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Journal of Bacteriology, October 2000, p. 5381-5390, Vol. 182, No. 19
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Gene Conservation and Loss in the
mutS-rpoS Genomic Region of Pathogenic
Escherichia coli
Corinne J.
Herbelin,
Samantha
C.
Chirillo,
Kristen A.
Melnick, and
Thomas S.
Whittam*
Institute of Molecular Evolutionary Genetics,
Department of Biology, Pennsylvania State University, University
Park, Pennsylvania 16802
Received 17 May 2000/Accepted 5 July 2000
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ABSTRACT |
The extent and nature of DNA polymorphism in the
mutS-rpoS region of the Escherichia coli genome
were assessed in 21 strains of enteropathogenic E. coli
(EPEC) and enterohemorrhagic E. coli (EHEC) and in 6 strains originally isolated from natural populations. The intervening
region between mutS and rpoS was amplified by long-range PCR, and the resulting amplicons varied substantially in
length (7.8 to 14.2 kb) among pathogenic groups. Restriction maps based
on five enzymes and sequence analysis showed that strains of the EPEC
1, EPEC 2, and EHEC 2 groups have a long mutS-rpoS region
composed of a ~6.0-kb DNA segment found in strain K-12 and a novel
DNA segment (~2.9 kb) located at the 3' end of rpoS. The
novel segment contains three genes (yclC, pad1,
and slyA) that occur in E. coli O157:H7 and
related strains but are not found in K-12 or members of the ECOR group
A. Phylogenetic analysis of the common sequences indicates that the
long intergenic region is ancestral and at least two separate deletion
events gave rise to the shorter regions characteristic of the E. coli O157:H7 and K-12 lineages.
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INTRODUCTION |
The acquisition of new genes by
horizontal transfer has played a major role in the adaptation and
ecological specialization of bacterial lineages (17). It has
been estimated, for example, that ~18% of the current genome of
Escherichia coli K-12 represents foreign DNA acquired by
horizontal transfers since the divergence of E. coli and
Salmonella enterica (18). Gene acquisitions have also contributed to the variation in virulence among strains and closely related bacterial species (11, 38). In E. coli and S. enterica, blocks of virulence genes, called
pathogenicity islands, have been acquired at different times, thus
generating a variety of pathogens with distinct virulence genes and
mechanisms of pathogenesis (12, 31, 32). In some cases, loss
of genes has been important in adaptive radiation and the evolution of
bacterial virulence. For example, Maurelli and coworkers
(25) present evidence that the universal deletion of the
lysine decarboxylase gene (cadA) has enhanced the virulence
of Shigella species because cadaverine, a product of the
reaction catalyzed by lysine decarboxylase, inhibits the activity of
Shigella enterotoxin.
One active region of genomic evolution is located between 61 and 62 min
in the E. coli genome (19). This region includes two essential genes (Fig. 1):
mutS, which encodes one of the four proteins required for
DNA mismatch repair (39); and rpoS, which encodes
a sigma factor (
38) that regulates many stationary-phase
and environmental stress response genes (13). Both
mutS and rpoS are highly conserved in sequence
between E. coli and S. enterica; however, the
nearby genomic regions have diverged in a variety of ways. In S. enterica, there is a 40-kb pathogenicity island (SPI-1 [Fig. 1])
that is inserted next to mutS and is required for epithelial
cell invasion (11, 27). SPI-1 has been detected in all
Salmonella groups but is absent in E. coli,
suggesting that the island was acquired early in the evolutionary
radiation of the salmonellae (31). Among different E. coli strains, the length of the genomic sequence between the
mutS and rpoS genes is variable (Fig. 1). The
region is 6.9 kb long in the E. coli K-12 genome
(1) but varies in length among pathogenic strains of
E. coli and Shigella (19, 20;
P. E. Carter, L. Butler, I. R. Booth, and F. M. Thomson-Carter, Abstr. 99th Gen. Meet. Am. Soc. Microbiol., p. 32, 1999). Alterations in this region have been correlated with an enhanced
mutation rate and are implicated in the emergence of new pathogenic
clones (19).
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FIG. 1.
The mutS-rpoS genomic region located at
61 min on E. coli K-12 chromosome. Primers designed from
fhlA (FP1) and rpoS (RP1) of the K-12 genomic
sequences (1) produce a long PCR amplicon of 10.9 kb.
