RESEARCH LETTER
Molecular characterization of newly identi¢ed IS3, IS4 and IS30
insertion sequence-like elements in Mycoplasma bovis and their
possible roles in genome plasticity
Inna Lysnyansky1, Michael J. Calcutt2, Idan Ben-Barak3, Yael Ron3, Sharon Levisohn1, Barbara A. Methé4
& David Yogev3
1
Mycoplasma Unit, Kimron Veterinary Institute, Beit Dagan, Israel; 2Department of Veterinary Pathobiology, University of Missouri-Columbia, Columbia,
MO, USA; 3Department of Membrane and Ultrastructure Research, The Microbiology Institute, The Hebrew University-Hadassah Medical School,
Jerusalem, Israel; and 4J. Craig Venter Institute, Rockville, MD, USA
Correspondence: David Yogev, Department
of Membrane and Ultrastructure Research,
The Microbiology Institute, The Hebrew
University-Hadassah Medical School,
Jerusalem 91120, Israel. Tel.: 1972 2 675 81
76; fax: 1972 2 678 40 10; e-mail:
[email protected]
Received 3 November 2008; accepted 25
February 2009
First published online April 2009.
DOI:10.1111/j.1574-6968.2009.01562.x
Editor: Reggie Lo
Keywords
Mycoplasma bovis ; IS; inverted repeat; directly
repeated sequences; target specificity.
Abstract
Insertion sequences (ISs) are mobile genetic elements widely distributed among
bacteria. Their impact on the bacterial genome is multifold, including transfer of
genetic information, shuttle of adaptive traits and influence on the genomic
content. As a result, ISs play an important role in the organization, plasticity and
evolution of bacterial genomes. In this study, four new IS elements: ISMbov7;
ISMbov4 and ISMbov5; and ISMbov6, related, respectively, to the IS3, IS4 and IS30
gene families, were identified and characterized with respect to inverted repeat (IR)
and directly repeated (DR) sequences, putative target specificity and motifs related
to transposase function. For instance, IS30-related ISMbov6 isoform elements were
shown to (1) contain an a-helix-turn-a-helix homeodomain (HTH), (2) generate
long DR and (3) possess target specificity for a palindromic sequence derived from
putative rho-independent transcription terminators. Members of the IS3 family,
which had not been documented previously in Mycoplasma bovis, contain HTH,
leucine zipper and AT-hook motifs, which may be involved in DNA binding. In
addition, the availability of the M. bovis PG45 genome sequence allowed us to
elucidate the genomic organization of 54 intact or truncated IS elements and their
possible effect on the expression of adjacent genes.
Introduction
Prokaryotic insertion sequences (ISs) are among the smallest transposable elements, often present in multiple copies
in a genome. They can be classified into c. 20 families based
on conservation of the catalytic site, similarities in genetic
organization, transposase sequences and terminal inverted
repeats (IRs) (Mahillon & Chandler, 1998). The impact of IS
elements on bacterial genomes is multifold, including
activation or repression of genes and DNA rearrangements
resulting in deletions, inversions and gene amplification
(Chandler & Mahillon, 2002). The resultant genomic plasticity contributes to adaptation of microorganisms to changing environments by dissemination of antibiotic resistance
genes (Boutoille et al., 2004), association with virulence
functions (Kunze et al., 1991) and acquisition of new
metabolic capabilities (Lichter et al., 1996; Schmid-Appert
et al., 1997). In addition, high homology among IS isoforms
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c
(full and truncated) allows them to be involved passively in
DNA homologous recombination.
Sequence analysis of whole genome sequences of Mollicutes available in public databases revealed a very wide range
of IS densities, ranging from none in Mycoplasma genitalium
(Fraser et al., 1995), Mycoplasma pneumoniae (Himmelreich
et al., 1996) and Mycoplasma mobile (Jaffe et al., 2004) to
high density in Mycoplasma mycoides ssp. mycoides SC PG1,
in which ISs are present in 97 copies that comprise 29% of
the genome (Bischof et al., 2006). In general, most transposases identified in mycoplasmas belong to the IS3, IS4 and
IS30 families, although representatives of the IS21, Tra5 and
mutator families have also been found (Chambaud et al.,
2001; Sasaki et al., 2002; Papazisi et al., 2003; Westberg et al.,
2004; Sirand-Pugnet et al., 2007).
Recently, three ISs, designated ISMbov1, ISMbov2 and
ISMbov3, were identified and characterized in the bovine
pathogen Mycoplasma bovis (Thomas et al., 2005). Their
FEMS Microbiol Lett 294 (2009) 172–182
173
Insertion sequence-like elements in Mycoplasma bovis genome
copy number and distribution were shown to differ among
M. bovis field strains (Miles et al., 2005; Thomas et al., 2005),
contributing to the genetic variability of the pathogen.
In this study, we compiled a comprehensive overview of
the abundance, genomic environment and diversity of IS
elements in M. bovis. Four new IS elements were identified
and characterized in the M. bovis genome: ISMbov4 and
ISMbov5 (related to the IS4 gene family); ISMbov6 (IS30
gene family); and ISMbov7 (IS3 gene family). In addition,
the availability of the draft genome sequence for strain PG45
made it possible for us to map multiple isoforms of the
different IS gene families (new and previously described)
and to identify specific examples in which these transposable
elements influence the genome plasticity of M. bovis.
Materials and methods
Mycoplasma strain, growth conditions and DNA
extraction
Mycoplasma bovis strain PG45 and M. bovis strain PG45
clonal variant #6 were propagated at 37 1C in 100 mL
standard mycoplasma broth medium supplemented with
phosphate pyruvate (Chopra-Dewasthaly et al., 2005).
Genomic DNA was extracted using the DNA Isolation Kit
for Blood/Bone marrow/Tissue (Roche Diagnostics GmbH,
Mannheim, Germany).
