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Infection and Immunity, October 2001, p. 6012-6021, Vol. 69, No. 10
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.10.6012-6021.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Ferric Dicitrate Transport System (Fec) of Shigella
flexneri 2a YSH6000 Is Encoded on a Novel Pathogenicity
Island Carrying Multiple Antibiotic Resistance Genes
Shelley N.
Luck,1
Sally A.
Turner,1
Kumar
Rajakumar,1,2
Harry
Sakellaris,1,* and
Ben
Adler1
Bacterial Pathogenesis Research Group,
Department of Microbiology, Monash University, Victoria
3800,1 and Department of Microbiology
and Infectious Diseases, Royal Children's Hospital, Parkville,
Victoria 3052,2 Australia
Received 24 April 2001/Returned for modification 12 June
2001/Accepted 18 July 2001
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ABSTRACT |
Iron uptake systems which are critical for bacterial survival and
which may play important roles in bacterial virulence are often carried
on mobile elements, such as plasmids and pathogenicity islands (PAIs).
In the present study, we identified and characterized a ferric
dicitrate uptake system (Fec) in Shigella flexneri
serotype 2a that is encoded by a novel PAI termed the
Shigella resistance locus (SRL) PAI. The
fec genes are transcribed in S. flexneri, and complementation of a fec deletion in
Escherichia coli demonstrated that they are functional.
However, insertional inactivation of fecI, leading to a
loss in fec gene expression, did not impair the growth
of the parent strain of S. flexneri in iron-limited culture media, suggesting that S. flexneri carries
additional iron uptake systems capable of compensating for the loss of
Fec-mediated iron uptake. DNA sequence analysis showed that the
fec genes are linked to a cluster of multiple antibiotic
resistance determinants, designated the SRL, on the chromosome of
S. flexneri 2a. Both the SRL and fec loci
are carried on the 66,257-bp SRL PAI, which has integrated into the
serX tRNA gene and which carries at least 22 prophage-related open reading frames, including one for a P4-like integrase. This is the first example of a PAI that carries genes encoding antibiotic resistance and the first report of a ferric dicitrate uptake system in Shigella.
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INTRODUCTION |
Pathogenicity islands (PAIs)
are increasingly recognized as playing a vital role in bacterial
virulence. PAIs are distinct virulence cassettes that often integrate
into tRNA genes and encode bacteriophage-like integrases. Such islands
may occupy large regions of the chromosome and often carry mobile
elements, such as insertion sequences and transposons
(25). PAIs have been found in many bacterial species,
including Yersinia spp. (9, 13),
enteropathogenic, enterohemorrhagic, and uropathogenic
Escherichia coli (24, 38, 51), Salmonella
enterica serovar Typhimurium (61), Vibrio cholerae (32), Helicobacter pylori
(14), and Shigella flexneri (2, 42, 57,
71). Some strains of uropathogenic E. coli and
S. enterica serovar Typhimurium may harbor at least five
PAIs (19, 74). A variety of virulence determinants may be
carried on PAIs, including genes encoding fimbriae, hemolysins
(31, 64), type III secretion systems (15,
27), and iron uptake systems (13, 42, 71, 75).
Various Shigella spp. produce the siderophores enterobactin
and/or aerobactin, which are involved in iron uptake (34,
50). The aerobactin locus in S. flexneri was
recently shown to be carried on the SHI-2 PAI (42, 71). This was the first report of an iron transport system being carried on
a PAI in Shigella.
A number of PAI-like elements in Shigella spp. have been
described. These include the SHI-2 PAI and a family of structurally related elements (42, 71) and the she PAI,
which also belongs to a larger family of structurally related elements
(2, 57). One of the characteristics of PAIs is their
tendency to excise spontaneously from their sites of integration in the
chromosome (26). In the S. flexneri 2a strain
YSH6000, the spontaneous loss of multiple antibiotic resistance is
accompanied by the deletion of an approximately 99-kb chromosomal
region (56). The deletion of this region also coincides
with a 50% decrease in contact hemolysis, a trait that correlates
closely with virulence in Shigella spp. (56).
