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

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 <i>niaD</i>) and a new assimilatory-type NiR gene (named <i>niiA</i>) are cloned, identified, and functionally characterized by 5' and 3' RACE, alignment, annotation, phylogenetic tree, and yeast mutant complementation analyses from <i>Pisolithus</i> sp.1, a dominant symbiotic ectomycorrhizal fungi (EMF) that can assist in phytoremediation. Assimilatory-type <i>niaD</i> and <i>niiA</i> 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 (<i>Pisolithus</i> sp.1 NR) and NiR (<i>Pisolithus</i> sp.1 NiR) of <i>Pisolithus</i> sp.1 are 6.07 and 6.38, respectively. The calculated molecular mass of <i>Pisolithus</i> sp.1 NR and <i>Pisolithus</i> 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 <i>Pisolithus</i> sp.1 NR is a functional NR that mediates Cr(VI) tolerance and reduction. The multiple alignment demonstrates that <i>Pisolithus</i> sp.1 NR is potentially a nicotinamide adenine dinucleotide phosphate-dependent flavin mononucleotide reductase and also Class II chromate reductase. Our results suggest that <i>Pisolithus</i> sp.1 NR plays a key role in Cr(VI) reduction in the EMF <i>Pisolithus</i> sp.1.