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Vol. 176, No. 11JOURNAL OF BACrERIOLOGY, June 1994, p. 3257-3268
0021-9193/94/$04.00+0
Copyright C) 1994, American Society for Microbiology
Transposon Tn5O9O of Plasmid R751, Which Carries an
Integron, Is Related to Tn7, Mu, and the Retroelements
PETER RADSTROM,1'2 OLA SKOLD,1 GOTE SWEDBERG,1 JOHN FLENSBURG,3 PAUL H. ROY,1
AND LARS SUNDSTROMl*
Department of Pharmaceutical Biosciences, Division of Microbiology, Uppsala University, Biomedicum, S-751 23
Uppsala,' Applied Microbiology, Lund University, S-221 00 Lund,2 and Medical Products Agency,
S-751 03 Uppsala,3 Sweden
Received 16 November 1993/Accepted 19 March 1994
Integrons confer on bacterial plasmids a capability of taking up antibiotic resistance genes by integrase-
mediated recombination. We show here that integrons are situated on genetic elements flanked by 25-bp
inverted repeats. The element carrying the integron of R751 has three segments conserved with similar
elements in Tn2l and Tn5O86. Several characteristics suggest that this element is a transposon, which we call
TnSO9O. TnS090 was shown to contain an operon with three open reading frames, of which two, tniA and tniB,
were predicted by amino acid similarity to code for transposition proteins. The product of tni4 (559 amino
acids) is a probable transposase with 25% amino acid sequence identity to TnsB from Tn7. Both of these
polypeptides contain the D,D(35)E motif characteristic of a protein family made up of the retroviral and
retrotransposon IN proteins and some bacterial transposases, such as those of TnS52 and of a range of
insertion sequences. Like the transposase genes in Tn552, Mu, and Tn7, the tnL4 gene was followed by a gene,
tniB, for a probable ATP-binding protein. The ends of TnSO90, like those of most other elements producing
D,D(35)E proteins, begin by 5'-TG and also contains a complex structure with four 19-bp repeats at the left
end and three at the right end. Similarly organized repeats have been observed earlier at the termini of both
Tn7 and phage Mu, where they bind their respective transposases and have a role in holoenzyme assembly.
Another open reading frame observed in TnSO90, tniC, codes for a recombinase of the invertase/resolvase
family, suggesting a replicative transposition mechanism. The data presented here suggest that TnS090, Tn7,
TnS52, and Mu form a subfamily of bacterial transposons which in parallel to many insertion sequences are
related to the retroelements.
The genetic flexibility of plasmids is augmented by integrons
(56), which are genetic systems for the mobilization of single
resistance genes by site-specific recombination (8, 41, 57). The
resistance genes acquired by integrons are organized as cas-
settes with flanking GTT sequences and with short palindromic
sequences adjacent to the 3' end of the genes. The 30 or so
inserted cassettes occurring among the integrons form a vari-
able region surrounded by conserved sequences in both direc-
tions. The 5' conserved sequence contains the gene int, coding
for a site-specific recombinase of the type defined by Argos et
al. (2, 42, 57). This integrase is necessary for the uptake of
resistance cassettes (11, 34). The segment 3' of the cassettes
carries a qac gene for an exporter protein mediating resistance
to antiseptics and disinfectants (45) and, in most studied
integrons, the sulI gene for a sulfonamide-resistant dihydro-
pteroate synthase (47, 57). The integrons occur on plasmids of
different incompatibility groups, implying that they are borne
on mobile elements, but the extent and organization of these
mobile elements have not been characterized previously. We
describe here in detail the element carrying the integron in the
IncP plasmid R751. Several independent lines of evidence
suggested that the element is a transposon, Tn5O90, which was
mapped to a position very close to that shown earlier for Tn402
(54); the two transposons may be identical.
The members of the Tn2l family of mercuric resistance
transposons are known to carry an integron which makes them
* Corresponding author. Mailing address: Department of Pharma-
ceutical Biosciences, Division of Microbiology, Uppsala University,
P.O. Box 581, Biomedicum, S-751 23 Uppsala, Sweden. Phone:
46-18-17 41 15. Fax: 46-18-50 27 90.
confer a varied antibiotic resistance phenotype on their hosts.
This integron is part of an internal element of Tn2J missing in
some otherwise related mercury resistance transposons, e.g.,
TnS0J (7, 24). The internal element of Tn2l consists of 11 kb
and resembles a transposon (here referred to as Tn5092)
because it is flanked by 25-bp inverted repeats (IRs) with
associated 5-bp direct repeats (7). Parts of Tn5092 are missing
or substituted for in related Tn2l-like transposons. The corre-
sponding element (called TnS093) of Tn5086, for instance,
lacks a large segment downstream of sulI (60), which explains
its smaller size (7 kb). By comparison of Tn5O90 of R751 with
Tn5092 and TnS093, we observed both common and unique
regions. The largest common region has not been studied
previously and was found here to contain probable transposi-
tion genes.
The protein which is responsible for the cutting and transfer
of DNA strands to move transposable elements is called
transposase; however, often one or more auxiliary proteins are
required for mobility (38). Some of these are suggested to
promote essential contacts between transposition proteins and
DNA, under binding of ATP (51). The degree of the multi-
functionality of transposition proteins seems to vary among
different mobile elements (38). At one extreme, those of Tn7
seem to be very specialized, as reflected in the four different
proteins that are required to make Tn7 transpose (48). In
contrast, Tn3 utilizes only one, very large transposase. One
further recombinase of Tn3 belongs to a site-specific resolution
system (55), which is explained by the need to separate the
cointegrates formed by replicative transposition (53). Such a
system seems unnecessary for transposons such as Tn7, which
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3258 RADSTROM ET AL.
is capable of cutting both donor strands and transposing
directly by a cut-and-paste mechanism (3).
Recently uncovered features of transposition proteins indi-
cate that some bacterial transposable elements do in fact
resemble those of eukaryotic cells (16, 31, 49). This rather
subtle sequence similarity was observed by comparison of the
transposase of TnS52 and those of a range of insertion (IS)
elements with the IN proteins of retroviruses and the long
terminal repeat-type retrotransposons (16, 49). A motif of
acidic amino acids has been identified in the most conserved
region, and there is evidence that these sustain both processing
and integration of retroviral DNA (31). Those transposons and
retroelements, with very few exceptions, furthermore have the
same first two nucleotides at their ends. By these criteria, we
found that Tn5O9O and Tn7 also belong to this greater family
of transposons.
MATERIALS AND METHODS
Bacterial strains and plasmids. Eschenichia coli J53 (F- pro
met) (50) was used as the host for conjugative plasmids, JM105
thi rpsL endA sbcB15 hspR4 A(lac-proAB) F'[traD36 proAB+
lacIqZAM15] was used as the host for M13mpl8 and -19, and
JM83 araA(lac-proAB)rpsL 4801acZAM15 was used for pUC18
and -19 (65). Plasmid clones were made in pUC18/19 (65).
Resistance plasmids and derivatives used were R388 and pSa
(64), R751 (44), pLMO20 (57), pLMO229 (59), N3 (6),
pBR322::Tn2l, and pBR322::TnSO86 (60). Subclones for se-
quencing of Tn5090 were made from pLKO603 (the 8.5-kb PstI
fragment from R751 including the dhfrIIc gene, cloned in
pUC19). Clones used for sequencing Tn2l and Tn5086 were
described earlier (60).
Materials. Media used were Iso-Sensitest (Oxoid, Basing-
stoke, United Kingdom), LuriaBertani broth, mineral salts
medium M9, and double-concentrated YT broth (50). T4 DNA
ligase was from New England BioLabs, Inc., Beverly, Mass.,
and other enzymes were from Boehringer GmbH, Mannheim,
Germany. The isotopically labeled compounds used were
[a-35S]dATP and [ot-32P]dCTP (New England Nuclear,
Dreieich, Germany).
Methods. The nucleotide sequences of R751 and R388 indi-
cated in Fig. 1 were determined from both strands. Most of
this sequence was obtained from randomly cloned re-
striction fragments, but as a complement we utilized the following
integron-specific oligonucleotides as sequencing primers: 5'-
CGAGATIGGTGCAGATCAC`17CTG-3' (292 to 315 in Fig.
2) 5'-CAGGGCGGCCI Gl TllTCGGA-3' (positions 1351 to
1371), 5'-TIGCTATCACATGGAGGAGCC-3' (3025 to 3045),
5'-TCTCGGGGTTGTGTCGCGCT-3' (3964 to 3983), 5'-
GGCTGTGAGCAATTATGTGC1TAGT-3' (5500 to 5524), 5'-
TACGCAGCAGGGCAGTCGCCCTAAA-3' (6268 to 6292),
5'-GCGCCGTTACCACCGCITGCGTT-3' (6413 to 6434), 5'-
GACGCACACCGTGGAAACGGAT-3' (6530 to 6551), and
5'-GGATCCAACCCCTCCGCTGCTA-3' (7542 to 7563). The
last unresolved region was characterized by automated sequenc-
ing, for which we used the primers 5'-TGGAGCAGGTGCA
GATAGACCATACG-3' (positions 884 to 909) and 5'-AT
GATCCGTTCCACGATGCCGCCATAGT-3' (1244 to 1271).
