Download Identification of an Insertion Sequence Located

Identification of an Insertion Sequence Located
in a Region Encoding Virulence Factors of
Streptococcus pyogenes
Andreas Berge, Magnus Rasmussen and Lars Björck
Infect. Immun. 1998, 66(7):3449.
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INFECTION AND IMMUNITY, July 1998, p. 3449–3453
0019-9567/98/$04.0010
Copyright © 1998, American Society for Microbiology. All Rights Reserved.
Vol. 66, No. 7
Identification of an Insertion Sequence Located in a Region
Encoding Virulence Factors of Streptococcus pyogenes
ANDREAS BERGE,* MAGNUS RASMUSSEN,
AND
LARS BJÖRCK
Section for Molecular Pathogenesis, Department of Cell and Molecular Biology, Lund University, Lund, Sweden
Received 18 August 1997/Returned for modification 25 November 1997/Accepted 28 April 1998
broth (27), supplemented with 100 mg of ampicillin (Sigma, St.
Louis, Mo.) per ml when cells contained plasmids. S. pyogenes
strains were grown in Todd-Hewitt broth (Difco, Detroit,
Mich.) in 5% CO2 at 37°C.
DNA methods and homology searches. Streptococcal DNA
was isolated by the method described by Pitcher et al. (22),
which was modified by adding a digestion with 1,000 U of
mutanolysin (Sigma) per ml and 100 mg of lysozyme (Sigma)
per ml in 0.1 M potassium phosphate buffer (pH 6.2) to replace
the initial incubation step. PCR was performed with Taq DNA
polymerase (Gibco-BRL, Gaithersburg, Md.). Standard ligation, transformation, and plasmid isolation procedures were
used (27). Ligase and restriction enzymes were obtained from
Gibco-BRL. Double-stranded DNA sequencing was done by
the dideoxy nucleotide chain termination method (28) with the
T7 sequencing kit (Pharmacia Biotech, Uppsala, Sweden) with
the use of synthetic oligonucleotides that hybridize to the obtained sequence. Southern blotting was performed according
to the methods of Sambrook et al. (27), with Hybond-N membranes (Amersham, Amersham, United Kingdom). The Megaprime DNA labeling system (Amersham) was used to generate
probes from PCR products. The oligonucleotides used in this
study (Fig. 1B) to generate different PCR products were
named as follows (with the source indicated in parentheses):
A16 (7), nucleotides (nt) 896 to 880; ES1 (1), nt 871 to 890;
1474 (7), nt 1114 to 1137; 1476 (7), nt 3960 to 3937; 3244 (Fig.
1), nt 601 to 624; 3266 (Fig. 1), nt 1265 to 1290; 3267 (Fig. 1),
nt 1604 to 1575; and 4419 (15), 59-TTA CCA/G TCA ACA
GGT/C GAA A/GCA GCT/A AAC CCA TTG-39, a partly
degenerate oligonucleotide hybridizing with the consensus sequences of SF1 and SF3 coding for the LPXTGE motif (30).
Homology searches were performed at the National Center for
Biotechnology Information by using the BLAST network service, the Wisconsin package (version 8; Genetics Computer
Group, Madison, Wis.), and the available sequences from the
Streptococcal Genome Sequencing Project, Department of
Chemistry and Biochemistry, The University of Oklahoma,
Norman, Okla.
Characterization of the sequence between the sic and scpA
genes. Chromosomal DNA from the AP1 strain was used as
the target in PCR with primers derived from the 39 end of sic
(ES1) and the strand complementary to the 59 end of scpA
(A16) (Fig. 1). PCR with these primers generated a product
with a length of 2,300 bp. The sequence revealed an open
reading frame (ORF) of 1,203 bp oriented in the direction
opposite that of the other genes of the mga regulon (Fig. 1B).
