New plasmid vectors for the construction of translational gene fusions in Bacillus subtilis

325

Gene, 65 (1988) 325-328 Elsevier GEN 02389

New plasmid vectors for the construction of translational gene fusions in BaciZZznsubtilis (Gene expression;

P-galactosidase;

E. coli; reporter

gene; plasmid

stability)

Juan C. Alonso Max-Planck-Inns&t fir Molekulare Genetik, Berlin (Germany) Received Revised

30 July 1987 12 January

1988

Accepted

1 February

Received

by publisher

1988 18 February

1988

SUMMARY

A series of plasmids has been constructed that can be used to fuse the IucZ gene of Escherichiu coli to Bacillus subtilis genes, in all three transcriptional reading frames. Gene expression can be monitored, after gene fusion, by measuring /?-galactosidase activity.

INTRODUCTION

The molecular characterization of gene regulation in E. coli has been greatly advanced by the utilization of fusions between the gene of interest and selected ‘reporter’ genes such as IacZ (Casadaban et al., 1980). In B. subtilis, a number of vectors have been constructed that permit fusions, at the transcriptional level, between the E. coli pGal with regulated gene products (Donnelly and Sonenshein, 1982; Zuber and Losick, 1983; Youngman et al.,

Correspondenceto: Dr. J.C. Alonso, Molekulare

Genetik,

Ihnestrasse

Max-Planck-Institut

73, D-1000

Berlin

fur

33 (Ger-

many) Tel. (030)8307274. Abbreviations: bp;

‘2~~2,

neomycin;

/IGal, ,%galactosidase; 5’-truncated

Pm, phleomycin;

0378-l 119/88/$03.50

gene

1ucZ

bp, base pair(s); kb, 1000 coding

for

BGal;

Nm,

a, resistance.

0 1988 Elsevier

Science Publishers

B.V. (Biomedical

1985). Such constructs have been widely used specifically to study the control of sporulation or served as promoter search plasmids (Leonhardt and Alonso, 1988). In post-transcriptional gene regulation, the control is exerted by regulating the initiation of protein synthesis. Protein-mediated translational repression is a well-documented mechanism by which prokaryotes regulate the synthesis of proteins from specific mRNAs (see Campbell et al., 1983, for a review). More recently, translational control by RNA molecules complementary to mRNA has become known (see Green et al., 1986, for a review). B. subtilis represents a useful prokaryotic organism in which to study the post-transcriptional regulation of development and differentiation. To study this type of regulation, translational fusion vectors have been constructed. These vectors as well as some of their uses are described in this communication. Division)

326

EXPERIMENTAL

from agarose gels by electroelution, and joined to the purified 3.5kb fragment of the B. subtilis plasmid pUBll0 (Gryczan et al., 1978) that had been isolated following digestion with EcoRI + PvuII. The ligated molecules were introduced into B. subtilis SB1207 (Viret and Alonso, 1987) via protoplast transformation, as described by Chang and Cohen (1979). Selection of transformants was for either Nm or Pm resistance (5 pg/ml and 0.1 pg/ml, respective-

AND DISCUSSION

(a) Vector construction

To take advantage of the /?GaI fusion system of E. coli for the study of gene regulation in B. subtilis,

the 3.1-kb EcoRI-DruI fragments of DNA from plasmids pNM480, pNM48 1 and pNM482 (Minton, 1984) were isolated. The fragments were recovered

ly). EcoRI GAA TTC ---

pBT141

Fig. 1. Structure

frames.

nucleotides

CGT

Sal1 CGA

Pst1 CCT GCA

GCC

TCC

CGG

GGA

TCC

GTC

GAC

CTG

CAG

CCA

AGC

pBTl43

GA ATT

CCC

GGG

CAT

CCG

TCG

ACC

TGC

AGC

CAA

GCT

ofpUB

sequence

BarnHI GGG ATC

G AAT

T@$m;XXX

10 derived IacZ fusion vectors pBT141, pBT142 and pBT143. The diagram common

the confines

The restriction

nucleotide

CCG

pBTl42

shows the components the pUB 110 marks

Sma1

to all vectors. The thick line represents of the PUB I 10 minireplicon

sites mark

of the polylinker

mark the only differences

either the truncation regions between

(Maciag

et al., 1988). The internal

of the antibiotic

of the three plasmids the three plasmids.

in the lower part (here ofpBT141)

E. co/i DNA, the thin line pUBll0 markers

or the initiation

is shown above. Unique xxx represents

DNA. The shaded area within

open arrows

restriction

represent

replication

protein,

sites are specified.

open reading RepU.