Most of the sequencing was done with autoradiographic detection
of radioactive labeling; however, a minor part was performed with
fluorescein-labeled primers, the AutoRead Sequencing kit, and
the ALF DNA Sequencer (Pharmacia LKB Biotechnology).
Sequence analysis was assisted by using a Vax-8200 computer, a
GraphOn 250 graphic terminal, and the Genetics Computer
Group software package (14). Oligonucleotides were automati-
cally synthesized in a Gene Assembler Plus and chromatograph-
ically purified by using the FPLC system or the SMART system
(Pharmacia LKB Biotechnology AB). Incompatibility determina-
tion ofpLMO20 was done by transformation into J53(N3) (6) and
selection on plates containing 0.1 mg of trimethoprim per ml.
After restreaking of colonies several times on the same medium,
the absence of the N3-specific marker gene, aadA2, was checked
on plates containing 20 ,ug of spectinomycin per ml. Agarose and
polyacrylamide gel electrophoresis and hybridizations were per-
formed essentially as described earlier (50). Probe fragments
were labeled by chain elongation primed by randomized hex-
anucleotides with [tx-32P]dCTP as the labeling component. DNA
sequencing was performed according to the dideoxynucleotide-
chain termination method (52).
Nucleotide sequence accession number. The nucleotide se-
quences reported in this paper have been assigned accession
numbers X12868, X12869, X58425, X72585, U09049, and
U09050 in the EMBL, GenBank, and DDBJ data bases.
RESULTS
Characterization and nucleotide sequence of the integron
element TnSO9O in R751. Restriction endonuclease mapping
predicted more extended similarities between Tn2J and R751
than those noticed earlier (Fig. 1) and indicated that R751
could carry a segment corresponding to the internal 11-kb
element of Tn2l. This segment of R751 would have a location
very similar to that of Tn4O2, described by Shapiro and Sporn
in 1977 (36, 54). The size of Tn4O2 was originally reported to
be approximately 7.5 kb, and it was roughly mapped to include
the leftmost BamHI site in Fig. 1, but not the Sall site to the far
right (36, 54). It was not established whether Tn4O2 is identical
to the element of Tn5O9O described below, but it seems very
likely.
An 8.3-kb PstI fragment from R751 was cloned and se-
quenced (Fig. 1 and 2). The analyzed sequence covered the
region of predicted homology with Tn2l. It was observed that
25-bp IRs identical to those flanking the 11-kb element in
Tn2l, flanked a 7.40-kb segment in R751. These IRs are
referred to as IRi for the integrase end and IRt for the opposite
end (Fig. 1 to 3; see Fig. 6). The 7.40-kb element, called
TnS090, was surrounded by sequences that were different from
those bordering the 11-kb integron-element (Tn5092) of Tn2J
(see Fig. 6) (7), indicating a distinct location. The internal
sequences were largely identical. TnS090 carried segments of
1.4 kb at the left end and 2.7 kb at the right end which were
identical to those of TnS092 or its smaller relative TnSO93. We
sequenced random fragments of Tn5092 covering about 60%
of its 2.7-kb region without observing any divergent nucleo-
tides (Fig. 1). In addition to the major conserved parts, there
was conservation of the exporter gene qacE (45), in the middle
of TnSO90. Between qacE and the flanking conserved segments
of TnSO9O there were unique sequences of 0.7 kb to the left
and 2.3 kb to the right.
The 3' conserved segment of integrons is an immobilized
cassette. The left 1.4-kb segment of TnSO9O (Fig. 1) carries the
integrase gene, int, which is related to the uptake of cassettes
and which has been studied previously for other integrons (42,
57). Only two nucleotide differences from the corresponding
part of Tn5093 (60) were observed, and these made the
hexamers of the main promoter (TTGACA and TAAACT) of
the operon of cassettes more similar to the E. coli consensus.
TnSO9O contains two tandemly inserted cassettes. The cassette
proximal to the promoter contains the dhfrIIc gene (17), which
encodes a dihydrofolate reductase of type Ilc. The cassette
resembles the dhfrIIb cassette in R388 (8, 57, 62) with a 60-bp
insertion 3' of the gene in R751 as a notable difference. The
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TRANSPOSON TN5090 OF PLASMID R751 3259
#6
#1 #2 #3 #4 #5
F H H H H H
II V
H H H
A " H H H
d I
I
IV
IRt
Il
-I--2.3kb-- 2.7
F1 H H H H H
lo o H it i %
I. .I I. E aI
11 -I IY lII
-->
_-
IRi int aadAl qacEAl sulI
AI
mow m
Y
H HH H H
)IIIII_4
-. L I I
>
IRi int dhfrVII qacEAl sulI
HH
HH
H H H H H
~H H i tt H
m. e0 I.0 '
. 1f 1
-_ -D - -_
IRi it dhfrIIb qacEAl sulI
orfA
I
IRt
2.7 k-
H H
y
5kb
FIG. 1. Maps of the regions containing integrons in plasmids R751 and R388 and in transposons Tn2l and Tn5O86. Sequences that are identical
in TnSO92 and at least one more instance are indicated by heavy lines. Horizontal bold arrows, genes previously known; and unfilled arrowheads,
genes borne on a cassette. Nucleotide sequences determined in this paper are underlined; sequences that are underlined and marked with letters
are from other sources as follows: A, reference 57; B, reference 62; C, reference 17; D, reference 45; E, reference 27; F, reference 60; and G,
reference 7. The conserved region of 2.7 kb to the right is boxed by a continuous line, and the 2.3-kb region unique to Tn5O9O is boxed by an
interrupted line. It should be noted that the tnp and mer ends of Tn21 and Tn5O86 occur far outside IRi and IRt, respectively, and are not shown.
cassette downstream of dhfrIIc contained an unidentified open
reading frame (ORF) (orfD; Fig. 3) that was found previously
in other integrons (39, 56).
Downstream of these movable cassettes and located on a
0.4-kb conserved segment, TnSO9O harbors an exporter gene,
qacE, mediating multiresistance (45). The corresponding
qacEAl of TnS092 and Tn5O93 (57, 60) mediates less efficient
export because of a 3' truncation of qacE which was generated
by the insertion of sulI (45). The sulI gene has so far been
exclusively found on integrons (47); however, the mechanism
for its insertion into qacE is unknown. The absence of sulI in
R751 revealed an 8-bp sequence downstream of qacE (GTTA
GATG in Fig. 2), which is an IR of the recombination core
sequence upstream of this gene (CATCTAAC in Fig. 2). IRs at
these positions are found in all known cassettes (25, 26a) and
indicated that qacE is borne on a cassette. There was also
sequence similarity further 3' of qacE to a part of thosestem-loops found downstream of other cassettes (similarity
ends at no. 6 in Fig. 1 and 2) (8). The idea that qacE has a
different origin than the 5' conserved segment was further
supported in that its codon usage (23) (Fig. 4) differed
markedly from those of all other ORFs in TnSO9O.
TnSO90 carries transposition genes. The conserved segment
of 2.7 kb to the right in TnSO9O was observed to carry two
ORFs, tniA and tniB, which were predicted to have functions
connected to transposition (Fig. 2 and 3). A third ORF, orf6,
follows just after tniB in TnSO90 but is absent in Tn5O92 and
Tn5093. All three ORFs seem to be transcribed from a
common promoter (Fig. 2). The orf6 indicated by its codon
preference a good likelihood of being translated in E. coli (data
not shown); however, the 405-amino-acid product could not be
identified by comparison with protein sequences in the data
bases. Probable functions of the gene products of both tniA
and tniB, however, could be identified. The tniA gene, directed
inward from the right terminal IR (IR,), was found to be a
gene for a basic 559-amino-acid protein which is likely to be a
transposase (Fig. 2) because of its approximately 25% amino
acid identity to both tnsB of Tn7 and P480 of TnS52 (Fig. 5).
R751
(Tn5090)
#7
IRi int dhfrIIc \qacE
orfD
#8
.I
H H
H H
H H H H
7:t'A: eX8
Z 'S m *S a
Y I ((
H H
H H
H H H H
t lat Ica: ZY.
V I((
Tn2l
(Tn5O92)
Tn5O86
(T5093)
R388
N~~~~~~~~~~ Il d
H
H
4J 14 M
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J. BACrERIOL.
PstI .. . ScI .. Sai
CT,GCAGCATCGCCATT,GCCGACG;ATGGCACCAGGTCGCCGGCGGTIGGCCACCGACCTCGACGCTCGCCACATICCACGA7CGCGCGGCGTCG;ACCATCGCCAGG,TCTIGACG.
NotI .. ** t
GCGGCCGCCCTGCCCTGGATCTCGCATCGCCArIs:TGGCGAT,GAGATCCACGGAGCGGCCATlwAGACCCGCCAATAA,CGACCCGGCCAAGAT!CA
IRt
3 p -1 . t3 RBH.