RNA isolation, reverse transcription (RT) and
RT-PCR
Total RNA was extracted from a mid-logarithmic-phase
culture of M. bovis PG45 using the RNeasy Mini kit
(Qiagen). To eliminate contaminating genomic DNA, total
RNA was subjected to DNAse I (FMI Fermentas) treatment,
as specified by the manufacturer. RT was performed with
the RevertAid Moloney murine leukemia virus reverse
transcriptase (FMI Fermentas), as described previously
(Flitman-Tene et al., 2003), using the 7A-R primer (5 0 CACC
AGCTAGGCCTTTAT 3 0 ) specific to ORF14. The resultant
cDNA product was subjected to PCR using Taq
DNA polymerase (AB, UK) and 5F (5 0 CTTGTT
TTATGCTTAAACTGG 3 0 ) primer specific for 63 nt from
the ISMbov3A gene. The PCR amplification program was as
follows: 30 cycles of denaturation at 95 1C for 30 s, primer
annealing at 52 1C for 30 s and extension at 72 1C for 30 s.
Nucleotide sequence analysis
Nucleotide sequence analysis was performed using DNASTAR
software, version 5.06/5.51, 2003 (Lasergene Inc., Madison,
WI), ExPASy Molecular Biology server of the Swiss Institute
of Bioinformatics (available at http://expasy.org/) and by
NCBI (http://www.ncbi.nlm.nih.gov). The prediction of the
FEMS Microbiol Lett 294 (2009) 172–182
IS secondary structures and a scan of IS sequences for sites/
signatures were performed using the PSIPRED server
(http://bioinf.cs.ucl.ac.uk/psipred/psiform.html) and Proscan server (http://npsa-pbil.ibcp.fr), respectively.
Results and discussion
Molecular properties of new M. bovis
IS30 -related elements
During our previous studies of the vsp locus in M. bovis
clonal variant #6 (Lysnyansky et al., 1999), an IS30-like
element, designated in this study as ISMbov6, was found.
ISMbov6 is 1017 bp in length, encoding a single ORF of
339 aa that shows 41% homology to the IS1630-like transposase of Mycoplasma penetrans (Sasaki et al., 2002). ISMbov6 exhibits only 17.2% identity to previously identified
ISMbov1 of M. bovis (Thomas et al., 2005) and, as such, is a
putative new member of the IS30 family, according to the
criteria of Mahillon & Chandler (1998). Further studies
revealed six HindIII-restricted genomic fragments that hybridized strongly with a DIG-labeled PCR probe complementary to ISMbov6 (data not shown). These fragments
were gel-excised, cloned and sequenced. Overlapping fragments, when needed, were cloned using a previously described genomic library (Lysnyansky et al., 1996). The
nucleotide sequences of ISMbov6-related fragments and
their flanking regions were determined and compared with
sequences currently available in GenBank and later also with
the whole genome sequence of M. bovis PG45. A schematic
representation of six ISMbov6-related elements and flanking
ORFs is shown in Fig. 1 and in Supporting Information,
Table S1.
A scan of the ISMbov6-related elements for identifiable
domains and motifs (http://www.ebi.ac.uk/InterProScan/)
revealed the presence of a homeodomain at the N-terminus
of the transposase (Fig. 2a). The homeodomain motif,
which consists of an a-helix, followed by an a-helix-turna-helix (HTH), has been found in many DNA-binding
proteins including recombinases and transcriptional regulators (Wintjens & Rooman, 1996). It has been shown that
many members of the IS30 family carry a predicted
H–HTH2 motif in their N-terminus regions. Site-directed
mutagenesis and deletions of amino acids from the
H–HTH2 motif of Escherichia coli IS30 revealed that this
motif was required for the insertion of IS30 specifically to IR
sequences as well as to a palindromic hot spot (Nagy &
Chandler, 2004). Protein sequence alignment and prediction
of the secondary structure for ISMbov1 of M. bovis did not
show the presence of the typical H–HTH2 domain (Nagy &
Chandler, 2004), suggesting a different mechanism of target
selection recognition for the two IS30 family members,
ISMbov6 and ISMbov1. In addition, several IS30-related
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174
I. Lysnyansky et al.
1
2
3
4
5
6
7
8
9
10
11
12
transposases, including IS30 itself but not ISMbov6, have an
N-terminal extension in the form of an additional HTH1,
which appears to be responsible for hot spot recognition
(Fig. 2a) (Nagy & Chandler, 2004).
Analysis of nucleotide sequences flanking the left and
right termini of ISMbov6 isoforms revealed the presence of
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c
Fig. 1. Genomic organization of
cloned IS30, IS4 and IS3-like
isoforms in Mycoplasma bovis
clonal variant #6 (1.1) and in PG45
strain (1.2–1.12). The location and
direction of ORFs flanking each
cloned IS-related genomic
fragment (not in scale) are shown
by open labeled arrows. Members
of the IS30 gene family (ISMbov1
and ISMbov6
), the IS4
, ISMbov3
family (ISMbov2
, ISMbov4
and ISMbov5
) and the IS3 family (ISMbov7
) are similarly colored. ORFs
and their gene products are
described in Table S1. The fusion of
the ORF14 and the 63 nt flanking
the ISMbov3A is shown by a black
rectangle. The position of PCR
primers 7A-R and 5F used for
RT-PCR of the orf14 gene is shown
in panel 1.5 by arrows. The status
of the IS element is presented
using the following codes: i,
interrupted; t, truncated; and
p, pseudogene.
almost perfect direct repeats (DRs) in the form of palindromes (Table 1). Interestingly, although ISs transpose to
different target sites, insertion into the genome is not a
random event. It has been shown recently that several
members of the IS30 gene family possess target specificity
upon insertion in the form of palindromic terminal DRs
FEMS Microbiol Lett 294 (2009) 172–182
175
Insertion sequence-like elements in Mycoplasma bovis genome
(a)
H
IS30
T
H1
H
H
T
H2
MRRTFTAEEKASVFELWKNGTGFSEIANILGSKPGTIFTMLRDTGGIKPHERKRAVAHLTLSEREEIRAGLSAKMSIRAIATALNRSPSTISREVQRNRGR
:::
ISMbov6
::
:
::: ::
::::::: :::
::
MNYTKNYNHLTYDERAFIEIQIKSGYSIRSIARQLNRSPSTVSRELKRNTAF
Consensus
Y..LT..ER..I......G.S.R.IA..LGRSPSTISRE..RN...
