Mechanisms of chlorate toxicity and resistance in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that often encounters hypoxic/anoxic environments within the host, which increases its tolerance to many conventional antibiotics. Toward identifying novel treatments, we explored the therapeutic potential of chlorate, a pro-drug that kills hypoxic/anoxic, antibiotic-tolerant P. aeruginosa populations. While chlorate itself is relatively nontoxic, it is enzymatically reduced to the toxic oxidizing agent, chlorite, by hypoxically induced nitrate reductase. To better assess chlorate's therapeutic potential, we investigated mechanisms of chlorate toxicity and resistance in P. aeruginosa. We used transposon mutagenesis to identify genes that alter P. aeruginosa fitness during chlorate treatment, finding that methionine sulfoxide reductases (Msr), which repair oxidized methionine residues, support survival during chlorate stress. Chlorate treatment leads to proteome-wide methionine oxidation, which is exacerbated in a ∆msrA∆msrB strain. In response to chlorate, P. aeruginosa upregulates proteins involved in a wide range of functions, including metabolism, DNA replication/repair, protein repair, transcription, and translation, and these newly synthesized proteins are particularly vulnerable to methionine oxidation. The addition of exogenous methionine partially rescues P. aeruginosa survival during chlorate treatment, suggesting that widespread methionine oxidation contributes to death. Finally, we found that mutations that decrease nitrate reductase activity are a common mechanism of chlorate resistance.

Isolation and identification of chlorate-reducing Hafnia sp. from milk.

Chlorate has become a concern in the food and beverage sector, related to chlorine sanitizers in industrial food production and water treatment. It is of particular concern to regulatory bodies due to the negative health effects of chlorate exposure. This study investigated the fate of chlorate in raw milk and isolated bacterial strains of interest responsible for chlorate breakdown. Unpasteurized milk was demonstrated to have a chlorate-reducing capacity, breaking down enriched chlorate to undetectable levels in 11 days. Further enrichment and isolation using conditions specific to chlorate-reducing bacteria successfully isolated three distinct strains of Hafnia paralvei. Chlorate-reducing bacteria were observed to grow in a chlorate-enriched medium with lactate as an electron donor. All isolated strains were demonstrated to reduce chlorate in liquid medium; however, the exact mechanism of chlorate degradation was not definitively identified in this study.

Impacts of co-contaminants and dilution on perchlorate biodegradation using various carbon sources.

This research investigates the biodegradation of perchlorate in the presence of the co-contaminants nitrate and chlorate using soluble and slow-release carbon sources. In addition, the impact of bio-augmentation and dilution, which results in lower total dissolved salts (TDS) and contaminant levels, is examined. Laboratory microcosms were conducted using actual groundwater and soils from a contaminated aquifer. The results revealed that both soluble and slow-release carbon sources support biodegradation of contaminants in the sequence nitrate > chlorate > perchlorate. Degradation rates, including and excluding lag times, revealed that the overall impact of the presence of co-contaminants depends on degradation kinetics and the relative concentrations of the contaminants. When the lag time caused by the presence of the co-contaminants is considered, the degradation rates for chlorate and perchlorate were two to three times slower. The results also show that dilution causes lower initial contaminant concentrations, and consequently, slower degradation rates, which is not desirable. On the other hand, the dilution resulting from the injection of amendments to support remediation promotes desirably lower salinity levels. However, the salinity associated with the presence of sulfate does not inhibit biodegradation. The naturally occurring bacteria were able to support the degradation of all contaminants. Bio-augmentation was effective only in diluted microcosms. Proteobacteria and Firmicutes were the dominant phyla identified in the microcosms.

Cytoprotective effect of taurine against sodium chlorate-induced oxidative damage in human red blood cells: an ex vivo study.

