Kok, Jan

van der Vossen, Josephus

Venema, Gerhardus

English

Shuttle vectors have been constructed which are suitable both for the selection of regulatory sequences and for gene cloning in Bacillus subtilis and Streptococcus lactis. The promoter screening vectors contain a promoterless chloramphenicol acetyltransferase gene; the insertion of suitable DNA fragments upstream of the gene restored the enzyme activity. With a related set of vectors, transcription termination signals can be selected

Dissertations of the University of Groningen

   University of GroningenConstruction of Cloning, Promoter-Screening, and Terminator-Screening Shuttle Vectors forBacillus subtilis and Streptococcus lactisvan der Vossen, Josephus; Kok, Jan; Venema, GerhardusPublished in:Applied and environmental microbiologyIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:1985Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):van der Vossen, J., Kok, J., & Venema, G. (1985). Construction of Cloning, Promoter-Screening, andTerminator-Screening Shuttle Vectors for Bacillus subtilis and Streptococcus lactis. Applied andenvironmental microbiology, 50(2), 540-542.CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Vol. 50, No. 2APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1985, p. 540-5420099-2240/85/080540-03$02.00/0Copyright C) 1985, American Society for MicrobiologyConstruction of Cloning, Promoter-Screening, andTerminator-Screening Shuttle Vectors for Bacillus subtilis andStreptococcus lactisJOS M. B. M. VAN DER VOSSEN,* JAN KOK, AND GERARD VENEMADepartment of Genetics, University of Groningen, 9751 NN Haren, The NetherlandsReceived 22 January 1985/Accepted 20 May 1985Shuttle vectors have been constructed which are suitable both for the selection of regulatory sequences andfor gene cloning in Bacillus subtilis and Streptococcus lactis. The promoter screening vectors contain apromoterless chloramphenicol acetyltransferase gene; the insertion of suitable DNA fragments upstream of thegene restored the enzyme activity. With a related set of vectors, transcription termination signals can beselected.Recently, we have described the construction of a set ofcloning vectors for lactic streptococci (9). The vectors usedin this system also replicate in Bacillus subtilis and Esche-richia coli. However, screening of regulatory sequences forgene expression with these vectors was not possible. In viewof the development of plasmid vectors which would allowthe proper expression of heterologous genes in lactic strep-tococci and to obtain information concerning the structure oflactic streptococcal promoters, the availability of vectors forscreening promoter and transcription termination sequencesis desirable.In this paper we describe the construction of a shuttlevector family which allows the isolation of promoter andtranscription termination signals in lactic streptococci. Themethod of detecting promoter activity in this system isanalogous to that described for screening promoter activityin B. subtilis by means of pPL603 (23).The starting material for the construction of the screeningvectors were (i) pGK3, which contains the largest ClaIfragment of the cryptic Streptococcus cremoris Wg2 plasmidpWVO1 and the erythromycin resistance (Em9 gene on aClaI-HpaII fragment of pE194 cop-6 (22) and (ii) the B.subtilis vector pPL608 (23) carrying the chloramphenicolacetyltransferase (CAT) gene from B. pumilus (Fig. 1).pGK3 and pPL608 were isolated from B. subtilis 8G-5 (2)after the cells were grown in TY broth (18) with 5 ,ug ofkanamycin per ml by the method of Ish-Horowicz and Burke(8) with some modifications (9). Restriction enzymes wereused as recommended by the manufacturer. Digested DNAwas analyzed in 0.8% agarose gels (11). pPL608 was com-pletely digested with PvuII and partially with EcoRI restric-tion endonucleases. The largest PvuII-EcoRI fragment, con-taining the SPO2 promoter and the CAT gene, was isolatedby electroelution from the agarose gel. After filling in theClaI-linearized pGK3 with the Klenow fragment of E. coliDNA polymerase I and the EcoRI cohesive end of thePvuII-EcoRI fragment mentioned above, these