Cloning, Prokaryotic Expression and Functional Characterization of NifH Gene from the Associative Nitrogen-Fixing...

Biological nitrogen fixation (BNF) is a unique mechanism in which microorganisms utilize the nitrogenase enzyme to catalyze the conversion of atmospheric nitrogen (N₂) to ammonia (NH₃). Fe protein, encoded by the nifH gene, is an essential component of the nitrogenase in Klebsiella variicola DX120E. However, the function of this gene in regulating nitrogen fixing activity is still unclear.

The objective of this study was to reveal the function of nifH gene in associative nitrogen-fixing bacteria Klebsiella variicola DX120E and micro-sugarcane system by immunoassay and gene editing.

In the current investigation, the nifH gene was cloned in a pET-30a (+) vector and expressed in Escherichia coli. The NifH protein was purified and used to immunize rabbit, and then the serum was collected and purified to obtain rabbit anti-NifH polyclonal antibodies. The CRISPR-Cas9 system was applied to produce nifH mutant strains, and the nitrogen-fixing enzyme activity, gene, and protein expression were analyzed.

Both in vitro and in vivo NifH proteins were detected by Western blotting, which were 43 and 32 kDa respectively. The expression of nifD and nifK genes was decreased, and nitrogenase activity was reduced in the nifH mutant strain.

The nifH gene mutant weakened the nitrogenase activity by regulating the expression of Fe protein, which suggests a potential strategy to study the nitrogen fixation-related genes and the interactions between endophytic nitrogen-fixing bacteria and sugarcane.