Perchlorate-induced stress responses of Escherichia coli and their implications for the habitability of Mars
Authors: Kloss Lea Doris Friedel , Heinz Jacob , Schulze-Makuch Dirk 
Published: September 2022

&lt;p&gt;Putative Martian microorganisms could have adapted to the dry, subzero environment of present-day Mars by resorting to hygroscopic salts that might ensure, at least temporarily, the existence of near-surface liquid brines&lt;sup&gt;1&lt;/sup&gt;. Of relevance for this are perchlorates (ClO&lt;sub&gt;4&lt;/sub&gt;&lt;sup&gt;-&lt;/sup&gt;), which are widespread on Mars&lt;sup&gt;2&lt;/sup&gt; and can absorb atmospheric water in a process called deliquescence while at the same time lowering its freezing point&lt;sup&gt;3&lt;/sup&gt;. However, they might impair microbial life due to the reduction of water activity and ion-specific characteristics harmful to cells such as chaotropicity. As chaotropic agents, perchlorates disrupt the hydrogen bonding network between water molecules and thus the cellular biochemistry by promoting the denaturation of macromolecules.&lt;/p&gt;
&lt;p&gt;Within the scope of this study, we aim to identify the perchlorate-specific stress response of the well-established model organism &lt;em&gt;Escherichia coli&lt;/em&gt; by exposing the bacterium to sodium perchlorate (NaClO&lt;sub&gt;4&lt;/sub&gt;) while additionally examining other solutes (e.g. glycerol, NaCl, guanidine hydrochloride, and hydrogen peroxide) separately that induce osmotic, ionic, chaotropic, and oxidative stress and comparing the individually occurring cellular responses.&lt;/p&gt;
&lt;p&gt;The growth medium DMSZ #1 (0.5% peptone, 0.3% meat extract, pH ~ 7.0) is being used as the basis for aerobic growth of &lt;em&gt;E. coli&lt;/em&gt; in liquid cultures at a temperature of 35 &amp;#176;C and is supplemented with the additional solutes of interest for stress induction. &lt;em&gt;E. coli&lt;/em&gt; is iteratively adapted to higher solute concentrations to quantify the various solute tolerances of the bacterium which guide further experiments. Cell growth and death are monitored by spectrophotometric measurement of the optical density at a wavelength of 600 nm (OD&lt;sub&gt;600&lt;/sub&gt;), as well as counting colony forming units (CFUs) and changes in cell morphology are observed by light microscopy.&lt;/p&gt;
&lt;p&gt;Based on our preliminary results and minimal inhibitory concentrations described in the literature&lt;sup&gt;4,5&lt;/sup&gt;, it seems like &lt;em&gt;E. coli &lt;/em&gt;can withstand higher concentrations of NaCl (up to 1 mol/kg) than NaClO&lt;sub&gt;4&lt;/sub&gt; (up to 0.15 mol/kg). This reduced salt tolerance for NaClO&lt;sub&gt;4&lt;/sub&gt; compared to NaCl has already been described for other organisms such as the halotolerant yeast &lt;em&gt;Debaryomyces hansenii&lt;/em&gt; and could possibly be linked to the chaotropicity of perchlorates, causing macromolecule destabilization&lt;sup&gt;6&lt;/sup&gt;. While final solute tolerances are still being determined, preliminary results suggest that &lt;em&gt;E. coli&lt;/em&gt; exhibits a filamentous cell structure at increasing NaClO&lt;sub&gt;4&lt;/sub&gt; concentrations, with cells clustered together lengthwise in a chain-like arrangement of varying lengths. This change in morphology could potentially be attributed to incomplete cell division&lt;sup&gt;7&lt;/sup&gt; and is in stark contrast to that of control cells in optimal growth medium, which dominantly appear as individual, rod-like cells. Cell filamentation triggered by NaClO&lt;sub&gt;4&lt;/sub&gt; exposure has already been observed for the thermophilic and desiccation-tolerant organism &lt;em&gt;Hydrogenothermus marinus&lt;sup&gt;8&lt;/sup&gt;&lt;/em&gt;.&lt;/p&gt;
&lt;p&gt;We are progressing to more precisely identify the biochemical processes involved in perchlorate-specific stress responses via proteome analysis. In addition, cell filamentation prompts further examinations, such as statistical chain-length evaluation, scanning electron microscope (SEM) imaging and testing for morphological reversibility upon NaClO&lt;sub&gt;4&lt;/sub&gt;-stress removal. Collectively, these results will help us understand the effects of perchlorate-induced stress and thereby allow us to further identify cellular processes critical for life to thrive in and adapt to perchlorate-rich environments like Martian brines.&lt;/p&gt;
&lt;p&gt;&amp;#160;&lt;/p&gt;
&lt;p&gt;&lt;strong&gt;References&lt;/strong&gt;&lt;/p&gt;
&lt;p&gt;1. Davila AF, Schulze-Makuch D. The Last Possible Outposts for Life on Mars. &lt;em&gt;Astrobiology&lt;/em&gt;. 2016;16(2):159-168. doi:10.1089/ast.2015.1380&lt;/p&gt;
&lt;p&gt;2. Clark BC, Kounaves SP. Evidence for the distribution of perchlorates on Mars. &lt;em&gt;International Journal of Astrobiology&lt;/em&gt;. 2016;15(4):311-318. doi:10.1017/S1473550415000385&lt;/p&gt;
&lt;p&gt;3. Zorzano M-P, Mateo-Mart&amp;#237; E, Prieto-Ballesteros O, Osuna S, Renno N. Stability of liquid saline water on present day Mars. &lt;em&gt;Geophys Res Lett&lt;/em&gt;. 2009;36(20). doi:10.1029/2009GL040315&lt;/p&gt;
&lt;p&gt;4. Cebri&amp;#225;n G, Arroyo C, Ma&amp;#241;as P, Cond&amp;#243;n S. Bacterial maximum non-inhibitory and minimum inhibitory concentrations of different water activity depressing solutes. &lt;em&gt;Int J Food Microbiol&lt;/em&gt;. 2014;188:67-74. doi:10.1016/j.ijfoodmicro.2014.07.011&lt;/p&gt;
&lt;p&gt;5. D&amp;#237;az-Rullo J, Rodr&amp;#237;guez-Valdecantos G, Torres-Rojas F, et al. Mining for Perchlorate Resistance Genes in Microorganisms From Sediments of a Hypersaline Pond in Atacama Desert, Chile. &lt;em&gt;Front Microbiol&lt;/em&gt;. 2021;12:723874. doi:10.3389/fmicb.2021.723874&lt;/p&gt;
&lt;p&gt;6. Heinz J, Doellinger J, Maus D, et al. Perchlorate-Specific Proteomic Stress Responses of Debaryomyces hansenii Could Enable Microbial Survival in Martian Brines. preprint available at&lt;em&gt; bioRvix.org&lt;/em&gt;. 2022. doi:10.1101/2022.05.02.490276&lt;/p&gt;
&lt;p&gt;7. Nguyen K, Kumar P. Morphological Phenotypes, Cell Division, and Gene Expression of Escherichia coli under High Concentration of Sodium Sulfate. &lt;em&gt;Microorganisms&lt;/em&gt;. 2022;10(2). doi:10.3390/microorganisms10020274&lt;/p&gt;
&lt;p&gt;8. Beblo-Vranesevic K, Huber H, Rettberg P. High Tolerance of Hydrogenothermus marinus to Sodium Perchlorate. &lt;em&gt;Front Microbiol&lt;/em&gt;. 2017;8:1369. doi:10.3389/fmicb.2017.01369&lt;/p&gt;