![[Feedback]](/icons/banner/feedbackACT.gif)
![[For Subscribers]](/icons/banner/subscriberACT.gif)
![[Archive]](/icons/banner/archiveACT.gif)
![[Search]](/icons/banner/searchACT.gif)
![[Contents]](/icons/banner/tocACT.gif)
Previous Article | Next Article 
Journal of Bacteriology, April 1999, p. 2286-2289, Vol. 181, No. 7
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Target Joining of Duplicated Insertion Sequence
IS21 Is Assisted by IstB Protein In Vitro
Sergio
Schmid,1,2
Bernard
Berger,3 and
Dieter
Haas1,3,*
Mikrobiologisches Institut,
Eidgenössische Technische Hochschule, CH-8092
Zürich,1 Ecole
d'Ingénieurs du Valais, CH-1950 Sion,2
and Laboratoire de Biologie Microbienne, Université
de Lausanne, CH-1015 Lausanne,3 Switzerland
Received 10 September 1998/Accepted 24 January 1999
 |
ABSTRACT |
Tandemly repeated insertion sequence IS21, located on a
suicide plasmid, promoted replicon fusion with bacteriophage 
in vitro in the presence of ATP. This reaction was catalyzed in a cell
extract containing the 45-kDa IstA protein (cointegrase) and the 30-kDa
IstB helper protein of IS21 after both proteins had been
overproduced in Escherichia coli. Without IstB, replicon fusion was inefficient and did not produce the 4-bp target duplications typical of IS21.
| |
TEXT |
Insertion sequence
IS21 occurs on the broad-host-range plasmid
R68 and contains two genes, istAB, in an operon
(22, 29). The istA gene encodes, in frame, a
46-kDa and a 45-kDa protein, termed transposase and cointegrase,
respectively (25). Transposase essentially catalyzes
transposition of single IS21 elements. Cointegrase rarely
carries out this reaction but is highly effective in another type of
transpositional recombination, i.e., replicon fusion between plasmids carrying an IS21 tandem duplication
[(IS21)2] and target replicons
(25). Formally, the products of this replicon fusion reaction are cointegrates. However, they are formed by a
cut-and-paste transpositional mechanism (20)
rather than by replicative transposition, which, for example, underlies
the move of bacteriophage Mu or transposon Tn3
(18, 26). The 30-kDa helper protein IstB contains an ATP-GTP
binding motif (7), as do the transposition helper proteins
MuB of phage Mu and TnsC of transposon Tn7 (3, 18). In the
absence of IstB, IS21 transposition cannot be detected and
(IS21)2-mediated cointegration occurs at very
low frequencies (21, 25).
Under optimal conditions in Escherichia coli, the frequency
of (IS21)2-dependent replicon fusion approaches
10
1, as measured by a mating-out assay (25).
This high frequency is made possible on the one hand by an
IS21 tandem with an optimal 4-bp spacer (5'-TATA-3') between
the two IS21 elements and on the other hand by the
overexpression of cointegrase and IstB in trans by using the
expression vector pJF118EH (25). When the overexpressed
istAB gene products are supplied in trans, a
fragment carrying the reactive IS21-IS21 junction
suffices to give replicon fusion (21, 25). Encouraged by the
high in vivo activity of cointegration, we have now set up an in vitro
system. Previously, we have shown that an istA gene product
(probably the cointegrase) can cleave the
IS21-IS21 junction at the inner 3'
ends of the IS21 elements in vitro (21). This
cleavage, which does not require IstB, results in a staggered cut,
exposing 3'-OH groups of the terminal nucleotides (A) in each inverted
repeat (see the IS21-IS21 junction of pME3940
[Fig. 1]). We have now obtained
evidence that strand transfer from the IS21-IS21
junction of (IS21)2 to DNA can occur in
vitro.
View larger version (21K):
[in this window]
[in a new window]
|
FIG. 1.
In vitro cointegration assay. The incubation mixture
contained concatemerized gtWES.B DNA ( , cos site),
the pVS1-derived suicide plasmid pME3940 carrying the reactive
IS21-IS21 junction on a 1.3-kb fragment from
pME3918 (25), and the supF (amber suppressor)
gene on a 0.21-kb fragment, as well as IS21 transposition
proteins in the cell extract. The DrdI cleavage site
(indicated by vertical arrows) at the artificial
IS21-IS21 junction may mimick the cuts made by
IstA (21). The assay of gtWES.B::pME3940
cointegrates is explained in the text. IS21L' and
IS21R', truncated left and right IS21 elements,
respectively; IR, inverted repeat; bla, ampicillin
resistance gene.
|
|
Components of the in vitro cointegration system.
