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Biological nitrogen fixation catalyzed by nitrogenase contributes greatly to the global nitrogen cycle. Nitrogenase expression is subject to regulation in response to nitrogen availability. However, the mechanism through which the transcriptional activator NifA regulates nitrogenase expression by interacting with PII nitrogen regulatory proteins remains unclear in diazotrophic proteobacteria lacking NifL. Here, we demonstrate that in Rhodopseudomonas palustris grown with ammonium, NifA bound deuridylylated PII proteins to form an inactive NifA-PII complex, thereby inhibiting the expression of nitrogenase. Upon nitrogen limitation, the dissociation of uridylylated PII proteins from NifA resulted in the full restoration of NifA activity, and, simultaneously, uridylylation of the significantly up-regulated PII protein GlnK2 led to the increased expression of NifA in R. palustris. This insight into how NifA interacts with PII proteins and controls nitrogenase expression sets the stage for creating highly efficient diazotrophs, reducing the need for energy-intensive chemical fertilizers and helping to diminish carbon emissions.

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Cyclic di-adenosine monophosphate (c-di-AMP) is a second messenger involved in diverse metabolic processes including osmolyte uptake, cell wall homeostasis, as well as antibiotic and heat resistance. This study investigates the role of the c-di-AMP receptor protein DarA in the osmotic stress response in Bacillus subtilis. Through a series of experiments, we demonstrate that DarA plays a central role in the cellular response to osmotic fluctuations. Our findings show that DarA becomes essential under extreme potassium limitation as well as upon salt stress, highlighting its significance in mediating osmotic stress adaptation. Suppressor screens with darA mutants reveal compensatory mechanisms involving the accumulation of osmoprotectants, particularly potassium and citrulline. Mutations affecting various metabolic pathways, including the citric acid cycle as well as glutamate and arginine biosynthesis, indicate a complex interplay between the osmotic stress response and metabolic regulation. In addition, the growth defects of the darA mutant during potassium starvation and salt stress in a strain lacking the high-affinity potassium uptake systems KimA and KtrAB can be rescued by increased affinity of the remaining potassium channel KtrCD or by increased expression of ktrD, thus resulting in increased potassium uptake. Finally, the darA mutant can respond to salt stress by the increased expression of MleN , which can export sodium ions.IMPORTANCEEnvironmental bacteria are exposed to rapidly changing osmotic conditions making an effective adaptation to these changes crucial for the survival of the cells. In Gram-positive bacteria, the second messenger cyclic di-AMP plays a key role in this adaptation by controlling (i) the influx of physiologically compatible organic osmolytes and (ii) the biosynthesis of such osmolytes. In several bacteria, cyclic di-adenosine monophosphate (c-di-AMP) can bind to a signal transduction protein, called DarA, in Bacillus subtilis. So far, no function for DarA has been discovered in any organism. We have identified osmotically challenging conditions that make DarA essential and have identified suppressor mutations that help the bacteria to adapt to those conditions. Our results indicate that DarA is a central component in the integration of osmotic stress with the synthesis of compatible amino acid osmolytes and with the homeostasis of potassium, the first response to osmotic stress.

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BACKGROUND: Rice bean (Vigna umbellata), an underrated legume, adapts to diverse climatic conditions with the potential to support food and nutritional security worldwide. It is used as a vegetable, minor food crop and a fodder crop, being a rich source of proteins, minerals, and essential fatty acids. However, little effort has been made to decipher the genetic and molecular basis of various useful traits in this crop. Therefore, we considered three economically important traits i.e., flowering, maturity and seed weight of rice bean and identified the associated candidate genes employing an associative transcriptomics approach on 100 diverse genotypes out of 1800 evaluated rice bean accessions from the Indian National Genebank. RESULTS: The transcriptomics-based genotyping of one-hundred diverse rice bean cultivars followed by pre-processing of genotypic data resulted in 49,271 filtered markers. The STRUCTURE, PCA and Neighbor-Joining clustering of 100 genotypes revealed three putative sub-populations. The marker-trait association analysis involving various genome-wide association study (GWAS) models revealed significant association of 82 markers on 48 transcripts for flowering, 26 markers on 22 transcripts for maturity and 22 markers on 21 transcripts for seed weight. The transcript annotation provided information on the putative candidate genes for the considered traits. The candidate genes identified for flowering include HSC80, P-II PsbX, phospholipid-transporting-ATPase-9, pectin-acetylesterase-8 and E3-ubiquitin-protein-ligase-RHG1A. Further, the WRKY1 and DEAD-box-RH27 were found to be associated with seed weight. Furthermore, the associations of PIF3 and pentatricopeptide-repeat-containing-gene with maturity and seed weight, and aldo-keto-reductase with flowering and maturity were revealed. CONCLUSION: This study offers insights into the genetic basis of key agronomic traits in rice bean, including flowering, maturity, and seed weight. The identified markers and associated candidate genes provide valuable resources for future exploration and targeted breeding, aiming to enhance the agronomic performance of rice bean cultivars. Notably, this research represents the first transcriptome-wide association study in pulse crop, uncovering the candidate genes for agronomically useful traits.

