An integrative vector exploiting the transposition properties of Tn1545 for insertional mutagenesis and cloning of genes from Gram-positive bacteria

Gene, 106 (1991) 21-27 0 1991 Elsevier Science

GENE

Publishers

B.V. All rights reserved.

21

0378-l 119/91/$03.50

06061

An integrative vector exploiting the transposition properties of Tn1.545 for insertional mutagenesis and cloning of genes from Gram-positive bacteria (Recombinant

DNA;

Patrick Trieu-Cuot,

multiple

cloning

site; erythromycin

resistance;

CCcile Carlier, Claire Poyart-Salmeron

kanamycin

resistance;

electroporation;

mobilization)

* and Patrice Courvalin

Unit& des Agents Antibacttkiens, Institut Pasteur, 75724 Paris Cedex 15 (France) Received by J.-P. Lecocq: 3 June 1991 Accepted: 2 July 1991 Received at publishers: 23 July 1991

SUMMARY

We have constructed and used an integrative vector, pAT112, that takes advantage of the transposition properties (integration and excision) of transposon Tn154.5. This 4.9-kb plasmid is composed of: (i) the replication origin of pACYC 184; (ii) the attachment site (art) of Tn1545; (iii) erythromycinand kanamycin-resistance-encoding genes for selection in Gram _ of the vector from and Gram+ bacteria; and (iv) the transfer origin of IncP plasmid RK2, which allows mobilization recipients. Integration of pAT112 requires the presence of the transposon-encoded Escherichia coli to various Gram+ integrase, Int-Tn, in the new host. This vector retains the insertion specificity of the parental element Tn1545 and utilises it to carry out insertional mutagenesis, as evaluated in Enterococcusfaecafis. Since pATl12 contains the pACYC184 replicon and lacks most of the restriction sites that are commonly used for molecular cloning, a gene from a Gram+ bacterium disrupted with this vector can be recovered in E. coli by cleavage of genomic DNA, intramolecular ligation and transformation. Regeneration of the gene, by excision of pAT112, can be obtained in an E. coli strain expressing the excisionase and integrase of Tn1545. The functionality of this system was illustrated by characterization of an IS3O-like structure in the chromosome of En. fuecalis. Derivatives pATl13 and pATl14 contain ten unique cloning sites that allow screening of recombinants having DNA inserts by !x-complementation in E. coli carrying the dM 15 deletion of 1acZcr. These vectors are useful to clone and introduce foreign genes into the genomes of Gram+ bacteria.

INTRODUCTION

Conjugative transposons Tn1545 (Courvalin and Carlier, 1986) and Tn916 (Franke and Clewell, 1981) have been proven to be powerful genetic tools in a large variety of Correspondence Institut

Pasteur,

to: Dr. P. Trieu-Cuot,

Tel. (33-1)456883 * Present

Unite des Agents

Antibacteriens,

25 rue du Dr. Roux, 75724 Paris Cedex

15 (France)

Laboratoire

mycin;

ernr, gene encoding

group; int-Tn, gene encoding kb, kilobase

18; Fax (33-1)45688319.

address:

Gram + bacteria (Caparon and Scott, 1987; Gaillard et al., 1986; Ivins et al., 1988; Wooley et al., 1989). Transposition of these elements proceeds by way of excision of a free, nonreplicative, covalently closed circular intermediate that is a substrate for integration (Scott et al., 1988). The inte-

de Microbiologic,

HBpital

Enfants Malades, 149 rue de Stvres, 75730 Paris Cedex Tel. (33-1)42738842; Fax (33-1)42738844.

Necker-

15 (France)

Abbreviations: 3’-aminoglycoside Tn1545;

aa, amino acid(s); Ap, ampicillin; phosphotransferase;

replication;

bp, base pair(s);

uphA-3, gene encoding

attTnZ545,

BHI, brain heart infusion;

unit(s); Cm, chloramphenicol;

attachment

site of

cfu, colony-forming

d, deletion; En., Enterococcus; Er, erythro-

Tra,

integrase

ofTn1545;

or 1000 bp; Km, kanamycin;

Mob, mobilizable; deoxyribonucleotide; rifampicin;

ErR methyltransferase;

oriT, origin

self-transferable;

plasmid-carrier

sequence;

MCS, multiple cloning site;

Nal, nalidixic acid; nt, nucleotide(s); oligo, oligoORF, open reading frame; oriR, origin of DNA of DNA

Sm, streptomycin;

pyranoside;

Inc, incompatibility IS, insertion

XGal,

R, resistance/resistant;

Rif,

SmR; Tc, tetracycline;

5-bromo-4-chloro-3-indolyl-/?-D-galacto-

xis-Tn, gene encoding state.

transfer,

Str, chromosomal excisionase

of T&545;