Previous studies have shown that Salmonella strains have a
40-kb pathogenicity island (SPI-1) between fhlA and
mutS (27) and that the genomic region between
mutS and rpoS in E. coli O157:H7 and
Shigella strains is variable in length (19,
20).
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The purpose of this study was to assess DNA polymorphism in the
mutS-rpoS region and to infer the evolutionary history of divergence of this region among pathogenic strains of E. coli. The study focuses on four groups of pathogenic strains
(45) representing enteropathogenic E. coli
(EPEC), a prominent cause of infantile diarrhea in the developing world
(29), and enterohemorrhagic E. coli (EHEC), a
major cause of food-borne illness (29). We used a
combination of restriction fragment length polymorphism (RFLP) analysis
and nucleotide sequencing to characterize the genetic variation in the
mutS-rpoS region among the EPEC and EHEC strains and
compared the variation to that in nonpathogenic strains isolated from
natural populations and strains closely related to laboratory strain
K-12.
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MATERIALS AND METHODS |
Bacterial strains.
Of the 27 strains used in this
study (Table 1), 21 were implicated in
diarrheal diseases. Nineteen were originally isolated from patients,
one (EDL-933) was isolated from hamburger implicated in an 1982 outbreak of hemolytic colitis, and DEC 8c was isolated from a calf with
scours. The laboratory strain K-12 and five ECOR (E. coli
reference collection) group A strains originally isolated from natural
populations (33) were also included. Twenty of the 21 pathogenic strains represent classical serotypes of EPEC and EHEC
(Table 1). The pathogenic strains have been classified previously into
four clonal groups (EPEC 1, EPEC 2, EHEC 1, and EHEC 2) based on
analysis by multilocus enzyme electrophoresis (MLEE)
(43-45). Another pathogenic strain included in this study (921-B4, serotype O111:H9), originally recovered from a disease outbreak in Finland (42), does not fall into one of the four groups (T. S. Whittam, unpublished data). All isolates are
epidemiologically unrelated. Bacteria were maintained in Luria-Bertani
broth supplemented with 20% glycerol at 
70°C.
Preparation of genomic DNA.
Genomic DNA was prepared from 1 ml of bacterial culture grown in Luria-Bertani broth (37°C, 16 h, 150 rpm) with a PUREGENE genomic DNA isolation kit (Gentra Systems
Inc., Minneapolis, Minn.) and was stored at 4°C.
Restriction enzyme analysis.
An amplicon extending from the
3'-end region of fhlA to the 5'-end region of
rpoS including the entire mutS gene was produced using primers fhlA FP1 and rpoS RP1 (Fig. 1). The
predicted PCR amplicon in K-12 contains eight open reading frames
(ORFs) (including mutS) and has a size of 10,950 bp. The
primers were designed on the fhlA and rpoS
sequences from the E. coli K-12 genome (GenBank accession
no. U29579). The primer sequences and positions in the K-12 genome
(indicated in parentheses) are as follows: fhlA FP1,
5'-CGCGCGGTATTGCTAACACG-3' (28461 to 28481); and
rpoS RP1, 5'-GATTCGCCAGACGATTGAAC-3' (39391 to
39411). The DNA was amplified with the Taq Plus Long PCR system
(Stratagene, La Jolla, Calif.). While kept on ice, 50 ng (in 1 µl) of
purified genomic DNA template was mixed with 49 µl of PCR mixture
containing 20 mM Tris-HCl (pH 9.2), 60 mM KCl, 2 mM MgCl2,
0.5 µM each primer, 250 µM each deoxynucleoside triphosphate
(dNTP), and 5 U of Taq Plus Long polymerase mixture. Samples were
heated at 94°C for 5 min and then subjected to 30 PCR cycles
consisting of 30 s at 94°C, 1 min at 56°C, and 20 min at
72°C. Amplicons were electrophoresed in 0.4% SeaKem Gold agarose gel
in 1× Tris-borate-EDTA (TBE) buffer with ethidium bromide at 0.5 µg/ml. The amplicons (2 µl) and the ladder (1 µg) were heated for
10 min at 65°C prior to gel loading, and electrophoresis was
performed under ~5 mm of buffer overlay at 1.5 V/cm for 20 h.
The fragment sizes were determined using the DNA ProScan molecular
weight software (DNA ProScan, Inc., Nashville, Tenn.) with lambda
DNA/HindIII (GIBCO-BRL, Life Technologies, Rockville,
Md.) as a standard.
