Skip to content
1887

Microbiology Society logo Microbiology

Volume 156, Issue 3

Cytocidal amino acid starvation of and acetolactate synthase (Δ) mutants is influenced by the carbon source and rapamycin Free

Abstract

The isoleucine and valine biosynthetic enzyme acetolactate synthase (Ilv2p) is an attractive antifungal drug target, since the isoleucine and valine biosynthetic pathway is not present in mammals, Δ mutants do not survive , mutants are avirulent, and both and mutants die upon isoleucine and valine starvation. To further explore the potential of Ilv2p as an antifungal drug target, we disrupted , and demonstrated that Δ mutants were significantly attenuated in virulence, and were also profoundly starvation-cidal, with a greater than 100-fold reduction in viability after only 4 h of isoleucine and valine starvation. As fungicidal starvation would be advantageous for drug design, we explored the basis of the starvation-cidal phenotype in both and Δ mutants. Since the mutation of , required for the first step of isoleucine biosynthesis, did not suppress the Δ starvation-cidal defects in either species, the cidal phenotype was not due to -ketobutyrate accumulation. We found that starvation for isoleucine alone was more deleterious in than in , and starvation for valine was more deleterious than for isoleucine in both species. Interestingly, while the target of rapamycin (TOR) pathway inhibitor rapamycin further reduced Δ starvation viability, it increased Δ and Δ viability. Furthermore, the recovery from starvation was dependent on the carbon source present during recovery for Δ mutants, reminiscent of isoleucine and valine starvation inducing a viable but non-culturable-like state in this species, while Δ and Δ viability was influenced by the carbon source present during starvation, supporting a role for glucose wasting in the cidal phenotype.

  • Received:
  • Accepted:
  • Revised:
  • Published Online:
Keyword(s): FRT, FLP recombination target , Nat, nourseothricin and TOR pathway, target of rapamycin pathway
SGM
Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.034348-0
2010-03-01
2025-11-19

Metrics

Loading full text...

Full text loading...

/deliver/fulltext/micro/156/3/929.html?itemId=/content/journal/micro/10.1099/mic.0.034348-0&mimeType=html&fmt=ahah

