UNASSIGNED: Glutamine synthetase (GS), glutamate synthase (GOGAT), and nitrate reductase (NR) are key enzymes involved in nitrogen assimilation and metabolism in plants. However, the systematic analysis of these gene families lacked reports in soybean (Glycine max (L.) Merr.), one of the most important crops worldwide.
UNASSIGNED: In this study, we performed genome-wide identification and characterization of GS, GOGAT, and NR genes in soybean under abiotic and nitrogen stress conditions.
UNASSIGNED: We identified a total of 10 GS genes, six GOGAT genes, and four NR genes in the soybean genome. Phylogenetic analysis revealed the presence of multiple isoforms for each gene family, indicating their functional diversification. The distribution of these genes on soybean chromosomes was uneven, with segmental duplication events contributing to their expansion. Within the nitrogen assimilation genes (NAGs) group, there was uniformity in the exon-intron structure and the presence of conserved motifs in NAGs. Furthermore, analysis of cis-elements in NAG promoters indicated complex regulation of their expression. RT-qPCR analysis of seven soybean NAGs under various abiotic stresses, including nitrogen deficiency, drought-nitrogen, and salinity, revealed distinct regulatory patterns. Most NAGs exhibited up-regulation under nitrogen stress, while diverse expression patterns were observed under salt and drought-nitrogen stress, indicating their crucial role in nitrogen assimilation and abiotic stress tolerance. These findings offer valuable insights into the genomic organization and expression profiles of GS, GOGAT, and NR genes in soybean under nitrogen and abiotic stress conditions. The results have potential applications in the development of stress-resistant soybean varieties through genetic engineering and breeding.

BACKGROUND: On a global scale, India holds the distinction of having the greatest number of tuberculosis (TB) cases caused by Mycobacterium tuberculosis (MTB) complex. The study aimed at evaluating the sensitivity, specificity, accuracy, cost, rapidity, and feasibility of the performance of the colorimetric nitrate reductase-based antibiotic susceptibility (CONRAS) test against the indirect proportion method (IPM) on Lowenstein-Jensen media as the gold standard.
METHODS: A comparative cross-sectional study was performed on 51 MTB isolates. Fresh subcultures were used for drug susceptibility testing by IPM on the Lowenstein-Jensen medium and the CONRAS method in liquid medium. Quality control for drug susceptibility testing was done using a known sensitive strain of MTB (H37Rv) and strains resistant to both isoniazid (INH) and rifampicin (RIF) - multidrug-resistant (MDR), mono-resistant to RIF, streptomycin (STM), and ethambutol (EMB). Statistical analysis was performed using MedCalc software (Version 20.027).
RESULTS: CONRAS, carried out in microfuge tubes, was cost-efficient and easy to perform/interpret with most results being available in 10 days compared to 42 days in the case of IPM. The sensitivity, specificity, and accuracy of RIF and INH were 100%, 97.37%, and 98.04 and 93.33%, 97.59%, and 96.08%, respectively, which translates into an almost perfect agreement between the two methods as indicated by κ value of 0.905 and 0.949, respectively, for the two drugs. The performance of CONRAS was less satisfactory for STM and EMB when compared to IPM.
CONCLUSIONS: CONRAS may serve as a useful test for the detection of MDR-TB because of its accuracy, low cost, ease of performance/interpretation, and rapidity when compared to IPM on LJ medium. It does not involve the use of expensive reagents and equipment, as is the case with molecular methods like GeneXpert and line probe assay, making it a suitable option for the detection of MDR-TB in resource-poor settings.