The principle
of the in vitro cointegration system shown in Fig. 1 is inspired by in
vitro systems devised for murine leukemia virus, the Ty element of
Saccharomyces cerevisiae, and Tn3 (1, 4,
9). The istA and istB genes were
overexpressed in E. coli ED8767 (metB recA56 hsdS supE
supF) (23), using the previously described
inducible tac promoter constructs pME3902, pME3913, pME3910, pME3944, and pME3945 (Table 1) (25).
Cells were grown aerobically at 37°C in L broth
(23) containing 100 µg of ampicillin per ml to a density
of about 5 × 108 per ml, induced with 1 mM
isopropyl-
-D-thiogalactopyranoside (IPTG) for 2 h,
harvested by centrifugation, and resuspended in 25 mM HEPES-1 mM EDTA,
pH 7.5, to a density of approximately 1012 per ml. Cells
were placed on ice; KCl and dithiothreitol were added to final
concentrations of 100 and 2 mM, respectively. Cells were shaken
gently at 0 to 4°C and treated successively with 250-µg/ml lysozyme
for 20 min and 10 mM MgCl2 for 30 min. Thereafter, cells were frozen in liquid N2 and lysed by thawing on ice.
Cell debris was removed by centrifugation in an Eppendorf 5415C
centrifuge at 14,000 rpm for 30 min, and the cell extract was stored in
10-µl aliquots (quick frozen in liquid N2) at 
70°C.
Under these conditions, extracts were stable for several months.
The donor plasmid pME3940 (Fig. 1 and 2)
was constructed essentially as follows. The 6.8-kb
Pseudomonas vector pME290, which is derived from pVS1
and cannot replicate in E. coli (10, 11), was
used to clone the IS21-IS21 junction region with
the 4-bp spacer 5'-TATA-3' from pME3918 (25) and the
supF gene from pBRG1310 (19) in
Pseudomonas aeruginosa PAO25 (6). Plasmid pME3940 was purified from strain PAO25 by centrifugation in a
CsCl-ethidium bromide density gradient (10).
View larger version (40K):
[in this window]
[in a new window]
|
FIG. 2.
Analysis of pME3940 insertions in gtWES.B. (a) The
map and genotype symbols are according to Leder et al.
(12) and Hendrix et al. (8), and the orientation
of pME3940 (indicated by a flag) is defined below. rep,
replication; sta, segregational stability; bla,
ampicillin resistance gene. (b) The insertion sites were sequenced by
the method of Manfioletti and Schneider (15), using
15-mer primers specific for either IS21 end. IstA/IstB,
transposition proteins provided by pME3902; IstA, cointegrase and
transposase provided by pME3944; IstB, helper protein provided by
pME3945; IstA + IstB, transposition proteins provided by a mixture
of ED8767/pME3944 and ED8767/pME3945 extracts; IstA(P45)/IstB,
cointegrase and helper protein provided by pME3913. The asterisk
indicates that there was a 14-bp deletion at the insertion site.
Underlined nucleotides in boldface indicate the target duplications.
|
|
Concatemerized gtWES.B DNA served as the target.
Phage gtWES.B was propagated on E. coli LE392
(supF58 supE44 hsdR514 galK2 galT22 metB1 trpR55 lacY1)
(12, 23). Phage DNA isolated by standard methods
(23) was purified on a Qiagen midicolumn according to the
instructions of the supplier and ligated at 200 µg/ml (1).
