Mini-Mulac transposons with broad-host-range origins of conjugal transfer and replication designed for gene regulation studies in Rhizobiaceae

41

Gene, 63 (1988) 41-52 Elsevier GEN 02298

Mini-MuZuc transposons with broad-host-range for gene regulation studies in Rhizobiaceae Iac fusions; selectable markers;

(Translational

origins of conjugal transfer and replication designed

Agrobacterium rhizogenes Ri origin;

mini-Mu transposon;

Rhizobium me~iloti;Agrobacter~um tumefacien~; Azorhizobium cau~inoda~ ORS57 1; replicon stability; recombi-

nant DNA).

P. Ratet, J. Schell and F.J. de Bruijn A~geilung ~e~e~~&~e G~ndlagen der P~anze~~~c~~ung, ~ax-P~anck-I~ti~t~r Zuchtungsfor~chung,Kiiln (F.R.G.) Received Revised

10 April

1987

22 September

Accepted

16 November

Received

by publisher

1987 1987 27 November

1987

SUMMARY

Novel mini-Mu derivatives were constructed, carrying a truncated lacZYA operon fused to the terminal 117 bp of the Mu S-end, for the isolation of translational lac fusions by mini-Mu-mediated insertion mutagenesis. Different selectable markers (chloramphenicol resistance; gentamycin resistance) were introduced to allow selection for mini-Mu insertions in different replicons and bacterial strains. A mini-Mulac derivative carrying the site for conjugal transfer of plasmid RP4 (oriT) and the origin of replication of the Agrobacterium rhizogenes Ri plasmid (o~~HR1) was constructed to enable one-step Iac-fusion mutagenesis of cloned (plasmid-borne) regions in Escherichia coli and efficient conjugal transfer of gene fusions to a variety of Gram-negative bacteria. The conjugation frequency, stability and copy number of replicons carrying mini-Mulac derivatives with oriT and oriRiHR1 in members of the Rhizobiaceae such as Rhizobium meliloti, Azorhizobium caulinodans ORS.571 and Agrobacterium tumefaciens C58 was examined.

Co~es~ondence Grundlagen

to: Dr. F.J. de Bruijn, der

Pflanzenziichtung,

Ziichtungsforschung,

5000 Koln

Abteihmg

Gene&he

Max-Planck-Institut 30 (F.R.G.)

t%r

Tel. (0049)221-

5062315.

fixation

gene; nod gene, nodulation

origin of DNA replication; tance; Ri, root inducing; tomycin;

Abbreviations:

A, absorbance;

Cb, carbenicillin;

my&n; GSH, glutamine of nodulation

synthetase

bp, base pair(s);

A, deletion;

site; moi.,

0378-I tl9/88~$~3.S0

AND

METHODS,

multiplicity

section

of infection;

0 1988 Elsevier

Cm, genta-

II; hsn, gene, host specificity

gene; kb, 1000 bp; Km, kanamycin;

see MATERIALS zation

Ap, ampicillin;

Cm, chIoramphenico1;

LB and LSO, b; mob, mobili-

niff;x gene, nitrogen

Science Publishers

B.V. (Biomedical

conjugal sensitive; XGal, joint;

transfer

Ti, tumor

function;

inducing,

[ 1, designates

plasmid-carrier

Tn, transposon;

Tra,

ts, temperature

AND METHODS, state;

Sm, strep-

gene; Sym, symbio-

Tm, trimethoprim;

TY, see MATERIALS

ari,

unit(s); R, resis-

‘, sensitivity;

dismutase

5-bromo-4-chloro-3-indolyl-8_D-galactoside;

phage state.

Division)

Rif, rifampicin;

sodAB gene, superoxide

tic; Tc, tetracycline;

gene; Nm, neomycin;

p.f.u., plaque-forming

section b;

: : , novel

( ), designates

pro-

42

transfer

INTRODUCTION

in the mini-Mulac

Groisman Bacterial gene regulation studies have benefitted greatly from the analysis of hybrid protein+galactosidase

fusions

(see Silhavy

Plasmid

vectors

fusion,

by cloning

carrying

of distinct

transcriptional (Casadaban

restriction

and translational

nals in front of a truncated described

and Beckwith,

for the construction

lational

Zac gene

fragments start sig-

IacZ gene, have been

et al., 1980; Minton,

Shapira et al., 1983). Transposon allow the generation

1985).

of lac gene

derivatives,

of transcriptional

fusions

by inserting

1983; which

Here we report derivatives

material

are specifically

gene or operon

host

multicopy

pBR322

of mini-Mulac adapted

in Rhizobiaceae.

for such studies

agrobacterial range,

et al., 1984;

1986; 1987a and b).