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The conserved cyanobacterial protein PipX is part of a complex interaction network with regulators involved in essential processes that include metabolic homeostasis and ribosome assembly. Because PipX interactions depend on the relative levels of their different partners and of the effector molecules binding to them, in vivo studies are required to understand the physiological significance and contribution of environmental factors to the regulation of PipX complexes. Here, we have used the NanoBiT complementation system to analyse the regulation of complex formation in Synechococcus elongatus PCC 7942 between PipX and each of its two best-characterized partners, PII and NtcA. Our results confirm previous in vitro analyses on the regulation of PipX-PII and PipX-NtcA complexes by 2-oxoglutarate and on the regulation of PipX-PII by the ATP/ADP ratio, showing the disruption of PipX-NtcA complexes due to increased levels of ADP-bound PII in Synechococcus elongatus. The demonstration of a positive role of PII on PipX-NtcA complexes during their initial response to nitrogen starvation or the impact of a PipX point mutation on the activity of PipX-PII and PipX-NtcA reporters are further indications of the sensitivity of the system. This study reveals additional regulatory complexities in the PipX interaction network, opening a path for future research on cyanobacteria.

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PII proteins are signal transduction proteins that belong to a widely distributed family of proteins involved in the modulation of different metabolisms in bacteria. These proteins are homotrimers carrying a flexible loop, named T-loop, which changes its conformation due to the recognition of diverse key metabolites, ADP, ATP, and 2-oxoglutarate. PII proteins interact with different partners to primarily regulate a set of nitrogen pathways. In some organisms, PII proteins can also control carbon metabolism by interacting with the biotin carboxyl carrier protein (BCCP), a key component of the acetyl-CoA carboxylase (ACC) enzyme complex, inhibiting its activity with the consequent reduction of fatty acid biosynthesis. Most bacteria contain at least two PII proteins, named GlnB and GlnK, with different regulatory roles. In mycobacteria, only one PII protein was identified, and the three-dimensional structure was solved, however, its physiological role is unknown. In this study we purified the Mycobacterium tuberculosis (M. tb) PII protein, named GlnB, and showed that it weakly interacts with the AccA3 protein, the α subunit shared by the three different, and essential, Acyl-CoA carboxylase complexes (ACCase 4, 5, and 6) present in M. tb. A M. smegmatis deletion mutant, ∆MsPII, exhibited a growth deficiency on nitrate and nitrite as unique nitrogen sources, and accumulated nitrite in the culture supernatant. In addition, M. tb PII protein was able to interact with the C-terminal domain of the ammonium transporter Amt establishing the ancestral role for this PII protein as a GlnK functioning protein.

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SbtB is a PII-like protein that regulates the carbon-concentrating mechanism (CCM) in cyanobacteria. SbtB proteins can bind many adenyl nucleotides and possess a characteristic C-terminal redox sensitive loop (R-loop) that forms a disulfide bridge in response to the diurnal state of the cell. SbtBs also possess an ATPase/ADPase activity that is modulated by the redox-state of the R-loop. To investigate the R-loop in the cyanobacterium Synechocystis sp. PCC 6803, site-specific mutants, unable to form the hairpin and permanently in the reduced state, and a R-loop truncation mutant, were characterized under different inorganic carbon (Ci) and light regimes. Growth under diurnal rhythm showed a role of the R-loop as sensor for acclimation to changing light conditions. The redox-state of the R-loop was found to impact the binding of the adenyl-nucleotides to SbtB, its membrane association and thereby the CCM regulation, while these phenotypes disappeared after truncation of the R-loop. Collectively, our data imply that the redox-sensitive R-loop provides an additional regulatory layer to SbtB, linking the CO2-related signaling activity of SbtB with the redox state of cells, mainly reporting the actual light conditions. This regulation not only coordinates CCM activity in the diurnal rhythm but also affects the primary carbon metabolism.

