Cloning vectors and antibiotic-resistance markers for Brevibacterium sp. R312

Gene, 105 (1991) 119-124 0 1991 Elsevier Science Publishers

GENE

B.V. All rights reserved.

119

0378-I 119/91/$03.50

06027

Cloning vectors and antibiotic-resistance (Cryptic

Brevibacterium-Escherichia

plasmid;

markers for Brevibacterium sp. R312

coli shuttle

vectors;

tetracycline

resistance;

recombinant

DNA)

C.K.N. Chan Kwo Chion, R. Duran, A. Arnaud and P. Galzy Chaire de Microbiologic Industrielle et de Genetique des Microorganismes, ENSA, 34060 Montpellier Cedex (France) Received by G. Wilcox: 8 March 1991 Revised/Accepted: 22 April/ 17 June 199 1 Received at publishers: 1 July 1991

SUMMARY

of several cryptic plasmids from coryneform strains was investigated in Brevibacterium sp. R312. Only the glutamicum pSR1 replicon was found to be suitable for establishing a host-vector system. Two pSR1 derivatives, pRPCG200 and pHYCG1, were used as cloning vectors. They carry a neomycin-resistance-encoding and a tetracycline-resistance-encoding gene, respectively. Replication

Corynebacterium

construction of cloning vectors for Brevibacterium sp. R3 12, a strain with industrial applications (Bui et al., 1982).

INTRODUCTION

Cloning vectors were constructed for several coryneform strains such as Arthrobacter sp. (Shaw and Hartley, 1988), B. lactofermentum (Santamaria et al., 1984; 1987; Smith et al., 1986; Takagi et al., 1986; Yeh et al., 1986), B.jlavum (Patek et al., 1989; Satoh et al., 1990), C. diphtheriae (Serwold-Davis et al., 1990), C. glutamicum (Miwa et al., 1985; Yoshihama et al., 1985), C. melassecofa (Takeda sp. (Vogt-Singer and et al., 1990) and Rhodococcus Finnerty, 1988; Desomer et al., 1990). Here we describe the

Correspondence to: Dr. A. Arnaud, et de Genetique

Chaire de Microbiologic

des Microorganismes,

ENSA,

Industrielle

34060 Montpellier

Abbreviations: encoding

Fax (33)67.61.25.80.

bla, P-lactamase-encoding

gene; ble, BeR-encoding

C., Corynebacterium; CAT, gene; Cm, Luria-Bertani encoding

uph, aminoglycoside

Ap, ampicillin;

gene; B., Brevibacterium; Be, bleomycin;

TcR-encoding

gene; bp, base pair(s);

chloramphenicol; kb, kilobase (medium); MCS, multiple cloning

gene;

replication;

phosphotransferaseBGal, /?-galactosidase;

Cm acetyltransferase;

Nm, neomycin; R, resistance/resistant;

cat, CAT-encoding or 1000 bp; LB, site(s); neo, NmR-

nt, nucleotide(s); on’, origin of DNA R., Rhodococcus; Tc, tetracycline; tet,

gene; Tn. transposon.

AND DISCUSSION

Different small cryptic plasmids were characterized in aa-producing coryneform strains: pBL1 in B. lactofermentum (Santamaria et al., 1984), pSR1 in C. gfutamicum (Yoshihama et al., 1985), pCG1 and pCG2 in C. glutamicum (Ozaki et al., 1984), pCC1 in C. calhmae (Sandoval et al., 1984), pBLlO0 in B. linens (Sandoval et al., 1985). Replications of pBL1, pSR1 and pCC1 in Brevibacterium sp. R3 12 were examined.

