Metallomics

Cadmium, being a highly toxic metal, perturbs cellular homeostasis by forming stable complexes with numerous thiol-active proteins, ultimately leading to severe liver and lung damage. Despite its well-documented toxicity, the molecular mechanisms governing cadmium export remain poorly understood. Given the chemical similarity between cadmium and copper, we investigated whether the canonical copper-exporting ATPases, ATP7A and ATP7B participate in cadmium handling. Upon Cd treatment in hepatocytes, ATP7B undergoes trafficking to lysosomes via the retromer complex, as also observed in the case of elevated copper, accompanied by the upregulation of acidic lysosomal populations. In contrast, ATP7A expressed in lung adenocarcinoma cells, though exhibit vesicular redistribution upon Cd exposure, does not mediate lysosomal sequestration, suggesting distinct deployment of late secretory pathways by the two copper ATPases in response to cadmium. We have also observed that ATP7B-/- hepatocytes exhibit increased sensitivity to Cd exposure compared to wild-type cells. Whereas, overexpressing the ATP7B amino-terminal copper-binding domain in bacteria alleviates cadmium-induced stress, indicating its capacity to sequester Cd. Caenorhabditis elegans lacking copper-ATPase cua-1, displayed increased Cd sensitivity, while mutants (glo-1-/-), deficient in lysosome-related organelles (LRO), and (lmp-1-/-), deficient in lysosomal membrane glycoprotein, showed reduced resistance to cadmium toxicity. Treatment of the worm with cadmium increases the abundance of lysosomes marked by elevation in lysosomal biogenesis and functional genes, reinforcing the importance of lysosomal pathways in cadmium detoxification. To summarise, we delineated the non-canonical role of copper ATPases and lysosomes in cadmium-induced cellular toxicity.

The facultative methylotroph model organism Methylorubrum extorquens AM1 is a known lanthanide user, which has shed light on the role of rare-earth metals in biochemistry. The characterization of a methanol dehydrogenase (MDH) protein which requires lanthanides as an enzymatic cofactor outlined the question of how these metals are acquired from the environment. It has been proposed that mesophilic organisms as M. extorquens AM1 can produce siderophore-like molecules, which chelate, transport and traffic rare-earth elements into the microbial cell. Therefore, we performed the bioinformatic and chemical investigation of M. extorquens AM1 by using genome mining, the CAS and arsenazo assay, molecular networking and chemical analytical techniques. Our results showed that indeed Methylorubrum extorquens AM1 harbored a gene cluster to produce metal chelators. The chemical analysis confirmed the production of the known hybrid hydroxamate-citrate siderophores schizokinen A and N-deoxyschizokinen A, which are very likely the side products of the transformation of schizokinen and N-deoxyschizokinen. The determination of the lanthanide chelation activity of the schizokinen siderophores series against three different lanthanides (La, Eu and Lu) showed no coordination activity, thus ruling out the involvement of schizokinen siderophores in rare-earth metal transport.

The production of reactive oxygen species (ROS) in response to cadmium (Cd) has been extensively studied, demonstrating that they play a key role in the plants response to this heavy metal. While the role of enzymes like RBOHs has been thoroughly studied, the function of other ROS-producing enzymes, such as peroxisomal glycolate oxidase (GOX), remains largely overlooked. Peroxisomal GOX is a core metabolic enzyme of the photorespiratory pathway occurring in chloroplasts, mitochondria and peroxisomes. Using Arabidopsis (Arabidopsis thaliana) mutants lacking the main peroxisomal GOX genes, GOX1 (gox1-1) and GOX2 (gox2-1) we explored their function in plant response to Cd. Although photosynthetic capacity appears to be affected to the same extent in both mutants under control and Cd stress conditions, GOX2 seems to play a greater role in ROS production in response to the metal. Transcriptomic analyses on WT and gox2-1 pointed to the mitochondrial electron transport chain (mETC) as a target of Cd stress. We further investigated the individual GOX1 and GOX2 functions in mETC regulation and redox state. Although oxidative ratio of mitochondria was higher in both mutants, it was more pronounced in the absence of GOX1. Furthermore, the mETC is affected in both mutants but the regulation of its components differs in each mutant. These results point out the different functions of the two photorespiratory GOX isoforms in Arabidopsis, leading to a better understanding of the photorespiratory pathway.