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New protein-fragment complementation assays (PCA) have successfully been developed to characterize protein-protein interactions in vitro and in vivo. Notably, the NanoBiT technology, employing fragment complementation of NanoLuc luciferase, stands out for its high sensitivity, wide dynamic range, and straightforward read out. Previously, we explored the in vitro protein interaction dynamics of the PII signalling protein using NanoBiT, revealing significant modulation of luminescence signals generated by the interaction between PII and its receptor protein NAGK by 2-oxoglutarate levels. In the current work, we investigated this technology in vivo, to find out whether recombinantly expressed NanoBiT constructs using the NanoLuc large fragment fused to PII and PII-interaction partners NAGK or PipX-fused to the NanoLuc Small BiT are capable of detecting the metabolic fluctuations in Escherichia coli. Therefore, we devised an assay capable of capturing the metabolic responses of E. coli cells, demonstrating real-time metabolic perturbation upon nitrogen upshift or depletion treatments. In particular, the PII-NAGK NanoBitT sensor pair reported these changes in a highly sensitive manner.

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Coordinated carbon and nitrogen metabolism is crucial for bacteria living in the fluctuating environments. Intracellular carbon and nitrogen homeostasis is maintained by a sophisticated network, in which the widespread signaling protein PII acts as a major regulatory hub. In cyanobacteria, PII was proposed to regulate the nitrate uptake by an ABC (ATP-binding cassette)-type nitrate transporter NrtABCD, in which the nucleotide-binding domain of NrtC is fused with a C-terminal regulatory domain (CRD). Here, we solved three cryoelectron microscopy structures of NrtBCD, bound to nitrate, ATP, and PII, respectively. Structural and biochemical analyses enable us to identify the key residues that form a hydrophobic and a hydrophilic cavity along the substrate translocation channel. The core structure of PII, but not the canonical T-loop, binds to NrtC and stabilizes the CRD, making it visible in the complex structure, narrows the substrate translocation channel in NrtB, and ultimately locks NrtBCD at an inhibited inward-facing conformation. Based on these results and previous reports, we propose a putative transport cycle driven by NrtABCD, which is allosterically inhibited by PII in response to the cellular level of 2-oxoglutarate. Our findings provide a distinct regulatory mechanism of ABC transporter via asymmetrically binding to a signaling protein.

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OBJECT: To obtain Pulmonary Inflammation Index scores from imaging chest CT and combine it with clinical correlates of viral pneumonia to predict the risk and severity of viral pneumonia using a computer learning model. METHODS: All patients with suspected viral pneumonia on CT examination admitted to The Affiliated Traditional Chinese Medicine Hospital of Southwest Medical University from December 2022 to March 2023 were retrospectively selected. The respiratory viruses were monitored by RT-PCR and categorized into patients with viral pneumonia and those with non-viral pneumonia. The extent of lung inflammation was quantified according to the Pulmonary Inflammation Index score (PII). Information on patient demographics, comorbidities, laboratory tests, pathogenetic testing, and radiological data were collected. Five machine learning models containing Random Forest(RF), Radial Basis Function Neural Network (RBFNN), Support Vector Machine (SVM), K Nearest Neighbour Algorithm (KNN), and Kernel Ridge Regression (KRR) were used to predict the risk of onset and severity of viral pneumonia based on the clinically relevant factors or PII. RESULTS: Among the five models, the SVM model performed best in ACC (76.75 %), SN (73.99 %), and F1 (72.42 %) and achieved a better area under the receiver operating characteristic curve (ROC) (0.8409) when predicting the risk of developing viral pneumonia. RF had the best overall classification accuracy in predicting the severity of viral pneumonia, especially in predicting pneumonia with a PII classification of grade I, the RF model achieved an accuracy of 98.89%. CONCLUSION: Machine learning models are valuable in assessing the risk of viral pneumonia. Meanwhile, machine learning models confirm the importance in predicting the severity of viral pneumonia through PII. The establishment of machine learning models for predicting the risk and severity of viral pneumonia promotes the further development of machine learning in the medical field.

