Summary
A recombinant cosmid carrying the sucrase gene (sacA) was obtained from a colony bank ofE. coli harboring recombinant cosmids representative of theB. subtilis genome. It was shown that thesacA gene is located in a 2 kbEcoRI fragment and that the cloned sequence is homologous to the corresponding chromosomal DNA fragment. A fragment of 2 kb containing the gene was subcloned in both orientations in the bifunctional vector pHV33 and expression was further looked for inB. subtilis andE. coli. Complementation of asacA mutation was observed in Rec+ and Rec- strains ofB. subtilis. Expression of sucrase was also demonstrated inE.coli, which is normally devoid of this activity, by SDS-polyacrylamide gel electrophoresis, specific immunoprecipitation and assay of the enzyme in crude extracts. The specific activity of the enzyme depended on the orientation of the inserted fragment. The saccharolytic activity was found to be cryptic inE. coli since the presence of the recombinant plasmids did not allow the transport of [U14C] sucrose and the growth of the cells.
It was shown also that the recombinant cosmid contained part of the neighboring locus (sacP) which corresponds to a component of the PEP-dependent phosphotransferase system of sucrose transport ofB. subtilis.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Aubert E, Fargette F, Fouet A, Klier A, Rapoport G (1981) Use of a bifunctional cosmid for cloning large DNA fragments ofBacillus subtilis. In: Hoch J-A, Chang S, Ganesan AT (eds) Molecular cloning and gene expression in Bacilli. Academic Press, New-york, in press
Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucl Acids Res 7:1513–1523
Bolivar F, Backman K (1979) Plasmids ofEscherichia coli as cloning vectors. In: Wu R (ed) Methods in enzymology; vol 68. Academic Press, New York, pp 245–267
Dedonder RA, Lepesant J-A, Lepesant-Kejzlarová J, Billault A, Steinmetz M, Kunst F (1977) Construction of a kit of reference strains for rapid genetic mapping inBacillus subtilis 168. Appl Environ Microbiol 33:989–993
Delobbe A, Haguenauer R, Rapoport G (1979) Studies on the transport of α-methyl-D-glucoside inBacillus subtilis 168. Biochimie 53:1015–1021
Destrée OHJ, Haenni A-L, Birnstiel ML (1971) Histone mRNA inXenopus laevis ovaries: identification of the H4 messenger. Nucl Acids Res 4:801–811
Dubnau D, Davidoff-Abelson R, Scher B, Cirigliano C (1973) Fate of transforming deoxyribonucleic acid after uptake by competentBacillus subtilis: phenotypic characterization of radiation-sensitive recombination-deficient mutants. J Bacteriol 114:273–286
Glatron M-F, Rapoport G (1972) Biosynthesis of the parasporal inclusion ofBacillus thuringiensis: half-life of its corresponding messenger RNA. Biochimie 54:1291–1301
Hohn B, Collins J (1980) A small cosmid for efficient cloning of large DNA fragments. Gene 11:291–298
Klier A, Kunst F, Rapoport G (1979) Structure of cloned ribosomal DNA cistrons fromBacillus thuringiensis. Nucl Acids Res 7:997–1010
Kunst F, Pascal M, Lepesant J-A, Walle J, Dedonder R (1974) Purification and some properties of an endocellular sucrase from a constitutive mutant ofBacillus subtilis Marburg 168. Eur J Biochem 42:611–620
Le Minor L, Coynault C, Rhode R, Rowe B, Aleksic S (1973) Localisation plasmidique du déterminant génétique du caractère atypique “saccharose+” desSalmonella. Ann Microbiol 124:295–306
Lepesant J-A, Kunst F, Lepesant-Kejzlarová J, Dedonder R (1972) Chromosomal location of mutations affecting sucrose metabolism inBacillus subtilis Marburg. Mol Gen Genet 118:135–160
Lepesant J-A, Billault A, Kejzlarová-Lepesant J, Pascal M, Kunst F, Dedonder R (1974) Identification of the structural gene for sucrase inBacillus subtilis Marburg. Biochimie 56:1465–1470
Lepesant J-A, Kunst F, Pascal M, Kejzlarová-Lepesant J, Steinmetz M, Dedonder R (1976) Specific and pleiotropic regulatory mechanisms in the sucrose system ofBacillus subtilis 168. In: Schlessinger D (ed) Microbiology. Am Soc Microbiol, Washington DC, pp 58–69
O'Farrell PH (1975) High resolution two dimensional electrophoresis of proteins. J Biol Chem 250:4007–4021
Primrose SB, Ehrlich SD (1981) Isolation of plasmid deletion mutants and study of their instability. Plasmid 6:193–201
Rambach A, Hogness D (1977) Translation ofDrosophila melanogaster sequences inEscherichia coli. Proc Natl Acad Sci USA 74:5041–5045
Rapoport G, Klier A, Billault A, Fargette F, Dedonder R (1979) Construction of a colony bank ofEscherichia coli containing hybrid plasmids representative of theBacillus subtilis 168 genome. Mol Gen Genet 176:239–245
Rigby PWJ, Diechmann M, Rhodes C, Berg P (1977) Labeling deoxyribonucleic acid to high specific activity in vitro by nick-translation with DNA polymerase I. J Mol Biol 113:237–251
Schmid K, Schupfner M, Schmitt R (1981) Analysis of the plasmid mediated sucrose metabolism inEscherichia coli K12. In: Abst. Lunteren Lectures on Molecular Genetics “Molecular Aspects of Gene Expression in Bacteria and Eukaryotic Cells” (Lunteren, The Netherlands).
Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503–517
Author information
Authors and Affiliations
Additional information
Communicated by A. Böck
Rights and permissions
About this article
Cite this article
Fouet, A., Klier, A. & Rapoport, G. Cloning and expression inEscherichia coli of the sucrase gene fromBacillus subtilis . Molec. Gen. Genet. 186, 399–404 (1982). https://doi.org/10.1007/BF00729460
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00729460