To estimate the length of the
mutS-rpoS genomic region and
to map the length variation, the long PCR amplicons were digested
with
five restriction enzymes,
EcoRV and
NdeI (New
England Biolabs,
Beverly, Mass.),
Csp45 (Promega, Madison,
Wis.), and
AccI and
NspI (GIBCO-BRL). A total of
2 µl of product was digested with
20 U of enzyme for 16 h at
37°C. Restriction fragments were electrophoresed
in a 0.75% SeaKem
GTG agarose gel (1× TBE, 5 h, 6 V/cm) with 1
µg of 1-kb DNA
ladder (GIBCO-BRL) as a size standard. DNA fragments
stained with
ethidium bromide were detected under UV illumination,
and fragment
sizes were estimated with DNA ProScan molecular weight
software.
RFLP in the
mutS-rpoS genomic region was further assessed by
digestion of 5 µl of the product (containing the long PCR amplicon)
with 10 U of four-base cutter restriction endonucleases
(
AluI
and
Sau3A1; Promega) at 37°C for 2 h. Restriction fragments were
electrophoresed in a 4% NuSieve GTG
agarose gel (1× TBE; 6 h,
6 V/cm) with 1 µg of the 100-bp DNA
ladder (GIBCO-BRL) as a size
standard.
Nucleotide sequencing of DNA located in the mutS-rpoS
intergenic region.
Strains DEC 1a and E2348/69 (EPEC 1), DEC 12e
(EPEC 2), and DEC 9f (EHEC 2) were used for nucleotide sequencing. The
region extending from the 5' end of ORF o388 (see Fig. 3) to
the 3' end of rpoS was amplified with the Taq Plus Long PCR
system using two primers, o388 FP2
(5'-CCGGAAGCAATCGACGCACT-3'; positions 34758 to 34778) and
rpoS RP2 (5'-GTGTTCGCCAGATTCAGGTT-3'; positions 38936 to 38956), designed from the E. coli K-12 genome. For
long PCR, 50 ng (in 1 µl) of purified genomic DNA template was mixed on ice with 49 µl of PCR mixture containing 20 mM Tris-HCl (pH 8.75),
10 mM KCl, 10 mM (NH4)2SO4, 2 mM
MgCl2, 0.1% Triton X-100, 0.1 mg of nuclease-free bovine
serum albumin per ml, 0.5 µM each primer, 250 µM each dNTP, and 5 U
of Taq Plus Long polymerase mixture. Samples were heated at 94°C for
3 min and then subjected to 30 PCR cycles consisting of 30 s at
94°C, 1 min at 56°C, and 6.5 min at 72°C.
Cycle sequencing PCR was performed with a Prism Ready Reaction
DyeTerminator cycle sequencing kit from Applied Biosystems.
Sequencing
gels were run on an Applied Biosystems 373A automated
sequencer. Raw
sequences of both DNA strands were analyzed and
concatenated by DNASTAR
(Madison, Wis.) software. Additional internal
sequencing primers were
sequentially designed as sequence data
were generated. All conflicting
and putative polymorphic nucleotides
sites were sequenced at least
three times on both strands with
multiple primers to eliminate
sequencing
errors.
Phylogenetic analysis.
For comparative purposes, sequences
of the mutS-rpoS genomic region from GenBank were included
in the analysis for E. coli K-12 (AE000357 and AE000358) and
O157:H7 strains (ECAJ6210) and for Shigella (AF055472).
Rates of synonymous and nonsynonymous substitutions were estimated by
the Nei-Gojobori method (30), and gene phylogenies were
constructed using the neighbor-joining method (40) in MEGA
(16).
Nucleotide sequence accession numbers.
The sequences
reported here were deposited in GenBank with accession numbers AF242208
to AF242211.
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RESULTS |
Size variation and DNA polymorphism in the mutS-rpoS
region.
The genomic region between mutS and
rpoS was amplified from strain K-12 as a single ~10.9-kb
PCR product as predicted from the genomic sequence (Fig. 1).
Application of the long PCR primers to 27 strains of E. coli
resulted in amplicons that ranged in size between 8.0 and 14.5 kb (Fig.
2B). A comparison of the amplified DNA
among strains of distinct phylogenetic groups defined previous by MLEE
(Fig. 2A) shows that the long PCR amplicons were consistent in size
within the major groups but different in size between groups (Fig. 2B).
