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Applied and Environmental Microbiology, November 2009, p. 6706-6711, Vol. 75, No. 21
0099-2240/09/$08.00+0     doi:10.1128/AEM.00905-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Insufficiency of Copper Ion Homeostasis Causes Freeze-Thaw Injury of Yeast Cells as Revealed by Indirect Gene Expression Analysis {triangledown}

Shunsuke Takahashi,1,{dagger} Akira Ando,1,{dagger} Hiroshi Takagi,2 and Jun Shima1*

National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan,1 Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan2

Received 22 April 2009/ Accepted 29 August 2009

Saccharomyces cerevisiae is exposed to freeze-thaw stress in commercial processes, including frozen dough baking. Cell viability and fermentation activity after a freeze-thaw cycle were dramatically decreased due to freeze-thaw injury. Because this type of injury involves complex phenomena, the injury mechanisms are not fully understood. We examined freeze-thaw injury by indirect gene expression analysis during postthaw incubation after freeze-thaw treatment using DNA microarray profiling. The results showed that genes involved in the homeostasis of metal ions were frequently contained in genes that were upregulated, depending on the freezing period. We assessed the phenotype of deletion mutants of the metal ion homeostasis genes that exhibited freezing period-dependent upregulation and found that the strains with deletion of the MAC1 and CTR1 genes involved in copper ion homeostasis exhibited freeze-thaw sensitivity, suggesting that copper ion homeostasis is required for freeze-thaw tolerance. We found that supplementation with copper ions during postthaw incubation increased intracellular superoxide dismutase activity and intracellular levels of reactive oxygen species were decreased. Moreover, cell viability was increased by supplementation with copper ions. These results suggest that insufficiency of copper ion homeostasis may be one of the causes of freeze-thaw injury.

* Corresponding author. Mailing address: National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan. Phone: 81-29-838-8066. Fax: 81-29-838-7996. E-mail: shimaj{at}affrc.go.jp

{triangledown} Published ahead of print on 11 September 2009.

{dagger} S.T. and A.A. contributed equally to this work.

Applied and Environmental Microbiology, November 2009, p. 6706-6711, Vol. 75, No. 21
0099-2240/09/$08.00+0     doi:10.1128/AEM.00905-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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