1887

Abstract

We identified and attempted to disrupt the homoserine and/or threonine biosynthetic genes encoding aspartate kinase (), homoserine kinase () and threonine synthase (); however, each gene proved recalcitrant to disruption. By replacing the endogenous promoters of and with the copper-repressible promoter, we showed that and were essential for the growth of in rich media, when ammonium was the nitrogen source, or when threonine was supplied as an amino acid instead of a dipeptide. Moreover, the severity of the growth defect associated with or repression increased with increasing incubation temperature. We believe this to be the first demonstration of threonine biosynthetic genes being essential in a fungus. The necessity of these genes for growth, particularly at physiologically relevant temperatures, makes threonine biosynthetic genes ideal anti-cryptococcal drug targets.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.2008/019729-0
2008-09-01
2019-10-22
Loading full text...

Full text loading...

/deliver/fulltext/micro/154/9/2767.html?itemId=/content/journal/micro/10.1099/mic.0.2008/019729-0&mimeType=html&fmt=ahah

References

  1. Arevalo-Rodriguez, M., Pan, X., Boeke, J. D. & Heitman, J. ( 2004; ). FKBP12 controls aspartate pathway flux in Saccharomyces cerevisiae to prevent toxic intermediate accumulation. Eukaryot Cell 3, 1287–1296.[CrossRef]
    [Google Scholar]
  2. Birrell, G. W., Giaever, G., Chu, A. M., Davis, R. W. & Brown, J. M. ( 2001; ). A genome-wide screen in Saccharomyces cerevisiae for genes affecting UV radiation sensitivity. Proc Natl Acad Sci U S A 98, 12608–12613.[CrossRef]
    [Google Scholar]
  3. Birrell, G. W., Brown, J. A., Wu, H. I., Giaever, G., Chu, A. M., Davis, R. W. & Brown, J. M. ( 2002; ). Transcriptional response of Saccharomyces cerevisiae to DNA-damaging agents does not identify the genes that protect against these agents. Proc Natl Acad Sci U S A 99, 8778–8783.[CrossRef]
    [Google Scholar]
  4. Care, A., Vousden, K. A., Binley, K. M., Radcliffe, P., Trevethick, J., Mannazzu, I. & Sudbery, P. E. ( 2004; ). A synthetic lethal screen identifies a role for the cortical actin patch/endocytosis complex in the response to nutrient deprivation in Saccharomyces cerevisiae. Genetics 166, 707–719.[CrossRef]
    [Google Scholar]
  5. Chayakulkeeree, M., Rude, T. H., Toffaletti, D. L. & Perfect, J. R. ( 2007; ). Fatty acid synthesis is essential for survival of Cryptococcus neoformans and a potential fungicidal target. Antimicrob Agents Chemother 51, 3537–3545.[CrossRef]
    [Google Scholar]
  6. Davidson, R. C., Blankenship, J. R., Kraus, P. R., de Jesus Berrios, M., Hull, C. M., D'Souza, C., Wang, P. & Heitman, J. ( 2002; ). A PCR-based strategy to generate integrative targeting alleles with large regions of homology. Microbiology 148, 2607–2615.
    [Google Scholar]
  7. Deutschbauer, A. M., Williams, R. M., Chu, A. M. & Davis, R. W. ( 2002; ). Parallel phenotypic analysis of sporulation and postgermination growth in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 99, 15530–15535.[CrossRef]
    [Google Scholar]
  8. Dunn, C. D., Lee, M. S., Spencer, F. A. & Jensen, R. E. ( 2006; ). A genomewide screen for petite-negative yeast strains yields a new subunit of the i-AAA protease complex. Mol Biol Cell 17, 213–226.
    [Google Scholar]
  9. Enyenihi, A. H. & Saunders, W. S. ( 2003; ). Large-scale functional genomic analysis of sporulation and meiosis in Saccharomyces cerevisiae. Genetics 163, 47–54.
    [Google Scholar]
  10. Fraser, J. A., Subaran, R. L., Nichols, C. B. & Heitman, J. ( 2003; ). Recapitulation of the sexual cycle of the primary fungal pathogen Cryptococcus neoformans var. gattii: implications for an outbreak on Vancouver Island, Canada. Eukaryot Cell 2, 1036–1045.[CrossRef]
