1887

Abstract

Use of green fluorescent protein (GFP) as a molecular reporter is restricted by several environmental factors, such as its requirement for oxygen in the development of the fluorophore, and its poor fluorescence at low pH. There are conflicting data on these limitations, however, and systematic studies to assess the importance of these factors for growing bacterial cultures are lacking. In the present study, homogeneous expression of the mut3* gene directed by a synthetic constitutive lactococcal promoter was demonstrated in batch cultures and in biofilms of DL1. A lower limit of oxygen concentration for maturation of the GFP fluorophore was determined: fluorescence was emitted at 01 p.p.m. dissolved oxygen (in conventionally prepared anaerobic media lacking reducing agents), whereas no fluorescence was detected in the presence of 0025 p.p.m. dissolved oxygen (obtained by addition of L-cysteine as reducing agent). When an anaerobically grown (non-fluorescent) >50 μm thick biofilm was shifted to aerobic conditions, fluorescence could be detected within 4 min, reaching a maximum over the next 16 min. It was not possible to detect any fluorescence gradients (lateral or vertical) within the >50 μm thick biofilm, and fluorescence development after the shift to aerobic conditions occurred throughout the biofilm (even at the substratum). This suggests that oxygen gradients, which might result in reduced GFP fluorescence, did not exist in the >50 μm thick biofilm of this organism. Production of lactic acid and the subsequent acidification in batch cultures of DL1 led to a decrease in fluorescence intensity. However, severe pH reduction was prevented when the bacterium was grown as a biofilm in a flowcell, and a homogeneous distribution of a strong fluorescence signal was observed. These findings show that GFP can be applied to studies of oxygen-tolerant anaerobic bacteria, that densely packed, flowcell-grown biofilms of do not develop oxygen gradients inhibitory to GFP fluorescence development, and that the often transient nature of GFP fluorescence in acid-producing bacteria can be overcome in flowcells, probably by the elimination of metabolic by-product accumulation.

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2001-05-01
2020-01-27
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References

