Characterization of Tn3926, a new mercury-resistance transposon from Yersinia enterocolitica

Gene. 40 (1985) 79-91 Elsevier GENE

1458

Characterization (Recombinant plasmid;

of Tn3926, a new mercury-resistance

DNA; restriction

complementation;

map; transposition

function;

transposon from Yersinia enterocolitica homologies

to Tn21, Tn501, Tnl721;

resolvase;

Hg2 +

Marie-Claire Lett”*, Peter M. Bennettb and Dominique J-M. Vidon” “Universite Louis Pasteur. Laboratoire de Bactt+iologie. FaculttG de Pharmacie, BP 10, 67048 Strasbourg Cede-x (France) Tel. 188)66.90.77, poste 707, and bDepartment of Bacteriology, Medical School. Universitll Walk, Bristol BS8 1 TD (U.K.J Tel. 272 24 161, ext. 919 (Received

April 25th, 1985)

(Revision

received

(Accepted

September

September

17th, 1985)

ISth, 1985)

SUMMARY

A new transposon coding for mercury resistance (HgR), Tn3926, has been found in a strain of Yersinia YE138A14. The element has a size of 7.8 kb and transposes to conjugative plasmids belonging to different incompatibility groups. A restriction map has been established. DNA-DNA hybridization indicates that Tn3926 displays homology with both Tn501 and Tn2I ; the greatest homology is shown with the regions of these transposons that encode Hg R. Weaker homology is observed between Tn3926 sequences and those regions of TnSOl and Tn2I that encode transposition functions. Complementation experiments indicate that the Tn3926 transposase mediates transposition of Tn21, albeit somewhat inefficiently, but not of Tn501, while the resolvase mediates resolution of transposition cointegrates formed via Tn21, Tn501, or TnZ721. enterocolitica,

INTRODUCTION

Mercury resistance in bacteria is both well-known and widespread. The earliest observations were reported 20 years ago and involved hospital isolates

* To whom correspondence

and

reprint

requests

should

be

addressed. Abbreviations:

Ap, ampicillin;

phenicol;

Hg, mercuric

nalidixic

acid;

resistant;

Rif, rifampicin;

tetracycline;

Nm, neomycin;

Cm, chloram-

nt, nucleotides;

Sm. streptomycin;

Tn, transposon;

soy agar; TSB, trypcase plasmid-carrier

bp, base pair(s);

ions; kb, 1000 bp; Km, kanamycin; Su, sulfamides;

Tp, trimethoprim;

soy broth;

0

1985 Elsevier

Science

Tc,

TSA, trypcase

::, novel joint;

[ 1,designates

state.

0378-I 119/85/$03.30

Nal,

R, resistance,

Publishers

of Staphylococcus aureus (Richmond and John, 1964). Subsequently, HgR bacteria were isolated from mercury-polluted soils (Tonomura and Kanzaki, 1969). In most cases, genetic studies of mercury resistance have shown that the determinants of this phenotype are plasmid-borne (Smith, 1967; Summers et al., 1975; Silver et al., 1976; Weiss et al., 1978; Ogawa et al., 1984). Plasmids conferring in a resistance to Hg2+ have been demonstrated wide range of species including S. aureus (Novick and Roth, 1968; Weiss et al., 1977) Pseudomonas sp. (Joly et al., 1976; Clark et al., 1977), Escherichia coli (Summers and Silver, 1972; Nakahara et al., 1977) and other enteric bacteria (Schottel et al., 1974). The prototype HgR determinant in Gram-negative bac-

teria is that carried (Tanakaet Silver,

on the IncFII

plasmid

RlOO

al., 1976; Miki et al., 1978; Summers

1978;

Silver

and

Kinscherf,

1982;

and

resistance

to mercuric

strains

of Pseudomonas

Friello

and Chakrabarty,

et

et al.,

1977;

1980), E. coli (Summers

al., 1980), Citrobacter, Klebsiella (Radford

constituents

of transposable

agar (Institut

agar or

Production).

(b) Genetic experiments

et al.,

et al., 1983) are

Transposition of HgK gene(s) was detected by mating strains containing both a plasmid encoding HgR (PLY 1 or pCL4) R388, pUB307) tive recipient

elements.

plasmid

selected either for acquisition

designated

[ R388]; TcR, [pUB307]).

by the target

(Sa,

mercury-sensi-

strain of E. coli. Transconjugants

encoded

which was the only resistant

and a target

with a plasmid-free,

We reported previously the isolation of a HgK strain of Yersiniu enterocolitica (Vidon et al., 1981), YE138A14,

Pasteur

ions in certain

(Stanisich

1981) and Proteus mirubilis (Tanaka

Mueller-Hinton

Misra

et al., 1985). It has also been shown that the genes determining

TSB on Luria broth (L broth) or on Hektoen

were

of HgR or a resistance (Cm”[ Sa] ; Tp”,

plasmid

The ratio of the number

of

one among more than 200 strains isolated from raw milk. Although YEl38A14 is resistant to mercuric chloride and merbromin, it is sensitive to the organomercurial derivatives sodium merthiolate and phenyl mercuric borate. This phenotype corresponds to the narrow spectrum of resistance found in a number of mercury-resistant enterobacteria. This paper reports that the HgR of Yersiniu enterocolitica 138A14 is carried on a transposon, Tn3926. This transposon forms part of a nonconjugative plasmid, named pLY1, present in YEl38A14. Some molecular properties of Tn3926 are presented and its relationship to Tn.501 (Bennett et al., 1978) and to Tn21 (Nisen et al., 1977; de la Cruz and Grinsted, 1982) is investigated.

the former to the number transposition frequency.

k1.KI’kRlALS

mutants of Tn21, Tn501 and Tnl721, experiments were executed as described by Grinsted et al. (1982). Derivatives of E. coli UB5201 containing the conjugal plasmid R388, a plasmid carrying the appropriate mutant transposon and a plasmid carrying

AND METHODS

(a) Media Bacteria TABLE Relevant

were grown on TSA (Biomtrieux)

or in

of the latter gave the Donor strains were

counterselected with Nal, Sm, or Rif, as appropriate. Crosses were carried out by growing both donor and recipient strains in mixed culture on Mueller-Hinton agar (18 h, 37’(Z), after which the cells were resuspended in 1 ml to a cell density of approx. 10”’ cells/ml. 0.1 ml of appropriate dilutions were spread on selective agar. The levels of antimicrobials used in selective agar were: mercuric chloride, 12 pg/ml; Km, 12 pg/ml; Nal, 40 pg/ml; Sm, 100 pg/ml; Rif, 100 pg/ml; Tc, 20 pg/ml; Cm, 25 Llgiml; Tp. 25 [[g/ml. Drugs were incorporated in TSA (except when Tp was used; then minimal agar with appropriate supplements or Mueller-Hinton agar was used). To test the ability of Tn3926 to complement tnpA