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IMPORTANCE: Small proteins containing fewer than 70 amino acids, which were previously disregarded due to computational prediction and biochemical detection challenges, have gained increased attention in the scientific community in recent years. However, the number of functionally characterized small proteins, especially in archaea, is still limited. Here, by using biochemical and genetic approaches, we demonstrate a crucial role of the small protein sP36 in the nitrogen metabolism of M. mazei, which modulates the ammonium transporter AmtB1 according to nitrogen availability. This modulation might represent an ancient archaeal mechanism of AmtB1 inhibition, in contrast to the well-studied uridylylation-dependent regulation in bacteria.

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AIM: To elucidate how proinsulin synthesis and insulin was affected by metformin under conditions of nutrient overstimulation. MATERIALS AND METHODS: Isolated human pancreatic islets from seven donors were cultured at 5.5 mmol/L glucose and 0.5 mmol/L palmitate for 12, 24 or 72 h. Metformin (25 µmol/L) was introduced after initial 12 h with palmitate. Proinsulin and insulin were measured. Expression of prohormone convertase 1/3 (PC1/3) and carboxypeptidase E (CPE), was determined by western blot. Adolescents with obesity, treated with metformin and with normal glucose tolerance (n = 5), prediabetes (n = 14), or type 2 diabetes (T2DM; n = 7) were included. Fasting proinsulin, insulin, glucose, 2-h glucose and glycated haemoglobin were measured. Proinsulin/insulin ratio (PI/I) was calculated. RESULTS: In human islets, palmitate treatment for 12 and 24 h increased proinsulin and insulin proportionally. After 72 h, proinsulin but not insulin continued to increase which was coupled with reduced expression of PC1/3 and CPE. Metformin normalized expression of PC1/3 and CPE, and proinsulin and insulin secretion. In adolescents with obesity, before treatment, fasting proinsulin and insulin concentrations were higher in subjects with T2DM than with normal glucose tolerance. PI/I was reduced after metformin treatment in subjects with T2DM as well as in subjects with prediabetes, coupled with reduced 2-h glucose and glycated haemoglobin. CONCLUSIONS: Metformin normalized proinsulin and insulin secretion after prolonged nutrient-overstimulation, coupled with normalization of the converting enzymes, in isolated islets. In adolescents with obesity, metformin treatment was associated with improved PI/I, which was coupled with improved glycaemic control.

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N-Acetyl-L-glutamate kinase (NAGK) catalyzes the rate-limiting step in the ornithine/arginine biosynthesis pathway in eukaryotic and bacterial oxygenic phototrophs. NAGK is the most highly conserved target of the PII signal transduction protein in Cyanobacteria and Archaeplastida (red algae and Chlorophyta). However, there is still much to be learned about how NAGK is regulated in vivo. The use of unicellular green alga Chlamydomonas reinhardtii as a model system has already been instrumental in identifying several key regulation mechanisms that control nitrogen (N) metabolism. With a combination of molecular-genetic and biochemical approaches, we show the existence of the complex CrNAGK control at the transcriptional level, which is dependent on N source and N availability. In growing cells, CrNAGK requires CrPII to properly sense the feedback inhibitor arginine. Moreover, we provide primary evidence that CrPII is only partly responsible for regulating CrNAGK activity to adapt to changing nutritional conditions. Collectively, our results suggest that in vivo CrNAGK is tuned at the transcriptional and post-translational levels, and CrPII and additional as yet unknown factor(s) are integral parts of this regulation.