  1. Andersen J. B., Sternberg C., Poulsen L. K., Bjørn S. P., Givskov M., Molin S. 1998; New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl Environ Microbiol64:2240–2246
    [Google Scholar]
  2. Bubert A., Sokolovic A., Chun S.-K., Papatheodorou L., Simm A., Goebel W. 1999; Differential expression of Listeria monocytogenes virulence genes in mammalian host cells. Mol Gen Genet261:323–336
    [Google Scholar]
  3. Cody C. W., Prasher D. C., Westler W. M., Prendergast F. G., Ward W. W. 1993; Chemical structure of the hexapeptide chromophore of the Aequorea green-fluorescent protein. Biochemistry32:1212–1218[CrossRef]
    [Google Scholar]
  4. Cook G. M., Russell J. B. 1994; The effect of extracellular pH and lactic acid on pH homeostasis in Lactococcus lactis and Streptococcus bovis . Curr Microbiol28:165–168[CrossRef]
    [Google Scholar]
  5. Cormack B. P., Valdivia R. H., Falkow S. 1996; FACS-optimized mutants of the green fluorescent protein (GFP. Gene173:33–38[CrossRef]
    [Google Scholar]
  6. Crameri A., Whitehorn E. A., Tate E., Stemmer W. P. C. 1996; Improved green fluorescent protein by molecular evolution using DNA shuffling. Nat Biotechnol14:315–319[CrossRef]
    [Google Scholar]
  7. Cubitt A. B., Heim R., Adams S. R., Boyd A. E., Gross L. A., Tsien R. Y. 1995; Understanding, improving and using green fluorescent proteins. Trends Biochem Sci20:448–455[CrossRef]
    [Google Scholar]
  8. Delagrave S., Hawtin R. E., Silva C. M., Yang M. M., Youvan D. 1995; Red-shifted excitation mutants of the green fluorescent protein. Biotechnology13:151–154[CrossRef]
    [Google Scholar]
  9. Ehrig T., O’Kane D. J., Prendergast F. G. 1995; Green-fluorescent protein mutants with altered fluorescence excitation spectra. FEBS Lett367:163–166[CrossRef]
    [Google Scholar]
  10. Ehrlich S. D., Bruand C., Sozhamannan S., Dabert P., Gros M. F., Janniere L., Gruss A. 1991; Plasmid replication and structural stability in Bacillus subtilis . Res Microbiol142:869–873[CrossRef]
    [Google Scholar]
  11. Elsliger M.-A., Wachter R. M., Hanson G. T., Kallio K., Remington S. J. 1999; Structural and spectral response of green fluorescent protein variants to changes in pH. Biochemistry38:5296–5301[CrossRef]
    [Google Scholar]
  12. Fernández de Palencia P.. Nieto C., Acebo P., Espinosa M., López P. 2000; Expression of green fluorescent protein in Lactococcus lactis . FEMS Microbiol Lett183:229–234[CrossRef]
    [Google Scholar]
  13. Freitag N. E., Jacobs K. E. 1999; Examination of Listeria monocytogenes intracellular gene expression by using the green fluorescent protein of Aequorea victoria. Infect Immun67:1844–1852
    [Google Scholar]
  14. Geoffroy M.-C., Guyard C., Quatannens B., Pavan S., Lange M., Mercenier A. 2000; Use of green fluorescent protein to tag lactic acid bacterium strains under development as live vaccine vectors. Appl Environ Microbiol66:383–391[CrossRef]
    [Google Scholar]
  15. Hansen M. C., White D. C, Palmer R. J. Jr. 2000; Flowcell culture of Porphyromonas gingivalis biofilms under anaerobic conditions. J Microbiol Methods40:233–239[CrossRef]
    [Google Scholar]
  16. Heim R., Tsien R. Y. 1996; Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer. Curr Biol6:178–182[CrossRef]
    [Google Scholar]
  17. Heim R., Prasher D. C., Tsien R. Y. 1994; Wavelength mutations and posttranslational autoxidation of green fluorescent protein. Proc Natl Acad Sci USA91:12501–12504[CrossRef]
    [Google Scholar]
  18. Heim R., Cubitt A. B., Tsien R. Y. 1995; Improved green fluorescence. Nature373:663–664
    [Google Scholar]
  19. Inouye S., Tsuji F. I. 1994; Evidence for redox forms of the Aequorea green fluorescent protein. FEBS Lett351:211–214[CrossRef]
    [Google Scholar]
  20. Jensen P. R., Hammer K. 1998; The sequence of spacers between the consensus sequences modulates the strength of prokaryotic promoters. Appl Environ Microbiol64:82–87
    [Google Scholar]
  21. Kimata Y., Iwaki M., Lim C. R., Kohno K. 1997; A novel mutation which enhances the fluorescence of green fluorescent protein at high temperatures. Biochem Biophys Res Commun232:69–73[CrossRef]
    [Google Scholar]
  22. Kolenbrander P. E., London J. 1993; Adhere today, here tomorrow: oral bacterial adherence. J Bacteriol175:3247–3252
    [Google Scholar]
  23. Lewis P. J., Errington J. 1996; Use of green fluorescent protein for detection of cell-specific gene expression and subcellular protein localisation during sporulation in Bacillus subtilis. Microbiology 142:733–740[CrossRef]
    [Google Scholar]
  24. Lewis P. J., Marston A. L. 1999; GFP vectors for controlled expression and dual labelling of protein fusions in Bacillus subtilis. Gene 227:101–109[CrossRef]
    [Google Scholar]
  25. Miesenböck G.. De Angelis D. A., Rothman J. E. 1998; Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins. Nature394:192–195[CrossRef]
    [Google Scholar]
  26. O’Sullivan D. J., Klaenhammer T. R. 1993; High- and low-copy-number Lactococcus shuttle cloning vectors with features for clone screening. Gene137:227–231[CrossRef]
    [Google Scholar]
  27. Palmer R. J. Jr, Caldwell D. E. 1995; A flowcell for the study of plaque removal and regrowth. J Microbiol Methods24:171–182[CrossRef]
    [Google Scholar]
  28. Patterson G. H., Knobel S. M., Sharif W. D., Kain S. R., Piston D. W. 1997; Use of the green fluorescent protein and its mutants in quantitative fluorescence microscopy. Biophys J73:2782–2790[CrossRef]
    [Google Scholar]
  29. Perry D., Wondrack L. M., Kuramitsu H. K. 1983; Genetic transformation of putative cariogenic properties in Streptococcus mutans . Infect Immun41:722–727
    [Google Scholar]
  30. Prasher D. C., Eckenrode V. K., Ward W. W., Prendergast F. G., Cormier M. J. 1992; Primary structure of the Aequorea victoria green-fluorescent protein. Gene111:229–233[CrossRef]
    [Google Scholar]
  31. Reid B. G., Flynn G. C. 1997; Chromophore formation in green fluorescent protein. Biochemistry36:6786–6791[CrossRef]
    [Google Scholar]
  32. Robey R. B., Ruiz O., Santos A. V. P., Ma J., Kear F., Wang L.-J., Li C.-J., Bernardo A. A., Arruda J. A. L. 1998; pH-dependent fluorescence of a heterologously expressed Aequorea green fluorescent protein mutant: in situ spectral characteristics and applicability to intracellular pH estimation. Biochemistry37:9894–9901[CrossRef]
    [Google Scholar]
  33. Scott K. P., Mercer D. K., Glover L. A., Flint H. J. 1998; The green fluorescent protein as a visible marker for lactic acid bacteria in complex ecosystems. FEMS Microbiol Ecol26:219–230[CrossRef]
    [Google Scholar]
  34. Siegumfeldt H., Rechinger K. B., Jakobsen M. 2000; Dynamic changes of intracellular pH in individual lactic acid bacterium cells in response to a rapid drop in extracellular pH. Appl Environ Microbiol66:2330–2335[CrossRef]
    [Google Scholar]
  35. Siemering K. R., Golbik R., Sever R., Haseloff J. 1996; Mutations that suppress the thermosensitivity of green fluorescent protein. Curr Biol6:1653–1663[CrossRef]
    [Google Scholar]
  36. Ward W. W. 1998; Biochemical and physical properties of green fluorescent protein. In Green Fluorescent Protein: Properties, Applications and Protocols pp45–75 Edited by Chalfie M., Kain S.. New York: Wiley;
    [Google Scholar]
  37. Yanisch-Perron C., Vieira J., Messing J. 1985; Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene33:103–119[CrossRef]
    [Google Scholar]
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