Bacterial pathogens must adapt to dynamic host tissue environments to proliferate. Accordingly, elegant regulatory systems evolved to overcome challenges presented by the host and satisfy nutritional requirements. Sulfur is an essential macronutrient and Gram-positive bacteria such as Staphylococcus aureus balance this nutritional requirement by employing the transcriptional repressor, CymR. Previous investigations defined the S. aureus CymR regulon by comparing transcripts generated in a cymR mutant cultured in cystine replete, rich medium to wild type cells. This study defines the S. aureus CymR-dependent and -independent sulfur-starvation response in chemically defined growth conditions. Results demonstrate that the sulfur starvation and sulfur replete CymR regulons exhibit considerable overlap, including previously noted connections between iron acquisition, oxidative stress, and sulfur metabolism. The link between iron acquisition, oxidative stress, and sulfur metabolism is validated further by the finding that sulfur-containing glutathione (GSH) mitigates heme and peroxide toxicity. In addition to GSH, Cys and thiosulfate fulfill the S. aureus sulfur requirement. Transcriptional responses to organic (cysteine, cystine, reduced and oxidized GSH) or inorganic thiosulfate were quantified, revealing sulfur source-specific expression patterns. Thiosulfate induced the largest number of differentially expressed genes. Consequently, the thiosulfate transporter (SAUSA300_RS10985) has been confirmed as essential for S. aureus growth when thiosulfate is the sulfur source. Furthermore, we demonstrate that a hypothetical protein operonic with SAUSA300_RS10985, SAUSA300_RS10980, supports maximal growth on thiosulfate. Collectively, a resourceful transcriptomics framework is provided which underscores the dynamic nature of S. aureus sulfur metabolism.

Glutathione (GSH) is a tripeptide that plays essential roles in redox regulation and stress responses across organisms. In Escherichia coli, the GSH-specific {gamma}-glutamyl cyclotransferase (ChaC) has been characterized biochemically, yet its physiological role remains unclear. Moreover, ChaC has been annotated as a regulator of the Na/H antiporter ChaA based on its genomic association, although experimental evidence supporting this function is limited. In this study, we investigated whether chaC and its co-transcribed gene, chaB, are involved in sodium transport or GSH metabolism. Gene expression analyses revealed that chaA, chaB, and chaC are upregulated under salt stress. Functional analyses using deletion mutants showed that loss of chaA reduced salt tolerance, whereas deletion of chaB enhanced tolerance and decreased intracellular sodium levels. In contrast, deletion of chaC had no significant effect on salt tolerance or sodium accumulation. Overexpression of cha genes further indicated that chaA, but not chaB or chaC, contributed to salt tolerance. Importantly, overexpression of chaC significantly reduced intracellular GSH levels, whereas chaB overexpression had no effect. These results indicate that ChaC primarily functions in GSH degradation rather than in cation transport, and that ChaB does not participate in GSH metabolism. Our findings clarify the distinct physiological roles of ChaC and ChaB and provide new insight into bacterial physiology regarding GSH metabolism and ion transport in E. coli.