I properties

of the bacterial

Strain

Species

strains

used Relevant

genotype

Source

or reference

BJ 6183

E. co/i

recBC sbcB end& gul met SW thi bio hsdR

B. Jarry

HB 101

E. co/i

pro leu thi lcrc gal recA rpsL (SmR)

G. Gerbaud

C 600

E. coli

thr leu thi lrrc tmA supE

G. Gerbaud

YE 13XAl4

Y. enrrrocolitica

this study. Vidon et al. (1981)

Nal 4

E. coli

W? F recA .sv CrrgC metA NalR

LIB 281

E. coli

pro, met, NalK

Bennett

and Richmond

(1976)

JC 6310

E. coli

hi\, try, IJS, luc, recA, rpsL (SmR)

Bennett

and Richmond

(1976)

UB 5201

E. coli

pro, met, recA, NalR

Bennett

et al. (1977)

UB 1832

E. coli

his, rry, l.1.3,lm, rpsL, rpoB, RifK

Sanchez

this study.

et al. (1982)

Tn3926

were constructed

E. coli UB1637.

Selection

which had acquired

number ance

crossed

with

was for transconjugants

linked resistance

marker on the mutant total number

and then

transposon.

to Tp and the The ratio of the

of such transconjugants

of transconjugants

which

to only Tp was taken

to the total acquired

resist-

samples

were

mounted

technique

(1969) as described

on grids

of Westmoreland

by Davis

using et al.

et al. (197 1). Phage

$X174 single- or double-stranded was used as internal standard.

DNA (5375 nt)

RESUI.TS AND DlSClJSSION

To test the ability of Tn3926 of TnZI, pCLi7

was transformed

to complement

tnpR

Tn.501 and Trill 721, experiments

were carried out as described Plasmid

DNA

the formamide

as the transposition

frequency. mutants

(e) Electron microscopy

(otherwise

by Diver et al. (1983). pACYC184:

into derivatives

:Tn3926)

of E. coii UB5201

containing one of the test plasmids pJOE562, pUB2589 or pUB2591. Resolution was seen to have occurred when linkage between the ApR determinant and the other resistance determinant(s) on the test plasmid was broken.

(c) Isolation of plasmid DNA (i) Rapid preparation. Rapid plasmid preparation was performed essentially as described by McCormick et al. (198 1). Plasmid DNA isolated by this method was used to determine plasmid size after electrophoresis in 0.7% (w/v) agarosc gels by comparison with standard plasmids. (iij Large-scale isolation of plasmid DNA Plasmid DNA was isolated using a Triton X-100 cleared-lysate procedure (Davis et al., 1980). 500 ml overnight cultures in L-broth were used as starting material. DNA was recovered from agarose scribed by Hubert et al. (1980).

(d) DNA-DNA

gels as de-

hybridization

DNA from 17; (w/v) agarose gels was transferred to Schleicher & Schuell nitrocellulose sheets according to the procedure of Southern (1975). Plasmid DNA probes were prepared by nick translation (Maniatis et al., 1975). Hybridization was carried out in heat-sealable plastic bags at 42°C for 20 h with a 3”P-labelled probe (1-5 x lo6 cpm) as described by Maniatis et al. (1975). Filters were autoradiographed at -2O’C for 48-168 h.

(a) Plasmid content of YE138A14 YE138A14 merbromin

is resistant

to mercuric

but not to merthiolate

chloride

and

nor to phenyl

mercuric borate. The strain contains two plasmids: one of 30 kb (PLY 1) and one of 13 kb (pLY2), as determined by agarose gel electrophoresis. Electron microscope contour length measurements gave values of 31.6 kb and 12.5 kb for pLY1 and pLY2, respectively, in good agreement with the values obtained from gel electrophoresis. HgK was not transferred by conjugation. To determine if HgR was related to extrachromosomal DNA, we extracted plasmid DNA from the agarose gel and used it to transform E. coli BJ6183. Hgn transformants were selected. These were recovered when pLYI, but not pLY2, was the transforming DNA, and such transformants contained a plasmid that comigrated with PLY 1. Plasmid DNA isolated from these transformants was able, in turn, to transform E. co/i HBlOl to HgK. (b) Mobilization and transposition of mercury resistance and isolation of the recombinant plasmids All attempts to transfer HgR by conjugation from YE138A14 to E. coli Na14 were unsuccessful, both at 37 “C and at 28’C. Consequently, mobilization of the HgR determinant by the R plasmids Sa, R388 and pUB307 was investigated (see MAT‘ERIALS AND METHODS, section b). HgR transconjugants were isolated from all crosses at a frequency of about lo-‘. Five of the recombinant plasmids, designated pCL4, pCL5, pCL6, pCL7 and pCL9 (see Table II), were chosen for further study. All but pCL.5 were larger than the parent plasmid and the phenotypes conferred by the recombinant plasmids were those of the recipient plasmid plus HgR (see Table II). Comparing the restriction digest of plasmids obtained during such crosses with those ofthe parent plasmids (not shown) we could determine that PLY 1 was not

TABLE

II

Plasmids.

host cells and bacterial

Plasmids

Relevant

pLYl

HgK

strains

used Construction

characteristics“

source this paper

PLY?

this paper gift of N. Dattab

Sa

IncW. Tra, SmR, Kmn, CmR, SuK

Ward

and Grinsted

(19X2)

R388

IncW, Tra, SuR. TpR

Ward

and Grinsted

(1982)

pUB307

IncP, Tra, NmiKm,

Bennett

pBR322

TcR. ApR

pACYCl84

TcR, CmR

pVSl

Sun. HgK

pCL4

IncW,

Ten

et al. (1977)

Gift of G. Loison Gift of V. Stanisich‘

Tra,

SmR. KmR, CmR, Sun,

Sa::Tn3Y26’

this paper

HgR pCL5

IncW, Tra, Smn. KmR, SuR, Hgn

Sad::Tn3Y26”

this paper

pCL6

IncW. Tra, Sun, Tpn, HgR

R388::TnSY26’

this paper

pCL7

IncP, Tra, NmK:KmK.

Ten, HgR

pUB307::Tn3926”

this paper

pCL9

IncP, Tra, NmR:KmR.