These findings suggested that the 99-kb region is a deletable genomic
element that carries multiple antibiotic resistance determinants. Preliminary sequence analysis of the 99-kb deletable element, which we
have termed the multiple resistance deletable element (MRDE),
demonstrated that the four antibiotic resistance determinants associated with the element are clustered within a 16-kb region (54) which we have termed the Shigella
resistance locus (SRL). We recently found that the loss of multiple
antibiotic resistance also occurs via a second type of spontaneous
deletion event involving a distinct 66-kb element contained within the
99-kb MRDE (66). In the present study, we demonstrate that
the 66-kb element is a PAI, termed the SRL PAI, that encodes a
functional ferric dicitrate uptake system.
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MATERIALS AND METHODS |
Bacterial strains, plasmids, and media.
Bacterial strains
and plasmids used in this study are listed in Table
1. Strains were grown routinely with
aeration at 37°C in either 2YT broth (40) or
Luria-Bertani broth (LB) (5) with the addition of
ampicillin (100 µg/ml), kanamycin (50 µg/ml), or tetracycline (12.5 µg/ml) when necessary.
Molecular techniques.
Plasmid DNA was isolated using a
modified alkaline lysis method (35), while genomic DNA was
isolated as described previously (5). Restriction digests
were carried out using enzymes supplied by Roche Molecular Biochemicals
or New England Biolabs. Transformation of E. coli and
S. flexneri strains was performed following electroporation (63) with a Bio-Rad gene pulser at 1.8 kV, 25 µF, and
200 
in 0.1-cm electroporation cuvettes.
RNA was extracted from
E. coli and
S. flexneri
strains for expression analysis of the
fec locus. Inocula
were prepared by
growing bacteria overnight in LB supplemented with
antibiotics
where necessary. Following centrifugation at 10,000 ×
g for 1
min, cells were washed in 1 ml of Fec medium
(
49) modified by
the addition of 2',2-dipyridyl (0.4 mM)
and citrate (1 mM). Fifty
milliliters of modified Fec medium was
inoculated with 100 µl
of each bacterial suspension and incubated
with aeration until
early exponential phase (4 h). Cells were
centrifuged at 1,300
×
g for 10 min, and the
supernatant was discarded. RNA was extracted
as described previously
(
62) and treated with DNase (Roche)
to remove DNA
contamination. RNA dot blots were probed with either
a
fecA
DNA fragment or a
recA fragment that served as a control
for
sample loading. Probes were derived by PCR amplification
(
fecA,
5'-GTTGTCGTCATAAGAGCGG-3' and
5'-GCTCCCATTTCGCTCGGC-3';
recA,
5'-CTACGCACGTAAACTGGGCG-3' and
5'-ACCGGTAGTGGTTTCCGGG-3') and
labeled with digoxigenin
(Roche) as recommended by the manufacturer.
The RNA concentration of
matched strain pairs SBA844-AA93, YSH6000-YSH6000T,
and
SBA1366-SBA1415 was standardized by absorbance at 260 nm.
The
equal loading of RNA on membranes was confirmed by dot blot
analysis
with the
recA-derived
probe.
PCR and sequencing.
Standard PCR, single strand-specific PCR
(sspPCR), and inverse PCR were performed as described previously
(47; PCR Applications Manual, 2nd ed., Roche
Molecular Biochemicals; J. Novak and L. Novak, Promega Notes Magazine
61:26-29, 1997). Long-range PCR was carried out with the
Expand long-range PCR kit (Roche).
Nucleotide sequencing of the PAI in
S. flexneri strain
YSH6000 was carried out by sequencing genomic clones, inverse PCR
products,
sspPCR products, and a long-range PCR product. Sequence
reactions
were conducted using the BigDye system (PE Biosystems Inc.).
Reaction
products were analyzed on an Applied Biosystems model 373A DNA
sequencing system. Sequence editing was carried out using Sequencher
3.0 for Macintosh. Sequence analysis and database comparisons
were
performed using BlastN and BlastX (
3). Analysis of
proteins
was carried out using previously described web-based analysis
tools (
28,
29,
43,
46,
60).
Assays of growth under iron-limited conditions.