the construction

which

study of gene regulation starting

(Groisman

and Casadaban,

(Table I) in Escherichia

Often the

is a rhizobial

carried

cloning

for the or

by a narrow

vector

such

as

coli, which can be

conjugally mobilized to Rhizobiaceae at low frequencies, if at all. After mini-Mufac mutagenesis in

and trans-

E. cofi, the putative

within

must be transferred to the appropriate rhizobial or agrobacterial strain to allow screening for proper fusions and regulation studies. This requires appro-

the

coding region of the gene of interest or its promoter, have been constructed (TnS-lac, Kroos and Kaiser, 1984; Tn3-Ho-Ho, Stachel et al., 1985; Tn917-fat, Perkins and Youngman, 1986). However, the most versatile vehicles for the isolation of fat gene fusions by insertional mutagenesis - especially of plasmidborne genes - are derivatives of bacteriophage Mu (Casadaban and Cohen, 1979; Casadaban and Chou, 1984; Castilho et al., 1984). These mini-Mulac vehicles combine the useful properties of Mu, namely high transposition frequency, headful packaging and relatively low insertional specificity (see Toussaint and Resibois, 1983) with the well established lac fusion methodology (see Silhavy and Beckwith, 1985). The mini-Mulac transposons usually consist of the left and right end extremities (c and S-end) of Mu, flanking non-Mu sequences, such as the (truncated) Zac operon, selectable marker (antibiotic resistance) genes and in some cases the Mu transposition functions. They are constructed by joining the truncated lac operon to the terminal 117 bp of the S end of Mu in such a way as to allow the isolation of transcriptional/translational fusions when the miniMulac inserts in a gene of interest in the S--f c end orientation (in the proper reading frame) (Casadaban and Cohen, 1979; Casadaban and Chou, 1984; Castilho et al., 1984). To isolate stable mini-Mulac insertion mutants, derivatives without the Mu transposition functions are usually used and the necessary functions for transposition and headful packaging are provided in trans by a helper Mu phage carrying a temperature inducible repressor gene (Mutts) (Casadaban and Cohen, 1979; Castilho et al., 1984). The utility of these mini-Mulac transposons has been further extended to in vivo gene cloning by the inclusion ofplasmid replicons and an origin ofconjugal

translational

gene-lac

fusions

priate selectable markers and an efficient conjugal mobilization system. In addition, it is desirable that the replicon carrying the proper gene-lac fusion exists as low- or single-copy number in the cell, to avoid artifacts due to multiple gene promoter copies (titration of activators or repressors). One of the miniMulac transposons described in this study, MudIIPR48, allows one-step mini-Mulac-mediated plasmid-gene fusion mutagenesis in E. coli and highfrequency conjugal transfer to and stable, low-copy maintenance of plasmid-borne fat-gene fusions in Rhizobiaceae.

MATERIALS

AND METHODS

(a) Strains and plasmids The bacterial strains and plasmids study are listed in Table I.

used in this

(b) Media and chemicals E. coli strains were grown in LB medium (Miller, 1972). TY (Beringer, 1974), or LSO (Elmerich et al., 1982) medium supplemented with 40 mg/ml of nico-

tinic acid and 0.2% (NH&SO,, were used for growth of Azorhizobium caulinodans 0RS57 1, R. rneliloti 1021 and A. tumefaciens C58. Antibiotics were added at the following concentrations: for E. coli: 10 pg Tc or Gm/ml, 20 pg Km or Cm/ml and 25 pg Ap/ml. For 0RS571: 10 pg Tc/ml, 50 pg Gm/ml, and 500 pg Cb/ml. For R. meliloti 1021: 10 pg

TABLE Bacterial

I strains

and plasmids Genotype,

Strains

antibiotic

resistance

Relevant

phenotype

Source

or use

or reference

E. coli recA1, e&Al,

JMlOS(Mucts)

gyrA96,

thi, hsdRl7,

supE44, reiA 1, A (lac- proAB)

RecA-

recipient

strain for mini-Mu

chromosomal

Yanisch-Perron

et al. (1985)

and Ratet and Richaud

integration

(1986)

(Mutts) A(lucIPOZYA)74,

MC1060

rpsL(SmR), MC4100

gaiK15, galEl6,

hsdR

strain for cloning

ex-

Casadaban

and Cohen (1979)

Casadaban

(1976)

periments

aruD 139, A(argF-luc)Ul69, rpsL150(SmR),

RecA _ Lac

reL4 1, flbB5301,

RecA recipient

strain for mini-Mu

chromosomal

integration

ptsF25, deoC1 MC4lOO(Mucts)

aruDl39,

A(argFJac)Ul69,

rpsL150(SmR),

reZAl,jIbB5301,

MC4100

(Mu&s)

lysogen for mini-

Ratet and Richaud

(1986)

