Replacement of branched-chain polyamine biosynthesis with thermospermine supports survival under both cold and heat stress in the hyperthermophilic archaeon <i>Thermococcus kodakarensis</i>

<i>N⁴</i>-bis(aminopropyl)spermidine (BCPA), a branched-chain polyamine, is uniquely found in bacterial and archaeal hyperthermophiles. In <i>Thermococcus kodakarensis</i>, BCPA is synthesized by BCPA synthase (BpsA), an aminopropyl transferase encoded by <i>bpsA</i>. This highly positively charged molecule is localized in both the nucleic acid and membrane fractions of <i>T. kodakarensis</i> cells. The <i>bpsA</i> deletion strain (DBP1), which lacks BCPA, failed to grow at 93°C and exhibited poor survival under repeated cold stress, indicating that BCPA is essential for membrane stability and function <i>in vivo</i>. Additionally, the expression of specific genes, including the cytoplasmic hydrogenase subunit <i>hyhL</i>, was absent in DBP1, suggesting a role for BCPA in gene regulation. To further investigate BCPA's function, we replaced <i>bpsA</i> in <i>T. kodakarensis</i> with <i>speE</i> from the hyperthermophilic archaeon <i>Pyrobaculum calidifontis</i>, enabling the production of norspermine instead of BCPA. The resulting KPS strain accumulated thermospermine as its major polyamine. Growth at 93°C was partially restored in KPS, and cold-stress survival improved significantly. Additionally, KPS exhibited biosurfactant (sophorolipid) tolerance comparable to that of the parental <i>T. kodakarensis</i> strain KU216 under thermal conditions. Furthermore, <i>hyhL</i> expression was restored in KPS, as confirmed by immunoblotting with anti-HyhL antisera, suggesting that thermospermine can functionally compensate for BCPA. Notably, mutant DBP1 cells lacking both BCPA and thermospermine did not survive repeated cycles of cold and heat stress. This observation suggests that these polyamines play a crucial role in long-term survival, potentially facilitating hibernation-like states in natural environments where extreme temperature fluctuations occur.IMPORTANCEAt the hot springs of Kodakarajima Island, surrounded by cold ocean water, diverse hyperthermophiles, including <i>Thermococcus, Thermotoga,</i> and <i>Thermus</i> species, naturally produce branched-chain polyamines (BCPAs) via a unique aminopropyltransferase BpsA, in addition to spermidine. In <i>Pyrobaculum calidifontis</i>, the <i>Pc-</i>SpeE enzyme produces norspermine <i>in vivo</i>. However, when the <i>speE</i> gene from <i>P. calidifontis</i> is introduced into <i>Thermococcus kodakarensis</i>, the transformant (Δ<i>bpsA::Pc-speE</i>) produces thermospermine instead of norspermine. This shift suggests that the product specificity of <i>Pc-</i>SpeE is influenced by factors inherent to the host organism. Interestingly, thermospermine appears to functionally substitute for BCPA, potentially by forming BCPA-like structures with bent nitrogen atoms. This structural mimicry could contribute to cellular stability under both heat and cold stress, highlighting a potential mechanism for temperature and stress adaptation in <i>T. kodakarensis</i>. These findings further suggest that while BCPA and thermospermine are distinct, they may play similar roles in stress resilience.