, kpsM,
kpsT, and kpsE from an Extraintestinal
Isolate of Escherichia coli (CP9, O4/K54/H5)
Thomas A.
Russo,1,2,3,*
Suzanne
Wenderoth,1,
Ulrike B.
Carlino,1,3
Joseph M.
Merrick,2,3 and
Alan J.
Lesse1,2,4,5
Department of
Medicine,1
Department of
Microbiology,2
The Center for Microbial
Pathogenesis,3
Department of
Pharmacology and Toxicology,4 and
the
Buffalo VA Medical Center,5 SUNY at Buffalo,
Buffalo, New York 14215
Received 12 June 1997/Accepted 8 November 1997
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ABSTRACT |
Group III capsular polysaccharides (e.g., K54) of extraintestinal
isolates of Escherichia coli, similar to group II capsules (e.g., K1), are important virulence traits that confer resistance to
selected host defense components in vitro and potentiate systemic infection in vivo. The genomic organization of group II capsule gene
clusters has been established as a serotype-specific region 2 flanked
by regions 1 and 3, which contain transport genes that are highly
homologous between serotypes. In contrast, the organization of group
III capsule gene clusters is not well understood. However, they are
defined in part by an absence of genes with significant nucleotide
homology to group II capsule transport genes in regions 1 and 3. Evaluation of isogenic, TnphoA-generated, group III
capsule-minus derivatives of a clinical blood isolate (CP9, O4/K54/H5)
has led to the identification of homologs of the group II capsule
transport genes kpsDMTE. These genes and their surrounding
regions were sequenced and analyzed. The genomic organization of these
genes is distinctly different from that of their group II counterparts. Although kpsK54DMTE are
significantly divergent from their group II homologs at both the DNA
and protein levels phoA fusions and computer-assisted
analyses suggest that their structures and functions are similar. The
putative proteins KpsK54M and KpsK54T appear to
be the integral membrane component and the peripheral ATP-binding component of the ABC-2 transporter family, respectively. The putative KpsK54E possesses features similar to those of the membrane
fusion protein family that facilitates the passage of large molecules across the periplasm. At one boundary of the capsule gene cluster, a
truncated kpsM (kpsMtruncated) and
its 5' noncoding regulatory sequence were identified. In contrast to
the complete kpsK54M, this region
was highly homologous to the group II kpsM. Fifty-three base pairs 3' from the end of kpsMtruncated was
a sequence 75% homologous to the 39-bp inverted repeat in the
IS110 insertion element from Streptomyces
coelicolor. Southern analysis established that two copies of this
element are present in CP9. These findings are consistent with the
hypothesis that CP9 previously possessed group II capsule genes and
acquired group III capsule genes via IS110-mediated
horizontal transfer.
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INTRODUCTION |
Over 80 serologically and chemically
unique capsular polysaccharides can be produced by Escherichia
coli (22, 31). Initially, these polysaccharides were
divided into group I and group II based on chemical, physical, genetic,
and microbiological distinctions (20, 21). Subsequently, the
division of the group II capsules into groups II and III (formerly I
and II) has been proposed (36).
Group I capsules are chemically and physically characterized by a high
molecular weight (>100,000), an acidic component usually consisting of
hexuronic acid or pyruvate, a low charge density and electrophoretic
mobility, and stability at pH 5 to 6 at 100°C. Group I capsules may
protect against desiccation and may contribute to adherence in enteric
disease-producing isolates of E. coli (17, 27,
30). However, a role in the pathogenesis of extraintestinal E. coli infection has not been demonstrated (45).
In contrast, epidemiologic and experimental evidence supports a role
for group II and group III capsules as virulence factors for
extraintestinal infection (10, 43, 44), and these capsules possess multiple similarities with the capsules of pathogenic strains
of Neisseria meningitidis and Haemophilus
influenza (26). The group II capsules are characterized
by a molecular weight of <50,000; hexuronic acids; N-acetyl
neuraminic acid, phosphate, or 2-keto-3-deoxyoctonic acid (KDO) as
acidic components; a higher charge density and electrophoretic
mobility; and a general lack of stability at pH 5 to 6 at 100°C.
Several group II capsules are linked to KDO-phosphatidic acid, which
may serve both as a recognition signal for transport across the
cytoplasmic membrane and as a membrane anchor (6, 7). The
genes that code for these capsules have been mapped near
serA (32, 33, 53), and these capsules are
coexpressed with a large number of O antigens. It was originally
believed that a given E. coli strain possessed only genes
for either a group I or a group II capsule. However, it has been
subsequently shown that three group II or III capsule (K1, K5, and
K54)-producing strains were also capable of producing the group I
capsule colanic acid (25, 46). This finding suggests the
possibility that many if not all strains of E. coli have the capability to produce a group I capsule, whereas only a subset can
produce a group II or group III capsule. The gene clusters coding for
the group II capsules K1, K4, K5, K7, K12, and K92 have been cloned and
extensively studied (particularly K1 and K5) and have a common
organization of three functional regions (38-40, 48).
