An integrative vector exploiting the transposition ... - Science Direct

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

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B.V. All rights reserved.

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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