The in vitro reaction was carried out in a 20-µl volume containing 14 µl of reaction buffer (25 mM HEPES [pH 7.5], 10 mM
MgCl2, 50 mM KCl, 1 mM dithiothreitol, 50 µg of
bovine serum albumin/ml, and 5% [wt/vol] polyvinylpyrrolidone K90,
amended with 1.8 mM ATP and 143 µM each dATP, dCTP, dGTP and dTTP
[21]), 4 to 5 µl of DNA (0.2 µg of pME3940 plus 1 µg of concatemeric gtWES.B), and 1 to 2 µl of a cell extract
containing overexpressed istAB gene products (protein
concentration, 20 to 30 mg/ml). This mixture was incubated on ice for
10 min and at 30°C for 60 to 120 min. The reaction was stopped by
mixing a 4-µl aliquot with a commercial packaging extract
(Gigapack II Gold; Stratagene). Packaging was performed as described by
the supplier. The phage lysate thus obtained was titrated
(23) on two maltose-grown indicator strains of E. coli, LE392 and CES200 (sbcB15 recB21 recC22 hsdR
hisG4 thi-1 leuB6 
[gpt-proA]62 argE3
thr-34::Tn10 lacY1 galK2 ara-14 kdgK51
mtl-1 xyl-5 rfbD1 tsx-33 rpsL31) (4). Phage
gtWES.B, which contains three amber mutations, makes plaques on
the amber suppressor strain LE392. On the sup0
strain CES200, gtWES.B will form plaques only when it contains a
pME3940 insertion providing the amber suppressor supF (Fig. 1). Thus, the frequencies of gtWES.B::pME3940
cointegrates can be determined (see footnote b of Table 1).
Roles of the istAB gene products in replicon fusion in
vitro.
Incubation with a cell extract prepared from E. coli ED8767 carrying pME3902 (expressing cointegrase, transposase,
and IstB) produced 3 × 104 cointegrates per h
(Table 1). A 2-h incubation raised the
cointegration frequency to 103. In these experiments,
typically several thousands of plaques were obtained per microgram of
DNA on the sup0 host CES200. A vector
(pJF118EH) control did not yield cointegrates (Table 1), indicating
that illegitimate recombination did not interfere. When IstB was
present, cointegrase (encoded by pME3913) gave a higher
cointegration rate than did transposase (encoded by pME3910) (Table 1),
suggesting that most of the cointegrates formed in a
cointegrase-transposase mixture are the result of cointegrase activity.
This situation has previously been observed in vivo (25).
However, the in vitro cointegration rates measured should be
interpreted cautiously since the amounts of the istAB gene
products in the cell extracts are not known. (In Coomassie blue-stained
gels, the IstA and IstB proteins were not conspicuous after expression
in E. coli carrying istAB constructs.)
Importantly, the pME3940 insertions (no. 1 to 7 [Fig. 2]) that were
obtained from several pME3902-dependent reactions all occurred at
different sites in gtWES.B; in each case, the junction sequence
5'-TATA-3' (identifiable as a DrdI restriction site [Fig.
1]) was lost from the donor pME3940, and each insertion was flanked by
a 4-bp target duplication (Fig. 2). IS21 is known to
generate target duplications of 4 bp normally and 5 bp exceptionally
(23, 25). An extract prepared from strain ED8767/pME3913,
which contained cointegrase and IstB but no transposase, also allowed
the formation of regular 4-bp target duplications (no. 13 and 14 [Fig.
2]).
In the absence of IstB, the istA construct pME3944
gave a low cointegration rate (Table 1) and none of the four
pME3940 insertions analyzed (no. 8 to 11 [Fig. 2]) was flanked by a
4-bp direct repeat. Addition of an extract of E. coli cells
carrying the istB expression plasmid pME3945 sharply
increased the cointegration rate (Table 1) and restored the typical
4-bp target duplication in the example checked (no. 12 [Fig. 2]). In
this extract, the IstB protein was clearly visible in Coomassie
blue-stained gels, representing a few percent of the total protein
(data not shown). An extract containing IstB, but not IstA, was
inactive in cointegration assays (Table 1). These results suggest that
IstB facilitates intermolecular strand transfer in vitro in the
presence of cointegrase. IstB may also be involved in the appropriate
alignment of target DNA with a putative donor-cointegrase complex,
since the formation of regular target duplications requires IstB.
When ATP and the four deoxynucleoside triphosphates (dNTPs) were
not included in the reaction buffer, no cointegrate formation was
detected. A control showed that the dNTPs inhibited nucleolytic degradation of the linear concatemers by the E. coli extract; it is not clear whether the dNTPs are involved
in the cointegration reaction. ATP stimulated cointegration about
1,000-fold (data not shown). A role for ATP is plausible considering
the presence of an ATP-GTP binding motif in IstB (7).
At 4°C, the cointegration activity of ED8767/pME3913 extracts decayed
rapidly and attempts to stabilize this activity by the addition of
compounds known to stabilize proteins were not successful. Moreover,
overexpressed istA gene products had a marked tendency to
form insoluble aggregates. The IS21 cointegration system,
therefore, has not reached the sophistication of some other in vitro
transposition systems based on purified components (2, 5, 13, 14, 17, 24, 27, 28).