Region 2, which is unique for a given capsular antigen, codes for genes
whose products are responsible for the synthesis of the K-specific
serotype. This region is flanked by regions 1 and 3, which are highly
conserved among the group II capsule gene clusters evaluated to date.
In fact, a DNA probe generated from region 1 in the K1 capsule gene
cluster was used to identify the K4, K5, K7, K12, and K92 capsule gene
clusters (13, 38). Region 1 contains six genes
(kpsFEDUCS), and region 3 contains two genes
(kpsMT); each region is organized in a single
transcriptional unit and is temperature regulated. These gene products
are needed for transport of the capsular polysaccharide across the
cytoplasmic membrane and assembly onto the cell's surface
(4).
Group III capsules were originally categorized as group II capsular
polysaccharides. Although these groups have similar biochemical and
physical characteristics (31), map to the same location on
the chromosome (32, 33, 53), confer resistance to selected host defense components in vitro, and potentiate systemic infection in
vivo (10, 43, 44), differences exist between them. Group III
serotypes K2, K3, K10, K11, K19, and K54 do not show temperature regulation of capsule expression, a characteristic which correlates with constitutive levels of CMP-KDO activity (14), whereas
group II capsules have increased capsule expression and CMP-KDO
activity at 37°C. Further, only the K2 capsule gene cluster, but not
those from K3, K10, K11, K19, and K54, possesses DNA sequences
homologous to group II capsule gene cluster regions 1 and 3 on the
basis of Southern analysis (12, 36). In a recent study that
described the cloning of a K10 and a different K54 (E. coli
A12b) capsule gene cluster, Southern analysis and complementation
studies were used to elucidate group III capsule gene organization
(36). The preliminary results of these analyses suggested
that a central serotype-specific region was flanked by two regions in
which there was homology between the K10 and K54 gene clusters.
Further, complementation studies demonstrated that group II
kpsK5D and
kpsK5E mutations, but not
kpsK5M or
kpsK5T mutations, were complemented
by subclones from the K10 and K54 capsule gene clusters. Therefore,
this finding suggested that, despite a lack of DNA homology, functional
homology exists, at least in part, between proteins involved in the
export of group II and group III capsular polysaccharides. The
combination of these findings has resulted in the designation of
serotypes K3, K10, K11, K19, and K54 (with or without K2) as group III
capsules (36) and has suggested that these gene clusters are
phylogenetically divergent from those of group II. In support of this
concept, a clonal group of clinical E. coli isolates was
recently identified from multiple geographic regions (23,
24). These strains were characterized in part by possession of
the papGJ96 (class I) and prsGJ96 (class III) genes, the O4-specific
antigen moiety of lipopolysaccharide, the H5 flagellar antigen, the F13
fimbrial antigen, and a group III capsule (K3, K10, and K54/96).
Researchers in our laboratory have been studying a clinical bacteremic
isolate of E. coli (CP9, O4/K54/H5) as a model pathogen for
extraintestinal infection (42). Its group III K54 capsular polysaccharide has been shown to be important for serum resistance in
vitro (44) and systemic infection in vivo (43)
but not for resistance to bactericidal permeability-increasing protein in vitro (45) or urinary tract infection in vivo
(41). Previously, we reported the construction and initial
characterization of TnphoA-generated, isogenic K54-minus
derivatives of CP9 that were used in these studies (42). In
this study, we describe the genomic location and novel organization of
a portion of the K54 capsule gene cluster, the DNA sequences of
kpsK54DMTE, and an analysis of these
genes.
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MATERIALS AND METHODS |
Strains.
The strains used for this study are listed in Table
1. The wild-type strain (CP9, O4/K54/H5),
a clinical blood isolate, and its K54 capsule-minus isogenic
derivatives have been previously described in part (23, 42).
Construction of capsule gene subclones.
Subclones of the K54
capsule gene locus 5' to the TnphoA insertions in CP9.29,
CP9.108, CP9.137, CP9.171, CP9.C43, CP9.C54, and CP9.C56 were obtained
by restricting whole-cell DNA with BamHI, which recognizes a
site located 3' to the kanamycin resistance gene in TnphoA
with or without XbaI (CP9.108, 137, 171, C43, C54, and C56)
or ApaI (CP9.29), neither of which restricts within
TnphoA. Ligations of these restrictions into pBSII SK
,
electroporation into XL1 Blue (Stratagene, La Jolla, Calif.), and
selection of ampicillin (100 µg/ml)- and kanamycin (40 µg/ml)-resistant transformants resulted in the identification of the
subclones p29.1, p108.1, p137.1, p171.1, pC43.1, pC54.1, and pC56.1. To
construct a second set of subclones, in which the active
phoA fusion was in the opposite orientation, p108.1, p137.1,
and p171.1 were restricted with BamHI and XbaI,
p29.1 was restricted with BamHI and ApaI, and the
inserts were purified by electroelution and ligated into pBSII KS.
