Molecular Components of Nitrogen Fixation Gene Cluster and Associated Enzymatic Activities of Non-Heterocystous Thermophilic Cyanobacterium Thermoleptolyngbya sp.

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.

Suppression ofhesAmutation on nitrogenase activity inPaenibacillus polymyxaWLY78 with the addition of high levels of molybdate or cystine

The diazotrophicPaenibacillus polymyxaWLY78 possesses a minimal nitrogen fixation gene cluster consisting of nine genes (nifB nifH nifD nifK nifE nifN nifX hesAandnifV). Notably, thehesAgene contained within thenifgene cluster is also found withinnifgene clusters among diazotrophic cyanobacteria andFrankia. The predicted product HesA is a member of the ThiF-MoeB-HesA family containing an N-terminal nucleotide binding domain and a C-terminal MoeZ/MoeB-like domain. However, the function ofhesAgene in nitrogen fixation is unknown. In this study, we demonstrate that thehesAmutation ofP. polymyxaWLY78 leads to nearly complete loss of nitrogenase activity. The effect of the mutation can be partially suppressed by the addition of high levels of molybdate or cystine. However, the nitrogenase activity of thehesAmutant could not be restored byKlebsiella oxytoca nifQorEscherichia coli moeBcompletely. In addition, thehesAmutation does not affect nitrate reductase activity ofP. polymyxaWLY78. Our results demonstratehesAis a novel gene specially required for nitrogen fixation and its role is related to introduction of S and Mo into the FeMo-co of nitrogenase.

Molecular phylogeny of the heterocystous cyanobacteria (subsections IV and V) based on nifD

The heterocystous cyanobacteria are currently placed in subsections IV and V, which are distinguished by cellular division in one plane (false branching) and in more than one plane (true branching), respectively. Published phylogenies of 16S rRNA gene sequence data support the monophyly of the heterocystous cyanobacteria, with members of subsection V embedded within subsection IV. It has been postulated that members of subsection V arose from within subsection IV. Therefore, phylogenetic analysis of nucleotide sequences of the nitrogen-fixation gene nifD from representatives of subsections IV and V was performed by using maximum-likelihood criteria. The heterocystous cyanobacteria are supported as being monophyletic, with the non-heterocystous cyanobacteria as their closest relative. However, neither subsection IV nor subsection V is monophyletic, with representatives of both subsections intermixed in two sister clades. Analysis of nifD does not support recognition of two distinct subsections.