GTTIGA7TGGGCGTAATIGGC7GT,GXLAGCCAGCTCCTGACAG=AAZTCAGAAGTGATCTGCACCAATCTCGACTATG,CTCAATACTCGTGTGCACCAA&GC AWIGCAMT
-35 p_> -10 RBS X
----- - - - - -.---.-.-.-.-kTTCCtAGAAAAGGCGraGCACTl
I P E Q G V A T
XCGGCTCAGGCCICTGGCT17TCT
A A Q A L G L S
3GGCGCTTGCCGGAACCGGTCGAG
G R L P E P V E
:AAAAACTr,CGAGT%2CCGGCGCGC
Q K L R V P A R
3CGACGCCTCCTGCCGTGACCGCG
G E P P A V T A
;CCATCGACGTGTTCACCCGCTG3C
A I D V F T R C
3GACTGAACGTGGAAATGGATTGG
G L N V E N D W
:TGGACTATCGCCCGCT3GGGACAG
L D Y R P L G Q
'GCGGCGACTACGATTCCGAAAAC
R G D Y D S E N
L P D A W
ACCI
00CGCCAGGTATACGTT'
R R Q V Y V
&GCGTCATCCACGAGCTA
R V I H E L
iATACCGT0GCCTTACGG0
N T V A L R
CCTG13GAGCAOGTGCAG
P L E Q V Q
GTGCTCGGCATGGTCGTC.
V L G M V V
&AGATGAGCGGCAAGCCC'
Q M S G K P
CCGCACTAlfGCGGCATC
P H Y G G I
iAGGcCGCCCTGACGCTm
K A A L T L
E R
CT6ATC
L I
,C76CAA
L Q
,ATCGCT
I A
ATAGAC
I D
ACGCTG
T L
GCTG
L L
GMGMAA
V E
CGCGAG
R E
A R R
CGGCGT&CC
R R A
AAGCGGTTC
K R F
AGCCTTI6AC
S L D
CATACG&TC
H T V
GAAGCGCCG
E A P
CWTACCTA
L Y L
CGGATCiTO
R I I
CTAGAGCGC
L E R
R A E I I S P L
CGGCAAGGCAGCGGCCTCGTGACG
R Q G S G L V T
CGACCAAGCAGAAGCGCAGCCTA
L T K Q K R S L
CCGCGCAAGGTCATCCGCCGGCGG
P R K V I R R R
kTCGACCTGATCGTGGTCGATGAC
I D L I V V D D
ICTGCCGTTTCGGTTGGCCTGTGC
S A V S V G L C
;ACAACGCGGCCGAGTTCAAGAGC
D N A A E F K S
0GCACGGCGATGCAGATGATTCAC
G T A M Q M I R
rGGCTCACATT0;GCGGTCGGCACC
W L T L A V G T
A Q S
GAT6TGGTG
D L V
GCGGCCTTT
A A F
GAAGGCCAG
E G Q
CGCGACCGG
R D R
CTCGTGCAT
L V H
DraII
GAGGCCCTG
E A L
GACGAACTG
D E L
TACCACGGT
Y H G
IA
C
c
c
C
AT1
-4
star
;GCG
A
CA&
E T V G H
;CCCGGCCAGTCCGGT
P G Q S G
rCACCGCGAAGTCACT
H R E V T
;GATGCCGCTCGTGAC
D A A R D
ICAACCTAT'rGGCCGC
Q P I G R
rTcGCCTGCGACAAG
V A C D K
;CCCGGGGTTGCGAG
R R G C E
;CCGGGAACGACCTTC
P G T T F
rTCGTGCACAACGGC
S V H N G
CTG,CTCCAACCGCCGGCCGCGCGCTGAGGCCGTGGCCGGGrCGTIGT,CGGCGTACCGGCCGTCGTCACACGCGCTACTTCGTTCCT ;GTCGATTlT£TtCTCCGATCCTCCGGCGCACGCT,G
LL Q P P A A R W A E A V A R V G V P A V V T R A T S F L V D F L P I L*R R T L
ACCCGCACCGGCTTTIGTCATCGACCACATCCACTACTACGCCGATGCGCTCAAGCCGTIGGATT,GCGCGGCGTGAACGCTGGCCGTCCTYYsTGATCCGGCGCGAT,CCGCGCGACATCAGC
T R T G F V I D N I H Y Y A D A L K P W I A R R E R W P S P L I R R D P R D I S
Drall
CGTATITCTGGGTCCTIGGAACCGGAGGGACAGCATTACCTIGGAAATTCCCTACCGTACCTTI"GTCGCATCCGGCT,GTCACCC'TGGGAACAACGGCAGCGCGC7'GGCGAAACTIGCGGCAGCAA
R I W V L E P E G Q H Y L E l P Y R T L S H P A V T L W E Q R Q A L A K L R Q Q
GGGCGCGAACAGGTGGATGAGTCGG;CGCTGTTCCCATOATCGGCAGATGCGTGAT7GACCAGCGCGCAGAAGGCCACACGCAAGGCGCGGCGTGACGCGGATCGCCGCCAGCAC
G R E Q V D E S A L F R H I G Q M R E X V T S A Q K A T R K A R R D A D R R O H
start tniB -
stop tniA - I
CT,CAAGACATCAGCTCGGCCGGACAAGCCCGTTCCGCCGGATACGGATATTGCCGACCCGCAGGCAGACAACTT-GCCACCCGCCAAACCGTTCGACCAGATTGAUAQTG]fTAGCCGTGG
L K T S A R P D K P V P P D T D I A D P Q A D N L P P A K P F D Q I E E W M
RBS
ACGAATATCCCATCATCGACCT,GTCCCACCTGCT,GCCGGCGGCCCAGGGCTTGGCCCGTCTTCCGGCGGACGAGCGCATCCAGCGCCTTCGCGCCGACCGCT,GGATCGGCTATCCGCGCG
D E Y P I I D L S H L L P A A Q G L A R L P A D E R I Q R L R A D R W I G Y P R
SphI.
CAGTCGAGGCGCTGAACCGGCT,GGAAGCCCTTTATGCGT,GGCCAAACAAGCAACGCATGCCCAACC7GCGCTlGGTITGGCCCGACCAACAATGGCAAGTCGATGATCGTCGAGAAGTTCC
A V E A L N R L E A L Y A W P N K Q R M P N L L L V G P T N N G K S M I V E K F
GCCGCACCCAiCCCGGCCAGCTCCGACGCCGiACCAGGAGCACATCCCGGTGTTGGT,CGTGCAGAT,GCCGTCCGAGCCGTCCGTGATCCGCTTCTACGTCCGCTGCCGCCGCGATGGGCG
R R T H P A S S D A D Q E H I P V L V V Q M P S E P S V I R F Y V A L L A A M G
SphI.
CGCCGCTGCGCCCACGCCCACGGTTGCGAGACACC,7MCTGCT_GCGCAAGGTCGGCGTGCGCATG;CTGGTGATCGACGAGCTGCACAACGTGriCGGCCGGCAACA
A P L R P R P R L P E M E L A L A L L R K V G V R M L V I D E L H N V L A G N
EcoRI.
