Gene, 102 (1991) 93-98 0

1991 Elsevier

GENE

Science

Publishers

93

B.V. 0378-l 119/91/$03.50

04096

A family of Corynebacterium glutamicum/Esc~erichia expression, and promoter probing (Recombinant

DNA;

expression

Bernhard J. Eikmanns”,

vector;

Eva Kleinertz”,

promoter

probe vector;

coli shuttle vectors for cloning, controlled gene

co-transformation)

Wolfgang Liehl b and Hermann Sahm”

“ Institut ftir Biotechnologie I, Forschungszentrum Jiilich GmbH, D-51 70 Jiilich (F.R. G.) and b Lehrstuhl ftir Mikrobiologie, Technische UniversitdtMiinchen, D-8000 Miinchen (F.R. G.) Tel. (49-89)2105-2378 Received by J.-P. Lecocq: 1 October Revised: 18 January 1991 Accepted: 6 March 1991

1990

SUMMARY

A new family of vectors including cloning vectors (pEK0; pECS), an expression vector (pEKExl), and promoter probe vectors (pEKpllacZ; pEKplCm), has been constructed. All these shuttle vectors are based on the replication origins of the corynebacterial pBL1 and the Escherichia coli ColEl plasmids, and thus are able to replicate in Corynebacterium glutamicum and E. coli. Plasmids pEK0 and pEC5 carry multiple restriction sites useful for gene cloning and the kanamycinor chloramphenicol-resistance-encoding gene from Tn903 or from Tn9, respectively. In C. glutamicum, both vectors are compatible with vectors containing the corynebacterial pHM1519 replicon. Based on plasmid pEK0, the expression vector pEKEx1 was developed to allow for isopropyl-@-D-thiogalactopyranoside-inducible expression of inserted genes in C. glutamicum and E. coli. Also based on pEK0, the promoter probe vectors pEKpllac2 and pEKplCm were constructed to carry the promoterless facZ or cut reporter genes downstream from useful cloning sites, for assaying the transcriptional activity of cloned fragments.

INTRODUCTION

Coryneform bacteria comprise a large group of pleomorphic Gram + bacteria. Some closely related species, including C. glutumicum, B. jlavum and B. lactofermentum, are widely used in the industrial production of amino acids (Kinoshita, 1985). Despite their economic importance only in recent years have efforts been made to apply recombinant DNA techniques to elucidate the Correspondenceto: Dr. B.J. Eikmanns, Forschungszentrum

Jiilich

(F.R.G.) Tel. (49-2461)61-3967;

GmbH,

Institut Postfach

ftir Biotechnologie 1913,

D-5170

1,

Jtilich

molecular biology of these organisms (reviewed in Martin, 1989). Cloning vectors for use in C. glutumicum and in E. coli, based on the oris of B. lactofermentum pBL1 or of C. glutamicum pHM1519 (= pSRl), have been developed previously (reviews: Martin et al., 1987; Martin, 1989). Also, Tsuchiya and Morinaga (1987) described vectors conferring inducible cat expression which can be used for controllable expression of genes inserted between the dNTP,

deoxyribonucleoside

galactopyranoside;

/r’-galactosidase;

Fax (49-2461)61-3870.

Ap, ampicillin; BHIS,

Corynebacrerium; CAT,

IPTG,

isopropyl$-D-thio-

or 1000 bp; Km, kanamycin;

MCS,

ori, origin of DNA replication; P, PolIk, Klenow (large) fragment of E. coli DNA polymerase I;

multiple cloning site; nt, nucleotide(s); promoter;

K, resistance/resistant; Abbreviations:

triphosphate;

kb, kilobase

bp, base pair(s); B., Brevibacrerium; BGal,

brain-heart

infusion

Cm acetyltransferase;

sorbitol

(medium);

Cm,

chloramphenicol;

C.,

transposon;

XGal,

RBS, ribosome-binding

site; Tc, tetracycline;

5-bromo-4-chloro-3-indolyl-B-D-galactopyranoside;

[ 1, denotes plasmid-carrier

state.