TcR, HgR

pUB307::T1r3926~

this paper

pCLl7

CmK, HgK, TcK

pACYC184::Tn3926

this paper

pUB78 1

ColEI ::TnSOl

p U B8 I0

Hgn CmK

pUBXlX

TcR, ApK

pUB2401

Cmn,

pUB2402

TcK, SmK:SpH,

pUB2406

Cmn

SmR/SpR,

HgR, SuR SuK, HgR

of pDS6501

Gift of J. Grinstcd

deletion

derivative’

of pJOE105

Gift of J. Grinsted

pACYC184

(rer::TnZly

pACYC184

(ccrt::Tn21)’

EcoRI deletion

Gift of J. Grinsted Gift of J. Grinsted

derivativek

of

Gift of J. Grinsted

I Gift of J. Grinsted

pBR322::TnZlA

ApK TcR

et al. (1978)

derivativeh

pUB240 pIJB3321

Bennett

deletion

carrying

the same Tn21 EcoRI

dele-

tion as pUB2406 ptiB2575

Tpn, TcR

R388::Tn1721

pJOE562

Apn. KmR

pBR322

derivative

tro and carrying flanked

by direct repeats

Cointegrate

ApR. TpR, SuR

pUB2589

constructed

generated

tion of Tn8/3

in vi-

a KmR determinant

R. Schmitt

via J. Grinsted

(Altenbuchner

and Schmitt,

19X3)

of Tnl721 by transposi-

J. Grinsted

from pBR322::Tn813

to R38X Apn. Tpn. SuR, KmR

pUB2591

Cointegrate tion

generated

of Tn1727

from

J. Grinsted

by transposipJOE529’

to

R388 I’ Symbols

for resistance

’ Dept. of Bacteriology, ‘ Dr. V.A. Stanisich,

phenotypes

Department

cl Plasmid

Sa was introduced

containing

mercuric

were selected probably

chloride

a deletion

into YEl38A14

’ Plasmid pCL6 was constructed

” pIJBXI0 (Grinsted repeats

Hammersmtth University, (Lesage

by Novick

Hospital,

Bundoora

Ducane

et al. (1976). Road,

WI2 OHS (U.K.)

(Australia).

et al., 1975). Transconjugants

YEl38Al4(Sa)

London

3083, Melbourne

were selected

on Hektoen

agar

were in turn crossed with E. co/i Nal4 and transconjugants

resistance. Sa. The Cmn gene is lost. The deletion

recombination

across

the short homologous

to occur with Sa itself in both UB28l by transforming

(Cohen

is not adjacent

sequences

and UB5201

flanking

(P.M. Bennett

et al., 1972) pLY1 into the rerA

to the Tn3Y26 insertion, the CmR gene (Ireland,

and V.G. Krishna,

strain

UB5201

(R388).

but 1983).

unpublished). Transformants

both plasmids were. in turn, crossed with E. coli JC6310. Transconjugants were selected for Sm and mercury resistance. pCL7 and pCL9 were constructed by mtroducing pCL3 in the ret strain UB5201 [pUB307] by conjugation. TranscoirJuganta

vvcre in turn. crossed of Glasgow).

La Trobe

by conjugation

of 5.2 kb of plasmid

We have also shovvn this deletion

follow those proposed

School,

and Km. These transconjugants

arose due to host-dependent

harbouring ” Plasmids

by plasmids Medical

of Microbiology,

for Nal and mercury

’ pCL5 contains

conferred

Royal Postgraduate

pDS6501

are intact,

with E. w/i UB1832.

Progeny

et al., 1982) is a deletion is a pACYCl84::TnSOl

but all other functions

of these crossed

derivative

of pDS6501

recombinant

plasmid.

have been lost or damaged.

were selected (obtained

for Rif, mercury

and Tc resistances.

from Prof. D. Sherratt,

The 6-kb deletion

is fully contained

Dept. of Genetics,

University

within Tn501. The inverted

a

b

a 12

1234

2

3

4

2 .6kb

Fig. 2. Comparison

of plasmids

(1 “b) slab gel electrophoresis Fig. I. Comparison

of plasmids

Sa, pCL3,

restriction

fragments

ofplasmids:

(1) Sa; (2) pCL4; (3) pCL5. (b)

autoradiographs

Southern

blot autoradiographs

of EcoRI

ffindII1,

plasmids

(1) Sa; (2) pCL4; (3) pCL5, 3”P-labeled PLY i DNA.

translated.

pCL5. digested

hybridized

(a) EcoRi DNAs

EeoRI,

of

to nick-

’ pUB818

(Grinsted

DNA

(4) SalI,

et al., 198 I) is a insertion

derivative

digested

by (1) EcoRI,

hybridized

k pUB2406

(de la Cruz fragment

’ pJOE529, generated

blot (2)

to nick-translated,

pCL4 DNA.

(c) Verification of Tn3926 on recombinant plasmids The relationship between pLY1 and pCL4 and pCL5 was investigated by DNA-DNA hybridization. “2P-labelled pLY1 was used as a DNA probe against Southern blot transfers of pCL4 and pCL5 digested with EcoRI (Fig. 1). Plasmid pLY1 did not hybridize to Sa DNA but did hybridize with two fragments each of pCL4 (one of 2.6 kb and one larger fragment) and two fragments of pCL.5 (one of 2.6 kb and one larger fragment). In the reciprocal hybridization using 12P-labelled pCL4 DNA against a Southern blot transfer of PLY 1 digested with EcoRI, pCL4 hybridized with two fragments of PLY 1 (one of 2.4 kb and one of 9.3 kb, Fig. 2). Comparison of restriction digests of Sa, pCL4 and pCL5 showed that the 2.6skb fragment

of pJOElO5.

of Tn I72i into a derivative

B pUR2401and pUB2402 (de la Cruz and Grinsted,

EroRl-EcoRI

by (I)

(b) Southern

----

et al., 1982) is a deletion

the IL’?gene, and in pUB2402

pJOE529

of pL.Yl

1 DNA digested

(4) S&I.

_

-~.I_--

encodes

(3) BarnHI,

(3) BarnHI,

‘*P-labeled

mobilized and that each HgR transconjugant contained a recombinant plasmid comprising the conjugative plasmid (Sa, R388 or pUB307) and part of pLYI. On the other hand the recombin~t plasmids showed the same DNA sequence inserted into two different sites in Sa (generating pCL4 and pCL5) and into two different sites in pUB307 (generating pCL7 and pCL9). Insertion into R388 generated pCL6. These data are consistent with the interpretation that the HgR determinant originating in YE138A14 and carried on pLY1 is part of a transposable element. The element is designated Tn3926. Tn3926 was, in turn, transposed from pCL6 to pACYC184 to generate pCL17 and its integrity was confirmed by showing that it could retranspose to R388 at a frequency of 10e5.

