Characterization of Tn1547, a composite transposon flanked by the IS16 and IS256-like elements, that confers vancomycin resistance in Enterococcus faecalis BM4281

Gene, 172(1996)1-S 0 1996 Elsevier Science B.V. All rights reserved.

GENE

037%1119/96/$15.00

09708

Characterization of Tn1547, a composite transposon flanked by the IS16 and IS256-like elements, that confers vancomycin resistance in Enterococcus faecalis BM428 1 (Glycopeptide;

Richard

inverted

Quintiliani

repeat; TnlO; Tn4001; transposase;

uanB)

Jr. and Patrice Courvalin

Unitt des Agents Antibacttriens, Centre National de la Recherche Scientijique EPJ0058,

Institut Pasteur, 75724 Paris Cedex 15, France

Received by F. Barany:

1995; Received at publishers:

18 September

1995; Revised/Accepted:

1 October/l

November

6 February

1996

SUMMARY

A 64-kb genetic element harboring a uanB vancomycin-resistance (Vm”) gene cluster was shown to translocate from the chromosome of Enterococcus faecalis BM4281 into the hemolysin (Hly) plasmid, pIP964. Sequence analysis of the resulting junction fragments indicated that the VmR genes were carried by a composite transposon, Tn1547, bounded by two distantly related insertion sequences (IS), designated IS256-like and IS16, in a direct orientation. IS256-like (1324 bp) was identical to IS256, except for two nucleotide (nt) transitions in the putative transposase gene. IS16 (1466 bp) contained a large open reading frame (ORF) that was 61% identical to the gene encoding the putative transposase of IS256. There was 58% identity between the deduced amino acid (aa) sequences of the putative transposases of IS256-like (390 aa) and IS16 (395 aa). IS16 was delineated by imperfect inverted repeats (IR) (18 out of 26 bp) which were related (23/26 bp identity) to their respective imperfect IR counterparts in IS256. The difference between the IS was not a barrier for transposition of Tn1547 which generated an 8-bp duplication at the target site. Dissemination of VanB-type

resistance

among

enterococci

results

from two mechanisms:

(i) large conjugative

elements

translocate

from

chromosome to chromosome following inter-strain transfer; and (ii) as described in this report, the VmR genes transpose from replicon to replicon within the same strain as part of composite transposons that are internal to the conjugative elements.

INTRODUCTION

The glycopeptide antibiotics vancomycin (Vm) and teicoplanin (Te) bind to the peptidyl-D-alanyl-D-alanine termini of peptidoglycan precursors at the cell surface and prevent transglycosylation and transpeptidation steps in Correspondence to: Dr. P. Courvalin, Unitt des Agents Antibacttriens, Institut Pasteur, 28, rue du Dr. Roux, 75724 Paris Cedex 15, France. Tel. (33-l) 45 68 83 20; Fax (33-l) 45 68 83 19; e-mail: [email protected] Abbreviations: aa, amino acid(s); Ap, ampicillin; aphA-3, gene encoding an aminoglycoside 3’-O-phosphotransferase; bp, base pair(s); Ef Enterococcus faecalis; Efa, Enterococcus faecium; Er, erythromycin; ermB, gene encoding a rRNA methylase; Fus, fusidic acid, Hly, hemolySSDI 0378-1119(96)00110-2

cell wall synthesis (Reynolds, 1989). Two classes of acquired resistance to glycopeptides in enterococci, VanA and VanB, can be distinguished on the basis of resistance or susceptibility to Te. Resistance to high levels of Vm and Te defines the VanA phenotype and is mediated by the vanR, S, H, A, X, Y, Z gene cluster (Arthur and sin; IR, inverted repeat(s); IR,, left IR; IR,, right IR; IS, insertion sequence(s) (Fiandt et al., 1972); kb, kilobase or 1000 bp; Km, kanamycin; nt, nucleotide(s); oligo, oligodeoxyribonucleotide; ORF, open reading frame; PCR, polymerase chain reaction; R, resistance/resistant; RBS, ribosome-binding site(s); Rif, rifampin; Sm, streptomycin; Te, teicoplanin; Tn, transposon; tnp, gene encoding a transposase; Tra, selftransferable; Vm, vancomycin; [I, denotes plasmid-carrier state; ::, novel junction (insertion).