The ORF is flanked by three sets of inverted repeats. nt 379 to
404 are complementary to nt 1861 to 1836 (IR1 in Fig. 1A) in
Chromosomal rearrangements and horizontal gene transfer
promote microbial evolution and can be facilitated by insertion
sequences (IS). These mobile genetic elements, by definition,
contain genes related only to insertion functions (4). Despite
this definition, the phenotype of the recipient bacterium can be
changed if the IS is inserted into a structural gene or if the
insertion in front of a gene affects the expression of a downstream gene(s) (11). IS can also mediate deletions, duplications, and inversions and cointegrate formation contributing to
changes in the bacterial genome. Virulence genes in pathogenic bacteria are often located together on the chromosome
in so-called pathogenicity islands (12). In gram-negative bacteria these islands can contain IS which promote rearrangements and the horizontal transfer of virulence genes (12).
Chromosomal regions in gram-positive bacteria also exhibit
similarities to pathogenicity islands, but so far they have not
been reported to contain IS (12).
Streptococcus pyogenes is an important human pathogen.
Among significant virulence factors are the M or M-like proteins (10, 26) and the C5a peptidase (17, 21, 33), encoded by
one to three emm-like genes and scpA, respectively. These
genes are colocated on the chromosome and are referred to as
the mga regulon. Their expression is controlled by the positive
transcriptional regulator Mga encoded by mga located upstream from emm (5, 20, 31) (Fig. 1B). In some strains of S.
pyogenes the emm-like genes and scpA are separated by a
3,300-bp DNA segment. In the strain of the M1 serotype studied here, this segment contains the gene coding for protein
SIC, sic (1). In this study, sequencing of the remaining part of
this segment led to the identification of an IS (IS1562) in a
chromosomal locus important for the virulence of S. pyogenes.
Bacterial strains, plasmids, and growth conditions. Sequencing of the segment described above was done with S.
pyogenes AP1 (strain 40/58 of the M1 serotype from the Institute of Hygiene and Epidemiology, Prague, Czech Republic).
Forty-eight additional strains of different M serotypes were
also obtained from this institute. A collection of strains of the
M1 serotype isolated in Sweden in 1988 to 1989 from patients
with septicemia were kindly provided by Stig Holm, Umeå
University, Umeå, Sweden (16). For direct cloning of PCR
products the pGEM-T vector (Promega, Madison, Wis.) was
used. The pGEM-T vector was transformed into competent
Escherichia coli JM109. Cells were grown in Luria-Bertani
* Corresponding author. Mailing address: Department of Cell and
Molecular Biology, Section for Molecular Pathogenesis, Lund University, P.O. Box 94, S-221 00 Lund, Sweden. Phone: 46-46-2224488. Fax:
46-46-157756. E-mail: [email protected].
3449
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An insertion sequence, IS1562, was identified in a Streptococcus pyogenes strain of the clinically important M1
serotype. IS1562 is located in the mga regulon between the genes coding for the M protein and the C5a
peptidase, both important virulence factors. The same or similar insertion sequences were found in most S.
pyogenes strains, but the chromosomal location differed among isolates.
INFECT. IMMUN.
NOTES
3450
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VOL. 66, 1998
NOTES
3451
FIG. 1. The nucleotide sequence containing IS1562, deduced amino acid sequence of the ORF protein, and the relation to other genes in the mga regulon. (A)
Nucleotide sequence and deduced amino acid sequence of the ORF from S. pyogenes AP1. The sequence of the PCR product generated with primers derived from
scpA (A16) and sic (ES1) (1, 7) as shown. The deduced amino acid sequence predicts a protein with a molecular mass of 46,382 Da. Boxes mark cysteine residues
arranged in a pattern resembling a zinc finger motif or an iron sulfur cluster (8, 9). Possible terminal inverted repeats are marked IR1. A star marks the end of the
homology to the promoter region of scpA in other strains. A sequence which matches the consensus sequence to which Mga binds (20) is shown. (B) Schematic
representation of the mga regulon of S. pyogenes AP1. The protein encoded by mga is a transacting positive regulator of transcription of the two emm-like genes, (emm1
and sph), sic, and scpA. The ORF is located on the opposite strand compared to the other genes in the mga regulon. Apart from the AP1 strain and other strains of
the M1 serotype, the ORF sequence is also found immediately upstream of scpA in strains of the M12 and M55 serotypes. Primers used to screen for the ORF sequence
and to identify the flanking genes are depicted (not in scale) in the positions at which they hybridize on the AP1 chromosome.