Cedex

1 (France) Tel. (33)67.61.22.15;

EXPERIMENTAL

(a) Plasmid carrying the pBL1 replicon Previous studies on the replication origin of pBL1 showed that the Hind111 site was not involved in plasmid replication (Yamaguchi et al., 1986), we thus chose this site for the construction of pBBLH22 (Table I). Although the pBL1 replicon replicates in many different coryneform strains (Bonamy et al., 1990), this was not the case in Brevibacterium sp. R3 12 (Table II). This result was surprising since pBL1 and pSR1 are compatible in B. lactofermentum (Morigana et al., 1987) and the pSR1 derivative

120 TABLE

I

Bacterial

strains

and plasmids

Strains

Description

B. luctofermentum

Source

of plasmid

pBL1

ATCC13869

C. cc&true

Source

of plasmid

pCC1

Arnaud et al. (1976) NRRL B-2243

C. lil~um

Source

C. ~iuta~l~cu~n

Strain

E. coli HBlOl

Host for the propagation

E. coli XL1 B

Host for pUC18

Source il

B. sp. R312

Size

Plasmids

of plasmid harboring

pCL1

NRRL

pSR1 cryptic

plasmid

of plasmids

BRL

derivatives

Stratagene

Description

Resistance

B-2243

ATCCl3058

Source”

(kb) pBL1

4.45

E. iuc@ermentum

pcc1

4.2

C. callunae cryptic

cryptic

plasmid

Santamaria

pSR1

3.0

C. @unzicum

pCL1 c.e

4.1

C. lih’um cryptic

pUCl8 pUC4K

2.1

AP

3.97

4,

Nm

Source

of Nm~-encoding

gene (undo)

pUC4KIXX

4.0

Ap, Be, Nm

Source

of NmR-encoding

gene (aphlf)

pHY 163PLK

2.56

Tc

Source

of TcK-encoding

plasmid

cryptic

plasmid

plasmid

Vector for z-complementation

This study Clontech

of BGal

Pharmacia and ble gene

Pharmacia Ishiwa and Shibahara

gene (fetal )

pBBLH22’

10.5

Cm, Nm

pBR328 derivative

pUCCK25’

8.0

AP, Nm

pUCl8

pUCCH1810h

8.0

Ap, Nm

pUCl8

pUCLX5 r pUCLX5 1 b.C*G

6.8

AP AP, Nm

pUCl8

derivative

carrying

pUCl8

derivative

carrying

pUCLB2” pUCLB213

6.8

in XbuI site pUCi8 derivative

carrying

pUC18 derivative

carrying

This study carrying

aphl (Nmn) gene in PstI site and pCC1

carrying

aphl (NmR) gene in PstI site and pCC1

in Kl>trI site derivative

in NindIII 8.0

This study

site

This study pCL1 in XbaI site

This study

uphl (NmR) gene in PstI site and pCL1 This study pCL1 in BamHI

site

This study

8.0

AP AP, Nm

6.8

AP, Nm

in BumHI site pUC8 derivative carrying

6.5

Nm

E. coli-Brevibacterium

5.2

Tc

(Nmu) gene pHY 163PLK derivative

4.5

TC

from pSV73 pHYCG 1 derivative

with the 0.7-kb PvuI fragment

6.8

Nm. Tc

pHYCG1

carrying

pRBR80 b.d

8.0

Cm, Nm

pUCEK 1

5.2

AP, Nm

pRPCG200 derivative carrying cat gene from pBR328 pUC18 derivative carrying aphi (NmR) gene in PstI site and the

pUCSS1

5.8

AP, Nm

1.3-kb EcoRI fragment from pSV73 pUC18 derivative carrying aphI (NmR) gene in PstI site and the

psv73

‘=g

b,d

in BlrrnHl

pRPCG200 pHYCG1

‘.<’ b,d

pHYCGP1 pHYCGT9

h+d

aphl (NmR) gene in Pstf site and pCL1 This study uphl (NmR) gene in &I

site and pSRl

site

Yeh (1988) sp.