moleculeswere ligated. Subsequently, protoplasts of B. subtilis PSL1(16) were exposed to the ligation mixture by the method ofChang and Cohen (3), and Emr chloramphenicol-resistant(Cmr) transformants were selected. Two types of transform-ants were obtained, one carrying plasmid pGKV1 (Fig. 1)and the other carrying pGKV2 (data not shown). The differ-* Corresponding author.ence between pGKV1 and pGKV2 concerns the orientationof the CAT gene. Both vectors transformed protoplasts of S.lactis to Emr (5 ,ug/ml) and Cmr (4 ,uglml) at a frequency ofapproximately 103 transformants per ,ug of DNA. The S.lactis protoplasts were prepared as described by Okamoto etal. (15), except that protoplasts were made by incubating thecells for 1 h in 30 mM Tris-hydrochloride buffer (pH 8.0)-3mM MgClr-25% sucrose-30 ,ug of lysozyme per ml. Trans-formation was performed by the method of Kondo andMcKay (10) with some modifications (9).Since fusion of the filled in recessed ends of the ClaI andEcoRI sites restored the EcoRI site, the SPO2 promoter wasdeleted in vitro by EcoRI digestion, resulting in pGKV10(Fig. 1) and pGKV20 with opposite orientations of the CATgene (data not shown). Both plasmids were transformed toB. subtilis PSL1 and gave rise to Emr Cms colonies. TheCAT gene was not expressed in either pGKV10 or pGKV20,indicating that no sequence on the pWVO1 part of the vectorpromoted the expression of the gene, or that the CAT genewas expressed along with the Emr gene under the Emrpromoter. This is in accord with the presence of a termina-tion signal downstream of the Emr gene (7).Although the two vectors pGKV10 and pGKV20 can beused to select for promoter activity on DNA fragmentsgenerated by EcoRI, more versatile derivates were made byreplacing the EcoRI-PstI fragment by a multiple cloning siteof double-stranded M13mpll (13), resulting in pGKV110(Fig. 1) and pGKV210 with opposite orientations of the CATgene (data not shown), which carries unique EcoRI, SmaI,XmaI, BamHI, SalI, and PstI sites upstream of and adjacentto the CAT gene. Transformation of S. lactis protoplastswith pGKV110 and pGKV210 gave rise to Emr Cm, trans-formants. The observations that pGKV1 and pGKV2 trans-formed B. subtilis and S. lactis cells to Cmr and thatpGKV110 and pGKV210 failed to do so indicate that theCAT gene is expressed in S. lactis under the control of the B.subtilis phage promoter. To examine whether promotersequences could be selected with the aid of the vectorpGKV110, MboI fragments of B. subtilis OG-1 (2) and S.cremoris Wg2 (17) DNA were cloned into the unique BamHIsite of pGKV110. Protoplasts of B. subtilis PSL1 wereexposed to the ligation mixture, and transformants wereselected on DM3 plates (3) with 5 jig of erythromycin and 5,ug of chloramphenicol per ml. Promoter selection efficiencyin B. subtilis was 500 to 1,000 Cmr transformants per jig of540TABLE 1. Cmr properties of pGKV1 and derivative plasmids"CAT activity Cmr CAT activity CmrPlasmid Source of insert (mU/mg of protein) (,ug/ml) for (mU/mg of protein) (4g/mi)for B. subtilis B. subtilis for S. lactis for S. lactispGKV110 None 0.1 <5 0 <3pGKV1 EcoRI of SPO2 2.5 30 0.1 4pGKV111 MboI of B. subtilis 0.5 30 0 <3pGKV112 MboI of B. subtilis 2.1 30 0 <3pGKV113 MboI of B. subtilis 2.0 30 0 <3pGKV114 MboI of B. subtilis 0.6 10 0 <3pGKV143 MboI of S. cremoris 0.8 30 0 <3pGKV144 MboI of S. cremoris 4.0 30 0.07 4pGKV145 MboI of S. cremoris 3.6 30 0.003 4pGKV146 Mbol of S. cremoris 0.9 30 0.002 4a CAT activity was assayed in cell extracts grown in TY (B. subtilis) or M17 (S. lactis) after induction of the CAT gene with 4 pg of chloramphenicol per mlwhen the cells were in the mid-logarithmic phase. Protein was measured by the method of Bradford (1). The maximum Cmr is given in micrograms per milliliter forB. subtilis and was determined as described by Williams et al. (23) in TY containing 0, 5. 