1.Abiotic stresses like nitrogen deficiency and soil salinity are major factors contributing to low crop yields. The use of selective biofertilizers alleviates both types of stress. In this study, we investigated the biofertilizer activity and plant growth-promoting properties (PGP) of Rhodococcus jialingiae RS1 through cytosolic proteome remodelling. We cultured RS1 under two conditions, i) without and ii) with 6% NaCl, in nitrogen-deficient defined Burks medium. Under dual stress of nitrogen limitation and salt stress, Orbitrap LC-MS/MS proteomics revealed one-quarter of the proteome remodelling, particularly the upregulation of ribosomal synthesis and protein repair systems. As expected, we found high expression of EctC, an ectoine synthase, a key enzyme in osmolyte biosynthesis. Additionally, ribosomal and translational-associated factors, including RpsL, RpsS, RpsT, RpsR1, RplV, RplL, RplA, and elongation factor Tuf, were highly expressed, suggesting enhanced translational fidelity under dual stress. High levels of DNA protection protein, Dps suggest dual stress may lead to DNA damage. Upregulation of chaperones, environmental sensors (KinE), and redox transcriptional factors like WhiB3, Hsp18, AhpC, and MetE suggests protein misfolding and oxidative stress. Metabolic modulations were evident through high expression of IlvA, NAD-dependent glutamate dehydrogenase, lipid/envelope-remodelling enzymes, cutinase/esterases, lipases, endopeptidases like NlpC/P60 and transport systems. In contrast, proteins involved in urease structural components (urea-G), nitrogen regulators and ammonium transporters (GlnK and Amt) were downregulated. Dual stress may lead to an energy crisis, prompting strategic shifts away from high-ATP-dependent ureolytic nitrogen-scavenging pathways towards lower-energy nitrogen-assimilating routes, such as IlvA-mediated deamination and NAD-dependent glutamate dehydrogenation. Genetic manipulations of the above-mentioned genes or their homologues across the genera of microbes, plants, and crops may enhance resilience to abiotic stresses. Our studies reveal stress-responsive genes and biochemical pathways that could be used to improve transgenic efficacy in nitrogen-limited, saline soil and other (a)biotic stresses. Global Proteome Profiling of Rhodococcus jialingiae RS1 to Develop Transgenics O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/724437v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@1719d80org.highwire.dtl.DTLVardef@1b6b59org.highwire.dtl.DTLVardef@24d367org.highwire.dtl.DTLVardef@1b33224_HPS_FORMAT_FIGEXP M_FIG C_FIG

{gamma}-Glutamyl cyclotransferases (GGCTs) belongs to class of cytosolic enzymes that are responsible for glutathione (GSH) degradation under stress conditions. They regulate GSH homeostasis through the {gamma}-glutamyl cycle which is responsible for maintaining the synthesis of GSH as well as its breakdown, enabling recycling of its constituent amino acids. Although GGCTs have been implicated in enhancing heavy metal (HMs) tolerance in plants, their role in biotic stress remains largely unexplored. Previously, OsGGCT1 was identified as a gene strongly upregulated in Fusarium stress. In this study, the GGCT1 homolog from Oryza sativa japonica was characterized for its role in conferring tolerance to Fusarium oxysporum (F.O.). Similar to abiotic factors, biotic stresses significantly impact crop yield and productivity. The rhizosphere harbors diverse microbial communities, including harmful pathogens such as F. oxysporum. Fusarium causes wilt disease in a variety of plant species, such as: tomato, legumes, rice, and Arabidopsis thaliana. Our results demonstrate that overexpression of OsGGCT1 enhanced tolerance to F. oxysporum in A. thaliana, primarily by reducing fungal spore accumulation. Transgenic plants showed elevated expression of OsGGCT1 along with AtGSH1 and AtGSH2, reduced levels of reactive oxygen species (ROS), improved growth and photosynthetic performance and enhanced activities of the antioxidant enzymes. OsGGCT1 serves as a key component in maintaining GSH homeostasis by supporting glutamate (Glu) regeneration necessary for sustained GSH biosynthesis. Overall, these findings identify OsGGCT1 as an important constituent of the GSH-mediated detoxification pathway against Fusarium oxysporum and provide valuable molecular insights for developing Fusarium-tolerant rice varieties with reduced fungal accumulation.