GCGTCAACCGCCGGGAATTCCTCAACCTG,CTGCGCTTCCTGCACGAATTGCGCTTGGTGGAGGGAGCACTGCT CGCTCCGATGACCAGTG
S V N R R E F L N L L R F L G N E L R I P L V G V G T R D A Y L A I R S D D Q L
AAAATCGCT+CGAGCCGATGAT,GCTGCCGGTATGxGGAGGCCAACGACGTTGCTGCTCACl7GCTAGGCCAGCTTCGCmCCGllxCCGC'GMCCGGCCTTCCCCAATTGCCACGCTGG
E N R F E P M M L P V W E A N D D C C S L L A S F A A S L P L R R P S P I A T L
ACATGGCTCGCTACCT,GCTC&ACACGCAGCGAGGGCACCATGGACGCCC GTGTGGCGCATCGTCGCCGTGGAGAGCGGCWACGAGGAAG CTGAA
DM A R Y L L T R S E G T I G E L A H L L M A A A I V A V E S G E E A X N H R T
-+ start orf6
.*t7 . . . * stop tniB -
TCAGCATGGCCGTTTACACCGGACCCC ATCiTTAGCTGCGCCCGCTGGCWCGCTGCATCCCGCCCCGAAAGAAGGCGAGGCGCTGTC
L S M A V Y T G P S E R R R Q P E R E L H *
RBS M K P A P R W P L H P A P K E G E A L S
CTCAT,GGCTCAACCGCGTGGCCCTTT13CTATCACAT,GGAGGAGCGACCcATGGAGTTGTAGcAGTAG TGCGCACCACTCTCGCTACCTGc
S W L N R V A L C Y H SE E P D L L E N D L G H G Q V D D L D T A P P L S L L A
GCrCCAGGAGCGGATcGAGc,mGAcTGGTGAGcTGTAcGGTGcGrTGGAc,GTAGcTT,GAGcTC74"ACTTGATGA CCAGA TTCCAGACGCCTTTGGAAACCTA
L L S Q R S G I E L D R L R C M S F A G W V P W L L D S L D D Q X P D A L E T Y
TG,CGTTTCAGCTCGGTrGTTGCTGCCAAGACTCCGCCGTAAGACGCGATCCATCACGAGClnGCGTG;CC:G7CCCAGCCAGCCGATAAACCGCGCCT,GTCCGoCT4CIsCCTIGAGCGA
A F Q L S V L L P R L R R K T R S I T S W R A W L P S Q P X N R A C P L C L S D
TCCGGAGAACCAAGCCGTAC7GCT,CGCG7GGAAGCTGCCCCTGATGCTGAGCTGCCC _CTG7GTGTGATCTA7GGGGCGT,GCCAGGGCGG SrA
P E N Q A V L L A W K L P L M L S C P L H G C W L E S Y W G V P G R P L G W E N
CGCCGACGCCGAACCGCGCA,CCGCCAGCG'ACGCGAT,X;CGGCGATGGACCAGCGTACCTCTGAGATGACAACCGGTCACGTGGAGCTC7CGCGCCGACGCATCCACGCCGGATTGTG
A D A E P R T A S D A I A A M D QR T W Q A L T T G H V E L P R R R I H A G L W
GTTTCGAC7TCVCCACGCrTCGATGAGCTGAACACCCCGCTTTCCGCGTGCGGAACCTG,CGCGGGGTATCCCCGCCAAGGCWGAAGGC'lr7CGGCTCCxCMGCTGGGGCA
P R L L R T L L D E L N T P L S A C G T C A G Y P R 0 V W E G C G H P L R AG
AAGTCTGTGG6CGACCGTATGaAACCCMTAAiTCCGATAGTACGGTTACAGAGCGGAGCGGGGCAACGGCAATCAGCTTGATTGAGGTGAGGGACATCAGCCCGCCAGGCGAGCAGGC
S L W R P Y E T L N P I V R L Q M L E A A A T A I S L I E V R D I SP P G E Q A
FIG. 2
120
240
rt tniA
360
2
480
42
600
82
720
122
840
162
960
202
1080
242
1200
282
1320
322
1440
362
1560
402
1680
442
1800
482
1920
522
2040
559/1
2160
41
2280
81
2400
121
2520
161
2640
201
2760
241
2880
281
3000
302
20
3120
60
3240
100
3360
140
3480
180
3600
220
3720
260
3840
300
3260 RADSTROM ET AL.
T D T P R
GAAGCGGCC6ATATG
E A A D M
GGAGGTAAAGGTAAG
G G K G K
CAGGTGTGCAAGGCT
O V C K A
CTACAAGGTGTGGGCI
L Q G V G
CCGTACCTOACCCTC(
P Y L T L
CGCCCTTGGCTGGAAC
R P W L E
CAGCATGGCATCCGG
Q H G I R
TCCAACCCTGACCAG(
S N P D Q
rCTIGCCTIGA7GAGGCTTGGGAGCGTGMCGCCGTCGIC,CGGAGATCATCAGTCCGTTCvGCGCAGICGGAGACGGTCGGGCACGGAACCC'C'ACGGA
3(3GXccrcre;G-
INNGXVazr-G
i1(cc:AXU36'G
AX,1T:A-G'G4
N'GWI13.6LG
XIGkc.C.I3(&;cl
r,ITITXM;C(
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TRANSPOSON TN5090 OF PLASMID R751 3261
AAAGCTATTsTGGTCCGAGCCCCAAACCGGGTTCACCAGTG,GCCTGCCGACGAAAGCGCCGAAGCCCGAGCCCATCAATCACTG.GCAGCGTGCAGTCCAGGCCATCGACGAGGCCATCAT
K L F W S E P Q T G F T S G L P T K A P K P E P I N H W Q R A V Q A I D E A IXI
TG,AAGCGCGA~CACAACCCCGAGACGG;CACGCTCGCTGTTC&GCTGGCTrCCTATGGTCGGCGCGATCCCGCTTCCTT,GGAACGG7nSCGCGCCACCTTCGTGAAGGAAGGCATCCCGCC
E A R H N P E T A R S L F A L A S Y G R R D P A S L E R L R A T F V K E G FP P
stop orf6
-I1
GGAATTTCTGTCACATTACCTGCCTIGATGCACCCTTTGCATCAAAAATG ACCC GTTAAGTGACAAATTIa &GATAGAGCTTCGGCTJIATCACATAATCGAACG
E F L S H Y L P D A P F A C L K Q N D G L S D K F *_35 p -10
AccI start tniC - BamHlI
TATACGTGACG9QTAAAAGGTGCTGATCGGCTACATGCGCGTATCGAAGGCGGACGGATCCCAGTCCACCAATTT,GCAACGCGATG.CGCTCATCGCCGCT-GGTGTGAGCCTTGCGCACC
RBS M L I G Y H R V S K A D G S Q S T N L Q R D A L I A A G V S L A H
TTTACGAGGATCT,GGCCTCGGGAGGCGCGATGATCGCCCAGGGTTG,GCTG,CTTGCCTGAAGGCGCTTCGTGAAGGGGACACGCT,GATCGTGTGGAAGCTCGATCGGC7'GCGTGATC
L Y E D L A S G R R D D R P G L A A C L K A L R E G D T L I V W K L D R L G R D
Dra&II .SA1I . HizidIII
TGCGCCACCTGATCAACACCGTGCACGACCTAACTGCGCGTAGCGISGGCICMAGGTCCTGACCGGTCACGGTGCGGCGGTCGACACGACGACTGCCGCCGGCAAGCTTGT,GTT4CGGTA
L R H L I N T V H D L T A R S V G L K V L T G H G A A V D T T T A A G K L V F G
TTTTT,GCCGGCGCTGGCCGATTCGAGCGTG6AGTTG.ATTTCCGAGCGAACAGCGG CCCTTCAAGATGACCGCCGCCA
I F A A L A E F E R E L I S E R T V A G L I S A R A R G R K G G R P F K M T A A
AGCTACGCCTGv.GCGAT,GGC'CAGCATGGGGCAACCGGAAACCAAGGTGGGCGATCTCTCAGACGATCCGAAGTTCGCCTTGCAGGGATC
K L R L A M A S M G Q P E T K V G D L C E E L G I T R Q T L Y R H V S P K G E L
. stop tniC
-.I Sall
GGCCAGACGGCGTAAAGCT,GCTCTCCCTCGGTrCAGCCGCATAAATGGAGGCGACCTGGAACGGGGCGCTGTTCAGTGCGGCAACGATCCGATTACCGGT,GTCGACCCAGAGCAGCCGTA
R P D G V K L L S L G S A A
GAGCTTTTGG;GAAAGCTGTCGTTCAACGCCGAGTTCAGCGGCAGTTTTAAGTTGTGATTTTATCCAATACTTTTGCGCAGCAAAACCATAAAGCCGCGACTTAAAAACTGTCCAGCGCA
SS* [F-stop qacE 5
GGAGATCGACG3mACATGTGTATATTTATGGCACG3AGAAGTTAACTCAACACTAACTATGAGCCCCATACCTA
HindIII
CAAAGCCCCACGCATC.AAGCTTTTGCCCATG,AAGCAACCAGGCAATGGCT ATAAGCGCCGAGTCCCGACGACCTCCGCATACAACACGAGATCGAC
HindlIl
GAGAAAGAAAATAAAATGCGATGCCATAACCGTAGCACGGAGGCAAGCTTAGTAAAGCCCTCGCTAGATTTTAATGCGGATGTTGCGATTACTTCGCCAACTATTGCGA
start qacE +-
TAACAAGAAAAAGCCAGCCTTTrCATGATATATCTCCCAATTT,GTGTAGGGCTTATTATGCAC CAGAGCTGACTATAGTTTGGCTGTGAGCAATTATGTGC
stop orfD |4-88
TTAGTGCATCTAACGGGCGAGGTAAGCCGACCGCAGAATGCGGGTCGGCTT'GACCGAAAT,GTTAGAGCCAGAAGCCAAAACGGATAACCGTAACTTGC,GACGGGCGCTAACTGCGAAAA
...
