Gene, 122 (1992) 187-192 0 1992 Elsevier Science Publishers

GENE

B.V. All rights reserved.

187

03781119/92/$05.00

06820

A series of shuttle vectors for Bacillus subtilis and Escherichia coli (Recombinant

Reinhold

DNA;

gene cloning;

reporter

genes; transcriptional

terminators)

Brtickner

~o~ekula~e Genetik der UniversitiitD-6900 Heidelberg, Germany: and .~ikrobieile Genetik, ~niversit~t Tiibingen. D-7400 Tiibingen, Germnny Received

by M. Salas:

1 March

1991; Revised/Accepted:

10 August/l7

August

1992; Received

at publishers:

3 September

1992

SUMMARY

A series of shuttle vectors for Bacillus subtilis and Escherichia coli was developed. These are derived from one basic construct composed of parts of the Gram+ plasmid pUI3110 and the Gramplasmid pBR322. They contain muitiple cloning sites flanked by transcriptional terminators. In one plasmid, a vegetative B. subtilis promoter drives transcription of inserted genes. For the construction of operon and gene fusions, the cat gene of PUB 112 and the IacZ gene of E. coli were employed as reporter genes. With these vectors, cloning and expression of genes as well as probing of regulatory signals can be performed in B. subtilis and E. coli.

INTRODUCTION

For gene cloning in B. subtiIis a large number of vectors have been constructed. Due to sometimes low transformation frequencies using B. subtilis as the primary host, it is often more convenient to conduct the initial cloning steps with a shuttle plasmid in E. coli and then transfer the recombinant plasmids to B. subtilis. Most of these vectors are based on small staphylococcal plasmids that replicate via an ssDNA intermediate by a rolling-circle mechanism

Correspondence to: Dr. R. Bruckner Genetik,

Auf der Morgenstehe

Tel. (49-7071)

296939;

at his present

Fax (49-7071)

Abbreviations:

aa,

/?-galactosidase

(encoded

Bmn-encoding

gene; Bm, bleomycin;

gene encoding protease

amino

address:

28, D-7400 Tiibingen,

acid(s);

Mikrobielle

Germany.

294634.

Ap, ampicillin;

B. Bacillus; BGal,

by [acZ); bla, gene encoding CAT,

p-lactamase;

Cm acetyltransferase;

epr, gene encoding

CAT, Cm, ~hlor~phenicol;

of B. subrilis; hmw, high molecular

ble, cat,

minor Ser

weight; kb, kilobase(

Km,

kanamycin;

MCS, multiple cloning site(s); M-O, minus on’; neo, Km-Nm

nucleotidyl

transferase-encoding

DNA replication; ss,

single

pyranoside;

gene;

R, resistance/resistant;

strand(ed);

XGal,

Nm,

neomycin;

SD, Shine-Da&rno

ori, origin

5-bromo-4-chloro-3-indolyl-&D-galacto-

’ (prime), gene truncated

at the indicated

side.

of

sequence;

(Gruss and Ehrlich, 1989; Novick, 1989). The appearance of ssDNA is believed to contribute to structural plasmid instabilities in B. subti~~s(Ehrlich et al., 1986), ~though a stable ssDNA vector system has been described recently (Haima et al., 1990). The occurrence of deletion formation has been found to be caused by erroneous replication termination (Michel and Ehrlich, 1986) and aberrant nickingclosing events (Ballester et al., 1989) mediated by the replication proteins of the plasmids. Topoisomerase-like activities were also implicated in illegitimate recombination (Lopez et al., 1984). Plasmids that do not replicate via ssDNA intermediates were reported to be more suitable as cloning vectors, but have not yet been employed frequently (Jam&-e et al., 1990). In B. subtiljs pUBll0 seems to be the most stable of the staphylococcal plasmids, possibly because its M-O is functional in this host (Boe et al., 1989). It is often observed that a fragment, which is stably maintained on a particular plasmid, causes structural instability after moving it to another vector. Sometimes, even inversion of the insert is detrimental. Mechanisms inherent to ssDNA replication may not be the only reason for this instability. Additional effects can be caused by the structural variability of the vectors. Cloning sites are located in different parts of the plasmids, the orientation of reporter

188 genes is variable and in most cases no transcriptional terminators have been included to protect essential plasmid functions from transcriptional readthrou~h initiated at cloned promoters. It has been shown in E. coli that cloning of strong transcriptional

promoters on plasmids is only possible if terminators are included (Gentz et al.,

198 1). The same may be true for Gram + plasmids. Therefore, I constructed a series of shuttle vectors that are derived from one pUB 1 lo-based shuttle construct, contain reporter genes in a fixed orientation and have transcriptional terminators flanking the MCS.