The average sizes based on electrophoresis were ~11.0 kb for E. coli K-12 and ECOR group A strains, ~14.5 kb for each EPEC 1, EPEC 2, and EHEC 2 strain, and ~8.0 kb for O157:H7 and other strains
of the EHEC 1 group (Fig. 2B). Strain 921-B4, an O111:H9 pathogen that
does not belong to the EPEC or EHEC groups (Fig. 2A), produced a
14.5-kb amplicon (Fig. 2B).
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FIG. 2.
(A) Neighbor-joining tree of 27 E. coli
strains based on genetic distances estimated from MLEE data. The clonal
groups based on electrophoretic type (ET) and strain names are, from
left to right: K-12, ECOR group A (ECOR 1, ECOR 10, ECOR 7, ECOR 2, and
ECOR 3), EPEC 2 (DEC 12e, 125-55, DEC 12c, DEC 12d, and DEC 11a),
921-B4, EHEC 2 (DEC 9e, DEC 8c, DEC 8b, 928/91, and CL 37), EPEC 1 (DEC
2a, D55, DEC 1a, E2348/69, and E851/71), and EHEC 1 (DEC 5d, OK-1,
86-24, EDL-933, and 93-111). (B) Resolution of long PCR amplicons
corresponding to the genomic region extending from mutS to
rpoS. The molecular weight marker (MW) is the lambda
DNA/HindIII ladder. (C) Restriction fragments (>200 bp
in length) of the mutS-rpoS long PCR products digested with
AluI. The molecular weight marker is a 100-bp DNA ladder.
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We digested the
mutS-rpoS amplicons with five six-base
cutter restriction enzymes to estimate more accurately the total length
and to create maps of the restriction sites (Table
2 and Fig.
3). Restriction digests with four of
the enzymes (
EcoRV,
NdeI,
AccI, and
Csp45) could not distinguish strains of the EHEC 2 and
EPEC
2 groups; however, some genetic differences between strains
of these
groups were obtained using
NspI (Table
2). The
NspI
digest also revealed that the
mutS-rpoS
sequence in strain 921-B4
differs from that of EPEC 2 and EHEC 2 strains (Table
2).
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FIG. 3.
Restriction maps of the mutS-rpoS chromosomal
region. Approximate locations of restriction sites for five restriction
enzymes: EcoRV (E), NdeI (N), AccI
(A), Csp45 (C), and NspI (Ns). The pattern of
restriction sites is conserved among strains of each pathogenic group
with the exception of the second NspI site in
mutS [(Ns)*], which is present in EPEC 2 strains but
absent in EHEC 2 strains. A distinct NspI map was obtained
for 921-B4 (not shown). The novel DNA segment found in EPEC and EHEC
strains is located at the 3' end of rpoS and is highlighted
with the gray bar.
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Comparisons of the restriction maps (Fig.
3) show that bacteria from
the EPEC 1, EPEC 2, and EHEC 2 groups share a distinct
DNA segment
located between
o454 and
rpoS; this ~2.9-kb
novel
DNA segment is not found in K-12 or strains of the ECOR group
A. The maps reveal that the genes occur in the same order in all
strains
of the EPEC 1, EPEC 2, and EHEC 2 groups (Fig.
3). In
addition, there
is an extra 400-bp segment at the 5' end of
mutS in EPEC 1 strains (Fig.
3). In contrast, strains from the EHEC
1 group have a
shorter
mutS-rpoS region with 3.1 kb less than
K-12 and its
relatives and ~6.0 kb less than the EPEC 1, EPEC
2, and EHEC 2 groups. Absence of the restriction fragments predicted
from the K-12
sequence for
NdeI,
EcoRV,
NspI, and
AccI between
positions 6797 bp (
NdeI) and 11793 bp (
NspI) indicates that part
of the genomic DNA between
mutS and
rpoS in EHEC 1 strains is
missing or
changed. The fact that restriction sites for
NspI (position
5843 in K-12) and
NdeI (position 5982 in K-12) as well as
several
upstream sites are intact suggests that the sequence from
mutS to ORF
o218 has been conserved in both
O157:H7 and DEC 5d (O55:H7)
strains (Fig.
3). The remaining DNA is
highly divergent compared
to the K-12 group (Fig.
3). A short segment
of DNA between
mutS and
rpoS in O157:H7 has been
reported previously (
4); Carter
et al., Abstr. 99th Gen. Meet. Am.
Soc. Microbiol.,
1999.