R312

shuttle

vector

aphl

carrying

Chan Kwo Chion et al. (1991) carrying

the 2.7-kb SlnlrI-BglII

fragment This study

derivative

aphll (NmR)

deleted

and ble genes

This study from

pUC4KIXX

1.8-kb SphI-S’maI .1 ATCC, American

Type Culture Collection,

Northern

Research

Regional

Center

Rockville,

fragment

MD; BRL, Bethesda

(U.S. Department

(1985)

uphl (NmR) gene in P.stI site and pBL1

carrying

in Hind111 site derivative

et al. (1984)

Sandoval et al. (1984) Yoshihama et al. (1985)

of Agriculture),

This study This study This study

from pSV73 Research

Peoria,

Laboratory,

IL; Pharmacia,

This study Gaithersburg, Uppsala,

MD; Clontech,

Sweden;

Stratagene,

Palo Alto, CA; NRRL, La Jolla, CA.

h See Table II. c See Fig. 1. ‘I See Fig. 3. e pCL1 cryptic

plasmid

was extracted

from C. lilium cells grown

at 30°C on 2000 ml of TGY medium

(5 mg Bacto tryptone/5

mg yeast extract/l

mg

glucose/l mg K,HPO, per ml). Cell wall was weakened with glycine (2% w/v) one generation before cell harvest, and plasmids were extracted as described by Santamaria et al. (1984). r Plasmids pUCLX5 and pUCLB2 were obtained by subcloning pCLl into pUC18 XbaI or EarnHI sites, respectively. Only the XbaI site close to NdeI was digested

(DNA methylation

of C. Iilium DNA or site preference

of restriction

endonuclease).

$ Plasmids pUCLX51 and pUCLB213 were constructed by ligation of pUCLX5 and pUCLB2 R%I-linearized with the 1.3-kb PsrI fragment from pUC4K previously purified by the Geneclean method (Vogelstein and Gillespie, 1979), by transforming E. caii XLlB and spreading the transformation mixture of LB agar containing

0.1 mg Nm/ml

and 0.1 mg Ap/ml.

121 has been shown to replicate in Brevibacterium (Chan Kwo Chion et al., 1991). (b) Plasmid carrying the pCC1 replicon Since the ori of pCC1 was not localized,

sp. R312

two separate

sites were selected to prevent destruction of this ori. Hind111 and KpnI sites were used to subclone pCC1 into pUCl8, then the aphl gene (NmR) was inserted in the PstI site to produce pUCCH1810 and pUCCK25 (Table I). But neither of these two plasmids seemed to replicate in Brevibacterium sp. R312 (Table II). It was previously reported that the pCC1 replicon seemed to have stability problems (Sandoval et al., 1984) and a very low plasmid copy number in Arthrobacter (Shaw, 1989).

TABLE

II

Replication

Plasmid

lines represent pCL1;

broad

of hybrid plasmids

pUC18; segments

NmR) from pUC4K.

double indicate

from pCL1 (see Table I). Single

lines (narrow

segments)

the 1.2-kb PstI fragment

The orientation

correspond (aphl

to gene;

of the aphl gene in pUCLB213

unknown.A,AlwNI;B,BamHI;H,HindIII;N,NdeI;P,PstI;

Sm,SmaI;

X, XbaI.

is

main

corynebacterial

tested”

cryptic

plasmids

in Brevi-

Replicon b

Replication’

pBBLH22

pBL1

_

pUCCK25 pUCCH1810

pcc1

_

pcc1

_

pUCLB213

pCLl

_

pCLl pSR1

_

pRPCG200

pSRl

+

pHYCGl

pSRl

+

pHYCGT9

pSR1

+

pRBR80

pSRl

+

pUCLX5 psv73

Fig. 1. Construction

of the sp. R312

bacterium

1

See Table I, footnote from

plasmid

DNA

1982). Plasmid

b. Methods:

+

all the plasmids

minipreparations

miniprep

(1~1; approx.

electroporation-competent

Brevibacterium

was then immediately

cuvette (BioRad; electrical

2 mm electrode

quickly

medium

sp. R312

cells (Chan

polypropylene

transferred

Kwo

test tube. The

into an electroporation

gap) and cooled in an ice bath. A single

pulse (12000 V/cm; 100 ohm; 25 pF) was applied to the cuvette

with a Genepulser were

et al.,

100 ng) was mixed with 40 ~1 of

Chion et al., 1991) in a 1.5-ml pre-cooled whole mixture

tested were obtained

of E. coli cells (Maniatis

connected

placed

(Hanahan, except

(BioRad).