GGCACGGT,GCCACCGAGGCGGCACAGCACTACGAAAATGC CCGCGC7rCCTACACAAGGGCCAGAGGCCAAAAAGACCACAAACCAGCGTAACGCCACCAGGCGAAG;CCCAGA
* start orfD . S*3
GAAACGGCAAACCCAGCGCTGCACAGCGTTTGAGAAACTGAATGCCGTTATACTGCAAAAGGG% ACGTACCGGGGC GCC
EcoRI .1| - stop dhfrllc
GTCCGCTGGAGCGACCAGl GTTGCGATCCGTTACCGGGTCCCACTGAATTCAGGCCACGCGTTCAAGCGCAGCCACAGGATAAATCTGTACTG
AACCGGGGTGAGACTCGGACTCGACGGCATAGCCTTCAGGGGTCAGTTTTGTGCAGTACCACCCGACAACTT,GACCCTGCCAAGCGGCGCCAGATrCI,CCACGCGAT,CTCCCAGGC
start dbfrllc 4-
CAAACGTGGCGTGCGATGGGAGCGCAAACT,GGCCAGCAACTAGAGTACT,GACTCCATTGTT,GTGTT,GGTCCATATCT,GACCmcrTrETT,GAATiTi-v-T-GAGATcTCAcCA
. **2
CGCTGCGTCGGT,GAAGCCCAACTTTGTTTTAGGGCGACTGCCCTGCTGCGTAACATCGTTGCT,GCTCCATAACATCAAACATCGACCCACGGCGTAACGCGCTrGCTTGGATGCCCG
-35 p_>
* start int
AGGCATAGACTGTACAAAAAAACAGTCATAACAAGCCATGAAAACCGCCACT GCGCCGTTACCACCGCTGCGTTCGGTCAGTC,GCCAGTTGCGTGAGCGCATACGCTACTTGCA
-10 EK T A T A P L P P L R S V K V L D Q L R E R I R Y L H
<-P
-10 -35
TTACAGrTTTCGAACCGAACAGGCTTATGTCAACTGGGTTCGTGCCTTCATCCGTTTCCACGGTGTGCGTCACCCGGCAACC'GGGCAGACGATCGAGGCATTTCTGTCCTGGCT
Y S L R T E Q A Y V N W V R A F I R F H G V R H P A T L G S S E V E A F L S W L
GGCGAACGAGCGCAAGGTTTCGGTCTCCACGCATCGTCAGGCATTGGCGGCCTGTTCTTCGAGGGTTCCGTTCCTGGCTTCAGGAGATCGGAAGACCTCG
A N E R K V S V S T H R Q A L A A L L F F Y G K V L C T D L P W L Q E I G R P R
*.
-. SphI
GCCGTCGCGGCGCTTGCCGGTGGTGCT,GACCCCGGATGAAGT,GGTTCGCATCCTCGGrr'GGAGCAGCATCGTTTTTGCC
P S R R L P V V L T P D E V V R I L G F L E G E H R L F A Q L L Y G T G E R I S
E G L Q L R V K D L D F D H G T I I V R E G K G S K D R A L M L P E S L A P S L
PvuIIGCGCGAGCAGCTIGTCGCGTGCACGGGCATGGTGGCTGAAGGACCAGGCCG6AGGGCCGCAGCGGCGTT'GCGCTTCCCGACG~CCCTTGAGCGGAAGTATCCGCGCGCCGGGCATTCCTGGCC
R E Q L S R A R A W W L K D Q A E G R S G V A L P D A L E R K Y P R A G H S W P
GTGGTTCTGGGTTTTTGCGCAGCACACGCATTCGACCGATCCACGGAGCGGTGTCGTIGCGTCGCCATCACATGTATGACCAGACCTTTCAGCGCGCCTTCAAACGTGCCGTAGAACAAGC
W F W V F A Q H T H S T D P R S G V V R R H H M Y D Q T F Q R A F K R A V E Q A
AGGCATCACGAAGCCCGCCACACCGCACACCCTCCGCCACTCGTTCGCGACGGCCTTGCTCCGCAGCGGTTACGACATTCGAACCGTG,CAGGATCTGCTCGGCCATTCCGACGTCTCTAC
G I T K P A T P H T L R H S F A T A L L R S G Y D I R T V Q D L L G H S D V S T
stop int
-fl
GAGTGTTCCCATG7:CTGAAAGTTGGCGGT ,GCCGGAG7:CGCTCCCGCTTGGAqnCGCT,GCCGCCCCTCACTAGTIGAGAGGTAGGGCAGCGCAAGTCAATCCTGGCGGATTCA
f i4 . 1 i3 ~ BaHI
CTACCCCTGCGCGAA,GGCCATCGGTGCCGCATCGAACGGCCGGTTGCGGAAAGTCCTCCCTGCGTCCGCTIGATGGCCGGCAGCAGCCCGTCGTTGCTGATGGATCCAACCCCTCCGCT,G
CTATAGTGCAGTCGGCTTCTGACG
_C_TA_GCCCTGCACAACCAGGCGCAGGCGNTGACCTACTCG CGCGGCTCGGGGGTAGT
( IRi-~~ ~ ~ ~ ~ ~ 8..
TCATCCACAGCCGTTCGATAGCGATGCCGCCGCGCGTCATCGCGTTGAAGTCGATGGTTCTCATGACGCCAAGGGAGAGCTGTCATAGATCGTTGAGCTGTAGCCCGACACCATCACCG
CGCACGGCAA,GCCGGCCAGACG
3960
340
4080
380
4200
405
4320
33
4440
73
4560
113
4680
133
4800
193
4920
207
5040
5160
5280
5400
5520
5640
5760
5880
6000
6120
6240
6360
28
6600
68
6720
108
6840
148
6960
188
7080
228
7200
268
7320
308
7440
7560
7680
7800
7827
FIG. 2. The nucleotide sequence of TnSO9O and its immediate surroundings in plasmid R751. Arrows indicate IRs; those of 25 bp at the limits
of Tn5O90 are denoted IRt and IRi, and those of 19 bp close to the ends are denoted tl to t3 and il to i4. The ORFs tniA, tniB, orf6, and tniC and
the previously observed int (42, 57) are shown with their translations. Locations of important branch points that are marked by # and numbered
are also shown in Fig. 1. Promoters are indicated by P and an arrow. The borders of cassettes are indicated by vertical arrows (note that cassette
genes and ORFs are translated from the opposite strand). The sequence shows two sequences determined previously, i.e., that for the qacE
multiresistance gene, which has recently been described in a separate study (45), and a revised version of that for the dhfrIIc gene (17). The
integrase gene segment of the R388 integron (from no. 2 to 1; see Fig. 1) was analyzed here and revealed complete identity to Tn5O9O.
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3262 RADSTROM ET AL.
A
1.4 0.7 0.4 2.3
C
2.7
B
w~
I
_-
l _ _
IRi int dhfrVII qacEAl sulI tniB
1.4 1 0.6 1 1.9 2.7
I I \~
I I ~~~~~~~~~~~~A
aadAl 4.2
0.9
tniAIRt
5%~N Tn5090
IRt (R751)
Tn5 093
(Tn5O86)
Tn5092
(Tn2l)
2 kb
B
IRL genetic cassettes |
inC dhfrI aadAl tnsE tnsD t1i nsA
sat
Tn7
2 kb
FIG. 3. Physical organization of TnSO9O and two related elements and similarities with Tn7. (A) Genes and conserved areas (hatched) among
Tn5O9O, Tn5O92, and TnSO93. ORFs are indicated by horizontal arrows, and those located on cassettes are marked with open arrowheads. Small
figures indicate the sizes in kilobases of the constant and variable segments. A, B, and C at the vertical arrows refer to the sections of Fig. 6 where
sequences at breakpoints are given. RS indicates the cassette insertion site 5' of int. (B) Map ofTn7 based on references 18, 32, and 58 and citations
therein, shown for comparison. Names of structures or putative functions in Tn7 corresponding to features of TnSO9O are boxed. Filled vertical
boxes indicate terminal repeats.
All three transposases carried the motif D,D(35)E earlier
observed in the latter protein and in a newly defined super-
family of bacterial transposases and IN proteins of long
terminal repeat retroelements and retrotransposons (16, 31,
49). Less certain similarity (18% identity) was observed with
transposase (A) of phage Mu (26), which seemed to contain
only the first D (D-269) and the E (E-392) of the complete
D,D(35)E. Those transposases of IS elements which carry the
D,D(35)E motif (16) had less similarity to the tniA gene
product than the level of similarity among TniA, TnsB, and
P480.
The second ORF predicted to take part in transposition,
tniB, is located between tniA and orf6 and in the same
orientation. It encodes a 302-amino-acid polypeptide which
was predicted to be isoelectric at neutral pH. This contrasts
with the basic products encoded by int, tniA, and tniC (see
below). The amino acid sequence of the tniB gene product
indicated two sites for potential binding and hydrolysis of
nucleoside triphosphates (22) (Fig. 5) and showed 21% iden-
tity to the TnsC protein of Tn7 (18) and 24% identity to the B
protein of bacteriophage Mu (37). Both TnsC and MuB are
ATP-binding proteins (20, 36), and p271 of TnSS2 is also very
likely to be one (49).