References

  1. Barclay B. J., Little J. G. 1977; Selection of yeast auxotrophs by thymidylate starvation. J Bacteriol 132:1036–1037
     [Google Scholar]
  2. Boer V. M., Amini S., Botstein D. 2008; Influence of genotype and nutrition on survival and metabolism of starving yeast. Proc Natl Acad Sci U S A 105:6930–6935
     [Google Scholar]
  3. Brauer M. J., Huttenhower C., Airoldi E. M., Rosenstein R., Matese J. C., Gresham D., Boer V. M., Troyanskaya O. G., Botstein D. 2008; Coordination of growth rate, cell cycle, stress response, and metabolic activity in yeast. Mol Biol Cell 19:352–367
     [Google Scholar]
  4. Bulder C. J. 1964; Induction of petite mutation and inhibition of synthesis of respiratory enzymes in various yeasts. Antonie Van Leeuwenhoek 30:1–9
     [Google Scholar]
  5. Chipman D., Barak Z., Schloss J. V. 1998; Biosynthesis of 2-aceto-2-hydroxy acids: acetolactate synthases and acetohydroxyacid synthases. Biochim Biophys Acta 1385401–419
     [Google Scholar]
  6. Crispens C. G. 1975; Section IV. Blood. In Handbook on the Laboratory Mouse pp 93–123 Springfield, IL: Charles C. Thomas;
     [Google Scholar]
  7. Culbertson M. R., Henry S. A. 1975; Inositol-requiring mutants of Saccharomyces cerevisiae. Genetics 80:23–40
     [Google Scholar]
  8. Cynober L. A. 2002; Plasma amino acid levels with a note on membrane transport: characteristics, regulation, and metabolic significance. Nutrition 18:761–766
     [Google Scholar]
  9. Daniel J., Dondon L., Danchin A. 1983; 2-Ketobutyrate: a putative alarmone of Escherichia coli. Mol Gen Genet 190:452–458
     [Google Scholar]
  10. Daniel J., Joseph E., Danchin A. 1984; Role of 2-ketobutyrate as an alarmone in E. coli K12: inhibition of adenylate cyclase activity mediated by the phosphoenolpyruvate : glycose phosphotransferase transport system. Mol Gen Genet 193:467–472
     [Google Scholar]
  11. Divol B., Lonvaud-Funel A. 2005; Evidence for viable but nonculturable yeasts in botrytis-affected wine. J Appl Microbiol 99:85–93
     [Google Scholar]
  12. Epelbaum S., Chipman D. M., Barak Z. 1996; Metabolic effects of inhibitors of two enzymes of the branched-chain amino acid pathway in Salmonella typhimurium. J Bacteriol 178:1187–1196
     [Google Scholar]
  13. Fradin C., Kretschmar M., Nichterlein T., Gaillardin C., d'Enfert C., Hube B. 2003; Stage-specific gene expression of Candida albicans in human blood. Mol Microbiol 47:1523–1543
     [Google Scholar]
  14. Fradin C., De Groot P., MacCallum D., Schaller M., Klis F., Odds F. C., Hube B. 2005; Granulocytes govern the transcriptional response, morphology and proliferation of Candida albicans in human blood. Mol Microbiol 56:397–415
     [Google Scholar]
  15. Gillum A. M., Tsay E. Y., Kirsch D. R. 1984; Isolation of the Candida albicans gene for orotidine-5′-phosphate decarboxylase by complementation of S. cerevisiae ura3 and E. coli pyrF mutations. Mol Gen Genet 198:179–182
     [Google Scholar]
  16. Goldstein A. L., McCusker J. H. 1999; Three new dominant drug resistance cassettes for gene disruption in Saccharomyces cerevisiae. Yeast 15:1541–1553
     [Google Scholar]
  17. Goldstein A. L., McCusker J. H. 2001; Development of Saccharomyces cerevisiae as a model pathogen. A system for the genetic identification of gene products required for survival in the mammalian host environment. Genetics 159:499–513
     [Google Scholar]
  18. Gray J. V., Petsko G. A., Johnston G. C., Ringe D., Singer R. A., Werner-Washburne M. 2004; “Sleeping beauty”: quiescence in Saccharomyces cerevisiae. Microbiol Mol Biol Rev 68:187–206
     [Google Scholar]
  19. Guldener U., Heck S., Fielder T., Beinhauer J., Hegemann J. H. 1996; A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Res 24:2519–2524
     [Google Scholar]
  20. Henry S. A., Donahue T. F., Culbertson M. R. 1975; Selection of spontaneous mutants by inositol starvation in yeast. Mol Gen Genet 143:5–11
     [Google Scholar]
  21. 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
     [Google Scholar]
  22. Ito-Harashima S., Hartzog P. E., Sinha H., McCusker J. H. 2002; The tRNA-Tyr gene family of Saccharomyces cerevisiae: agents of phenotypic variation and position effects on mutation frequency. Genetics 161:1395–1410
     [Google Scholar]
  23. Kell D. B., Kaprelyants A. S., Weichart D. H., Harwood C. R., Barer M. R. 1998; Viability and activity in readily culturable bacteria: a review and discussion of the practical issues. Antonie Van Leeuwenhoek 73:169–187
     [Google Scholar]
  24. Kingsbury J. M., McCusker J. H. 2008; Threonine biosynthetic genes are essential in Cryptococcus neoformans. Microbiology 154:2767–2775
     [Google Scholar]
  25. 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
     [Google Scholar]
  26. 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
     [Google Scholar]
  27. 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
     [Google Scholar]
  28. Kirsch D. R., Whitney R. R. 1991; Pathogenicity of Candida albicans auxotrophic mutants in experimental infections. Infect Immun 59:3297–3300
     [Google Scholar]
  29. Landstein D., Chipman D. M., Arad S. M., Barak Z. 1990; Acetohydroxy acid synthase activity in Chlorella emersonii under auto- and heterotrophic growth conditions. Plant Physiol 94:614–620