Modified Fec
medium (49) for growth under iron-limited conditions
consisted of LB containing 0.4 mM 2',2-dipyridyl, 1 mM citrate, and
kanamycin when required. Inocula were prepared by growing strains
overnight in 2.5 ml of LB or LB containing kanamycin for the
maintenance of plasmids. To remove exogenous iron, bacteria were
centrifuged at 10,000 × g for 1 min and resuspended in
modified Fec medium. Following a second wash in modified Fec medium,
bacterial suspensions were standardized by absorbance at 600 nm. Fifty
milliliters of the modified Fec medium was inoculated with 0.1 ml of
the standardized bacterial suspension. Aerated cultures were incubated
at 37°C. Two-milliliter samples were taken over a 24-h period (2, 4, 6, 8, 12, and 24 h), and the absorbance at 600 nm was measured.
Four cultures of each strain were grown simultaneously. Viable counts were also performed at 0 and 24 h to compare with absorbance readings.
Nucleotide sequence accession number.
Nucleotide sequences
have been deposited in GenBank under the accession number AF326777.
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RESULTS |
Identification and functional analysis of the fec
iron transport locus in S. flexneri 2a.
Sequencing
of the regions surrounding the SRL using marker-rescued clones
(described below) revealed a locus that was homologous to the ferric
dicitrate transport (fec) genes located at min 97.3 of the
E. coli K-12 genome (68). Like the E. coli fec locus, the Shigella locus consists of two
operons carrying the regulatory genes, fecI and
fecR, and the downstream structural genes,
fecABCDE. The S. flexneri fec genes showed
more than 99% nucleotide identity with the E. coli K-12
genes, but there were differences in the regions flanking the locus.
The E. coli K-12 locus is flanked upstream by IS1
and downstream by an IS911 element that is insertionally disrupted by an IS30 and a truncated IS2. In
contrast, the S. flexneri fec locus was flanked downstream
by an intact IS911. The sequence directly downstream of
IS911 is identical in E. coli and S. flexneri. Upstream of the S. flexneri fec locus were
the first 61 bp of IS1 followed by remnants of
IS3, IS629, and IS903-like elements.
Prior to this report, the fec locus had been identified only
in E. coli strains (36, 53, 65, 72), although
fecA, fecD, and fecE homologs had been
observed in H. pylori (69). This is the first
report of an intact ferric dicitrate transport system in a
Shigella sp.
The ferric dicitrate iron transport system has been well characterized
in
E. coli K-12 (
17). It is capable of
maintaining
the growth of
E. coli under iron-limited
conditions in the absence
of other iron uptake systems. Because of the
high similarity between
the
S. flexneri and
E. coli
fec loci, the function of the SRL
PAI
fec locus was
tested in an
E. coli 
fec strain, AA93
(
49).
In LB alone, all strains grew equally well (data not
shown). However,
the growth rate of AA93 under iron-limited conditions
(LB supplemented
with 0.4 mM 2,2'-dipyridyl and 1 mM citrate) was
greatly reduced
(Fig.
1A).
Complementation of AA93 with the cloned
S. flexneri fec
locus (pSBA491), but not the cloning vector alone, restored
its ability
to grow under iron-limited conditions (Fig.
1A), demonstrating
that the
S. flexneri fec locus is functional.
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FIG. 1.
Growth of E. coli (A) and S.
flexneri (B) under iron-limiting conditions in medium
supplemented with citrate. (A) SBA844 (solid line) is a
fec strain complemented with the S. flexneri
fec locus, and SBA845 (broken line) is the
fec strain carrying an empty vector. (B) S.
flexneri strains used were the wild-type SBA1366 (solid line),
the fecI::kan strain SBA1415
(short dashes), and the MRDE deletant strain YSH6000T (long dashes).
Error bars represent 2 standard deviations.
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Although the
fec genes are functional in
E. coli,
it was necessary to establish their ability to function in
S. flexneri.
FecI is a member of the sigma 70 factor subclass that
responds
to extracytoplasmic stimuli and regulates extracytoplasmic
functions
(
37). In
E. coli, the
fecI
gene is essential for the transcription
of the
fecABCDE
operon (
48). Therefore, to study the function
of the
fec locus in
S. flexneri, we inactivated the
fecI gene
by inserting the
kan cassette from
pUC4-KIXX into the unique
XhoI
site within
fecI.