Mu lysate production

pstF25, deoC1, (Mutts) M8820(Muc

+)

uraD 139, A(uruCOIBA-leu)7679, A(proAB, urgF-lucIPOZYA)XIII,

recipient

strain for mini-Mu

trans-

Casadaban

(1975)

duction

rpsL (SmR), (Mu) s17-1

recA, pro, hsdR. thi, chromosomally integrated Km

RP4-2-Tc

Tra + strain for conjugal

transfer

Simon et al. (1983)

: : Mu-

: : Tn7, TpR SmR

Rhizobiaceae ORS.571

Azorhizobium cuulinodans

CbR

Dreyfus

and Dommergues

(1981) Rm1021

SmR

C58

RifR, nopaline

type GV3 101

Rhizobium meliloti

Meade

Agrobucterium tumefaciens

Holsters

et al. (1982) et al. (1980)

[pTiC58] Plasmids pFR97

ApR

pMCl403

pFRlO0

pBR322

pPR3

ApR ApR, KmR, CmR

pFR210

KmR, oriRSF1010,

~45-2

ApR, GmR

derivative

et al. (1983)

Ratet and Richaud

(1986)

Ratet

and Richaud

(1986)

cloning vector

Ratet and Richaud

(1986)

pBR322

Koncz

MudIIPR3 oriPl5A

Shapira

derivative in pFRlO0 derivative

et al. (1984)

pSUP5011

ApR, CmR, KmR, mob +

Tn5-mob in pBR325

Simon (1984)

pLJbB 11

KmR

oriRiHR1

Jouanin

pJRDl84

ApR, TcR

cloning vector

pBR322

ApR, TcR

ColEl

derived

pACYCl84

CmR, TcR

cloning

vector

pRK2013

KmR, Tra + , mob + , incP

Tra + helper plasmid

pRK290

TcR,

Tra- , mob + , incP

wide host range cloning

vector

pLAFR1

TcR, cos, Tra -, mob + , incP

wide host range cosmid

cloning

in pHSG262

et al. (1985)

Heusterspreute cloning vector

Bolivar Chang

for triparental

et al. (1985)

et al. (1977) and Cohen (1978)

Ditta et al. (1980)

mating Ditta et al. (1980) vec-

Friedman

et al. (1982)

tor pVSP9A

TcR, KmR

R. meliloti nigh-IucZ gene fusion in pWB5 (pRK290

derivative)

Sundaresan

et al. (1983)

T. Nixon and F.M. Ausubel, unpublished

pFB682

CmR

R. mehioti glnA (GSI) locus in

pFB691

R. meliloti GSII locus in pBR322

pLRSA2

ApR TcR

ORS571

ni$A in pLAFR1

Pawlowski

pPR53

TcR, CmR, GmR

pLRSA2

: : MudIIPR46

P. R. and F.J. de B., unpublished

ApR

A. tumefaciens gbtA (GSI) locus in

F.J. de B., unpublished

pACYCl84

pPR56

pFRD184

F.J. de B., unpublished et al. (1987)

P. R. and F.J. de B., unpublished

44

Tc/ml,

50 pg Cm/ml,

and 250 pg Sm/ml. For

A. tumefaciens C58: 10 pg Tc/ml, 100 pg Gm/ml and

100 pg R.if/ml. For detection of ,%galactosidase activity the media were supplemented with 30 pg XGal/ml. (c) DNA isolation

Maxiprep plasmid DNA was prepared as described by Ish-Horowitz and Burke (198 1). ‘Miniprep’ plasmid DNA was prepared as described by de Bruijn and Lupski (1984). Chromosomal DNA was prepared as described by Meade et al. (1982). (d) Restriction endonuclease transformations

analysis,

ligations and

Conditions used for DNA manipulations and transformations have been described by Maniatis et al. (1982). The enzymes used in these analyses were used according to the specifications of the manufacturers (Boehringer, Mannheim; Bethesda Research Laboratories, Bethesda, MD; New England Biolabs, MA). (e) Conjugal transfer

Plasmids were transferred from E. coli strains to Rhizobiaceae strains using strain S17-1 (Simon et al., 1983) or using the helper plasmid pRK2013 (Table I), in triparental mating experiments (Ditta et al., 1980). (f) Construction

of 1~2

gene fusions

Phage manipulation, mini-Mu transduction and screening for mini-Mu- induced gene fusions were carried out as described by Castilho et al. (1984) and Ratet and Richaud (1986), or as described in RESULTS AND DISCUSSION sections c, d and e.

48, respectively)

were constructed as outlined in Fig. 1 and described in the legend.