tube containing

1985), then incubated

at 30°C without

150 min before plate spreading (0.1 mg/ml),

to the Pulse Controller

in a polypropylene

for

The cells

1 ml of LB agitation

onto LB agar petri plates containing

pHYCG1

and

pHYCGT9

(0.1 mg/ml) was used. The tranformation efficiencies IO’ transformants per pg of plasmid DNA used.

for

for Nm

which

Tc

were always

about

nonreplicating

plas-

’ See Table I and Figs. 1 and 3. ‘ Plus symbol, mids.

Fig. 2. Stability bacterium

replicating

plasmids;

of pSV73 in Brevibacterium

sp. R312 in the stationary

103-fold in LB medium without overnight. number 20 number

30 of

generar~ons

40

minus symbol,

Similar

subcultures

of generations

sp. R312. A culture of Brevi-

phase containing

Nm and incubated were performed

from the first inoculation

pSV73 was diluted

at 28°C with agitation afterwards was plotted

daily. The against

the

% of NmK colonies obtained by transferring 100 colonies isolated on LB agar onto LB agar with 0.1 mg Nm/ml. C.F.U., colony-forming units.

122 (c) Isolation

iilium and

of pCL1 from Corynebacterium

plasmids

construction of hybrid vectors carrying the pCL1 replicon Plasmid pCL1 has not been previously described in the literature; we estimated its size to be 4.1 kb by electrophoresis and its restriction map is shown in Fig. 1. Two separate sites were chosen for the construction of pUCLB213 and pUCLX.51 (Fig. 1); however, these two

BclI/

BarnHI

a.

did not

AatE+

-.

I

XmaI

+ Barn

sp. R312

(d) Expression of the fetal gene (TcR) from pAMa The only functional replicon in Brevibacterium sp. R3 12 is the pSR1 replicon in pSV73 and pRPCG2OO (Table II).

Sm YXb

in Brevibacterium

replicate

(Table II). Further studies are underway to verify the replication of these ptasmids in other coryneform bacteria.

HI

XboI

pBR32B

t

AatE+

NheI

E

1

At

XbaI/

I

PV Sal1 I+

t

Fig.

3. Insertion

cryptic

XhoI

Xhol

of NmR, CmR and TcK genes into E. ~oli-Breyibffcreriut~ sp. R312 shuttle vectors

pSR1 plasmid.

The single lines represent

genes from pUC4KIXX. pSV73 (digested

pUC4KIXX SolI/

E. coli plasmids.

The MCS of pRPCG200

by XmaI + BglII) with pHYl63PLK

is derived (digested

Open segments

from pSP73

(Chan

by XmaI + BumHI),

represent

(see Table I). The blackened

uphi (NmR) gene from pUC4K

segments

represent

the

or uphIi (NmR and BeR)

Kwo Chion et al., 1991). Plasmid pHYCG1 was obtained by ligating transforming into E. coli HB 101, and spreading the transformation

mixture onto LB agar + 20 pg Tc/ml. Plasmid pHYCGT9 was obtained by ligating pHYCG1 (linearized by SalI) with pUC4KIXX (digested by XhoI), transforming E. coli HB 101, and selecting at 20 pg Tc/ml and 0.1 mg Nm/ml). The cut gene (CmR) was inserted into pRPCG200 by ligation of pRPCG200 (digested by A&II +XhuI) Restriction sites separated E, EcuRI:

Ev; EcoRV;

and pl3R328 (eat) (digested by AnrII + NheI), transforming E. cali HBIOI, and selecting at 50 pg Cm/ml and 0.1 mg Nmiml. by a slash are sites of ligation which did not regenerate any of the parental sites. At, AafII; B, BumHI; Be, BclI; Bg, Bgltt;

K, KpnI; N, NdeI; Nh, NheI; I’s, PsfI; Pv, Pvul; SC, SacI;

Si, .SnlI; Sm, SmaI;

Sp, SphI; Xb, XbuI; Xh, Xhol.

