Perchlorate-induced proteomic stress responses of Debaryomyces hansenii and their consequences for the habitability of Martian brines
Authors: Heinz Jacob , Doellinger Joerg , Maus Deborah , Schneider Andy , Lasch Peter , Grossart Hans-Peter , Schulze-Makuch Dirk 
Published: September 2022

&lt;p&gt;&lt;strong&gt;Introduction: &lt;/strong&gt;While perchlorate (ClO&lt;sub&gt;4&lt;/sub&gt;&lt;sup&gt;-&lt;/sup&gt;) salts occur only occasionally in natural environments on Earth, they seem to be widely distributed on Mars [1]. The hygroscopicity of these salts and their potential to reduce the freezing point of pure water might enable the provision of liquid water by the formation of temporarily stable liquid brines close to the surface of Mars [2]. However, low water activities and the enhanced chaotropicity (i.e. the potential of biomacromolecule destabilization) of perchlorate pose a huge challenge for putative microorganisms in these brines [3]. Here we present the results of the first study investigating perchlorate-specific stress responses (i.e., with a significant distinction compared to general salt stress) of a halotolerant model organism, &lt;em&gt;Debaryomyces hansenii&lt;/em&gt;, with an untargeted proteomic approach to provide fundamental understanding of the required cellular adaptation mechanisms for life in perchlorate-rich habitats on Mars.&lt;/p&gt;
&lt;p&gt;&lt;strong&gt;Methodology&lt;/strong&gt;: Growth experiments with D&lt;em&gt;. hansenii&lt;/em&gt; were conducted in liquid growth media DMSZ #90 containing either NaCl, NaClO&lt;sub&gt;4&lt;/sub&gt; or no additional salt. Water activities (a&lt;sub&gt;w&lt;/sub&gt;) of the growth media were measured with the Rotronics&amp;#174; &amp;#8216;HC2-AW-USB&amp;#8217; a&lt;sub&gt;w&lt;/sub&gt; meter. Proteomics analyses of the grown cultures were conducted using the SPEED (Sample Preparation by Easy Extraction and Digestion) protocol described previously [4].&lt;/p&gt;
&lt;p&gt;&lt;strong&gt;Results:&lt;/strong&gt; While &lt;em&gt;D. hansenii &lt;/em&gt;grew at NaCl concentrations of up to 4.0 mol/kg (a&lt;sub&gt;w&lt;/sub&gt; = 0.854), growth in perchlorate-containing media was obtained only at NaClO&lt;sub&gt;4&lt;/sub&gt; concentrations up to 2.5 mol/kg (a&lt;sub&gt;w&lt;/sub&gt; = 0.926) [5]. The proteomics data [6] indicated that the chaotropicity of perchlorate is likely to be the major factor of the reduced tolerance of &lt;em&gt;D. hansenii&lt;/em&gt; towards NaClO&lt;sub&gt;4&lt;/sub&gt; compared to NaCl, while the perchlorate&amp;#180;s oxidative properties seem to play only a subordinated role in growth limitation. This is consistent with previous studies finding a high kinetic barrier for the reduction reaction of the perchlorate anion in solution [7].&lt;/p&gt;
&lt;p&gt;The chaotropic activity of perchlorate leads to a destabilization of biomacromolecules such as proteins. Consequently, cells of &lt;em&gt;D. hansenii&lt;/em&gt; showed upregulation of metabolic pathways involved in glycan biosynthesis, protein glycosylation and the requisite protein transport mechanisms when exposed to perchlorate stress, presumably in order to increase protein stability. Furthermore, the biosynthesis and reorganization of cell wall components, e.g. chitin, was significantly upregulated compared to NaCl-induced stress, indicating the necessity of stabilizing the cell envelope. In addition to these perchlorate-specific stress responses, many other metabolic pathways were expressed similarly in NaCl and NaClO&lt;sub&gt;4&lt;/sub&gt;-stressed cells, such as signaling pathways, elevated energy metabolism, or osmolyte biosynthesis.&lt;/p&gt;
&lt;p&gt;&lt;strong&gt;Conclusions:&lt;/strong&gt; To counteract perchlorate-induced chaotropic stresses, cells initiate metabolic pathways that stabilize biomacromolecules and cell envelopes. We hypothesize that these stress responses would also be relevant for putative Martian microorganisms, which likely developed chaotropic defense strategies in order to counteract the relatively high perchlorate concentrations in the Martian regolith. Chaotropic-specific adaptations might be the evolutionary development of stabilized confirmations of biomacromolecules in which structures with covalent bounds and cross-linking are favored over looser electrostatic interactions, hydrogen bonding or hydrophobic effects. Additionally, cell components susceptible to chaotropic stress might be stabilized by the attachment of polymers similar to stabilization effects via protein glycosylation as observed in our experiments. Characteristic biomarkers might result from these adaptions which will likely prompt further investigations.&lt;/p&gt;
&lt;p&gt;&lt;strong&gt;References: &lt;/strong&gt;&lt;/p&gt;
&lt;p&gt;[1] Clark, B.C. and Kounaves, S.P. (2016) &lt;em&gt;IJA,&lt;/em&gt; &lt;em&gt;15&lt;/em&gt;, 311&amp;#8211;318; doi: 10.1017/S1473550415000385.&lt;/p&gt;
&lt;p&gt;[2] Mart&amp;#237;n-Torres, F.J., et al. (2015) &lt;em&gt;Nat. Geosci.&lt;/em&gt; 8, 357&amp;#8211;361; doi: 10.1038/ngeo2412.&lt;/p&gt;
&lt;p&gt;[3] Hallsworth, et al. (2007) &lt;em&gt;Environ. Microbiol.,&lt;/em&gt; 9, 801&amp;#8211;813; doi: 10.1111/j.1462-2920.2006.01212.x.&lt;/p&gt;
&lt;p&gt;[4] Doellinger, J., et al. (2020) &lt;em&gt;MCP,&lt;/em&gt; &lt;em&gt;19&lt;/em&gt;, 209&amp;#8211;222; doi: 10.1074/mcp.TIR119.001616.&lt;/p&gt;
&lt;p&gt;[5] Heinz, J., et al. (2021) &lt;em&gt;Life,&lt;/em&gt; &lt;em&gt;11&lt;/em&gt;; doi: 10.3390/life11111194.&lt;/p&gt;
&lt;p&gt;[6] Heinz, J., et al. (2022) preprint at &lt;em&gt;biorxiv.org&lt;/em&gt;; doi: 10.1101/2022.05.02.490276.&lt;/p&gt;
&lt;p&gt;[7] Urbansky, E.T. (1998) &lt;em&gt;Bioremediat. J.,&lt;/em&gt; &lt;em&gt;2&lt;/em&gt;, 81&amp;#8211;95; doi: 10.1080/10889869891214231.&lt;/p&gt;
&lt;p&gt;&amp;#160;&lt;/p&gt;