[ 1, denotes

22 ration-excision system of these transposons is structurally and functionally related to that of lambdoid phages (Poyart-Salmeron et al., 1989; 1990). Excision and integration occur by reciprocal site-specific recombination

and is devoid of most of the restriction sites that are commonly used for molecular cloning. This vector is intended for insertional

mutagenesis

and

cloning

of genes

from

between nonhomologous nt sequences of 6 or 7 bp, designated overlap sequences (Caparon and Scott, 1989; Poyart-Salmeron et al., 1989; 1990). Excisive recombination requires two transposon-encoded proteins, an excisionase (Xis-Tn) and an integrase (Int-Tn), whereas Int-Tn alone is sufficient for integration (Poy~-Salmeron et al., 1990). Excision can, but does not always restore the original target sequence (Clewell et al., 1988; Poyart-Salmeron et al., 1989). In Escherichia coii, these elements excise at high frequencies when cloned on multi-copy plasmids (Courvalin and Carlier, 1987; Gawron-Burke and Clewell, 1984). Based on this property, a cloning strategy which allows regeneration of an insertionally inactivated gene from a Gram + bacterium after excision of Tn916 in E. cofi has been proposed (Gawron-Burke and Clewell, 1984). This strategy was successfully used to clone in E. coli a mutacin-associated gene from Streptococcus mutans (Cautield et al., 1990). Conjugative transposons are large DNA segments (2 16.4 kb) moderately convenient for fine-scale genetic analysis. The aim of the present study was to construct and use a small 4.9-kb integrative vector which utilizes the transposition properties (integration and excision) of Tn1545. This vector, designated pAT112, was designed to allow easy recovery in E. coli of an insertionally inactivated gene and its utility was investigated in a strain of En. faecalis. Two derivatives of pATl12 were also constructed for integration of recombinant DNA into the chromosome of Gram+ hosts.

(HP)

/ ,

/

/

/

MCS

E

SC

K

SIX

3

pATl13

1

I

1

1

1

II

sp

P

Sl

Xb

R

pATll4

I

I

I

I

I

I

Fig. 1. Structure pATll4.

of the

integrative

The components

PstI-SmaI

fragment

(a) Structure of integrative vectors pAT112, pAT113 and pATl14 The structure of integrative vectors pAT112, pATl13 and pAT114 is shown in Fig. 1. The plasmids were designed to possess the replication properties of the Gram- replicon pACYC184 and the integrative properties of the pneumococcal transposon Tn154.5 (Figs. 1 and 2). Since the three hybrids contain the origin of transfer plasmid RK2 of incompatibility group IncP, they are transferable by conjugation to various Gram+ bacteria provided that the E. coli donor contains a co-resident self-transferable IncP plasmid (Trieu-Cuot et al., 1991). They all confer resistance to Er and Km in E. cofi (selective concentrations, 150 pg Er/ml and 50 pg Km/ml), in Gram+ aerobes (8 pg Er/ml and 50 pg Km/ml) and in Gram + anaerobes (8 pg Er/ml and 500 pg Km/ml). Plasmid pATl12 has a length of 4.9 kb

conjugative

transposon

(Trieu-Cuot

et al.,

containing

1

s/x

K

SE

E

I

I

I

I

pAT1 I3 and

fragment

and Yakobson,

containing

the KmR gene

pJH I (Trieu-Cuot

fragment

frag-

1988); (ii) a 0.7-kb

(Rose,

containing

and

and Courvalin,

the erm gene of the

to as anTnI54S.

0.32-kb KpnI-EcoRl

(v)a

of Tni.545

in the attachment

Plasmid

a unique AvaI restriction

KpnI, PstI, SacI, SalI, SmaI, SphI,XbaI.

site but no sites for BarnHI,

a 0.45-kb HpaI fragment

gene of pUC19

fragment

coll~guration

and

pAT112 has a size of 4.9 kb and

contains

and pATl14,

It

’

pATl12,

HindHI, reporter

sp

Tn154.5 from Srrepfococcirs pneumoniae BM4200 1990);

the termini

hence referred

P

1

are: (i) a 1.4-kb EcoRI-XbaI

plasmid

1983); (iv) 1.3-kb BamHI-KpnI

\ \

’

ariT of RK2 (Guiney

containing

uphA- from the enterococcal

.

St

’

pACYCl84

1983); (iii) a 1.3-kb Clai-Hind111 AND DISCUSSION

Xb

vectors

of pATll2

ariR of plasmid

ment containing

RESULTS

‘\,

containing

(pATI 13) or pUCl8

To construct

EcoRI, pAT1 I3

the MCS and the 1ucZr (pATll4)

(Stewart

et ai.,

1986) was inserted at the junction of the DNA fragments carrying a&4-S and ori?? Symbols: uphA-3, gene encoding 3’-aminoglycoside phosphotransferase;

attTnl545,

attachment

site of Tn1545;

erm, gene encoding

ErR methyltransferase; lucZr, gene encoding P-galactosidase (a-peptide); MCS. multiple cloning site; oriR, origin of replication; oriT, origin of transfer. transfer

Arrows

indicate

at oriT (Grinter,

the direction

of transcripti

1981). Heavy

black triangles

and left(L) termini ofTn154.5

(attTn1545).