Sodium chlorate (NaClO3) is a common non-selective herbicide that is also used in paper and pulp mills and is produced as a by-product during drinking water disinfection by chlorine dioxide. Here, we report the effect of dietary antioxidant taurine on NaClO3-induced cytotoxicity in human red blood cells (RBC). RBC were treated with 5 mM NaClO3, either alone or in presence of 1, 2.5 and 5.0 mM taurine. Incubation of RBC with NaClO3 alone caused hemolysis, increased oxidation of lipids and proteins, methemogobin level and decreased total sulfhydryl and glutathione content. It lowered the activities of antioxidant enzymes thioredoxin reductase, glutathione peroxidase, catalase and glutathione reductase, while Cu-Zn superoxide dismutase activity was increased. The antioxidant capacity of RBC was impaired. This strongly suggests that NaClO3 causes the induction of oxidative stress condition in RBC. The specific activities of lactate dehydrogenase, glucose 6-phosphate dehydrogenase and plasma membrane bound enzymes, were also greatly altered. However, prior treatment of RBC with taurine conferred significant protection against NaClO3-induced oxidative damage and also improved the antioxidant defence system of cells. These results were supported by electron microscopy images of RBC. Treatment with NaClO3 alone converted the normal biconcave discoidal RBC to acanthocytes and echinocytes but this transformation was greatly prevented in the presence of taurine. Thus, taurine mitigates the cytotoxicity of NaClO3 in human RBC and can function as an effective chemoprotectant.

Arabidopsis PIC30 encodes a major facilitator superfamily transporter responsible for the uptake of picolinate herbicides.

Picloram is an auxinic herbicide that is widely used for controlling broad leaf weeds. However, its mechanism of transport into plants is poorly understood. In a genetic screen for picloram resistance, we identified three Arabidopsis mutant alleles of PIC30 (PICLORAM RESISTANT30) that are specifically resistant to picolinates, but not to other auxins. PIC30 is a previously uncharacterized gene that encodes a major facilitator superfamily (MFS) transporter. Similar to most members of MFS, PIC30 contains 12 putative transmembrane domains, and PIC30-GFP fusion protein selectively localizes to the plasma membrane. In planta transport assays demonstrate that PIC30 specifically transports picloram, but not indole-3-acetic acid (IAA). Functional analysis of Xenopus laevis oocytes injected with PIC30 cRNA demonstrated PIC30 mediated transport of picloram and several anions, including nitrate and chloride. Consistent with these roles of PIC30, three allelic pic30 mutants are selectively insensitive to picolinate herbicides, while pic30-3 is also defective in chlorate (analogue of nitrate) transport and also shows reduced uptake of 15NO3- . Overexpression of PIC30 fully complements both picloram and chlorate insensitive phenotypes of pic30-3. Despite the continued use of picloram as an herbicide, a transporter for picloram was not known until now. This work provides insight into the mechanisms of plant resistance to picolinate herbicides and also shed light on the possible endogenous function of PIC30 protein.

(Per)chlorate in Biology on Earth and Beyond.

Respiration of perchlorate and chlorate [collectively, (per)chlorate] was only recognized in the last 20 years, yet substantial advances have been made in our understanding of the underlying metabolisms. Although it was once considered solely anthropogenic, pervasive natural sources, both terrestrial and extraterrestrial, indicate an ancient (per)chlorate presence across our solar system. These discoveries stimulated interest in (per)chlorate microbiology, and the application of advanced approaches highlights exciting new facets. Forward and reverse genetics revealed new information regarding underlying molecular biology and associated regulatory mechanisms. Structural and functional analysis characterized core enzymes and identified novel reaction sequences. Comparative genomics elucidated evolutionary aspects, and stress analysis identified novel response mechanisms to reactive chlorine species. Finally, systems biology identified unique metabolic versatility and novel mechanisms of (per)chlorate respiration, including symbiosis and a hybrid enzymatic-abiotic metabolism. While many published studies focus on (per)chlorate and their basic metabolism, this review highlights seminal advances made over the last decade and identifies new directions and potential novel applications.

Comparative Transcriptomics of Rice Genotypes with Contrasting Responses to Nitrogen Stress Reveals Genes Influencing Nitrogen Uptake through the Regulation of Root Architecture.

The indiscriminate use of nitrogenous fertilizers continues unabated for commercial crop production, resulting in air and water pollution. The development of rice varieties with enhanced nitrogen use efficiency (NUE) will require a thorough understanding of the molecular basis of a plant's response to low nitrogen (N) availability. The global expression profiles of root tissues collected from low and high N treatments at different time points in two rice genotypes, Pokkali and Bengal, with contrasting responses to N stress and contrasting root architectures were examined. Overall, the number of differentially expressed genes (DEGs) in Pokkali (indica) was higher than in Bengal (japonica) during low N and early N recovery treatments. Most low N DEGs in both genotypes were downregulated whereas early N recovery DEGs were upregulated. Of these, 148 Pokkali-specific DEGs might contribute to Pokkali's advantage under N stress. These DEGs included transcription factors and transporters and were involved in stress responses, growth and development, regulation, and metabolism. Many DEGs are co-localized with quantitative trait loci (QTL) related to root growth and development, chlorate-resistance, and NUE. Our findings suggest that the superior growth performance of Pokkali under low N conditions could be due to the genetic differences in a diverse set of genes influencing N uptake through the regulation of root architecture.

