Tetraether archaeal lipids promote long-term survival in extreme conditions

Abstract

The sole unifying feature of the incredibly diverse Archaea is their isoprenoid-based ether-linked lipid membranes. Unique lipid membrane composition, including an abundance of membrane-spanning tetraether lipids, impart resistance to extreme conditions. Many questions remain, however, regarding the synthesis and modification of tetraether lipids and how dynamic changes to archaeal lipid membrane composition support hyperthermophily. Tetraether membranes, termed glycerol dibiphytanyl glycerol tetraethers (GDGTs), are generated by tetraether synthase (Tes) by joining the tails of two bilayer lipids known as archaeol. GDGTs are often further specialized through the addition of cyclopentane rings by GDGT ring synthase (Grs). A positive correlation between relative GDGT abundance and entry into stationary phase growth has been observed, but the physiological impact of inhibiting GDGT synthesis has not previously been reported. Here, we demonstrate that the model hyperthermophile Thermococcus kodakarensis remains viable when Tes (TK2145) or Grs (TK0167) are deleted, permitting phenotypic and lipid analyses at different temperatures. The absence of cyclopentane rings in GDGTs does not impact growth in T. kodakarensis, but an overabundance of rings due to ectopic Grs expression is highly fitness negative at supra-optimal temperatures. In contrast, deletion of Tes resulted in the loss of all GDGTs, cyclization of archaeol, and loss of viability upon transition to the stationary phase in this model archaea. These results demonstrate the critical roles of highly specialized, dynamic, isoprenoid-based lipid membranes for archaeal survival at high temperatures.

Keywords: GDGT; archaea; archaeol; tetraether lipids; tetraether synthase; thermophily.

Figure 1.. Tetraether lipid biosynthetic pathway in T. kodakarensis.

The C20 isoprenoid chains of archaeol are covalently linked to generate GDGT (also termed GDGT-0), through the activity of TK2145, Tk-Tes, with GTGT and macrocyclic archaeol (MA) as intermediate or side products, respectively. Production of cyclized-tetraether lipids (GDGT-1, 2, 3, 4), is catalyzed by TK0167, Tk-Grs. Archaeol-1 and archaeol-2 are detected only in strains ectopically expressing Tk-Grs or those with disruption of native Tk-Tes activities.

Figure 2.. Long-term survival of T. kodakarensis demands Tes (TK2145) catalyzed tetraether lipid production but not Grs (TK0167) catalyzed cyclization, .

(A) The genomic locus of TK0167, predicted to encode Tk-Grs (blue), shown with flanking genes (green) in the parental strain (TS559) (Top) and in the TK0167 partial deletion strain (Δgrs; AL010) (Bottom). (B) The genomic locus of TK2145, predicted to encode Tk-Tes (blue), shown with flanking genes (green) in the parental strain (TS559) (Top) and in the TK2145 deletion strain (Δtes; AL016) (Bottom). (C) LC-MS extracted ion chromatograms of one replicate of the acid hydrolyzed lipid extracts from the parent strain TS559, the Δtes strain, and the Δtes strain complemented with TK2145. GDGT production was lost upon deletion of Tk-Tes and restored with ectopic expression of TK2145. (D) LC-MS extracted ion chromatograms of one replicate of the acid hydrolyzed lipid extracts from the parent strain TS559, the Δgrs strain, and the Δgrs strain complemented with TK0167. Deletion of Tk-Grs resulted in the loss of cyclized GDGTs and cyclization was restored with ectopic expression of TK0167. (E and F) Exponential growth rates of T. kodakarensis at 85°C (E) and 95°C (F) are not significantly impacted by deletion of TK2145 (Tk-Tes; strain AL016) or TK0167 (Tk-Grs; AL010), however, deletion of Tk-Tes (TK2145) dramatically impacts survival upon entry into stationary phase.

Figure 3.. Loss of hyperthermophily due to loss of tetraether lipid synthesis is restored via ectopic complementation of Tk-Tes activities.

(A & B) The growth of triplicate biological cultures of T. kodakarensis strains TS559 (black), Δtes (dark blue), Δtes + pTS543 (blue), and pTS543 + pTS543-TK2145 (light blue) were monitored via changes in optical density (at 600nm) at 85°C and 95°C, respectively, revealing the rescue of reduced survival during transition to stationary phase due to the lack of Tk-Tes activities. (C & D) Lipid analyses of triplicate biological cultures of T. kodakarensis strains grown at 85°C and 95°C reveals that ectopic expression of Tk-Tes activities restores tetraether lipid synthesis to strains lacking TK2145 on the genome and an unanticipated increase in cyclized GDGTs. Panel C and D show diether and tetraether lipid production, respectively.

Figure 4.. Dramatic increases in cyclized GDGTs from ectopic Tk-Grs expression coincide with growth defects at supra-optimal temperature.

(A & B) Triplicate biological cultures of T. kodakarensis strains TS559 (black), Δgrs (dark red), Δgrs + pTS543 (red), and Δgrs + pTS543-TK0167 (orange) were monitored for changes in optical density (at 600nm) at 85°C and 95°C, respectively. Ectopic expression of Tk-Grs (orange) resulted in impaired growth at 95°C. (C & D) Lipidome analyses of triplicate biological cultures of T. kodakarensis strains grown at 85°C and 95°C. Ectopic TK-Grs expression increases cyclized-GDGT levels ~50-fold (to ~20% of extracted core lipids). Panel C and D show diether and tetraether lipid production, respectively.