2 Courvalin, 1993). The vanA gene encodes a D-Ala:D-Ala ligase that synthesizes D-alanyl-D-lactate which is incorporated peptides

into peptidoglycan precursors that bind glycowith reduced affinity. VanA-type resistance is

mediated by the transposon Tn1546 or related elements, which belong to the Tn3 family (Arthur and Courvalin, 1993). Strains with the VanB phenotype have various levels of resistance

to Vm but remain

susceptible

have from

the mobility

of the vanB gene cluster we

studied the transposition of the resistance genes the chromosome of Enterococcus fuecalis (Ef)

BM4281

into a resident

RESULTS

AND DISCUSSION

hemolysin

plasmid.

to Te

(Quintiliani et al., 1993). VanB-type resistance is mediated by the vanR,, Sg, Y,, W, B, X, gene cluster (Evers and Courvalin, 1996) which, like the vanA operon, directs synthesis of peptidoglycan precursors terminating in D(Evers et al., 1994). The vanB gene cluster

alanyl-D-lactate

ther characterize

is usually carried by large conjugative elements (90-250 kb) which are transferable from chromosome to

(a) Translocation of the uanB resistance gene cluster from the chromosome of EfBM4281 into hemolysin plasmid pIP964 Trans conjugant Ef BM4281 (Table I) results from transfer of a 250-kb element conferring VanB-type resistance from the chromosome of Eju BM4120 into that of

Ef JH2-2

(Quintiliani

and

Courvalin,

1994).

Plasmid

chromosome between Enterococcus spp., suggesting that vanB resistance genes are carried by conjugative transpo-

pIP964, which conjugates between enterococci at a high frequency (10-l per donor) and mediates production of

sons (Quintiliani

hemolysin

TABLE

and Courvalin,

1994). In order

to fur-

(Table I), was introduced

into

BM4281

I

Bacterial

strains

and plasmids

Strain=

Relevant

E. co/i JM83 EfJH2-2 Efa BM4107

ara, A(lac-proAB), strA ($80 1acZAM 15) FusR, RifR, spontaneous mutant of JH2 FusR, RifR, spontaneous mutant of BM4105

EfBM4113

ErR, KmR, SmR, integration genes into the chromosome

EfBM4110 Ef BM41 lO[pIP964]

SmR SmR Hly+,

Efa BM4120

KmR, VmR, wild VanB-type

Ef BM4281

Ef BM4304

FusR, RifR, VmR, 250-kb element of BM4120 in the 400-kb @I fragment of JH2-2 by conjugation FusR, RifR, VmR, Hly+, conjugation BM4281 x BM4110[pIP964] ErR, KmR, Sms, VmR, Hly+, 250-kb element of BM4281[pIP964] in the

(1994) This study This study

Ef BM4305

400-kb @I fragment of BM4113 by conjugation ErR, KmR, SmR, VmR, pIP824 (pIP964: :Tn1547);

This study

Ef BM4281 [pIP964]

propertiesb

conjugation

Reference

Vieira and Messing (1982) Jacob and Hobbs (1974)

of pAT112 carrying of BM4110

HO238

or source

the aphA-3 and ermB

x BM4110

strain

conjugation

Leclercq et al. (1989) Trieu-Cuot et al. (1991) Courvalin

and Carlier

Courvalin Quintiliani

and Carlier (1986) and Courvalin

(1994) Quintiliani

(1986)

and Courvalin

BM4281[pIP964]xBM4113 Ef BM4260

FusR, RifR, Vms, 90-kb element of UMH in the 610-kb SfiI fragment of JH2-2 by conjugation FusR, RifR, VmR, 250-kb element of BM4120 in the 290-kb @I fragment

Ef BM4274

of JH2-2 by conjugation FusR, RifR, VmR, conjugation BM4120 x BM4107 ErR, KmR, SmR, VmR, wild VanB-type strain

Efa BM4306 Ef UMH-1 Plasmids pIP964 pIP824 (pIP964: pAT425 pAT426 pAT427 pAT428

:Tn1547)

Tra, Hly + VmR, Tra, transposition of TnZ547 into the cylB gene of pIP964 ApK, pUClSfi2.2-kb BglII fragment of pIP824 (pIP964: :Tn1547) containing the left extremity of IS256-like ApR, pUClSfi2.2-kb Sau3AI fragment of pIP824 (pIP964: :Tn1547) containing IS256-like ApR, Bluescript@ SKR1.2-kb HpaII fragment of pIP824 (pIP964: :Tn1547) containing the left extremity of IS16 ApR, pUClSfi1.3.kb Sau3AI fragment of pIP824 (pIP964: :Tn1547) containing the right extremity of IS16

“Ef, Enterococcus faecalis; Efa, Enterococcus faecium. b Cl, in vitro insertion;

x . genetic cross,

Quintiliani (1994) Quintiliani (1994) Quintiliani Schwalbe

and Courvalin and Courvalin et al. (1993) et al. (1991)