Tn,

pUL330

DUC~K derived

m

a

derived

pUC derived

SBSmKSaE

\ Hindlll +

0

DD

HSBSmKSaE

Fig. 1.

Dral+HindllllPollk

Ligation

Y

/ Hindlll + Sal1

fragment

S B Sm K Sa E

Pollk

pUC4K

A

[P/EL

pEKExCAT

ii EBSPH

t I I I

a

h , I . I

I--

IS/PI

[eg/Sml

derived

derived

derived

pkk232-B

pJFllBut

pEK0

[PV/S]

B

”

! 1 , i

I I

Fig. 2.

m

1

r

f

H c Nx

1

Y tWmP

%lK

tacP II \I II \\ II 11 , I SBSmKSa

)

B

[H/PI

ESaKSmBSPH

B [Sm/H]

B

m Ek

pEKpllacZ-tat

pEKpllacZ-horn

Tn5

derived

derived

derived

pWST1

piWiT

X’b ‘e sm ‘K

ID/E’-‘1Bc

u

pEKplCm-tat

pEKplCm-horn

‘1 \ ’

1 \

BSPH

taCP

‘5

B (Sm,“];

\

I1

‘I I(

, ,

/

t2P

‘H’Sm21K’H1

95 respective

promoter

and the cat structural

TABLE

gene. However,

those and most of the cloning vectors have disadvantages as they are relatively large and contain only one or few suitable cloning sites. Thus, one aim of this study was the

Bacterial

Relevant

supE44,

C. glutamicum

ATCC13059

of

(3)

AS019

Plasmids KmR, ori of pBL1

(4)

pUC18

(5)

pUC4K

ApR ApR, KmR

pJFl18ut

Apa,

(6) tacP,

IacIQ,

derivative

of

(7)

pJFl18EH cat

pKK232-8

ApR, promoterless

pKK223-3

ApR, tacP

pEK-horn-thrB

KmR, homP

(10)

piWiT

TcR, 1acZ

(11)

pWST1

KmR, ori of pHM1519

(12)

pUCpUL

ApR, ori of pBLl

(13)

pEK0

KmR, ori of pBL1

(13)

pEKEx 1

KmR, lacIQ, tacP, ori of pBL1

pEKpllacZ

KmR,

(8) (9)

lacZ,

promoterless

(14)

ori of

(14)

pBLl cat, ori of pBL1

KmR, promoterless

pEKplCm pEC5

CmR, ori of pBL1

(14)

pWLT17

KmR, ori of pHM1519

(14)

il (1) Hanahan

(1985); (2) American

and Schein (1990); (4) Santamaria

Type Culture

(14)

Collection;

(3) Liebl

et al. (1985); (5) Yanisch-Perron

et al.

(1985); (6) Vieira and Messing (1982); (7) Fiirste et al. (1986); (8) Brosius Brosius and Holy(1984);

of C. glutamicum-E. coli shuttle vector pEK0. The 3.7-kb SalI-Hind111 the ColEI

fragment

replicon

(10) Eikmanns

et al. (1991); (11) von

et al. (1986); (12) Liebl et al. (1989b); (13) this work,

Fig. 1; (14) this work,

Since ApR is not expressed

mutant derivative

pUL330

(1984);(9)

in pUCpUL.

(1) (2)

restriction-deficient

R163

Wilcken-Bergmann

carrying

1, endA 1,

wild type

C. glutamicum

DISCUSSION

the Sal1 and Hind111 sites of pUCl8

recA

ATCC13032

(a) Construction of the basic vector pEK0 The C. glutamicum/E. coli shuttle vector pEK0 was constructed as outlined in Fig. 1. The 6.1-kb plasmid has a multiple cloning region with single PstI, BglI, SalI, BumHI, KpnI, SacI, EcoRI restriction sites, replicates in C. glutumicum and E. coli, and confers Km resistance to both organisms. Thus, pEK0 is a small and versatile shuttle vector for gene cloning.