Digestion of the long PCR amplicons with the four-base cutting enzyme
AluI resolves the 27
E. coli isolates into 11 different
RFLP types (Fig.
2C). A phylogenetic analysis (not shown)
based
on the presence and absence of restriction sites showed that the
EPEC and EHEC strains can be separated into three groups consistent
with the previously defined phylogeny based on MLEE (Fig.
2A).
RFLP
data generated by
Sau3A1 revealed a similar phylogeny (data
not shown). The RFLP analysis shows that the O111:H9 strain (921-B4)
belongs to a branch between the EPEC 2 and EHEC 2 groups, consistent
with the MLEE-based dendrogram (Fig.
2A); however, the RFLP analysis
could not resolve the relationship between EPEC 2 and EHEC 2 strains
(C. Herbelin, S. D. Reid, and T. S. Whittam, Abstr. 99th Gen.
Meet. Am. Soc. Microbiol., p. 237,
1999).
Sequence analysis of genes in mutS-rpoS region.
We
sequenced genes located in the mutS-rpoS intervening region
with genomic DNA isolated from four strains, DEC 1a and E2348/69 (EPEC
1 group), DEC 9f (EHEC 2), and DEC 12e (EPEC 2). The novel DNA sequence
found in these strains contains the same ORFs (yclC, pad1, and slyA) found in the mutS-rpoS
region of E. coli O157:H7 (Carter et al., Abstr. 99th Gen.
Meet. Am. Soc. Microbiol., 1999). The 5' end of the intergenic sequence
between o454 and the yclC-slyA segment contains
100 bp with more than 90% identity to the 3'-end sequence of
rpoS from S. enterica serovar Typhi and a
sequence with homology to sequence directly downstream from the 3' end of rpoS in serovars Typhimurium and Typhi. This sequence
appears to be a remnant of an ancestral inversion (see Discussion) and is oriented in the opposite direction of the E. coli rpoS
gene (Fig. 1).
Although the restriction analysis indicates substantial variation in
the size and gene content of the
mutS-rpoS region among
pathogenic groups, sequence analysis reveals that individual genes
are
highly conserved (Fig.
4). Pairwise
comparison of the sequences
shows that the percentage of polymorphic
nucleotides ranges from
1.3 to 3.7% for genes in the
mutS-rpoS region (Table
3).
This
level of nucleotide polymorphism is intermediate between those
for
the highly conserved
rpoS sequences and the more variable
mutS sequences that flank the region (Table
3).
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FIG. 4.
Polymorphic codons (pc) of four protein-coding genes in
the mutS-rpoS region. Boxes highlight amino acid
replacements. (A) Three out of 22 pc predict replacements (I191V,
K202T, and A206T) in o454 (1,362 bp); (B) 5 out of 86 pc
predict replacements (A36D, T58I, A63T, T83A, and D119E) in
yclC (1,425 bp); (C) 6 out of 22 pc predict replacements
(C12C, K37T, R52H, T70I, M124T, and H179Y) in pad1 (591 bp);
(D) 6 out of 21 pc predict replacements (A2T, A81P, I98V, A119G, M126V,
and T135A) in slyA (405 bp).
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The degree of selective constraint on sequence divergence can also be
seen in comparison of the differences at synonymous
(
dS) and nonsynonymous
(
dN) sites. For all comparisons (Table
3),
dS exceeds
dN
(
dN dS < 0), a pattern
indicating the past
action of purifying selection against mutations
resulting in amino
acid replacements. These results indicate that the
genes between
mutS and
rpoS have levels of
polymorphism similar to those for
mutS and
rpoS
and are conserved at the amino acid level, with
divergence attributable
to the accumulation of silent substitutions
(Fig.
4).
To analyze the history of sequence divergence in
mutS-rpoS
region, sequence data corresponding to the ORFs common to each
group of
strains were combined. For comparison of K-12 to the
EPEC and EHEC
strains, we combined the coding sequences for two
adjacent genes,
o258 and
o454 (Fig.
3), which covered 2,136 nucleotides
and included 48 variable sites. We inferred a
neighbor-joining
phylogenetic tree for the
o258-o454 genomic
region from the divergence
at synonymous sites for the 712 codons of
the combined genes (Fig.
5A). The
phylogeny shows that the EPEC 1 strains (DEC 1a and E2348/69)
are most
divergent, differing from the sequences of the other
three strains at
more than 2% of the synonymous sites. The
o258-o454 sequence of K-12 shares its most recent ancestor with the homologous
region of DEC 9f (EHEC 2) and DEC 12e (EPEC 2) strains, which
are
themselves very similar (Fig.
