Abstract
Escherichia coli strain HB101 harboring an expression plasmid bearing calf prochymosin gene under the control of thetac promoter was grown in the presence of IPTG with or without novobiocin at 28 and 40 °C, respectively. The differential rates of synthesis of prochymosin inclusion, and, for comparison, of β-lactamase and β-galactosidase, as well as plasmid copy number, were determined during the first hours of steady state growth. At 28 °C the induced expression of prochymosin gene was almost blocked. Addition of novobiocin did not alleviate this effect. In fact, it strengthtened it, and we conclude that both these additive inhibitory effects are a consequence of the decrease in negative superhelical tension of plasmid DNA to an insufficient level. At 40 °C the differential rate of prochymosin synthesis was markedly enhanced. Since the copy number of the expression plasmid increased approximately to the same extent, we conclude that an increase in gene dose is the cause. The stimulation of cloned heterologous gene expression at 40 °C and inhibition at 28 °C may be conveniently used in biotechnological-scale cultivations of some recombinant bacteria.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Battistoni A., Carri M.T., Mazzetti A.P., Potilio G.: Temperature-dependent protein foldingin vivo—lower growth temperature increases yield of two genetic variants ofXenopus laevis Cu,Zn-superoxide dismutase inEscherichia coli.Biochem. Biophys. Res. Comm. 186, 1339–1344 (1992).
Bertrand-Burggraf E., Oertel P., Schnarr M., Daune M., Granger-Schnarr M.: Effect of induction of SOS response on expression of pBR322 genes and on plasmid copy number.Plasmid 22, 163–168 (1989).
Čejka K., Holubová I., Hubáček J.: Curing effect of chlorobiocin onEscherichia coli plasmids.Mol. Gen. Genet. 186, 153–155 (1982).
Chalmers J.J., Kim E., Telford J.N., Wong E.Y., Tacon W.C., Shuler M.L., Wilson D.B.: Effects of temperature onEscherichia coli overproducing β-lactamase or human epidermal growth factor.Appl. Environ. Microbiol. 56, 104–111 (1990).
Crosa J.H., Falkow S.: Plamids, pp. 267–282 in P. Gerhardt (Ed.):Manual of Methods of General Bacteriology. American Society for Microbiology, Washington (DC) 1981.
Dobrogosz W.J.: Enzymatic activity, p. 365–392 in P. Gerhardt (Ed.):Manual of Methods for General Bacteriology. American Society for Microbiology, Washington (DC) 1981.
Drlica K.: Biology of bacterial deoxyribonucleic acid topoisomerases.Microbiol. Rev. 48, 273–289 (1984).
Drlica K.: Bacterial topisomerases and the control of DNA supercoiling.Trends Genet. 6, 432–437 (1990).
Engberg B., Nordström K.: Replication of R-factor R1 inEscherichia coli K-12 at different growth rates.J. Bacteriol. 123, 179–186 (1975).
Gellert M.: DNA topisomerases.Ann. Rev. Biochem. 50, 879–910 (1981).
Goldstein E., Drlica K.: Regulation of bacterial DNA supercoiling: Plasmid linking numbers vary with growth temperature.Proc. Nat. Acad. Sci. USA 81, 4046–4050 (1984).
Hooper D.C., Wolfson J.S., McHugh G.L., Swartz M.D., Tung C., Swartz M.M.: Elimination of plasmid pMG110 fromEscherichia coli by novobiocin and other inhibitors of DNA gyrase.Antimicrob. Agents Chemother. 25, 586–590 (1984).
Jones P.G., Krah R., Tafuri S.R., Wolffe A.P.: DNA gyrase, C57.4, and the cold shock response inEscherichia coli.J. Bacteriol. 174, 5798–5802 (1992).
Kaprálek F., Ječmen P., Sedláček J., Fábry M., Zadražil S.: Fermentation conditions for high-level expression of thetac-promoter-controlled calf prochymosin cDNA inEscherichia coli HB101.Biotech. Bioeng. 37, 71–79 (1991).
McHugh G.L., Swartz M.N.: Elimination of plasmids from several bacterial species by novobiocin.Antimicrob. Agents Chemother. 12, 423–426 (1997).
Mizukami T., Komatsu Y., Hosoi N., Itoh S., Oka T.: Production of active human interferon-β inE. coli. I. Preferential production by lover culture temperature.Biotechnol. Lett. 8, 605–610 (1986).
Piatak M., Lane J.A., Laird W., Bjorn M.J., Wang A., Williams M.: Expression of soluble and fully functional ricin A chain inEscherichia coli is temperature-sensitive.J. Biol. Chem. 263, 4837–4843 (1988).
Schein C.H., Noteborn M.H.M.: Formation of soluble recombinant proteins inEscherichia coli is favored by lower growth temperature.Bio/Technology 6, 291–29413 (1988).
Seo J.-H., Bailey J.E.: Effects of recombinant plasmid content on growth properties and cloned gene product formation inEscherichia coli.Biotech. Bioeng. 27, 1668–1674 (1985).
Son K.H., Jang J.H., Kim J.H.: Effect of temperature on plasmid stability and expression of cloned cellulase gene in a recombinantBacillus megaterium.Biotechnol. Lett. 9, 821–824 (1987).
Spangler R., Zhang S., Krueger J., Zubay G.: Colicin synthesis and cell death.J. Bacteriol. 163, 167–173 (1985).
Sykes R.B., Nordström K.: Microiodometric determination of β-lactamase activity.Antimicrob. Agents Chemother. 1, 94–99 (1972).
Taylor D.E., Brose E.C.: Modified Birnboim-Doly method for rapid detection of plasmid copy number.Nucl. Acids Res. 16, 9056 (1988).
Uhlin B.E., Nordström K.: R plasmid gene dosage effects inEscherichia coli K-12: copy mutants of the R plasmid R1drd-19.Plasmid 1, 1–7 (1977).
Uhlin B.E., Nordström K.: Preferential inhibition of plasmid replicationin vivo by altered DNA gyrase activity inEscherichia coli.J. Bacteriol. 162, 855–857 (1985).
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Kaprálek, F., Tichý, P.J., Fábry, M. et al. Effects of temperature and novobiocin on the expression of calf prochymosin gene and on plasmid copy number in recombinantEscherichia coli . Folia Microbiol 43, 63–67 (1998). https://doi.org/10.1007/BF02815545
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF02815545