Borderon et al. (1982) This study This study This study This study This study

by

3 conjugation resulting used

with

BM4110[pIP964]

transconjugant

BM4281[

as a vanB donor

BM4113

in mating

at a frequency

agar

plates

and

Vm (10 pg/ml).

containing

transconjugants

of each

Strains

of lop8

BM4304

36.4

of VmR

VanB-type 10-l

after digestion

and

resistance

BM4305

to JH2-2 at

per donor,

respectively.

were analyzed

8.4

for DNA

electrophoresis

with SfI

1994) and by agarose

12.5

(4 of 20 clones).

by zero integrated-field

DNA

Courvalin,

classes

BM4304

and BM4305

rearrangements of total

of VmR on blood

which were either hemo-

class,

and

Ef

Sm (1000 pg/ml)

or non-hemolytic

(Table I), retransferred frequencies

transfer

phenotypic

were obtained

with

per donor

Er (8 pg/ml), Two

lytic (16 of 20 clones) A representative

and

of lo-*

The

was, in turn,

experiments

(Table I) as a recipient

was obtained

(Table I). pIP964]

(Quintiliani

and

gel electrophoresis

of

purified plasmid DNA digested with EcoRI, followed in each case by Southern hybridization (Fig. 1 and data not

shown).

revealed

The

SfiI

a 250-kb

restriction

chromosomal

identified

by the replacement

BM4113

by a 650-kb

vanB

probe

as

(Quintiliani plasmid

previously

from

described and

to a vanB probe.

indicate

acquired

These

from

results

did not

BM4281 [pIP964] element

mediating

2.7

of to a

BM4281 profiles

of

BM4110[pIP964]

and

has

for

1994). The EcoRI

BM4304

was

fragment

hybridized

(Table I) were indistinguishable

copy of the 250-kb

which

of a 400-kb that

3.3

of BM4304

insertion

fragment

and Courvalin, DNA

profile

that

hybridize BM4304

a chromosomal VmR and that the

0.6

presence of pIP964 did not affect the frequency of conjugal transfer of this element ( lop8 per donor; Quintiliani and

Courvalin,

some.

The

hemolytic

1994)

from

SfiI profiles

chromosome

of total

transconjugant

BM4305

BM4113

were indistinguishable.

pIP964,

the

BM4305, fragment

designated pIP824 that was replaced

EcoRI

DNA

profile

and

to chromofrom

the non-

from

recipient

Compared of

plasmid

to that DNA

of

from

(Table I), had lost a 1.3-kb by seven fragments with a

total size of 65 kb (Fig. 1). One of these fragments,

with

a size of 12.5 kb, hybridized to probes specific for vanB and the 1.3-kb fragment establishing that the VmR genes of BM4281

had translocated

into the 1.3-kb EcoRI frag-

ment of pIP964 as part of a 64-kb provisionally designated Tn1547.

element

that

was

(b) Cloning and sequence analysis of the junction fragments between Tn1547 and pIP964 The junction fragments composed of DNA from Tnl547 fused to that of pIP964 were cloned in Escherichiu coli JM83 (Table I) and analysis of their sequences indicated that the VmR genes were carried by

Fig. 1. Analysis (pIP964::Tn1547)

of EcoRI restriction DNA by agarose

profiles of pIP964 and pIP824 gel electrophoresis (left) and by

Southern hybridization (right) with a oanB-specific probe. The common fragments are indicated by lines. The size in kb of the additional fragments in pIP824

(pIP964::Tn1547)

is indicated

on the right of the gel.

The arrowhead on the left indicates the 1.3-kb EcoRI target fragment (poorly visible) in which Tn1547 translocated. The 12.5-kb additional fragment of pIP824 hybridized with the uanB probe. Methods: Plasmid DNA purified by C&l-ethidium bromide ultracentrifugation was digested with EcoRI, fractionated by 0.7% agarose gel electrophoresis, transferred by vacuum onto a nylon membrane (Schleicher and Schuell, Dassel, Germany) and hybridized with a uanB probe (Evers et al., 1994) which was 32P-labeled by nick translation (Nick translation kit, Amersham,

Les Ulis, France).

a composite transposon sequences (IS) designated

flanked IS256-like

by two insertion and IS16 (Fig. 2).

(i) IS2564ike element Screening of nt sequence databases indicated that the left end of Tnl547, as defined in Fig. 2A, was nearly identical to the 1324-bp IS256 first isolated in Staphylococcus

Tn 7547

A

IS 16

IS 2564ike B

s

A

s

‘S pAT425

s

vanRBSB y, W HBB XB

I

I

:/

f”P

ffv

‘9

>

‘97

pAT427 pAT426

pAT426

B Target

Target duplication

Fig. 2. Structure

IS 256.like

of Tnf.547.