    [Google Scholar]
  11. Giaever, G., Chu, A. M., Ni, L., Connelly, C., Riles, L., Véronneau, S., Dow, S., Lucau-Danila, A., Anderson, K. & other authors ( 2002; ). Functional profiling of the Saccharomyces cerevisiae genome. Nature 418, 387–391.[CrossRef]
    [Google Scholar]
  12. Gietz, R. D., Schiestl, R. H., Willems, A. R. & Woods, R. A. ( 1995; ). Studies on the transformation of intact yeast cells by the LiAc/SS-DNA/PEG procedure. Yeast 11, 355–360.[CrossRef]
    [Google Scholar]
  13. Goldstein, A. L. & McCusker, J. H. ( 1999; ). Three new dominant drug resistance cassettes for gene disruption in Saccharomyces cerevisiae. Yeast 15, 1541–1553.[CrossRef]
    [Google Scholar]
  14. Hinnebusch, A. ( 1992; ). General and pathway-specific regulatory mechanisms controlling the synthesis of amino acid biosynthetic enzymes in Saccharomyces cerevisiae. In The Molecular and Cellular Biology of the Yeast Saccharomyces: Gene Expression, pp. 319–414. Edited by E. W. Jones, J. R. Pringle &. J. R. Broach. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
  15. Hoffman, C. S. & Winston, F. ( 1987; ). A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene 57, 267–272.[CrossRef]
    [Google Scholar]
  16. Jones, E. W. & Fink, G. R. ( 1982; ). Regulation of amino acid and nucleotide biosynthesis in yeast. In The Molecular Biology of the Yeast Saccharomyces: Metabolism and Gene Expression. Edited by J. N. Strathern, E. W. Jones & J. R. Broach. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  17. Kingsbury, J. M., Yang, Z., Ganous, T. M., Cox, G. M. & McCusker, J. H. ( 2004a; ). Cryptococcus neoformans Ilv2p confers resistance to sulfometuron methyl and is required for survival at 3 °C and in vivo. Microbiology 150, 1547–1558.[CrossRef]
    [Google Scholar]
  18. Kingsbury, J. M., Yang, Z., Ganous, T. M., Cox, G. M. & McCusker, J. H. ( 2004b; ). A novel chimeric spermidine synthase-saccharopine dehydrogenase (SPE3–LYS9) gene in the human pathogen Cryptococcus neoformans. Eukaryot Cell 3, 752–763.[CrossRef]
    [Google Scholar]
  19. Kingsbury, J. M., Goldstein, A. L. & McCusker, J. H. ( 2006; ). Role of nitrogen and carbon transport, regulation, and metabolism genes for Saccharomyces cerevisiae survival in vivo. Eukaryot Cell 5, 816–824.[CrossRef]
    [Google Scholar]
  20. Kotre, A. M., Sullivan, S. J. & Savageau, M. A. ( 1973; ). Metabolic regulation by homoserine in Escherichia coli B-r. J Bacteriol 116, 663–672.
    [Google Scholar]
  21. Loftus, B. J., Fung, E., Roncaglia, P., Rowley, D., Amedeo, P., Bruno, D., Vamathevan, J., Miranda, M., Anderson, I. J. & other authors ( 2005; ). The genome of the basidiomycetous yeast and human pathogen Cryptococcus neoformans. Science 307, 1321–1324.[CrossRef]
    [Google Scholar]
  22. Martin-Rendon, E., Farfan, M. J., Ramos, C. & Calderon, I. L. ( 1993; ). Isolation of a mutant allele that deregulates the threonine biosynthesis in Saccharomyces cerevisiae. Curr Genet 24, 465–471.[CrossRef]
    [Google Scholar]
  23. McDade, H. C. & Cox, G. M. ( 2001; ). A new dominant selectable marker for use in Cryptococcus neoformans. Med Mycol 39, 151–154.[CrossRef]
    [Google Scholar]
  24. Mountain, H. A., Bystrom, A. S., Larsen, J. T. & Korch, C. ( 1991; ). Four major transcriptional responses in the methionine/threonine biosynthetic pathway of Saccharomyces cerevisiae. Yeast 7, 781–803.[CrossRef]
    [Google Scholar]
  25. Nazi, I., Scott, A., Sham, A., Rossi, L., Williamson, P. R., Kronstad, J. W. & Wright, G. D. ( 2007; ). Role of homoserine transacetylase as a new target for antifungal agents. Antimicrob Agents Chemother 51, 1731–1736.[CrossRef]