(Sch6ffl

(2) HindIII,

PLY1 and pCL4. (a) Agarose of PLY

insertion

and Grinsted,

Deletion

overlaps

part of the mpA gene of Tn1721.

pJOE105

of pBR322.

1982) are recombinants

of pACYC184

and TnZI.

In pUB2401

insertion

is into

is into the cat gene. 1982) was generated

after EcoRI

digestion

of pUB2401.

This plasmid

has lost the inner

of Tn21. from the pBR322 derivative

KmK by virtue of the insertion was kindly supplied

plasmid

pJOElO0

(Schbf?l et al., 1981), carries Tni727.

of the KmR gene of Tn5 into the H&d111 site of TnJOl.

by R. Schmitt.

Tn1727

a derivative carries

ofTnSOI

which

the res site of TnSOI.

84 EBt3H

h

p

BhB

h I

E

B

h I

P

hhBB Illi

t

I

/

,

hBB

H

h I

II

I

I

/

EP iI

E

i

&_

..---

.__

Fig, 3. Restriction E, EcoRI; PwII, acetate,

fragments

_-__p-*

Tn 3926

map of pCLl7.

H, HirzdIII;

were obtained

generated

___

h, HitidIl;

Length

of restriction

from Miles or BRL. Digestions were analysed

10 mM disodium

EDTA,

fragments

B, Rgll; P, PvuII. EcoRI, Hindll, on horizontal

were performed

agarose

pH S.3) or Tris borate

HindIll,

were purchased

as described

of pCL17

Comparison of restriction digests of pACYC 184 and pCL17 indicated that the latter was generated from the former by the insertion of a single 7.8-kb DNA sequence with the restriction map presented in Fig. 3. The insertion, Tn3926, has two EcoRI sites, one site each for HindIII and PvuII, six Hind11 sites and seven BglI sites. of Tn3926 to TnSUZ and Tn2I

Transposons Tn21 and Tn501 also confer resistance to mercuric ions and merbromin (Nisen et al., 1977; de la Cruz and Grinsted, 1982; Stanisich et al., 1977; Bennett et al., 1978). The relationship of Tn3926 to these elements was examined, therefore, using DNA-DNA hybridization. (1) Hybridization to Tn501 “‘P-labelled pLY1 DNA was hybridized to Southern blots of pUB78 1, a ColEl : : Tn501 derivative, digested with BglI and PvuIl, and BglI and E’coRI. Plasmid pLY1 hybridized with four BgZI-generated fragments of pUB781 (A,B,C,D), four PvuII

pACYC

location

184

----.--__*

of the boundorics

from Boehringer-Mannheim;

by Grinsted

slab gels using either Tris acetate

to Sa.

(e) Hybridization

is given in kb. The precise

buffer (Petrocheiiou

was derived entirely from Tn3926. Furthermore, the fact that, in addition to the 2.6-kb EcoRI fragment, both pCL4 and pCL5 display only one other EcoRI fragment with sequence homology to PLY 1 led us to conclude that one of the two EcoRI sites of Tn3926 must be located at or very close to one end of the clement. These results con~rmed the conclusion that DNA sequences, i.e., Tn3926, had transposed from PLY 1

(d) Characterization

~------_

et al. (1978). Restriction

buffer

is uncertain. Bg/I, and endonucleasc-

( 150 mM Tris OH. 50 mM sodium

et al., lir’lh}.

fragments (A,B,D,E), live fragments generated with BglI plus PvuII (A,B,C,E,F) and three generated with BglI plus EcoRI (B,D,E) (Fig. 4A). The degree of hybridization to the various fragments was not uniform and appeared to be most efficient with Bg0 fragment C, PvuII fragment A, BglI-PvuII fragment E and Bg!I-EcoRI fragment E, all of which include either part of, or the entire gene for mercu~ reductase. Less intense hybridization was observed between PLY 1 sequences and fragments of pUB78 1 derived from the transposition region of Tn5OI. A reciprocal hybridization indicated that “Plabelled pUB781 DNA hybridized to seven fragments of pCL17 generated with Hind11 plus EcoRI (fragments A,B,C,D,G,H,I, Fig. 4B). With the exception of fragment A, all of these fragments are derived from Tn3926. Fragments A,C,D and H show more intense hybridization than do fragments B,G and I. Fragment A contains the replication region of pACYC184 which is related to that of ColEl (Stuber and Bujard, 198 l), and so hybridization between pUB781 (otherwise ColEl : : Tn501) and pCLl7 (otherwise pACYC184 : : Tn3926) was to be expected. Hybridization to other fragments of pCL17 confirms that there is homology between Tn3926 and Tn501. The transposition functions of Trill 721 have been shown to be very closely related to those of Tn501 (Altenbuchner et al., 1981; Grinsted et al., 1982). In agreement with this result and results reported above we found that Tn3926 displayed homology with Tn1721 (not shown). (2) Hybridization to Tn 2 1 Tn3926 was also compared with Tn21 which is known to show homology with Tn.501 (Grinsted et al., 1982). ASP-labelled pLY1 was hybridized

to

85

1 b

a

4

3

2 a

a

b

b

a

Tn 501 res

pvu

ttlpR

A*

fl

a

b

tnpA

Fig. 4. Hybridization

of Tn3926

to TnSOI. (A) Hybridization

is shown on the bottom. hybridization).

Agarose

Length

has been described

to pCL17

of restriction

fragments

(1“()) slab gel electrophoresis

I b, Zh, 3b, 4b. (B) Hybridization pCLl7

bctwecn

in Fig. 3. Agarose

is shov,,tn in lanes (b).

between

pUB781

pCLl7

is given in kb.

