Geraldy Lie Stefanus Liman

Replicative DNA synthesis is distributed among several DNA polymerases (DNAPs) in both Bacteria and Eukarya. The division of synthesis permits specialized activities of unique DNAPs to facilitate rapid replication in a continuous manner on the leading strand and discontinuous synthesis of the lagging strand with repetitive priming and maturation of Okazaki fragments. Many archaea encode two evolutionarily unrelated DNAPs, PolB and PolD, but how leading and lagging strand synthesis responsibilities are partitioned between each polymerase has yet to be resolved in vivo. In the model archaeon Thermococcus kodakarensis, Pol B is dispensable, demonstrating PolD can independently support replicative synthesis, but does not exclude the possibility that PolB normally participates in key aspects of DNA replication. Here, we combine phenotypic analyses of strains encoding variant DNAPs, high-throughput sequencing, and purified replication assays to establish that PolB actively facilitates lagging strand synthesis in vivo. Pol B outperforms PolD at strand displacement and Okazaki fragment maturation, demonstrating that specialized DNAPs share replicative synthesis duties in most Archaea. Our findings define a foundational division of labor between PolB and PolD in Archaea and establish the use of specialized replicative DNAP activities for strand-specific synthesis across all life.

Enhancement of the cyclization of membrane lipids GDGTs (glycerol dialkyl glycerol tetraethers) is a critical strategy for archaea to adapt to various environmental stresses. However, the physiological function of membrane lipid cyclization remains unclear. Here, we reported that the GDGT ring synthases mutant, deficient in GDGT cyclization, inhibited archaellum formation and reduced cell motility in thermoacidophilic crenarchaeon Sulfolobus acidocaldarius . This inhibition was caused by decreased transcription of the archaellum operon, likely due to cleavage of the C-terminal domains in transmembrane proteins ArnRs, the transcription factors that regulate archaellum operon expression. The transcriptomic and proteomic analysis showed deficiency of GDGT cyclization broadly impacted the expression of membrane associate proteins, including respiratory chain proteins, and decreased cellular ATP concentration. Moreover, phylogenetic analysis demonstrated that the correlation between GDGT cyclization and archaellum formation is widespread among (hyper)thermophilic archaea, and this was further verified in the euryarchaeon Thermococcus kodakarensis. Our findings suggested that archaea modify their membrane lipids to profoundly alter cellular appendages and cell physiology to adapt to environmental fluctuations.

Inteins (intervening proteins), mobile genetic elements removed through protein splicing, often interrupt proteins required for DNA replication, recombination, and repair. An abundance of in vitro evidence implies that inteins may act as regulatory elements, whereby reduced splicing inhibits production of the mature protein lacking the intein, but in vivo evidence of regulatory intein excision in the native host is absent. The model archaeon Thermococcus kodakarensis encodes 15 inteins, and we establish the impacts of intein splicing inhibition on host physiology and replication in vivo. We report that a decrease in intein splicing efficiency of the recombinase RadA, a Rad51/RecA homolog, has widespread physiological consequences, including a general growth defect, increased sensitivity to DNA damage, and a switch in the mode of DNA replication from recombination-dependent replication toward origin-dependent replication.

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.