A,AvaI;

and of DNA depict

B, BarnHI;

right (R) C, Clc21;

E, EcoRI; H, NindIII; Hp, HpaI; K, KpnI; P, PsrI; SC. SacI; Sl, SalI; S, SmaI; Sp, SphI; X&, XbaI; X, XmaI. The restriction sites in parentheses were destroyed by DNA polymerase and fused to form the vectors.

23 Rightextremity SO GGTACCA~~TCPTGTTUTTAGTAGTAGTAC~T~TTTACTACTTATTTAC~~CTGA~~T~GACATG DR2 Du2 *

160 240

~TA~AT~TCTAG~ATCC~AT f_----

~M~TAT~~T~TTAG~~TATAC~~GT~atttt --

320

~CT~CTPATCA~CTTTAT~~TC~CCACTC~TTTACTACTMTTTACTACTTAT~TGA~TTTGA * DR2 DR2 TACGACGATTTATCCGAATTC

341

EcoRl Left Fig. 2. Nucleotide

sequence

1988) of the corresponding

extremity

of the 341-bp KpnI-EcoRI region in circular

fragment

intermediates

AGG) and the right (5’-GGTACCAGGAGCGTCTTGTTGCTTAG) Presence

of the

not shown).

1I-bp directly repeated nt sequences

Imperfect

inverted

repeats

containing

extremities

DR2 in both arms of anTnl545

and DR2 ofarrTnl.745

The DNA fragment

arrTnl545.

was obtained

by amplification

(Saiki et al.,

of Tn154.5 using oligos specific for the left (5’-GAATTCGGATTAAATCGTCGTATCAA-

are depicted

of the element.

Right and IeR extremities

was found to be essential

by horizontal

refer to integrated

for efficient integration

arrows. The 6-bp variable

of pATI

elements. (data

overlap region is shown in lower-case

letters.

+ bacteria in E. coli. Plasmids pATI and pATI are 5.4 kb in length and contain ten unique cloning sites (Fig. 1) which allow screening of derivatives containing DNA inserts by cc-complementation in E. coli carrying the dM15 deletion of 1acZa. They are therefore adapted to stable inte~ation of foreign DNA into the genome of Gram + bacteria. In each vector, a DNA fragment having two different restriction sites compatible with the MCS can be inserted in either orientation relative to that of the luc promoter. Therefore, pATll3 and pATI are transcriptional fusion vectors permitting expression in E. co/i of genes from Gram + bacteria with promoter sequences not recognized by E. coli I%o’~ RNA polymerase. Gram

(b) Mutagenesis of Entemewcus fueculis with pAT112 The genome of various En. faecalis strains was mutagenesed with pATl12 using two transfer methods (conjugation and electroporation) and two compiementation systems (Tn916 or pAT145) to provide the transposonencoded integrase Int-Tn in trans (Tables I and II). The efficiency of PAT 112 integration depended upon the combination used to deliver and rescue the vector (Table II). With both transfer methods, the inte~ation frequency of pATl12 in En. fuecalis BM4111 (Tn916) was two orders of magnitude lower than that obtained with En. faecalis BM4112 (pAT145). Overproduction of Int-Tn in cells harbouring pATI relative to those containing Tn916 is the most likely cause for this difference in integration frequency. This implies that the level of synthesis of integrase in the recipient is a rate-limiting factor for integration of pAT112. The frequencies of integration obtained following delivery of pATl12 by mating or electroporation are not comparable because they simply reflect the fact that the number of ErR electrotransformants recovered per plate was approx. 102fold greater than that of ErR transconjugants for a given recipient, BM4111 or BM4112. Finally, the presence of the

tr~sposon-encoded integrase in the new host was found to be essential for integration of PAT112 since ErR clones were not obtained when En. faecafis BM4110 (no complementation system) was used as a recipient. Taken together, these resuits indicate that obtention of numerous and nonredundant mutants in a given species will be greatly facilitated by (i) the construction of a strain harbouring pAT145, or any functionally similar replicon, and (ii) the availability of an efficient electroporation procedure for introduction of pATl12. For inte~ation of foreign DNA, transfer by conjugation of PAT 113 and PAT 114 derivatives carrying inserts from an E. coli donor to a Gram+ recipient Tn916 represents a convenient alternative. (c) Physical analysis of pATll2