To introduce the
fecI mutation into the
S. flexneri chromosome, a 5.1-kb PCR product containing
fecI::
kan,
fecR, and
the 5'
end of
fecA was cloned into the suicide vector pCACTUS
and
subsequently introduced by electroporation into SBA1366 (YSH6000,
SRL
). A double-crossover mutant, SBA1415, was
selected by growth
at 42°C in the presence of sucrose. The mutation
was confirmed
by PCR using primers within, and external to, the pCACTUS
construct
(data not
shown).
To test whether the Fec iron transport system had a role in the growth
of
S. flexneri 2a YSH6000 under iron-limited
conditions,
the
fecI::
kan
mutant was compared to its parent (SBA1366) and
strain YSH6000T,
which has undergone a spontaneous excision of
the 99-kb element.
When strains were cultured under iron-limited
conditions in medium
supplemented with citrate, there was no significant
difference in
growth rate between any of the strains (Fig.
1B);
neither mutation nor
loss of the
fec locus had any effect on the
growth of
S. flexneri 2a strain
YSH6000.
There were several possible explanations for the lack of
phenotypic difference between the wild-type strain and the
fecI mutant,
including the possibility that the
fec locus is not transcribed
in
S. flexneri
or that
fecI may not be essential for transcription
of
the
fec structural genes in
S. flexneri. Both of
these hypotheses
were tested by RNA dot blot analysis of the
expression of
fec in YSH6000, YSH6000T, AA93, SBA844,
SBA1366, and SBA1415 cells
grown under iron-limited conditions (see
Materials and
Methods).
Although analysis with a
recA probe confirmed that equal
amounts of RNA were loaded for each pair on the dot blot,
fecA mRNA
was undetectable in the
S. flexneri
fecI mutant, SBA1415, but
readily detectable in the isogenic
parent strain, SBA1366, and
in the wild-type strain YSH6000 (Fig.
2). As expected,
fecA
transcript
was undetectable in the negative control strains YSH6000T
and
AA93, which do not carry the
fec locus, but was detected
in an
AA93 strain complemented with the
S. flexneri fec
locus (SBA844).
These results demonstrated that the
fec
locus is expressed in
YSH6000 in a
fecI-dependent manner and
therefore suggest that
this strain carries additional iron uptake
systems that compensate
for the
fec mutation when grown in
laboratory culture media.
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FIG. 2.
Transcriptional analysis of fecA in
S. flexneri and E. coli. An RNA dot blot
compares matched strain pairs (E. coli SBA844-AA93 and
S. flexneri YSH6000-YSH6000T and SBA1366-SBA1415) for
transcription of fecA under iron-limited conditions.
Lanes: 1, undiluted sample; R, samples treated with RNase to ensure
that there was no DNase contamination. SBA844, YSH6000, and SBA1366
carry the S. flexneri fec locus, while AA93 and
YSH6000T do not. SBA1415 carries the fec locus with a
kan cassette inserted in fecI.
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Southern hybridization showed that the
fec locus is present
in a single copy in YSH6000 and absent in YSH6000T (data not shown).
Therefore, if a second iron uptake system exists in YSH6000, it
must
belong to another class. Recently, aerobactin-mediated iron
uptake systems were found on proposed PAIs on the chromosome of
several
S. flexneri serotypes (
42,
71). To test whether
such
a system existed in YSH6000, we examined this strain for the
presence
of
iucA, the first gene in the aerobactin
biosynthesis operon.
A PCR product was amplified from strains YSH6000
and YSH6000T
using primers designed from the SHI-2 PAI
iucA
region. The sequence
of the PCR product from YSH6000T confirmed that an
iucA gene identical
to that from the SHI-2 PAI was
present in YSH6000T. Therefore,
it seems possible that an
aerobactin locus and/or other types
of iron uptake systems may
have compensated for the loss of
fec function in
SBA1415.
The fec locus is present on a PAI.