(b) Integration

of

Mud11

derivatives

into

the

chromosome

The mini-Mulac carrying plasmids were transformed into E. co/i MC4lOO(Mucts) (Table I) by selecting for CmR. Tr~sfo~ants were purified on Cm-containing plates, liquid cultures were grown up and Mu phage production was induced, as described in MATERIALS AND METHODS, section f. The resulting mixed lysate was used to infect E. coli JM108(Mucts) (Table I) and CmR transduct~ts were selected and purified. Due to the recA mutation of JM 108, regeneration of mini-Mulac-carrying plasmids occurs at a very low frequency only (Castilho et al., 1984). Most CmR transductants were found to be Lac -- and ApS, suggesting that they resulted from mini-Mu&c transposition into the chromosome of JM108(Mucts) in a region devoid of promoters and translation initiation sites. Because instability of JM108(Mucts)(mini-Mu&) double lysogens was observed (not shown), a culture of this strain was induced and the mixed lysate obtained was used to infect E. coli MC4100 (Table I). A number of CmR, Lac -, temperature-sensitive transductants, representing putative MC4100(Mucts)(mini-Mulac) double lysogens, were purified. To verify the double lysogen status of these purified tr~sduct~ts and to confirm that the mini-Mu& derivatives had retained their ability to transpose and create lac gene fusions, liquid cultures were grown up at 28 ‘C, partially induced at 37°C (30 min) and plated on LB-XGal plates (MATERIALS AND METHODS, section b). Transductant strains yielding Lac + clones, representing those cases where the mini-Mulac had transposed with the help of the induced Mutts helper phage and created a (translational) fusion to a chromosomal E. coli gene, were retained for further analysis. Thus strains MC4lOO(Mucts)(MudIIMC4lOO(Mucts)(MudIIPR46) and PR13), MC4100(Mucts)(MudIIPR48) were constructed.

RESULTS AND DISCUSSION

(a) Construction

of ~udIIPRl3,

40, 46 and 48

The piasmids carrying the mini-Mulac transposons MudIIPR13,40,46, and 48 (pPR13,40,46 and

(c) Frequency of transposition ation in ~sc~~~j~~~u cd

and k-fusion

gener-

To determine the transposition and lac-fusion generation frequency of the different mini-Mulac-

45 TABLE II

Frequencies of transposition and kc-fusion generation by Mud11derivatives Donor strains

p.f.u. a

Transposition frequency b

% kc-gene fusions”

Transposition into the E. coli chromosome MC4100(Mucts)(MudIIPR13) MC4lOO(Mucts)(MudIIPR46) MC4lOO(Mucts)(MudIIPR48)

1.5 x IO8 1 x loa 5 x 10’

8.7 x 10m6 3.7 x 10-e 6 x lo-’

4.3 4.6 5.4

Transposition into a plasmid MC4100(Mucts)(MudIIPR13)[pPR56] MC4100(Mucts)(MudIIPR46)[pPR56] MC4lOO(Mucts)(MudIIPR48)~pFR56]

2 x 10s 1.1 x 10s 3 x 10’

9 x 1o-6 2.5 x 10-G 6 x lo-’

11 10 7.5

a Plaque-forming units; titered on MC4100 (see Table I). b Transposition frequency expressed as number of CmR per p.f.u. c % of lac-gene fusions (Lac + CmR transductants).

derivatives in E. coli, cultures of strains MC4100(Mucts)(MudIIPR13), MC4100(Mucts) (MudIIPR46) and MC4100(Mucts)(MudIIPR48) were grown up to an A,, of approx. 0.2 and induced at 45 ‘C for 30 min. The resulting lysates were used to infect cells of E. caii MC4100 at an m.o.i. of 0.001 and the infected cells were plated on LB Cm Xgal plates (MATERIALS AND METHODS, section b). In addition, their phage titer (p.f.u.) was determined (MATERIALS AND METHoDs, section f). The results are summarized in Table II. The transposition frequency of the different Mud11 derivatives, expressed as number of CmR transductants/p.f.u., varied between 6 x lo-’ and 8.7 x 10e6. MudIIPR48 transposition appeared to be the lowest. This is not likely to be due to the larger size of MudIIPR48, since the transposition frequency of other derivatives of Mu of the same size appears to be unaffected (Faelen et al., 1986). It is more likely due to the nature of the sequences cloned between the Mu ends (R.M. Harshey, personal communication). The three different Mud11 derivatives yield Lac + colonies (luc-fusions) at similar frequencies (approx. 5 y0 ; Table II), comparable to those found for other Mud11 transposons (Castilho et al., 1984).

