nifH gene expression and diversity in geothermal springs of Tengchong, China

Abstract

Terrestrial hot springs have been suggested to harbor diverse diazotrophic lineages by using DNA-based nifH gene phylogenetic analysis. However, only a small amount of diazotrophs were ever confirmed to perform nitrogen fixation. In order to explore the compositions of active diazotrophic populations in hot springs, the in situ expression and diversity of nifH and 16S rRNA genes were investigated in the sediments of hot springs (pH 4.3-9.1; temperature 34-84°C) in Tengchong, China, by using high-throughput sequencing. The results showed that active diazotrophs were diverse in the studied Tengchong hot springs. The main active diazotrophs in high-temperature hot springs were affiliated with Aquificae, while those in low-temperature hot springs belonged to Cyanobacteria and Nitrospirae. Such dominance of Aquificae and Nitrospirae of diazotrophs has not been reported in other ecosystems. This suggests that hot springs may harbor unique active diazotrophs in comparison with other type of ecosystems. Furthermore, there were significant differences in the phylogenetic lineages of diazotrophs between hot springs of Tengchong and other regions, indicating that diazotrophs have geographical distribution patterns. Statistical analysis suggests that the expression and distribution of nifH gene were influenced by temperature and concentrations of ammonia and sulfur seem in Tengchong hot springs. These findings avail us to understand element cycling mediated by diazotrophs in hot spring ecosystems.

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

“…Following quality ltering, 7,206,573 (~5%) of the retained reads mapped to the T-2 T genome. Although Jinze Hot Spring is not the most nitrogen-depleted in this region (23), several reads mapped to the nif cluster, including the nitrogenase reductase nifH, the α chain of the nitrogenase (nifD), and both genes encoding the β chain of the nitrogenase (nifK)(Fig. 1A).…”

“…Integral roles for Aqui caceae in several biogeochemical cycles in terrestrial geothermal springs have been proposed for decades (5,6,8,30,33), but very little direct evidence for their role in the nitrogen cycle has been obtained until recently (5,23,24,37). Nitrogen xation in strain T-2 T brings the total number of cultivated Aqui caceae shown to x nitrogen to three isolates, with T-2 T being the rst con rmed diazotrophic Aqui caceae isolate with genome data available.…”

“…Although nif genes have been noted in the family before (20,22), limited information on the structure and expression of these gene clusters is available. Recently, expression of nifH as a proxy for diazotrophic activity has been reported for Aqui caceae inhabiting various geothermal springs in Tengchong, particularly high-temperature springs (23), and several springs with > 30% of the population predicted to be diazotrophic, comprised largely of Aqui caceae (23). This is similar to previous observations for springs in Yellowstone National Park, USA (5,38), where Thermocrinis was the most abundant genus in Perpetual Spouter Hot Spring, and most nifH sequences recovered from metatranscriptomes were assigned to this genus (5).…”

“…Here, we also recover metatranscriptomic reads from Jinze Hot Spring mapping to the nif cluster of T-2 T . However, as nitrogen xation can be repressed in the presence of bioavailable nitrogen (5,39), and the abundance of nifH transcripts was negatively correlated with available ammonium in Tengchong (23), future studies to address nitrogen xation could be focused on the most nitrogen-limited springs, e.g., Dagunguo, Gumingquan, Jiemeiquan, Xianrendong, and Rehaitiyanqu (23,25). Such experiments could include bulk 15 N 2 incorporation or acetylene reduction assays, or taxonomically focused experiments such as 15 N 2 stable isotope probing followed by uorescence in situ hybridization and metatranscriptomics.…”

“…To date, only the genomes of Hydrogenobacter thermophilus (20,21) and Thermocrinis albus (22) have been reported to encode a gene cluster for a molybdenum-iron nitrogenase. Although abundant nifH transcripts in situ in Yellowstone National Park, USA (5) and Tengchong, China (23) were assigned to members of the Aqui caceae, only two isolates belonging to the family have recently been shown to x dinitrogen (24).…”