(A) Tn1547

site

Target duplication

1%

(64 kb) is delineated

by IS256like

and IS16 (open boxes) and carries

nunR,,

S,,

Y,, W, H,,

B, X,

genes.

The intervening regions between the IS and cvlB of pIP964 are indicated by double and single lines, respectively. The orientation of the canB gene cluster is arbitrary. The terminal IR, and IR, of the IS are indicated by black triangles. The arrows indicate the ORFs for the putative transposases. A, AoaII; B, BglII; H, HpaII; S, Sau3AI. Closed boxes indicate limits of the inserts of recombinant plasmids. (B) Partial nt sequence of the 1.3-kb EcoRI fragment of pIP964 before transposition of Tn1547. The 8-bp target site is in uppercase letters and boxed. The nt numbering is that of cylB (Gilmore et al., 1990). The nt sequence of 700 bp of pIP964 containing the target site for Tn1.547 was identical to a portion of pADl cy1B (Fig. 2 and data not shown). (C) Sequence of the junction fragments in pIP824 (pIP964::Tn1547). The 8.bp target duplication is indicated in uppercase letters and boxed. The IR, of IS256-like and the IR, of IS16, which constitute the terminal IR of Tn1547, are indicated in uppercase letters and underlined. Methods: The 1.3.kb EcoRI fragment of pIP964 was purified (Sephaglas BandPrep kit, Pharmacia, St. Quentin-en-Yvelines, France), labeled with [a-3ZP]dCTP (Amersham, Les Ulis, France) by random primer labeling (Megaprime DNA labeling kit, Amersham) and used as a probe in Southern hybridization with BglII-digested pIP964 and pIP824 (pIP964::Tn1547) DNA (data not shown). The probe hybridized to a 2.2.kb BglII fragment of PIP824 (pIP964::Tn1547) which was purified and cloned into BarnHI-cut and dephosphorylated pUCl8 DNA (Appligene, Illkirch, France) to generate

pAT425.

Sequencing

of 1.1 kb of the pAT425

insert

by the dideoxynucleotide

chain

termination

method

(Sanger

et al., 1977) with

[C(-35S]dATP (400 Ci/mol; Amersham, UK) and the Sequenasem version 2.0 DNA sequencing kit (US Biochemical Corp., Cleveland, OH, USA) revealed a sequence identical to that of cylB (nt 1411~1600, numbering of Gilmore et al., 1990) fused to the left extremity of IS256-like (up to the BglII site at nt 915 of IS256-like, Table I, Fig. 2). To complete the IS256-like sequence, pIP824 (pIP964::Tn1547) DNA was partially digested with Sau3A1, cloned into BarnHI-cut and dephosphorylated pUC18, and the inserts carrying the right extremity of IS256-like identified using a PCRgenerated probe internal to IS256-like. The probe was amplified with Tuq DNA polymerase (Perkin Elmer-Cetus, Norwalk, CT, USA) using pAT425 as a template. The oligo primers were selected on the sequence of IS256-like determined from pAT425. Sequencing of the 2.2-kb insert of pAT426 indicated that it included the entire IS256-like element and 446 bp of cylB on the left (Sau3AI site at nt 1857-1411, numbering of Gilmore et al., 1995) and 470 bp of the Tn1547 internal region on the right (Table I, Fig. 2). The right junction was determined by sequencing the inserts of plasmids selected by screening the same libraries with a PCR-generated probe specific for the c$B-related sequence predicted to be at the right junction. PCR was performed with oligo primers selected from the sequence of cylB using pIP964 as a template. Sequencing of the 1.2-kb insert of pAT427 indicated that it included the right extremity of IS16 up to the Sau3AI site at nt 544 (IS16 numbering) and 233 bp of cyIB (Suu3AI site at nt 11851418, numbering of Gilmore et al., 1990). The sequence of IS16 was completed by sequencing 700 bp of the 1.3.kb insert of pAT428 (Table I. Fig. 2) which was identified by screening a library constructed by cloning partially HpaII-digested pIP824 (pIP964::Tnf547) DNA into the CluI site of the vector Bluescript@ SK+ (Stratagene, La Jolla, CA, USA) with a PCR-generated probe internal to IS16. pAT428 overlapped with pAT427 and contained the left extremity of IS16 up to the HpaII site at nt 570 (Table I and Fig. 2). Partial sequencing of the vanB gene cluster of Tnf547 was performed by sequencing the inserts of plasmids selected by screening the above libraries with PCR-generated probes internal to the EfV5X3 t:mR,, S,, H,, B and X, genes. PCR was performed using oligo primers chosen from the sequence of the mnB gene cluster (Evers and Courvalin, 1996) using V583 DNA as a template. The V583 sequence was also used for selection of oligo primers for PCR mapping of the VmK gene cluster of Tn1547.

aureus (Byrne et al., 1989). This module, designated IS256-like, differed from IS256 by T552 (Trp) to C (Arg) and A916 (Lys) to G (Arg) transitions (Fig. 3). The latter nt substitution resulted in a BglII site not present in IS256.