Alicycliphilus: current knowledge and potential for bioremediation of xenobiotics.

Alicycliphilus is a promising candidate for participating in the development of novel xenobiotics bioremediation processes. Members of the Alicycliphilus genus are environmental bacteria mostly found in polluted sites such as landfills and contaminated watercourses, and in sewage sludges from wastewater treatment plants. They exhibit a versatile metabolism and the ability to use oxygen, nitrate and chlorate as terminal electron acceptors, which allow them to biodegrade xenobiotics under oxic or anoxic conditions. Pure cultures of Alicycliphilus strains are able to biodegrade some pollutants such as industrial solvents (acetone, cyclohexanol and N-methylpyrrolidone), aromatic hydrocarbons (benzene, toluene and anthracene), as well as polyurethane varnishes and foams, and they can even transform Cr(VI) to Cr(III). In addition, Alicycliphilus has also been identified in bacterial communities involved in wastewater treatment plants for denitrification, and the degradation of emerging pollutants such as triclosan, nonylphenol, N-heterocyclic aromatic compounds (indole and quinoline), and antibiotics (tetracycline and oxytetracycline). This work summarizes the current knowledge on the Alicycliphilus genus, describing its different metabolic characteristics, focusing on its xenobiotic biodegradation abilities and examining the distinct pathways and molecular bases that sustain them. We also discuss the progress made in genetic manipulation and 'omics' analyses, as well as Alicycliphilus participation in novel bioremediation strategies.

Concurrent Haloalkanoate Degradation and Chlorate Reduction by Pseudomonas chloritidismutans AW-1T.

Haloalkanoates are environmental pollutants that can be degraded aerobically by microorganisms producing hydrolytic dehalogenases. However, there is a lack of information about the anaerobic degradation of haloalkanoates. Genome analysis of Pseudomonas chloritidismutans AW-1T, a facultative anaerobic chlorate-reducing bacterium, showed the presence of two putative haloacid dehalogenase genes, the l-DEX gene and dehI, encoding an l-2-haloacid dehalogenase (l-DEX) and a halocarboxylic acid dehydrogenase (DehI), respectively. Hence, we studied the concurrent degradation of haloalkanoates and chlorate as a yet-unexplored trait of strain AW-1T The deduced amino acid sequences of l-DEX and DehI revealed 33 to 37% and 26 to 86% identities with biochemically/structurally characterized l-DEX and the d- and dl-2-haloacid dehalogenase enzymes, respectively. Physiological experiments confirmed that strain AW-1T can grow on chloroacetate, bromoacetate, and both l- and d-α-halogenated propionates with chlorate as an electron acceptor. Interestingly, growth and haloalkanoate degradation were generally faster with chlorate as an electron acceptor than with oxygen as an electron acceptor. In line with this, analyses of l-DEX and DehI dehalogenase activities using cell-free extract (CFE) of strain AW-1T grown on dl-2-chloropropionate under chlorate-reducing conditions showed up to 3.5-fold higher dehalogenase activity than the CFE obtained from AW-1T cells grown on dl-2-chloropropionate under aerobic conditions. Reverse transcription-quantitative PCR showed that the l-DEX gene was expressed constitutively independently of the electron donor (haloalkanoates or acetate) or acceptor (chlorate or oxygen), whereas the expression of dehI was induced by haloalkanoates. Concurrent degradation of organic and inorganic halogenated compounds by strain AW-1T represents a unique metabolic capacity in a single bacterium, providing a new piece of the puzzle of the microbial halogen cycle.IMPORTANCE Halogenated organic and inorganic compounds are important environmental pollutants that have carcinogenic and genotoxic effects on both animals and humans. Previous research studied the degradation of organic and inorganic halogenated compounds separately but not concurrently. This study shows concurrent degradation of halogenated alkanoates and chlorate as an electron donor and acceptor, respectively, coupled to growth in a single bacterium, Pseudomonas chloritidismutans AW-1T Hence, besides biogenesis of molecular oxygen from chlorate reduction enabling a distinctive placement of strain AW-1T between aerobic and anaerobic microorganisms, we can now add another unique metabolic potential of this bacterium to the roster. The degradation of different halogenated compounds under anoxic conditions by a single bacterium is also of interest for the natural halogen cycle in different aquatic and terrestrial ecosystems where ample natural production of halogenated compounds has been documented.