The

The shaded

the eighth codon of the IacZ gene (8th aa of BGal),

327

No activity above the background (plasmid-free cells) was detected in cells containing the vector pBT143 (Table I). More than lOOO-foldhigher level of BGal activity was observed in case of the fused plasmid pBT251, but the synthesis of /IGal activity dropped about 14-fold when synthesis of incA was permitted.

The new vectors obtained (pBT141, pBT142 and pBT143; Fig. 1) differ from each other by the presence of 3-, 4- or 5-bp inserts between the polylinker and the ‘IucZ gene. These differences permit the fusion of cloned genes to the ‘1acZ in all three translational reading frames in B. subtilis. The predicted nucleotide sequences of the polylinker regions contained within the coding region of the ‘1acZ gene are given for each plasmid in Fig. 1. To further enhance the usefulness of these new vectors, we made the transfer system accessible to monomeric plasmid transformation by inserting the PmR gene (Semon et al., 1987) into the chromosome of selected B. subtilis strains. Since these strains now share homology with the PUB 1lo-derived plasmid vectors, they can be used as highly efficient recipients for competent cell transformation of plasmid molecules (plasmid facilitation). No integration of the plasmid into the chromosome occurs (Canosi et al., 1981).

The stability of plasmids pBT141, pBT142 and pBT143 was studied and it was determined that more than 99% of the cells retained antibiotic resistance after approx. 100 generations of growth in non-selective medium. The antibiotic-resistant colonies were shown to still contain plasmids of the correct corresponding M,. Also, no plasmid structural instability was observed after fusing the 5’-terminal region of the pC194 initiation replication protein RepH to BGal.

(b) Expression

(d) Conclusions

of a gene fused to 1acZ

To illustrate the use of the /IGal fusion system, the plasmids pBT141 and pBT143 were used to generate functional translational fusions to the pC194 repH gene (Alonso and Tailor, 1987). Plasmid pC194 specifies a positively trans-acting replication-initiation protein, RepH, and a negatively truns-acting product IncA. By cloning, in frame, the 5’ end of the repH gene to the truncated form of the 1acZ gene, one can analyze RepH regulation by a simple biochemical assay. The promoter and the first eleven codons of the repH gene are contained either in the Hinff-B (nucleotide coordinates 1184-712) fragment, which allows the expression of repH and incA, or in the Hi&I-AccI fragment (nucleotide coordinates 1184-774 of the pC194 map; Horinouchi and Weisblum, 1982) that only allows the expression of repH. The former was cloned into pBT141 and the latter into pBT143, generating plasmids pBT253 and pBT25 1, respectively (see Alonso and Tailor, 1987). By using this approach we have shown that the synthesis of the pC194 replication-initiation protein (RepH) is post-transcriptionally regulated (Alonso and Tailor, 1987). Cells containing either pBT253, pBT251 or the vector plasmid were used for BGal assay in glucose-containing medium.

(c) Plasmid stability

The vectors described here permit the fusion of genes cloned in B. subtilis to the E. coli ’ 1acZ gene in all three translational reading frames. Thus, this translational fusion should greatly facilitate the investigation and characterization of regulatory systems in B. subtilis, a bacterium capable of multiple forms of differentiation and development.

TABLE

I

Synthesis

of b-galactosidase

by the pBT plasmids

BGal units a

Plasmid none

0.240

pBTl43

0.252

pBTl51

327.1

pBT253

23.7

a B. subtih cells SB1207 (Viret and Alonso, or bearing

plasmids

TY medium (Rottllnder exponential

andTrautner,

activity

1970) till the middle ofthe

phase. The cells were centrifuged,

buffer pH 7.0 (Na,HPO,, mM; MgSO,

60 mM; NaH,PO,

1 mM; /I-mercaptoethanol

was assayed

1987), plasmid-free

pBT143, pBT251 or pBT253, were grown in

according

resuspended

in Z

40 mM; KC1 10

50 mM) and the BGal

to Miller (1972).