(ii ) IS16 The right end of Tn1547 consisted of a module, designated ISZ6, distantly related to IS256 (Figs. 2 and 3). IS16 contained a single large ORF which displayed 61% nt identity with the gene for the putative transposase of

3

IS256-like

Left IR GATRAAGTCCGTATAATTGTGT-GT~GGCCATAT~C------AGTCCTTTTACGGTAC~TGTTTTT~CGAC

IS16

III I III1 GATTACGTCCATATAATTGTT-GTGAAGCATC

IIIIIIIIIIIIIlIII

Left

II

I I I I I

II

M

IR

IS16

CATT?TACTTTTGAAAGCGAAGRAATTCAAGCAATTATATCGGCGGGCGTGATA~CGC GAN.TA. A. .NE. . ..FE.....

IS16

IIIIIIIIIIlII

II

IIIIIlIIII

IIIII

IIIIII

IIIII

I

IS16

IS256-like IS16

IS256-like IS16

IS256-like I.516

ISZSG-like IS16

IS256-like IS16

I

I

I

IIIIIIII

IIIII

I II

I III1

I

III

III

RYORNE

III1

IIII

II

I II/I

I II

I IIIIIIII

I

III/II

120 4

II

IIIIII

233

I III/III

II

IIlIIIl

II

II

II

IIIIIIIII

II

84 353

IlIIIIIIIIII

Illll .

.

II/I

I

124

RKVSKI”

473

IIIIIIIIIIllllIIIlIlIIIII

IIIIIIlIIIIlIIIII

II

4RO 124

GACGGTRRGTTTAGTCCTACTATCTTTGARAGATATATCAGCG~GCG~GCT~GATCG=TGCCATGA~G-TGGT=ATTT~TGGTGTCTCCACTCGC-GT~CT-CAGTT I.A.I..“I........T.T. .K . I . . . . ..S

s ” s K s FVSSLTE C G K E E I., GAAGAACTT---TGTGGTAAATCCGTCTCTAAGTCCTTCGTTTCTAGCTTAAT~GT~TATTA~~AG

III

IIIIII/IIIIIlIIlIII

III1

II

Q

L

E

P

IIIII

I II II

IIIIII

I III

II

II

II

GAACTGCTRACGGATGGTGCGA~GTTTCTAAATCATT~G~TC-TC~GATG-CAGTTGGATCCATTTG=**TTGAGTGGAG-CCGGAG~CTAG~GGCTCGG~TATCCA~T L.TD.AT.......N.“K..D.F.F..R..S.

IIII

III

II

IIIII

IIIIII

I III

I IIIII

II

I

I

III1

I

IIIIIIII

IIIII

I

IIIIII

Illll

III

203 710

IIIIIIII

II

TTCATGTGTGATGCCCTGTATATGAAAGTAAGGG-TCATCGTA~TGTCTC-GGTG~ATATA~~GGTATCGGCA~GATTCTGA~GACGACGTACGATTCTTGGT~TGATGTT G”Y.G...DS..R.T.L..D” F.C.A..M....NH.I”..

IIIII

IIIIII

I

II

III

IIIII

III

IIII

I

IIIII

IIII

III

II

IllIIIIIlII

IIIIIIII

IIIII

II

II

I

CARGACGGCGAATCGGAAGATAATTGGGATACCGTTTTTCGGCTGTT~GC QSF”Q...F.“K...........K.“. .D....DN.D.“.

SWQRCQ”HFLRN1 K S F T N ” AAATCCTTCACCAACGTAAGTTGGCAAAGATGCCAAGTTCTAT~TTTA~~AC~A~T~~T

II

I

III

II

lIIIIIlIIIIIIIII

IIIII

F

I IIIIIIII

T

T

III

I

II

III

III

I I I III

AAGRACTTTTTAGGTGCGAG~TGGC-GATGCCAAGCC~GCC~A~~TTTGAG-TATTTTTGAT~G~~~~-G-G~TTCT~CTGA~GT-GACGAG~G~GAGTATCT~SD”KDEL.S... LGA......A.......OKL...V. N