Characterisation of chlorate reduction in the haloarchaeon Haloferax mediterranei.

BACKGROUND: Haloferax mediterranei is a denitrifying haloarchaeon using nitrate as a respiratory electron acceptor under anaerobic conditions in a reaction catalysed by pNarGH. Other ions such as bromate, perchlorate and chlorate can also be reduced. METHODS: Hfx. mediterranei cells were grown anaerobically with nitrate as electron acceptor and chlorate reductase activity measured in whole cells and purified nitrate reductase. RESULTS: No genes encoding (per)chlorate reductases have been detected either in the Hfx. mediterranei genome or in other haloarchaea. However, a gene encoding a chlorite dismutase that is predicted to be exported across the cytoplasmic membrane has been identified in Hfx. mediterranei genome. Cells did not grow anaerobically in presence of chlorate as the unique electron acceptor. However, cells anaerobically grown with nitrate and then transferred to chlorate-containing growth medium can grow a few generations. Chlorate reduction by the whole cells, as well as by pure pNarGH, has been characterised. No clear chlorite dismutase activity could be detected. CONCLUSIONS: Hfx. mediterranei pNarGH has its active site on the outer-face of the cytoplasmic membrane and reacts with chlorate and perchlorate. Biochemical characterisation of this enzymatic activity suggests that Hfx. mediterranei or its pure pNarGH could be of great interest for waste water treatments or to better understand biological chlorate reduction in early Earth or Martian environments. GENERAL SIGNIFICANCE: Some archaea species reduce (per)chlorate. However, results here presented as well as those recently reported by Liebensteiner and co-workers [1] suggest that complete perchlorate reduction in archaea follows different rules in terms of biological reactions.

Genetic dissection of chlorate respiration in Pseudomonas stutzeri†PDA reveals syntrophic (per)chlorate reduction.

Genes important for growth of Pseudomonas stutzeri PDA on chlorate were identified using a randomly DNA bar-coded transposon mutant library. During chlorate reduction, mutations in genes encoding the chlorate reductase clrABC, predicted molybdopterin cofactor chaperon clrD, molybdopterin biosynthesis and two genes of unknown function (clrE, clrF) had fitness defects in pooled mutant assays (Bar-seq). Markerless in-frame deletions confirmed that clrA, clrB and clrC were essential for chlorate reduction, while clrD, clrE and clrF had less severe growth defects. Interestingly, the key detoxification gene cld was essential for chlorate reduction in isogenic pure culture experiments, but showed only minor fitness defects in Bar-seq experiments. We hypothesized this was enabled through chlorite dismutation by the community, as most strains in the Bar-seq library contained an intact cld. In support of this, Δcld grew with wild-type PDA or ΔclrA, and purified Cld also restored growth to the Δcld mutant. Expanding on this, wild-type PDA and a Δcld mutant of the perchlorate reducer Azospira suillum PS grew on perchlorate in co-culture, but not individually. These results demonstrate that co-occurrence of cld and a chloroxyanion reductase within a single organism is not necessary and raises the possibility of syntrophic (per)chlorate respiration in the environment.

Genome and proteome analysis of Pseudomonas chloritidismutans†AW-1T that grows on n-decane with chlorate or oxygen as electron acceptor.