Colletotrichum sublineola (Cs), the hemibiotrophic fungus that causes sorghum anthracnose, impacts sorghum grain and biomass crop production worldwide. Although nutrient availability is known to influence development in filamentous fungi, including Colletotrichum species, how in vitro nutrient limitation reprograms the Cs cellular state remains unclear. We cultured Cs on full-strength, half-strength, and one-tenth-strength potato dextrose agar (PDA) to define responses across a nutrient gradient. Nutrient limitation induced a pronounced high-sporulation phenotype, with one-tenth-strength PDA producing the strongest conidiation response, followed by half-strength PDA. To study the underlying molecular programs in each condition, we employed a multiplexed metabolite, protein, and lipid extraction (MPLEx) protocol for global proteomics and metabolomics. Global proteomics resulted in 4,590 protein identifications, including 204 unique to one-tenth-strength PDA. Among them are proteins linked to sporulation, vesicular transport, glycosylphosphatidylinositol (GPI)-anchor biosynthesis, and common in fungal extracellular membrane (CFEM)-domain proteins. Differential abundance and pathway analyses revealed a broad reduction of central carbon and energy metabolism, including glycolysis/gluconeogenesis, pentose phosphate, pyruvate metabolism, and glyoxylate pathways, together with increased ribosome-related processes, cAMP signaling, and cell-surface remodeling in one-tenth-strength PDA conditions. In addition, correlative metabolomics supported selective metabolic depletion and resource reallocation toward stress adaptation, membrane remodeling, and conidiation, supporting proteomics findings. Together, these data support a starvation-adapted Cs developmental state associated with enhanced sporulation, cellular pathway reprogramming, and potential virulence linked preparedness under nutrient-limited growth conditions in vitro. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=101 SRC="FIGDIR/small/724728v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@f6ceb2org.highwire.dtl.DTLVardef@17c4836org.highwire.dtl.DTLVardef@68e995org.highwire.dtl.DTLVardef@1bf3983_HPS_FORMAT_FIGEXP M_FIG C_FIG

Mitochondrial morphology and function are critical determinants of neuronal function and survival, with disruptions in mitochondrial dynamics often preceding the overt neuronal dysfunction seen in neurodegenerative diseases such as Alzheimers disease, Huntingtons disease and Parkinsons disease. The kynurenine pathway accounts for 95% of dietary tryptophan catabolism and many of the metabolites are neuroactive, including redox-active 3-hydroxykynurenine (3-HK). 3-HK is present under normal physiological conditions in the central nervous system (CNS) and is elevated during inflammation. While supraphysiological levels of 3-HK have been associated with neurotoxicity, the effects of physiological concentrations on neuronal cells, and specifically their mitochondria, remain poorly understood. Here we assessed viability, ATP levels and redox status to determine cellular health and function in neuronal cells exposed to physiological levels of 3-HK, alongside confocal imaging and transcriptomic profiling, finding significant alterations in mitochondrial function and morphology. Interestingly, a biphasic influence of 3-HK on mitochondrial morphology was observed, with an elongated network as well as decreased surface area and volume being observed only at the lowest concentration of 3-HK, reflecting normal physiological levels. At the highest 3-HK concentration tested, reflecting an inflammatory situation, an increased number of mitochondria were present, accompanied by increased activation of caspase-3/7 and enhanced production of mitochondrial superoxide. These results highlight a previously unknown role for 3-HK in regulating mitochondrial function and structure, possibly through altered fission and fusion events, suggesting that subtle changes in kynurenine pathway metabolism may contribute to early mitochondrial dysfunction in neurological disease.