Fig. I. Construction

hsdR 17,

gyrA96, thi-I, relA 1

we are interested in isolating proof structures which determine the

terium was ligated between

Reference

characteristics

Strains E. coli DH5

as reporter genes has been reported by Morinaga et al. (1987) and by Cadenas et al. (1988), respectively. The aim of the present study was to develop promoter probe shuttle vectors carrying the promoterless 1ucZ gene or the promoterless cut gene which allow screening or selection of transcription initiation sequences (blue colonies or CmR) and easy determination of promoter strength (BGal or CAT activity, respectively). AND

and plasmids

or source”

efficiency of transcription initiation. For this purpose it is necessary to have promoter probe vectors. Construction of such vectors using the CmR gene cat or the KmR gene aphll

EXPERIMENTAL

strains

Strain/plasmid

construction of improved C. glutamicum/E. coli shuttle vectors for gene cloning and inducible gene expression. To increase our knowledge of gene expression and regulation in C. glutamicum moters for investigation

I

Fig. 2.

of pUL330

(Tomizawa

in C. glutamicum (Ozaki et al., 1984) the ApR gene from pUCpUL

the pBL1 ori for Corynebac-

providing

and Som, 1984) and the ApR gene, resulting was replaced

by the KmR gene from Tn903

(Oka et al., 1981). For this purpose a 1.3-kb PstI fragment of pUC4K carrying the aminoglycoside 3’-phosphotransferase-encoding gene and conferring Km resistance was PolIk-blunted and ligated with the 4.8-kb DraI-Hind111 PolIk-blunted fragment derived from pUCpUL. (PolIk blunting was done in the presence

of the four dNTPs.)

Abbreviations:

B, BamHI;

Bc, BclI; Bg, BglI; C, ClaI; D, DraI; E, EcoRI;

EV, EcoRV;

Mlul; N, NruI; Nd, NdeI; P, PstI; Pv, PvuII; S, SalI; Sa, SacI; SC, ScaI; Sm, SmaI; X, XhoI; Xb, XbaI; Xm,XmnI; with the respective Fig. 2. Diagrams pEKpllacZ-tat pWLT17

enzymes,

PolIk-blunted,

of (A) the expression

and ligated without vector

and -horn, (C) the promoter

(E). For construction

of pEKEx1

pEKEx1

regeneration

and derivative

probe vector pEKplCm a 2.6-kb ScaI-EcoRV

of either restriction

pEKExCAT,

and derivatives

fragment

sites double-restricted

site.

(B) the promoter

pEKplCm-tat

K, KpnI; M,

H, HindIII;

[/I, designates

probe

vector

pEKpllacZ

and -horn, and of the cloning vectors

from pJFI 18ut was ligated into PstI-EcoRI-cleaved

and

derivatives

pEC5 (D) and

and PolIk-blunted

pEK0.

lacIQ, tacP, an MCS, and the transcription terminators of the E. coli rrnB operon. For construction of pEKExCAT the promoterless cut gene from pKK232-8 was isolated as PstI-PvuII fragment, PolIk-blunted, and ligated into SalI-cleaved and PolIk-blunted pEKEx1. Plasmid pEKpllac2 was constructed by inserting the promoterless IacZ gene isolated as 3.3-kb XbaI-XmnI (PolIk-blunted) fragment from plasmid

The pJFl18ut

piWiT

fragment

contains

into EcoRI + PolIk-cleaved

by cloning the promoterless

pEK0. For construction

cut gene from pKK232-8

of pEKpllacZ-tat

and -horn see footnote

as 1.4-kb SmaI-PvuII PolIk-blunted

For construction ofpEKplCm-tat and -horn see footnote b in Table IV. For construction PolIk-blunted, and ligated with the MCS of pUC18. Plasmid pWLT17 was derived containing

the pBDl0

Bacillus KmR by the 1.3-kb HindHI-SmaI

deletion of the 1.3 kb DraI-EcoRV Fig. 1, legend.

fragment

(containing

fragment

ofpEC5 plasmid pEKplCm-tat from pWSTl by substitution

Tn5 KmR determinant

ApR) gave rise to pWLT17.

b in Table III. Plasmid into BgiI + EcoRI-cleaved

which functions

Only the relevant

pEKplCm

was constructed

and PolIk-blunted

pEK0.