    [Google Scholar]
  26. O'Barr, T. P. & Everett, K. A. ( 1971; ). Effect of l-homoserine on the growth of Mycobacterium tuberculosis. Infect Immun 3, 328–332.
    [Google Scholar]
  27. Ory, J. J., Griffith, C. L. & Doering, T. L. ( 2004; ). An efficiently regulated promoter system for Cryptococcus neoformans utilizing the CTR4 promoter. Yeast 21, 919–926.[CrossRef]
    [Google Scholar]
  28. Pascon, R. C., Ganous, T. M., Kingsbury, J. M., Cox, G. M. & McCusker, J. H. ( 2004; ). Cryptococcus neoformans methionine synthase: expression analysis and requirement for virulence. Microbiology 150, 3013–3023.[CrossRef]
    [Google Scholar]
  29. Payne, S. H. & Loomis, W. F. ( 2006; ). Retention and loss of amino acid biosynthetic pathways based on analysis of whole-genome sequences. Eukaryot Cell 5, 272–276.[CrossRef]
    [Google Scholar]
  30. Perfect, J. R., Toffaletti, D. L. & Rude, T. H. ( 1993; ). The gene encoding phosphoribosylaminoimidazole carboxylase (ADE2) is essential for growth of Cryptococcus neoformans in cerebrospinal fluid. Infect Immun 61, 4446–4451.
    [Google Scholar]
  31. Ramos, C. & Calderon, I. L. ( 1992; ). Overproduction of threonine by Saccharomyces cerevisiae mutants resistant to hydroxynorvaline. Appl Environ Microbiol 58, 1677–1682.
    [Google Scholar]
  32. Rees, W. D., Grant, S. D., Hay, S. M. & Saqib, K. M. ( 1994; ). Threonine synthesis from homoserine as a selectable marker in mammalian cells. Biochem J 299, 637–644.
    [Google Scholar]
  33. Roberg, K. J., Bickel, S., Rowley, N. & Kaiser, C. A. ( 1997; ). Control of amino acid permease sorting in the late secretory pathway of Saccharomyces cerevisiae by Sec13, Lst4, Lst7 and Lst8. Genetics 147, 1569–1584.
    [Google Scholar]
  34. Sambrook, J., Fritsch, E. F. & Maniatis, T. ( 1989; ). Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  35. Sherman, F., Fink, G. R. & Lawrence, C. W. ( 1974; ). Methods in Yeast Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  36. Suliman, H. S., Appling, D. R. & Robertus, J. D. ( 2007; ). The gene for cobalamin-independent methionine synthase is essential in Candida albicans: a potential antifungal target. Arch Biochem Biophys 467, 218–226.[CrossRef]
    [Google Scholar]
  37. Suvarna, K., Bartiss, A. & Wong, B. ( 2000; ). Mannitol-1-phosphate dehydrogenase from Cryptococcus neoformans is a zinc-containing long-chain alcohol/polyol dehydrogenase. Microbiology 146, 2705–2713.
    [Google Scholar]
  38. Toffaletti, D. L., Rude, T. H., Johnston, S. A., Durack, D. T. & Perfect, J. R. ( 1993; ). Gene transfer in Cryptococcus neoformans by use of biolistic delivery of DNA. J Bacteriol 175, 1405–1411.
    [Google Scholar]
  39. Vallim, M. A., Fernandes, L. & Alspaugh, J. A. ( 2004; ). The RAM1 gene encoding a protein-farnesyltransferase β-subunit homologue is essential in Cryptococcus neoformans. Microbiology 150, 1925–1935.[CrossRef]
    [Google Scholar]
  40. Wach, A., Brachat, A., Pohlmann, R. & Philippsen, P. ( 1994; ). New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast 10, 1793–1808.[CrossRef]
    [Google Scholar]
  41. Yang, Z., Pascon, R. C., Alspaugh, A., Cox, G. M. & McCusker, J. H. ( 2002; ). Molecular and genetic analysis of the Cryptococcus neoformans MET3 gene and a met3 mutant. Microbiology 148, 2617–2625.
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.2008/019729-0
Loading
/content/journal/micro/10.1099/mic.0.2008/019729-0
Loading

Data & Media loading...

Supplements

vol. , part 9, pp. 2767 - 2775

Primers used in this study [ PDF] (74 kb)



PDF
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