U U
A
A
C C
A
U
G
U
U
G
U
U A
A-U
G G
G-C
U
A
A-U
A-U
U
G
A-U
U
G
U-A
U-A
A-U
G-C
A:A
A-U
A-U
A-U
G-C
U-A
C-G
A-U
A-U
ISMbov6B
U-A
G-C
G-C
C-G
U:G
orf33
A-U
C-G
U-A
G-C
A-U
A-U
U-A
A-U
A-U
G-C
AAAUAAUUU-82nt-ACAGAA
UUUU
A-U
GAAUAAGU-14nt-CUAAAAA
UUUU
A-U
K *
A-U
E *
C-G
A-U
ISMbov6A
A-U
UCG
U
A
U
A-U
A-U
orf21
C
A
U
C
C-G
U-A
A-U
G-C
GUCUAACUAAAA-123nt-GGACAA
UUUU
A-U
U-A
A-U
V
*
A-U
C-G
ISMbov6C
A-U
AAUUAAA-14nt-CUAAAA
UUUU
A-U
C:A
N *
orf35
G-C
U-A
ISMbov6
A-U
C-G
vspJ
C-G
G-C
A-U
C-G
A-U
A-U
CAAUAAUUA-14nt-UAAUAA
G-C
Q *
UAGAUUU
(b)
U
ISMbov6D
ISMbov7E
ISMbov6E
orf69
Fig. 2. (a) Protein sequence alignment and a secondary structure prediction for Mycoplasma bovis ISMbov6-related transposases. The alignment of the
N-terminal region of ISMbov6 and IS30 from Escherichia coli was performed manually using the sequence consensus predicted by Nagy & Chandler
(2004). The secondary structure prediction of ISMbov6 was made by the PSIPRED server (http://bioinf.cs.ucl.ac.uk/psipred/psiform.html). Conserved
amino acids are marked by a colon. Amino acids encoding HTH are in bold and positions of the HTH1 within IS30 and HTH2 motifs within IS30 and
ISMbov6 are overlined. (b) Insertion of ISMbov6 into IR sequences resembling transcriptional terminators of the neighboring genes. Predicted RNA
secondary structures for putative rho-independent transcription terminators are shown. The C-terminal amino acid (single-letter code) and a stop codon
() are shown to the left of each secondary structure. The secondary structures were derived by deleting the sequence of ISMbov6 and one copy of the
DR. For ISMbov6D and ISMbov6E, the appropriate sequences flanking these elements are not present.
(Olasz et al., 1997; Kiss et al., 2007). For example, sites
targeted by IS30 of E. coli and IS1655 of Neisseria meningitidis are characterized by 24 and 19-bp nearly palindromic
consensus sequences (Olasz et al., 1998; Kiss et al., 2007).
During transposition, IS30 and IS1655 of N. meningitidis
insert into the central portion of these palindromic targets
and duplicate a 2- or a 3-bp sequence, respectively (Olasz
et al., 1998; Kiss et al., 2007). In contrast, Mycoplasma
fermentans IS1630 possesses target specificity for a palindromic sequence that is derived from rho-independent
transcription terminators of neighboring genes (Calcutt
et al., 1999a, b). IS30-related ISMbov6 shows 40% identity
and has more features in common with M. fermentans
IS1630 than with the prototype IS30. Both ISMbov6 and
IS1630 (1) possess similar IRs (42% identity for IRR and
50% for IRL); (2) generate long DRs upon insertion (from
FEMS Microbiol Lett 294 (2009) 172–182
19 to 26 bp for IS1630 and from 22 to 36 bp for ISMbov6);
(3) possess target specificity for a palindromic sequence with
different lengths and without consensus; and (4) generate
duplication of symmetrical IR sequences that are derived
from rho-independent transcription terminators of neighboring genes (Fig. 2b). In the case of ISMbov6E, the
sequence that was disrupted by IS-element insertion cannot
be deduced (IRR and DRR are missing), but the presence of
the stem–loop structure downstream of orf69 is consistent
with this insertion site encoding a transcription terminator
(Fig. 2b). ISMbov6 and IS1630 probably possess the same
specificity for target site and mechanism (as yet unknown)
of transposition. Their target specificity can present some
selective advantages, because palindromic transcription terminators are common features that are usually present in
bacterial genomes. In addition, transcription terminators
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176
usually lie outside the gene unit and therefore minimize
the risk of gene inactivation, which could be deleterious
for the host and, as a consequence, for the mobile element.
Molecular characterization of new M. bovis
IS4 - like elements
Our previous study revealed a new IS-like element, designated ISMbov4, within the vsp locus of PG45 clonal variant
#6 (Fig. 1.1) (Lysnyansky et al., 1999). The ISMbov4 ORF
spans 1434 bp, showing 91% homology to the N-terminal
portion of the MAG4100 transposase (pseudogene) of
Mycoplasma agalactiae (Sirand-Pugnet et al., 2007), and
was found by Southern blot analysis (data not shown) to be
present in nine and eight copies in the M. bovis clonal
variant #6 and PG45 genomes, respectively (Fig. 1 and
Table S1). Interestingly, comparison of genomic organization of the vsp locus between M. bovis clonal variant #6 and
PG45 revealed the presence of ISMbov4 in the former
(Fig. 1.1 and 1.12). The presence of ISMbov4 in M. bovis
clonal variant #6, which is a derivative of M. bovis PG45,
suggests that at least this element is active and can transfer.
ISMbov4 differed markedly from the recently characterized ISMbov2 and ISMbov3 (33.8% and 16.6% identity in
amino acid sequence, respectively) (Thomas et al., 2005),
indicating that it represents a new member of the IS4 family
of bacterial mobile elements.
At this point in our research, the preliminary whole
genome sequence of M. bovis PG45 became available to
extend the genomic and molecular data regarding ISs. Three
additional IS4-related elements, designated ISMbov5, ISMbov5A and ISMbov5B, respectively, were identified (Fig. 1.7
and 1.9). ISMbov5B is 1388 bp in length, encoding an ORF
of 462 aa, whereas ISMbov5 and ISMbov5A are truncated
copies. ISMbov5B shows only 32% aa identity to ISMbov4,
but 91% identity (from 1 to 289 aa) and 94% identity (from
322 to 452 aa) to the N- and C- termini of the M. agalactiae
transposase pseudogenes MAG6150 and MAG6160, respectively (Sirand-Pugnet et al., 2007).