was digested with NruI + HindHI, of the 6.1-kb SalI-NdeI fragment

in C. glutumicum and in E. coli. Subsequent

restriction

sites are shown. For abbreviations

see

96 (b) Construction pEKEx1

and assessment

of expression

vector

To develop a vector system for inducible gene expression in C. glutumicum we constructed pEKEx1 (Fig. 2A). This vector carries the luc repressor gene lacZQ and upstream thereof, and in the opposite orientation, the tat promoter (tucP) followed by unique f&RI, BamHI, SalI, PstI cloning sites. The tacP/lucZQ system was chosen as controllable promoter, since this system was previously shown to be functional in B. lactofermentum (Tsuchiya and Morinaga, 1987), a close relative of C. glutumicum (Kinoshita, 1985). To assess the functionality

of pEKEx1

a promoterless

cut

gene was ligated into pEKEx1, resulting in pEKExCAT (Fig. 2A), and the specific CAT activity of E. coli and of C. glutamicum clones harbouring pEKExCAT was determined before and after induction with IPTG (Table II). As reference, the specific CAT activities of clones harbouring pEC5 (see section d), a plasmid carrying also the cut gene under transcriptional control of tacP but lacking &lo are given. The results in Table II demonstrate the suitability of pEKEx1 for controlled gene expression. Such a gene control system might be especially useful for cloning of genes which exert negative effects on growth or are toxic in case of uncontrolled expression. Furthermore, this system offers the possibility of modulating the concentration of a desired gene product by adding various concentrations of IPTG (Filrste et al., 1986). (c) Construction and assessment of promoter probe vectors pEKpllacZ and pEKplCm Based on vector pEK0 we constructed the promoter probe vectors pEKpllacZ (Fig. 2B) and pEKplCm (Fig. 2C). In pEKpllacZ unique restriction sites for PstI, SalI, BumHI, and KpnI are located in front of the 1uc.Z reporter gene while plasmid pEKplCm carries BamHI, &z/I, and PstI cloning sites in front of the cut reporter gene.

In particular the BamHI sites allow cloning of Sau3Adigested chromosomal C. glutumicum DNA for identification of promoter-active sequences, and adjacent sites allow re-isolation of the promoters. In both vectors the region between the multiple cloning site and the reporter gene contains translational stop codons in all three reading frames followed by an RBS. The stop signals prevent possible translational readthrough from inserted fragments which could interfere with the expression of the reporter genes. C. glutumicum cells harbouring pEKpllacZ or pEKplCm are KmR, form white colonies on LB plates containing XGal, and are sensitive to Cm at < 1 pg/ml. Thus, there is no readthrough transcription from promoters in front of the MCS. To test the utility of pEKpllacZ and pEKplCm known promoters were cloned into the probing sites of both vectors resulting in pEKpllacZ-tat and -horn (Fig. 2B) or in pEKplCm-tat and -horn (Fig. 2C), respectively. As shown in Table III, pEKpllacZ-tat and -horn conferred /IGal activity to C. glutamicum and led, depending on promoter activity, to pale or deep-blue colonies on LB plates containing XGal (not shown). As shown in Table IV, cloning of tacP or homP into pEKplCm conferred CAT activity to C. glutumicum and CmR up to 120 pg Cm/ml. The copy numbers of pEKpllacZ and pEKplCm derivatives were estimated to be similar since transformation to E. co/i DH5 with DNA from the same amount of recombinant C. glutumicum cells resulted in about the same number of transformants. This ruled out major errors in evaluation of promoter strength caused by different indicator gene dosage. The results show that both promoter probe vectors

TABLE

III

Specific

/?Gal

harbouring

activity

pEKpllacZ

of Corynebacterium glutamicum ATCC13032 or derivatives

front of the promoterless

II

Specific

CAT activity medium

of Corynebacterium glutamicum ATCCl3032 pEKExCAT

or pECS during growth

before and 1 h after addition

promoters

on

b

fiGa

activity

protein) b

pEKpllacZ

none

pEKpllacZ-tat

IacP

1.220

pEKpllacZ-horn

homP

0.040

- IPTG

+ IPTG

was isolated