Mycobacterium abscessus (Mab) is an opportunistic pathogen afflicting individuals with underlying lung disease such as Cystic Fibrosis (CF) or immunodeficiencies. Current treatment strategies for Mab infections are limited by its inherent antibiotic resistance and limited drug access to Mab in its in vivo niches resulting in poor cure rates of 30-50%. Mab\'s ability to survive within macrophages, granulomas and the mucus laden airways of the CF lung requires adaptation via transcriptional remodeling to counteract stresses like hypoxia, increased levels of nitrate, nitrite, and reactive nitrogen intermediates. Mycobacterium tuberculosis (Mtb) is known to coordinate hypoxic adaptation via induction of respiratory nitrate assimilation through the nitrate reductase narGHJI. Mab, on the other hand, does not encode a respiratory nitrate reductase. In addition, our recent study of the transcriptional responses of Mab to hypoxia revealed marked down-regulation of a locus containing putative nitrate assimilation genes, including the orphan response regulator nnaR (nitrate/nitrite assimilation regulator). These putative nitrate assimilation genes, narK3 (nitrate/nitrite transporter), nirBD (nitrite reductase), nnaR, and sirB (ferrochelatase) are arranged contiguously while nasN (assimilatory nitrate reductase identified in this work) is encoded in a different locus. Absence of a respiratory nitrate reductase in Mab and down-regulation of nitrogen metabolism genes in hypoxia suggest interplay between hypoxia adaptation and nitrate assimilation are distinct from what was previously documented in Mtb. The mechanisms used by Mab to fine-tune the transcriptional regulation of nitrogen metabolism in the context of stresses e.g. hypoxia, particularly the role of NnaR, remain poorly understood. To evaluate the role of NnaR in nitrate metabolism we constructed a Mab nnaR knockout strain (MabΔnnaR ) and complement (MabΔnnaR+C ) to investigate transcriptional regulation and phenotypes. qRT-PCR revealed NnaR is necessary for regulating nitrate and nitrite reductases along with a putative nitrate transporter. Loss of NnaR compromised the ability of Mab to assimilate nitrate or nitrite as sole nitrogen sources highlighting its necessity. This work provides the first insights into the role of Mab NnaR setting a foundation for future work investigating NnaR\'s contribution to pathogenesis.

Amaryllidaceae alkaloids are structurally diverse natural products with a wide range biological properties, and based on the partial identification of the biosynthetic enzymes, norbelladine would be a common intermediate in the biosynthetic pathways. Previous studies suggested that norbelladine synthase (NBS) catalyzed the condensation reaction of 3,4-dihydroxybenzaldehyde and tyramine to form norcraugsodine, and subsequently, noroxomaritidine/norcraugsodine reductase (NR) catalyzed the nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reduction of norcraugsodine to generate norbelladine. However, recent studies have highlighted possible alternative Amaryllidaceae alkaloid biosynthetic pathways via the formation of isovanillin and vanillin from the 4-O- and 3-O-methylation reactions of 3,4-dihydroxybenzaldehyde, respectively. Herein, we focused on NpsNBS and NpsNR, which were initially identified from Narcissus pseudonarcissus, and explored their substrate recognition tolerance by performing condensation reactions of tyramine with various benzaldehyde derivatives, to shed light on the Amaryllidaceae alkaloid biosynthetic pathway from the viewpoint of the enzymatic properties. The assays revealed that both NpsNBS and NpsNR lacked the abilities to produce 4\'-O- and 3\'-O-methylnorbelladine from isovanillin and vanillin with tyramine, respectively. These observations thus suggested that Amaryllidaceae alkaloids are biosynthesized from norbelladine, formed through the condensation/reduction reaction of 3,4-dihydroxybenzaldehyde with tyramine.