harbouring

insertions in Enterococcus

faecalis In each of the four integration systems described in Table II, ten ErR clones of En. faecalis originating from the same experiment were selected for further studies. Total DNA from these clones was digested with E;coRI or Hind111 and analysed by Southern hybridization using a DNA probe specific for the erm gene to detect the restriction fragment(s) carrying pAT112. The hybridization patterns obtained in the four groups of transcipients studied indicated that, in every experiment, a single copy of pATl12 had inserted at ten different loci (Fig. 2 shows part of this analysis). (d) Stability of pAT112 insertions in Enterococcus fuecdis BM4111 In En. faecalis BM4111, resident Tn916 can catalyze integration and excision of a related element defective for these functions. We therefore studied segregation and structural stability of pAT112 insertions in this strain. Unrestricted total DNA (5 pg) from six mutants of En.~ecal~s B&I4111 with pATl12 at different loci in the

24 TABLE

I

Bacterial Strain”

strains

and plasmids

or plasmid

Relevant

properties”

Reference

Bacteria E. coli JM83

ara, A(iac-proAB), strA, @So IacZAM 15

Messing

K802N

hsdR ~,

Trieu-Cuot

DHl

F ~, recA I, endA 1) gyrA96, thi, hsdR 1I, supE44, relA I,

hsdM+, gal-, tnet ~, supE, NalR, Rip A:

(1983) et al. (1991)

Low (1968)

En. faecalis BM4110

StrR

Courvalin

BM4111

StrR, TcR; (BM41 lO::Tn916)

This paper

BM4112

KmR, StrR; (BM4110

[pATl45])

Storrs

pRK24

ApK, TcR; Tra + , Mob

’ , IncP, trpE

Thomas

and Smith (1987)

pHG327 pHG329

ApR, IacZr

but rap + )

Stewart

et al. (1986)

pAT145

KmR; pATl87-102.2-kb

pAT295

CmR; pHSG57602-kb

and Carlier

(1986)

et al. (1991)

Plasmids + (as pUCl8,

ApR, 1ucZz + (as pUCl9. but rap + )

Stewart

EcoRI fragment Sau3A

fragment

int-Tn transcribed

containing containing

from spat promoter

xis-Tn and int-Tn transcribed

Storrs

et al. (1986) et al. (1991)

from lac

promoter

Poyart-Salmeron This paper

pATl14

; oriR pACYC184, attTn1545 ErR, KmR, Mob + ; oriR pACYCl84, attTn1545, lacZa’, MCS of pUCl9 ErK, KmR, Mob + ; oriR pACYCl84, attTnl.54.5, lacZa’, MCS of pUCl8

pAT307

pATll2::2.6-kb

pAT308

pAT307QpHG327

This paper This paper

pAT309

pHG327Q2.6-kb

pATl12

ErR, KmR, Mob+

pATl13

.I Bacteria

were grown

b rap, repressor

in BHI broth

EcoRI

enterococcal

EcoRI enterococcical or agar. All incubations

of primer; spat, hybrid promoter

DNA fragment DNA fragment were carried

of the phage SPOl-26

et al. (1989)

This paper This paper

This paper out at 37°C.

early gene promoter

and the fat operator;

a, in vitro insertion;

:: in vivo insertion

(novel joint).

TABLE

II

Integration Transfer

of plasmid

pATll2

in Enterococcusfaecalis

Recipient

system

Conjugatiot+

Integration

En. fuecalis BM4110

< 10 - ‘(’

En. faecalis BM4111

4x lo-’ 1.5 X 10-7

En. faecalis BM4112 Electroporation”

En. faecalis BM41 IO


En. faecalis BM4111

4x lomh

En. faecalis BM4112

2x lo-”

.’ Values are means of three independent within one order h Filter

matings

matings

frequency”

and were reproducible

of magnitude. E. co/i JM83[pRK24+pATll2]

between

faecalis were as described

(Trieu-Cuot

and

et al., 1991) and integration

En. fre-

quency was expressed as the number of transconjugants per donor cfu after mating. Transconjugants were selected on BHI agar containing Nal (50 pg/ml)

and Er (8 pg/ml).

i_ En. faecalis cells were transformed with 2.5 pg of plasmid DNA by electroporation (Cruz-Rodz and Gilmore, 1990) using a BioRad Gene Pulser apparatus and integration frequency was expressed as the number of electrotransformants per pg of DNA per viable cell. Electrotransformants

were selected

on BHI agar containing

Er (8 pgiml).