5A). The results suggest that
the
o258-o454 region was present in the ancestral genome prior
to the divergence of the EPEC and EHEC 2 groups and the K-12 lineage.
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FIG. 5.
Phylogenetic trees of the genomic region between
mutS and rpoS. The trees were constructed by the
neighbor-joining algorithm, with genetic distance measured by the
number of synonymous substitutions per 100 synonymous sites. (A) Gene
phylogeny for the combined coding sequences o258 and
o454 found in K-12, EPEC, and EHEC strains; (B) gene
phylogeny for the combined sequences of yclC,
pad1, and slyA found in E. coli
O157:H7, EPEC, and EHEC strains.
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To compare the O157:H7 sequence with those of the EPEC and EHEC 2 groups, the coding sequences of three genes (
yclC,
pad1,
and
slyA) were combined. The combined
sequence is 2,421 bp long
(807 codons), with a total of 133 variable
nucleotide sites including
17 that predict amino acid differences. A
neighbor-joining tree,
constructed using divergence at synonymous
sites, separates the
five strains into three divergent branches
differing at ~6% of
the synonymous sites based on this region (Fig.
5B). The EPEC
1 strains (DEC 1a and E2348/69) are closely related to
each other,
as are the EPEC 2 (DEC 12e) and EHEC 2 (DEC 9f) strains
(Fig.
5B). Although the O157:H7 sequence joins with the EPEC 1 branch
in the phylogeny (Fig.
5B), this node is not supported by a significant
bootstrap value. In this case, the analysis suggests that the
yclC-slyA region was also present in the common ancestral
strain
prior to the radiation of the pathogenic
lineages.
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DISCUSSION |
A key observation of this study is the substantial size
variation occurring in the mutS-rpoS region between clonal
groups of pathogenic E. coli. Strains from the EHEC 1 group,
including O157:H7 and O55:H7, have a distinctively short intervening
region. In a previous study, LeClerc et al. (19) observed a
similar length for the mutS-rpoS region in O157:H7 and
O55:H7 strains, which are closely related. They found larger deletions
at the 3' end of mutS in the "mutator" phenotype of an
O157:H7 strain, which is characterized by a higher frequency of
mutations that confer antibiotic resistance. They also reported the
presence of a novel DNA sequence (~2.7 kb) in the
mutS-rpoS region in nonmutator strains of O157:H7 serotype,
as well as in O55:H7 and two related ECOR strains. Here we also found
the novel DNA sequence (~2.9 kb) reported by LeClerc et al.
(19) in the mutS-rpoS region of EPEC groups and
EHEC 2 strains but not in strain K-12 or related isolates of the ECOR
group A. The extent to which the length and composition of the
mutS-rpoS region influence local or genomewide mutation
rates remains to be elucidated.
Evolutionary model.
Several lines of evidence support the idea
that the ancestral E. coli had a long mutS-rpoS
region that contained both of the segments that are now found
separately in E. coli K-12 and O157:H7 strains. First, a
long genomic region, with the genes in identical order, is found in the
two highly divergent EPEC lineages. Individual gene phylogenies (Fig.
5) are compatible with the phylogeny of the pathogenic clones (Fig.
2A). The phylogenies each have four deep branches, consistent with the
idea that the sequences were present in the most recent common ancestor
of the pathogenic groups. Second, the genes of the regions are
conserved, with an average rate of synonymous substitution that greatly
exceeds the nonsynonymous rate (Table 3), indicating that the coding
sequence is under purifying selection. The pattern of synonymous codon
usage is similar to that found for the majority of E. coli
conserved genes, an observation that is counter to the hypothesis that
the region was recently acquired as foreign DNA (20). The
sequence analysis showed that most of the divergence at the sequence
level is silent and that the function of the proteins has been, for the
most part, conserved. Both observations support the idea that the
genomic region is old.
The hypothesis that the primitive
mutS-rpoS genomic region
contained all of the genes now found in the various
E. coli
lineages
implies that sequence divergence was accompanied by major
deletions
that eventually gave rise to the shortened intergenic region
now
seen in strains K-12 and O157:H7. An evolutionary scenario for
these changes is outlined in Fig.
