G-quadruplex structures in 16S rRNA regions correlate with thermal adaptation in prokaryotes.

G-quadruplex (G4) structure is a nucleic acid secondary structure formed by guanine-rich sequences, playing essential roles in various biological processes such as gene regulation and environmental stress adaptation. Although prokaryotes growing at high temperatures have higher GC contents, the pattern of G4 structure associated with GC content variation in thermal adaptation remains elusive. This study analyzed 681 bacterial genomes to explore the role of G4 structures in thermal adaptation. Our findings revealed a strong positive correlation between G4 patterns in the region encoding 16S rRNA genes and optimal growth temperatures (Topt), whereas genomic GC content and G4 patterns did not show significant correlations with Topt. Evolutionary analysis showed distinctive differences in G4 stability between Thermotoga (Topt ≥ 80°C) and Pseudothermotoga (60°C ≤ Topt < 80°C) species, with Thermotoga species exhibiting higher G4 stability, indicating stronger selective pressure for G4 structures. In vitro spectroscopy analysis showed that base mutations at key sites resulted in the absence of G4 structural stability and integrity in Thermotoga compared to Pseudothermotoga. Collectively, this study suggests that the G4 structures in 16S rRNA regions emerged as key indicators of thermal adaptation in prokaryotes and contributes to our understanding of the molecular basis of evolutionary adaptation.