(d) Methodology for the isolation of lae fusions to genes cloned in different replicons

Depending on the size of the target plasmid, the mini-Music and the purpose of the experiment, two basic methods for the isolation of Zacfusions to plasmid-borne genes are available. The first method involves the mini-Mulac plasmid transduction technique described by Castilho et al. (1984) and Ratet and Richaud (1986). If the gene to be analyzed is expressed in E. coli, direct screening for Lac + colonies on XGal plates can be used to identify proper translational &-gene fusions in the target plasmid. If it is not expressed in E. co&, but the target plasmid contains a multicopy vector replicon, proper translational &c-gene fusions can often still be identified due to random transcriptional readthrough on the plasmid template which results in light blue colonies on XGal plates. Otherwise the mini-MuZ~c mutagenized region must be transferred to the bacterial species of origin for further analysis. For the lac-fusion mutagenesis of larger plasmids, such as cosmids derived from the cloning vector pLAFR1 or other pRK290 derivatives (Table I), an alternative protocol can be used. The target plasmid is transformed or conjugally mobilized into the desired E. coli MC4100(Mucts)(MudIIPR) doubly lysogenic strain and the plasmid-containing bacteria

46

E BHBSPE

PH

P E

pFR97

Y A

Ap’

Cm’

pPR13 E

SPE

BE

PH

PE

pJROl8L

E+P EBSSsP

S

pPRb0

B partial

ori T

B

psuPsol1 F=l 6

B

t-tacz

Gmr

9 A

orif

Ap’

Cm’

pPRL9

Bprrtial

oriRiHR1

B

_B B

PLJbB”

t----Gmr QlillD

YA pPR 48 E

I I XbBpB

BE

Fig. 1. Construction

of piasmids

BumHI,

H; SalI, S; PsfI, P; Smal,

B; HindHI,

S end termini antibiotic

pPR13, carrying of pFR97

of the mini-Mu’s

resistance

pPR13,

pPR40,

MudIIPR13,

was constructed

(Table I), which contains

I

and pPR48.

striped

by replacing

the truncated

IIll

luc operon.

Ap’

/d-m

PHES

BSPEPHPE

The restriction

endonuclease

code used is as follows: EcoRI,

X; S&l, Ss; BgZII, Bg. The blackened

boxed regions/arrows

of transcription

Cm’ !,I/

oriRiHR1

I B

Sm; X&II, Xb; X&I,

The vertically

genes and their direction

pPR46

OUT

denote

(when known). the BumHI-Sal1 To construct

structural

gene sequences

A scale in kb is provided fragment

plasmid

regions

denote

for the Inc operon

at the top right corner.

of pPR3 (Table I) by the BarnHI-Sal1

pPR33,

pFR210

E;

the c and and

Plasmid fragment

(Table I) DNA was digested

with

are partially

induced

for 24h. Plasmids insertion

for Mu transposition

which have obtained

can be transferred

and XGal to screen for lac fusions.

at 37°C

summarized

a mini-Mulac

to the desired rhizobial

or agrobacterial strain, provided the vector or the mini-Mu/at carries an origin of conjugal transfer

Mu-lac

mating’ of the induced,

pRK20 13 (helper plasmid, functions)

bacterial

species,

et al. (198 1). Rhizobial

which are relevant pPR39.

To establish

by Ditta

to the construction

to form plasmid

of plasmid partial

pSUP5011

digestion

into pPR46, between fragments

pPR40.

-48) and test the replicon

of pPR33,

(Table I) carrying restriction

oriT and CmR of pPR46, contained

sites flanking

In order to introduce on’Twas

fragment

of plasmid

after partial

in the MudIIPR

derivatives

E: 0.117 kb; B, E-Xb ([UC): 6.2 kb; Xb-Bg:

stability of the oriRiHR1 of

this experiment since it does not carry the oriRiHR1 allowing replication in Rhizobiaceae. No stable

the RSFlOlO

ori, a PstI-EcoRI

of plasmid

segment,

the GmR gene in pPR39,

(otir)

of RP4 into pPR40,

the origin of replication

with BumHI,

shown (based on MudIIPR48,

the oriRiHR1

of

into the Sal1 site a BamHI

fragment

the GmR and CmR genes, after of the A. rhizogenes Ri plasmid

was cloned into the BumHI

to form plasmid

fragment

the GmR gene, to form plasmid

the GmR gene was then recloned

transfer

site of pPR40 located between

pPR46. To introduce

a 200-bp &I-EcoRI

TcR gene containing

~45-2 (Table I), containing

the origin of conjugal

of pPR46

polylinker

1986). Only the first two of these fragments,

in Fig. 1. The BarnHI-XbaI

pLJB 11 (Table I) carrying

digestion

of and

recipient were spotted on TY plates, conjugated overnight at 28°C and GmR transconjugants were selected at 28’ C on minimal LSO plates (see MATERIALS AND METHODS, section b). The resulting mobilization frequencies are listed in Table III. Apparently pPR48 can be conjugally transferred and maintained at a frequency quite comparable to the pPR53 control plasmid. Plasmid pPR46 serves as negative control for examining replication/stability in