Iron
transport systems have been identified on several PAIs, including the
high-pathogenicity island (HPI) of Yersinia spp. and
E. coli (10, 33, 59), Salmonella
pathogenicity island 1 of S. enterica serovar
Typhimurium (30), and
PAI-VICFT073 in uropathogenic E. coli, which carries a putative iron transport system
(23). Several of these systems, as well as the
aerobactin iron transport system on the SHI-2 PAI of S. flexneri (42, 71), have roles in virulence. Based on
the findings presented below, we demonstrated that the S. flexneri fec locus also is carried on a PAI that in addition
carries the multiple antibiotic resistance genes of the SRL.
Southern hybridization analysis demonstrated that the
fec
locus is present in
S. flexneri 2a YSH6000 but is absent in
the
spontaneous, antibiotic-sensitive derivative YSH6000T, suggesting
that the
fec locus and the SRL are physically linked. This
was
confirmed by DNA sequencing, which showed that the tetracycline
and
chloramphenicol resistance determinants characterized by Rajakumar
et
al. (
57) are on the same
BamHI fragment as the
fec locus
(Fig.
3). We have
found that the
fec locus is present in a variety
of
Shigella strains. Thirty-five of 55
Shigella
strains examined
by high-stringency Southern analysis carried the
fecA gene. Long-range
PCR analysis of a single sample strain
from each
Shigella species
with the same antibiotic
resistance profile as strain YSH6000
confirmed that in each case the
fec locus is linked to the tetracycline
resistance gene, as
it is in strain YSH6000 (data not shown).
This suggests that the SRL
PAI is present in all species of
Shigella.
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FIG. 3.
Genetic organization of the SRL PAI. A map of the SRL
PAI sequence from bp 1 to 31,987 (A) and 31,988 to 66,257 (B) is shown.
The ORFs are represented by boxes above the line (forward orientation)
or below the line (reverse orientation). The unlabeled open
boxes represent ORFs of unknown function. IS elements (grey boxes) have
been designated A through M (Table 3). Boxes with light grey horizontal
lines represent the CP4 prophage-related ORFs, while boxes with
diagonal black lines represent 933L prophage-related ORFs. Boxes with
black horizontal lines represent ORFs that have homologs on both the
933L and CP4 prophages. Sequence data were derived from the genomic
subclones pSBA509 and pSBA361 and PCR-derived products indicated below
each map. The G+C content of the SRL PAI was plotted using a window
size of 100 bp.
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The antibiotic resistance determinants of strain YSH6000 delete
spontaneously from the chromosome at a frequency of
10
5 to 10
6
(
66), suggesting that the SRL, and therefore the closely
linked
fec locus, may be carried on a mobile element. To
gain a better
understanding of the element, the regions flanking the
SRL were
sequenced using plasmid clones obtained by marker rescue of
the
resistance determinants encoded by the SRL.
BamHI
fragments of
27.6 kb, carried on pSBA361, and 24 kb, carried on pSBA509
(Fig.
3), were cloned by selection for tetracycline and ampicillin
resistance,
respectively. The remainder of the element was sequenced
from
DNA fragments derived by inverse PCR, sspPCR, and long-range PCR
(Fig.
3).
Analysis of the DNA sequence showed that a large genetic element has
inserted into the 3' terminal region of the
serX tRNA
gene
in YSH6000 (Fig.
4A). The element is
bounded on the
serX-distal
side by a 14-bp direct repeat
(DR) of the 3'-terminal 14-bp sequence
of
serX (Fig.
4A).
The DNA sequences upstream of
serX and downstream
of the
serX-distal DR are almost identical to sequences that are
contiguous with the
serX gene in
E. coli, a
species that is closely
related to
S. flexneri. Notably, the
3' termini of tRNA genes
commonly serve as integration sites for PAIs
and prophages (
25).
In addition, the 3' terminus of
serX has sequence similarity to
a P4
att site
(Fig.
4B), implying that it may act as an integration
site for
prophage-like or PAI-like elements. The sequence of the
genetic element
revealed the presence of a P4 bacteriophage-like
integrase gene 161 bp
upstream of the 14-bp
serX-distal DR. The
integration of the
element into the 3' end of a tRNA gene, the
presence of an integrase
gene near one boundary of the element,
and the recent finding that the
element undergoes spontaneous,
integrase-mediated, precise excision to
restore the
serX locus
as it is organized in
E. coli (
66) led us to conclude that the
element is a
PAI, which we have termed the SRL PAI.