Microbial ammonia oxidation, the first and rate-limiting step of nitrification, plays a central role in soil nitrogen cycling. It is most relevant in agricultural soils as nitrifiers compete with crops for ammonia-based fertilizers. Therefore, synthetic nitrification inhibitors are widely used alongside fertilizers to reduce the activities of dominant drivers of this process, i.e. ammonia-oxidizing archaea (AOA) and bacteria (AOB). However, the physiological responses of ammonia oxidizers remain poorly resolved. Here the response of the AOA Nitrososphaera viennensis to the nitrification inhibitors 3,4-dimethylpyrazole phosphate (DMPP) and allylthiourea (ATU) were investigated using a combination of functional genomics, physiological assays, and relief experiments. The results overturn earlier assumptions that DMPP and ATU act by chelating free copper. Both compounds affected ammonia oxidation and triggered broader shifts in energy metabolism and stress-response pathways, which diverged markedly between the two inhibitors. We propose a competitive inhibition of the ammonia monooxygenase complex with DMPP as it can be alleviated by additional ammonia and elicits activation of urea acquisition, while ATU acted as a non-competitive inhibitor generally inducing quiescence. Both modes of inhibition were associated with clear transcriptomic and proteomic signals that will be advantageous for the identification of mechanisms of other nitrification inhibitors in the future. Key word: Ammonia-oxidizing archaea, nitrification, nitrification inhibitors, archaea, nitrogen cycle

Per- and polyfluoroalkyl substances (PFAS) are new pollutants in the environment whose effects on bacterias physiology is not well understood. In this study, we show that exposure to PFAS causes membrane depolarization in Salmonella enterica serovar Typhi. This works as a metabolic uncoupler that breaks down proton motive force without immediately killing the cells. This disturbance results in a significant elevation of intracellular NADH and NAD levels while preserving redox equilibrium, signifying an augmented metabolic flux. At the same time, we see that {beta}-oxidation pathways are turned on, which suggests that the cells are shifting toward breaking down fats to make up for the lack of energy. Even though there are more reducing equivalents, ATP levels go down, which is what happens when respiration is uncoupled. This puts the cells in a state of "pseudo-starvation." This metabolic stress triggers the SpoT-dependent stringent response, leading to the accumulation of (p)ppGpp. Genetic analysis employing {Delta}relA and {Delta}relA{Delta}spoT mutants confirm that SpoT is necessary for this adaptive response. Functionally, PFAS-treated populations show an enhanced proportion of persister-like cells, which connects exposure to environmental pollutant in the environment to antibiotic tolerance. Our findings reveal a previously unidentified mechanism by which PFAS alters bacterial metabolism and stress responses, facilitating persistence through membrane depolarization, metabolic reconfiguration, and stringent response activation. This study underscores the potential influence of environmental pollutants on bacterial survival mechanisms and antibiotic resistance.

Meloidogyne incognita is a major plant-parasitic nematode responsible for substantial yield losses in tomato worldwide. Current control strategies rely heavily on chemical nematicides, which raise environmental concerns and face increasing regulatory restrictions, underscoring the need for sustainable alternatives. Here, we show that foliar application of an aqueous extract from cavalcade (Centrosema pascuorum) enhances tomato resistance against M. incognita. Pre-inoculation treatment with cavalcade extract prior to inoculation with root-knot nematodes (RKN) significantly reduced root gall formation, delayed nematode development, and limited second-stage juvenile penetration compared with untreated infected controls, whereas post-inoculation application conferred partial protection. Transcriptomic analyses revealed the activation of multiple defense-related pathways, including salicylic acid- and jasmonic acid-associated signaling and phenylpropanoid metabolism, supported by the upregulation of PR1 and PAL. Additional induction of lipid transfer proteins, leucine-rich repeat receptor-like kinases, resistance proteins, mitochondrial calcium uniporter, and GA2ox5 suggests coordinated activation of pathogen recognition, calcium signaling, and hormone-regulated defense networks. These findings demonstrate that cavalcade extract primes broad-spectrum defense responses in tomato and highlight its potential as an environmentally sustainable strategy for nematode management.

