Proteomic and Metabolomic Profiling of Deinococcus radiodurans Recovering After Exposure to Simulated Low Earth Orbit Vacuum Conditions

Abstract

The polyextremophile, gram-positive bacterium Deinococcus radiodurans can withstand harsh conditions of real and simulated outer space environment, e.g., UV and ionizing radiation. A long-term space exposure of D. radiodurans has been performed in Low Earth Orbit (LEO) in frames of the Tanpopo orbital mission aiming to investigate the possibility of interplanetary life transfer. Space vacuum (10 ^{- 4}-10 ^{- 7} Pa) is a harmful factor, which induces dehydration and affects microbial integrity, severely damaging cellular components: lipids, carbohydrates, proteins, and nucleic acids. However, the molecular strategies by which microorganisms protect their integrity on molecular and cellular levels against vacuum damage are not yet understood. In a simulation experiment, we exposed dried D. radiodurans cells to vacuum (10 ^{- 4}-10 ^{- 7} Pa), which resembles vacuum pressure present outside the International Space Station in LEO. After 90 days of high vacuum exposure, survival of D. radiodurans cells was 2.5-fold lower compared to control cells. To trigger molecular repair mechanisms, vacuum exposed cells of D. radiodurans were recovered in complex medium for 3 and 6 h. The combined approach of analyzing primary metabolites and proteins revealed important molecular activities during early recovery after vacuum exposure. In total, 1939 proteins covering 63% of D. radiodurans annotated protein sequences were detected. Proteases, tRNA ligases, reactive oxygen species (ROS) scavenging proteins, nucleic acid repair proteins, TCA cycle proteins, and S-layer proteins are highly abundant after vacuum exposure. The overall abundance of amino acids and TCA cycle intermediates is reduced during the recovery phase of D. radiodurans as they are needed as carbon source. Furthermore, vacuum exposure induces an upregulation of Type III histidine kinases, which trigger the expression of S-layer related proteins. Along with the highly abundant transcriptional regulator of FNR/CRP family, specific histidine kinases might be involved in the regulation of vacuum stress response. After repair processes are finished, D. radiodurans switches off the connected repair machinery and focuses on proliferation. Combined comparative analysis of alterations in the proteome and metabolome helps to identify molecular key players in the stress response of D. radiodurans, thus elucidating the mechanisms behind its extraordinary regenerative abilities and enabling this microorganism to withstand vacuum stress.

Keywords: Deinococcus radiodurans; dehydration; high vacuum exposure; metabolomics; molecular stress response; proteomics.

FIGURE 1

Effect of vacuum on survival and growth of D. radiodurans. (A) OD₆₀₀ measurements at t₀ (inoculation timepoint) and the harvesting timepoint for the control cells and vacuum exposed cells. (B) Changes in OD₆₀₀ values between t₀ and the harvesting timepoint per hour of cultivation shown as ratio per hour. (C) Colony forming units (CFUs), counted for the control cells and vacuum exposed cells. In case with CFU, samples for plating were picked at t₀. Error bars always show the standard error based on the measurements of four replicates. Error bars at t₀ represent the measurement error of the instrument.

FIGURE 2

Metabolic response of D. radiodurans to vacuum (targeted approach). (A) Principal component analysis of 32 metabolites. The plot was created in R with the pca3d package. All data was z-scored, and the most impactful loadings are indicated as arrows. (B) Heatmap of measured metabolites with a corresponding dendrogram created in R with the heatmap.2 package. Metabolite data was z-scored before plotting and the dendrogram was drawn by the hclust function.

FIGURE 3

Heatmap of untargeted metabolites with the corresponding dendrogram. Data was z-scored before plotting.

FIGURE 4

Proteomics analysis of the vacuum exposed and control cells after 6 h of recovery in a complex medium. (A) Volcano plot of all 1166 proteins that were identified in every replicate of every condition and timepoint. The y-axis plots the negative log₁₀ corrected p-value (q-value) of the ANOVA. The x-axis shows the log₂ fold change. All proteins with a fold change below 1.5 are indicated in brighter colors. (B) STRING database analysis from selected proteins. Proteins with a corrected p-value below 0.05 were divided into two groups. A post hoc test confirmed if there is a difference at the 6 h timepoint between control and vacuum exposed cells. 107 proteins were identified as higher abundant in vacuum exposed cells (orange group) and 105 proteins as less abundant (blue group). The STRING database was able to map 104 of the higher abundant ones and 98 of the lower abundant ones. Nodes are uploaded proteins and edges are interactions between proteins. The null hypothesis tests if the number of interactions could be assigned to any random set of proteins. The p-value for the orange set is 6.0^∗10^-7, the one for the blue set is 0.26. (C) KEGG pathway annotations were added to the uploaded proteins. The number of proteins from both sets of proteins which belong to several KEGG pathways is shown in the spider plot.

FIGURE 5

Proteomics and Metabolomics comparison of the TCA cycle between vacuum exposed and the control cells after 6 h of recovery in a complex medium. Metabolites are rounded rectangles and proteins are circles. Molecules in orange were more abundant after the vacuum exposure (ANOVA and post hoc test), blue molecules were less abundant. Gray ones were measured, but no statistical difference was calculated. In addition, the sizes of protein circles mirror their fold changes (vacuum 6 h/control 6 h).

FIGURE 6

(A) Logarithmic fold change of all measured tRNA ligases between vacuum exposed and the control cells after 6 h of recovery in complex medium. The ones with statistically significant differences (ANOVA and post hoc test) are high lightened in color. (B) Normalized intensities of measured Clp and Lon proteases after 6 h of recovery between the vacuum exposed and the control cells. Statistically significant differences are indicated with a^∗.

FIGURE 7

Normalized intensities of measured histidine kinases between vacuum exposed and control cells after 6 h of recovery in a complex medium. Statistically significant differences are indicated with a^∗.

FIGURE 8

Summary of affected molecular components in D. radiodurans during the early stage of recovery from vacuum stress in a complex medium.