In addition, analysis of the nucleotide sequences that
flank each M. bovis IS4-related element revealed several
features. ISMbov4 isoforms possess 13-bp IRs with two
mismatches (Table 1). ISMbov2 isoforms possess 29-bp IRs
that are identical to the 30-bp IRs of ISMmy1 (Westberg
et al., 2002). Interestingly, similar to ISMmy1, the
C-terminal region of the ISMbov2-related elements contained six nucleotides, including a stop codon (ATATAG)
that overlaps the IRR. Location of the translation termination codon within the IR has been observed in other IS
elements and is suggested to play a role in the regulation of
transposition activity (Mahillon & Chandler, 1998). Analysis
of the nucleotide sequences that flank each ISMbov3-related
element revealed that different copies, like their IS1634
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I. Lysnyansky et al.
homologs from M. mycoides ssp. mycoides SC type strain
PG1 (Vilei et al., 1999), produce DRs of variable length in
the genome, ranging from 16 bp (ISMBov3C) to 145 bp
(ISMBov3B) (Table 1). DRs of four of the six ISMbov3
isoforms (ISMbov3B, ISMbov3D, ISMbov3E and ISMbov3G)
were found within the encoding sequences of neighboring
genes orf9, orf47, orf57 and orf73, respectively (Fig. 1, 1.3,
1.8, 1.9 and 1.11), without the formation of a premature
stop codon or gene inactivation. ISMbov3, together with
IS1634, ISMhp1 of Mycoplasma hyopneumoniae and IS1549
of Mycobacterium smegmatis (Plikaytis et al., 1998; Minion
et al., 2004; Vasconcelos et al., 2005), are the only IS4-related
members capable of producing long variable DRs. As
suggested by Vilei et al. (1999), these elements might use
the same peculiar mechanism of creation of staggered nicks
at variable distances in the target DNA sequence as the initial
step of transposition. The IRs of the ISMbov3 isoforms
contain 17 bp, including the complete 13-bp sequence of
IRs of the IS1634 element (Table 1).
In contrast to the ISMbov3 isoforms, both ISMbov4- and
ISMbov2-related elements possess 6-bp-long AT-rich DRs
with consensus 5 0 NTNT/AAN3 0 and 5 0 ATANAT3 0 , respectively (Table 1 and Fig. 3a and b). No conserved bases were
found in the flanking sequences of ISMbov4- and ISMbov2related DRs. The target consensus sequence of ISMbov4 is
less defined and apparently lacks the palindrome structure
that exists in the target sites of ISMbov2 (Fig. 3b). It is
known that several IS4-related transposases, such as IS10
(Bender & Kleckner, 1992) or IS231A (Hallet et al., 1994),
possess specificity for short well-determined palindromic
sequences (5 0 ATNTWWWWW3 0 , where W indicates an A
or a T). The use of short AT-rich consensus sequences as the
target site for transposition might allow insertions of
ISMbov4 and ISMbov2 almost at random into the
mycoplasma genome, which is 71% A1T in the case of
M. bovis.
Molecular characterization of M. bovis IS3 -like
elements
Screening of the PG45 genome in silico revealed the presence
of nine isoforms, designated ISMbov7 and ISMbov7A-H,
belonging to the IS3 family (Fig. 1.8–1.11, Table S1).
ISMbov7, ISMbov7A-C and ISMbov7H possess 26-bp IRs
with three mismatches and form 3-bp DRs upon insertion.
Other ISMbov7-related elements (ISMbov7E to ISMbov7G)
show different changes within the IRs and lack DRs
(Table 1).
The ISMbov7-related elements, similar to most IS3 family
members, contain a DNA-binding domain (DBD) that may
specifically recognize the terminal IRs during transposition.
The DBD in IS3 transposases contains two motifs: an HTH
motif (between codons 26 and 51 in ISMbov7-related
FEMS Microbiol Lett 294 (2009) 172–182
177
Insertion sequence-like elements in Mycoplasma bovis genome
elements) that is essential for transposase activity, as shown
for IS911 from Shigella dysenteriae (Rousseau et al., 2004),
and a leucine zipper (LZ) motif that is involved in DNA
binding and ensures interaction between the transposase
and its IRs (Fig. 3c) (Mahillon & Chandler, 1998). The
predicted LZ motif of ISMbov7-related elements is located
between codons 84–105 (Fig. 3c).
Amino acid sequence analysis of the HTH motif from
distinct subgroups of IS3 family (IS2, IS3, IS151, IS150 and
IS407) performed by Rousseau et al. (2004) revealed the
presence of a conserved tryptophan residue (W) within the
last a-helix domain. Notably, in all the ISMbov7 isoforms, a
W residue was identified within the HTH motif at position
48, but it was not present in the corresponding position
(between codons 29 and 56) in the closely related ISMmyc2
transposase of M. mycoides ssp. mycoides LC (Fig. 3c). In
Mycoplasma pulmonis IS1138 (Bhugra & Dybvig, 1993), the
HTH motif was not identified, but a W residue is present at
position 49 (Fig. 3c). Point mutations in the W residue of
IS911, resulting in conversion of the amino acid to alanine
Table 1. Characterization of inverted and directly repeated sequences of the IS30, IS4 and IS3-related elements in M. bovis
____________________________________________________________________________________________________________________
IS designation
Inverted repeat (IRL/DRR)
Direct repeat (DRL/DRR)
____________________________________________________________________________________________________________________
ISMbov6
ISMbov6A
GCTTGATTGTAAGTTCAAGTGCAACA
: : :::: ::::::::::::::::
GGTATTTACTTTCAAGTTCACGTTGT
AACAAAATTCAGaTAGTGTAaCTGAATTTTGTT
GACGAAGGTTTTAAGTCTTCGTC
ISMbov6B
AAAGCAAAGTCCGCTTTGCTTT
ISMbov6C
AATATTtGAACAAATAATTATGTTTGTTCgAATATT
ISMbov6D
IRR is within the ISMbov7E-i
GACGCTcAAGTTCCTTaAGCGTC
ISMbov6E
IRR is missing
AACAGACTTATTCGACAAGTCTGTT†
ISMbov1A -i
ISMbov1B -p
ISMbov1C
ISMbov1D
–
IRR is missing
ATA
:::
TAT
IRL is missing
–
–
AAACAAAATGAACA
ATTTATTTTTTGTT
ISMbov1E-i
ISMbov1F
ISMbov1G
ISMbov1H-t
ISMbov1I
ISMbov1K
ISMbov1L
ISMbov1M
ISMbov2A
ISMbov2B-p
ISMbov2C
ISMbov2D-p
ISMbov2E
ISMbov2F -i
ISMbov2G-p
ISMbov2H
ISMbov3A-i
ISMbov3B
–
–
TAAAAAATTCCCAATTTTGGACACTATAT
:::::::::::::::::::::::::::::
ATTTTTTAAGGGTTAAAACCTGTGATATA
TAAAAAATTCtCAATTTTGGACACTATAT
:::::::::: ::::::::::::::::::
ATTTTTTAAGgGTTAAAACCTGTGATATA
Like ISMbov2A
Like ISMbov2A
IRR is missing
Like ISMbov2A
IRL is missing
TCCTACGTTTCCCACTT
:::::::::::::: ::
AGGATGCAAAGGGTCAA
ISMbov3C
ISMbov3D
ISMbov3E
ISMbov3F
ISMbov3G
FEMS Microbiol Lett 294 (2009) 172–182
–
AAACCATATATAAT
TTCTAATTTTTGTT
–
ATTATAAAAAATAC
CTCTAACATTAAGC
–
AAAGGCAAGTCAAT
GTACAT
ATACAT
ATTTAT
ATGTAT
ATATA
ATACTT
–
ATAAAT
–
145 bp (TAAGTC...TTCAAA)
ATACACAAAATATTTA
65 bp(AGTAAA...TGTTTT)
100 bp(AAAATT...AGTTTC)with one mismatch
at position 51
–
100 bp(AAAAA...TATTGG)