Gold is widely distributed in the biosphere, and higher plants growing on geochemically anomalous substrates can accumulate significant amounts of gold. This study reports, for the first time from Goa, the detection, spectroscopic characterisation, and X-ray diffraction analysis of phytoformic gold -- biologically sequestered crystalline gold -- in the above-ground dry litter ash of six tree species (Acacia auriculiformis, Alstonia scholaris, Anacardium occidentale, Artocarpus heterophyllus, Ficus benghalensis, Syzygium cumini) growing on mining dumps within the North Goa Banded Iron Formation (BIF) Belt of the Western Dharwad Craton. Microgravimetric analysis of aqua regia-extracted heavy ash fractions revealed gold concentrations of 275-1100 ppm, two to five orders of magnitude above the crustal background ([~]0.004 ppm). Fourier Transform Infrared (FTIR) spectroscopy of 0.22{square}m membrane-filtered crude extracts confirmed the tetrachloroaurate(III) complex [AuCl{square}]{square} as the dominant dissolved gold species, with the diagnostic 1400-1700{square}cm{square}1 absorption envelope present in all six species. UV-Visible spectrophotometry confirmed chloroauric acid formation with a universal {lambda}max at 372.5{square}nm across all species. Powder X-ray diffraction (XRD) of heavy ash fractions yielded the characteristic FCC metallic gold reflections Au(111), Au(200), and Au(220) in all five species analysed. Application of the Debye-Scherrer equation to the Au(111) reflection (2{theta} = 38.2{degrees}, Cu K) established crystallite sizes of 17.7-31.8{square}nm, confirming that phytoformic gold exists as nanoscale crystalline particles in all species. Ficus benghalensis produced the largest and most crystalline gold nanoparticles (31.8{square}nm) and uniquely exhibited strawberry-shaped isomorphic auriferous siliceous biominerals designated phytoauroliths. The described low-cost protocol -- ashing, aqua regia extraction, membrane filtration, and multi-technique spectroscopic and diffraction confirmation -- constitutes a validated method for rapid biogeochemical gold anomaly detection. Applications in gold phytoextraction and mining waste phytoremediation are discussed.

The yeast secreted proteome plays critical biological roles and influences product and production parameters in industrial fermentation. Systematic profiling of the response of the yeast secretome to intrinsic and extrinsic factors is therefore essential for understanding these functions and for optimizing manufacturing processes. Here, we characterized the yeast secretome under diverse proteosynthetic stress conditions, including glycosylation deficiency, oxidative, reductive, and thermal stresses. The secretome was predominantly composed of conventionally secreted proteins, while a subset of proteins appeared to be secreted via unconventional pathways. Distinct secretome profiles were observed in response to different stressors, driven by a combination of altered intracellular proteomes, altered canonical secretion, and altered cell lysis and unconventional protein secretion, while reflecting the underlying metabolic state of the cells. Heat stress did not impact protein glycosylation but did cause similar protein misfolding stress to N-glycosylation deficiency. Intriguingly, canonically intracellular chaperone BiP was abundant in the secretome in particular stress conditions where its activity would be beneficial. BiP interacted with probable extracellular client proteins in vitro, consistent with it acting as a functional extracellular chaperone/holdase in conditions such as reductive stress in which client proteins could be misfolded outside the cell.

Aphanomyces euteiches, the causative agent of Aphanomyces root rot (ARR), is of major concern for pea and other legume crops globally. This oomycete pathogen causes substantial decreases in crop yields, is unaffected by most fungicides, and persists in the soil for many years via its resilient oospores. Given the significance of pea crops in sustainable agriculture, namely the ability to fix nitrogen and act as a sustainable protein source, solutions to ARR are of high importance. We used RNA-seq in a novel strain of Pseudomonas donghuensis to identify two biosynthetic gene clusters under GacA/S control that are involved in producing bioactive molecules capable of inhibiting A. euteiches. Based on similarity to other reported clusters in Pseudomonas, the first is predicted to encode for a pseudoiodinine compound, while the second is predicted to produce the siderophore 7-hydroxytropolone. Individual knockouts of each cluster showed loss of inhibitory action of P. donghuensis NRC29 against A, euteiches in vivo. This is the first report highlighting the potential of P. donghuensis and the products of the two identified biosynthetic pathways as biocontrol agents for A. euteiches. Further investigations into the efficacy of P. donghuensis NRC29 and its metabolites in inhibiting A. euteiches in field trials will be of high value in developing sustainable strategies for ARR mitigation. ImportanceModern fungicidal treatments for control of root rot in pulse crops are ineffective for control of A. euteiches, leaving limited strategies for management of A. euteiches infected fields. We describe a novel P. donghuensis strain with potential for biocontrol against this persistent pathogen. Given the economic value of peas and other pulses globally, further work into harnessing the bioactive metabolites produced by this strain into a practical in-field treatment will be valuable.