genome was introduced by transformation into restrictiondeficient E. coli K802N and clones containing the excised vector were selected on Km. Transformants were obtained at a very low frequency with a single DNA preparation and all contained intact pATl12. Stability of pATl12 in the same enterococcal mutants was also assessed by replica plating of a minimum of 100 colonies after growth for approx. 100 generations in antibiotic-free broth. No clone susceptible to Er was found which corresponds to a frequency of pAT112 loss lower than lo- 3. Total DNA extracted from the mutants every 20 generations during this growth period was analyzed by Southern hybridization as described in section c. Migration of pATl12 from one locus to another was only observed with the En. faecalis mutant that produced excised vectors (data not shown). These results demonstrate that integrative recombination of pATl12 and related vectors in En. faecalis BM4111 can be reversible, and that the rare excision events are followed by reinsertion of the vector at a new site of the host genome. Plasmid pAT145, which does not encode Xis-Tn, is therefore more appropriate than Tn916 to obtain stable p AT 112generated mutants. In addition, this replicon can be lost

25 spontaneously

in the absence of selective pressure (data not

shown). (e) Recovery of DNA fragments after integration of pATll2 and insertion specificity of the vector Recovery in E. coli of a DNA fragment after integration of pATl12 by using its replication properties is facilitated by the fact that the vector is devoid of most of the restriction sites commonly used for cloning. To study the specificity of insertion of the vector, we cloned into E. coli various enterococcal DNA fragments following pATl12 insertion. Total DNA of twelve mutants of En. faecalis BM4111 and BM4112, six of each isolated in the same experiment, was digested with EcoRI or Hind111 to obtain the smaller pAT112 hybrid, as determined by Southern analysis (Fig. 3). Following self-ligation, DNA was introduced by transformation into E. coli K802N and clones were selected on Km and Er. The plasmid content of twelve E. coli transformants representative of the En. faecalis mutants studied was purified and the vector-target junctions were sequenced (data not shown). Integration of Tn1545 and related elements occurs by reciprocal site-specific recombination between nonhomologous regions of attTn and of the target 12345678910

kb

4lIlmb (

(

12

3

Fig. 3. Integration DNA

4

5

purified (Courvalin

and Carlier,

ferred onto a Nytran fragment intragenic 1989). The two bands digestion

8

of BM4112

1986), digested

by agarose

9

IO

kb

(

5.00

+

4.50

a

2.x5

Total

resistant

2.85

genomic

to Er were

with EcoRI (panel A) or

gel (0.8%) electrophoresis,

trans-

membrane, and hybridized with a 560-bp SspI DNA to erm labeled in vitro with 32P (Sambrook et al., in panel A, lane 6, result from incomplete

since a single band

Hind111 digestion.

7

in En. faecalis BM4112.

of pATl12

from ten electrotransformants

Hind111 (Panel B), resolved

6

14.40

Molecular

is present

in panel

sizes (kb) are indicated

EcoRI

B, lane 6, following on the right margin.

site (Poyart-Salmeron et al., 1989; 1990). Therefore, knowledge of the nt sequences of attTn1.545 in pATl12 and of the vector-target junctions enables deduction of those of the target sites prior to integration (Fig. 4). Analysis of the data revealed that the specificity of integration of pAT112 is similar in En. faecafis BM4111 and BM4112. The twelve targets contained, on each side of the recombining site, a ‘I-bp-long segment which exhibits extensive similarity with the corresponding region in attTn1545 (Fig. 4). Similar motives are present in all the Tn1545/Tn916 integration sites characterized

so far (Caillaud

and Courvalin,

1987;

Clewell et al., 1988; Caparon and Scott, 1989; PoyartSalmeron et al., 1990) indicating that pATl12 has retained the specificity of insertion of the parental element. (f) Regeneration of a DNA fragment by excision of pATl12 One of the BM4112::pAT112 mutants was resistant to low levels of Er suggesting that, in this strain, the erm gene of pAT112 is transcribed counterclockwise relative to a chromosomal promoter. This mutant was selected to illustrate the strategy proposed to regenerate a DNA fragment by excision of pATl12. Recovery in E. co/i of the enterococcal fragment containing pAT112 was obtained following EcoRI digestion. The vector-target junctions of the EcoRIgenerated pATl12 derivative, designated pAT307, were sequenced and the nt sequence of the target site before integration was deduced (Fig. 4, insertion 1). Plasmid pAT308 was then constructed by inserting the EcoRIlinearized and dephosphorylated plasmid pHG327 into the unique EcoRI site of pAT307. To recover the insertionally inactivated enterococcal gene, excision of pATll2 from pAT308 was catalyzed by providing in tram Xis-Tn and Int-Tn. To do so, pAT308 was introduced by transformation into E. coli DHI harbouring pAT295 (pHSG576Qxis-Tn + int-Tn) and clones were selected on Ap and Cm. The plasmids from eight transformants obtained independently have been purified and used to transform E. coli JM83 cells that were plated on BHI agar containing Ap and XGal. In every experiment, the plasmid content of a lac - transformant resistant to Ap and susceptible to Cm and Km was analysed by agarose gel electrophoresis following digestion with EcoRI. All clones were found to contain a single plasmid, designated pAT309, consisting of pHG327 plus the 2.6-kb fragment of En. faecalis BM4112 chromosome. Partial sequencing of the enterococcal insert in these plasmids revealed the presence of either of the two expected hexanucleotides at the excision site of pATl12 (Fig. 5) and at approximately similar ratios (5/8 vs. 3/8). Precise regeneration of the enterococcal DNA fragment occurred in all plasmids that contained the TAACAT motif. This analysis also revealed the presence of the 3’-OH terminus of an ORF having the expected location and orientation relative to erm in