6. The
cladogram to the left
depicts the branching pattern for the clonal
frames of the pathogenic
groups supported by previous data from MLEE
(
45). In this phylogeny,
the ancestral lineages leading to
EPEC 1 strains split first followed
by the O157:H7 lineage (EHEC 1),
the K-12 and ECOR group A lineages,
and finally the split of the EPEC 2 and EHEC 2 groups. Given that
EPEC 1 is the most basal group, the
primitive ancestor is posited
to have a long intergenic region with
both
f265-o454 and
yclC-slyA segments. This
ancestral arrangement of genes is conserved as
lineages diverge and is
found in the contemporary EHEC 2 and EPEC
groups. To account for the
shortened regions, two major deletions
are hypothesized to have
occurred: the loss of
yclC-slyA in the
branch leading to the
ECOR group A and the loss of
f265-o454 in
the branch leading
to EHEC 1. The
yclC-slyA deletion must have
happened before
the recent radiation of the ECOR group A. The
f265-o454
deletion also must have occurred before the most recent
common ancestor
of O157:H7 and other members of the EHEC 1 group
(
8). This
deletion must also have preceded the divergence of
EHEC 1 from related
ECOR strains (ECOR 37 and 42) (
26,
35)
that have the same
proximal borders as O157:H7 based on colony
hybridizations
(
20). Finally, we hypothesize that the EPEC 1
group acquired
a small insert upstream of
mutS; however, this
could be an
ancient remnant that was lost near the base of the
cladogram. The
nature of this event can eventually be resolved
by sequencing of this
region and comparison to more divergent
outgroups.
View larger version (25K):
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|
FIG. 6.
Evolutionary model of the mutS-rpoS genomic
region. (A) The left side is a cladogram of the phylogeny of the
groups; the right side shows a diagram of the genes in the
mutS-rpoS region. The EPEC 1, EPEC 2, and EHEC 2 strains
have a conserved ancestral sequence in both the f265-o454
and yclC-slyA gene segments. The model predicts two
independent deletions of gene segments: the loss of
yclC-slyA in the branch leading to K-12 and the ECOR group A
strains and the loss of the f265-o454 segment in the branch
leading to O55:H7, O157:H7, and other EHEC 1 strains. It is not clear
if the additional DNA upstream of mutS in the EPEC 1 strains
is acquired (as marked here) or is ancestral and has been lost early in
divergence. (B) Orientation of the genes for ancestral E. coli and Salmonella. The location of a short sequence
with high homology to the Salmonella rpoS gene is marked as
the rpoS remnant.
|
|
The evolutionary model (Fig.
6) is a parsimonious explanation for the
contemporary DNA polymorphism in the
mutS-rpoS region.
Other
possible scenarios would require multiple gains and losses
of the
entire region or pieces of the region in independent lineages.
Moreover, the source of the imported DNA would have to be such
that the
mutations in the sequences would be consistent with the
chromosomal
background that followed the divergence of the clonal
groups
(
17). These alternative models cannot be ruled out at
this
point. The simple model (Fig.
6), requiring two independent
deletions,
can be tested against these alternatives by examining
the
mutS-rpoS region in other groups of
E. coli.
Testing the model with Salmonella genomic
sequences.
One prediction from the phylogenetic analysis is that
the long intergenic region was present in the most recent ancestor of the E. coli groups. To test this idea, we did a BLAST
comparison of the mutS-o454 segment of K-12 and the
o454-slyA segment from EPEC1 against the unfinished
microbial genomes of S. enterica serovars Typhimurium,
Typhi, Paratyphi A, and Enteritidis. The purpose of this search was to
determine if homologous sequences occur in S. enterica and
to infer the extent to which the arrangement has been preserved in the
100 million years of separation of these bacterial species. The search
produced 30 sequences with significant alignments (results not shown).
These sequences were assembled into a single contig with DNASTAR. The
National Center for Biotechnology Information ORF finder and subsequent
BLAST searches were used to identify homologous genes in E. coli.
The search of the unfinished
Salmonella genomes yielded two
important results. First, homologs to all of the genes in the
mutS-rpoS intervening region of
E. coli occur in
Salmonella genomes,
and the sequences can be assembled into
a contig. This finding
supports the evolutionary model that the
ancestral
mutS-rpoS region
contained both
f265-o454 and
yclC-slyA segments and that the
short
mutS-rpoS regions of K-12 and O157:H7 groups are
derived states.
The level of sequence divergence also supports the
hypothesis
that these segments are ancestral; for example, the
yclC genes
from
E. coli O157:H7 and EPEC 1 are
0.84% divergent in amino acid
sequence and 2.5% divergent from the
Salmonella yclC homolog.