Growth of Pseudomonas chloritidismutans AW-1T on C7 to C12 n-alkanes with oxygen or chlorate as electron acceptor was studied by genome and proteome analysis. Whole genome shotgun sequencing resulted in a 5 Mbp assembled sequence with a G + C content of 62.5%. The automatic annotation identified 4767 protein-encoding genes and a putative function could be assigned to almost 80% of the predicted proteins. The distinct phylogenetic position of P. chloritidismutans AW-1T within the Pseudomonas stutzeri cluster became clear by comparison of average nucleotide identity values of sequenced genomes. Analysis of the proteome of P. chloritidismutans AW-1T showed the versatility of this bacterium to adapt to aerobic and anaerobic growth conditions with acetate or n-decane as substrates. All enzymes involved in the alkane oxidation pathway were identified. An alkane monooxygenase was detected in n-decane-grown cells, but not in acetate-grown cells. The enzyme was found when grown in the presence of oxygen or chlorate, indicating that under both conditions an oxygenase-mediated pathway is employed for alkane degradation. Proteomic and biochemical data also showed that both chlorate reductase and chlorite dismutase are constitutively present, but most abundant under chlorate-reducing conditions.

Chlorate reduction in Shewanella algae†ACDC is a recently acquired metabolism characterized by gene loss, suboptimal regulation and oxidative stress.

Previous work on respiratory chlorate reduction has biochemically identified the terminal reductase ClrABC and the chlorite detoxifying enzyme Cld. In Shewanella algae ACDC, genes encoding these enzymes reside on composite transposons whose core we refer to as the chlorate reduction composite transposon interior (CRI). To better understand this metabolism in ACDC, we used RNA-seq and proteomics to predict carbon and electron flow during chlorate reduction and posit that formate is an important electron carrier with lactate as the electron donor, but that NADH predominates on acetate. Chlorate-specific transcription of electron transport chain components or the CRI was not observed, but clr and cld transcription was attenuated by oxygen. The major chlorate-specific response related to oxidative stress and was indicative of reactive chlorine species production. A genetic system based on rpsL-streptomycin counter selection was developed to further dissect the metabolism, but ACDC readily lost the CRI via homologous recombination of the composite transposon's flanking insertion sequences. An engineered strain containing a single chromosomal CRI did not grow on chlorate, but overexpression of cld and its neighbouring cytochrome c restored growth. We postulate that the recently acquired CRI underwent copy-number expansion to circumvent insufficient expression of key genes in the pathway.

High-affinity nitrate/nitrite transporters NrtA and NrtB of Aspergillus nidulans exhibit high specificity and different inhibitor sensitivity.

The NrtA and NrtB nitrate transporters are paralogous members of the major facilitator superfamily in Aspergillus nidulans. The availability of loss-of-function mutations allowed individual investigation of the specificity and inhibitor sensitivity of both NrtA and NrtB. In this study, growth response tests were carried out at a growth-limiting concentration of nitrate (1 mM) as the sole nitrogen source, in the presence of a number of potential nitrate analogues at various concentrations, to evaluate their effect on nitrate transport. Both chlorate and chlorite inhibited fungal growth, with chlorite exerting the greater inhibition. The main transporter of nitrate, NrtA, proved to be more sensitive to chlorate than the minor transporter, NrtB. Similarly, the cation caesium was shown to exert differential effects, strongly inhibiting the activity of NrtB, but not NrtA. In contrast, no inhibition of nitrate uptake by NrtA or NrtB transporters was observed in either growth tests or uptake assays in the presence of bicarbonate, formate, malonate or oxalate (sulphite could not be tested in uptake assays owing to its reaction with nitrate), indicating significant specificity of nitrate transport. Kinetic analyses of nitrate uptake revealed that both chlorate and chlorite inhibited NrtA competitively, while these same inhibitors inhibited NrtB in a non-competitive fashion. The caesium ion appeared to inhibit NrtA in a non-competitive fashion, while NrtB was inhibited uncompetitively. The results provide further evidence of the distinctly different characteristics as well as the high specificity of nitrate uptake by these two transporters.

Phenotypic and genotypic description of Sedimenticola selenatireducens strain CUZ, a marine (per)chlorate-respiring gammaproteobacterium, and its close relative the chlorate-respiring Sedimenticola strain NSS.