The ORF tniC codes for a recombination protein of a type
different from those encoded by int, tniA, and tniB and was
found in the unique 2.3-kb region of Tn5O90. The putative
promoter was observed in the region between orf6 and tniC,
and the latter gene may thus have its own regulation separate
from the operon of tniA, tniB, and orf6. The product of tniC
consists of 207 amino acids when the most probable translation
start (GTG) is utilized. With its 67% identity to the resolvase
of TnS393 (10) and 47% identity to Gin of phage Mu (46), it
belongs to the invertase/resolvase family of site-specific recom-
binases (Fig. 5) (21). The identity to most transposon re-
A
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TRANSPOSON TNSO90 OF PLASMID R751 3263
int dhfr orfD qacE tniC orff6 tniB tniA
0.00 7.58 3.43 8.71 4.58 3.63 2.02 0.81
0.00 5.89 6.98 6.92 3.87 6.81 6.72
0.00 8.58 5.92 5.06 4.60 3.10
0.00 9.12 8.13 9.72 9.70
0.00 4.97 2.74 3.48
0.00 2.47 3.79
FIG. 4. Correspondence analysis of codoi
usage was analyzed by the Genetics Comput
RESPOND (14) using the statistics describes
The tniB gene was truncated to include o
Tn5O9O, Tn5092 and TnSO93. D-squared v
likelihood of a common origin for the genes
solvases, e.g., that of Tn21 (24), was lo,
most conserved residues of invertases/rc
including the serine 9 corresponding
Ser-9 in Gin (30). There was also a
binding motif (43) close to the 3' end.
Tn5O9O ends have a complex structui
at the 25-bp terminal IRs of TnSO9O bc
This characteristic of ends has been obs
IS elements (16, 19), transposons Mu,
after end processing, also for retrovi
posons. Beginning eight nucleotides i
there were 19-bp repeated sequences th
at the left end (il, i2, i3, and i4) and thre
(tl, t2, and t3) (Fig. 7). The repeats i3 a
most diverged from the others (see cons
7B). The repeats at one end were in ti
with respect to those occurring at the o
the t2 repeat at the right end. Similarly (
to the ends of both bacteriophage M
identified as the binding sites for the r
MuA and TnsB (1, 13).
Further sequences external to IR, a
pLMO20 (classified here; see Material
an integron (57) which was found he
element flanked by both IR, and IR.
nucleotide sequencing and was found
stream of the unique SacI site close tc
inverted repeats, IRt and IRE, in pLMO,
direct repeats (CTGTT; see Fig. 6A ai
probably a target site duplication. The
however, lacked the transposition genm
ably as a result of the presence of an IS
observed to be joined to an EcoRII re
gene, as shown previously for IncN
elements carry the IS6100 (33) 152 bp
We sequenced the IRi borders of
containing elements. These elements ii
revealed unique border sequences (Fig
IR; border sequence was documented I
Some TnSO9O-like elements have persist
location. One example is IncW plasmid
to be flanked outside IRi by the same se4
sequences flanking Tn5092 and TnS5U
(Fig. 6). Nevertheless, we have shown a variety of other
sequence contexts harboring Tn5O90-related elements, which
warrants their identification as a family of transposable ele-
int ments flanked by 25-bp IRs. TnSO9O probably represents a
dhfr complete version of these transposons, while other elements,
e.g., that in R388, have lost major parts in the course of
orfD plasmid evolution (Fig. 1 and 6).
qacE
tniC
orf6
DISCUSSION
In this work, a type of elements carrying integrons is
0.00 1.27 tniB identified as potential transposons related primarily to a few
0.00 tniA other mobile elements in bacteria and also remotely to the
retroelements. By detailed study of a range of plasmids of
usagerinpTn0o9. Codon different incompatibility groups (e.g., W, P, N, and F), wedrb Groutphrogram Rl.(3). confirm here that the 25-bp IRs IRi and IR, are the true endsInby Granthampat al.mon(. of this family of elements.nly the part common to A distinguishing feature of the retroviral type of mobile
elements is the dinucleotide 5'-TG which always occurs at their
termini (31, 63). The ends of Tn509O as well as the ends of Tn7
(32), of Mu (28), and of Tn552 (49) carry 5'-TG, which is also
found at the ends of most IS elements coding for a transposase
wer, around 30%. The of the retroviral superfamily (16, 19). The 5'-TG ends of
esolvases were present, retroviruses and retrotransposons are exposed only after the
to the DNA-reactive removal of one or two terminal nucleotides (38, 63). The ends
helix-turn-helix DNA- of both Mu and Tn7 have an unusually complex structure with
several iterations of the target sequence for binding of trans-
re. The conserved ends posase (1, 11, 32). These repeated sequences in Mu were
egin by 5'-TG (Fig. 6). recently shown to take part in assembly of the active tetrameric
served earlier for some form of the transposase MuA (4). This assembly process was
TnSS2, and Tn7, and, described as a key regulatory step in Mu transposition. We
iruses and retrotrans- have observed here similar 19-bp repeats close to the ends of
nward from each end Tn5090 which could indicate a relationship between the trans-
iat occurred four times position mechanisms of these transposable elements. This
e times at the right end would also mean that the transposase of Tn5090 is a multi-
ind i4 seemed to be the meric protein which requires the 19-bp repeats near each end
,ensus sequences in Fig. in order to assemble (3, 15).
he inverted orientation Recently, the Xanthomonas mercury resistance transposon
Ipposite end, except for Tn5053 (29) was reported to carry IRs of 25 bp similar but not
organized repeats close fully identical to those of TnS092 (the 11-kb element in Tn21
,u and Tn7 have been [7])and to generate 5-bp direct repeats of the target sequence.
espective transposases, Both Tn5092 and the integron-containing element of pLMO20
(Fig. 6) are bracketed by 5-bp target duplications. The absence
and IRt. IncN plasmid of target duplications around Tn5090 could be due to an
s and Methods) carries adjacent deletion removing the bordering segment on one side.
re to be borne on an The available sequence for the right end of TnSO53 (722 bases)
. IRt was localized by is 79% identical to the right end of TnSO9O and contains the
to occur 1.8 kb down- three 19-bp repeats (tl, t2, and t3; Fig. 7) and the 5' part of a
) sulI (57). Both 25-bp tniA-like (81%-amino-acid-identity) gene.
20 were flanked by 5-bp It has been shown that retroviral integration mechanistically
nd B); this sequence is resembles the transposition of bacterial mobile elements (38).
element of pLMO20, The recent finding (16, 49) of a structural similarity among the
es of Tn5090, presum- probable transposase P480 of TnSS2, some IS transposases,
,6100 (25). The IRt was and the retroviral transposases, commonly called integrases or
striction endonuclease IN proteins, supports this view. The D,D(35)E motif in the
plasmid N3 (6). Both active regions of all of these transposases is highly conserved
from the end of IRt. (38). The motif also includes a few additional amino acids of
a few more integron- more relaxed conservation. Some of these are aspartic acid
n R388 and pLMO229 residues. There is proof obtained by mutagenesis that the two
6A). Another unique aspartic acid residues and the glutamate of D,D(35)E in IN
previously in R46 (56). proteins are critical both for cytoplasmic end processing of the
ted in the same plasmid reversely replicated viral genomes and for their subsequent
I pSa, which was found insertion into a host chromosome (31). The related bacterial
quence as in R388. The transposases await similar analysis. The interactions between
93 were also identical the three acidic amino acids and DNA may be similar to those
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MSMATDT ... PRIPEQGVATLPDEAWZRARRR.AEIISPLAQSET .V 40
MWQINEVVLFDNDPYRILAIEDGQVVWMQI SADKGVPQARAELLLMQYLDEGRLVRTDDPYVHLDLEZPSVDSV8FFQR=DYIKLPXXNSKDRFDPKV 10 0
MKNKZKYLTNFSEAZRKE&TQK.YNISKPF....I L 31
GHAADMAAQALGLSRRQVYVLIRRARQGSGLVTDLVPGQSGG .....................GKG. RLPEPVERVIBHLLQRMFLTKQKR. SL&AF 117
RSZLVEHVVQEHXVTKATr6RYWQRaQT PNALXPDYKNSGAPGERRSATGTAKI GRAREYOT=TVPEIMILFRLTIEKLNQKGTKTTJY 20 0
.KQSLSSISKSKGIALSTLYRWNKLYKEQG. .LTGLIH.NTRV...... .QNIXDZI..KLALKNKRNBIATI 101
EVTQVC ..... KAQKLRVPARNTVALRIASLDPRKVIRRR. EGQDAARDLQGV. GGEPPAVTAPLXQVQXDNTVIDLIVVDDRDRQP. IGRPYLTLAI 209
RR}tVDLFAQYFPRXPQEDYPTLRQFRYFYDREYPKAQRLKSRVKAGVYKKDVRPLSSTATSQALGPGSRYEX1_ATIADXYLVDHHDRQKIIXlRPTLYXVI 30 0
HRKIANYC..... IENNFYKPSYKQVYSIIKAMPKSVIDFSH. QGEKYQNKYDL ..IQIRESSRPN3IWQA2HTLLDIYILDQKGN... INRPWLTIIM 190
IS3 .. .GPNQKWAGDITYLRT...
Copia .. .RPLFVHS2VCGPIT...
MoMLV ... RPGTHWEIDFTEVKP...
