Functional Insights of Salinity Stress-Related Pathways in Metagenome-Resolved Methanothrix Genomes

Area of Science:

• Microbiology
• Environmental Science
• Genomics

Background:

• Methanogenic archaea, particularly *Methanothrix*, are crucial for stable anaerobic bioreactor function.
• Previous studies implicated *Methanothrix harundinacea* in forming stable granules in saline conditions (up to 20 g/L Na+).

Purpose of the Study:

• To reconstruct and analyze *Methanothrix* metagenome-assembled genomes (MAGs) from saline granular sludge.
• To elucidate the genetic mechanisms underlying salinity tolerance in *M. harundinacea*.

Main Methods:

• Metagenome-assembled genome reconstruction from granular sludge.
• Genome annotation and metabolic pathway analysis.
• Identification of salinity stress-related genes and pathways.

Main Results:

• Three *Methanothrix* MAGs were reconstructed, including a new subspecies of *M. harundinacea* (MAG_279).
• MAG_279 possesses genes for producing compatible solutes (Nε-acetyl-β-lysine, ectoine) and modifying glycoconjugates via N-glycosylation for salinity resistance.
• Isoprenoid production for cell membrane stabilization was also identified as a stress response.

Conclusions:

• A novel, halotolerant acetoclastic methanogen subspecies of *M. harundinacea* was identified, capable of adapting to both low (5 g/L Na+) and high (20 g/L Na+) salinity.
• Genomic insights reveal specific adaptations, including EPS modification and compatible solute production, enhancing survival under high salinity.
• This study advances understanding of *M. harundinacea*'s ecological role in extreme environments and its potential for high-salinity anaerobic digestion.