IIIII

II

I

I

I I

Ill1

323

III1

IIIIIII

IS16

ATTTTGTCTTTTCCTG-TATCCGCCG~GGATTCGTA=~CC~TG~GTTGG~CGGT~G*CG~GAGATTCGCCGGAGAG~CGAG~GA*CGGA~TT~CC-TATC~TTCC I

I

II

IIIII

960 284

1070

IIIIII

GCCTCTGMCTTGAATTGACACGTG-G-GAGCACT~CTTGG~~A~GGTTGTGATT~-TT~GTGCTGCTTGCGACATTTTAG-CGGCTTTG~GA~GCTA~TC* FLEK.GCDS.L.A..DI.EN.....I. ASELE.T..R.EH

I

III

I

I

I

IllIll

I

Illlll

III/

III1

I Ill

I IIIIII

IllIIIIIIII/

IIIII

1187

II II II

III

RRYINFDR’ GAVLMDLHDEWIYSS A N R I. I GCCAATCGCTTRATTGGAGCCGTTCTTATGGRCCTATGGACCTACATGATG*TGGATTTATT~TT~~G-TA~ATC~TT~GA~~GTAG-TGGT~~ATTGTATAG~A---------

II

III

IIlI/III

I

II

IIIIII

I I

III

III

I

IIIIIIIIIIIII

I

IIIIII

IIII

GACACTGAGTGGCTGGCTTCTCCACG-TAGAA~CTGTGCGAGT~TCT

1080 324

362

FPNQTS

I IIIII

LSFPE.IRR.IRT..“L....E.I....RV......IN.

ATCACTCGGATAATTGGAACACTTTTGATGGAAAAA IT.I..TL..EKDT..LA.P...LE.

840 244

IIIIIIII

TCGATTGATTCATGRTTATATCGATCAACCAAAATATTCA

IIIII

IIIIII

III

oQPKVSKACASLDoCFEDAF*

NLAREAKNRLIHDYI F T D I TTCACAGATATTAACTTAGCGCGTGAGGCTAAAAA

IIII

I

830

283 950

PKKNSRSFREAVKGIFK AAAUAAATTCRAGATCTTTCAGAGAAGCTGTTAAAGGW.TTTTTAAG

I II

I

720 204

243

LOGTEL"ISDAHKGL"SAIR ETWTTFFEYLKERG OSGESE SAAAGTGGCG~GCG~GAGACCTGGAC~~ATTT~~~G~TACCT-G~CGCGGTTTA~~GG~A~GG~~TCG~~A~TTCTGATG~G~A~~GGA~~AG~~~~~GCCATTAGA

III

600 164

EGSE..F

DREIIGFMI T K D G RAARAGCTGTCATATAGCGATSGGAATAACCARRGATGGCT~ATCGG~TTCATGATT

KVREENRVLSKSCHIAIGI LMTDVLYI TTAATGACCGATGTACTCTATAT-GTACGAGMG-TCGAGTACTCTC

II

163 590

MVNERQNRLLSEKNYPY AAAAAAATTATCCTTAC

II

360 84

..H

EMYVSGVST

KALMASML

IIIIIIIIIIIIIIIIIIIIIIIIII

240 44

TLM.IM......E..N‘!

LNQEVRRREKIIRI Y TV Q G N S H N R L K S TN L I E R ---TATACCGTACAAGGAARTTCCCACAATCGACTAARGACT-GAGTACC*TCT*TTG~~GACTG~T~~G~GTA~GCAG~GAG-GATTATT~G~ATCTTC~~C~TC-CATCA

IS16

113

IIIII

AAAAACACTTATGACTATCATGTTTAATCAGCTGATGGWXXACAACGAAATCAA

IS256-like

IS256-like

I I

I

~GCCCTCATGGCTTCRGTTGG-TGTATCTTGTG

GATGGCCATTTTTCACCCACAGTCTTTGAACGTTAT&LACGAAACG

IIII

I

44

TATATTCAGGCAARTGCCTATGAACGTTCAGRAGAACGGC*T~A~-G-TGG*TACTACGAT~GTTCGTT~AC~CTCG~~GGTACGTTAG~CTT~ATG~TC~ACG~C~AGA D ., I . . . ..N A... S E.........