D
DVVTRCVLGMVVTLZAPSAVSVGLCLVHVACDKRPWLEGLNVM ... DWQMSGKPLLLYLINAAZFKBEALRRGCEQHGIRLDYRPLAQPHYGGIVXRIII 306
DVFSRMITGFYXGVNNPBYVVAMQAFVNACSDKTAICAQHDIZISSSmPCVGLPDVLLA2RG. ZLM8HQVZALVSSFNVRVESAPPRRGDAKGIV5STF 399
DDYSRAIAGYFISFDAPNAQNTALTLHQAIWNKN ........ NT...NWPVCQIPEKFYTPSHGSDYrTSHMZQVAIDLKINLMFSKVGVPRGRGKI5RFF 279
IS3
Copia
MoMLV
... IVHTDRGGQYCSADYQAQLKRHNLRGSMSAKGCCYDNACV5SFFH ...
YLYIDNGRXYLBNEMRQFCVKKGISYHLTVPHTPQLNOVSZMIR ...
... VLGTPNGPAFVSQVSQSVAKLLGIDWKLHCAYRPQSSGQVIRMR...
D E
TniA GTAMQMIHDELPGTTFSMPDQR ......... GDYDSENKAALTLRELZRWLT LAVGTYHGSVH ..NGLLQPPAARWAEAVARVGVPAVVTRATSFLVDF
TnsB RTLQAE KSFAPGIVEGSRIKsHGETDYRLDASLSVFEFTQI ILRTILFRNNHVMKYDDAPDLPsIPVQLWQWGMQHRTGSLRAVEQEQLRAL
P480 QTVNQTFLEQLPG.YINNNDTs ......... SDL IDFQNFFEKLRYFLIEDYNQKEH. .SAIQSTPINRWNSNHFFPNMPSSLEQLDLLLLEI
TniA LPILRRTLTRTGFVIDHIHYYADALKPWIARRYERWPSFLXRRDPRDISRIWV.EPEGQHYLEIPYRTLSHPAVTLWQR.....................
TnsB LPRRKVSISSFQVNLWGLYYsGSEI. LREGWLQRSTDIARPQHLEAAYDPVLVDTIYLFPQVGSRVFWRCNLTERSRQFKGLSFWEVWDIQAQEKHNKA
P480 PK. .SRKXHsDGIHFQGFRYsNTNLTAYVG EYVLIRYNPNDtNAEIRVFYRDEFLCTAIS. PDLADYSIDIKEIQ
TniA
TnsB
P480
394
499
360
473
597
431
.QALALRQQGREQVDE8ALFRMIGQMREVT8AQKATRIA3RDADRRQHLKTSARPDKPVPPDTDIADPQADNLPPAKPFDQIEEW- 559
NAKQDELTKRRELEAFIQQTIQKANKLTPSTTEPKSTRISQXKTNKRAVTSERKKRAEHLKPSSGDEA4VI0PFNAVEADDQEDYSLPTYVPELFQDPPEKDES-
........^SQIRRKHLKQNIASPSSZTDLXIREEXSYGY8PQZTTKnU YRND* 48 0
B
Tn5090 TniB MDEYPIIDLSHLLPA ..........................AQGLARLPAD ............ Z.RIQRLRADRWIQ. YPRAV
Tn7 TnsC MSATRIQAVYRDTGVEAYRDNPFXEALPPLQESVNSAASLKSSLQLTSSDLQKSRVIRAHTICRIPDDYFQPLGTHLLLs .RXsvMIRGGYVGR -KTG
Mu B MNISDIRAGLRTLVENEETTFKQIALESGLSTGTXSSFXNDKYNGDEERVS
TniB EARLELYAWPNKQRM...................PNLVTN SMIVEKFRRTHPAS SDADQZHIPVLWQMPSEPSVIRFYVAL
TnsC DLQKHLQNGYERVQTGELETFRF3EARSTA Q LLIgCTSaSRLHRILATAYQVIYHRELNVFQVVYLKIDCSHNGSLKEICLNF
B QMLQRWLEKY.AVAELPEPPRFV3TQTVKQIWTSMRFASLTESIAVVCffl4. ....AAREYRRTM MITITPSCAV LC.
NTP-motif 1
TniB
TnsC
B
LAAMGAPL RPRPRLPEMEQLALALLRKV... GV}RZDYZLHNVLRGN8VNRRZFLELLRFLGUELRIPLVGVGTRD&YLAIRSDDQLENR...
FRALDRAI4SNYERRYGLKRHGIETNI LSSQIANAHALGL GZZIQHLSRSRBGGSQZMLFFVTMVNIIGVPVMLIGTPKAREIFEADLRSARRGAG
LTELAFELGM DAPRRKGPLSRALRRRLEGTQG. LVIIDZADLG& ZVLEELRLLQESTRIGLVLMQNHRVYSNMTGGNRTVEFARL
NTP motif 2
TniB FEPMMLPVWE .ANDDCCSLLASFAASLPLRRPSPIATLDMARYLLTRSEGTIGELA.LAALIVAVESGZZAINH ...............RTLSM
TnsC JGAXFWDPIQQTQRGKPNQEWIATDNLWQLQLL.QRKDALLSDEVRDVWYELSQVDVKFLQRAAMITAGLLRQVYQDELKPVHPMLE
B FSRI AKRTAINKTKKADVKLIADAW.. QINGEKELELLGQQ IAQKPGALRILNHSLRLAAMTAHGKGaRVNEDYLRQAFRELDLDVDISTLL
Tn5O92 TniB AC* 286
Tn5093 TniB ACRQPLAQPRHRGRTASDLZATGPSHPDMDLDRRTDVRFRVLGVRL* 330
Tn5O9O TniB AVYTOPS3RRRQFERELM- 302
TnsC ALRSGIPZRIARYSDLVVPZIDKRLIQLQLDIAAIQEQTPEEKALQELDTEDQ... 440
B RN* 312
C
Tn5O9O TniC
Tn5393 TnpR
Mu Gin
TniC
TnpR
Gin
M.XGzMaDVRaBG8Q8PNLQ3DALIAAaVSLAHLY3DL&8GRRDDRPGLALCLRAL3EQDTLIVWKLDRIaRDLRLXTVHDLTARsVGLKVLT?
S
HGaAVDATTTAGKLVGXA SZaRITLVAGMIBARAMGZEQRPOM4TAAK PQPETKVGDLCEZaXTRQTLYRHVSPKOZLRPD
KOAQI LDAtSItXATLAFRLARDI _V14DTV LCKI LaERVTLYYVGPOULDH
.SI.. DTSSPMGtFFVHMGALaZMzRNLXIRGLRN--PKLWRIGQPPKLTHAEWEQAGRLL&Q. GIPRKQVALIYDVALSTLYKKHPAKRAHIEN
* . . --- . *...- --..- **-**-** *- ** ***---*--* .. . ... .. . .... . .. ..
I-.---- .------- Ihelix helix
TniC GVKLLSLGSAA* 207
TnpR OKHVLGLT* 204
Gin DDRIN* 193
702
43
98
51
116
187
135
204
287
221
284
387
310
96
96
92
196
196
188
3264 RADSTROM ET AL.
A
J. BACTERIOL.
Tn5O90 TniA
Tn7 TnsB
Tn552 P480
TniA
TnsB
P480
TniA
TnsB
P480
TniA
TnsB
P480
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TRANSPOSON TN5090 OF PLASMID R751 3265
FIG. 5. The amino acid sequences of the translated ORFs tniA, tniB, and tniC of Tn5O9O (see Fig. 2 and 3) aligned with those of recombination
proteins studied earlier. Amino acids identical in at least two of three polypeptides are indicated by boldface letters. Amino acids identities between
Tn5O9O and an aligned sequence are marked with small dots, and identities with both sequences are marked with larger dots. (A) The product of
tniA aligned with the transposases TnsB from Tn7 (1, 18) and P480 from Tn552 (49). The alignment of the conserved D,D(35)E motif (31, 49)
of the retroelement integrase superfamily includes short segments of the transposases/IN proteins of IS3, Copia, and Moloney murine leukemia
virus (16, 49). (B) The protein encoded by tniB aligned with the ATP-binding proteins TnsC of transposon Tn7 (18) and B of bacteriophage Mu
(37). The sites potentially binding nucleoside triphosphates (22) are underlined. A vertical arrow marks the sequence divergence corresponding
to the end of the 2.7-kb segment. (C) Comparison of the probable resolvase encoded by tniC in R751 with the resolvase TnpR of transposonTn5393 (10) and the invertase Gin of bacteriophage Mu (46). The catalytic serine 9 of Gin (30) and the helix-turn-helix motif for DNA binding
(43) are marked.
between the E. coli DNA polymerase I and DNA involving the
binding of a metal ion (40).
The gene tniA codes for the probable transposase of TnSO9O,
which was observed to contain the D,D(35)E motif. The tniA
gene product is related to the transposases of Tn552 and Tn7
not only by this motif but also by a fairly high amino acid
similarity for other parts of the polypeptides. The D,D(35)E
regions among these related transposases appear to be as
closely related to the IN proteins of retroelements (31) as to
the IS element transposases (e.g., that of IS3 [16]) carrying the
motif. Some similarity was also observed with MuA trans-
posase, which may carry an incomplete D,D(35)E motif. If the
other aspects of similarity with Tn7 and Tn5O9O are consid-
ered, it seems plausible that Mu could have a related trans-
posase.