The tight regulation of iron homeostasis is of great importance for cellular health. An increase in intracellular iron levels results in the formation of free radicals, which damages macromolecules and membranes, eventually resulting in cell death by Ferroptosis. Recently, we showed that patients with mutations in VPS41 display a severe neurodegenerative phenotype with iron deposition in the brain. VPS41 is well known as subunit of the HOPS complex required for fusion of late endosomes and autophagosomes with lysosomes. However, VPS41 has also been identified as inhibitor of Ferroptosis and regulator of redox homeostasis. How VPS41 exerts these functions and if these are dependent on the HOPS complex is unknown. Here we show that depletion of VPS41 results in increased intracellular iron levels, ROS formation and mitochondrial fission. Our findings indicate an important role for VPS41 in the regulation of iron homeostasis and mitochondrial fission and suggest Ferroptosis as a possible cause for neurodegeneration in VPS41 patients.

Artemisinin is an effective antimalarial drug sourced from Artemisia annua, but its low and variable yields require enhancement either semi-synthetically or in-planta to meet the global demand for treatment. Though essential enzymes have been identified in the artemisinin biosynthetic pathway, including an essential Cytochrome P450 monooxygenase (CYP71AV1), there are still many unknowns. Cytochrome P450 reductase 1 (herein, AaCPR1), has been experimentally confirmed as an electron transfer partner for CYP71AV1 in its three step oxygenation of key artemisinin precursors. However, the recent discovery of a highly related CPR, herein AaCPR2, introduces the possibility that another, potentially more catalytically favourable interaction, could exist for CYP71AV1. Therefore, enzyme kinetics and differential scanning fluorimetry (DSF) were used in the characterisation of both AaCPR1 and AaCPR2 to determine the existence and source of their catalytic differences. Tested enzyme activity under cytochrome c and NADPH concentrations revealed that AaCPR1 had lower Km and higher kcat/Km values, while AaCPR2 had higher Vmax and kcat values. This suggests that AaCPR1 is more effective at reducing cytochrome c when substrate conditions are limiting, whereas AaCPR2 is more effective than AaCPR1 at reducing cytochrome c when substrate conditions are saturating. This implies a functional partitioning of the two enzymes on the basis of substrate availability. The DSF results provided deeper insight into the different protein-ligand interactions between the two enzymes. AaCPR2 reached lower maximum melting temperatures across all tested conditions, whereas AaCPR1 had higher maximum melting temperatures. Thus, AaCPR1 exhibits higher thermal stability and has a higher temperature threshold than AaCPR2. This contributes to the notion that the AaCPRs are functionally divergent also on the basis of temperature. The cumulative differences in melting behaviour between the two enzymes led to the hypothesis that AaCPR1 and AaCPR2 exhibit different domain motions that may lead to preferential catalysis for one redox partner over another. This was further supported by the prediction of a highly variable loop region between the two enzymes at the connecting domain just after the flexible hinge. If such loops are highly mobile, as predicted, then the residue differences therein could provide a bio-structural basis for the kinetic and thermal/biophysical differences observed between AaCPR1 and AaCPR2. These data support that AaCPR1 and AaCPR2 possess fundamental biophysical differences despite their high degree of relatedness. Ultimately, these differences suggest differential metabolic functions of the two enzyme in artemisinin biosynthesis and/or other important secondary metabolic processes.