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178
I. Lysnyansky et al.
Table 1. Continued
ISMbov4
ISMbov4A
ISMbov4B
ISMbov4C
ISMbov4D
ISMbov4E
ISMbov4F
ISMbov4G
ISMbov4H*
TATAAAAGTTATA
:: ::::::: ::
ATTTTTTCAAAAT
ISMbov7
ISMbov7A
ISMbov7B
ISMbov7C
ISMbov7D-p-t
ISMbov7E-i
TACACTAGGACAAAAAAAGTAGACAT
:: :::::::::: :::: :::::::
ATTTGATCCTGTTATTTTTATCTGTA
TATAAAAGTTATA
:: :::::: ::
GAATCAACATCATTCTTTCAAAAT
CTCTAA
TTTAAT
TTATAA
TTAAAT
ATGAAA
CTGTAA
TTAAAA
ATTTAG
ATAAAT
AAA
TAA
TAA
GAA
–
–
–
TACACTAGGAC-AAAAAAGTAGACAT
IRR is missing
ISMbov7F -p
TACACTAGGAC-AAAAAAGTAGACAT
–
:: :::::::: : :::::::::: :
ATTTGATCCTGTTGTTTTCATCTGGA
ISMbov7G*-p
ACAAAAAAAGCAGACAT
–
:::: :::: ::::::
ATTTGATCCTGTTATTTTTATCTGTA
ISMbov7H
TACACTAGGACAAAAAAAGTAGACAT
GCT
:: :::::::::: :::: :::::::
ATTTGATCCTGTTATTTTTATCTGTA
____________________________________________________________________________________________________________________
IRR is longer than IRL.
w
DRL is only identified on the basis of the palindromic sequence.
i, interrupted gene; p, pseudogene containing a premature stop codon; t, truncated gene.
(A) or phenylalanine (F), had a marked effect on the DNAbinding activity of these mutants in comparison with the
wild-type W (Rousseau et al., 2004). However, mutants in
which the W residue was transformed to tyrosine (Y)
exhibited only slight changes in the DNA-binding activity
and appeared to be as active as the wild-type protein in
catalyzing strand cleavage and transfer (Rousseau et al.,
2004). The presence of Y instead of W in the second
helix of ISMmyc2 (Fig. 3c) supports the possibility of
in vivo functional complementation (helix packing) by the
Y residue. Notably, ISMbov7, ISMmy2 and IS1138 all possess
a third predicted helix domain adjacent to the HTH region,
as was mentioned for IS911 of S. dysenteriae (Fig. 3c)
(Rousseau et al., 2004).
In addition, ISMbov7-related elements possess the small
DNA-binding motif (AT-hook) that was first described in
the high-mobility group I non-histone chromosomal protein HMG-I(Y) (Reeves & Nissen, 1990). The AT-hook is a
small motif centered around a glycine–arginine–proline
(GRP) tripeptide (Reeves & Nissen, 1990). In ISMbov7related elements, the AT-hook motif is localized between
codons 73 and 83 (Fig. 3d). The AT-hook motif was also
identified within ISMmy2 and IS1138 elements (Fig. 3d).
Noteworthy, this motif was also identified in the transposase
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c
protein of several mariner family transposons, both bacterial
and eukaryotic, suggesting that it may act with the majorgroove-interacting HTH motif of these proteins to
additionally contact the DNA minor groove (Aravind &
Landsman, 1998).
The effect of IS-like elements on M. bovis genes
In this study, eight IS-like elements were found to be
inserted within M. bovis structural genes, which would be
predicted to disrupt their expression. IS4-like element
ISMbov4A was inserted together with ISMbov2D within
ORF11 (Fig. 1.4). In addition, ISMbov2E and ISMbov1E
were found within ORF16 and ISMbov4B and ISMbov4G in
ORF17 and ORF44, respectively (Fig. 1.5 and 1.8). ORF16
and ORF17 show 85% and 94% identity to CDS19 and
CDS17 of the M. agalactiae integrative conjugative element,
respectively, and ORF44 demonstrates 61% identity to
MAG2400, a predicted lipoprotein of M. agalactiae (Marenda et al., 2006; Sirand-Pugnet et al., 2007) (Table S1).