26 Right extremity

Left extremity

-----

-y---f atttta

GATAATTAGAAATTTATACTTTGTTT

-Ty_-AAAAA

** ** *** I atgtta IAAAACA>$TTTGT;A~TT~~G~G l

T~CTEG~=TCGZ~TGTTAA~T~~A l

l **

l **

l

~TcG&TTTTT~A&ATTTTTTT * l* GTTACAATTGCTA%CCT~A~T~~~

*

l

**

aatata

TTATTCACTGTG;&T&T;A;T:;; ~ATTA~CCT~GGZ~~T~CT=~~T~~~ l ** AT$iTAG;~TCT%G;CTTTTTTTT

B

t**

GTAiATtiTidGZ'iiTGATGG *et* AAAATAhA&&CCSCTAGAG~T *et** l * l l AAAAACAGAXOiATTTTCCGiACAG~T

****

* ** * *

AAZATAGTATCCTTTTTATATMCG

IX

IGiAAAGAGTCTT~GGAATCMGT l **** l l ** * *t CGTTTACGGkA ~CAAACAAAA t***** t* * l

**

l

l

*t*

l

l

****

l

l

l

l

.

l

*

*

AAFAAZACCTGACGATTAACGTCAGT

*tt

t***

***

CATTATAGAGTCTTTTGTCTTTTTTT

112

AMAACAGAAAATTT&GAAC!iGT I&aat I***** * I

.

ofintegration

****

AMATTAAGAAAGAGGT;TT;&T

** **

.

. .

.

-

l

I

. . .

.

. . . . .

.

l

.

12 in En. faecalis strains BM4111 (A) and BM4112 (B) (see Table I). Sequence

ofpAT

on purified double-stranded

DNA (Sambrook

AAG) and the right (5’-CGTGAAGTATCTTCCTACAGT) relative to Int-Tn cleavage

and boxed. Horizontal

denote terminal

are indicated

of TnZ.545 as primers.

sites (Poyart-Salmeron

imperfect

by asterisks.

inverted

Blackened

determination

ofthe vector-target

et al., 1989) using oligo specific for the left (5’-GGATAAATCGTCGTATCA-

extremities

aligned with that of attTnl545 arrows

*

17

Ill

the termini of attTnl545

*

l

tagaai l **** ctttta * l * gtaata

It0

was performed

l

t;a;;g

IY

Fig. 4. Specificity

****et**

l *****

AAAAATTTCATAAAAARATCTTAAAA l **** * ** l l * l * * AAAAACAACM4AUdGCAAAGCTTAC

l

junctions

AAAAATCTAGTTATC

repeats

squares

The nt sequences

et al., 1989; 1990). Overlap

in attTn1545. denote

Identical

positions

nt, in pairwise

with a minimum

of the target

regions

sites were deduced

are presented

comparisons,

score of identity

in lower-case

between

and

letters

every target and

of eight out of twelve target

DNAs.

Tn1.545 and can be introduced into Gram+ hosts by electroporation or by mobilization from E. cofi. Integration of these plasmids requires the presence of the transposonencoded integrase in the recipient. Plasmid pATl12 lacks most of the restriction sites that are commonly used for cloning and was constructed for insertional-mutagenesis in Gram + hosts and subsequent cloning and regeneration of the mutated gene in E. coli. The functionality of this system

pAT307. This ORF was truncated by cloning and could code for the C-terminal segment of a protein that shares significant aa similarity with the corresponding moiety of the transposase of IS30 from E. coli (Dalrymple et al., 1984) (Fig. 5). This ORF is currently being further characterized. (g) Conclusions We have constructed and used integrative vectors that should facilitate genetic analysis and manipulation of a large variety of Gram+ bacteria. These vectors capitalize on the transposition properties (integration and excision) of

E. coli (IS30)

Q Y TQHELD L V A R Q Y F P K K T C LA NTNGLI * * t * t:*t: *: SSVSNQRNH KSMDFREVNQTFI 'N S N G I RRNGLP GRATTCTAACGGGATTCGGCGTMTGGACT~C~T~T~ATTTTA~~GT~TCA~~TTTATTTCCAGTGT~~TCMCGTMTCAT

En.faecalis E.