TABLE 1. Peptide sequences homologous to the ORF protein
identified with the blastp and tfasta programs
Organism
Amino acid
%
GenBank
Amino acid positions with
ReferIdentity
accession
positions in homology to
ence
(%
no.
the ORF
the CDSa
similarity)
protein
Y. enterocolitica Z18920
S. haemolyticus
U35635
ORF 1
ORF 2
ORF 2
ORF 2
S. aureus
L14017
N. meningitidis
B. fragilis
B. vulgatus
B. fragilis
a
35
3
2
No CDS
1–401
37 (78)
112–152
51–71
93–119
145–154
122–162
267–287
302–328
348–357
46 (58)
47 (71)
33 (53)
50 (70)
TABLE 2. Chromosomal DNA from different strains of S. pyogenes
used as targets in PCR amplifications with oligonucleotides derived
from the ORF sequence, the emm or emm-like genes,
or scpA (see Fig. 1B)
51–71
93–119
145–154
267–287
302–328
348–357
47 (71)
33 (53)
50 (70)
Straina OF
Z49092
14
12–25
57–76
89–116
140–153
234–253
268–295
42 (78)
45 (60)
32 (53)
X72301
24
62–83
96–118
223–255
54–75
89–111
240–272
45 (72)
43 (56)
33 (57)
X71444
13
62–83
96–118
223–255
54–75
89–111
240–272
45 (72)
43 (56)
33 (57)
59–83
96–118
51–75
89–111
40 (72)
43 (56)
X76949
32
homologies to Z18920 (Yersinia enterocolitica) and U35635
(Staphylococcus haemolyticus) were found in different reading
frames of these sequences. However, IS use programmed
translational frameshift as a method to control expression and
can consequently translate a protein from different reading
frames (6). Only the Y. enterocolitica and the S. haemolyticus
sequences showed homology regions distributed over the entire ORF protein sequence, including the putative zinc finger
motif. Finally, the sequence determined in this study was compared to the sequences from the Streptococcal Genome Sequencing Project determined with a strain of the M1 serotype,
and an almost identical sequence (different in 6 of 2,307 bases)
was found. The region of homology is located upstream from
scpA in the sequencing project. Within the ORF protein sequence, 400 of 401 amino acid residues were identical. No
other significant homologies were identified.
In search of the ORF sequence in other strains of S. pyogenes. Some strains of S. pyogenes express an apolipoproteinase
called the opacity factor (OF) (29). With primer pairs 32443267 and A16-3267 within the mga regulon (Fig. 1B), none of
these OF-positive strains generated PCR products (Table 2).
In contrast, most of the OF-negative strains gave rise to bands
by PCR (Table 2), and in strains of M serotypes 1, 12, and 55,
the ORF sequence was found immediately upstream the scpA
gene. Southern blotting experiments, following digestion of
chromosomal DNA with XbaI, EcoRI, ClaI, and HindIII and
probing with scpA and the ORF sequence, verified these results (Fig. 2). In OF-positive strains several bands hybridized
with the ORF probe, as exemplified by strain AP4 (Fig. 2), and
multiple bands were also seen in several of the OF-negative
strains. The results demonstrate that sequences homologous to
CDS, coding region defined by the authors of the databank entry.