EXPERIMENTAL

AND DISCUSSION

(a) Shuttle vectors pRB373 and pRB374 A shuttle vector for E. coli and B. subtilis, pRB273, has been constructed previously (Bruckner et al., 1984). The bia gene and the ori for E. coli originated from pBR322 (Sutcliffe et al., 1979). The Nm-KmR and 13mK genes, neo and ble, and the plus ori for Gram’ bacteria had been derived from PUB 110 (McKenzie et al., 1986; Semon et al., 1987). It is not clear wether the M-O is also contained in pRB273. It would be present (Viret and Alonso, 1988) or absent (Boe et al., 1989). Since the vector was found to be structurally stable with various inserts, I sought to develop a series of shuttle plasmids based on pRB273. To improve the versatility of the vector a second transcriptional terminator, TI of E. colz’ rrnB (Brosius et al., 19X1), was introduced. At Tf as well as at to of phage I also present on the vector, more than 9004 of RNA synthesis is terminated in B. subtilis (Peschke et al., 1985). Subsequently, the b/a gene and the MCS of pRB273 were reptaced by the corresponding region of pUCl8 (YanischPerron et al., 1985). In the resulting vector, pRB373 (Fig. 1), eleven unique restriction sites flanked by two transcriptional terminators are available for cloning. Transformants with the plasmid can be either selected with Ap (100 yg/ml) in E. coli or Km (5 pg/ml) in B. subtifis. To express genes lacking their own promoters, a B. subtilj.~ promoter (veg11) fragment (Peschke et al., 1985) was inserted into pRB373 directing transcription towards the MCS (Fig. 1). The vegll promoter was found to initiate transcription in both B. subtilis and E. coli (Peschke et al., 1985). Genes can therefore be expressed in both hosts provided their ribosome-binding sites are also functional. (h) Promoter and terminator probe vectors pRB394 and pRB395 As the reporter gene for the const~~tion of a promoter probe vector a deletion derivative of the pUB 112 cat gene on pCR94 was chosen (Bruckner and Matzura, 1985). In

this cat variant

the promoter

and the regulatory

inverted

repeat had been deleted resulting in constitutive cat gene expression, provided a promoter is cloned in front of the gene (Bruckner and Matzura, 1985). An AccI restriction site located in the cat coding region was removed by changing a TTG (Leu) triplet, the G of which is part of the AccI recognition sequence GTATAC, to TTA (Leu) according to the method of Nakamaye and Eckstein (1986). The mutated cat gene was subsequently cloned adjacent to the MCS of pRB373 yielding the promoter probe pRB394 (Fig. 1), which contains eleven unique cloning sites located at the 5’-end of the promoterless cat gene. Transcription initiated at promoters cloned into the MCS is terminated at Tf behind the reporter gene. The presence of promoters can be selected by plating transformants of B. subtilis on media with 5 pg Cm/ml or on 10 pg Cm/ml for E. coli. In pRB394 containing B. subtilis cells one can detect a specific CAT activity of 0.06 units (ymol of Cm acetylated/ min~mg of protein), which does not allow pRB394 transformants to grow on media supplemented with 5 ,ug Cm/ ml. The vector has been successfully used to identify a promoter from Clostridium pqfringens (Steffen and Matzura, 1989). To convert the promoter probe plasmid to a terminator probe vector, the veg11 promoter fragment was inserted yielding pRB395 (Fig. 1). It directs high levels of cat gene expression in both B. subtilis (2.8 CAT units) and E. cofi. Termination activity of fragments cloned into the MCS between the promoter and the cut gene is indicated by reduced cnt gene expression. (c) Translational fusion vectors pRB381 and pRB382 To analyse translational signals, the truncated ‘laczfrom pMCl871 (Shapiraet al., 1983),lacking transcriptional and tr~slational initiation signals, was introduced into pRB373 (Fig. 1). The sequence of the MCS-‘EacZ fusion was confirmed by sequencing according to Sanger et al. (1977). In pRB381 five restriction sites within the MCS remained unique (Fig. 1) and are available for construction of translational fusions to ‘1acZ. Successful fusion of translation initiation signals to the truncated ‘1acZ gene can be monitored on XGal plates in E. coli EacZ deletion strains and in B. subtilis, since the endogenous /?Gal level in B. subtilis is low and only detectable during stationary phase (Errington and Vogt, 1990). With the aid of pRB381 gene fusions can be created and the expression of putative open reading frames is easily detectable. On pRB381 expression of ‘IacZ is only achieved if translation as well as transcription initiation signals are cloned. To be independent of the simultaneous cloning of promoters, the vegH promoter was inserted into pRB381. With the resulting plasmid, pRB382, translation initiation signals can be tested and their relative strength compared.

