1887

Abstract

The cellular distribution of the cephamycin biosynthetic enzyme lysine 6-aminotransferase (LAT) has been studied in hyphae by confocal microscopy using the S65T mutant of green fluorescent protein (GFP) as a reporter. LAT mediates the first committed step in the biosynthesis of the secondary metabolite cephamycin C by . The enzymic activity of LAT varies with time during the growth of in liquid medium. To investigate if this temporal variation occurs uniformly amongst all hyphae, was transformed with a plasmid containing the LAT-encoding gene translationally fused to the GFP-encoding gene. The LAT–GFP fusion product displayed fluorescence spectral characteristics of GFP, and showed similar temporal characteristics of LAT activity compared to the wild-type strain of . The transformed strain exhibited a heterogeneous distribution of fluorescence in mycelia grown in liquid cultures. This distribution varied significantly as the batch progressed: only a fraction of the mycelia fluoresced in the early growth phase, whereas nearly all hyphae fluoresced by the late growth phase. Thereafter, a non-uniform distribution of fluorescence was again observed in the declining growth phase. A large fraction of the non-fluorescent cells in the declining growth phase were found to be non-viable. Observations of colonies grown on solid agar also showed variation of LAT–GFP expression at different stages of growth. These observations in the solid phase can be explained in terms of nutrient deprivation and signalling molecules. The results suggest that physiological differentiation of mycelia leading to cephamycin C biosynthesis is both temporally and spatially distributed. The findings also revealed that the observed heterogeneity was independent of the position of individual cell compartments within the hypha. The potential of GFP as a reporter for the quantitative study of cephamycin biosynthesis at the cellular level has also been demonstrated.

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-146-8-1869
2000-08-01
2019-10-15
Loading full text...

Full text loading...

/deliver/fulltext/micro/146/8/1461869a.html?itemId=/content/journal/micro/10.1099/00221287-146-8-1869&mimeType=html&fmt=ahah

