Cloning of Nitrate Reductase and Nitrite Reductase Genes and Their Functional Analysis in Regulating Cr(VI) Reduction in Ectomycorrhizal Fungus Pisolithus sp.1

Liang Shi; Binhao Liu; Xinzhe Zhang; Yuan Bu; Zhenguo Shen; Jianwen Zou; Yahua Chen

doi:10.3389/fmicb.2022.926748

Cloning of Nitrate Reductase and Nitrite Reductase Genes and Their Functional Analysis in Regulating Cr(VI) Reduction in Ectomycorrhizal Fungus Pisolithus sp.1

Front Microbiol. 2022 Jul 7:13:926748. doi: 10.3389/fmicb.2022.926748. eCollection 2022.

Authors

Liang Shi¹, Binhao Liu¹, Xinzhe Zhang¹, Yuan Bu¹, Zhenguo Shen¹, Jianwen Zou², Yahua Chen^{1

2

3

4

5}

Affiliations

¹ College of Life Sciences, Nanjing Agricultural University, Nanjing, China.
² College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing, China.
³ Jiangsu Collaborative Innovation Center for Solid Organic Waste Resource, Nanjing Agricultural University, Nanjing, China.
⁴ National Joint Local Engineering Research Center for Rural Land Resources Use and Consolidation, Nanjing Agricultural University, Nanjing, China.
⁵ The Collaborated Laboratory of Plant Molecular Ecology (between College of Life Sciences of Nanjing Agricultural University and Asian Natural Environmental Science Center of the University of Tokyo), Nanjing Agricultural University, Nanjing, China.

Abstract

Assimilatory-type nitrate reductase (NR) and nitrite reductase (NiR) are the key enzymes that involve in nitrate assimilation and nitrogen cycling in microorganisms. NR and NiR with NADH or NADPH and FMN or FAD domains could be coupled to the reduction process of hexavalent chromium [Cr(VI)] in microorganisms. A new assimilatory-type NR gene (named niaD) and a new assimilatory-type NiR gene (named niiA) are cloned, identified, and functionally characterized by 5' and 3' RACE, alignment, annotation, phylogenetic tree, and yeast mutant complementation analyses from Pisolithus sp.1, a dominant symbiotic ectomycorrhizal fungi (EMF) that can assist in phytoremediation. Assimilatory-type niaD and niiA were 2,754 bp and 3,468 bp and encode a polypeptide with 917 and 1,155 amino acid residues, respectively. The isoelectric points of NR (Pisolithus sp.1 NR) and NiR (Pisolithus sp.1 NiR) of Pisolithus sp.1 are 6.07 and 6.38, respectively. The calculated molecular mass of Pisolithus sp.1 NR and Pisolithus sp.1 NiR is 102.065 and 126.914 kDa, respectively. Yeast mutant complementation analysis, protein purification, and activities of NR and NiR under Cr treatment suggest that Pisolithus sp.1 NR is a functional NR that mediates Cr(VI) tolerance and reduction. The multiple alignment demonstrates that Pisolithus sp.1 NR is potentially a nicotinamide adenine dinucleotide phosphate-dependent flavin mononucleotide reductase and also Class II chromate reductase. Our results suggest that Pisolithus sp.1 NR plays a key role in Cr(VI) reduction in the EMF Pisolithus sp.1.

Keywords: Cr(VI) reduction; ectomycorrhizal fungi; nitrate reductase; phytoremediation; tolerance; yeast.