Cyanobacteria are diverse photosynthetic microorganisms of great interest for fundamental science and sustainable biotechnological applications. However, their polyploidy makes genetic manipulation challenging and time-consuming. The development of CRISPR/Cas tools has greatly accelerated genome editing and metabolic engineering of few cyanobacterial model species. In this work, we extend the CRISPR/Cas12a system for targeted gene deletion in the non-model cyanobacterium Cyanothece PCC 7425, interesting for its ability to perform intracellular calcium carbonate (CaCO3) biomineralization, nitrogen fixation, etc. We demonstrate for the first time its tractability to gene knockout by generating deletion mutants of four genes (cax3-cax4, gor, and sodB) acting in metabolism and/or response to stresses, using Cas12a mediated homologous recombination. Importantly, full chromosome segregation was rapidly achieved after a single round of selection in all cases. All mutants were genotypically and phenotypically characterised. Moreover, biochemical analysis in the case of{Delta} sodB mutant further confirmed its targeted deletion. Overall, CRISRPR/Cas12a provides a rapid and efficient system for genome editing in Cyanothece PCC 7425, establishing this organism as a versatile model for studying oxidative stress pathways, metal toxicity and moreover, the still poorly known mechanism(s) of intracellular CaCO3 biomineralization. Key PointsO_LIRapid and efficient CRISPR/Cas12a editing established in Cyanothece PCC 7425. C_LIO_LIFully segregated knockout mutants obtained after single selection round. C_LIO_LIPlatform for nuclear waste bioremediation and other biotechnological applications. C_LI

Manufacturing and storage processes can expose microbes to oxidative stress, reducing viability and limiting their use in biotechnological applications. Here, we evaluate graphene quantum dots (GQDs) containing hydroxyl and carboxyl groups as protective additives that mitigate peroxide-induced oxidative stress in Escherichia coli. GQDs did not adversely affect bacterial growth under basal conditions and restored growth in the presence of hydrogen peroxide. Using the membrane-partitioning fluorescent probe C11-BODIPY, we found that GQDs reduced peroxide-induced oxidation in bacterial membranes. We further used redox-sensitive roGFP2 probes to monitor intracellular oxidative stress and found that GQDs suppressed intracellular hydrogen peroxide accumulation and attenuated disruption of glutathione redox homeostasis. Together, these results show that GQDs protect bacteria by limiting peroxide-driven oxidative damage at both membrane and intracellular levels. This work supports the potential use of GQDs as protective additives for microbial formulations that are susceptible to oxidative stress.

Chitosan is an oligosaccharide derived from chitin that is protonated at acidic pH to form a polycation. Its positive charge promotes the interaction with negatively charged components of the yeast cell surface, which has been associated with increased cell permeability and growth inhibition. In this study, we investigated the interaction of chitosan with the cell surface and its permeabilizing capacity in three yeast species displaying distinct susceptibility profiles, Saccharomyces cerevisiae, Candida albicans and Debaryomyces hansenii. We evaluated the correlation between differential susceptibility and chitosan association at the cell surface, as well as cell permeabilization, by integrating growth analyses with surface-binding assays, including FITC-conjugated chitosan to monitor surface association and cellular integration over time, and ultrastructural examination by transmission electron microscopy (TEM). Our results showed that chitosan exhibited varying effects on the growth and permeability of each yeast strain, with D. hansenii being the most susceptible. Furthermore, we observed the incorporation of chitosan onto the cell surface and confirmed its role as a permeabilizing agent. Finally, we used chitosan-induced permeabilization as a method to measure the activity of selected enzymes in situ, demonstrating its potential for studying metabolic functions in permeabilized yeast cells. Overall, our findings establish chitosan as a strain-dependent antifungal agent and a useful tool for functional biochemical analyses in yeast.