+

(a ColEl ::TnSOI

and pLY1 labelled with “I’. The map ofTn501

(Bennett

digested

by a mixture

+

derivative)

digested

of HilldIl

by

et al.. 1978; Altenbuchner

(A, EcoRI, +. PvuII. V, Bgll. Asterisks:

is shown in la, _?a, 3a, 4a, and hybridization

( I ‘I,,) slab gel electrophoresis

B*

E*

D*

l

Rx/I-Portll; (3) PvuII; (4) Bg/I-EcoRI,

b

of “P-1abelled

+ EcoRI and pUB781

bands

(1) Bgil; (2) ct al., 19x1) that shop

a

pLYl to plJB7XI

in

labelled with “P, The map of

is shown in lanes (a) and hybridization

of “P-labellcd

pUB7XI

X6

1 a

2 a

b

b

UB2402

A

C

F

t-

Tn21A

E

pUB2406

0

Fig. 5. Hybridization

ofTn3926

to Tn21. Hybridization

PLY I DNA. The map of pUB2402

EcoRI). pUB2406

Agarose

and pUB2406

(I “,,) slab gel electrophoresis

is shown

in lanes

8.7

5

ofpUB

(shown is shown

t

(1) and pUB2406

at the bottom) in lanes

(2), both digested

have been described

la, 2a and hybridization

by EcoRI, with nick-translated (1982); (7, I with pUB2402 and

by de la Cruz and Grinsted of 72P-labelled

PLY

lb, 2b.

Southern blots of EcoRI-digested pUB2402 and pUB2406 (pACYC184 : : Tn21 derivatives). Plasmid pLY1 displayed homology with fragments A,C,D and G of pUB2402 and with fragments A and B of pUB2406 (Fig. 5). Since pLY1 did not hybridize to

pACYC184 itself (not shown), then the hybridization noted in these experiments indicated hybridization of Tn21 sequences. Hybridization was most efficient with fragments D and G from pUB2402 and fragment B from pUB2406, fragments known to carry

87

sequences

encoding

the mercuric

Tn21 (de la Cruz and Grinsted, hybridization

to fragments

reductase

gene of

1982). In contrast,

A and C from pUB2402

and fragment A from pUB2406, which quences derived from the transposition

carry segenes of

end-products

position functions

of mutations

in the trans-

of Tu22, TnSOZ and Tn1721

by

of pUB810

pVSS82, ment

which carries

such as Tn21,

Tn5UI

and Tnf 721

transpose in a two-stage process which uses transposase function ft3zpA) for the first stage to generate cointegrate molecules (Arthur and Sherratt, 1979; Shapiro, 1979; Grinsted et al., 1982; Diver et al., 1983), which are resolved in the second stage to yield the transposition end product, together with a DNA molecule indistinguishable from the original transposon donor (Arthur and Sherratt, 1979; Shapiro, 1979). This second stage involves a site-specific recombination system comprising a tnpR gene which encodes the resolution enzyme, resolvase, and a res site, the site at which the resolvase acts. The hybridization experiments clearly indicated homology between Tn3926 and those sequences of Tn501, Tn1721 and Tn2I encoding transposition functions. Hence experiments were undertaken to determine if Tn3926 encodes functions which can complement one or more of the functions encoded by Tn21, Tn501

and Tn1721.

(1) Transpusase (tnpA) function Plasmid pUB8 18 carries a copy of Tn 1721 with a deletion that has removed part of the tnpl4 gene. The remainder of the element still encodes TcR and can transpose if complemented (Grinsted et al., 1982). We were unable to detect any complementation mediated by PLY 1 or pCL6 (frequency < 6 x lo-‘), although in control experiments pUB2575, which carries Tn1721, did complement to give a transposition frequency for the TcR of pUB818 of 2 x 10-h. Plasmid pUB810 carries a deleted Tn501. Both end-points of the deletion are within the element but it has inactivated both the HgR determinant and the transposition functions. When appropriately complemented, however, the deleted element generates cointegrates, rather than normal transposition

(containing

1.5 x lo-‘). Tn501

of the

and cointegrates

a frequency Transposons

1979; Shapiro, is a self-trans-

into R388 or pUB307,

of detection

transposition

pUB810

Tn 3924

DNA

missible plasmid, then, such cointegrates

tively (limit (f) Complementation

and Sherratt,

the recipient

pUB8 10) encode CmR and can be conjugally transferred. Neither pLY1 nor pCL6 mediated cointegration

Tn21 was less efficient.

(Arthur

1979). When

of about

intact,

respec-

In contrast, did comple-

residual

element

on

with R388 were formed at 10m4, as expected

(Grinsted

et al., 1982). Hence, neither pLY 1 nor pCL6, both of. which carry Tn3926, mutant

TnSOf

complement

or Tnl721

transposition

elements.

of

in contrast,

complementation of a mutant Tn21 was detected. Plasmid pUB2406 is a deletion derivative of the pACYC184: : Tn21 recombinant pUB2401 (Grinsted et al., 1982) and plasmid pUB3321 contains the same EcoRI deletions of Tn21 as pUB2406 but is a pBR322 : : Tn21 derivative. The deletion removed only Tn21 sequences, but left the ends of the element intact. Both the transposon encoded drug-resistance genes and the transposition functions were inactivated. Nonetheless, the residual element can be complemented to transpose by wild-type Tn2f transposition functions (Grinsted et al., 1982), to generate cointegrates comprising pUB2406 or pUB3321 and the recipient DNA molecule. In the presence of PLY 1, pUB2406 formed cointegrates with R388 (frequency, 1.9 x 10-7), while pCL6 mediated cointegration of pUB2406 and pUB307 (frequency, 1.3 x 10dh). Further pCL17, a pACYCl84: : Tn3926 recombinant also mediated cointegration of pUB3321 and R388 (frequency 4 x IO-“). That the transconjugants generated in these crosses did harbour cointegrates was confirmed by demonstrating that the CmK determinant of pUB2406 was linked to the TpR determinant of R388 because the former marker always cotransferred with the TpK determinant in conjugal crosses, irrespective ofwhich marker was selected. The transconjugants examined each contained a single plasmid, all of which were indistinguishable in size but which were larger than either pUB2406 or pUB3321 or R388. Restriction enzyme digests of some of these (data not shown) displayed fragment proliles which could be distinguished but which were all consistent with the recombinant plasmids being cointegrates of ~1182406 or pUB332 1 and R388 generated by trans-

xx position

of the deleted Tn21 present

on the former

plasmid.

tation of tnpR gene function. to indicate

that Tn3926

These results are taken

does possess

and therefore, by implication, (2) Resolvase (tnpR) function Plasmid

pUB2589

is

cointegrate

of R388

and

Tn813, a tnpR derivative and which encodes resolvase cons

is

pBR322

ofTn2l

mediated

by

(g) Conclusions

(Diver et al., 1983) When the missing

supplied

to yield

to be tested.

a transposon-generated

TpRApRTcR.

activity

resolve

that remains

a tnpR gene,

a res site, a conclusion

the

R388: :Tn813

two (TpK)

repliand

(1) Two plasmids, PLY 1 and pLY2, with sizes of 30 kb and 13 kb, respectively, have been identified within