10, 30, and 100 ,ug of chloramphenicol per ml. Resistance in S. lactis wasdetermined by plating a 105-diluted culture on M17 plates with 0, 1, 2, 3. and 4 ,ug of chloramphenicol per ml, respectively.DNA, irrespective of the source of the DNA. Direct trans-formation of the ligation mixture to S. lactis MG1363 (5)proved to be unsuccessful. However, it was possible totransform S. lactis protoplasts to Emr with plasmids derivedfrom the Emr Cmr B. subtilis transformants. The results inTable 1 show that none of the B. subtilis promoters wereactive in S. lactis, with the exception of the SPO2 promoter.Table 1 also shows that S. cremoris promoters were active inB. subtilis and that the majority of these promoters alsofunctioned in S. lactis, although at a substantially reducedlevel. Whether the difference in promoter activity betweenthe two bacterial species is caused by intrinsic differences inthe structure of the promoters or is attributable to differentstability of the mRNA or the gene product remains to beestablished.Reinserting the SP02 promoter on a 0.18-megadalton (Md)EcoRI fragment into the EcoRI site of pGKV110 andpGKV210 restored the CAT activity; B. subtilis cells har-boring both plasmids pGKV11 (Fig. 1) and pGKV21 withopposite orientations of the CAT gene (data not shown) wereCmr. Since the BamHI site of the multiple cloning site onplasmid pGKV11 is located between the promoter and theribosomal binding site of the CAT gene (4, 6, 19), it may beexpected that the insertion of DNA fragments carrying atranscription termination sequence in this site will abolishCAT expression and therefore that pGKV11 may be used asa transcription termination selection vector. To test this,MboI fragments of pPLP1 were inserted in the BamHI site ofplasmid pGKV11. pPLP1 is a recombinant plasmid, con-structed in our laboratory (J. van Randen, unpublishedresults), carrying the penicillinase gene of B. licheniformison a AhallI fragment (14) inserted into the HindlIl site ofpPL608. The transcription termination signal of the penicil-linase gene on this plasmid is located on a 0.4-Md MboIfragment. Among the Emr B. subtilis transformants, Cmscolonies were isolated with a frequency of 5%. Thesecolonies either contained a plasmid consisting of pGKV11with a 0.4-Md insert (pGKV12) or a plasmid consisting ofpGKV11 carrying a 0.7-Md insert (pGKV13). Restrictionenzyme analysis and Southern hybridization (data notshown) indicate that pGKV12 contained the 0.4-Md MboIfragment harboring the transcription termination signal ofthe penicillinase gene, which is a complementary repeatfollowed by oligodeoxythymidylate (14). Transformation ofS. lactis protoplasts with the recombinant plasmid pGKV12gave rise to Emr Cm' transformants, indicating that a Bacil-lus transcription termination signal also functioned in S.lactis. The sequence of the 0.7-Md insert in pGKV13 is onlypartially known (12). The lack of CAT expression suggeststhe presence of a transcription terminator in the unknownsequence.Some of the vectors constructed can also be used asregular cloning vehicles. This applies to the vectors pGKV1and pGKV2, carrying unique HindlIl and HpaII sites in theCAT gene, and to pGKV11 and pGKV21, carrying a uniqueHindIlI site in the CAT gene.r-t1 * 2EmnRpGK3 1.9MdO & XOm 1 2-' -* 3KeMR ori SP02 CATprom~~~~.....O 1 -'2 4- -EmR CAT SPO2I promo o -X 2 _ - 3lxO 1 --'2 *- 3EmR CAT0~~~~~O 1 -'2 4- 43.EmR CAT2 pEmR CAT K0arompPL608 3.3MdpGKVI 3.1 MdpGKV10 2.9MdpGKV1lO 2.9MdpGKV11 3.1 MdpWVOi pPL608 'Y ClaI/Pvu I fusion sitepEl9d. cop-6 dsM13mp1lFIG. 1. Vector construction strategy and restriction endonucle-ase maps of PGK3, pPL608, pGKV1, pGKV10, pGKV110, andpGKV11. The origin of the plasmid segments is indicated in the key.Only relevant restriction sites for construction and use of therecombinant vectors are given. Unique restriction sites of themultiple cloning site derived from double-stranded M13mpll areEcoRl, SmaI, XmaI, BamHI, Sall, and PstI, in the order given. Fordetails, see text.VOL. 50, 1985 NOTES 541.L0:0(LAPPL. ENVIRON. MICROBIOL.This work was supported by the Biomolucular Engineering Pro-gram of the Commission of the European Communities.We thank Henk Mulder for preparing the figure.LITERATURE CITED1. Bradford, M. M. 1976. A rapid and sensitive method for thequantitation of microgram quantities of protein utilizing theprinciple of protein dye binding. Anal. Biochem. 72:248-254.2. Bron, S., and G. Venema. 