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Flow cytometry studies of recombinant Escherichia coli in batch and continuous cultures: DNA and RNA contents; light-scatter parameters

  • Applied Genetics and Regulation
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Abstract

Flow cytometry has been used to study the contents of macromolecular compounds and light-scatter parameters in batch and continuous cultures of a recombinant Escherichia coli strain that forms protein inclusion bodies. Changes in relative DNA and RNA contents and cell mass as estimated by forward-angle light scatter were detected and tightly correlated in batch culture. In addition, heterogeneity of wide-angle light scatter (WALS), which we related to the presence of cellular inclusion bodies, was observed. In contrast, the relative RNA content and cell mass did not change during continuous culture, and homogeneity of WALS was found. In addition, unexpected changes in relative DNA content were observed after 67 h of culture, indicating a change in bacterial physiology.

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References

  • Bailey J (1993) Host-vector interactions in Escherichia coli. Adv Biochem Eng Biotechnol 48:29–52

    Google Scholar 

  • Barbotin J, Sayadi S, Nasri M, Berry F, Thomas D (1990) Improvement of plasmid stability by immobilization of recombinant microorganisms. Ann NY Acad Sci 589:41–53

    Google Scholar 

  • Boye E, Steen H, Skarstad K (1983) Flow cytometry of bacteria: a promising tool in experimental and clinical microbiology. J Gen Microbiol 129:973–980

    Google Scholar 

  • Brownlie L, Stephenson JR, Cole JA (1990) Effect of growth rate on plasmid maintenance by Escherichia coli HB101 (pAT153). J Gen Microbiol 136:2471–2480

    Google Scholar 

  • De Taxis du Poët P, Dhulster P, Barbotin J, Thomas D (1986) Plasmid inheritability and biomass production: comparison between free and immobilized cell cultures of Escherichia coli BZ18/pTG201, without selection pressure. J Bacteriol 165:871–877

    Google Scholar 

  • Doran P, Bailey J (1986a) Effects of hydroxyurea on immobilized and suspended yeast fermentation rates and cell cycle operation. Biotechnol Bioeng 28:1814–1831

    Google Scholar 

  • Doran P, Bailey J (1986b) Effects of immobilization on growth, fermentation properties, and macromolecular composition of Saccharomyces cerevisiae attached to gelatin. Biotechnol Bioeng 28:73–87

    Google Scholar 

  • Fouchet P, Jayat C, Hechard Y, Ratinaud MH, Frelat G (1993) Recent advances of flow cytometry in fundamental and applied microbiology. Biol Cell 78:95–109

    Google Scholar 

  • Frelat G, Laplace-Builhe C, Grunwald D (1989) Microbiol analysis by flow cytometry: present and future. In: Yen A (ed) Flow cytometry. Advanced research and clinical applications. CRC Press, Boca Raton, Fla., pp 255–279

    Google Scholar 

  • Georgiou G, Telford J, Shuler M, Wilson D (1986) Localization of inclusion bodies in Escherichia coli overproducing β-lactamase or alkaline phosphatase. Appl Environ Microbiol 52:1157–1161

    Google Scholar 

  • Ingraham J, Maaloe O, Neidhart F (1983) Growth of the bacterial cell. Sinauer Associates, Sunderland, Mass

    Google Scholar 

  • Kell D, Ryder H, Kaprelyants A, Westerhoff H (1991) Quantifying heterogeneity: flow cytometry of bacterial cultures. Antonie van Leeuwenhoek 60:145–158

    Google Scholar 

  • Koizumi JI, Aiba S (1989) Oscillatory behavior of population density in continuous culture of genetic-engineered Bacillus stearothermophilus. Biotechnol Bioeng 34:750–754

    Google Scholar 

  • Kramer J, Singleton F (1992) Variations in rRNA content of marine Vibrio spp. during starvation-survival and recovery. Appl Environ Microbiol 58:201–207

    Google Scholar 

  • Kumar P, Schügerl K (1990) Immobilization of genetically engineered cells: a new strategy for higher stability. J Biotechnol 14:255–272

    Google Scholar 

  • Locher G, Sonnleitner B, Fiechter A (1992) On-line measurement in biotechnology: techniques. J Biotechnol 25:23–53

    Article  CAS  PubMed  Google Scholar 

  • Maaloe O (1979) Regulation of the protein synthesizing machinery-ribosomes, tRNA, factors and so on. In: Goldberger RF (ed) Biological regulation and development. Plenum Press, New York, pp 487–542

    Google Scholar 

  • Manin C, Barbotin J, Thomas D, Lazzaroni J, Portalier R (1989) Production of alkaline phosphatase by immobilized growing cells of Escherichia coli excretory mutants. Appl Microbiol Biotechnol 32:143–147

    Google Scholar 

  • Primrose S, Derbyshire P, Jones I, Robinson A, Ellwood D (1984) The application of continuous culture to the study of plasmid stability. Biotech Med Environ 14:213–328

    Google Scholar 

  • Scheper T, Hitzmann B, Rinas U, Schügerl K (1987) Flow cytometry of Escherichia coli for process monitoring. J Biotechnol 5:139–148

    Google Scholar 

  • Schoner R, Ellis L, Schoner B (1985) Isolation and purification of protein granules from Escherichia coli cells overproducing bovine growth hormone. Bio/Technology 3:151–154

    Google Scholar 

  • Srienc F, Arnold B, Bailey J (1984) Characterization of intracellular accumulation of polyhydroxybutyrate in individual cells of Alcaligenes eutrophus H16 by flow cytometry. Biotechnol Bioeng 26:982

    Google Scholar 

  • Stephens ML, Lyberatos G (1988) Effect of cycling on the stability of plasmid-bearing microorganisms in continuous culture. Biotechnol Bioeng 31:464–469

    Google Scholar 

  • Wanner U, Egli T (1990) Dynamics of microbial growth and cell composition in batch culture. FEMS Microbiol Rev 75:19–44

    Google Scholar 

  • Wittrup K, Mann M, Fenton D, Tsai L, Bailey J (1988) Single-cell light scatter as a probe of refractile body formation in recombinant Escherichia coli. Bio/technology 6:423–426

    CAS  Google Scholar 

  • Wood T, Peretti S (1990) Depression of protein synthetic capacity due to cloned-gene expression in Escherichia coli. Biotechnol Bioeng 36:865–878

    Google Scholar 

  • Zabriskie D, Arcui E (1986) Factors influencing productivity of fermentations using recombinant microorganisms. Enzyme Microb Technol 8:706–717

    Google Scholar 

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Fouchet, P., Manin, C., Richard, H. et al. Flow cytometry studies of recombinant Escherichia coli in batch and continuous cultures: DNA and RNA contents; light-scatter parameters. Appl Microbiol Biotechnol 41, 584–590 (1994). https://doi.org/10.1007/BF00178494

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  • DOI: https://doi.org/10.1007/BF00178494

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