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FIG. 4.
Structure of the SRL PAI and the DR. (A) The SRL PAI is
represented by the open box. The shaded boxes represent the 14-bp DR.
The right-hand DR is the last 14 bp of serX. (B) The
14-bp DR and the potential att site. The lower sequence
shows the 3' end of the S. flexneri YSH6000
serX gene. The region representing the 14-bp DR is
underlined. The upper sequence represents the P4 core
att site. *, conserved nucleotide.
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Interestingly, there was a significant discrepancy in the lengths of
the 66.2-kb SRL PAI and the previously described 99-kb
deletable
element carrying multiple antibiotic resistance in YSH6000
(
56). This discrepancy has been explained by the recent
finding
that the SRL PAI is entirely contained within a yet-larger
distinct
genetic element, termed the MRDE, which is flanked by
IS
91-like
elements and is also capable of precise excision
from the chromosome
(
66) (Fig.
5). This is similar to the
Y. pestis 6/69 HPI, which
is contained within a larger, deletable
102-kb chromosomal region
and which also carries genes for hemin
utilization. The boundaries
of the 102-kb region are defined by two
IS
100 elements which mediate
the spontaneous deletion of the
HPI and flanking chromosomal DNA
in this strain (
9).
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FIG. 5.
Overview of the genetic organization of the S.
flexneri 2a YSH6000 chromosome surrounding the SRL PAI. The
three deletable elements in the region corresponding to min 23 of the
E. coli chromosome are shown. The dashed line represents
the chromosomal region identical to E. coli, while the
solid line represents the 99-kb MRDE. The grey box denotes the SRL PAI,
while the white box is the SRL. The MRDE is flanked by
IS91-like sequences.
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Genetic organization of the SRL PAI.
The SRL PAI is 66,257 bp
in length (Fig. 3), beginning 161 bp upstream of the int
gene and ending at the 3' terminus of the intact serX gene.
The SRL PAI contains 59 open reading
frames (ORFs) (Table 2; Fig. 3), excluding the ORFs associated
with insertion sequences, and has an average G+C content of 49.8 mol%. Although the overall G+C content is not significantly different from
that of the S. flexneri chromosome (51 mol%),
significant deviations occur in the regions homologous to
Tn2603, Tn10, and the fec locus, which
have G+C contents of 57, 39, and 58 mol%, respectively. This is
consistent with the fact that Tn2603 and Tn10 are
laterally acquired elements and suggests that the fec locus
may also have been laterally acquired by the PAI relatively recently.
The deduced product of the
int gene, located near the left
boundary of the SRL PAI, has significant sequence similarity at
the
amino acid level to several integrase proteins from the P4
prophage Int
family, including those from the CP4-57 cryptic prophage
of
E. coli (49% similar) and the
V. cholerae PAI (52%
similar).
Integrases from the other
S. flexneri PAIs,
she PAI and SHI-2,
showed 47 and 45% similarity to the SRL
PAI Int, respectively.
The putative Int protein encoded on the SRL PAI
possesses a conserved
motif (R, HXXR, Y) necessary for the function of
P4-like integrases
(
1,
4), suggesting that the integrase
may be
functional.
Twelve ORFs on the SRL PAI have significant sequence similarity,
ranging from 45 to 88%, to ORFs carried on the CP4-57 prophage
(Fig.