Periplasmic nitrate reductase NapA from Campylobacter jejuni (C. jejuni) contains a molybdenum cofactor (Moco) and a 4Fe-4S cluster and catalyzes the reduction of nitrate to nitrite. The reducing equivalent required for the catalysis is transferred from NapC → NapB → NapA. The electron transfer from NapB to NapA occurs through the 4Fe-4S cluster in NapA. C. jejuni NapA has a conserved lysine (K79) between the Mo-cofactor and the 4Fe-4S cluster. K79 forms H-bonding interactions with the 4Fe-4S cluster and connects the latter with the Moco via an H-bonding network. Thus, it is conceivable that K79 could play an important role in the intramolecular electron transfer and the catalytic activity of NapA. In the present study, we show that the mutation of K79 to Ala leads to an almost complete loss of activity, suggesting its role in catalytic activity. The inhibition of C. jejuni NapA by cyanide, thiocyanate, and azide has also been investigated. The inhibition studies indicate that cyanide inhibits NapA in a non-competitive manner, while thiocyanate and azide inhibit NapA in an uncompetitive manner. Neither inhibition mechanism involves direct binding of the inhibitor to the Mo-center. These results have been discussed in the context of the loss of catalytic activity of NapA K79A variant and a possible anion binding site in NapA has been proposed.

A membrane-aerated biofilm-coupled Fe/C supported sludge system (MABR-Fe/C) was constructed to achieve in situ electron production for NO3--N reduction enhancement in different Fe/C loadings (10 g and 200 g). The anoxic environment formed in the MABR-Fe/C promoted a continual Fe2+release of Fe/C in 120 d operation (average Fe2+concentrations is 1.18 and 2.95 mg/L in MABR-Fe/C10 and MABR-Fe/C200, respectively). Metagenomics results suggested that the electrons generated from ongoing Fe2+ oxidation were transferred via the Quinone pool to EC 1.7.5.1 rather than EC 1.9.6.1 to complete the process of NO3--N reduction to NO2--N in Acidovorax, Ottowia, and Polaromonas. In the absence of organic matter, the NO3--N removal in MABR-Fe/C10 and MABR-Fe/C200 increased by 11.99 and 12.52 mg/L, respectively, compared to that in MABR. In the further NO2--N reduction, even if the minimum binding free energy (MBFE) was low, NO2--N in Acidovorax and Dechloromonas preferentially bind the Gln-residues for dissimilatory nitrate reduction (DNR) in the presence of Fe/C. Increasing Fe/C loading (MABR-Fe/C200) caused the formation of different residue binding sites, further enhancing the already dominant DNR. When DNR in MABR-Fe/C200 intensified, the TN in the effluent increased by 3.75 mg/L although the effluent NO3--N concentration was lower than that in MABR-Fe/C10. This study demonstrated a new MABR-Fe/C system for in situ electron generation to enhance biological nitrogen removal and analyzed the NO3--N reduction pathway and metabolic mechanism, thus providing new ideas for nitrogen removal in electron-deficient wastewater.

OBJECTIVE: To explore the effect of intestinal nitrates on the growth of Klebsiella pneumoniae and its regulatory mechanisms.
METHODS: K. pneumoniae strains with nitrate reductase narG and narZ single or double gene knockout or with NarXL gene knockout were constructed and observed for both aerobic and anaerobic growth in the presence of KNO3 using an automated bacterial growth analyzer and a spectrophotometer, respectively. The mRNA expressions of narG and narZ in K. pneumoniae in anaerobic cultures in the presence of KNO3 and the effect of the binary regulatory system NarXL on their expresisons were detected using qRT-PCR. Electrophoretic mobility shift assays (EMSA) and MST analysis were performed to explore the specific regulatory mechanisms of NarXL in sensing and utilizing nitrates. Competitive experiments were conducted to examine anaerobic growth advantages of narG and narZ gene knockout strains of K. pneumoniae in the presence of KNO3.
RESULTS: The presence of KNO3 in anaerobic conditions, but not in aerobic conditions, promoted bacterial growth more effectively in the wild-type K. pneumoniae strain than in the narXL gene knockout strain. In anaerobic conditions, the narXL gene knockout strain showed significantly lowered mRNA expressions of narG and narZ (P < 0.0001). EMSA and MST experiments demonstrated that the NarXL regulator could directly bind to narG and narZ promoter regions. The wild-type K. pneumoniae strain in anaerobic cultures showed significantly increased expressions of narG and narZ mRNAs in the presence of KNO3 (P < 0.01), and narG gene knockout resulted in significantly attenuated anaerobic growth and competitive growth abilities of K. pneumoniae in the presence of KNO3 (P < 0.01).
CONCLUSIONS: The binary regulatory system NarXL of K. pneumoniae can sense changes in intestinal nitrate concentration and directly regulate the expression of nitrate reductase genes narG and narZ to promote bacterial growth.