328 ACKNOWLEDGEMENTS

I thank Dr. W. Messer for providing the plasmids pNM4g0, pNM48 1 and pNM482, Dr. G. Tiraby for the gift of phleomycin, and Dr. N. Sueoka for communication of results prior to publication. The excellent technical assistance of R. Tailor is acknowledged.

REFERENCES Alonso, J.C. and Tailor, R.H.: Plasmid pC194 replication and its control in Ba&&s subtilis. Mol. Gen. Genet. 210 (1987) 476-484.

Canosi, U., Iglesias, A. and Trautner, T.A.: Plasmid transformation in Bacillus subtilts:effects ofinsertion ofBacillus subtilis DNA into pC194. Mol. Gen. Genet. 181 (1981) 434-440. Casadaban, M.J., Chou, J. and Cohen, S.N.: In vitro gene fusions that join an enz~atic~ly active ~-galactosidase segment to amino-terminal fragments of exogenous proteins: Escherichia coli plasmid vectors for the detection and cloning of translational initiation signals. J. Bacterial. 143 (1980) 971-980. Campbell, K.M., Stormo, G.D. and Gold, L.: Protein-mediated translational repression. In Beckwith, J., Davies, J. and Gallant, J.A. (Eds.), Gene Function in Prokaryotes. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1983, pp. 185-210. Chang, A.C.Y. and Cohen, S.N.: High frequency transformation of Bacillus sub&s protoplasts by plasmid DNA. Mol. Gen. Genet. 168 (1979) 11l-l 15. Donnel1y;C.E. and Sonenshein, A.L.: Genetic fusion ofE. cobfat genes to a B. subtib’spromoter. In Ganesan, A., Hoch, J. and Chang, S. (Eds.), Molecular Cloning and Gene Regulation in Bacilli. Academic Press, New York, 1982, pp. 63-72.

Green, P.J., Pines, 0. and Inouye, M.: The role of antisense RNA in gene regulation. Annu. Rev. Biochem. 55 (1986) 569-597. Gryczan, T.J., Contente, S. and Dubnau, D.: Characterization of Staphyl~o~~ aureus plasmids introduced by ~~sfo~ation into Bacillus sub&s. J. Bacterial. 134 (1978) 318-329. Horinouchi, S. and Weisblum, B.: Nucleotide sequence and functional map of pC194, a plasmid that specifies inducible chloramphenicol resistance. J. Bacterial. 150 (1982) 8 15-825. Leonhardt, H. and Alonso, J.C.: Construction of a shuttle vector for inducible gene expression in Esche~~hia coli and Badus subtiks. J. Gen. Microbial. 134 (1988) 605-609. Maciag, I.E., Viret, J.-F. and Alonso, J.C.: Replication and incompatibility properties of plasmid pUBll0 in Bacillus subtilts. Mol. Gen. Genet. (1988) in press. Miller, J.H.: Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1972. Minton, N.: Improved plasmid vectors for the isolation of translational Iac gene fusions. Gene 3 1 (1984) 269-273. Semon, D., Movra, N.R., Smith, T.F., El Alama, N. and Davis, J.: Plasmid-determined bleomycin resistance in Staphylococcus aureus. Plasmid 17 (1987) 46-53. Rottlander, E. and Trautner, T.A.: Genetic and transfection studies with B. ~~~~Z~phage SP50, I. Phage mutants with restricted growth on B. subtilis strain 168. Mol. Gen. Genet. 108 (1970) 47-60. Viret, J.-F. and Alonso, J.C.: A new mutator strain of Bacillus subtilis. Mol. Gen. Genet. 208 (1987) 353-456. Youngman, P., Zuber, P., Perkins, J.B., Sandman, K., Igo, M. and Losick, R.: New ways to study developmental genes in spore-forming bacteria. Science 228 (1985) 285-291. Zuber, P. and Losick, R.: Use of a 1acZ fusion to study the role of the spo0 genes of Bacillus subhks in developmental regulation. Cell 35 (1983) 275-283. Communicated by R.E. Yasbin.