Size of PCR product (bp) generated with
primer pair indicated
3244-3267 A16-3267 A16-4419 ES1-3244 3244-4419 3266-3267
AP1
Umeå
AP12
AP55
AP6
AP23
AP53
AP5
AP57
AP2
AP4
AP25
AP58
2
2
2
2
2
2
2
2
2
1
1
1
1
1,000
1,000
1,000
1,000
1,000
1,000
1,000
1,500
1,500
1,500
1,500
1,600
1,600
500
500
500
500
500
500
500
500
500
2,800
2,800
2,800
2,800
350
350
350
350
350
350
350
350
350
350
350
a
The number denotes the M protein serotype. Umeå refers to eight strains of
the M1 serotype isolated from patients with septicemia (16).
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21 of 26 nt. Two other possible inverted repeats are the sequences at nt 411 to 429 and 2217 to 2199 (15 of 19 nt hybridize) and sequences at nt 385 to 394 and 1830 to 1821, which
match perfectly.
The nucleotide sequence obtained had the highest homology
to GenBank entry J05229, coding for the C5a peptidase from
an S. pyogenes strain of the M12 serotype. The sequence in Fig.
1 is 98% homologous to nt 1 to 887 in J05229. nt 1 to 380 show
extensive homology to the region upstream from scpA in the S.
pyogenes strains in which the gene sequence is known. When nt
1 to 380 were excluded, no homologies to any sequence in the
database, except the previously mentioned scpA and sic sequences, were found (1).
The translation of the ORF predicts a protein with 401
amino acids and a molecular mass of 46,382 Da. Five of the
eight cysteines in the protein are located in the COOH-terminal region, and the distribution of these cysteines (C-X2-CX25-C-X2-C-X5-C) resembles DNA-binding zinc finger motifs
(9) or iron sulfur clusters (8). Significant homologies with the
protein were found only among peptides encoded by other IS
or a sequence of unknown function (Z18920) (Table 1). The
3452
NOTES
INFECT. IMMUN.
the ORF are found, in variable numbers, in most S. pyogenes
strains. As mentioned above, primer pair 3244-3267 failed to
generate PCR products in OF-positive strains. However,
primer pair 3266-3267 generated bands with expected sizes,
showing that a region similar to the ORF sequence encoding
the putative zinc finger motif is present in these strains.
Whether these sequences are part of an IS also in OF-positive
strains is under investigation.
Conclusions. In the AP1 strain of the M1 serotype, the
sequence between the genes encoding protein SIC and the C5a
peptidase was determined. It was found to contain an ORF
with homology to six proteins encoded by IS in other bacterial
species. IS encode transposases responsible for the transposition of the IS (11). Three sets of inverted repeats flanking the
ORF were found, but none was flanked by target site duplications. The homology to the promoter region of scpA in other
strains ends at nt 380, suggesting that the repeats (marked IR1
in Fig. 1) are used by the IS. The sequence fulfills the criteria
for an IS (4) and was designated IS1562.
One IS has previously been described in S. pyogenes, IS1239
(18). It is located 103 bp upstream from the gene encoding the
streptococcal superantigen SSA (25) in a strain of the M15
serotype. IS can enhance the transcription of downstream
genes (11), suggesting that the two IS thus far identified in S.
pyogenes may contribute to virulence. IS can also form compound transposons by flanking other genes. It is possible that
both IS1562 and sic are remains of a compound transposon
inserted in the mga regulon. Finally, horizontal gene transfer
has been implicated in playing an important role in the pathogenicity and epidemiology of S. pyogenes (19, 23, 34). Consequently, the property of IS to facilitate recombinational events
(11), and thereby genetic flexibility and microbial adaptation,
could also promote the virulence of this important human
pathogen.
Nucleotide sequence accession number. The GenBank accession number of the nucleotide sequence determined in this
study is AF064540.
This work was supported by grants from the Swedish Medical Research Council (project 7480); the Medical Faculty, Lund University;
the Foundations of Kock and Österlund; the Göran Gustavsson Foundation for Research in Natural Sciences and Medicine; and Actinova,
Ltd.
We are indebted to Arne Olsén for kindly providing the modification of the method used for isolation of streptococcal DNA and to
Kristin Persson for excellent technical assistance.
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VOL. 66, 1998
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NOTES