ISMbov2F, together with ISMbov7A, interrupted ORF41,
which shows 80% homology to a hypothetical protein of
M. agalactiae (Fig. 1.8 and Table S1). IS3-like elements
ISMbov7C and ISMbov7F were inserted within ORF48 and
FEMS Microbiol Lett 294 (2009) 172–182
179
Insertion sequence-like elements in Mycoplasma bovis genome
(a)
(b)
ISMbov4
ISMbov4A
ISMbov4B
ISMbov4C
ISMbov4D
ISMbov4E
ISMbov4F
ISMbov4G
ISMbov4H
CTCTAA
TTTAAT
TTATAA
TTAAAT
ATGAAA
CTGTAA
TTAAAA
ATTTAG
ATAAAT
Consensus
NTNAAN
T
ISMbov2A
ISMbov2B
ISMbov2C
ISMbov2D
ISMbov2E
ISMbov2F
ISMbov2H
Consensus
GTACAT
ATACAT
ATTTAT
ATGTAT
ATATAT
ATACTT
ATAAAT
ATANAT
(c)
H
Predicted H-HTH
IS911
H1
T
H2
LZ
1-46 MKKRNFSAEFKRESAQLVVDQNYTVADAAKAMDVGLSTMTRWVKQL
H
H1
T
63-94:EQIEIRELRKKLQRIEMENEILKKGYRALDVR
H2
ISMbov7
1-53 MAKQLSVNEWKYLFEKYEKYRSGELTKKCFLNEMMKIKNVEHISDDQWKRLVN
ISMmy2
1-57 MKRGKQLSVNEWIELFKHYKDYLSHNITKKEFYYIYSKIRNSGDSLPSQKAMEYLSK
IS1138
1-60 MKQLKPEQWKKWFSLYEEFYDGKINIKKYIFLVNKNIGKEWKNTYVKSWFFKKYSAFQKD
77-112:GSGRPKKTKSNDEILDEFLNDLNKEDLIKIIKIIST
95-130:KQRNEVIKEIDREVLEWLITDLFKEEILKKHKVDNL
130-158:KISWKEFLFSKSTYYSWKKPKLAEPKKDQ
DNA binding domain (DBD)
(d)
70
AT-hook motif
85
ISMbov7
ESMSGRSPKKGKGSGRPKKTKSNDE
ISMmy2
ESQTGRAPKKGKGSGRPRKNKEEYD
IS1138
ISQTGKSTANKKNNGRPPKRKEVNE
Fig. 3. Target sites and consensus sequence determined for ISMbov4-related elements (a) and ISMbov2 (b). Gray boxes represent identical nucleotides.
Six-base pair DRs generated upon ISMbov4 (a) and ISMbov2 (b) insertion were considered in generating the consensus of target sites. The most
frequently found nucleotide (from 70% to 100%) is specified below as a consensus. The opposing arrows indicate IRs within the consensus of ISMbov2related elements. (c) Protein sequence alignment of the DBD of the IS3 elements. The position and amino acid residues comprising the helix (H)-turn (T)helix (H) motifs (H, H1 and H2) in ISMbov7 of Mycoplasma bovis, IS911 of Shigella dysenteriae, IS1138 of Mycoplasma pulmonis and ISMmy2 of
Mycoplasma mycoides ssp. mycoides LC are indicated by bold and overlined. The number of amino acids is shown on the left. The conserved tryptophan
(W) residue is underlined. The LZ regions of ISMbov7, IS911, IS1138 and ISMmy2 within the DBD are shown. (d) Amino acid sequence alignment of the
DNA-binding motif (AT-hook). Gray boxes represent identical nucleotides. The position of the AT-hook motif of ISMbov7, ISMmy2 and IS1138 is shown.
ORF68, respectively, which correspond to hsdR- and hsdMlike structural genes of restriction modification (R–M) type
I and type III systems (Fig. 1.9, 1.10 and Table S1).
ISMbov1L interrupted ORF70 that encodes the M. bovis
ortholog of ICEF-IA ORF9 (Fig. 1.10) (Marenda et al.,
2006). Insertion of an IS element within another IS element
was also found, with no apparent relationship with a specific
IS family. For example, ISMbov7A was identified within the
C-terminal region of ISMbov2F (Fig. 1.8); ISMbov2B was
inserted into ISMbov3A (Fig. 1.5); and interruption of
ISMbov7E, possibly as a result of insertion of ISMbov6D,
was also observed (Fig. 1.10).
IS elements may also influence genome variability by
affecting the expression of neighboring genes. For example,
a short sequence of 63 bp, which lies downstream to
ISMbov3A and contains its IRR, was found to be fused inframe to ORF14 (Fig. 1.5). ORF14 exhibits 90% and 52%
FEMS Microbiol Lett 294 (2009) 172–182
homology to the DNA polymerase I of M. agalactiae
(Sirand-Pugnet et al., 2007) and to the exonuclease of
M. fermentans strain II-29/1, respectively (Calcutt et al.,
1999a). Interestingly, both ORF14 and the M. fermentans
exonuclease lack the N-terminal region of 132 aa, which
provides the polymerase domain of the DNA polymerase I
enzyme. The fusion of the 63-bp sequence to the truncated
DNA polymerase I gene in M. bovis raised the possibility
that it may provide the transcription signals that are missing
in the truncated gene. To explore this issue, RT analysis,
using primer 7A-R (Fig. 1.5) complementary to the encoding region of ORF14, was performed. The resultant cDNA
was used as a template in a PCR reaction with primers 7A-R
and 5-F, specific to the ORF14 and to the 63 nt, respectively.
A positive PCR product of 190 bp was identified and
sequenced (Fig. 4, lane 2). The data support the notion that
in-frame fusion of the short sequence found downstream to
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c
180
I. Lysnyansky et al.
M
1
2
3
bp
et al., 2001) and M. hyopneumoniae (six copies in strain 232,
17 in strain 7448 and 25 in strain J) (Minion et al., 2004;
Vasconcelos et al., 2005). To the best of our knowledge,
the M. bovis genome represents the first example in the
Mollicutes, in which multiple transposases belonging to
three distinct IS families were identified.
Acknowledgements
190–
Fig. 4. RT-PCR analysis of the fused orf14 gene. Primers 7A-R and 5F
(Fig. 1.5) were used in PCR amplification of the orf14 gene with
Mycoplasma bovis PG45 genomic DNA as a positive control (lane 1) and
with the corresponding cDNA (lane 2). An identical 0.19-kb PCR product
was detected in both reactions. PCR reaction was also performed directly
on the RNA preparation as a negative control (lane 3) to confirm the
absence of DNA contamination. The lane marked as M indicates a 100bp ladder.
ISMbov3A with the M. bovis exonuclease gene may provide
transcriptional signals needed for its expression.
Unfortunately, the role of IS elements in the antigenic
variation of mycoplasmas has not yet been documented. The
antigenic variations of the major family of M. bovis variable
surface lipoproteins (Vsp) have been deciphered and explained by reported recombination mechanisms (Lysnyansky et al., 2001a, b). The IS elements within the vsp locus are
not involved in the Vsp phase variation. However, in other
bacteria, the role of ISs in reversible antigen inactivation was
described (Bartlett et al., 1988; Bartlett & Silverman, 1989;
Sokol et al., 1994; Hammerschmidt et al., 1996; Ziebuhr
et al., 1999).