This set of constructs has been designated p29.2, p108.2, p137.2, and p171.2. These plasmids are described in detail in Table 1.
Subclones of the K54 capsule gene locus 3' to the Tn
phoA
insertions in CP9.108 and CP9.171 were obtained by restricting
whole-cell
DNA with
ClaI, which recognizes a site 5' to the
kanamycin resistance
gene in Tn
phoA. Ligations of these
restrictions into pBSII SK
,
electroporation into XL1 Blue
(Stratagene), and selection of ampicillin-
and kanamycin-resistant
transformants resulted in the identification
of the subclones p108.3
and p171.3. Each contains the right 6.7
kb of Tn
phoA and
either 3.0 kb (p108.3) or 2.0 kb (p171.3) of
chromosomal DNA 3' to the
respective Tn
phoA insertions.
Identification of a cosmid clone containing capsule genes.
Whole-cell DNA was purified from CP9 as described previously
(42), and DNA fragments (30 to 50 kb) were ligated into the unique BamHI site of the 8.8-kb cosmid cloning vector pWE15
(Clontech Laboratories, Palo Alto, Calif.). The ligation mix was
packaged into lambda phage in vitro and transduced into E. coli NM554, and the resultant CP9-derived DNA library was
amplified once. The amplified library was screened for clones
containing capsule genes via colony filter hybridization as described
previously (16). The probe used for detection was generated
by digesting p171.1 with PvuI/XbaI, purifying the
1.3-kb restriction product via electroelution, and subsequent
radioactive labelling with [
-32P]dCTP by random
oligonucleotide priming. Approximately 1,000 colonies of NM554
containing the CP9 DNA library were screened, and a cosmid clone
(cos9a) was detected. Cos9a was confirmed to contain capsule gene DNA
via Southern analysis (42), with the p171.1
PvuI/XbaI 1.3-kb fragment as the probe.
DNA sequencing, determination of TnphoA insertion
sites, and analysis of capsule genes.
DNA sequence was determined
by the dideoxy chain termination method of Sanger et al.
(47) with the capsule gene subclones (p29.1, p108.1, p108.3,
p137.1, p171.1, p171.3, and pC56.1) and cos9a as the DNA templates. DNA
sequencing of the capsule gene subclones p29.1, p108.1, p137.1, p171.1,
and pC56.1 was initially with a TnphoA' fusion joint primer
(5' AATATCGCCCTGAGC 3'), which established the location for
a given TnphoA insertion. Sequencing of capsule gene
subclones p108.3 and p171.3 was initially with the TnphoA
primer (5' CATGTTAGGAGGTCACAT 3'). Subsequent DNA sequence was determined with primers derived from the deduced sequences of the
capsule gene subclones or the cosmid cos9a. A consensus sequence was
generated by assembling and editing the DNA sequence obtained from 76 overlapping but independent sequencing reactions with AssemblyLIGN
1.0.2 (Oxford Molecular Group, Beaverton, Oreg.). Both strands of the
capsule gene sequence submitted in this report were sequenced. The
organization of the assembled subclone sequences and that of the cosmid
cos9a sequence were in agreement. Sequence analysis, comparisons, and
CLUSTAL alignments were performed, in part with MacVector (version 6.0;
Oxford Molecular Group). Comparisons were also performed via BLAST
analysis of the nonredundant GenBank, EMBL, DDBJ, and PDB sequences.
Percentages of similarity and identity were determined by the GAP
program of the Wisconsin Sequence Analysis Packages (Genetics Computer
Group, Madison, Wis.). The PROSITE database was used for motif searches
(2). SignalP V1.1 was used for identification of signal
sequences (28). A terminator sequence search was performed
by the method of Brendel and Trifonov adapted for the Wisconsin
Sequence Analysis Packages (TERMINATOR) (5).
Capsule loci based on XbaI DNA fragments.
We
previously reported, based on Southern analysis of pulsed-field gel
electrophoretically separated DNA from CP9 and
TnphoA-generated isogenic capsule-minus derivatives, that
the K54 capsule genes were located on at least three different
XbaI DNA fragments (capsule loci [cl] 1, 2, and
3). These fragments were linked within the transducing range of
bacteriophage T4 (100 to 150 kb) (42). cl1 was
estimated to be a 10.3-kb fragment, and strains CP9.29, CP9.108,
CP9.137, CP9.171, and CP9.C56 had insertions within this locus.
Sequence analysis from the present study confirmed this finding and
indicated that cl1 was approximately 6.0 kb (Fig. 1). The fragment identified as
cl3 and containing the TnphoA insertion in
CP9.C54 was proven by sequence analysis to also be cl1.