References

  1. Aharonowitz, Y. & Demain, A. L. ( 1979; ). Nitrogen nutrition and regulation of cephalosporin production in Streptomyces clavuligerus. Can J Microbiol 25, 61-67.[CrossRef]
    [Google Scholar]
  2. Alexander, D. C. & Jensen, S. E. ( 1998; ). Investigation of the Streptomyces clavuligerus cephamycin C gene cluster and its regulation by the CcaR protein. J Bacteriol 180, 4068-4079.
    [Google Scholar]
  3. Brana, A. F., Manzanal, M. B. & Hardisson, C. ( 1982; ). Mode of cell wall growth of Streptomyces antibioticus. FEMS Microbiol Lett 13, 231-235.[CrossRef]
    [Google Scholar]
  4. Brana, A. F., Wolfe, S. & Demain, A. L. ( 1986; ). Relationship between nitrogen assimilation and cephalosporin synthesis in Streptomyces clavuligerus. Arch Microbiol 146, 46-51.[CrossRef]
    [Google Scholar]
  5. Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W. & Prasher, D. C. ( 1994; ). Green fluorescent protein as a marker for gene expression. Science 263, 802-805.[CrossRef]
    [Google Scholar]
  6. Chater, K. F. & Bibb, M. J. ( 1997; ). Regulation of bacterial antibiotic production. In Biotechnology, vol 7, Products of Secondary Metabolism, pp. 57-105. Edited by H. Kleinkauf & H. von Dohren. Weinheim: VCH.
  7. Chater, K. F. & Losick, R. ( 1997; ). Mycelial life style of Streptomyces coelicolor A3(2) and its relatives. In Bacteria as Multicellular Organisms, pp. 149-182. Edited by J. A. Shapiro & M. Dworkin. New York: Oxford University Press.
  8. Chung, J. D. & Stephanopoulos, G. ( 1995; ). Studies of transcriptional state heterogeneity in sporulating cultures of Bacillus subtilis. Biotechnol Bioeng 47, 234-242.[CrossRef]
    [Google Scholar]
  9. Chung, J. D., Stephanopoulos, G., Ireton, K. & Grossman, A. D. ( 1994; ). Gene expression in single cells of Bacillus subtilis: evidence that a threshold mechanism controls the initiation of sporulation. J Bacteriol 176, 1977-1984.
    [Google Scholar]
  10. Davey, H. M. & Kell, D. B. ( 1996; ). Flow cytometry and cell sorting of heterogeneous microbial populations: the importance of single-cell analyses. Microbiol Rev 60, 641-696.
    [Google Scholar]
  11. Detterbeck, S., Morandini, P., Wetterauer, B., Bachmair, A., Fischer, K. & MacWilliams, H. K. ( 1994; ). The ‘prespore-like cells’ of Dictyostelium have ceased to express a prespore gene: analysis using short-lived beta-galactosidases as reporters. Development 120, 2847-2855.
    [Google Scholar]
  12. Drouin, J. F., Louvel, L., Vanhoutte, B., Vivier, H., Pons, M. N. & Germain, P. ( 1997; ). Quantitative characterization of cellular differentiation of Streptomyces ambofaciens in submerged culture by image analysis. Biotechnol Tech 11, 819-824.[CrossRef]
    [Google Scholar]
  13. Gramajo, H. C., Takano, E. & Bibb, M. J. ( 1993; ). Stationary-phase production of the antibiotic actinorhodin in Streptomyces coelicolor A3(2) is transcriptionally regulated. Mol Microbiol 7, 837-845.[CrossRef]
    [Google Scholar]
  14. Han, L., Khetan, A., Hu, W. S. & Sherman, D. H. ( 1999; ). Time-lapsed confocal microscopy reveals temporal and spatial expression of the lysine ϵ-aminotransferase gene in Streptomyces clavuligerus. Mol Microbiol 34, 878-886.[CrossRef]
    [Google Scholar]
  15. Heim, R., Cubitt, A. B. & Tsien, R. Y. ( 1995; ). Improved green fluorescence. Nature 373, 663-664.
    [Google Scholar]
  16. Hopwood, D. A., Bibb, M. J., Chater, K. F. & 7 other authors (1985). Genetic Manipulation of Streptomyces: a Laboratory Manual. Norwich: John Innes Foundation.
  17. Illing, G. T., Normansell, I. D. & Peberdy, J. F. ( 1989; ). Protoplast isolation and regeneration in Streptomyces clavuligerus. J Gen Microbiol 135, 2289-2297.
    [Google Scholar]
  18. Ingram, C., Brawner, M., Youngman, P. & Westpheling, J. ( 1989; ). xylE functions as an efficient reporter gene in Streptomyces spp.: use for the study of galP1, a catabolite-controlled promoter. J Bacteriol 171, 6617-6624.