Conclusions
This study presents an overview of the abundance, genomic
environment and diversity of IS elements in the M. bovis
PG45 genome. Four new IS elements (ISMbov4, ISMbov5,
ISMbov6 and ISMbov7) were identified and characterized
with respect to IR, DR and structural domains. Overall, 54
full and truncated IS elements (27 copies of IS4, 18 copies of
IS30 and nine copies of IS3-related gene families) were
found in M. bovis PG45, comprising about 6.4% of its
genome. The density of ISs in M. bovis PG45 is much higher
than in the closely related species M. agalactiae strain PG2
(10 copies, many of which are pseudogenes) (Sirand-Pugnet
et al., 2007) or other phylogenetically related mycoplasmas,
such as Mycoplasma synoviae strain 53 (14 copies) (Vasconcelos et al., 2005), M. pulmonis (16 copies) (Chambaud
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c
This research was supported by Research Grant Award No.
IS-3404-03C from BARD, the United States – Israel Binational Agricultural Research and Development Fund and by
the Program for Prevention of Animal Infectious Diseases
(USDA ARS 58-1940-5-519).
References
Aravind L & Landsman D (1998) AT-hook motifs identified in a
wide variety of DNA-binding proteins. Nucleic Acids Res 26:
4413–4421.
Bartlett DH & Silverman M (1989) Nucleotide sequence of IS492,
a novel insertion sequence causing variation in extracellular
polysaccharide production in the marine bacterium
Pseudomonas atlantica. J Bacteriol 171: 1763–1766.
Bartlett DH, Wright ME & Silverman M (1988) Variable
expression of extracellular polysaccharide in the marine
bacterium Pseudomonas atlantica is controlled by genome
rearrangement. P Natl Acad Sci USA 85: 3923–3927.
Bender J & Kleckner N (1992) IS10 transposase mutations that
specifically alter target site recognition. EMBO J 11: 741–750.
Bhugra B & Dybvig K (1993) Identification and characterization
of IS1138, a transposable element from Mycoplasma pulmonis
that belongs to the IS3 family. Mol Microbiol 7: 577–584.
Bischof DF, Vilei EM & Frey J (2006) Genomic differences
between type strain PG1 and field strains of Mycoplasma
mycoides subsp. mycoides small-colony type. Genomics 88:
633–641.
Boutoille D, Corvec S, Caroff N et al. (2004) Detection of an IS21
insertion sequence in the mexR gene of Pseudomonas
aeruginosa increasing beta-lactam resistance. FEMS Microbiol
Lett 230: 143–146.
Calcutt M, Lavrrar JL & Wise KS (1999a) IS1630 of Mycoplasma
fermentans, a novel IS30-type insertion element that targets
and duplicates inverted repeats of variable length and sequence
during insertion. J Bacteriol 181: 7597–7607.
Calcutt MJ, Kim MF, Karpas AB, Muhlradt PF & Wise KS (1999b)
Differential post-translational processing confers intraspecies
variation of a major surface lipoprotein and a macrophageactivating lipopeptide of Mycoplasma fermentans. Infect
Immun 67: 760–771.
Chambaud I, Heilig R, Ferris S et al. (2001) The complete genome
sequence of the murine respiratory pathogen Mycoplasma
pulmonis. Nucleic Acids Res 29: 2145–2153.
FEMS Microbiol Lett 294 (2009) 172–182
181
Insertion sequence-like elements in Mycoplasma bovis genome
Chandler D & Mahillon J (2002) Insertion sequences revisited.
Mobile DNA II, Vol. II, (Crag NL, Craigie R, Gellert M &
Lamboxitz A, eds), pp. 305–366. ASM Press, Washington.
Chopra-Dewasthaly R, Zimmermann M, Rosengarten R & Citti C
(2005) First steps towards the genetic manipulation of
Mycoplasma agalactiae and Mycoplasma bovis using the
transposon Tn4001mod. Int J Med Microbiol 294: 447–453.
Flitman-Tene R, Mudahi-Orenstein S, Levisohn S & Yogev D
(2003) Variable lipoprotein genes of Mycoplasma agalactiae are
activated in vivo by promoter addition via site-specific DNA
inversions. Infect Immun 71: 3821–3830.
Fraser CM, Gocayne JD, White O et al. (1995) The minimal gene
complement of Mycoplasma genitalium. Science 270: 397–403.
Hallet B, Rezsohazy R, Mahillon J & Delcour J (1994) IS231A
insertion specificity: consensus sequence and DNA bending at
the target site. Mol Microbiol 14: 131–139.
Hammerschmidt S, Hilse R, van Putten JP, Gerardy-Schahn R,
Unkmeir A & Frosch M (1996) Modulation of cell surface
sialic acid expression in Neisseria meningitidis via a
transposable genetic element. EMBO J 15: 192–198.
Himmelreich R, Hilbert H, Plagens H, Pirki E, Li BC &
Herrmann R (1996) Complete sequence analysis of the
genome of the bacterium Mycoplasma pneumoniae. Nucleic
Acids Res 24: 4420–4449.
Jaffe JD, Stange-Thomann N, Smith C et al. (2004) The complete
genome and proteome of Mycoplasma mobile. Genome Res 14:
1447–1461.
Kiss J, Nagy Z, Toth G, Kiss GB, Jakab J, Chandler M & Olasz F
(2007) Transposition and target specificity of the typical IS30
family element IS1655 from Neisseria meningitidis. Mol
Microbiol 63: 1731–1747.
Kunze ZM, Wall S, Appelberg R, Silva MT, Portaels F &
McFadden JJ (1991) IS901, a new member of a widespread
class of atypical insertion sequences, is associated with
pathogenicity in Mycobacterium avium. Mol Microbiol 5:
2265–2272.
Lichter A, Manulis S, Valinsky L, Karniol B & Barash I (1996)
IS1327, a new insertion-like element in the pathogenicityassociated plasmid of Erwinia herbicola pv. gypsophilae. Mol
Plant Microbe In 9: 98–104.