CP9.C54 has subsequently been shown to contain a truncated form of
TnphoA, a finding which led to the incorrect interpretation
that cl1 and cl3 were separate loci.
cl2 was an estimated 18.5 kb, and strain CP9.C43 had a
TnphoA insertion within this locus. Sequence analysis has
demonstrated that the TnphoA insertion, responsible for the K54
phenotype in CP9.C43, is in a novel DNA sequence
which has no identifiable homology with any known capsule gene. This
sequence is not part of the K54 capsule gene cluster reported here
(Fig. 1). This data, in conjunction with the information described
below, suggests that the TnphoA insertion in CP9.C43 and
cl2 are located 3' to the end of
kpsK54E (bp 6132).
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FIG. 1.
(A) Schematic diagram of the K54 group III capsule gene
sequence described in this study. From left to right are (i) the
sequence homologous with the K-12 genome and its intersection (section
269, bp 6222) with a sequence unique to CP9 (90° arrow); (ii) bp 0 to
345, which are 85 to 90% homologous to the 5' noncoding region of
kpsK1,5M (including the JUMPstart
site as marked); (iii) a truncated kpsM (bp 346 to 476 and
501 to 526 are designated kpsMtruncated) that is
85 to 90% homologous to the corresponding region of
kpsK1,5M; (iv) an IS110
element (bp 581 to 626) 53 bp from the 3' end of
kpsMtruncated; and (v) the shaded region marked
from 0 to 7012, representing the capsule gene sequence submitted in
this report. The region from bp 627 to 1644 is unidentified but is
probably capsule gene sequence. This region is followed by
kpsK54DMTE, with their respective
ORFs and reading frames depicted below. The 0.9 kb 3' to
kpsK54E (bp 6133 to 7012) plus 0.5 kb
is unidentified K54 capsule gene sequence. Prior to sequence analysis,
the K54 cl were defined as XbaI fragments
(44). The defined location of cl1 (6.0 kb) and
the presumed location of cl2 are marked above. (B) The lines
represent various inserts of subclones used for sequence analysis and
promoter localization. Insert sizes are as marked. Length is
proportional, and location corresponds to the schematic diagram above.
The insert in p29 consists of the first 154 bp of
kpsK54D and an 8.9-kb region 5' to
the start of kpsK54D. The insert in
p108 contains the first half of
kpsK54M, all of
kpsK54D, and 1.2 kb 5' to
kpsK54D (bp 452 to 3878). The insert
in p137 consists of two-thirds of
kpsK54D and the 1.2 kb 5' to it (bp
452 to 2801). The insert in p171 (bp 3894 to 5288) covers the first
half of kpsK54E, all of
kpsK54T, and the last half of
kpsK54M. The dotted lines at the
leftward boundaries of cos9a and p29.1 represent extension into K-12
homologous sequence beyond what is depicted above.
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Southern analysis.
Whole-cell DNA was prepared as described
previously (42) and restricted with AccI as
suggested by the manufacturer (New England Biolabs, Beverly, Mass.).
Southern hybridization was performed as described previously
(42) with the following modifications. A Robbins Scientific
model 1000 hybridization oven was used. Salmon sperm DNA (180 µl of
150 µg/ml stock) was added to 10 ml of prehybridization solution,
which was removed and replaced with 10 ml of hybridization solution
(5× SSC [1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate]-1% sodium dodecyl sulfate). An oligonucleotide was made from the ISCP9110 sequence (bp 581 to 626), labelled with
[
-32P]dATP with T4 polynucleotide kinase according to
the manufacturer's instructions (Gibco BRL, Gaithersburg, Md.), and
used as a probe. After hybridization at 65°C for 18 h, the blot
was washed once at 65°C with 1× SSC-0.1% sodium dodecyl sulfate for
3 min, followed by five washes at 25°C with 6× SSC-1% Sarkosyl for
5 min.
Alkaline phosphatase assays.
Alkaline phosphatase assays
were performed as previously described except that a Beckman DU 640B
spectrophotometer was used to record the hydrolysis rates of
p-nitrophenyl phosphate (46). The baseline
activity of CP9 is negligible and therefore was not accounted for in
this calculation. PhoA activity from each of the measured constructs
represents the mean of five independent evaluations.
Nucleotide sequence accession number.
The accession no. of
the nucleotide sequence shown in Fig.
2 is AF007777.
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FIG. 2.
Nucleotide sequence and deduced amino acid sequence of
kpsK54D,
kpsK54M,
kpsK54T, and
kpsK54E. Arrows identify putative
transcriptional start sites, solid triangles identify the insertion
site of active TnphoA fusions, the open triangle identifies
the insertion site of an inactive TnphoA fusion, and the
underlined regions identify the inverted repeats of a strong
theoretical rho-independent RNA polymerase terminator. The JUMPstart
site, the truncated kpsM
(kpsMtruncated), and
ISCP9110 are marked and identified by the dotted
lines.
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RESULTS AND DISCUSSION |
Location, organization, and analysis of the K54 capsule gene
locus.