    [Google Scholar]
  19. Jensen, S. E. & Demain, A. L. ( 1995; ). Beta-lactams. In Genetics and Biochemistry of Antibiotic Production, pp. 239-268. Edited by L. C. Vining & C. Stuttard. London: Butterworth-Heinemann.
  20. Khetan, A. (1998). Analysis of temporal and spatial control of cephamycin C biosynthesis in Streptomyces clavuligerus. PhD thesis, University of Minnesota.
  21. Khetan, A. & Hu, W. S. ( 1999; ). Metabolic engineering of antibiotic biosynthetic pathways. In Manual of Industrial Microbiology and Biotechnology, pp. 717-724. Edited by A. L. Demain & J. Davies. Washington, DC: American Society for Microbiology.
  22. Khetan, A., Malmberg, L. H., Sherman, D. H. & Hu, W. S. ( 1996; ). Metabolic engineering of cephalosporin biosynthesis in Streptomyces clavuligerus. Ann NY Acad Sci 782, 17-24.[CrossRef]
    [Google Scholar]
  23. Khetan, A., Malmberg, L. H., Kyung, Y. S., Sherman, D. H. & Hu, W. S. ( 1999; ). Precursor and cofactor as a check valve for cephamycin biosynthesis in Streptomyces clavuligerus. Biotechnol Prog 15, 1020-1027.[CrossRef]
    [Google Scholar]
  24. Kremer, L., Baulard, A., Estaquier, J., Poulaingodefroy, O. & Locht, C. ( 1995; ). Green fluorescent protein as a new expression marker in mycobacteria. Mol Microbiol 17, 913-922.[CrossRef]
    [Google Scholar]
  25. Lewis, P. J. & Errington, J. ( 1996; ). Use of green fluorescent protein for detection of cell-specific gene expression and subcellular protein localization during sporulation in Bacillus subtilis. Microbiology 142, 733-740.[CrossRef]
    [Google Scholar]
  26. Lewis, P. J., Nwoguh, C. E., Barer, M. R., Harwood, C. R. & Errington, J. ( 1994; ). Use of digitized video microscopy with a fluorogenic enzyme substrate to demonstrate cell- and compartment-specific gene expression in Salmonella enteritidis and Bacillus subtilis. Mol Microbiol 13, 655-662.[CrossRef]
    [Google Scholar]
  27. McAdams, H. H. & Arkin, A. ( 1997; ). Stochastic mechanisms in gene expression. Proc Natl Acad Sci USA 94, 814-819.[CrossRef]
    [Google Scholar]
  28. McDaniel, R., Ebert-Khosla, S., Hopwood, D. A. & Khosla, C. ( 1993; ). Engineered biosynthesis of novel polyketides. Science 262, 1546-1550.[CrossRef]
    [Google Scholar]
  29. Madduri, K., Shapiro, S., DeMarco, A. C., White, R. L., Stuttard, C. & Vining, L. C. ( 1991a; ). Lysine catabolism and α-aminoadipate synthesis in Streptomyces clavuligerus. Appl Microbiol Biotechnol 35, 358-363.
    [Google Scholar]
  30. Madduri, K., Stuttard, C. & Vining, L. C. ( 1991b; ). Cloning and location of a gene governing lysine ϵ-aminotransferase, an enzyme initiating β-lactam biosynthesis in Streptomyces spp. J Bacteriol 173, 985-988.
    [Google Scholar]
  31. Malmberg, L. H. & Hu, W. S. ( 1991; ). Kinetic analysis of cephalosporin biosynthesis in Streptomyces clavuligerus. Biotechnol Bioeng 38, 941-947.[CrossRef]
    [Google Scholar]
  32. Malmberg, L. H., Hu, W. S. & Sherman, D. H. ( 1993; ). Precursor flux control through targeted chromosomal insertion of the lysine ϵ-aminotransferase (lat) gene in cephamycin C biosynthesis. J Bacteriol 175, 6916-6924.
    [Google Scholar]
  33. Malmberg, L. H., Hu, W. S. & Sherman, D. H. ( 1995; ). Effects of enhanced lysine ϵ-aminotransferase activity on cephamycin biosynthesis in Streptomyces clavuligerus. Appl Microbiol Biotechnol 44, 198-205.[CrossRef]
    [Google Scholar]
  34. Martin, S. M. & Bushell, M. E. ( 1996; ). Effect of hyphal micromorphology on bioreactor performance of antibiotic-producing Saccharopolyspora erythraea cultures. Microbiology 142, 1783-1788.[CrossRef]
    [Google Scholar]
  35. Muller, W. H., van der Krift, T. P., Krouwer, A. J., Wosten, H. A., van der Voort, L. H., Smaal, E. B. & Verkleij, A. J. ( 1991; ). Localization of the pathway of the penicillin biosynthesis in Penicillium chrysogenum. EMBO J 10, 489-495.
    [Google Scholar]
