Eukaryotic Cell
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Eukaryotic Cell, June 2004, p. 752-763, Vol. 3, No. 3
1535-9778/04/$08.00+0     DOI: 10.1128/EC.3.3.752-763.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Novel Chimeric Spermidine Synthase-Saccharopine Dehydrogenase Gene (SPE3-LYS9) in the Human Pathogen Cryptococcus neoformans{dagger}

Joanne M. Kingsbury,1 Zhonghui Yang,1 Tonya M. Ganous,1 Gary M. Cox,2 and John H. McCusker1*

Department of Molecular Genetics and Microbiology,1 Department of Medicine, Duke University Medical Center, Durham, North Carolina 277102

Received 8 March 2004/ Accepted 15 April 2004

The Cryptococcus neoformans LYS9 gene (encoding saccharopine dehydrogenase) was cloned and found to be part of an evolutionarily conserved chimera with SPE3 (encoding spermidine synthase). spe3-lys9, spe3-LYS9, and SPE3-lys9 mutants were constructed, and these were auxotrophic for lysine and spermidine, spermidine, and lysine, respectively. Thus, SPE3-LYS9 encodes functional spermidine synthase and saccharopine dehydrogenase gene products. In contrast to Saccharomyces cerevisiae spe3 mutants, the polyamine auxotrophy of C. neoformans spe3-LYS9 mutants was not satisfied by spermine. In vitro phenotypes of spe3-LYS9 mutants included reduced capsule and melanin production and growth rate, while SPE3-lys9 mutants grew slowly at 30°C, were temperature sensitive in rich medium, and died upon lysine starvation. Consistent with the importance of saccharopine dehydrogenase and spermidine synthase in vitro, spe3-lys9 mutants were avirulent and unable to survive in vivo and both functions individually contributed to virulence. SPE3-LYS9 mRNA levels showed little evidence of being influenced by exogenous spermidine or lysine or starvation for spermidine or lysine; thus, any regulation is likely to be posttranscriptional. Expression in S. cerevisiae of the full-length C. neoformans SPE3-LYS9 cDNA complemented a lys9 mutant but not a spe3 mutant. However, expression in S. cerevisiae of a truncated gene product, consisting of only C. neoformans SPE3, complemented a spe3 mutant, suggesting possible modes of regulation. Therefore, we identified and describe a novel chimeric SPE3-LYS9 gene, which may link spermidine and lysine biosynthesis in C. neoformans.

* Corresponding author. Mailing address: Department of Molecular Genetics and Microbiology, Box 3020, Duke University Medical Center, Durham, NC 27710. Phone: (919) 681-6744. Fax: (919) 684-8735. E-mail: mccus001{at}mc.duke.edu.

{dagger} Supplemental material for this article may be found at http://ec.asm.org/.

Eukaryotic Cell, June 2004, p. 752-763, Vol. 3, No. 3
1535-9778/04/$08.00+0     DOI: 10.1128/EC.3.3.752-763.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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