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Aims This study aimed to investigate the effect of metal ions on lipopeptide production by Bacillus subtilis strain FJAT-4 and the mechanism of negative regulation by Ca2+. Methods and Results The quantitative measurement of lipopeptides in response to K+, Na+, Mg2+ and Ca2+ addition was carried out by LC-MS. The contents of fengycin and surfactin varied within the range of 116.24–129.80 mg/L and 34.03–63.11 mg/L in the culture media containing K+, Na+ and Mg2+, while the levels were 0.86 and 0.63 mg/L in the media containing Ca2+. Ca2+ at a high concentration (45 mM) did not adversely affect the growth of strain FJAT-4, but caused significant downregulation of lipopeptide synthesis-related gene expression, corresponding to a decrease in lipopeptide production. This inhibition by Ca2+ was further investigated by proteomic analysis. In total, 112 proteins were upregulated and 524 proteins were downregulated in the presence of additional Ca2+ (45 mM). Among these differentially expressed proteins (DEPs), 28 were related to phosphotransferase activity, and 42 were related to kinase activity. The proteomics results suggested that altered levels of three two-component signal-transduction systems (ResD/ResE, PhoP/PhoR and DegU/DegS) might be involved in the control of expression of the fen and srfA operons of FJAT-4 under high calcium stress. Conclusions The Ca2+ at the high concentration (45 mM) triggers a decrease in lipopeptide production, which might be attributed to the regulation of three two-component signal-transduction systems ResD/ResE, PhoP/PhoR and DegU/DegS. Significance and Impact of the Study The regulatory effect of calcium on the expression of genes encoding lipopeptide synthetases can be applied to optimize the production of lipopeptides.

Shigella, the causative agent of bacillary dysentery, subvert cytoskeletal and trafficking processes to invade and replicate in epithelial cells using an arsenal of bacterial effectors translocated through a type III secretion system. Here, we review the various roles of the type III effector IpgD, initially characterized as phosphatidylinositol 4,5 bisphosphate (PI4,5P2) 4-phosphatase. By decreasing PI4,5P2 levels, IpgD triggers the disassembly of cortical actin filaments required for bacterial invasion and cell migration. PI5P produced by IpgD further stimulates signaling pathways regulating cell survival, macropinosome formation, endosomal trafficking and dampening of immune responses. Recently, IpgD was also found to exhibit phosphotransferase activity leading to PI3,4P2 synthesis adding a new flavor to this multipotent bacterial enzyme. The substrate of IpgD, PI4,5P2 is also the main substrate hydrolyzed by endogenous phospholipases C to produce inositoltriphosphate (InsP3), a major Ca2+ second messenger. Hence, beyond the repertoire of effects associated with the direct diversion of phoshoinositides, IpgD indirectly down-regulates InsP3-mediated Ca2+ release by limiting InsP3 production. Furthermore, IpgD controls the intracellular lifestyle of Shigella promoting Rab8/11 -dependent recruitment of the exocyst at macropinosomes to remove damaged vacuolar membrane remnants and promote bacterial cytosolic escape. IpgD thus emerges as a key bacterial effector for the remodeling of host cell membranes.

p38γ and p38δ (p38γ/p38δ) regulate inflammation, in part by controlling tumor progression locus 2 (TPL2) expression in myeloid cells. Here, we demonstrate that TPL2 protein levels are dramatically reduced in p38γ/p38δ-deficient (p38γ/δ2/2) cells and tissues without affecting TPL2 messenger ribonucleic acid (mRNA) expression. We show that p38γ/p38δ posttranscriptionally regulates the TPL2 amount at two different levels. p38γ/p38δ interacts with the TPL2/A20 Binding Inhibitor of NF-κB2 (ABIN2)/Nuclear Factor κB1p105 (NF-κB1p105) complex, increasing TPL2 protein stability. Additionally, p38γ/p38δ regulates TPL2 mRNA translation by modulating the repressor function of TPL2 30 Untranslated region (UTR) mediated by its association with aconitase-1 (ACO1). ACO1 overexpression in wild-type cells increases the translational repression induced by TPL2 30UTR and severely decreases TPL2 protein levels. p38δ binds to ACO1, and p38δ expression in p38γ/δ2/2 cells fully restores TPL2 protein to wild-type levels by reducing the translational repression of TPL2 mRNA. This study reveals a unique mechanism of posttranscriptional regulation of TPL2 expression, which given its central role in innate immune response, likely has great relevance in physiopathology, MCIN/AEI/10.13039/501100011033 Grants PID2019-108349RB-100 (to J.J.S.-E. and A.C.) and SAF2016-79792R (to J.J.S.-E. and A.C.)

An increasing number of Bacillus strains have been identified, and the removal capacity of zearalenone (ZEN) was determined; however, they failed to reveal the detoxification mechanism and transformation product. Here, Bacillus subtilis Y816, which could transform 40 mg/L of ZEN within 7 h of fermentation, was identified and studied. First, the biotransformation products of ZEN and 17-β-estradiol (E2) were identified as ZEN-14-phosphate and E2-3-phosphate by HPLC-TOF-MS and NMR, respectively. An intracellular zearalenone phosphotransferase (ZPH) was found through transcriptome sequencing analysis of B. subtilis Y816. The phosphorylated reaction conditions of ZEN by ZPH were further revealed in this work. Furthermore, the phosphorylated conjugates showed reduced estrogenic toxicity compared with their original substances (ZEN and α/β-zearalenol) using an engineered yeast biosensor system. The first report on the phosphorylated conjugated mode of ZEN in B. subtilis Y816 will inspire new perspectives on the biotransformation of ZEN in Bacillus strains.

