Synthesis of nitrogenase by Paenibacillus sabinae T27 in presence of high levels of ammonia during anaerobic fermentation.

Mingyang Wang,Sanfeng Chen,Qin Li,Pengxi Liu,Xiaojuan He,Haowei Zhang

doi:10.1007/s00253-021-11231-z

Mingyang Wang, Sanfeng Chen + Show 4 more

Open Access

https://doi.org/10.1007/s00253-021-11231-z

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Abstract

Biological nitrogen fixation is usually inhibited by fixed nitrogen. Paenibacillus sabinae T27, a Gram-positive, spore-forming diazotroph, possesses high nitrogenase activity and has 3 copies of nifH (nifH, nifH2, nifH3), a copy of nifDK, and multiple nifHDK-like genes. In this study, we found that P. sabinae T27 showed nitrogenase activities not only in low (0-3 mM) concentrations of NH4+ but also in high (30-300 mM) concentrations of NH4+, no matter whether this bacterium was grown in a flask or in a fermenter on scale cultivation. qRT-PCR and western blotting analyses supported that Fe protein and MoFe protein were synthesized under both low (0-3 mM) and high (30-300 mM) concentrations of NH4+. Liquid chromatography-mass spectrometry (LC-MS) analysis revealed that MoFe protein was encoded by nifDK and Fe protein was encoded by both nifH and nifH2. The cross-reaction suggested the purified Fe and MoFe components from P. sabinae T27 grown in both nitrogen-limited and nitrogen-excess conditions were active. This is the first time to report that diazotrophs show nitrogenase activity in presence of high (30-300 mM) concentrations of NH4+. Our study will provide a clue for studying the mechanisms of nitrogen fixation in presence of the high concentration of NH4+. KEY POINTS: • P. sabinae T27 can synthesize active nitrogenase in presence of high levels of ammonia. •Fe and MoFe proteins of nitrogenase purified in absence of ammonia are the same as those purified from the high concentration of ammonia. • Fe protein is encoded by nifH and nifH2, and MoFe protein is encoded by nifDK.

Highlights

Biological nitrogen fixation catalyzed by nitrogenase is a high energy-intensive process, and nitrogenase synthesis and activity are inhibited by ammonium (NH4+)
Paenibacillus sabinae T27, a Gram-positive, spore-forming diazotroph (N2-fixing microorganism, showed nitrogenase activities in low (0-4 mM) concentration of NH4+, and in high (30-300 mM) concentration of NH4+, no matter whether the cells of this bacterium were grown in flask or in fermentor on scale cultivation. qRT-PCR and western blotting analysis supported that Fe protein and MoFe protein were synthesized under both low (0-4 mM) and high (30-300 mM) concentration of NH4+
Liquid chromatography-mass spectrometry(LC-MS)analysis revealed that MoFe protein purified form cultures grown in nitrogen-limited condition or nitrogen-excess condition was encoded by nifDK and Fe protein was encoded by both nifH and nifH2

Summary

Methods

P. sabinae T27 was grown overnight in LD medium (per liter contains: 2.5 g NaCl, 5 g yeast and 10 g tryptone) at 30°C with shaking. The cultures were transferred to a 100 mL amounts in 250 mL bottles or 500 mL in 1 L bottles containing 10 mmol/L NH4Cl supplemented in nitrogen-limited medium. Nitrogen-limited medium contained (per liter) 10.4 g Na2HPO4, 3.4 g KH2PO4, 26 mg CaCl2·2H2O, 30 mg MgSO4, 0.3 mg MnSO4, 36 mg Ferric citrate, 7.6 mg Na2MoO4·2H2O, 10 mg p-aminobenzoic acid, 5 μg biotin, 2 mM glutamate and 4 g glucose as carbon source. P. sabinae T27 were cultured in a 7.5 L fermentor with a working volume of 5 L. Temperature and pH were controlled at 30°C and 6.8, respectively. During the fermentation pH was automatically controlled by addition of KOH. The cell pellet was stored in liquid nitrogen for further use

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