3). Although the physical spacing of the SRL PAI ORFs differed
from
that of their homologues in CP4-57, their order and orientation
are
conserved, with the exception of
orf2 and
orf3 on
the SRL
PAI, which are inverted compared to their CP4-57 homologues,
yfjI and
alpA, respectively. A number of these
SRL PAI ORFs, including
orf16,
orf39, and
orf48, are truncated in comparison to their
CP4-57
homologues. Of the 12 ORFs homologous to CP4-57 ORFs, seven
have
homologues in CP4-44, another cryptic prophage in
E. coli K-12 (
8). Similarity between the SRL ORFs and ORFs on
CP4-44
ranged from 87 to 99%. The SRL PAI also carries homologues of
ORFs L0007 to L0015 from a third prophage, 933L, situated on the
enterohemorrhagic
E. coli (EHEC) locus of enterocyte
effacement
(LEE) PAI. Two of these ORFs, ORFs 53 and 54, are also
common
to CP4-44. Thus, a total of 22 SRL PAI ORFs, comprising just
under
20% of the PAI sequence, appear to have a prophage origin. ORF
47 of the SRL PAI is homologous to autotransporter protein Antigen
43 (87% similarity), encoded by the cryptic prophage CP4-44, YpjA
(37%
similarity), encoded by a CP4-57 ORF of unknown function,
and
Sap (72% similarity), encoded by an ORF of unknown function
on
the
she PAI of
S. flexneri 2a.
The SRL PAI carries six intact insertion sequences and seven IS
remnants that have undergone deletions (Table
3). IS elements
and their remnants,
including IS
1, IS
200, IS
600,
IS
629, and IS
1328,
which are present on the SRL
PAI, are commonly found in other
PAIs (
7,
21,
39,
71). The
SRL PAI also carries an ORF,
designated
shf, that is almost
identical to the previously described
shf ORF carried on the
virulence plasmids of
S. flexneri serotype
2a strain YSH6000
(
55) and serotype 5 (
11). The
shf
ORFs have
sequence characteristics that are common to some mobile
elements,
such as retroviral integrases and IS transposases.
Close homologues
of
shf (>83% similarity at the
protein level) are also found on
the plasmids pAA2 of enteroaggregative
and diffusely adhering
E. coli (
18) and
pO157 of EHEC (
12), as well as a chromosomal
locus
bearing two overlapping genes,
sat1 and
sat2, in
S. enterica serovar Typhimurium. In
Shigella
and
E. coli,
shf is part of a
larger locus
that includes a hexosyltransferase homologue,
capU,
an
msbB homologue, and an IS
911 remnant (Fig.
6).
View larger version (19K):
[in this window]
[in a new window]
|
FIG. 6.
Genetic organization of the shf locus in
S. flexneri and E. coli, showing the
extent of the conserved shf locus in several bacterial
strains. pMYSH6000 is carried by YSH6000, the strain that contains the
SRL PAI. pWR100 is borne by S. flexneri 5 strain M90T
(70). pAA2 is from enteroaggregative E.
coli strain O42 (18), and pO157 is carried by EHEC
O157:H7 (12). shf and capU
are conserved in all loci, although they are truncated in the S.
flexneri chromosome due to an IS1 insertion. An
IS911 is found upstream of three of the loci, although
it is intact only on the S. flexneri SRL PAI chromosomal
locus and is oriented in the reverse direction on pAA2 in
enteroaggregative E. coli. In addition, the
plasmid-borne loci exhibit a conserved organization with regard to
shf, capU, virK, and
msbB2, with pO157 having an alternative gene in place of
virK.
|
|
| |
DISCUSSION |
This study confirms our hypothesis that multiple antibiotic
resistance in S. flexneri strain YSH6000 is encoded by a
PAI. While our previous work suggested that such a PAI might be
approximately 99 kb in length (56), we have shown that the
SRL PAI is only 66.2 kb in length and is located on a distinct 99-kb
element which, like the PAI, is capable of excision from the chromosome
(66). Our conclusion that the antibiotic resistance
determinants of the SRL are carried on a PAI is based on several
characteristic sequence features and genetic properties common to other
PAIs. These include a chromosomal insertion site in the 3' terminus of
a tRNA gene, the presence of short DRs at the boundaries of the
element, the presence of a P4-like integrase gene near one end of the
element, and the recent demonstration that the element undergoes
precise, integrase-mediated excision from the chromosome (66). In addition, the SRL PAI typically contains a large
number of IS elements and transposons. A striking feature of the PAI is
the large number of ORFs that are related to the CP4 group of prophages
and the 933L prophage, which is associated with the EHEC LEE PAI. Of
particular interest is the almost complete conservation in the
organization and orientation of homologous ORFs in the SRL PAI and CP4,
suggesting very strongly that the SRL PAI shares a common ancestry with
these prophages.