The tniB gene codes for a protein with the sequence motifs
GPTNNGKS and MLVIDE (Fig. SB), which indicate a site for
binding of nucleoside triphosphates (22). Transposons Tn7
(TnsC) and bacteriophage Mu (B) encode separate proteins
which make use of ATP for transpositional recombination (18,
20, 37). A gene for a potential ATP-binding protein, P271, has
also been described for TnSS2 (49). The genes for transposase
and ATP-binding protein in the three mentioned transposons
are consecutive and located close together, which indicates a
possibility of translational cooperativity for expression. The
product of tniB is weakly related to both TnsC (21% identity)
of Tn7 and to the B protein (24% identity) of Mu. MuB
increases the efficiency of Mu transposition and affects the
choice of target sites (35). It may belong to the proteins named
molecular matchmakers which are supposed to utilize ATP for
the creation of transient conformational changes in proteins or
DNA (51) which could temporarily make possible critical
A
pLMO20
Tn2l /Tn5O86
R751
pLMO229
R388/pSa
pLMO20/N3 IS6100
Tn2l/Tn5O86 tniA <-
R751 tniA <-
R388
Tn2l
Tn5O86
Tn2l
R751
sulI ->
sulI ->
sulI ->
4.2 kb
tniC <-
<- int
<- int
<- int
<- int
<- int
-> ecoRII
<- mer
4.2 kb
<- tniB
<- tniB
<- tniB
GG TTCCCGGTTCAAA TGCACTGAACG
AGAGAATGAGGAACACCAGA TGTCGTC&G&GCGGCC&CTG&&CG
-IRj - )
B
DR
CAACTGGTC&GTWCQCTTCTG&AA&TGAC& CGIrTTGTATATAATCATGA
CAACTGGTGCAGTCGTCTTCTGAAAATGACA TCCATGCCCAGCCCGTGCGC
CACTGGTGCAGTCGTCTTCTGAAAATGACA TCTTGGCCGGGTCGTTATTG
IRt
( ti
C
ATGTCTTGGTTGCGC G&Q&GTTGCGATATCCTCCACTTCCATCATCAAC
ATGTCS O G G&TATCTGTTGATTTGCACCCAAAT
CAOTGGGTCAATTTTAGATGCAACTCAACAGCACATCGCSOOTGTGCGATG
FIG. 6. Nucleotide sequences at branch points of the integron-carrying elements in R751, Tn5O86, Tn2l (7), pLMO20, N3 (6), pLMO229,
R388, and pSa (see Fig. 1 and 3). The locations of A, B, and C are marked in Fig. 3. Note that the sequences are given for the opposite strand
with reference to Fig. 2. (A and B) Branch points at the left and right ends, respectively, of R751, Tn2l, and pLMO20 are indicated. Bases which
belong to the integron-carrying elements are indicated by boldface letters, and 5-bp direct repeats of the target sequences are underlined. An
insertion of IS6 (19) in pLMO20 with an associated 8-bp target site repeat (CTGCACTG) is indicated. (C) Coinciding branch points; the sequence
at the transition between the 2.7- and 2.3-kb segments in Tn5O9O (see Fig. 1, 2, and 3) and those at the junction between the 2.7- and 1.9-kb
segments in Tn5O93 which contains a 4.2-kb insertion in TnSO92 (60) and marks the rightmost (Fig. 1) end of homology with R388. Boldface letters
indicate identity in at least three sequences.
DR rIS26
TAACAGCCTTTCTGGCTGTT TGTCGTTTCAQ G QG C&CTG&&C
tnpR <- CCCTCGGCTACCACCTCATTGTGTTC3AG&&GACGCTGCACQACGQ
GCGCCCATACGCGCTTGACC TGTCGTTTTC&GAAGACCTGCACAACG
VOL. 176, 1994
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3266 RADSTROM ET AL.
A
L
Tn5O9O
R
L
Tn7
R
L
Mu
R
--
-70 -moil ±2 ±3 ±4
t'12 t-3M
--
->A
L1 L2 L3
-I -
Rlb > R3 mRw.
R2I
Li L2 L3
RI 2Ri 32 R33
B
TCAGAAGACGGCTGCACTG
TCAGAAGCCGACTGCACTA
TCAGAAGACGACTGCACCA
TCAGGAGCTGGCTGCACAA
TCAGAAGTGATCTGCACCA
TCAGRAGncRrCTGCACyR
<- 10 bp
TCAGGCAACGACGGGCTGC
TCAGCGGACGCAGGGAGGA
TCAGRRGncGrCtGcAcnR
ii
i2
tl
t2
t3
il-2, tl-3
i3
i4
all
FIG. 7. The multiple repeats of 19 bp which are clustered at the
ends of Tn5O9O, Tn5O92, and Tn5O93 and of the corresponding
element in pLMO20. (A) Organization of multiple repeats at the left
(L) and right (R) ends of Tn5O9O and of the 22-bp repeats similarly
occurring near the ends of Tn7 (1, 32) and Mu (4, 13, 28). Transposon
sequences are marked by a straight line, and surroundings are indi-
cated by a zigzag line. (B) The nucleotide sequences of the seven
copies of the 19-bp repeats of Tn5O9O. Consensus sequences are given
in boldface letters. The two most conserved parts of the repeats are
separated by about 10 bp, as indicated, which corresponds to one
helical turn of DNA.
macromolecular interactions. The direct contact between an
activated molecule of MuB and Mu transposase stimulates
DNA strand transfer; however, ATP hydrolysis does not
appear to be essential (5, 61). A similar function of the product
of tniB could be expected. Few transposons are known to carry
a separate gene for an ATP-binding protein, supporting the
idea that TnSO9O is related to Tn7, Mu, and Tn552.
The tniB gene starts only two nucleotides downstream of
tniA and thus reads in the -1 phase (9) with respect to the
latter. A small part of the 3' end of tniB in Tn5O9O does not
belong to the conserved segment of 2.7 kb, which makes the
products of the tniB genes in Tn5O92 and TnSO93 contain
different C-terminal amino acid sequences. We observed that
orf6 (Fig. 2 and 3) has its most likely translation start codon
(GTG) just at the 3' end of tniB in Tn5O9O, in the -1 phase.
Starting from this codon, orf6 encodes a neutral polypeptide of
405 amino acids. A probable operon promoter was found
between IRt and the start of tniA. This operon includes both
tniA and tniB and probably also orf6. The overlap of the
promoter with repeat t2 suggests that expression of the operon
could be regulated by binding of the tniA gene product to that
sequence.
The tniC product may be required in a resolution step in
transposition of Tn5O90. Its relatively strong similarity to the
invertase branch of the family including Gin of Mu (47% [46]),
compared with the weaker similarity with most transposon
resolvases, such as, e.g., that of Tn2l (about 30%), is unusual.
However, recent data show that the structures of resolvases
may be sufficiently diverse to also include TniC. The putative
resolvase encoded by the recently studied transposon Tn5393
(10) is the protein that most closely resembles TniC (67%
amino acid identity). Furthermore, the potential resolvase of
Tn552, BinL (49) is 42% identical to TniC and similarly related
to Gin of Mu. We have shown here that Tn5O9O, Tn7, and
TnS52 carry genes for related transposases, which could indi-
cate that donor cleavage and strand transfer occur similarly for
these elements. There are no direct data yet available suggest-
ing which mechanism is used for TnSO9O and Tn552; however,
candidate resolvases in both elements suggest replicative trans-
position. The structurally related Tn7 moves by direct trans-
position (3), which is notable. It could be mentioned that an
interrupted gene identical to tniC but deleted for 408 bp of the
central part was found by a computer search 3' of the LCR-1
P-lactamase gene in transposon Tn1412 (12).
Our findings show that integrons are located on a new type
of transposable elements, although it is still open to specula-
tion whetherthe integrons and their carrier elements have
evolved in concert. The result of codon usage analysis (23) is
consistent with a common origin for tni4, tniB, and int (Fig. 4).
tniC and orf6, however, seem to have slightly different codon
usage than tniA and tniB (Fig. 4). From a comparative view, it
is noteworthy that Tn7, besides being related to Tn5O9O, also
seems to contain a similar but distinct variant of the integron
integrase and similarly inserted cassettes (57-59). We observed
that the Tn7 genes tnsA-E (32) and int have a very similar
codon usage, but one that is distinct from that of TnSO9O. This
indicates that there may be an old connection between the
integrase systems (termed integrons) and their respective
transposons. Expression and purification of proteins from the
ORFs of Tn5O9O are in progress. The links between Tn5O9O
and its transposon and retroelement relatives merit further
exploration.
ACKNOWLEDGMENTS
We thank V. Nikiforov, Russian Academy of Sciences, Moscow,
Russia, for mutual communication of data prior to publication; and
Birgitta Nilsson, Carl-Gustaf Thulin, and Katarina Erixon for skillful
technical assistance.
This work was supported by the Swedish Medical Research Council.
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