K54-deficient strains generated by TnphoA
insertion mutagenesis (42) were used to identify clones
carrying the group III capsule genes (see Materials and Methods), which
were in turn used to identify a cosmid (pcos9a) carrying the wild-type
genes. Sequencing of the region and identification of the sites of
TnphoA insertion were carried out. The K54 group III capsule
gene locus is depicted in Fig. 1. As expected, since group II or III
capsule gene sequences have not been detected in E. coli
K-12, a search of GenBank did not identify any DNA or protein homology
of this capsule gene locus with the deposited E. coli K-12
sequence. Novel loci of unique DNA not present in laboratory strains of
E. coli have been termed "pathogenicity islands," and
this sequence likely represents a portion of such a locus. One of the
two boundaries of this novel CP9 DNA sequence with E. coli
sequence from the K-12 genome was established (Fig. 1). The boundary
was contiguous with the third base (bp 6222, section 269, accession no.
AE000379) of a 178-amino-acid open reading frame (ORF) (bp 6220 to
6756) of unknown identity from the complete E. coli K-12
genome. Interestingly, this novel CP9 sequence is 150 bp 3' to the
phenylalanine tRNA (bp 5996 to 6071). The points of insertion of
several pathogenicity islands are within various tRNAs (15).
This location was consistent with the genetic linkage of the K54
capsule genes with serA (32), and its point of
insertion is identical with that of the K5 capsule gene cluster.
Four homologs of group II capsule transport genes were recognized in
the K54 capsule gene locus described in this study (Fig.
1).
kpsK54D (bp 1645 to 3387, Fig.
2),
kpsK54M (bp 3457 to 4254,
Fig.
2),
kpsK54T (bp 4269 to 4916, Fig.
2),
and
kpsK54E (bp 4888
to 6132, Fig.
2)
were identified. However, while in group II capsule
gene loci,
kpsD and
kpsE are in region 1 along with four
other
genes (
kpsFEDUCS), and
kpsMT are in region
3, in the CP9 (K54)
capsule gene locus, these four genes are grouped
together (
kpsDMTE).
These findings demonstrate that the
organization of the K54 capsule
transport genes in CP9 is unequivocally
different from that of
the corresponding regions in strains with group
II capsule genes
(
39). Further, this data confirms the
prediction, from complementation
studies, that functional homologs of
kpsK5D and
kpsK5E existed
in the K10 and K54
capsule gene clusters (
36).
The putative molecular weights, estimated pIs, guanosine-plus-cytosine
content, and presence or absence of an identifiable
Shine-Dalgarno or
signal sequence of
kpsK54DMTE and
their comparison
with
kpsK1,5DMTE are
summarized in Table
2 (
9,
34,
35,
49,
54). The guanosine-plus-cytosine content of
kpsK54DMTE ranged from 37 to 43%,
compared to the 51% observed for
E. coli K-12, and
suggested that these genes were acquired by horizontal
transfer from an
unknown species. A nucleic acid subsequence analysis
program and a
manual search failed to identify any highly conserved
Shine-Dalgarno
sequences. The reason for this is unknown. However,
since these genes
are in essence "foreign DNA," perhaps their
mRNAs possess
sequence elements with complementarity to parts
of the 16S rRNA that
are distinct from those recognized by Shine-Dalgarno
sequences, which
in turn serve as translational enhancers.
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TABLE 2.
Comparisons of putative molecular weight, pI, and GC
content and the presence or absence of Shine-Dalgarno and signal
sequences between kpsK54DMTE
and kpsK1,5DMTE
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The ORF (ORF1) from bp 1645 to 3387 encoded
kpsK54D. It is 50% homologous at the
nucleotide level and has 33% identity and
54% similarity at the
predicted protein level with the group II
genes
kpsK1,5D. The limited homology with
GumB (16% identity,
32% similarity) and OtnA (17% identity, 32%
similarity) may represent
common functional regions involved with
transport (
4). The
identification of a putative signal
sequence (Fig.
3), the presence
of active
Tn
phoA fusions within
kpsK54D, a hydrophilic hydropathy
profile, and secondary-structure predictions similar to those
of
Kps
K1,5D (data not shown) suggest that KpsD has a
periplasmic
location and a function similar to that of its group II
counterpart,
despite sequence divergence.
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FIG. 3.
CLUSTAL alignment of the predicted amino acid sequences
of E. coli kpsK54D (this study),
kpsK1D, and
kpsK5D. The boxed sequence identifies
amino acid residues that are functionally similar (lighter shading) or
identical (darker shading). Numbers above the sequences are residue
numbers. The predicted signal sequence of KpsK54D is
identified.
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ORF2 (bp 3457 to 4254) encoded
kpsK54M. No significant homology was
detected at the nucleotide level; however, it has 39%
identity and
52% similarity at the predicted protein level with
the group II genes
kpsK1,5M. Other homologs in
Actinobacillus pleuropneumoniae (CpxB),
N. meningitidis (CtrC),
H. influenzae (BexB), and
Salmonella typhi (VexB) revealed amino acid identities
from
23 to 24% and similarities from 37 to 34%. All of these homologs
have
been implicated as the integral membrane component of the
ABC-2
transporters of capsular polysaccharide across the cytoplasmic
membrane
(
1,
18,
37). In
kpsK54M,
the identification of
the ABC-2 transporter system integral membrane
protein signature
(Fig.
