Abstract
Thermoleptolyngbya is a genus of non-heterocystous cyanobacteria that are typical inhabitants of hot spring microbial mats. These filamentous cyanobacteria are capable of nitrogen fixation. In this study, we examined the genome sequences of five publicly available Thermoleptolyngbya strains to explore their nitrogen fixation gene cluster. Analysis of the nitrogen-fixation clusters in these extremophilic strains revealed that the cluster is located in a single locus in Thermoleptolyngbyace. The average nucleotide and amino acid identities of the nitrogen-fixation cluster combined with phylogenetic reconstructions support that nitrogen fixation genes in Thermoleptolyngbyaceae are closely related to one another but also heterogeneous within the genus. The strains from Asia, and China more specifically, generate a separate clade within the genus. Among these strains Thermoleptolyngbya sp. PKUAC-SCTB121 has been selected for experimental validation of clade's nitrogen fixation capacity. The acetylene reduction experiments of that strain shown that the strain can reduce acetylene to ethylene, indicating a fully functional nitrogenase. The activity of nitrogenase has been tested using different gas compositions across 72 h and exhibited a two-phase trend, high nitrogenase activity at the beginning of the assay that slowed down in the second phase of the analysis.