In conclusion, IstB of IS21 and MuB of phage Mu may have
comparable functions. MuB, a DNA binding protein with
ATPase activity, stimulates intermolecular strand transfer
(16). In the absence of MuB, the MuA transposase can carry
out slow intramolecular strand transfer but hardly any intermolecular
strand transfer. MuB is important for the selection of proper target
DNA sites and may serve as a scaffold which directs the assembly of the transposition complex (18, 24). In our in vitro system, IstB stimulated the intermolecular joining reaction at least 100-fold. The
cointegrates formed in the absence of IstB may be the products of
residual, imprecise strand transfer by the IstA protein(s). Alternatively, the cleaved IS21-IS21 donor
(21) may be joined to the target by illegitimate
recombination brought about by some proteins in the E. coli
cell extract. Irrespective of this mechanistic aspect, target capture
is an important function of IstB.
| |
ACKNOWLEDGMENTS |
We thank C. Reimmann and T. Seitz for discussions.
This work was supported by grants from the Swiss National Science
Foundation, the International Brachet Foundation, the Roche Research
Foundation, and the Eidgenössische Technische Hochschule Zürich, where this study was begun.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Laboratoire de
Biologie Microbienne, Université de Lausanne, CH-1015 Lausanne,
Switzerland. Phone: 41 21 692 56 31. Fax: 41 21 692 56 35. E-mail:
Dieter.Haas{at}lbm.unil.ch.
| |
REFERENCES |
| 1.
|
Brown, P. O.,
B. Bowerman,
H. E. Varmus, and J. M. Bishop.
1987.
Correct integration of retroviral DNA in vitro.
Cell
49:347-356[Medline].
|
| 2.
|
Chalmers, R. M., and N. Kleckner.
1994.
Tn10/IS10 transposase purification, activation, and in vitro reaction.
J. Biol. Chem.
269:8029-8035[Abstract/Free Full Text].
|
| 3.
|
Craig, N. L.
1996.
Transposition, p. 2339-2362.
In
F. C. Neidhardt, R. Curtiss III, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella: cellular and molecular biology, 2nd ed. ASM Press, Washington, D.C.
|
| 4.
|
Eichinger, D. J., and J. D. Boeke.
1988.
The DNA intermediate in yeast Ty1 element transposition copurifies with virus-like particles: cell free Ty1 transposition.
Cell
54:955-966[Medline].
|
| 5.
|
Goryshin, I. Y., and W. S. Reznikoff.
1998.
Tn5 in vitro transposition.
J. Biol. Chem.
273:7367-7374[Abstract/Free Full Text].
|
| 6.
|
Haas, D.,
A. Schamböck,
T. Leisinger, and B. W. Holloway.
1977.
The genetic organization of arginine biosynthesis in Pseudomonas aeruginosa.
Mol. Gen. Genet.
154:7-22[Medline].
|
| 7.
|
Haas, D.,
B. Berger,
S. Schmid,
T. Seitz, and C. Reimmann.
1996.
Insertion sequence IS21: related insertion sequence elements, transpositional mechanisms, and application to linker insertion mutagenesis, p. 238-249.
In
T. Nakazawa, K. Furukawa, D. Haas, and S. Silver (ed.), Molecular biology of pseudomonads. ASM Press, Washington, D.C.
|
| 8.
|
Hendrix, R. W.,
J. W. Roberts,
F. W. Stahl, and R. A. Weisberg.
1983.
Lambda II.
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
|
| 9.
|
Ichikawa, H., and E. Ohtsubo.
1990.
In vitro transposition of transposon Tn3.
J. Biol. Chem.
265:18829-18832[Abstract/Free Full Text].
|
| 10.
|
Itoh, Y.,
J. M. Watson,
D. Haas, and T. Leisinger.
1984.
Genetic and molecular characterization of the Pseudomonas plasmid pVS1.
Plasmid
11:206-220[Medline].
|
| 11.
|
Itoh, Y., and D. Haas.
1985.
Cloning vectors derived from the Pseudomonas plasmid pVS1.
Gene
36:27-36[Medline].
|
| 12.
|
Leder, P.,
D. Tiemeier, and L. Enquist.
1977.
EK2 derivatives of bacteriophage lambda useful in the cloning of DNA from higher organisms: the gtWES system.
Science
196:175-177[Abstract/Free Full Text].
|
| 13.
|
Leschziner, A. E.,
T. J. Griffin IV, and N. D. F. Grindley.
1998.