Denitrification is fragile to toxic substances, while currently there are few regulation strategies for toxic substance-stressed denitrification. This study proposed a combined bio-promoter composed of basic bio-promoter (cytokinin, biotin, L-cysteine, and flavin adenine dinucleotide) and phosphomolybdic acid (PMo12) to recover cadmium(II) (Cd(II)) stressed denitrification. By inhibiting 58.02% and 48.84% of nitrate reductase and nitrite reductase activities, Cd(II) caused all the influent nitrogen to accumulate as NO3--N and NO2--N. Combined bio-promoter shortened the recovery time by 21 cycles and improved nitrogen removal efficiency by 10% as the synergistic effect of basic bio-promoter and PMo12. Basic bio-promoter enhanced antioxidant enzyme activities for reactive oxygen species clearance and recovered 23.30% of nicotinamide adenine dinucleotide for sufficient electron donors. Meanwhile, PMo12 recovered electron carriers contents, increasing the electron transfer activity by 60.81% compared with self-recovery. Bio-promoters enhanced the abundance of denitrifiers Seminibacterium and Dechloromonas, which was positively correlated with rapid recovery of denitrification performance.

The impact of heavy metal ions on the biodenitrification process remains unknown, which is the key to understand the nitrogen cycle in estuarine areas. Here, denitrification rate and the abundance of five denitrifying enzyme genes (narG, nirK, napA, norB and nosZ) in Liaohe Estuary sediments were examined, and the community structure of nirK denitrifying bacteria was also analyzed. The results demonstrate a significant positive correlation between heavy metal content (Cu2+, Zn2+, and Cr) and the denitrification rate, and the abundance of napA/norB (periplasmic nitrate reductase and nitric-oxide reductase) in sediments. The dominant narG denitrifiers were Pseudomonas, Hydrogenophaga, and Serratia known to be tolerant to heavy metal pollution. Sediment particle size, NO3-, NO2-, Zn2+, and Cd2+ were the key factors influencing the denitrifying community structure. These findings suggest that heavy metals may enhance the aerobic denitrification process in sediments and mitigate the adverse effects of high dissolved oxygen levels.

The soil-water interface is replete with photic biofilm and iron minerals; however, the potential of how iron minerals promote biotic nitrate removal is still unknown. This study investigates the physiological and ecological responses of photic biofilm to hematite (Fe2O3), in order to explore a practically feasible approach for in-situ nitrate removal. The nitrate removal by photic biofilm was significantly higher in the presence of Fe2O3 (92.5%) compared to the control (82.8%). Results show that the presence of Fe2O3 changed the microbial community composition of the photic biofilm, facilitates the thriving of Magnetospirillum and Pseudomonas, and promotes the growth of photic biofilm represented by the extracellular polymeric substance (EPS) and the content of chlorophyll. The presence of Fe2O3 also induces oxidative stress (•O2-) in the photic biofilm, which was demonstrated by electron spin resonance spectrometry. However, the photic biofilm could improve the EPS productivity to prevent the entrance of Fe2O3 to cells in the biofilm matrix and mitigate oxidative stress. The Fe2O3 then promoted the relative abundance of Magnetospirillum and Pseudomonas and the activity of nitrate reductase, which accelerates nitrate reduction by the photic biofilm. This study provides an insight into the interaction between iron minerals and photic biofilm and demonstrates the possibility of combining biotic and abiotic methods to improve the in-situ nitrate removal rate.