Cross-Stress Adaptation in a Piezophilic and Hyperthermophilic Archaeon From Deep Sea Hydrothermal Vent

Abstract

Hyperthermophiles, living in environments above 80°C and usually coupling with multi-extreme environmental stresses, have drawn great attention due to their application potential in biotechnology and being the primitive extant forms of life. Studies on their survival and adaptation mechanisms have extended our understanding on how lives thrive under extreme conditions. During these studies, the "cross-stress" behavior in various organisms has been observed between the extreme high temperature and other environmental stresses. Despite the broad observation, the global view of the cross-stress behavior remains unclear in hyperthermophiles, leaving a knowledge gap in our understanding of extreme adaptation. In this study, we performed a global quantitative proteomic analysis under extreme temperatures, pH, hydrostatic pressure (HP), and salinity on an archaeal strain, Thermococcus eurythermalis A501, which has outstanding growth capability on a wide range of temperatures (50-100°C), pH (4-9), and HPs (0.1-70 MPa), but a narrow range of NaCl (1.0-5.0 %, w/v). The proteomic analysis (79.8% genome coverage) demonstrated that approximately 61.5% of the significant differentially expressed proteins (DEPs) responded to multiple stresses. The responses to most of the tested stresses were closely correlated, except the responses to high salinity and low temperature. The top three enriched universal responding processes include the biosynthesis and protection of macromolecules, biosynthesis and metabolism of amino acids, ion transport, and binding activities. In addition, this study also revealed that the specific dual-stress responding processes, such as the membrane lipids for both cold and HP stresses and the signal transduction for both hyperosmotic and heat stresses, as well as the sodium-dependent energetic processes might be the limiting factor of the growth range in salinity. The present study is the first to examine the global cross-stress responses in a piezophilic hyperthermophile at the proteomic level. Our findings provide direct evidences of the cross-stress adaptation strategy (33.5% of coding-genes) to multiple stresses and highlight the specific and unique responding processes (0.22-0.63% of coding genes for each) to extreme temperature, pH, salinity, and pressure, which are highly relevant to the fields of evolutionary biology as well as next generation industrial biotechnology (NGIB).

Keywords: archaea; cross-stress adaptation; extreme adaptation; extremophile; hyperthermophile; piezophile; proteomic analysis.

FIGURE 1

The statistical analysis of DEPs responding to different environmental stresses. (A) Correlation matrix of different stress responses based on DEPs. Spearman’s method was used to calculate the matrix with the “cor” function in the R program. Matrix elements have been scaled so that the smallest negative element is –1, the largest positive element is +1, and all elements retain their sign. (B) Three-dimensional plots of PCA of DEPs. The type of stress culture condition was used as the environmental variable. Black dots and red arrows present the strength and direction of the effects of environmental variables. The green dots indicate each significant DEP. (C) Clustering heatmap of significant DEPs. Up- and down-expressed proteins are shown in red and blue, respectively. Raw data of whole proteomic data were log₂-transformed and then clustered and reordered by both rows and columns using the Euclidean distance method and complete linkage cluster method. Symbols of tested conditions are as follows: “HpH,” pH 8.8 vs. pH 7.0; “LpH,” pH 4.4 vs. pH 7.0; “LS,” 1.5% NaCl vs. 2.3% NaCl; “HS,” 4.5% NaCl vs. 2.3% NaCl; “LT,” 65°C vs. 85°C; “HT,” 95°C vs. 85°C; “HP,” 85°C at 40 MPa vs. 85°C at 10 MPa; “HP@95°C,” 40 MPa at 95°C vs. 10 MPa at 95°C; “HT@10 MPa,” 95°C at 10 MPa vs. 85°C at 10 MPa; “HT@40 MPa,” 95°C at 40 MPa vs. 85°C at 40 MPa.

FIGURE 2

(A) Venn diagram of DEP numbers under different stress conditions. Temperature, union of significant DEPs at both low and high temperature stress; NaCl, union of significant DEPs under LS and HS; pH, union of significant DEPs under LpH and HpH; Pressure, significant DEPs at HP stress. Percent of the total DEPs in all tested stresses is shown in parenthesis. (B) COG classification of DEPs. The following COG categories were included: G, carbohydrate transport and metabolism; H, coenzyme transport and metabolism; Q, secondary metabolite biosynthesis, transport, and catabolism; E, amino acid transport and metabolism; F, nucleotide transport and metabolism; J, translation, ribosomal structure, and biogenesis; K, transcription; L, replication, recombination, and repair; O, post-translational modification, protein turnover, and chaperones; I, lipid transport and metabolism; M, cell wall/membrane/envelope biogenesis; U, intracellular trafficking, secretion, and vesicular transport; T, signal transduction mechanisms; P, inorganic ion transport and metabolism; B, chromatin structure and dynamics; D, cell cycle control, cell division, and chromosome partitioning; V, defense mechanisms; C, energy production and conversion; N, cell motility; R, general function prediction only; S, function unknown. The numbers of up- or down-regulated proteins are indicated as bars with positive or negative numbers on the histogram, respectively.

FIGURE 3

Proposed molecular functions and biological processes of T. eurythermalis A501 responding to different stresses. All the presented functions and processes were enriched with p-values less than 0.05. Universal responding processes are shown with a yellow background. The unique responses to each stress have a light green background. The compatible solutes and chaperonin are in pink. Presented in brackets is the number of DEPs in enriched pathways under each condition versus the all predicted protein-encoding genes in the complete genome of T. eurythermalis A501. MCP, methyl-accepting chemotaxis protein; MBH, membrane-bound hydrogenase; MBX, membrane-bound complex; FHL1 and FHL2, formate hydrogen lyase complexes 1 and 2; SOR, superoxide reductase; Rdr, rubrerythrin; VOR, ketoisovalerate oxidoreductase; POR, pyruvate oxidoreductase; IOR, indolepyruvate oxidoreductase; OGOR, oxoglutarate oxidoreductase; DIP, di-myo-inositol phosphate; Tre, Trehalose; cDPG: cyclic 2,3-diphosphoglycerate.

FIGURE 4

Energy metabolism responds to different environmental stresses. (A) Changes in energy metabolism responding to low and high salinity stresses. All genes of a complex responding to one stress in the same direction are shown in this graph. Membrane-bound complexes and oxidoreductases are in yellow. Sodium and proton antiporters are in purple. Formate dehydrogenase complexes are in green. Signal transduction and motility are shown in blue. Others are in gray. (B) Clustering heatmap of energy metabolism genes responding to different stresses. Significant DEPs were clustered in a heatmap. Up-expressed and down-expressed proteins and non-DEPs are presented in red, green, and white, respectively. MBH includes complexes of membrane-bound hydrogenase (Mbh) and Na⁺/H⁺ antiporter (Mrp). MBX includes complexes of membrane-bound oxidoreductase (Mbx) and Na⁺/H⁺ antiporter (Mrp). FHL1 includes complexes of formate dehydrogenase (Fdh1), membrane-bound hydrogenase (Mfh1), and Na⁺/H⁺ antiporter (Mrp). FHL2 includes complexes of formate dehydrogenase (Fdh2), membrane-bound hydrogenase (Mfh2), and Na⁺/H⁺ antiporter (Mrp). HYD: hydrogenase, including coenzyme F420 hydrogenase (Frh) and [NiFe] hydrogenase (Hyp). FLA, flagellum.