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Photosynthetic organisms must cope with environmental challenges, like those imposed by the succession of days and nights or by sudden changes in light intensities, that trigger global changes in gene expression and metabolism. The photosynthesis machinery is particularly susceptible to environmental changes and adaptation to them often involves redox-sensing proteins that are the targets of reactive oxygen species generated by photosynthesis activity. Here we show that EngA, an essential GTPase and ribosome-assembly protein involved in ribosome biogenesis in bacteria and chloroplasts, also plays a role in acclimatization to environmentally relevant stress in Synechococcus elongatus PCC7942 and that PipX, a promiscuous regulatory protein that binds to EngA, appears to fine-tune EngA activity. During growth in cold or high light conditions, the EngA levels rise, with a concomitant increase of the EngA/PipX ratio. However, a sudden increase in light intensity turns EngA into a growth inhibitor, a response involving residue Cys122 of EngA, which is part of the GD1-G4 motif NKCES of EngA proteins, with the cysteine conserved just in the cyanobacteria-chloroplast lineage. This work expands the repertoire of ribosome-related factors transmitting redox signals in photosynthetic organisms and provides additional insights into the complexity of the regulatory interactions mediated by EngA and PipX.

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The control of starch granule initiation in plant leaves is a complex process that requires active enzymes like Starch Synthase 4 and 3 (SS4 or SS3) and several noncatalytic proteins such as Protein Involved in starch Initiation 1 (PII1). In Arabidopsis leaves, SS4 is the main enzyme that control starch granule initiation, but in its absence, SS3 partly fulfills this function. How these proteins collectively act to control the initiation of starch granules remains elusive. PII1 and SS4 physically interact, and PII1 is required for SS4 to be fully active. However, Arabidopsis mutants lacking SS4 or PII1 still accumulate starch granules. Combining pii1 KO mutation with either ss3 or ss4 KO mutations provide new insights of how the remaining starch granules are synthesized. The ss3 pii1 line still accumulates starch, while the phenotype of ss4 pii1 is stronger than that of ss4. Our results indicate first that SS4 initiates starch granule synthesis in the absence of PII1 albeit being limited to one large lenticular granule per plastid. Second, that if in the absence of SS4, SS3 is able to initiate starch granules with low efficiency, this ability is further reduced with the additional absence of PII1.

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Excess ammonium imposes toxicity and stress response in cyanobacteria. How cyanobacteria acclimate to NH4+ stress is so far poorly understood. Here, Synechocystis sp. PCC6803 S2P homolog Slr1821 was identified as the essential regulator through physiological characterization and transcriptomic analysis of its knockout mutant. The proper expression of 60% and 67% of the NH4+ activated and repressed genes, respectively, were actually Slr1821-dependent since they were abolished or reversed in ∆slr1821. Synechocystis 6803 suppressed nitrogen uptake and assimilation, ammonium integration and mobilization of other nitrogen sources upon NH4+ stress. Opposite regulation on genes for assimilation of nitrogen and carbon, such as repression of nitrogen regulatory protein PII, PII interactive protein PirC and activation of carbon acquisition regulator RcbR, demonstrated that Synechocystis 6803 coordinated regulation to maintain carbon/nitrogen homeostasis under increasing nitrogen, while functional Slr1821 was indispensable for most of this coordinated regulation. Additionally, slr1821 knockout disrupted the proper response of regulators and transporters in the ammonium-specific stimulon, and resulted in defective photosynthesis as well as compromised translational and transcriptional machinery. These results provide new insight into the coordinated regulation of nutritional fluctuation and the functional characterization of S2Ps. They also provide new targets for bioengineering cyanobacteria in bioremediation and improving ammonium tolerance in crop plants.

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The PII superfamily consists of widespread signal transduction proteins found in all domains of life. In addition to canonical PII proteins involved in C/N sensing, structurally similar PII-like proteins evolved to fulfill diverse, yet poorly understood cellular functions. In cyanobacteria, the bicarbonate transporter SbtA is co-transcribed with the conserved PII-like protein, SbtB, to augment intracellular inorganic carbon levels for efficient CO2 fixation. We identified SbtB as a sensor of various adenine nucleotides including the second messenger nucleotides cyclic AMP (cAMP) and c-di-AMP. Moreover, many SbtB proteins possess a C-terminal extension with a disulfide bridge of potential redox-regulatory function, which we call R-loop. Here, we reveal an unusual ATP/ADP apyrase (diphosphohydrolase) activity of SbtB that is controlled by the R-loop. We followed the sequence of hydrolysis reactions from ATP over ADP to AMP in crystallographic snapshots and unravel the structural mechanism by which changes of the R-loop redox state modulate apyrase activity. We further gathered evidence that this redox state is controlled by thioredoxin, suggesting that it is generally linked to cellular metabolism, which is supported by physiological alterations in site-specific mutants of the SbtB protein. Finally, we present a refined model of how SbtB regulates SbtA activity, in which both the apyrase activity and its redox regulation play a central role. This highlights SbtB as a central switch point in cyanobacterial cell physiology, integrating not only signals from the energy state (adenyl-nucleotide binding) and the carbon supply via cAMP binding but also from the day/night status reported by the C-terminal redox switch.

