Dynamic evolution of nitrogen cycle and pH homeostasis genes enabled the diversification of Pampinifervens gen. nov. (Aquificaceae)

Abstract Background: Members of the phylum Aquificota were initially documented more than a century ago and play important roles in hydrogen, sulfur, and carbon cycles in geothermal systems as ubiquitous and abundant chemolithoautotrophs; however, their roles in the nitrogen cycle are not well defined. Results: Here, we show that a novel Aquificaceae isolate from Tengchong, China, fixes nitrogen in pure culture and that nitrogen fixation (nif) genes are transcribed in situ. We propose the name Pampinifervens diazotrophicum gen. et sp. nov. to accommodate this organism, with strain T-2T as the nomenclatural type. Analysis of metagenomes from physicochemically diverse springs in Tengchong showed that Pampinifervens dominates the Aquificota populations in most springs, consistent with a wide pH growth range for strain T-2T (pH 6.0-9.5) and another isolate, strain T-8T (pH 5.5-10.0). Despite the role of P. diazotrophicum in nitrogen fixation, strain T-8T does not fix nitrogen, but respires nitrate. Analysis of 104 metagenome-assembled genomes from 84 metagenomes globally, including at least 16 sympatric Pampinifervens species and other species from Canada, Japan, Malaysia, and Russia, revealed the variable presence of nif and denitrification genes. In contrast, genes for hydrogen chemolithotrophy, sulfur chemolithotrophy, and autotrophy were more conserved. Genes required for homeostasis were also present in the genus, with a proliferation of these genes in species recovered from Tengchong, China, that likely allowed them to diversify from neutral and alkaline bicarbonate-buffered springs into acidic sulfuric acid-buffered springs. Conclusions: Together, this work presents a widespread but overlooked genus of Aquificaceae and evolutionary patterns that suggest an important role for nitrogen metabolism and pH homeostasis in the local speciation of Pampinifervens in Southwest China. We suggest that Pampinifervens and possibly other Aquificaceae have underappreciated but diverse roles in nitrogen cycling.