  36. Nestaas, E. & Wang, D. I. C. ( 1981; ). A new sensor, the filtration probe, for quantitative characterization of the penicillin fermentation. 1. Mycelial morphology and culture activity. Biotechnol Bioeng 23, 2803-2813.[CrossRef]
    [Google Scholar]
  37. Petrich, A. K., Leskiw, B. K., Paradkar, A. S. & Jensen, S. E. ( 1994; ). Transcriptional mapping of the genes encoding the early enzymes of the cephamycin biosynthetic pathway of Streptomyces clavuligerus. Gene 142, 41-48.[CrossRef]
    [Google Scholar]
  38. Romero, J., Martin, J. F., Liras, P., Demain, A. L. & Rius, N. ( 1997; ). Partial purification, characterization and nitrogen regulation of the lysine ϵ-aminotransferase of Streptomyces clavuligerus. J Ind Microbiol Biotechnol 18, 241-246.[CrossRef]
    [Google Scholar]
  39. Russo-Marie, F., Roederer, M., Sager, B., Herzenberg, L. A. & Kaiser, D. ( 1993; ). β-Galactosidase activity in single differentiating bacterial cells. Proc Natl Acad Sci USA 90, 8194-8198.[CrossRef]
    [Google Scholar]
  40. Schauer, A., Ranes, M., Santamaria, R., Guijarro, J., Lawlor, E., Mendez, C., Chater, K. & Losick, R. ( 1988; ). Visualizing gene expression in time and space in the filamentous bacterium Streptomyces coelicolor. Science 240, 768-772.[CrossRef]
    [Google Scholar]
  41. Sun, J., Kelemen, G. H., Fernandez-Abalos, J. M. & Bibb, M. J. ( 1999; ). Green fluorescent protein as a reporter for spatial and temporal gene expression in Streptomyces coelicolor A3(2). Microbiology 145, 2221-2227.
    [Google Scholar]
  42. Takano, E., Gramajo, H. C., Strauch, E., Andres, N., White, J. & Bibb, M. J. ( 1992; ). Transcriptional regulation of the redD transcriptional activator gene accounts for growth-phase-dependent production of the antibiotic undecylprodigiosin in Streptomyces coelicolor A3(2). Mol Microbiol 6, 2797-2804.[CrossRef]
    [Google Scholar]
  43. Tolker-Nielsen, T., Holmstrom, K., Boe, L. & Molin, S. ( 1998; ). Non-genetic population heterogeneity studied by in situ polymerase chain reaction. Mol Microbiol 27, 1099-1105.[CrossRef]
    [Google Scholar]
  44. Tsien, R. Y. ( 1998; ). The green fluorescent protein. Annu Rev Biochem 67, 509-544.[CrossRef]
    [Google Scholar]
  45. Ward, J. M., Janssen, G. R., Kieser, T., Bibb, M. J., Buttner, M. J. & Bibb, M. J. ( 1986; ). Construction and characterisation of a series of multi-copy promoter-probe plasmid vectors for Streptomyces using the aminoglycoside phosphotransferase gene from Tn5 as indicator. Mol Gen Genet 203, 468-478.[CrossRef]
    [Google Scholar]
  46. Webb, C. D., Decatur, A., Teleman, A. & Losick, R. ( 1995; ). Use of green fluorescent protein for visualization of cell-specific gene expression and subcellular protein localization during sporulation in Bacillus subtilis. J Bacteriol 177, 5906-5911.
    [Google Scholar]
  47. Wrigley-Jones, C. A., Richards, H., Thomas, C. R. & Ward, J. ( 1993; ). Stability of plasmid vector plJ303 in Streptomyces lividans TK24 during laboratory-scale fermentations. Biotechnol Bioeng 41, 148-155.[CrossRef]
    [Google Scholar]
  48. Yoon, H. S. & Golden, J. W. ( 1998; ). Heterocyst pattern formation controlled by a diffusible peptide. Science 282, 935-938.[CrossRef]
    [Google Scholar]
  49. Yu, H., Serpe, E., Romero, J. & 8 other authors ( 1994; ). Possible involvement of the lysine ϵ-aminotransferase gene (lat) in the expression of the genes encoding ACV synthetase (pcbAB) and isopenicillin N synthase (pcbC) in Streptomyces clavuligerus. Microbiology 140, 3367–3377.[CrossRef]
    [Google Scholar]
  50. Zhang, J., Wolfe, S. & Demain, A. L. ( 1989; ). Ammonium ions repress δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine synthetase in Streptomyces clavuligerus NRRL 3585. Can J Microbiol 35, 399-402.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-146-8-1869
Loading
/content/journal/micro/10.1099/00221287-146-8-1869
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error