YE138A14,

a

HgK

wild-type

strain

of

pBR322 : : Tn813 (ApRTcR), and the former plasmid can be conjugally transferred independently of the

Y. enterocolitica. The 30-kb plasmid, PLY 1, encodes resistance to mercuric ions and merbromin. The Hg”

latter. Plasmid pUB2591 is a cointegrate of R388 and pJOE529, generated by transposition of the Tn1727 element (a derivative of Tn501 constructed in vitro

gene(s)

and encoding KmR) carried on pJOE529, and encodes TpKKmRApR. Resolution generates two plasTpKKmK and the other mids, one encoding ApRKmR. Again the former plasmid can be conjugally transferred independently of the latter. Plasmid pJOE562 is a derivative of pBR322 constructed in vitro. It encodes ApRKmK and the KmR determinant is flanked by direct repeats of the Tn1721 res site (Altenbuchner and Schmitt, 1983). Recombination across the duplicate res sites deletes the Kmn gene, which, because it is not longer linked to an origin of replication, is not recovered. Plasmid pCLl7 was transformed into UB5201 containing pUB2589, pUB2591 or pJOE562, and retention of linkage of “cointegrate” markers was determined. In all cases linkage of “cointegratc” markers was stable in the absence of pCLl7 (Table III), while in the presence of pCLl7 the link was severed (Table III), indicative of complemen-

TABLt Integrity Plnsmid

was

belonging

mobilized

by conjugative

to different incompatibility

cally Sa and R388 (both IncW) and pUB307 (IncP). The transconjugants from these experiments contained recombinant plasmids which all bore the same insert located at different sites on the different target plasmids. This recombination occurred in a recA strain, as well as in a Ret + strain. All these results together supported the conclusion that mercury resistance encoded by PLY 1 constitutes part of a transposon now called Tn3926. (2) Tn3926 is a 7.8-kb transposon which contains two EcoRI sites, one located very close to one end of the element and 2.6 kb from the other site. A Hind111 site is located between these EcoRI sites, 0.5 kb from the EcoRI site located at the end of the element (Fig. 3). These data indicate that Tn3926 is. in these respects, similar to Tn2613, another transposon encoding HgK (Tanaka et al., 1983). Other parts of the sequence are not obviously related to Tn2613. The restriction pattern differs from that of the prototype HgR transposon, Tn501, a transposon of almost the same size as Tn3926.

III of plasmids

carrying

two directly

repeated

ret sites in the presence

Linked resistance

Source

determinants

repented

of pCLl7

of directly

Linkage

YESyitc

second -pc1.17

of .ApK to marker’ tpC‘LI7

pJOE562

ApK. KmK

Tnl72l

50:50

I) 100

PUB2589

ApK, 1‘P K Ap’. TpK

‘I 1121

5050

0 100

Tn501

48.5lJ

13 50

pUB2591

I’ Loss of linkage of ApK and KmK (pJOE562) (pl!B2589

plasmids

groups, specifi-

and plJB2591)

was dctcrmined

WBS determined

by conjugal

transfer

by loss of Km” but retention of TpK unlinked

to Ap’

of ApK. Loss of linkage of ApK and Tp”

89

(3)

Tn3926

displayed

Tn501 and Tnl721

homology

with

as shown in hybridization

ments (Figs. 4 and 5). It hybridized

Tn21,

ACKNOWLEDGEMENTS

experi-

most strongly to

We

are

indebted

Schneider, F. Lacroute

zation

mation.

We thank

electron

microscopy

showed

a strong homology

and the left-hand the two EcoRI

sequences

between

of Tn3926,

sites (Fig. 4B). Together

Tn501

defined

by

to

M.

Nguyen-Juilleret,

and A. Mercenier

for materials

J. Menissier study.

Gerbaud,

above data this suggests that the genes for HgR en-

bacterial

coded by Tn3926

advice and a gift of plasmids.

on the left of the ele-

ment (Fig. 3). Homology

between Tn3926

was strong and involved

sequences

the latter element,

and Tn21

at both ends of

one end of which accommodates

genes for Hg R, including a mercury reductase gene. Our data are consistent with the previously observed homology between the mercury resistance genes of Tn501 and Tn21 (Tanaka et al., 1983). (4) The hybridization data clearly indicate homology between sequences carried by Tn3926 and sequences encoding transposition functions on Tn21, and Tn501 (and Tnl721), the transposition systems of which are known to be related (Grinsted et al., 1982; Diver et al., 1983). However, while Tn3926 was able to complement a tnpA mutant of Tn21, complementation of analogous mutants of Tn.501 and Tn 1721 was not observed. However, in that the transposase of Tn21 complements tnpA mutants of Tn501 and Tn1721, but not vice versa, despite the fact that these systems are related (Grinsted et al., 1982) the genetic results are consistent with the interpretation that the tnpA gene of Tn3926 is related to the equivalent genes of these other transposons. Transposon Tn3926 also encodes a function that can substitute for the resolvase functions of Tn21, Tn.501 and Tnl721 (Table 3), which are interchangeable (Diver et al., 1983). The data indicate that the transposition systems of all four elements are related and that there is a common progenitor sequence for all four transposons. Hence Tn3926 is clearly related to the family of transposons, the prototypes of which are Tn21 and Tn501, but is distinct from both. Our data suggest that Tn3926 is more closely related to Tn21 than to Tn.501 in that the former element displays homology with the latter throughout its sequence, and the transposition functions of Tn3926 can substitute for the equivalent gene products of Tn21.

N. Datta strains,

is indebted

or infor-

for her help in the

We are grateful

with the

are located

C.

G. Loison, R. Jund, A. Labigne-Roussel,

the fragments of TnSOl which encode the genes for mercury resistance (Fig. 4A). The reciprocal hybridi-

and V. Stanisich

we thank

to G.

for a gift of

J. Grinsted

for fruitful

One of us (M.C.L.)

to FEBS and EMBO

for financial

sup-

port to stay at the Medical School of Bristol, and to AH. Linton for his hospitality. This research was supported in part by the A.T.P. ‘Microbiologie’ No. 1482 from the Centre National de la Recherche Scientilique.

REFERENCES Altenbuchner,

J.. Choi,

Richmond,

(Tc) are related. Altenbuchner, specific

Schmitt,

recombinations

Bennett,

Tn1721:

deletions

D.-J.: Dissection

Mol. Gen. Genet.

site-

and inversions.

of the transposition

site specific

M.H.: Translocation acid carrying

J. and Richmond,

of TnA does not generate

deletions.

of a discrete

an rrntp gene between

in Eschen’chicr coli. J. Bacterial.

