Abstract
Hot spring ecosystems are analogous to some thermal environments on the early Earth and represent ideal models to understand life forms and element cycling on the early Earth. Denitrification, an important component of biogeochemical nitrogen cycle, is highly active in hot springs. Nitrite (NO2−) reduction to nitric oxide (NO) is the significant and rate-limiting pathway in denitrification and is catalyzed by two types of nitrite reductases, encoded by nirS and nirK genes. NirS and NirK were originally considered incompatible in most denitrifying organisms, although a few strains have been reported to possess both genes. Herein, we report the functional division of nirS and nirK in Thermus, a thermophilic genus widespread in thermal ecosystems. Transcriptional levels of nirS and nirK coexisting in Thermus antranikianii DSM 12462T were measured to assess the effects of nitrite, oxygen, and stimulation time. Thirty-nine Thermus strains were used to analyze the phylogeny and distribution of nirS and nirK; six representative strains were used to assess the denitrification phenotype. The results showed that both genes were actively transcribed and expressed independently in T. antranikianii DSM 12462T. Strains with both nirS and nirK had a wider range of nitrite adaptation and revealed nir-related physiological adaptations in Thermus: nirK facilitated adaptation to rapid changes and extended the adaptation range of nitrite under oxygen-limited conditions, while nirS expression was higher under oxic and relatively stable conditions.
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Funding
This work was financially supported by National Natural Science Foundation of China (Nos 91951205, 31970122, 31600298 and 31470139). W.J.L was also supported by Guangdong Province Science and Technology Innovation Strategy Special Fund (Grant no. 2018B020206001).
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Fig S1
Standard curves (a-c) and melting curves (d) of quantitative real-time PCR. I indicates nirS, II indicates nirK, and III indicates gyrB. (PNG 2661 kb)
Fig S2
Standard curve for nitrite concentration and absorbance. (PNG 870 kb)
Fig S3
Growth curves of Thermus brockianus SYSU G00112 (a), Thermus oshimai SYSU G00132 (b), Thermus brockianus SYSU G00253 (c), Thermus antranikianii DSM 12462T (d), Thermus arciformis SYSU G00537 (e) and Thermus caldifontis SYSU G00608 (f). Cells were cultured with shaking (150 r/min) before exponential phase, and then NaNO2 solutions with increasing concentrations (1, 2, 5, and 10 mM) were added to the R2A broth for standing incubation. Equal volumes of sterile water were added to control samples and cultured with shaking. Error bars show the standard deviation of the mean (n = 3). (PNG 2449 kb)
Table S1
Strains used in this study (XLSX 10 kb)
Table S2
Genes and primers used for qPCR analysis (XLSX 10 kb)
Table S3
Amino acid sequences of NirS and NirK (XLSX 13 kb)
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Liu, RR., Tian, Y., Zhou, EM. et al. Distinct Expression of the Two NO-Forming Nitrite Reductases in Thermus antranikianii DSM 12462T Improved Environmental Adaptability. Microb Ecol 80, 614–626 (2020). https://doi.org/10.1007/s00248-020-01528-3
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DOI: https://doi.org/10.1007/s00248-020-01528-3