4), a similar
hydropathy profile (hydrophobic protein with
six transmembrane
regions), and secondary-structure predictions
similar to those of
Kps
K1,5M (data not shown) support the notion
that
Kps
K54M is also a member of this family.
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FIG. 4.
CLUSTAL alignment of the predicted amino acid sequences
of E. coli kpsK54M (this study),
kpsK5M, and
kpsK1M. The boxed sequence identifies
amino acid residues that are functionally similar (lighter shading) or
identical (darker shading). The ABC-2 transporter system integral
membrane protein signature is marked and corresponds to amino acid
residues 190 to 224 (1, 18). Numbers above the sequences are
residue numbers.
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ORF3 (bp 4269 to 4916) encoded
kpsK54T. It is 65 to 62% homologous
at the nucleotide level and has 51 to 45% identity and
66 to 62%
similarity at the predicted protein level with the group
II genes
kpsK1,5T. Other homologs in
A. pleuropneumoniae (CpxA),
N. meningitidis (CtrD),
H. influenzae (BexA), and
S. typhi (VexC)
showed
amino acid identities from 47 to 24% and similarities of
59 to 36%.
These proteins are the peripheral ATP-binding components
of the ABC-2
transporter protein family. The identification of
Walker motifs (Fig.
5), an ABC transporter signature
sequence,
a similar hydrophilic hydropathy profile, and
secondary-structure
predictions similar to those of
Kps
K1,5T support the notion that
Kps
K54T is
also a member of the ABC-2 transporter family, and
its structure seems
conserved in comparison with that of Kps
K1,5T.
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FIG. 5.
CLUSTAL alignment of the predicted amino acid sequences
of E. coli kpsK54T (this study),
kpsK5T, and
kpsK1T. The boxed sequence identifies
amino acid residues that are functionally similar (lighter shading) or
identical (darker shading). The ATP-binding domain Walker A (residues
38 to 45), and Walker B (residues 145 to 151) motifs (34)
and the ABC-2 transporter signature sequence (residues 125 to 139) are
marked (1, 18). Numbers above the sequences are residue
numbers.
|
|
ORF4 (bp 4888 to 6132) encoded
kpsK54E. No significant homology was
detected at the nucleotide level; however, it has 31%
identity and
46% similarity at the predicted protein level with
the group II genes
kpsK1,5E. Other homologs in
A. pleuropneumoniae (CpxC),
N. meningitidis (CtrB),
H. influenzae (BexC), and
S. typhi (VexD) showed
amino acid identities from 27 to 20% and similarities
from 40 to 32%.
Analysis of the putative Kps
K54E protein via hydropathy
profiles and secondary-structure predictions suggests that this
protein
is similar to the membrane fusion protein family (
4,
11).
These proteins are believed to interact with ABC-type transport
proteins (and others) and perhaps outer membrane proteins to facilitate
substrate transport of large molecules. Kps
K54E has a
number of
features of this family, including (i) a hydrophilic
amino terminus
located in the cytoplasm (amino acids 1 to 60 of
Kps
K54E with
an excess of basic over acidic residues of a
net +11), (ii) a
hydrophobic amino terminus region that may both span
and anchor
the protein in the cytoplasmic membrane, (iii) a
hydrophilic,
largely alpha-helical periplasmic region (supported by
the presence
of active Tn
phoA fusions at amino acid residue
136), and (iv)
a hydrophobic carboxy terminus. However, the
Kps
K54E hydrophobic
carboxy terminus is significantly
smaller than in the membrane
fusion protein family, and the
conservation of residues in this
region is absent. Kps
K1,5E
possess a similar predicted structure
(
4) except that
Kps
K1E has a deletion of amino acid residues
1 to 71 and
Kps
K5E has a deletion of residues 37 to 71 (Fig.
6).
View larger version (82K):
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|
FIG. 6.
CLUSTAL alignment of the predicted amino acid sequences
of E. coli kpsK54E (this study),
kpsK1E, and
kpsK5E. The boxed sequence identifies
amino acid residues that are functionally similar (lighter shading) or
identical (darker shading). Numbers above the sequences are residue
numbers.
|
|
Based on DNA and protein homologies, the sequence from bp 1645 to 6132 clearly comprised genes involved in K54 transport.
Located 5' to the
end of this cluster is 1.0 kb (bp 626 to 1644)
of sequence which has no
identifiable DNA or protein homology.
No Tn
phoA insertions
have been mapped to this region. A 369-bp
ORF was identified from bp
1247 to 1615. Whether this ORF or another
sequence in this 1.0-kb
region codes for products involved with
capsule transport or synthesis
is unclear.