189

B9”-

SOl[ BornIll Xbol Ps\l IOC z ’ 1 TWTG.CCCTAGCA~C~GACGl.CCG.l+G~Ss /3-g,blV~jPro AspGlu Lru Thr Scr Arg Cys Ala

. Fig. 1. Restriction

by black bars and their orientation 374, 394, 395 is identical

is indicated

to that of pUC18,

HIS

Leu Scr

.

map of the shuttle vectors. Thin lines represent

and NmR, neo, BmR, ble, CmR, cat), IacZ, terminators

Hind111

PUB 110 DNA, thick lines pBR322 DNA. Location

(T,, r,, or t, of I cop RNA; Szybalski

by arrows.

Restriction

the MCS of pRB381

sites indicated

and 382 including

(d) Shotgun cloning In a shotgun cloning experiment B. subtilis chromosomal DNA partially restricted with Sau3AI was introduced into pRB373 and a gene for a secreted protease (epr) was iso-

and Szybalski,

of resistance

genes (ApR, bla, KmR

1979) and the vegII promoter

in the MCS are unique. The nt sequence

(P) is marked

of the MCS of pRB373,

the start of /IGal (p-gal) is given. Map units represent

kb.

lated on a 7.5kb fragment (Bruckner et al., 1990). In another attempt BclI-cleaved B. subtilis DNA was used and a gene for /IGal was isolated on a 4.5kb fragment. It seemed to be identical to the one identified by Zagorec and Stein-

190 metz (1991; unpublished results; D. le Coq, pers. commun.). An 8-kb genomic DNA fragment of ~~u~~~~Qc~cc~~ ~y~~sus cloned in E. coli into pRB373, which was suspected

It has been proposed,

that a functional

M-O is necessary

to contain a sucrase gene, could be tested for complementation of a SUCAmutation in B. subtilis after transfer into the

for stable plasmid maintenance in B. sub?i~j.~ (Bron et al., 1988). In other studies a stabilizing function of this structure has not been detected (Boe et al., 1989). During previous vector constructions two pUBll0 deletion deriva-

respective mutant (unpublished results). These examples demonstrate that fairly large fragments can be cloned into

tives, pRB 165 and pRB 103, had been obtained et al., 1984). On pRB165 the M-U is present,

pRB373. With the promoter probe vector pRB394 promoters were isolated by shotgun cloning DNA from bacilli, staphylo-

may

cocci and lactobacilli using B. subtilis or E. coli as the primary host (unpublished; Gaier, 1991). By using high (up

ative, is lost in 70% of the cells (Table I). Instability is obviously caused by insertion of pBR322 DNA, which may lead to accumulation of hmw DNA and impair growth of

to 50 pg/ml) concentrations

of Cm, strong promoters

(Brilckner whereas it

be deleted on pRB103 (see section a). Both plasmids are stably maintained for at least 60 generations without selection. However, the shuttle pRB373, a pRB 103 deriv-

could

be isolated.

plasmid-harbo~ng hardt and Alonso,

(e) Stability of the vectors The segregational stability of the vectors was tested over a period of 60 generations. As summarized in Table I, the loss of constructs differed markedly. Plasmids cont~ning

Stability of the vectors carrying insertions was also tested. As an example, gene epv (Bruckner et al., 1990) was chosen. Plasmid pHSl consists of eprcloned into pRB373 (Bruckner et al., 1990). On the pRB373-delved pHS2 the epr promoter is deleted reducing protease production about 200-fold. The pRB394 derivative pEC1 contains a 700-bp epr promoter fragment and the pRB381-based pBL5 the epr’-‘ZacZ gene fusion of placB (Bruckner et al., 1990). On pBL6 a frameshift mutation was introduced at the epi‘facZ junction, which prevents expression of the fiGa part of the Epr-PGal fusion protein found in pBL5 containing cells. Four of the plasmids with insertions are less stable than

either the cat(pRB394, pRB395) or ‘lac (pRB381, pRB382) gene were less stable than the basic shuttle vectors (pRB373, pRB374). The larger size (3 kb more) of the ‘Zac plasmids pRB381 and pRB382 may account for their decreased segregational stability. The cat plasmids pRB394 and pRB395 with insertions of only 0.7 kb were least stable. Considering the origin of the cat gene (the Gram + ssDNA plasmid PUB 112), this result is even more surprising.

TABLE

cells (Gruss 1991).

and Ehrlich,

1988; Leon-

1

Stability of the plasmids Plasmid”

in BaciNus strbtilisDB403

Size

Segregational

stabilityb

Structural

(generations)

stability’

(generations)

(kb) 20

40

60

20

40

60

pBR313

5.8

91

78

30

100

100

100

pBR374

5.9

93

15

28

100

100

100

pBR394

6.5

14