     [Google Scholar]
  30. LaRossa R. A., Van Dyk T. K. 1987; Metabolic mayhem caused by 2-ketoacid imbalances. Bioessays 7:125–130
     [Google Scholar]
  31. LaRossa R. A., Van Dyk T. K., Smulski D. R. 1987; Toxic accumulation of α-ketobutyrate caused by inhibition of the branched-chain amino acid biosynthetic enzyme acetolactate synthase in Salmonella typhimurium. J Bacteriol 169:1372–1378
     [Google Scholar]
  32. Liebmann B., Muhleisen T. W., Muller M., Hecht M., Weidner G., Braun A., Brock M., Brakhage A. A. 2004; Deletion of the Aspergillus fumigatus lysine biosynthesis gene lysF encoding homoaconitase leads to attenuated virulence in a low-dose mouse infection model of invasive aspergillosis. Arch Microbiol 181:378–383
     [Google Scholar]
  33. Lorenz M. C., Bender J. A., Fink G. R. 2004; Transcriptional response of Candida albicans upon internalization by macrophages. Eukaryot Cell 3:1076–1087
     [Google Scholar]
  34. McCusker J. H., Clemons K. V., Stevens D. A., Davis R. W. 1994; Genetic characterization of pathogenic Saccharomyces cerevisiae isolates. Genetics 136:1261–1269
     [Google Scholar]
  35. Mills D. A., Johannsen E. A., Cocolin L. 2002; Yeast diversity and persistence in Botrytis-affected wine fermentations. Appl Environ Microbiol 68:4884–4893
     [Google Scholar]
  36. 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
     [Google Scholar]
  37. Nicholas R. O., Berry V., Hunter P. A., Kelly J. A. 1999; The antifungal activity of mupirocin. J Antimicrob Chemother 43:579–582
     [Google Scholar]
  38. Noble S. M., Johnson A. D. 2005; Strains and strategies for large-scale gene deletion studies of the diploid human fungal pathogen Candida albicans. Eukaryot Cell 4:298–309
     [Google Scholar]
  39. Oliver J. D. 2005; The viable but nonculturable state in bacteria. J Microbiol 43:special issue93–100
     [Google Scholar]
  40. 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
     [Google Scholar]
  41. Reuss O., Vik A., Kolter R., Morschhauser J. 2004; The SAT1 flipper, an optimized tool for gene disruption in Candida albicans. Gene 341:119–127
     [Google Scholar]
  42. Rhodes D., Hogan A. L., Deal L., Jamieson G. C., Haworth P. 1987; Amino acid metabolism of Lemna minor L.: II. Responses to chlorsulfuron. Plant Physiol 84:775–780
     [Google Scholar]
  43. Saldanha A. J., Brauer M. J., Botstein D. 2004; Nutritional homeostasis in batch and steady-state culture of yeast. Mol Biol Cell 15:4089–4104
     [Google Scholar]
  44. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
  45. Schneper L., Duvel K., Broach J. R. 2004; Sense and sensibility: nutritional response and signal integration in yeast. Curr Opin Microbiol 7:624–630
     [Google Scholar]
  46. Shaner D. L., Singh B. K. 1993; Phytotoxicity of acetohydroxyacid synthase inhibitors is not due to accumulation of 2-ketobutyrate and/or 2-aminobutyrate. Plant Physiol 103:1221–1226
     [Google Scholar]
  47. Sherman F., Fink G. R., Lawrence C. W. 1974 Methods in Yeast Genetics Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
  48. Tkacz J. S., DiDomenico B. 2001; Antifungals: what's in the pipeline. Curr Opin Microbiol 4:540–545
     [Google Scholar]
  49. Unger M. W., Hartwell L. H. 1976; Control of cell division in Saccharomyces cerevisiae by methionyl-tRNA. Proc Natl Acad Sci U S A 73:1664–1668
     [Google Scholar]
  50. Van Dyk T. K., Smulski D. R., Chang Y. Y. 1987; Pleiotropic effects of poxA regulatory mutations of Escherichia coli and Salmonella typhimurium, mutations conferring sulfometuron methyl and α-ketobutyrate hypersensitivity. J Bacteriol 169:4540–4546
     [Google Scholar]
  51. 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
     [Google Scholar]
  52. Warburg O. 1956; On the origin of cancer cells. Science 123:309–314
     [Google Scholar]
  53. Whitcomb C. E. 1999; An introduction to ALS-inhibiting herbicides. Toxicol Ind Health 15:231–239
     [Google Scholar]
  54. 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]
  55. Zaman S., Lippman S. I., Zhao X., Broach J. R. 2008; How Saccharomyces responds to nutrients. Annu Rev Genet 42:27–81
     [Google Scholar]
  56. Ziegelbauer K. 1998; Decreased accumulation or increased isoleucyl-tRNA synthetase activity confers resistance to the cyclic β-amino acid BAY 10–8888 in Candida albicans and Candida tropicalis. Antimicrob Agents Chemother 42:1581–1586
     [Google Scholar]
  57. Ziegelbauer K., Babczinski P., Schonfeld W. 1998; Molecular mode of action of the antifungal β-amino acid BAY 10–8888. Antimicrob Agents Chemother 42:2197–2205
     [Google Scholar]
/content/journal/micro/10.1099/mic.0.034348-0
Loading
Cytocidal amino acid starvation of Saccharomyces cerevisiae and Candida albicans acetolactate synthase (ilv2Δ) mutants is influenced by the carbon source and rapamycin
Microbiology 156, 929 (2010); https://doi.org/10.1099/mic.0.034348-0
/content/journal/micro/10.1099/mic.0.034348-0
/content/journal/micro/10.1099/mic.0.034348-0
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF

Supplementary material 2

PDF

Supplementary material 3

PDF

Supplementary material 4

PDF

Most cited Most Cited RSS feed

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