“GHFSPTVFE IS256-like

IIIII

I

FTTRVGTLELKVPRTR NRQSQRNGVVERS QAKEYERTE Y I TATATTCAAGC-GAATATGAACGRRCRGAAARCCGAC-GTC~~G-~GGCTATTA~GAG~GCAG~TTTA~GACACGTGTAGGCACGCTAG*TT-GTA~~~AG~~ACG~

/IIIIIII

4

LTTVFNQLMENQRTE

CATTTTACACTG-GCGRGAGATTCRAAGCATTATTGGACG~TT~T

IS256-like

v

AAAAATATTTTAACAACGGTATTTACTCAACTAATGG-TCAACGAACAGAA

IS256-like

I

Q

AAAAAAGCCATTGATCTCAGTTAGGAGTAC~TGTTTTTTGCAG-CATACTCG~T~CTGATATC~~ACTC~GTA RBS

HFTLKSEEIaSIIEYS”KDD”SKN1

IIIIIIII

T

AAAAACATACCCA-GGAGGACTTTTACaACCCMGTA

I

II

1200 364

390

II

NSNNI*

I III

I

1320 395

IS256-like IS16

TCTTTCTGAGGAGAGACATCAAGAGTCGCTGCGCTGCGCTACTGCTCCTGACCTCTCTCCTCAG-G-GGCATGGCAG~AGGTTCAC~CGC-CATTGTGCTTTTGATGAGGTTG~C

IS256-like

Right IR TTTTACACAGGAGTCTGGACTTGACT

IS16

lIIIIIIII

II

I IIllllllllI

TTTTACACAAGATTGTGGACTACT Right IR

1440

1324 1466

Fig. 3. Comparison of the sequences of IS16 and IS256-like. Identity between the IS is shown by a vertical line and dashes indicate gaps introduced to optimize the alignment. The terminal IR are in bold characters and labeled left and right IR, respectively. The ORF for the putative transposase of IS16 is preceded by a potential RBS (GAATAGGACT) with complementarity (underlined) to five bases of the 3’-OH terminus of Bacillus subtilis 16s rRNA (3’-OH UCUUUCCUCC). The start (ATG) and stop (TAG) codons are indicated by a double underline and an asterisk, respectively. The deduced aa sequences of the putative IS256 and IS16 transposases are presented above and below the nt sequences, respectively, and are aligned with the first nt of each codon. Dashes in the aa sequence indicate gaps introduced to optimize the alignment. Amino acid identity is indicated by a dot. The deduced sequence of the IS256-like putative transposase differed from that of IS256 (Gilmore et al., 1990) by Trpr5r to Arg and LysZ7* to Arg substitutions (shown in bold characters). The sequence of IS16 and IS2564ike has been submitted to GenBank and has been assigned the accession No. U35366.

b

A.

B.

C.

D.

IS256 IS256

(IR,) (IR,)

AGTCAAGTCCAGACTCCTGTGTAAAA

IS16 IS16

(IR,) (IR,)

AGTCAAGTCCACAATCTTGTGTAAAA

IS256 IS16

(IR,) (IR,)

GATAAAGTCCGTATAATTGTGTAAAA

suggest that the vanB operon

(17/26)

among

GATTACGTCCATATAATTGTGTAAAA

(c) Relationship between Tn1547 and the 250-kb chromosomal element of BM4120

(18/26)

To

AGTCAAGTCCACAATCTTGTGTAAAA

(IR,) (IR,)

GATAAAGTCCGTATAATTGTGT~

IS256 IS16

(IR,) (IR,)

AGTCAAGTCCAGACTCCTGTGTAAAA

determine

Fig. 4. Sequence

(23/26)

GATTACGTCCATATAATTGTGTAAAA (23/26)

AGTCAAGTCCACAATCTTGTGTAAAA

similarity

between

the terminal

IR of IS256-like

if serial,

conjugative

transfer

of the

250-kb element of BM4120 from chromosome to chromosome involved transposition events mediated by IS16 or IS256-like,

E.

at least

(18/26)

GATAAAGTCCGTATAATTGTGTAAAA

IS256 IS16

is highly conserved

some enterococci.

and

IS16. Conserved nt are shown in bold letters. The nt identity is indicated in parentheses on the right. The IR of IS256-like are identical to those

Southern

hybridizations

were performed

with IS16 and IS256-like probes and total DNA of BM4120, recipients JH2-2 and BM4113 and transconjugants (data not shown). DNA

The probes

of BM4110[pIP964]

and

recipients.

The

to IS16

of IS256 (Byrne et al., 1989).

probe detected

IS256. The corresponding

390 and

of three transconjugants (BM4274, BM4281, BM4304) that carried the 250-kb element inserted in either the 290or the 400-kb S$I chromosomal fragment of Ef JH2-2 or

395 aa respectively, were 58% identical (Fig. 3). IS16 was delimited by imperfect IR (18 out of 26 bp) which were

BM4113 (Table I and data not shown). The probe also hybridized to a 1.8-kb AvaII fragment of transconjugant

related (23/26 bp identity)

BM4306,

deduced

aa sequences,

to their respective

counterparts

in IS256 (Fig. 4). The nt sequence between the 3’ extremity of the putative transposase genes of IS16 and IS256like and the corresponding right IR (IR,) were not significantly related in size (144 and 24 bp, respectively) or in sequence (Fig. 3). IS16 ( 1466 bp) was larger than IS256 (1324 bp) mostly because of the size difference of the unrelated segments.

