NAD⁺ is a central metabolite participating in core metabolic redox reactions. The prokaryotic NAD synthetase enzyme NadE catalyzes the last step of NAD⁺ biosynthesis, converting nicotinic acid adenine dinucleotide (NaAD) to NAD⁺. Some members of the NadE family use l-glutamine as a nitrogen donor and are named NadE^Gln. Previous gene neighborhood analysis has indicated that the bacterial nadE gene is frequently clustered with the gene encoding the regulatory signal transduction protein PII, suggesting a functional relationship between these proteins in response to the nutritional status and the carbon/nitrogen ratio of the bacterial cell. Here, using affinity chromatography, bioinformatics analyses, NAD synthetase activity, and biolayer interferometry assays, we show that PII and NadE^Gln physically interact in vitro, that this complex relieves NadE^Gln negative feedback inhibition by NAD⁺. This mechanism is conserved in distantly related bacteria. Of note, the PII protein allosteric effector and cellular nitrogen level indicator 2-oxoglutarate (2-OG) inhibited the formation of the PII-NadE^Gln complex within a physiological range. These results indicate an interplay between the levels of ATP, ADP, 2-OG, PII-sensed glutamine, and NAD⁺, representing a metabolic hub that may balance the levels of core nitrogen and carbon metabolites. Our findings support the notion that PII proteins act as a dissociable regulatory subunit of NadE^Gln, thereby enabling the control of NAD⁺ biosynthesis according to the nutritional status of the bacterial cell.

The urgent need for sustainable agriculture places biological nitrogen fixation at the forefront of current biotechnological research. Plant growth‐promoting rhizobacteria play crucial roles in agriculture by enhancing nutrient absorption, regulating hormonal balance, and providing reduced nitrogen to plants. Among these, diazotrophic bacteria, such as Azotobacter vinelandii , stand out for their ability to fix atmospheric nitrogen and release it in bioavailable forms. In this issue of The FEBS Journal , scientists mapped the interaction between NifL and NifA proteins, which regulate nitrogen fixation in A . vinelandii and many other Proteobacteria. This understanding will allow for engineering bacteria to enhance nitrogen delivery to plants by improving nitrogen fixation.

Here we describe a novel immunogenic method to detect COVID-19. The method is a chromogenic magnetic bead-based ELISA which allows inexpensive and quantitative detection of human IgG or IgM antibodies against SARS-CoV-2 in serum or whole blood samples in just 12 minutes. As a proof of concept, we compared the performance of our new method to classical ELISA. Person correlation between optical densities obtained using the two methods was 0.98, the color intensity observed in the novel method correlated with antibody titers determined by classical ELISA. The novel magnetic bead-based ELISA correctly classified all 6 positive COVID-19 samples tested and showed 100% specificity as judged by the analysis of a cohort of 26 negative samples. The magnetic bead-based ELISA performed better than classic ELISA to discriminate COVID-19 positive serum with low antibody titer. The chromogenic magnetic bead-based ELISA method described here can be applied to both point of care and high throughput analysis. The method is readily adaptable to be used with other protein and peptide-based antigens.

Glutamine synthetase (GS), encoded by glnA, catalyzes the conversion of L-glutamate and ammonium to L-glutamine. This ATP hydrolysis driven process is the main nitrogen assimilation pathway in the nitrogen-fixing bacterium Azospirillum brasilense. The A. brasilense strain HM053 has poor GS activity and leaks ammonium into the medium under nitrogen fixing conditions. In this work, the glnA genes of the wild type and HM053 strains were cloned into pET28a, sequenced and overexpressed in E. coli. The GS enzyme was purified by affinity chromatography and characterized. The GS of HM053 strain carries a P347L substitution, which results in low enzyme activity and rendered the enzyme insensitive to adenylylation by the adenilyltransferase GlnE.

The demand for sustainable, low-cost bioprocesses has encouraged the development of alternative enzyme production strategies. This study investigated the purification and characterization of a crude enzymatic extract (CEE) rich in α-amylase from Coprinus comatus, using wheat milling by-products as substrate. The CEE was obtained by submerged culture, followed by biomass removal, centrifugation, and ultrafiltration to yield a partially purified enzyme (PE). CEE and PE were evaluated for chemical composition, antioxidant and antibacterial activity, toxicity (Artemia salina assay), and enzymatic performance. Toxicity assays confirmed that CEE was non-toxic. Antioxidant activity reached 213.34 µmol TE·gDE⁻¹ (DPPH assay), with a total phenolic content of 8.01 mg GAE·gDE⁻¹. No antibacterial activity was detected. CEE hydrolyzed 96.31% of starch in 180 min, releasing 10.85 g·L⁻¹ glucose, while PE achieved 98% hydrolysis and released 14.5 g·L⁻¹. Optimal α-amylase activity occurred at 50 °C and pH 5.5 (CEE) or 5.0 (PE). Calcium ions improved the enzymatic activity and thermal stability. CEE retained over 60% activity after 721 days under refrigeration (4 °C) or freezing (0 °C). Although lyophilization enhanced enzyme concentration, it increased production costs. SDS-PAGE revealed bands of ~67 kDa (α-amylase) and ~35 kDa (glucoamylase). These findings support the use of CEE as a sustainable, low-cost biocatalyst for industrial use.