Physiological and Biochemical Characterization of AnNitA, the Aspergillus nidulans High-Affinity Nitrite Transporter

Abstract

High-affinity nitrite influx into mycelia ofAlthough nitrate and ammonium are the major sources of nitrogen for bacteria, fungi, algae, and plants, nitrite may also represent a significant nitrogen source under some circumstances. For example, in waterlogged anaerobic soils as well as in aerobic soils where gaseous anaerobic pockets exist, substantial levels of nitrite may accumulate (14). Phytoplankton has been shown to excrete significant amounts of nitrite when nitrate uptake exceeds the capacity for the assimilation of the nitrite generated (20). Conditions such as low light, low temperature, or a disruption of the nitrate reductase activity (40) may result in nitrite efflux that matches nitrate uptake in a 1:1 stoichiometry. This nitrite may subsequently be taken up and converted to ammonium by assimilatory nitrite reductase, followed by assimilation into organic nitrogenous compounds within root plastids or chloroplasts (2). As well as being a source of nitrogen for a range of organisms, there are associated agricultural, environmental, and even medical issues involved in nitrite biogeochemistry (16,37).There is a substantial amount of information available for the nitrite assimilatory enzyme system, nitrite reductase, as well as certain nitrate uptake systems that also transport nitrite as well as nitrate. These bispecific permeases (together with those that transport nitrate only, such as Chlamydomonas reinhardtii NRT2.2) are characterized by 12 transmembrane ␣-helical domains (Tms), a nitrate signature, and a major facilitator superfamily (MFS) motif and belong to the nitrate NNP (nitrate nitrite porter) subgroup (TC 2.A.1) of the MFS (21,25,33).In contrast, there are transporters that transport nitrite but not nitrate, for which much less is known. These transporters include Escherichia coli EcNirC (3, 12, 13), Aspergillus nidulans AnNitA (40), and C. reinhardtii CrNar1, which possesses no fewer than six paralogs (18, 23). Such proteins belong to a family quite separate from the MFS, known as the formate/ nitrite transporter (FNT) family (TC 2.A.44), and many FNT members are thought to transport nitrite and/or the structurally related formate. FNT family members are found in the microbial kingdoms of archaea, eubacteria, protista, and fungi. Most members of the FNT family probably possess 6 Tms and are much smaller (at the primary protein sequence level) than NNP proteins. For example, the A. nidulans AnNitA protein possesses 310 amino acid residues, compared to the AnNrtA high-affinity nitrate transporter, which has 507 amino acid residues. Recently, a structural analysis of the purified FocA proteins (members of the FNT family) from E. coli (7,41) and Vibrio cholerae (38) suggested that these proteins are pentameric formate channels, with each FocA subunit being made up of six transmembrane ␣-helices. FocA channels, with affinities for formate that may be in the millimolar to tens of millimolar range (38), are thought to export formate generated from pyruvate during anaerobic respiration. The extent ...

Comparative genomics reveals high biological diversity and specific adaptations in the industrially and medically important fungal genus Aspergillus

“…Bioinformatic analysis in the present study revealed that the majority of the aspergilli contain one gene encoding an FNT family protein, but a few species ( A. flavus , A. oryzae , A. sidowii , A. fischeri , and A. versicolor ) have two such genes. In A. nidulans , the FNT protein NitA has been shown to be a high affinity but low capacity nitrite transporter compared to NNP proteins and is expressed under the same conditions as the NNP proteins [ 191 ].…”

Characterization of nitrite uptake in Arabidopsis thaliana: evidence for a nitrite‐specific transporter

“…This finding signifies the existence of an additional transport mechanism for nitrite, independent of the AtNAR2.1 gene. Likewise, in A. nidulans the NitA gene encodes a nitrite-specific transporter that appears to function independently of any NAR2-like polypeptide (Unkles et al, 2011), and also the nitrite transport system III in C. reinhardtii (coded by CrNRT2.3) which is independent of the CrNAR2 gene (Rexach et al, 1999). Kotur et al (2012) reported that all NRT2 transporters in Arabidopsis, with the exception of AtNRT2.7, require NAR2.1 for functional nitrate transport.…”

“…The uptake of nitrite from the external environment may occur through nitrate–nitrite bispecific transporters, examples of which are NRT2.1/NAR2 in C. reinhardtii (Galvan et al ., ), NARU in Escherichia.coli (Jia et al ., ), NRTA and NRTB in Aspergillus nidulans (Wang et al ., ), or by ‐specific transporters as reported in C. reinhardtii (Galvan et al ., ), E. coli (Jia & Cole, ; Jia et al ., ), Hansenula polymorpha (Serrani & Berardi, ), A. nidulans (Wang et al ., ; Unkles et al ., ), Neurospora crassa (Gao‐Rubinelli & Marzluf, ) and Nostoc ANTH (Bhattacharya et al ., ). Nitrite transporters in A. nidulans (NitA) and E. coli (NirC) belong to a family of formate–nitrite transporters.…”

The C-terminal Six Amino Acids of the FNT Channel FocA Are Required for Formate Translocation But Not Homopentamer Integrity

“…Meanwhile, genome analyses have revealed that the FNT superfamily of anion channels is widespread in bacteria and archaea, with thousands of examples ( Waight et al, 2013 ). FNT proteins are also found in certain single-celled eukarya ( Unkles et al, 2011 ; Marchetti et al, 2015 ; Wu et al, 2015 ; Yamano et al, 2015 ). Many of the microorganisms in which these FNT proteins are found are pathogens ( Waight et al, 2010 ; Czyzewski and Wang, 2012 ; Lü et al, 2012b ; Marchetti et al, 2015 ; Wu et al, 2015 ).…”