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The use of nitrogen (N) fertilizers in agriculture has a great ability to increase crop productivity. However, their excessive use has detrimental effects on the environment. Therefore, it is necessary to develop crop varieties with improved nitrogen use efficiency (NUE) that require less N but have substantial yields. Orphan crops such as millets are cultivated in limited regions and are well adapted to lower input conditions. Therefore, they serve as a rich source of beneficial traits that can be transferred into major crops to improve their NUE. This review highlights the tremendous potential of systems biology to unravel the enzymes and pathways involved in the N metabolism of millets, which can open new possibilities to generate transgenic crops with improved NUE.

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Smart cities assure the masses a higher quality of life through digital interconnectivity, leading to increased efficiency and accessibility in cities. In addition, a huge amount of data is being exchanged through smart devices, networks, cloud infrastructure, big data analysis and Internet of Things (IoT) applications in the various private and public sectors, such as critical infrastructures, financial sectors, healthcare, and Small and Medium Enterprises (SMEs). However, these sectors require maintaining certain security mechanisms to ensure the confidentiality and integrity of personal and critical information. However, unfortunately, organizations fail to maintain their security posture in terms of security mechanisms and controls, which leads to data breach incidents either intentionally or inadvertently due to the vulnerabilities in their information management systems that either malicious insiders or attackers exploit. In this paper, we highlight the importance of data breaches and issues related to information leakage incidents. In particular, the impact of data breaching incidents and the reasons contributing to such incidents affect the citizens' well-being. In addition, this paper also discusses various preventive measures such as security mechanisms, laws, standards, procedures, and best practices, including follow-up mitigation strategies.

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Porcine epidemic diarrhea virus (PEDV) is a serious infectious causative agent in swine, especially in neonatal piglets. PEDV genotype 2 (G2) strains, particularly G2a, were the primary causes of porcine epidemic diarrhea (PED) outbreaks in Vietnam. Here, we produced a plant-based CO-26K-equivalent epitope (COE) variant from a Vietnamese highly virulent PEDV strain belonging to genotype 2a (COE/G2a) and evaluated the protective efficacy of COE/G2a-GCN4pII protein (COE/G2a-pII) in piglets against the highly virulent PEDV G2a strain following passive immunity. The 5-day-old piglets had high levels of PEDV-specific IgG antibodies, COE-IgA specific antibodies, neutralizing antibodies, and IFN-γ responses. After virulent challenge experiments, all of these piglets survived and had normal clinical symptoms, no watery diarrhea in feces, and an increase in their body weight, while all of the negative control piglets died. These results suggest that the COE/G2a-pII protein produced in plants can be developed as a promising vaccine candidate to protect piglets against PEDV G2a infection in Vietnam.

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The emergence of mutant strains such as Omicron has increased the uncertainty of COVID-19, and all countries have taken strict measures to prevent the spread of the disease. The spread of the disease between countries is of particular concern. However, most COVID-19 research focuses mainly on the country or community, and there is less research on the border areas between two countries. In this study, we analyzed changes in the total nighttime light intensity (TNLI) and total nighttime lit area (TNLA) along the Sino-Burma border and used the data to construct an epidemic pressure input index (PII) model in reference to the Shen potential model. The results show that, as the epidemic became more severe, TNLI on both sides of the border at the Ruili border port increased, while that in areas far from the port decreased. At the same time, increases and decreases in TNLA occurred in areas far from the port, and PII can indicate the areas where imported cases are likely to occur. Along the Sino-Burma border, the PII model showed low PII in the north and south and high PII in the central region. The areas between Dehong and Lincang, especially the Ruili, Wanding, Nansan, and Qingshuihe border ports, had high PII. The results of this study offer a reference for public health officials and decision makers when determining resource allocation and the implementation of stricter quarantine rules. With updated epidemic statistics, PII can be recalculated to support timely monitoring of COVID-19 in border areas.