Cob

was illustrated by the characterization of an IS30-like structure in the chromosome of an En. faecalis strain. Plasmids pATl13 and pAT114, which contain ten unique restriction sites, are adapted to the introduction of foreign genes into

(IS30)

R

P

R

K

T

L

K

*t*:t

F

K

T

P

::t*

K

E

I

l

.

I

E

R

G

V

A

L

T

A

Q t

L

N t

N

100

D End

t

En.foecnlis

FLSYVQEA I P R K S L N Y RTPIEI ATTCCAAG~TCATTGAATTACAGAACACCMTT~~TATTTTT~~TATGTA~~~TTTTA~M~M~

F

En.faecalis

CAAAAAAAAAATTTATTTTTTMTTTTCTTTTTGTTMCT~CGMTTTTTT~TGTTTTTCTM~T~~~~T~M

Y

S

N

L

I End GACAAATCATMTTT

200 300

taacat

En. faecalis

400

TAAMTTMCAMCGAGTTCGTAGTATAGCACGGATTTTTTCTCATGTAAACATCATTAMTAAACA~AATTTA

AAACGTTGTTTT taaaat

En.faecalis Fig. 5. Sequence site of pATll2 1). Numbering and compared

418

TACATATTTCTCAGCTM

of an enterococcal are indicated

DNA fragment

by superscript

after integration

and subscript

lower-case

and excision ofpAT

12. The two alternative

letters, The DNA strand

is complementary

hexanucleotides

detected

to that presented

at the excision

in Fig. 4 (insertion

begins at the first nt of the EcoRI site. The deduced aa sequence of the C-terminal moiety of the putative protein encoded is presented with that of the transposase oflS30 from E. coli (Dalrymple et al., 1984). Asterisks and colons indicate identical and homologous (I-L-V-M,

D-E, R-K, Q-N, S-T and F-Y) residues,

respectively.

The aa are aligned

with the second

nt of each codon.

27 Grinter,

the genome of Gram + bacteria. These vectors are routinely used in our laboratory for complementation and expression

N.J.:

Analysis

of chromosome

mobilization

between plasmid RP4 and a fragment Plasmid

of genes in En. faecalis.

using

ofbacteriophage

hybrids

i, carrying

ISI.

5 (1981) 267-276.

lvins, B.E., Welkos, Tn916

S.L., Knudson,

G.B. and Leblanc,

in Bacillus anthracis. Infect.

mutagenesis

D.J.: Transposon Immun.

56 (1988)

176-181. Low, B.: Formation recipient strains

ACKNOWLEDGEMENTS

This work was supported Institut

by a grant (CCAR)

from the

Pasteur.

of merodiploids in matings with a class of Recof Escherichia coli K12. Proc. Nat]. Acad. Sci. USA

60 (1968) 160-167. Messing, J.: New Ml3 vectors

for cloning. Methods

Enzymol.

Poyart-Salmeron, C., Trieu-Cuot, P., Carlier, C. and Molecular characterization of two proteins involved of the conjugative

REFERENCES Caillaud,

F. and Courvalin, shuttle

P.: Nucleotide

transposon

sequence

Tnl545.

of the ends of the

Mol.

Genet.

209 (1987)

of a gene that regulates

M.G. and Scott, J.R.: Identification

expression

of M protein,

streptococci. Caparon,

the major virulence

Proc. Natl. Acad.

transposon

Tn916 involves

determinant

of group A

and insertion

mechanism.

Cell

P.W., Shah, G.R. and Hdllingshead, to inactivate

and

isolate

analysis

Bacterial.

170 (1988) 3046-3052.

Courvalin,

of termini of the conjugative

P. and Carlier, C.: Transposable P. and Carlier,

Mol. Gen. Genet. Cruz-Rodz, plasmid

multiple antibiotic

M.S.:

prokaryotic

shuttle transposon.

mobile

efficiency

introduction

of

genetic

W.: Nucleotide

element

IS30.

sequence

EMBO

of the

J. 3 (1984)

A.E. and Clewell,

resistance

transposon

of ‘conjugal’ teriol. Gaillard,

transfer

D.B.: Evidence

for a chromosome

borne

(Tn916) in Streptococcusfuecalis that is capable in the absence

of conjugative

plasmid.

J. Bac-

J.L., Berche, P. and Sansonetti,

a tool to study

the role of hemolysin

monocytogenes. Infect. Immun. inactivated

P.: Transposon

C. and

in the virulence

as

of Listeriu

D.B.:

DNA

Regeneration

fragments

of insertionally

after excision

of trans-

poson Tn916 in Escherichiu co/i: strategy for targeting and cloning genes from Gram-positive bacteria. J. Bacterial. 159 (1984) 214-221. Guiney, D.G. and Yakobson, transfer Acad.

origin

E.: Location

of the broad

and nucleotide

host range

Sci. USA 80 (1983) 3595-3598.

plasmid

sequence RK2. Proc.