Second, the orientation of the
f265-o454 and
yclC-slyA segments,
relative to
mutS and
rpoS (Fig.
6B), has changed in such a
way
that there have been at least two inversions since
S. enterica and
E. coli shared a common ancestor. The
order of the homologous
genes in the
f265-o454 and
yclC-slyA segments is conserved; however,
each of these
segments lies in the opposite orientation relative
to
mutS
and
rpoS in
S. enterica (Fig.
6B). This inverted
arrangement
may also account for the remnant
Salmonella rpoS
sequence located
between
o454 and
yclC in the
E. coli genome. We suggest that this
remnant is a piece of
rpoS that was carried with the ancient inversion
that
resulted in the present orientation of
yclC-slyA in
E. coli.
Putative function of the yclC-slyA genes.
The
observation that the novel DNA is conserved in the EPEC and EHEC
pathogenic groups suggests that the products of yclC, pad1, and slyA function in pathogenesis.
Comparison to homologous proteins yields few insights into the role of
these genes in pathogenic E. coli. For example, in
Saccharomyces cerevisiae, the yclC gene encodes a
transmembrane voltage-gated Cl
protein with 13 hydrophobic domains (14), and the pad1 gene encodes phenylacrylic acid decarboxylase (PAD), which confers resistance to phenylacrylic acids (6). The predicted
242-amino-acid PAD polypeptide is 48% identical to the product of
dedF of E. coli (6). It is a
single-copy gene in the yeast genome and not essential for viability
(6). pad-related genes have also been described
for Bacillus subtilis, Bacillus pumilus, and
Lactobacillus plantarum (4).
A function of SlyA in pathogenesis is suggested by results from
Salmonella, where it has been shown to play a role in
bacterial
survival in the intracellular environment of host macrophages
(
3,
22). In
Salmonella infection, SlyA regulates
expression
of multiple proteins during stationary phase and upon
phagocytosis
by macrophages (
3). Its expression is required
for the destruction
of M cells but not for invasion or colonization of
the murine
small intestine (
7). A homologous gene has been
found at 37
min on the
E. coli K-12 genome, and its product
confers a hemolytic
phenotype by activating expression of
clyA, which encodes a member
of the RTX toxin family
(
23,
24,
34).
Moreover, SlyA is distantly related to a broad family of bacterial
regulatory proteins affecting diverse aspects of bacterial
physiology,
such as repression of microcin production, intrinsic
multiple
antibiotic resistance, and repression of growth in
E. coli. The family includes MrpA, HpcR, MarA, and Prs from
E. coli,
Hpr from
B. subtilis, and PecS from
Erwinia
chrysanthemi (
41).
Globally, members of this broad
family play a role in the internal
economy of the cell and govern
functions crucial for survival,
inactivation of deleterious exogenous
compounds, cytotoxicity
to the host, and acquisition of a resistant
phenotype. Despite
the sequence similarity, the nature of expression of
yclC,
pad1,
or
slyA, as well as the
function of the proteins in pathogenesis
and bacterial survival,
remains to be
evaluated.
Conclusion.
The mutS-rpoS region has diverged
dramatically among pathogenic groups of E. coli,
accumulating many point mutations in conserved genes, as well as
undergoing changes in gene content. Strains of three pathogenic groups
(EPEC 1, EPEC 2, and EHEC 2) contain a full array of genes between
rpoS and mutS which is hypothesized to reflect
the primitive state found before E. coli separated from
Salmonella. The evolutionary model proposed here invokes two
separate deletion events that resulted in the shorter
mutS-rpoS genomic region, characteristic of the E. coli O157:H7 and K-12 lineages, and may contribute to the
ecological specialization of these bacteria.
| |
ACKNOWLEDGMENTS |
We thank Andrew Clark and Heidi Waldrip for use of the DNA
ProScan program and Sheila Plock for assistance with the Applied Biosystems 373A automated sequencer. Preliminary sequence data were
obtained from The Institute for Genomic Research website at
http://www.tigr.org.
This research was supported by Public Health Service grant AI 42391.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: IMEG, Department
of Biology, 208 Mueller Laboratory, Pennsylvania State University, University Park, PA 16802-3500. Phone: (814) 863-1970. Fax: (814) 865-9131. E-mail: tswl{at}psu.edu.
| |
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Top
Abstract
Introduction