an AvaII fragment

did not hybridize

obtained

by intra-species

Target site of Tn1547

The sequences flanking Tn1547 were identical to a region of cylB of plasmid pAD1 (Gilmore et al., 1990), a gene required for expression of the hemolytic phenotype. Thus, insertional inactivation of a cylB-related gene in pIP964 by Tn1547 accounted for the non-hemolytic phenotype of BM4305 harboring pIP824 (pIP964::Tn1547). Tn1547 was flanked by an 8-bp direct repeat of the target sequence (Fig. 2B, C). The 8-bp target duplications also result from transposition of IS256-based elements (Byrne et al., 1989; Rice et al., 1995). These results confirm that translocation of the VmR genes into pIP964 was due to transposition of Tn1547. (iv) The vanB gene cluster of Tn1547

The sequence of the vanB gene of Tn1547 was identical to that of prototypic VanB-type strain Ef V583 (Evers et al., 1994) except for an A 969 (Asp) to G (Ser) transition. The partial nt sequence of the vanR,, vans,, vanH, and vanX, genes of Tn1547 was identical to that of the corresponding genes of strain V583 (data not shown). In addition, PCR mapping of the Tn1547 VmR gene cluster indicated that the organization of the operon, including the vanY, and vanW genes (Evers and Courvalin, 1996), was similar to that of V583 (data not shown). These data

conjugal

transfer

of

the VmR determinant from Efa BM4120 to Efa BM4107 (Table I and data not shown). The hybridization profile of BM4120 revealed the presence of a minimum of four copies of IS16, of which one was located on a fragment that co-migrated with the 1.8-kb fragment in the transconjugants. By contrast, the IS16 probe hybridized to a differently sized (9 kb) AvaII fragment of BM4305 (BM4113 [pIP964::Tn1547]).

(iii)

of 1.8 kb in total DNA

These

data

indicate

that

conjugative transfer of the 250-kb element did not involve ISlh-mediated transposition. Analysis with an IS256-like probe of total DNA digested with PstI + XbaI indicated that this IS was also not involved in conjugal transfer of the 250-kb chromosomal element (data not shown). Taken together, these results indicate that Tnf547 is internal to the 250-kb conjugative element of BM4120. In the hybridizations described above, the IS16 and IS256-like probes each hybridized to a single fragment of BM4305 (BM4113[pIP964::Tn1547]) indicating that the transposon carries only one copy of each IS. However, the IS256-like probe hybridized to two fragments of BM4120, BM4274, BM4281 and BM4304 indicating that a second copy of this IS was present in the donor and in the transconjugants harboring the 250-kb element (data not shown). (d) Sequences related to IS16 and IS256-like in VanB-type clinical isolates of enterococci Southern hybridization profiles consistent with the presence of Tnl547-related structures were detected in two of five transconjugants derived from VanB-type clinical isolates of enterococci from various origins (data not shown). Two contained IS256-related elements but not IS16 (data not shown) whereas the remaining transconju-

7 gant Ef BM4260, element

which

containing

Ef UMH-1

harbors

van&related

a 90-kb sequences

(Table I), did not contain

related sequences. Analysis of Efand

chromosomal originating

IS16-

in

Efu clinical isolates harboring

sequences

in the majority

strains. Of the eleven remaining IS256-related sequences and sequences tion,

related

both

Vm-susceptible Tn1547-like

IS16

uanB-

(21 of 32) of the

isolates, ten contained one did not contain

to either IS (data not shown). and

IS256-like

Efu. These structures

data

were indicate

are present

strains, the vanB gene cluster IS16 or IS256-like.

in certain

is not necessarily

ACKNOWLEDGEMENTS

We thank C. Carlier for help with experiments and M. Arthur for helpful critical reading of the manuscript.

in

a

although VanB-type linked

to

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(e) Tn1547, a mixed composite transposon bounded by distantly related IS The structural organization of Tn1547 is characteristic of that of a composite transposon, but distinguished by the distant relationship of its terminal IS (39% divergence of the putative transposase genes). Composite transposons are often flanked by IS that are, or are presumed, to be identical (Galas and Chandler, 1989). Among these elements TnlO is distinguished by the significant divergence (2.5%) of its IS which could result from the accumulation of mutations in ISIO-left since there is no selective pressure to maintain both IS functional (Foster et al., 1981). By contrast,

it appears

unlikely

the conjugation suggestions and

In addi-

detected that,

in Ef (Woodford

plasmid

or IS256-

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that diver-

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