Two (per)chlorate-reducing bacteria, strains CUZ and NSS, were isolated from marine sediments in Berkeley and San Diego, CA, respectively. Strain CUZ respired both perchlorate and chlorate [collectively designated (per)chlorate], while strain NSS respired only chlorate. Phylogenetic analysis classified both strains as close relatives of the gammaproteobacterium Sedimenticola selenatireducens. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) preparations showed the presence of rod-shaped, motile cells containing one polar flagellum. Optimum growth for strain CUZ was observed at 25 to 30 °C, pH 7, and 4% NaCl, while strain NSS grew optimally at 37 to 42 °C, pH 7.5 to 8, and 1.5 to 2.5% NaCl. Both strains oxidized hydrogen, sulfide, various organic acids, and aromatics, such as benzoate and phenylacetate, as electron donors coupled to oxygen, nitrate, and (per)chlorate or chlorate as electron acceptors. The draft genome of strain CUZ carried the requisite (per)chlorate reduction island (PRI) for (per)chlorate respiration, while that of strain NSS carried the composite chlorate reduction transposon responsible for chlorate metabolism. The PRI of strain CUZ encoded a perchlorate reductase (Pcr), which reduced both perchlorate and chlorate, while the genome of strain NSS included a gene for a distinct chlorate reductase (Clr) that reduced only chlorate. When both (per)chlorate and nitrate were present, (per)chlorate was preferentially utilized if the inoculum was pregrown on (per)chlorate. Historically, (per)chlorate-reducing bacteria (PRB) and chlorate-reducing bacteria (CRB) have been isolated primarily from freshwater, mesophilic environments. This study describes the isolation and characterization of two highly related marine halophiles, one a PRB and the other a CRB, and thus broadens the known phylogenetic and physiological diversity of these unusual metabolisms.

Microbial metabolism of oxochlorates: a bioenergetic perspective.

The microbial metabolism of oxochlorates is part of the biogeochemical cycle of chlorine. Organisms capable of growth using perchlorate or chlorate as respiratory electron acceptors are also interesting for applications in biotreatment of oxochlorate-containing effluents or bioremediation of contaminated areas. In this review, we discuss the reactions of oxochlorate respiration, the corresponding enzymes, and the relation to respiratory electron transport that can contribute to a proton gradient across the cell membrane. Enzymes specific for oxochlorate respiration are oxochlorate reductases and chlorite dismutase. The former belong to DMSO reductase family of molybdenum-containing enzymes. The heme protein chlorite dismutase, which decomposes chlorite into chloride and molecular oxygen, is only distantly related to other proteins with known functions. Pathways for electron transport may be different in perchlorate and chlorate reducers, but appear in both cases to be similar to pathways found in other respiratory systems. This article is part of a Special Issue entitled: Evolutionary aspects bioenergetic systems.

Microbial preference for chlorate over perchlorate under simulated shallow subsurface Mars-like conditions.

The Martian surface and shallow subsurface lacks stable liquid water, yet hygroscopic salts in the regolith may enable the transient formation of liquid brines. This study investigated the combined impact of water scarcity, UV exposure, and regolith depth on microbial survival under Mars-like environmental conditions. Both vegetative cells of Debaryomyces hansenii and Planococcus halocryophilus, alongside with spores of Aspergillus niger, were exposed to an experimental chamber simulating Martian environmental conditions (constant temperatures of about - 11 °C, low pressure of approximately 6 mbar, a CO2 atmosphere, and 2 h of daily UV irradiation). We evaluated colony-forming units (CFU) and water content at three different regolith depths before and after exposure periods of 3 and 7 days, respectively. Each organism was tested under three conditions: one without the addition of salts to the regolith, one containing sodium chlorate, and one with sodium perchlorate. Our results reveal that the residual water content after the exposure experiments increased with regolith depth, along with the organism survival rates in chlorate-containing and salt-free samples. The survival rates of the three organisms in perchlorate-containing regolith were consistently lower for all organisms and depths compared to chlorate, with the most significant difference being observed at a depth of 10-12 cm, which corresponds to the depth with the highest residual water content. The postulated reason for this is an increase in the salt concentration at this depth due to the freezing of water, showing that for these organisms, perchlorate brines are more toxic than chlorate brines under the experimental conditions. This underscores the significance of chlorate salts when considering the habitability of Martian environments.

Double knockout of two target genes via genome co-editing using a nitrate transporter gene nrtA and a putative thiamine transporter gene thiI as selectable markers in Aspergillus oryzae.