1972. Ultraviolet inactivation andexcision-repair in Bacillus subtilis. I. Construction of a trans-formable eight-fold auxotrophic strain and two ultraviolet-sensitive derivatives. Mutat. Res. 15:1-10.3. Chang, S., and S. N. Cohen. 1979. High frequency transforma-tion of Bacillus subtilis protoplasts by plasmid DNA. Mol. Gen.Genet. 168:111-115.4. Duvall, E. J., D. M. Williams, P. S. Lovett, C. Rudolph, N.Vasantha, and M. Guyer. 1983. Chloramphenicol-inducible geneexpression in Bacillus subtilis. Gene 24:171-177.5. Gasson, M. J. 1983. Plasmid complements of Streptococcuslactis NCDO 712 and other lactic streptococci after protoplast-induced curing. J. Bacteriol. 154:1-9.6. Harwood, C. R., D. M. Williams, and P. S. Lovett. 1983.Nucleotide sequence of a Bacillus pumilus gene specifyingchloramphenicol acetyltransferase. Gene 24:163-169.7. Horinouchi, S., and B. Weisblum. 1980. Posttranscriptionalmodification of mRNA conformation: mechanism that regulateserythromycin-Induced resistance. Proc. Natl. Acad. Sci.U.S.A. 77:7079-7083.8. Ish-Horowicz, D., and J. F. Burke. 1981. Rapid and efficientcosmid cloning. Nucleic Acids Res. 9:2989-2999.9. Kok, J., J. M. B. M. van der Vossen, and G. Venema. 1984.Construction of plasmid cloning vectors for lactic streptococciwhich also replicate in Bacillus subtilis and Escherichia coli.Appl. Environ. Microbiol. 48:726-731.10. Kondo, J. K., and L. L. McKay. 1982. Transformation ofStreptococcus lactis protoplasts by plasmid DNA. Appl.Environ. Microbiol. 43:1213-1215.11. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecularcloning: a laboratory manual. Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.12. Matsumura, M., Y. Katakura, T. Imanaka, and S. Aiba. 1984.Enzymatic and nucleotide sequence studies of a kanamycin-inactivating enzyme encoded by a plasmid from thermophilicbacilli in comparison with that encoded by plasmid pUB110. J.Bacteriol. 160:413-420.13. Messing, J. 1983. New M13 vectors for cloning. MethodsEnzymol. 101:20-78.14. Neugebauer, K., R. Sprengel, and H. Schaller. 1981. Penicil-linase from Bacillus licheniformis: nucleotide sequence of thegene and implications for the biosynthesis of secretory proteinin Gram-positive bacterium. Nucleic Acids Res. 9:2577-2588.15. Okamoto, T., Y. Fujita, and R. Irie. 1983. Protoplast formationand regeneration of Streptococcus lactis cells. Agric. Biol.Chem. 47:259-263.16. Ostroff, G. R., and J. J. Pene. 1983. Molecular cloning withbifunctional plasmid vectors in Bacillus subtilis: isolation of aspontaneous mutant of Bacillus subtilis with enhancedtransformability for Escherichia coli-propagated chimeric plas-mid DNA. J. Bacteriol. 156:934-936.17. Otto, R., W. M. de Vos, and J. Gavrieli. 1982. Plasmid DNA inStreptococcus cremoris Wg2: influence of pH on selection inchemostats of a variant lacking a protease plasmid. Appl.Environ. Microbiol. 43:1272-1277.18. Rottlander, E., and T. A. Trautner. 1970. Genetic and transfec-tion studies with Bacillus subtilis phage SP50. J. Mol. Biol.108:47-60.19. Schoner, R. G., D. M. Williams, and P. S. Lovett. 1983.Enhanced expression of mouse dihydrofolate reductase in Ba-cillus subtilis; Gene 22:47-57.20. Shaw, W. V. 1975. Chloramphenicol acetyltransferase fromchloramphenicol-resistant bacteria. Methods Enzymol. 43:737-755.21. Southern, E. M. 1975. Detection of specific sequences amongDNA fragments separated by gel electrophoresis. J. Mol. Biol.98:503-517.22. Weisblum, B., M. Y. Graham, T. Gryczan, and D. Dubnau.1979. Plasmid copy number control: isolation and characteriza-tion of high-copy-number mutants of plasmid pE194. J. Bacte-riol. 137:635-643.23. Williams, D. M., E. J. Duvall, and P. S. Lovett. 1981. Cloningrestriction fragments that promote expression of a gene inBacillus subtilis. J. Bacteriol. 146:1162-1165.542 NOTES

Construction of Cloning, Promoter-Screening, and Terminator-Screening Shuttle Vectors for Bacillus subtilis and Streptococcus lactis

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Construction of Cloning, Promoter-Screening, and Terminator-Screening Shuttle Vectors for Bacillus subtilis and Streptococcus lactis

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