P.M., Grinsted,

recombination

175 (1979) 267-274.

P.M. and Richmond,

replicons

R. and

190 (1983) 300-308.

piece of deoxyribonucleic Bennett,

R.: Transposon

a transposon-encoded

system.

J.. Schmitt,

TnSOl (Hg) and Tn172/

generates

A. and Sherratt,

process:

Grinsted,

Res. 37 (19X I ) 2X5-289.

Genet.

J. and

Mol. Gen. Genet. Arthur,

C.-L.,

M.H.: The transposons

126 (1976)

l-6.

M.H.: Transposition Mol. Gen. Genet.

154

(1977) 205-211. Bennett,

P.M., Grinsted,

J., Choi. C.L. and Richmond.

Characterization

of TnSOl a transposon

ance to mercuric

ions. Mol. Gen. Genet.

Bacterial. Cohen.

coli

by plasmids

and organomer-

in P.~e~~/~nror~~. J.

132 (1977) 1X6-196.

S.N., Chang,

antibiotic E.

determined

resist-

I59 (1978) 101-106.

Clark, D.L., Weiss. A.A. and Silver. S.: Mercury curia1 resistances

M.H.:

determining

A.C.Y.

rcsistancc

by R factor

and

Hsu,

in bacteria DNA.

L.: Nonchromosomal

genetic

Proc.

transformation

Natl. Acad.

of

Sci. IJSA 69

( 1972) 21 lo-21 14. de la Cruz. F. and Grtnstcd, terization

of TnZI,

RlOO-1. J. Bacterial.

J.: Genetic

a multiple

homology

method in nucleic

chnrac-

transposon

from

151 (19X2) 222-228.

Davis, R.W., Simon, M. and Davidson. heteroduplex

and molecular

resistance

for mapping acids.

N.: Electron regions

Methods

microscope

of base sequence

Enzymol.

21 D (1971)

413-428. Davis,

R.W., Botstein,

Genetics. Harbor,

Cold

D. and Roth, J.R.: Advanced

Spring

Harbor

NY, 1980. pp. 116-l 17.

Laboratory,

Bacterial

Cold

Spring

YO

Diver.

W.P.,

Grinsted,

J..

Fritzmger,

.Alte~~buch~ler. J., Rogowsky, quences

of and complementation

TnSOl and Tn1721. Friello,

Mol. Gen. Genet.

fects and Maintenance York,

A.M.:

in Pseudomonasputida,

K.R. (Eds.), Plasmids’and

Brown,

N.L..

R.: DNA se-

by the rnpR genes of Tn21,

D.A. and Chakrabarty,

resistance

D.C..

P. and Schmitt.

191 (1983)

in Stuttard,

Mechanisms.

ganic

RX31 b. Gcnc

C. and Rozee, Ef-

Press, New

IYXO, pp. 249-260.

Grinsted,

J., Bennett,

Regional RPl

preference

and

S. and Richmond,

of insertion

its derivatives.

of Tn501

Mol.

Gen.

M.H.:

and TnR02 into

Genet.

166 (1978)

3t3-320. Grinsted.

J., de la Cruz,

Complementation

F., Altenbuchner,

of transposition

TnZI, TnSOI and Tnf721. Hubert,

transcription

expressed Ireland,

of the yeast

R.:

of Tn3,

8 (1982) 276-286.

C.R.: Detailed

restriction

IncW

plasmid

chloramphenicol

F.: Measure

of asymme-

OMP-decarboxylase

gene

enzyme

pSa,

map of crowngall-

showing

sensitivity.

J. Bacterial.

to antibiotics

Pseudomonas

tory concentrations Lesage.

P. and Barjot,

155 (1983)

J.: The resistance

of

and heavy metal: minimal inhibi-

and genetic

transfers.

Ann. Microbial.

Gerbaud.

G.R.

and

Chabbert,

et structure

resistance

isole chez ~uly~~~nella ordone;.

(Paris)

Y.A.:

chez E. co/i K-12 d’un

Carte

plasmide

Ann.

de

j~icrobiol.

E.: Plasmid

mediated

by transposon

cointegrates Tn.?

H., Heffron. and

mutants.

F. and

their

resolution

Gene

I5 (1981)

T., Jeffre, A. and Kleid. D.G.: Nucleotide operator

USA 72(1975) resistance

of phage

1.. Proc.

sequence

Natl.

Acad.

of Sci.

1184-1188.

Miki, T.. Easton,

A.M.

and

Rownd,

genes of the R plasmid

R.H.:

NR

Mapping

of the

I, Mol. Gen. Genet. 158

(1978)217-224. D. and Silver. S.: Mercuric

and Mitsuhashi, Agents

S.: Mercury

genes from domains

of

resistance

and R plasmtds

ampicillin

in

Antt-

I I (1977) 999-1003.

R.P.

inorganic

occurring resistance

adjacent

S.N.: Site

to the termini of a

element Tn.3. J. Mol. Biol.

and salts

Roth,

C.:

Plasmid-linked

resistance

soil bacteria. Richmond,

10 (1976) 753-761.

to mercuric

J. Bacterial.

D.C.: Trans-

and phenyl mercuric

M.H. and John, M.: Co-transduction phage of the genes responsible

thesis

and resistance

to mercury

ions in

I 110-l 112.

147 (1981)

coccal

by a staphylo-

for peniciilinase

salts.

Nature

syn-

202 (1964)

1360-1361. J., Bennett,

P.M. and Richmond,

e/r-B, the gene encoding

the B-subunit

enterotoxin

coli, when

FEMS

of E4wichia

Microhiol.

Lctt. 13

R.: The tctracyclinc Tn1771

have three

Schottel,

M.: Expression

Volatilisation

cloned

in pACYClY4. J. and Schmitt,

transposons

38-base-patr

repeats

Tn17.?/

and

and generate

tivc-

Mol. Gcn. Gcnct.

IX1

(19XI ) X7-Y4.

A., Clark, D.. Silver. S. and Hedges.

of mercury

of

of the heat-labile

(lW1) l-5.

rcsistancc

direct rcpcats.

J.. Mandal.

Shapiro.

and organomercurials

R factor systems

R W:

determined

in enteric bacteria.

Nature 351

Molecular

model for the transpositi~)n

cation ofba~t~riophage

JA.:

Mu and other transposable

metals

J. and Weiss, A.: Bacterial

determined J.M.