Erlich,

H.A.:

phages.

Nucleic Acids Res.

S.J., Higuchi,

R., Horn,

Primer-directed

enzymatic

DNA polymerase.

E.F. and Maniatis,

Manual,

Cold Spring

T.: Molecular

2nd ed. Cold Spring Harbor

Harbor,

Scott, J.R., Kirchman,

Science

Cloning.

Laboratory

A

Press,

NY, 1989. P. and Caparon,

of the conjugative

Stewart,

G.S.A.B.,

Storrs,

M.G.: An intermediate

transposon

Lubinsky-Minsk,

in trans-

Tn916. Proc. Natl. Acad. Sci.

Plasmid

Conjugative teriol. Thomas,

ofthe Nat].

S., Jackson,

a pBR322 copy number

C.G., Cassel,

A. and

of pUC8 for cloning and

15 (1986) 172-181.

M.J., Poyart-Salmeron, transposition

C., Trieu-Cuot, ofTn916

P. and Courvalin,

requires

of the transposon-encoded

P.:

the excisive and integraintegrase

Int-Tn.

J. Bac-

173 (1991) 4347-4352. C.M., and Smith, CA.: Incompatibility

group P plasmids:

and use in genetic manipulation.

gen-

Annu. Rev. Micro-

biol. 41 (1987) 77-101. Trieu-Cuot,

P.

and

Courvalin,

P.:

Nucleotide

Sfreptococcus faecalis gene encoding Trieu-Cuot,

otide sequence Trieu-Cuot, Shuttle

Tn1545.

vectors

bacteria. Wooley,

of

the

phos-

resistance

P.: Nucle-

gene of the conjugative

Acids Res. 18 (1990) 3660.

C., Poyart-Salmeron, a multiple

C. and

cloning

Courvalin,

site and a lucZr

P.: gene

of DNA from Escherichia coli to Gram-positive

102 (1991) 99-104.

R.C., Pennock,

Transfer

Nucleic

containing

transfer

Gene

C., Carlier, C. and Courvalin,

of the erythromycin

P., Carlier,

for conjugal

sequence

the 3’5”-aminoglycoside

type III. Gene 23 (1983) 331-341.

P., Poyart-Salmeron,

transposon

52 (1986) 50-55.

Clewell,

streptococcal

mutagenesis

Stoffel, S., Scharf,

and

J., Fritsch,

photransferase

145 (1981) 494-502.

Gawron-Burke,

Sambrook,

etics, evolution,

2145-2149. Franke,

of lambddid

of pACYC184.

of DNA with a thermostable

tive activities

224 (1990) 152-154.

P. and Arber,

sequence

D.H.,

K.B.

Kuhn, J.: pHG165:

Enterococcus fuecalis by electropo-

DNA into glycine treated B., Caspers,

Mullis,

expression.

High

to those

P.: The Tn1545 is

USA 85 (1988) 4809-4813.

resistance

205 (1986) 291-297.

C.: Tn154.5: a conjugative

Mol. Gen. Genet.

Dalrymple,

C.:

Tn916. J.

206 (1987) 259-264.

A.L. and Gilmore,

ration.

G.T.,

Laboratory

transposon

related

transposon

4 (1990) 1513-1521.

Rose, R.E.: The nucleotide

position

in Streptococcus pneumoniae. Mol. Gen. Genet. Courvalin,

gene from

58 (1990) 4126-4135.

S.E., Ike, Y., Jones, J.M. and Gawron-Burke,

Sequence

C. and Courvalin,

239 (1988) 487-494.

S.K.: Use of transposon

a mutacin-associated

Streptococcus murans. J. Bacterial. Clewell, D.B., Flanagan,

and functionally

amplification

59 (1989) 1027-1034. Tn916

P., Carlier,

system of the conjugative

Mol. Microbial.

with other site-

EMBO J. 8 (1989) 2425-2433.

C., Trieu-Cuot,

integration-excision

Saiki, R.K., Gelfand,

of the conjugative

a novel recombination

Tn1.545: homologies

Courvalin, P.: in the excision

16 (1988) 355.

Sci. USA 84 (1987) 8677-8681.

M.G. and Scott, J.R.: Excision

Cautield,

Poyart-Salmeron, structurally

110-115. Caparon,

transposon

specific recombinases.

conjugative

lOl(l983)

20-78.

ofTnlS45

22 (1989) 169-174.

A., Ashton,

R.J., Davies,

A. and Young,

M.:

and Tn916 to Clostridiumacetobutylicum. Plasmid