cloned into the BamHI

to form plasmid

frequencies

serted in a pLAFRI-derived, stable, wide-host-range replicon (Table I) were transformed into E. coli S 17-1 (Table I) and mated with R. meliloti 102 1, AZ. caulinodans ORS571 and A. tumefaciens C58 (Table I). Strain S 17-1 provides the necessary transfer helper functions for the mobilization of replicons carrying oriT (Simon et al., 1983). Cultures of S 17-l harboring the plasmids mixed with cultures of the

are shown (separately) fragment

transfer

MudIIPR48 (Fig. 1) in different members of the Rhizobiaceae, plasmids pPR46, pPR48 (Fig. 1) and pPR53, a control plasmid carrying MudIIPR46 in-

the KmR gene and Pl5A ori (Ratet and Richaud,

by BarnHI-X&I

of pPR40 with EarnHI,

a BamHI

carrying

the conjugal

carrying the oriT of RP4 (MudIIPR46

plasmids

transconju-

fragment

carrying

Using the Sal1 and XhoI polylinker

of pPR13

to

and the desired

as described

a PstI-PstI

replaced

Zac fusions

to generate

cons carrying Mud11 derivatives

[pPR56] and MC4100(Mucts)(MudIIPR48)[pPR56] were grown up and heat-induced. The resulting lysate was titered and used to transduce strain MC4100. Cells were plated out on LB plates containing Ap to select for the target plasmid pPR56 (Table I), Cm to select for the MudIIPR derivatives

pPR33 was subsequently

the ability of all three

confirming

transfer

The ability of MudIIPR13, -46 and -48 to transpose into and generate lac fusions to plasmid-borne genes was tested using a plasmid carrying the glnA (glutamine synthetase I) locus of A. tumefaciens (pPR56, Table I). Strains MC4100(Mucts)(MudII PR13)[pPR56], MC41OO(Mucts)(MudIIPR46)-

fragment

quency of 5-10x,

Tra + , providing

(e) Frequency of transposition into and generation of luc fusions to plasmid-borne genes

and a PstI-PstI

to

(f) Conjugal transfer frequency and stability of repli-

gants, carrying the plasmid : : mini-Mulac derivatives, can be selected for using the mini-Mulac antibiotic resistance (CmR and/or GmR) and the plasmid vector markers. These transconjugants can then be screened for lac expression to identify translational Zac-gene fusions and used for gene regulation studies.

fragment

due

at a fre-

plasmid

or agrobacterial

EcoRI + Pstl. This generates

clones

were found

plasmid : : mini-

culture,

E. coli strain

containing

recipient

Lac’

a

E. coli

containing

Table II.

pPR56 : : Mud11 lac fusions mini-Mulac transposons plasmid-borne genes.

such as oriT (RP4). This is achieved by carrying out a ‘triparental

in

The results are

pPR48.

site located

The sizes of the restriction

from left to right) are as follows:

Mu S-end up to B,

1.3 kb; Bg-B: 1.6 kb; B-B (oriT): 1.7 kb; B-P: 3.2 kb; P-H: 0.4 kb: H-E: 0.9 kb; E-S: 1.0 kb;

S-B, S, P: 2.5 kb; B, S, P-E: 0.5 kb; E-P, H: 1.3 kb; P, H-Muc-end:

1.0 kb.

48

TABLE

III

Conjugal

transfer

Plasmids

a

frequencies Recipient

of MudIIPR

carrying

replicons

cells (hosts)”

ORS571

TABLE

IV

Stability

of a MudIIPR48

Plasmid a

Rml021

carrying

Recipient

in Rhizobiaceae

cells a

C58 ORS571

Number

replicon

Rml021

C58

of Gm R colonies ’ “i, GmR or TcR colonies”

pPR46

0(<10_8)

O(
0(<10_8)

pPR48

1om2

5 x 1om2

10-l

pPR48

pPR53

lo-*

2 x 10-z

10-1

pVSP9A

a The recipient

strains

and

plasmids

used

are described

in

Table I.

conjugation

100%

95%

94%

a The strains tested and plasmids b % GmR and TcR colonies

b The numbers rhizobial

< 1%

specify the number

or agrobacterial

colony

of GmR transconjugants forming

per

unit after 12-15 h of

rhizobial

or agrobacterial

absence

of antibiotics.

used are described

per colony-forming

96°C) loo”,, in Table 1. unit of the

strain tested after 48 h ofgrowth

in the

at 28°C.