Genome co-editing technology is effective in breeding filamentous fungi for applications in the fermentation industry, achieving site-directed mutagenesis, the status of non-genetically modified organisms (non-GMOs), and wild-type-like growth phenotype. Prior to this study, thiI gene was found as a selectable marker for such genome co-editing in the filamentous fungus Aspergillus oryzae, while it cannot be reused via marker recycling. Therefore, we aimed to identify another marker gene to knock out another target gene via genome co-editing in A. oryzae. In this study, we focused on the membrane transporter gene nrtA (AO090012000623), which promotes uptake of nitrate (NO3-). It is known that, in nrtA knockout strain, chlorate (ClO3-), an analog of nitrate with antifungal activity, cannot be imported into the cytosol, which enables the mutant to grow in the presence of chlorate. Based on this information, knockout of the target gene wA was attempted using both nrtA- and wA-specific single-guide RNAs via genome co-editing with KClO3 supplementation in A. oryzae laboratory strain RIB40 and industrial strain KBN616. Resultantly, wA knockout mutant was generated, and nrtA was identified as a selectable marker. Moreover, this genome co-editing system using nrtA was compatible with that using thiI, and thus, a double knockout mutant of two target genes wA and yA was constructed in RIB40 while maintaining non-GMO status and wild-type-like growth. As nrtA homologs have been found in several industrial Aspergillus species, genome co-editing using homolog genes as selectable markers is plausible, which would contribute to the widespread breeding of industrial strains of Aspergilli.

Substrate-dependent modulation of the enzymatic catalytic activity: reduction of nitrate, chlorate and perchlorate by respiratory nitrate reductase from Marinobacter hydrocarbonoclasticus 617.

The respiratory nitrate reductase complex (NarGHI) from Marinobacter hydrocarbonoclasticus 617 (Mh, formerly Pseudomonas nautica 617) catalyzes the reduction of nitrate to nitrite. This reaction is the first step of the denitrification pathway and is coupled to the quinone pool oxidation and proton translocation to the periplasm, which generates the proton motive force needed for ATP synthesis. The Mh NarGH water-soluble heterodimer has been purified and the kinetic and redox properties have been studied through in-solution enzyme kinetics, protein film voltammetry and spectropotentiometric redox titration. The kinetic parameters of Mh NarGH toward substrates and inhibitors are consistent with those reported for other respiratory nitrate reductases. Protein film voltammetry showed that at least two catalytically distinct forms of the enzyme, which depend on the applied potential, are responsible for substrate reduction. These two forms are affected differentially by the oxidizing substrate, as well as by pH and inhibitors. A new model for the potential dependence of the catalytic efficiency of Nars is proposed.

Functional assessment of the Medicago truncatula NIP/LATD protein demonstrates that it is a high-affinity nitrate transporter.

The Medicago truncatula NIP/LATD (for Numerous Infections and Polyphenolics/Lateral root-organ Defective) gene encodes a protein found in a clade of nitrate transporters within the large NRT1(PTR) family that also encodes transporters of dipeptides and tripeptides, dicarboxylates, auxin, and abscisic acid. Of the NRT1(PTR) members known to transport nitrate, most are low-affinity transporters. Here, we show that M. truncatula nip/latd mutants are more defective in their lateral root responses to nitrate provided at low (250 µm) concentrations than at higher (5 mm) concentrations; however, nitrate uptake experiments showed no discernible differences in uptake in the mutants. Heterologous expression experiments showed that MtNIP/LATD encodes a nitrate transporter: expression in Xenopus laevis oocytes conferred upon the oocytes the ability to take up nitrate from the medium with high affinity, and expression of MtNIP/LATD in an Arabidopsis chl1(nrt1.1) mutant rescued the chlorate susceptibility phenotype. X. laevis oocytes expressing mutant Mtnip-1 and Mtlatd were unable to take up nitrate from the medium, but oocytes expressing the less severe Mtnip-3 allele were proficient in nitrate transport. M. truncatula nip/latd mutants have pleiotropic defects in nodulation and root architecture. Expression of the Arabidopsis NRT1.1 gene in mutant Mtnip-1 roots partially rescued Mtnip-1 for root architecture defects but not for nodulation defects. This suggests that the spectrum of activities inherent in AtNRT1.1 is different from that possessed by MtNIP/LATD, but it could also reflect stability differences of each protein in M. truncatula. Collectively, the data show that MtNIP/LATD is a high-affinity nitrate transporter and suggest that it could have another function.