The SRL PAI represents the first example of a PAI that contains
multiple antibiotic resistance genes. One other mobile genetic element
that encodes antibiotic resistance, the SXT element of V. cholerae, has some features in common with the SRL PAI. The SXT
element is capable of integrase-mediated, site-specific integration into and excision from the chromosome and carries all of the genes required for conjugative self-transfer to new hosts. However, although
the SRL PAI undergoes site-specific, integrase-mediated excision from
the chromosome, it does not appear to carry any of the genes required
for conjugative transfer. Therefore, the SRL PAI appears to be quite
a distinct type of genetic element. Whether the SRL PAI is capable
of being transferred laterally to new hosts is unknown. However, the
finding that the fec and SRL loci are linked in several
tested strains from each of the four Shigella spp. suggests
that the SRL PAI has disseminated throughout the genus
Shigella.
In addition to antibiotic resistance, the SRL PAI encodes an iron
uptake system. Iron is an essential nutrient in bacteria, where it is a
component of the electron transport system (45) and an
essential cofactor for a variety of enzymes. While iron is readily
available in the environment, in the human host it is stored in
tissues, such as the liver, or chelated by extracellular proteins, such
as transferrin and lactoferrin (22, 41). Intracellular pathogens, such as Shigella, can scavenge iron from within
the cells they invade, but they must also obtain iron from the
extracellular environment of the host. To achieve this they produce
extracellular high-affinity, low-molecular-weight iron chelators,
called siderophores (16, 45). E. coli and some
S. flexneri and Shigella boydii strains
produce the catechol siderophore enterobactin (50), while some S. flexneri, S. boydii, and
Shigella sonnei strains also produce the dihydroxamate
siderophore aerobactin (34). In the present study
we have identified a third type of siderophore system, a ferric
dicitrate system, in S. flexneri 2a. The fec system in YSH6000 is only the second example of iron uptake genes carried on a PAI in Shigella. However, iron uptake genes are
also carried on PAIs in S. enterica serovar Typhimurium,
Yersinia spp., and some pathogenic strains of E. coli (13, 33, 75).
Our work also describes the first example of a ferric dicitrate uptake
system in the genus Shigella. Until now, this type of iron
uptake system has been found only in the commensal strains E. coli B and E. coli K12, E. coli strains causing bovine mastitis (36), and EHEC
O157:H7 strain EDL933 (65). Although it was demonstrated
that the fec locus is functional and is expressed in
S. flexneri strain YSH6000, we could not demonstrate any
alteration in the growth rate of a fecI mutant strain grown
in iron-limited culture media. This finding suggests that YSH6000
expresses additional iron uptake systems that are capable of
compensating for the loss of Fec function. This is consistent with
previous reports of siderophore production in S. flexneri
and the presence of iucA, one of the genes involved in
aerobactin synthesis, in strain YSH6000. Indeed the presence of
multiple iron uptake systems in a single strain is not unusual. For
example, E. coli strains may have up to five iron(III)
transport systems (20). The possession of more than one
iron uptake system may confer on bacteria a greater ability to survive
in different niches outside or inside the host. Since the Fec system is
expressed in nonpathogenic E. coli strains, which unlike
Shigella spp. do not invade intestinal cells, its primary
role in S. flexneri may be in the uptake of iron from the
intestinal lumen, where exogenous citrate is available for the
chelation of iron.
In conclusion, our discovery of the SRL PAI in S. flexneri
has revealed yet another type of genetic element that may be involved in the lateral transfer of antibiotic resistance. In addition, this
element also encodes the first ferric dicitrate uptake system known to
exist in Shigella spp. Future studies will address whether the SRL PAI is naturally mobilized to new bacterial hosts.
| |
ACKNOWLEDGMENTS |
This work was supported by a project grant from the National
Health and Medical Research Council, Canberra, Australia.
We acknowledge the excellent technical assistance of Ian McPherson and
Vicki Vallance.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology, Monash University, VIC 3800, Australia. Phone: 613 9905 4838. Fax: 613 9905 4811. E-mail:
harry.sakellaris{at}med.monash.edu.au.
Editor:
D. L. Burns
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Infection and Immunity, October 2001, p. 6012-6021, Vol. 69, No. 10
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.10.6012-6021.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
