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Journal of Bacteriology, December 2008, p. 8096-8105, Vol. 190, No. 24
0021-9193/08/$08.00+0     doi:10.1128/JB.01180-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Proteasomal Components Required for Cell Growth and Stress Responses in the Haloarchaeon Haloferax volcanii ^,

Guangyin Zhou, David Kowalczyk, Matthew A. Humbard, Sunil Rohatgi, and Julie A. Maupin-Furlow^*

Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida 32611-0700

Received 20 August 2008/ Accepted 6 October 2008

Little is known regarding the biological roles of archaeal proteases. The haloarchaeon Haloferax volcanii is an ideal model for understanding these enzymes, as it is one of few archaea with an established genetic system. In this report, a series of H. volcanii mutant strains with markerless and/or conditional knockouts in each known proteasome gene was systematically generated and characterized. This included single and double knockouts of genes encoding the 20S core 1 (psmA), β (psmB), and 2 (psmC) subunits as well as genes (panA and panB) encoding proteasome-activating nucleotidase (PAN) proteins closely related to the regulatory particle triple-A ATPases (Rpt) of eukaryotic 26S proteasomes. Our results demonstrate that 20S proteasomes are required for growth. Although synthesis of 20S proteasomes containing either 1 or 2 could be separately abolished via gene knockout with little to no impact on growth, conditional depletion of either β alone or 1 and 2 together rendered the cells inviable. In contrast, the PAN proteins were not essential based on the robust growth of the panA panB double knockout strain. Deletion of genes encoding either 1 or PanA did, however, render cells more sensitive to growth on organic versus inorganic nitrogen sources and hypo-osmotic stress and limited growth in the presence of L-canavanine. Abolishment of 1 synthesis also had a severe impact on the ability of cells to withstand thermal stress. This contrasted with what was seen for panA knockouts, which displayed enhanced thermotolerance. Together, these results provide new and important insight into the biological role of proteasomes in archaea.

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* Corresponding author. Mailing address: Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 32611-0700. Phone: (352) 392-4095. Fax: (352) 392-5922. E-mail: jmaupin{at}ufl.edu

Published ahead of print on 17 October 2008.

Supplemental material for this article may be found at http://jb.asm.org/.

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Journal of Bacteriology, December 2008, p. 8096-8105, Vol. 190, No. 24
0021-9193/08/$08.00+0     doi:10.1128/JB.01180-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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