Lysnyansky I, Rosengarten R & Yogev D (1996) Phenotypic
switching of variable surface lipoproteins in Mycoplasma bovis
involves high-frequency chromosomal rearrangements.
J Bacteriol 178: 5395–5401.
Lysnyansky I, Sachse K, Rosenbusch R, Levisohn S & Yogev D
(1999) The vsp locus of Mycoplasma bovis: gene organization
and structural features. J Bacteriol 181: 5734–5741.
Lysnyansky I, Ron Y, Sachse K & Yogev D (2001a)
Intrachromosomal recombination within the vsp locus of
Mycoplasma bovis generates a chimeric variable surface
lipoprotein antigen. Infect Immun 69: 3703–3712.
Lysnyansky I, Ron Y & Yogev D (2001b) Juxtraposition of an
active promoter to vsp genes via site-specific DNA inversions
generates antigenic variation in Mycoplasma bovis. J Bacteriol
183: 5698–5708.
FEMS Microbiol Lett 294 (2009) 172–182
Mahillon J & Chandler M (1998) Insertion sequences. Microbiol
Mol Biol R 62: 725–774.
Marenda M, Barbe V, Gourgues G, Mangenot S, Sagne E & Citti C
(2006) A new integrative conjugative element occurs in
Mycoplasma agalactiae as chromosomal and free circular
forms. J Bacteriol 188: 4137–4141.
Miles K, McAuliffe L, Persson A, Ayling RD & Nicholas RA
(2005) Insertion sequence profiling of UK Mycoplasma bovis
field isolates. Vet Microbiol 107: 301–306.
Minion FC, Lefkowitz EJ, Madsen ML, Cleary BJ, Swartzell SM &
Mahairas GG (2004) The genome sequence of Mycoplasma
hyopneumoniae strain 232, the agent of swine mycoplasmosis. J
Bacteriol 186: 7123–7133.
Nagy Z & Chandler M (2004) Regulation of transposition in
bacteria. Res Microbiol 155: 387–398.
Olasz F, Farkas T, Kiss J, Arini A & Arber W (1997) Terminal
inverted repeats of insertion sequence IS30 serve as targets for
transposition. J Bacteriol 179: 7551–7558.
Olasz F, Kiss J, Konig P, Buzas Z, Stalder R & Arber W (1998)
Target specificity of insertion element IS30. Mol Microbiol 28:
691–704.
Papazisi L, Gorton TS, Kutish G et al. (2003) The complete
genome sequence of the avian pathogen Mycoplasma
gallisepticum strain R(low). Microbiology 149: 2307–2316.
Plikaytis BB, Crawford JT & Shinnick TM (1998) IS1549 from
Mycobacterium smegmatis forms long direct repeats upon
insertion. J Bacteriol 180: 1037–1043.
Reeves R & Nissen MS (1990) The A.T-DNA-binding domain of
mammalian high mobility group I chromosomal proteins. A
novel peptide motif for recognizing DNA structure. J Biol
Chem 265: 8573–8582.
Rousseau P, Gueguen E, Duval-Valentin G & Chandler M (2004)
The helix-turn-helix motif of bacterial insertion sequence
IS911 transposase is required for DNA binding. Nucleic Acids
Res 32: 1335–1344.
Sasaki Y, Ishikawa J, Yamashita A et al. (2002) The complete
genomic sequence of Mycoplasma penetrans, an intracellular
bacterial pathogen in humans. Nucleic Acids Res 30:
5293–5300.
Schmid-Appert M, Zoller K, Traber H, Vuilleumier S & Leisinger
T (1997) Association of newly discovered IS elements with the
dichloromethane utilization genes of methylotrophic bacteria.
Microbiology 143: 2557–2567.
Sirand-Pugnet P, Lartigue C, Marenda M et al. (2007) Being
pathogenic, plastic, and sexual while living with a nearly
minimal bacterial genome. PLoS Genet 3: e75.
Sokol PA, Luan MZ, Storey DG & Thirukkumaran P (1994)
Genetic rearrangement associated with in vivo mucoid
conversion of Pseudomonas aeruginosa PAO is due to insertion
elements. J Bacteriol 176: 553–562.
Thomas A, Linden A, Mainil J, Bischof DF, Frey J & Vilei EM
(2005) Mycoplasma bovis shares insertion sequences with
Mycoplasma agalactiae and Mycoplasma mycoides subsp.
mycoides SC: evolutionary and developmental aspects. FEMS
Microbiol Lett 245: 249–255.
2009 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
182
Vasconcelos ATR, Ferreira HB, Bizarro CV et al. (2005) Swine and
poultry pathogens: the complete genome sequences of two
strains of Mycoplasma hyopneumoniae and a strain of
Mycoplasma synoviae. J Bacteriol 187: 5568–5577.
Vilei EM, Nicolet J & Frey J (1999) IS1634, a novel insertion
element creating long variable-length direct repeats which is
specific for Mycoplasma mycoides subsp. mycoides small-colony
type. J Bacteriol 181: 1319–1323.
Westberg J, Persson A, Pettersson B, Uhlen M & Johansson KE
(2002) ISMmy1, a novel insertion sequence of Mycoplasma
mycoides subsp. mycoides small colony type. FEMS Microbiol
Lett 208: 207–213.
Westberg J, Persson A, Holmberg A et al. (2004) The genome
sequence of Mycoplasma mycoides subsp. mycoides SC type
strain PG1T, the causative agent of contagious bovine
pleuropneumonia (CBPP). Genome Res 14: 221–227.
Wintjens R & Rooman M (1996) Structural classification of HTH
DNA-binding domains and protein-DNA interaction modes.
J Mol Biol 262: 294–313.
Ziebuhr W, Krimmer V, Rachid S, Lössner I, Götz F & Hacker J
(1999) A novel mechanism of phase variation of virulence in
2009 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
I. Lysnyansky et al.
Staphylococcus epidermidis: evidence for control of the
polysaccharide intercellular adhesin synthesis by alternating
insertion and excision of the insertion sequence element IS256.
Mol Microbiol 32: 345–356.
Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Table S1. Gene products encoded by genes flanking IS
elements in Mycoplasma bovis.
Please note: Wiley-Blackwell is not responsible for the
content or functionality of any supporting materials supplied by the authors. Any queries (other than missing
material) should be directed to the corresponding author
for the article.
FEMS Microbiol Lett 294 (2009) 172–182