pPR46-containing transconjugants were found. This could be attributed to the absence of the oriRiHR1, but also to failure of the oriT of MudIIPR46 to function. The latter possibility was ruled out by mutagenizing a narrow-host-range replicon (pACYC184, Table I) carrying the R. meliloti gZnA (GSI) region (pFB682; Table I) with MudIIPR46, isolating a Lac+, GSIglnA: : MudIIPR46 insertion and conjugally transferring this plasmid back to R. meliloti using the triparental mating protocol (see RESULTS AND DISCUSSION, section d). GmR transconjugants, due to single or double recombination between pFB682 and chromosomal borne glnA sequences were obtained at frequences of 10 - 3 and 10 - ‘, respectively (not shown). In fact, a chromosoma1 R. melilotigh4 insertion mutant was thus isolated carrying a translational glntl-lac fusion, which is being analysed for regulatory properties (F.J. de B., S. Rossbach, P.R. and J.S., in preparation). Thus MudIIPR46-tagged plasmids can be efficiently conjugally transferred to Rhizobiaceae with the oriT intact. To test the stability of pPR48 in the rhizobial and agrobacterial strains, pPR48-containing cultures were grown to early log phase in TY medium supplemented with Gm, diluted 1 : 100 in TY medium without antibiotics and grown to saturation. As a control the same strains harboring the wide-hostrange pRK290 derivative pVSP9A (TcR, Table I) were used. From the saturated cultures, bacteria were plated for single colonies on TY plates and screened for the GmR or TcR phenotypes, respec-

tively. The results are shown in Table IV. Replicons carrying the oriRiHR1 are very stable in A. tumefuciens (96%; see also Jouanin et al., 1985), and in R. meliloti (100%). The stability of the oriRiHR1 replicons is comparable to that of pRK290 derivatives in A. tumefuciens and R. meliloti (100x, 94%). In AZ. caulinoduns ORS571, however, plasmid pPR48 is highly unstable (< 1%) unlike pVSP9A (95 %). At 37’ C, the normal growth temperature for ORS57 1, the loss of the pPR48 plasmid is even more extreme, reflecting possibly a temperature sensitive replication characteristic of the Ri plasmid, in analogy to that observed for Ti (tumor inducing) and Sym (symbiotic) plasmids of agrobacterial and rhizobial species (Van Larebeke et al., 1974; iurkowski and Lorkiewicz, 1978). Similar results were observed with MudIIPR48 inserts in other pBR322 derivatives. To carry out gene regulation studies with lac gene fusions carried by oriRiHRI-derived replicons in rhizobia or agrobacteria, one must determine if these replicons can stably coexist with naturally occurring plasmids in these bacterial species. A. tumefaciens harbors the Ti plasmid (Van Larebeke et al., 1974). Compatibility of the oriRiHR1 with the Ti plasmids of both octopine and nopaline types of A. tumefaciens has been reported (Jouanin et al., 1986). R. meliloti harbors two megaplasmids involved in symbiotic interactions with plants, so-called Sym plasmids (see Long, 1984). To analyse the compatibility of replicons based on the oriRiHR1 with the Sym plasmids of R. meliloti

49

1021, cultures of 1021 (pPR48) were used to infect the roots of Medicago saliva plants. Normal nitrogen fixing (Fix + ) nodules were formed, confirming the compatiblity of otiRiHR1 and ori of the symbiotic mega plasmid. One month after infection bacteria were reisolated from surface-sterilized nodules as described (Pawlowski et al., 1987) and screened for the presence of pPR48 (GmR). Approximately 85% of the R. meli~oti colonies screened were GmR, further confirming the stability of the pPR48 replicon in R. meliloti. (g) Copy number of MudIIPR4tkarrying replicons To determine the copy number of oriRiHRTcontaining replicons, we mutagenized a pPR322derived plasmid carrying the R. meliloti glutamine synthetase (GSII, glnZZ) gene (pFB691; Table I) with MudIIPR48 and isolated a MudIIPR48 insertion in the cloned glnZZ region (pFB6911; not shown). We transferred pFB6911 into R. meliloti by triparental conjugation, isolated total DNA, digested it with Hind111 and hybridized a Southern blot carrying ZZindIII fragments with the R. meliloti glnZZ gene probe (pFB691). By comparing the intensity of the hybridizing bands corresponding to the chromosomal glnZZgene, with those corresponding to the plasmid-borne, MudIIPR4&containing, glnZZ gene we estimated that the oriRiHR1 replicon must be present in the cell at appro~mately the same copy number as the chromosome (one to two copies per cell; not shown). (h) Conclusions We have described the construction of novel Mud11 derivatives, which are particularly useful for gene regulation studies in members of the Rhizobiaceae and therefore represent a valuable addition to the various mini-Mulac fusion transposons constructed by Casadaban and coworkers (see INTRODUCTION). The special features of the Mud11 transposons described here include: (1) Selectable CmR and GmA markers GmR is a good marker for rhizobial and agrobacterial strains. It also allows the introduction of MudII-carrying replicons into mutant strains already containing Tn5 (KmR, NmR, SmR) and the construction of double mutants.