However, 5' to this region, a sequence homologous to the initial 20%
of
kpsM and its entire 5' noncoding regulatory region
(0.35 kb) was identified. The sequence homologous to the 5' coding
region of
kpsM (bp 346 to 476 and 501 to 526) has been designated
kpsMtruncated (Fig.
1 and
2). Over the
entire
kpsMtruncated sequence
and its 5'
noncoding regulatory region, an 85 to 90% DNA sequence
homology to its
K1 and K5 counterparts was observed. Previous
investigators, using
Southern analysis, did not identify any regions
of homology with a
variety of strains that contained group III
capsule gene clusters
(including a different K54 serotype) when
probes containing
kpsK5M were used (
12,
36).
The reasons for
this discrepancy are unclear. However, the degree of
homology
of
kpsMtruncated with
kpsK1,5M was notable, since the
complete
kpsK54M possessed no
significant homology at the nucleotide level
to
kpsK1,5M. This finding suggested the
possibility that CP9 originally
possessed a group II capsule gene
locus and that the K54 capsule
genes were subsequently acquired
by horizontal transfer. Therefore,
the DNA sequence immediately 3' to
kpsMtruncated was analyzed
for
potential insight into the evolution of this group III capsule
gene
locus. A 51-bp region (bp 581 to 626) identified 53 bp 3'
to the end of
kpsMtruncated (Fig.
1 and
2) was 75%
homologous
with the IS
110 insertion sequence identified from
Streptomyces coelicolor. The area of homology coincided with
a 39-bp inverted
repeat that has been identified within this element
(
8) and
the 12 bp 5' to the repeat (bp 1073 to 1122, accession no.
Y00434).
Runs of cytosines appear to be the target site
for this element,
and the 11-bp region 5' to this element contained two
runs of
cytosines (5' CCCGTTTCCCCC 3'). These findings were
consistent
with the hypothesis that CP9 previously possessed group II
capsule
genes and acquired group III capsule genes via
IS
110-mediated
horizontal transfer. A second prediction of
this model would be
the presence of a second
IS
CP9110 element at the 3' end of the
transferred segment. Southern analysis of
AccI-restricted
chromosomal
DNA isolated from CP9 and the closely related strain J96
(
23)
was performed under high-stringency conditions, with
the IS
CP9110 element used as an oligonucleotide
probe. Two copies of the IS
CP9110 element were
detected in the chromosomes of CP9 and J96 on identically
sized
restriction fragments (Fig.
7). One of
the fragments was
the size predicted from the known sequence of the K54
capsule
gene cluster containing IS
CP9110 (1.4 kb). Insertion elements
are well-known mediators of the evolution of
genomic organization,
and in other studies researchers have shown their
role in the
horizontal transfer of genes involved in polysaccharide
synthesis
(
3,
55). The origin of the group III genes,
however, remains
speculative.
View larger version (33K):
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|
FIG. 7.
Southern analysis of DNA from CP9 and J96 to detect the
copy number of ISCP9110 element.
AccI-restricted whole-cell DNA from CP9 and J96 was
separated by conventional electrophoresis, blotted onto nylon, and
subjected to Southern analysis under high-stringency conditions as
described in Materials and Methods, with the
ISCP9110 element as the probe. Lane 1, J96; lane
2, CP9.
|
|
The 1.3 kb downstream from bp 6132 (bp 6133 to 7012 plus 0.5 kb 3' to
the submitted sequence) appeared, at least in part,
to code for genes
involved in capsule synthesis or transport.
Although no homology to any
known capsule genes was identified
in this region, the transposon
insertion in the K54 capsule-deficient
strain CP9.C56 was located at bp
6179, which is 3' to
kpsK54E.
Further
evidence for the presence of these additional genes is
suggested by the
isolation of the capsule-negative strain CP9.C43
(
42), which
contains a Tn
phoA insertion in an 18.5-kb
XbaI
fragment,
cl2 (Fig.
1).
cl2 and the
cl1 fragment sequenced here are cotransducible
by phage T4,
suggesting that the gene defined by CP9.C43 insertion
and
cl2 are located 3' to the end of
kpsK54E.
Transcriptional organization of the K54 capsule gene locus.
CP9.29, CP9.108, CP9.137, and CP9.171 possessed active phoA
fusions with genes in the K54 capsule gene locus. The transposon insertion in CP9.29 (bp 1799) and that in CP9.137 (bp 2801) were within kpsK54D, the transposon
insertion in CP9.108 (bp 3878) was within
kpsK54M, and the transposon insertion
in CP9.171 (bp 5288) was within
kpsK54E (Fig. 1 and 2). These active
fusions confirm the direction of transcription.
kpsK54D and
kpsK54M are separated by 69 bp,
kpsK54M and
kpsK54T are separated by 14 bp, and
kpsK54T and
kpsK54E overlap by 29 bp. No
predicted promoter regions were identified. Bp 6135 to 6157, located
just 3 bp 3' to kpsK54E, were
consistent with a strong theoretical rho-independent RNA polymerase
terminator. These base pairs formed a hairpin structure which was
followed by a 6-bp poly(T) sequence (bp 6163 to 6169). The free energy
of the stem-loop (
G [25°C]) was calculated to be
10.2 kcal (52). The K1 K5 capsule gene cluster regions 1 and 3 are organized as a single transcription unit. The organization of
these regions for the K54 genes is not yet known.