123 This replicon is stable except in the absence of antibiotics (Fig. 2). HindIII, PstI and SmaI sites could not be used with pRPCG200 because of the aphl gene (Fig. 3);

that the replication

origin ofpSR1

included the SphI, EcoRI

pHYCG 1 was therefore constructed to use these restriction enzymes and also to test for expression of the tetal gene in strain R312 (Fig. 3). This gene, originally from Streptococcus faecalis (Ishiwa and Shibahara, 1985), is functional in Brevibacterium sp. R312 since it can be used as a selectable marker during transformation with pHYCG1 (Table II).

sites and the PvuI fragment. Thus, pRPCG200 cloning sites which could be used were BglII, EcoRV, KpnI, BamHI, XbaI and Sal1 clustered in a polycloning site with the Bg/II site located immediately downstream from the SP6 RNA polymerase promoter. The pHYCG1 cloning sites that could be used were BglII, HindIII, PstI, SalI, SmaI and XbaI. The copy number of the pSR1 derivatives was high in Brevibacterium sp. R312 since the plasmid minipreparations yielded as much plasmid as did E. coli mini-

(e) Insertion of the uphZZ gene in pHYCG1 The aphZZ gene (Beck et al., 1982) inserted in pHYCG1 to obtain pHYCGT9 (Fig. 3) was functional in E. coli but

preparations (not shown). This is in agreement with results obtained by Miwa et al. (1984) for pHM1519, a plasmid that is identical to pSR1 (Martin et al., 1987). No promoter characterization has yet been reported for pSR1.

not in Brevibacterium sp. R3 12 because only Brevibacterium TcR transformants were obtained. However, the aphZ gene has been used as a selectable marker for cloning vectors in B. lactofermentum (Santamaria et al., 1984), C. glutamicum (Thierbach et al., 1988) and a great number of coryneform strains (Schafer et al., 1990).

REFERENCES Beck, E., Ludwig, G., Auerswald, E.A., Reiss, B. and Schaller, otide sequence and exact localization of the neomycin transferase Bonamy,

(f) Insertion of the cat gene in pRPCG200 The cat gene from pBR328 was inserted into pRPCG200 (Fig. 3). The resulting pRBR80 plasmids, carrying aphl and cat genes, yielded only Brevibacterium sp. R3 12 NmR transformants. This indicates that the cat gene does not function in Brevibacterium sp. R312, although it is expressed in B. lactofermentum and C. glutamicum (Miwa et al., 1985) and in B. stationis (Kurusu et al., 1990) contrary to the CmR-encoding gene of pC194 (Smith et al., 1986; Britz and Best, 1986). Moreover, the CmR-encoding gene from Streptomyces acriminici is only functional in B. lactofermenturn through the pBR322 TcR gene promoter (Santamaria et al., 1987). Further studies are underway to determine whether the absence of Brevibacterium sp. R3 12 CmR transformants was due to a non-active CAT protein or to nonrecognition of the cat promoter by Brevibacterium RNA polymerase. (g) Cloning sites of pRPCG200 and pHYCG1 This study was undertaken to improve the usefulness of restriction sites on pRGCG200 and pHYCG1. Shaw and Hartley (1988) showed that the small Sac1 fragment of pRS 1 (Fig. 3) was indispensable for plasmid replication in Arthrobacter. The present study also showed that SphI, EcoRI and PvuI fragments are indispensable for plasmid replication in Brevibacterium sp. R3 12. There was no Brevibacterium transformant with pUCEK1 and pUCSS1 constructed by, respectively, subcloning EcoRI and SphI-SmaI fragments of pSV73 into pUC18, and by inserting the aphl gene in a second step. Moreover, pHYCGP1, obtained by deleting the small PvuI fragment from pHYCG1, did not replicate in Brevibacterium sp. R3 12. These results indicate

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