(2) The oriT mobilization sysrem This allows high-frequency conjugal transfer of replicons to a variety of Gram-negative bacteria and the isolation of mini-Mulac insertions in large plasmids, which cannot be transduced by mini-Mu duction. (3) The ori RiHRZ This allows stable, low-copy-number replicon maintenance in rhizobial and agrobacterial strains. Its compatibility with Ti and Sym plasmids makes regulation studies involving vir, nod, and niffix genes possible, using MudIIPR48carrying replicons in merodiploid strains. (4) Multiple restriction cleavage sites These allow convenient physical mapping of the MudIIPR insertion sites and their orientation. These sites include EcoRI, Z3amH1, PstI, ZZiBdIII, XbaI, BgUI and Sal1 (Fig. 1). Moreover, the nucleotide sequence of the junction between the mutated gene and the mini-Mu-S end can be determined using a primer homologous to the known Mu-S end sequences (black box, Fig. 1; Adachi et al., 1987), or the lac primer used for sequencing inserts in M13mp derivatives (Yanisch-Perron et al., 1985). The XbaI and BgZII sites just downstream from 1acA and the Z?stI site in front of the CmR gene (Fig. 1) are convenient for recloning complete Zac-gene fusions, without or with an additional selectable marker (GmR), in other vectors. (5) The sizes of the MudZZPR derivatives MudIIPR13, -46 and -48 are approx. 9.2, 13.7 and 21.5 kb, respectively (Fig. 1). For efftcient homologous recombination to occur between flanking mini-Mulac sequences, at least 1 to 2 kb of duplicated sequences are required. This allows the maximum size of the target plasmid to be approx. 27.8, 25.0 and 15.5 kb for MudIIPRl3, -46 and -48mediated mutagenesis, respectively, assuming maximum Mu packaging size to be 39 kb. No constraints on the minimum size of the target plasmid exist. If the length of the mini-Mulac cointegrate structure is shorter than 39 kb, some flanking host DNA will be packaged which will be lost upon cointegrated resolution in M8820 (Mu).

50

(6) The absence of Mu transposition functions MudIIPR den’vatives This precludes

secondary

transposition

on the

in Rhizo-

biaceae (see Plessis et al., 1985) or Mu-induced rearrangements in MudIIPR carrying plasmids. The initial transposition appears

frequency

not to be altered

of MudIIPR13 by the region

reduction

observed,

possibly

between

of transposition

and 46

frequency

due to the sequences

P.R. gratefully acknowledges the commission of the European Communities for financial support.

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MudIIPR13 has been used to create translational lac fusions to the AZ. caulinodans nigh and nifD genes and to study their regulation (Pawlowski et al., 1987). It has also been used to create Zac fusions in the sodAB genes of E. cofi (Carlioz and Touati, 1986), the pectate lyase genes of Erwinia chrysanthemi (A. Diolez, personal communication) and the nod genes of Rhizobium (Horvath et al., 1987). MudIIPR46 has been used to construct an AZ. caulinodans 0RS571 nfA-lac fusion (pPR53) (P.R., J.S. and F.J. de B., in preparation). MudIIPR46-induced insertions in genes carried by pBR322 replicons can also be recombined easily into the chromosome of the Rhizobium or Agrobacterium via single or double crossover after oriT mediated conjugal transfer. This has been used to create AZ. caulinodans ORS57 1 @LA + , nifA : : mini-Mulac merodiploids and n$A : : mini-Mulac chromosomal insertions (unpublished data) and to create an R . meliloti glnA : : mini-Mulac strain (RESULTS AND DISCUSSION, section f). MudIIPR48 has been used to construct an A. tumefaciens gInA-lac gene fusion (P.R. and F.J. de B., unpublished; RESULTS AND DISCUSSION, section e). The instability of oriRiHR1 containing replicons in AZ. caulinodans ORS57 1 was unexpected, even though other replicons, such as RSFlOlO (Bagdasarian et al., 1981)-derived plasmids are also very unstable in this strain (P.R., unpublished data). It may reflect the phylogenetic differences between this strain and rhizobial or agrobacterial strains (Jarvis et al., 1986; B. Dreyfus, J.L. Garcia and M. Gillis, in preparation).

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