Alkaline phosphatase assays on the subclones containing active
Tn
phoA insertions in either of two orientations were
performed.
In constructs in which the capsule gene insert was in the
opposite
orientation to the vector
lacZ promoter (p29.1,
p108.2, p137.2,
and p171.2), PhoA activity likely reflects
transcription from
an insert promoter; however, the possibility of
read-through transcription
from a cryptic promoter cannot be excluded.
In constructs with
the insert in the same orientation as the
lacZ promoter, PhoA
activity reflects transcription from the
insert and potentially
the vector promoter. The similar degrees of PhoA
activity seen
with p29.1 (15.2 ± 1.6) (mean ± standard
error) and p29.2 (17.7
± 2.1) suggested that this insert, not the
vector, contained a
promoter that is responsible for its
phoA gene fusion activity.
The inserts in p108 and p137 do
not appear to contain promoters,
based on the low level of PhoA
activity produced by p108.2 (0.59
± 0.2) and p137.2 (0.61 ± 0.1). p171 may possess a region with
promoter activity, since p171.2
produced 1.93 ± 0.1 U of PhoA
activity. The extent of these
inserts (Fig.
1) and their activity
or lack of activity suggest that an
essential promoter element
is upstream of bp 452, which is located
within
kpsMtruncated.
Although the insert in p29
contains about 8 kb of DNA upstream
of bp 452, it seems likely that the
promoter for these genes lies
within the 741 bp 5' to the initiation
site for
kpsMtruncated (bp 345), since the K5
region 3 promoter has been mapped to this
location (
50). No
stem-loop terminators were identified between
this region and the start
of
kpsK54D (bp 1645). Also present in
this region is the JUMPstart sequence (bp 279 to 317), which has
been
implicated in the regulation of a variety of polysaccharide
genes and
genes encoding secreted products (
19,
29). This
sequence is
present 5' to region 3 in both the K1 and K5 capsule
gene sequences, in
the promoter-operator region of the
cps genes
coding for the
group I colanic acid capsule (
51), and in the
5' noncoding
region of several lipopolysaccharide gene clusters
(
19) and
the
hly and
tra genes (
29). It has
been shown that
this site is required for the up-regulation of
hly genes and
kpsK5 region 2 genes by
RfaH via antitermination (
29,
50). The effect
of RfaH on K54
capsule gene activity has not yet been evaluated.
However, in previous
studies we have demonstrated that RcsA is
a negative regulator of group
III capsule genes, which we have
now identified as
kpsK54DME (
46). Although
the precise promoter-operator
region for these group III capsule genes
has not been definitively
established, it is intriguing that the
JUMPstart site is present
within both the insert in p29 and the
promoter-operator region
of the
cps genes encoding for the
group I colanic acid capsule.
Further, RcsA is a positive regulator of
group I capsule production
and interacts with the promoter-operator
region of the
cps genes
(
51). Since RcsA
divergently regulates the group 1 colanic acid
and the K54 capsular
polysaccharides, it is tempting to speculate
that this regulation is
mediated by RcsA either via direct interaction
with the JUMPstart site
or by interaction with protein complexes
assembled there.
In summary, the genomic location, the novel organization of a portion
of the group III, K54 capsule gene cluster, and the
DNA sequences of
kpsK54DMTE have been determined.
Despite a divergence
of these genes at the nucleotide and protein
levels, analysis
suggests that their function is similar to that of
described homologs.
Further, this study lends insight into the
phylogenetic evolution
of a group III capsule gene cluster. Findings
support the hypothesis
that CP9 previously possessed group II capsule
genes and acquired
group III capsule genes via
IS
110-mediated horizontal transfer.
Future studies will be
focused on the remaining genes in the K54
capsule gene cluster, in
particular those that do not possess
homology to reported capsule
genes. The products of these genes
may function differently from those
responsible for group II capsule
expression.
| |
ACKNOWLEDGMENTS |
This work was supported by Research for Health in Erie County
(T.A.R.) and the Office of Research and Development, Department of
Veterans Affairs (A.J.L.).
We appreciate the continued support of Tim Murphy.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Medicine, Division of Infectious Diseases, 3435 Main St., Biomedical Research Building, Room 141, Buffalo, NY 14214. Phone: (716) 829-2674. Fax: (716) 829-2158. E-mail: trusso{at}acsu.buffalo.edu.
Present address: Cornell University Medical School, New York, NY
10021.
| |
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J Bacteriol, January 1998, p. 338-349, Vol. 180, No. 2
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
