Abstract
A system is described that enables the cloning of genes specifying detrimental proteins inEscherichia coli. The system is based on pUC plasmids and was developed for the expression of theBacillus subtilis csaA gene, which is lethal when expressed at high levels. Suppressor strains that tolerate the presence of plasmids for high-level expression ofcsaA were isolated, which contained small cryptic deletion variants of the parental plasmid in high copy numbers. The cryptic plasmids consisted mainly of the pUC replication functions and lacked thecsaA region and selectable markers. The co-resident, incompatible, cryptic plasmids enabled the maintenance of thecsaA plasmids by reducing their copy number 20-fold, which resulted in a concomitant 3- to 7-fold reduction in the expression of plasmid-encoded genes. Strains carrying these cryptic endogenous plasmids proved to be useful for the construction of pUC-based recombinant plasmids carrying other genes, such as theskc gene ofStreptococcus equisimilis, which cannot be cloned in high copy numbers inE. coli. Several strategies to reduce production levels of heterologous proteins specified by plasmids are compared.
This is a preview of subscription content, log in via an institution to check access.
References
Bron S (1990) Plasmids. In: Harwood CR, Cutting SM (eds) Molecular biological methods forBacillus. Wiley and Sons, Chichester, pp 75–174
Bron S, Meijer W, Holsappel S, Haima P (1991) Plasmid instability and molecular cloning inBacillus subtilis. Res Microbiol 142:875–883
Carter P, Bedouelle U, Winter G (1985) Improved oligonucleotide site-directed mutagenesis using M13 vectors. Nucleic Acids Res 13:4431–4443
Casadaban MJ (1976) Transposition and fusion of thelac genes to selected promoters inEscherichia coli using bacteriophage Lambda and Mu. J Mol Biol 104:541–555
Glaser P, Kunst F, Arnaud M, Coudart M-P, Gonzales W, Hullo M-F, Ionescu M, Lubochinsky B, Marcelino L, Moszer I, Presecan E, Santana M, Schneider E, Schweizer J, Vertes A, Rapoport G, Danchin A (1993)Bacillus subtilis genome project: cloning and sequencing of the 97 kb region from 325° to 333°. Mol Microbiol 10:371–384
Gruss A, Ehrlich D (1989) the family of highly interrelated single-stranded deoxyribonucleic acid plasmids. Microbiol. Rev 53:231–241
Lopilato J, Bortner S, Beckwith J (1986) Mutations in a new chromosomal gene ofEscherichia coli K-12,pcnB, reduce plasmid copy number of pBR322 and its derivatives. Mol Gen Genet 205:285–290
Martinez E, Bartolomé B, de la Cruz F (1988) pACYC184-derived cloning vectors containing the multiple cloning site andlacZα reporter gene of pUC8/9 and pUC18/19 plasmids. Gene 68:159–162
Müller J, Reinert H, Malke H (1989) Streptokinase mutations relievingEscherichia coli K-12(prlA4) of detriments caused by the wild-typeskc gene. J Bacteriol 171:2202–2208
Müller J, Walter F, van Dijl JM, Behnke D (1992) Suppression of the growth and export defects of anEscherichia coli secA(Ts) mutant by a gene cloned fromBacillus subtilis. Mol Gen Genet 235:89–96
Novick RP (1987) Plasmid incompatibility. Microbiol Rev 51:381–395
Schauder B, Blöcker H, Frank R, McCarthy JEG (1987) Inducible expression vectors incorporating theEscherichia coli atpE translational initiation region. Gene 52:279–283
Takashita S, Sato M, Toba M, Masahashi W, Hashimot-Goto T (1987) High-copy-number and low-copy-number plasmid vectors forlacZα complementation and chloramphenicol or kanamycin resistance. Gene 61:63–74
Trieu-Cuot P, Courvalin P (1983) Nucleotide sequence of theStreptococcus faecalis plasmid gene encoding the 3′5″-aminoglycoside phosphotransferase type III. Gene 23:331–341
van Dijl JM, Smith H, Bron S, Venema G (1988) Synthesis and processing ofEscherichia coli TEM-β-lactamase andBacillus licheniformis α-amylase inE. coli: The role of signal peptidase I. Mol Gen Genet 214:55–61
Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103–119
Author information
Authors and Affiliations
Additional information
Communicated by W. Goebel
Rights and permissions
About this article
Cite this article
Müller, J., van Dijl, J.M., Venema, G. et al. Cloning of heterologous genes specifying detrimental proteins on pUC-derived plasmids inEscherichia coli . Molec. Gen. Genet. 252, 207–211 (1996). https://doi.org/10.1007/BF02173221
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF02173221