Tn552 transposase catalyzes concerted strand transfer in vitro.
Proc. Natl. Acad. Sci. USA
95:7345-7350[Abstract/Free Full Text].
|
| 14.
|
Maekawa, T.,
K. Yanagihara, and E. Ohtsubo.
1996.
A cell-free system of Tn3 transposition and transposition immunity.
Genes Cells
1:1007-1016[Abstract].
|
| 15.
|
Manfioletti, G., and C. Schneider.
1988.
A new and fast method for preparing high quality lambda DNA suitable for sequencing.
Nucleic Acids Res.
16:2873-2884[Abstract/Free Full Text].
|
| 16.
|
Maxwell, A.,
R. Craigie, and K. Mizuuchi.
1987.
B protein of bacteriophage Mu is an ATPase that preferentially stimulates intermolecular DNA strand transfer.
Proc. Natl. Acad. Sci. USA
84:699-703[Abstract/Free Full Text].
|
| 17.
|
May, E. W., and N. L. Craig.
1996.
Switching from cut-and-paste to replicative Tn7 transposition.
Science
272:401-404[Abstract].
|
| 18.
|
Mizuuchi, K.
1992.
Transpositional recombination: mechanistic insights from studies of Mu and other elements.
Annu. Rev. Biochem.
61:1011-1051[Medline].
|
| 19.
|
Phadnis, S. H.,
H. V. Huang, and D. E. Berg.
1989.
Tn5supF, a 264-base-pair transposon derived from Tn5 for insertion mutagenesis and sequencing DNAs cloned in phage .
Proc. Natl. Acad. Sci. USA
86:5908-5912[Abstract/Free Full Text].
|
| 20.
|
Reimmann, C., and D. Haas.
1987.
Mode of replicon fusion mediated by the duplicated insertion sequence IS21 in Escherichia coli.
Genetics
115:619-625[Abstract/Free Full Text].
|
| 21.
|
Reimmann, C., and D. Haas.
1990.
The istA gene of insertion sequence IS21 is essential for cleavage at the inner 3' ends of tandemly repeated IS21 elements in vitro.
EMBO J.
9:4055-4063[Medline].
|
| 22.
|
Reimmann, C.,
R. Moore,
S. Little,
A. Savioz,
N. S. Willetts, and D. Haas.
1989.
Genetic structure, function and regulation of the transposable element IS21.
Mol. Gen. Genet.
215:416-424[Medline].
|
| 23.
|
Sambrook, J.,
E. F. Fritsch, and O. Maniatis.
1989.
Molecular cloning: a laboratory manual, 2nd ed.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
|
| 24.
|
Savilahti, H.,
P. A. Rice, and K. Mizuuchi.
1995.
The phage Mu transpososome core: DNA requirements for assembly and function.
EMBO J.
14:4893-4903[Medline].
|
| 25.
|
Schmid, S.,
T. Seitz, and D. Haas.
1998.
Cointegrase, a naturally occurring, truncated form of IS21 transposase, catalyzes replicon fusion rather than simple insertion of IS21.
J. Mol. Biol.
282:571-583[Medline].
|
| 26.
|
Sherratt, D.
1989.
Tn3 and related transposable elements: site-specific recombination and transposition, p. 163-184.
In
D. E. Berg, and M. M. Howe (ed.), Mobile DNA. American Society for Microbiology, Washington, D.C.
|
| 27.
|
Stellwagen, A. E., and N. L. Craig.
1997.
Avoiding self: two Tn7-encoded proteins mediate target immunity in Tn7 transposition.
EMBO J.
16:6823-6834[Medline].
|
| 28.
|
Ton-Hoang, B.,
P. Polard, and M. Chandler.
1998.
Efficient transposition of IS911 circles in vitro.
EMBO J.
17:1169-1181[Medline].
|
| 29.
|
Willetts, N. S.,
C. Crowther, and B. W. Holloway.
1981.
The insertion sequence IS21 of R68.45 and the molecular basis for mobilization of the bacterial chromosome.
Plasmid
6:30-52[Medline].
|
Journal of Bacteriology, April 1999, p. 2286-2289, Vol. 181, No. 7
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Kehrenberg, C., Aarestrup, F. M., Schwarz, S.
(2007). IS21-558 Insertion Sequences Are Involved in the Mobility of the Multiresistance Gene cfr. Antimicrob. Agents Chemother.
51: 483-487
[Abstract]
[Full Text]
Top
Abstract
Text