Biodegradation

and

and replielctnents.

resistance

to toxic

by extrachromosomal

R factors,

Kaplan,

Proc.

Symp.

Falkow,

Silver, S. and Kinscherf,

Appl.

A.M. (Eds.), Science,

London,

in

3rd Int. 1076, pp.

T.G.: Genetic and biochemical

microbiol

transformation

mercurial

compounds,

degradation

and detoxification in Chakrabarty,

and Detoxification

CRC Press, Boca Raton, cobalt.

Science

Southern,

EM.:

fragments Stanisich.

VA,

ions

that

Stiibcr.

and

(Ed.).

of Environmental

Bio-

Pollutants.

to mercury.

nickel and

1114-I116.

of specific

sequences

P.M. and Richmond,

among

DNA

J. Mol. Biol. YX

on

ILL, R.. Data, for bacterial

Btt,jard.

a

non

conjugative

and

Summers,

A.O. and Silver, S.: Mercury of Escherichia

w/i.

to mercuric plasmid

in

I ?Y ( 1977) 121?7-I 233.

II.: Org;rnrratn)n pBR322

Sci. USA 78 (lY81) 167-171. strain

M.H.: Characteri-

unit encodingresistance

Acad.

122X-1276.

S.N., Curtiss

occurs

m plasmids

95

nomenclature

Bennett.

I>. and

signals

in Str~~lz~/o~o~~~irsu~~~~‘cII.T. J. Bacterial. R.C.. Cohen,

A.M.

by g!el electrophoresis.

zation ofa translocatton

bearing

S.: Uniform

Detection

basis for

of mercury

FL, 1982, pp. 85-103.

156 (1967)

separated

to

R.P., Clowes,

IN. and

locatable

resistance

(1068)1335-1342. Novick,

Ag. Chemo.

A.J.. Oliver, J., Kelly, W.J. and Reanney,

P c~‘t&/uo~r~\ trenr~irrmm J. Bacterial.

D.J., Chou. J. and Cohen,

P.D., Kopecko,

H.

I17 (1977) 957-998. Novick,

Antimicrob.

(1975) 5Cl.LiI7 I.. Kozukue,

from clinical lesions in Japan.

Chemother.

specific DNA deletion transposable

structural

Tn501: functional

T.. Sarai, Y., Kondo,

EJcherichirr coli isolated Nisen.

A.. Godette,

Gene 34 (1985) 253-262.

H., Ishikawa,

microb.

L., Schmidt,

reductase

R 100 and transposon

the enzyme. Nakahara.

M.H.: R plasmtd

Smith. D.H.: R factors mediate resistance

Brown, N.L., Haberstroh,

plasmid

plasmid

899-917.

the rightward

Misr3,T.K..

(H&r) loci of the IncM

( 1984) 31 l-320. selection pressure.

Sharpley.

103-l 18. Maniatis,

and

J. and Richmond,

Silver. S., Schottel.

W.. Ohtsubo,

Ohtsubo,

Phystcal

(Omr) and inor-

Proc. Nat]. Acad. Sci. USA 76 (1979) 193%3937.

126A (1975) 435-448. M., Wishart.

A.0:

resistance

( t 974) 335-337.

genetique

McCormick,

Rev. 40 (1976) 168-189.

Summers.

in vivo in the absence of antibiotic

by inducible

l27B (1976) 57-58. D.D.,

resistance 32

V.. Grinsted,

transfer

base-pair R., Henri,

and

SchiifEl, I.., Arnold. W.. I’tihler. A., Altenbuchner.

ends of deletion

722-727. Joly, B., Cluzel,

(Paris)

Putrocheilou,

Sanchez,

in yeast or E. co/i. Curr. Genet. 2 (1980) 103-107.

suppressive causing

J. and Schmitt.

of mnp.4 mutants

Plasmid

J.C., Bach, M.L. and Lacroute,

trical

mercury

Radford,

P.M., Higginson,

Bacterial.

CL.

genetic map ofthe organomercury

mercury

Environmental Academic

a proposal.

H.I., Tollc.

189-193.

Transposable

Transposons:

plasmids: Ogawa.

01 transcripti~~llal

pACYC184. resistance J. Bactcriol.

Proc.

Natt.

II) ;I plasmid-

I 12 ( 1972)

91

Microbiology, Summers,

A.O.

metals.

Washington, and

resistance

from

Escherichiu coli. Plasmid Tanaka,

M., Cramer,

endonuclease Bacterial.

map

poson. Tonomura, organic

of of

R plasmid

of

R.: EcoRI

J. Bacterial.

153 (1983)

K. and Kanzaki, mercurials

NR

I. J.

an ancestral

of complex

mercury

trans-

Murphy,

by cell-free extract

decomposition

of

of a mercury-resistant

tance in Yersinia enterocolitica. Ann. Microbial.

and genetic analysis

R388, Sa, and R7K. Plasmid S.D.

resistances

S.: Mercury

and

in the

7 (1982)

(Paris)

132B

and pseudomonal

and

plasmids

in

132 (1977) 197-208. resistance plasmids,

- 1978. American

B.C., Szybalski,

with enteric, in Schlessinger,

Society for Micro-

W. and Ris, H.: Mapping in heteroduplex

by J-P. Lecocq.

of

DNA molecules

lambda by electron microscopy.

1343-1348.

Communicated

Mercury

by

DC, 1978, pp. 121-124.

and substitutions

of bacteriophage (1969)

S.:

J.L., Clark, D.L., Belier, R.G. and Silver,

Washington,

deletions

Silver,

determined

and organomercurial

D. (Ed.), Microbiology biology, Westmoreland,

1432-1438.

F.: The reductive

pseudomonad. Biochim. Biophys. Acta 184 (1969) 227-229. Vidon, D.J.-M., Lett, M.-C. and Delmas, C.L.: Mercury resis(1981) 225-230.

A.A.,

organomercurial

staphylococcal

T. and Sawai, T.: Evolution from

Weiss,

Weiss, A.A., Schottel,

restriction

R plasmid

127 (1976) 619-636. transposons

J.: Physical

Staphylococcus aureus. J. Bacterial.

3 (1980) 35-47.

of the composite

Inc-W group plasmids 239-250.

G.A.: Transposition

a transferable

J.H. and Rownd,

M., Yamamoto,

resistance

transformation

32 (1978) 637-672.

A.O., Weiss, R.B. and Jacoby,

mercury

Tanaka,

S.: Microbial

Ant-m. Rev. Microbial.

Summers,

Ward, J.M. and Grinsted,

DC, 1975, pp. 219-226

Silver,

Science 163