The wide variety of protein structures and functions results from the diverse properties of the 20 canonical amino acids. The generally accepted hypothesis is that early protein evolution was associated with enrichment of a primordial alphabet, thereby enabling increased protein catalytic efficiencies and functional diversification. Aromatic amino acids were likely among the last additions to genetic code. The main objective of this study was to test whether enzyme catalysis can occur without the aromatic residues (aromatics) by studying the structure and function of dephospho-CoA kinase (DPCK) following aromatic residue depletion. We designed two variants of a putative DPCK from Aquifex aeolicus by substituting (i) Tyr, Phe and Trp or (ii) all aromatics (including His). Their structural characterization indicates that substituting the aromatics does not markedly alter their secondary structures but does significantly loosen their side chain packing and increase their sizes. Both variants still possess ATPase activity, although with 150-300 times lower efficiency in comparison with the wild-type phosphotransferase activity. The transfer of the phosphate group to the dephospho-CoA substrate becomes heavily uncoupled and only the His-containing variant is still able to perform the phosphotransferase reaction. These data support the hypothesis that proteins in the early stages of life could support catalytic activities, albeit with low efficiencies. An observed significant contraction upon ligand binding is likely important for appropriate organization of the active site. Formation of firm hydrophobic cores, which enable the assembly of stably structured active sites, is suggested to provide a selective advantage for adding the aromatic residues. This article is protected by copyright. All rights reserved.

The high osmolarity glycerol (HOG) pathway, comprising a two-component system and the Hog1 mitogen-activated protein kinase (MAPK) cascade, plays a pivotal role in eukaryotic organisms. Previous studies suggested that the biological functions of some key genes in the HOG pathway varied in filamentous fungi. In this study, we characterized a putative MAPK kinase kinase gene, Ssos4, in Sclerotinia sclerotiorum, which encoded a phosphotransferase in the MAPK cascade. Compared with the wild-type progenitor HA61, the deletion mutant ∆Ssos4-63 exhibited impaired mycelial growth, sclerotia formation, increased hyphal branches, and decreased virulence. The deficiencies of the deletion mutant ∆Ssos4-63 were recovered when the full-length Ssos4 gene was complemented. Deletion of Ssos4 increased the sensitivity to osmotic stresses and cell wall agents and the resistance to fludioxonil and dimethachlon. Intracellular glycerol accumulation was not induced in the deletion mutant ∆Ssos4-63 when treated with fludioxonil and NaCl and the phosphorylation of SsHog1 was also cancelled by the deletion of Ssos4. Consistent with the glycerol accumulation and increased expression levels of SsglpA and Ssfps1, controlling glycerol synthesis and close of glycerol channel under hyperosmotic stress, respectively, were detected in the wild-type strain HA61 but not in the deletion mutant ∆Ssos4-63. Moreover, the relative expression level of Sshog1 significantly decreased, whereas the expression level of Ssos5 increased in the deletion mutant ∆Ssos4-63. These results indicated that Ssos4 played important roles in mycelial growth and differentiation, sclerotia formation, virulence, hyperosmotic adaptation, fungicide sensitivity, and the phosphorylation of SsHog1 in S. sclerotiorum.

Nucleic acids research / Special publication 49(7), 3634-3650 (2021). doi:10.1093/nar/gkab136, Published by Information Retrieval Ltd., London

Background The C-3′,4′-dideoxygenation structure in gentamicin can prevent deactivation by aminoglycoside 3′-phosphotransferase (APH(3′)) in drug-resistant pathogens. However, the enzyme catalyzing the dideoxygenation step in the gentamicin biosynthesis pathway remains unknown. Results Here, we report that GenP catalyzes 3′ phosphorylation of the gentamicin biosynthesis intermediates JI-20A, JI-20Ba, and JI-20B. We further demonstrate that the pyridoxal-5′-phosphate (PLP)-dependent enzyme GenB3 uses these phosphorylated substrates to form 3′,4′-dideoxy-4′,5′-ene-6′-oxo products. The following C-6′-transamination and the GenB4-catalyzed reduction of 4′,5′-olefin lead to the formation of gentamicin C. To the best of our knowledge, GenB3 is the first PLP-dependent enzyme catalyzing dideoxygenation in aminoglycoside biosynthesis. Conclusions This discovery solves a long-standing puzzle in gentamicin biosynthesis and enriches our knowledge of the chemistry of PLP-dependent enzymes. Interestingly, these results demonstrate that to evade APH(3′) deactivation by pathogens, the gentamicin producers evolved a smart strategy, which utilized their own APH(3′) to activate hydroxyls as leaving groups for the 3′,4′-dideoxygenation in gentamicin biosynthesis.

Myxococcus xanthus generates polyphosphates (polyPs) during starvation and forms fruiting bodies through the activity of polyphosphate kinase (Ppk). M. xanthus polyP:AMP phosphotransferase (Pap), a class II Ppk2, catalyzes the transfer of the terminal phosphate from polyP to AMP to yield ADP, but its enzymatic properties have not been investigated in detail. In this study, we found that Pap was activated by Mn2+ or Mg2+ and required higher concentrations of these ions in reactions with longer polyPs to demonstrate maximum activity. The Km of Pap for polyP700-1000 was significantly lower than that for shorter polyPs, but the highest catalytic constant (kcat) was observed for polyP60-70. When Pap was incubated with polyP60-70 and AMP for 3 h, it first generated ADP and then gradually produced ATP, suggesting that M. xanthus Pap also has polyP:ADP phosphotransferase activity similar to that of class III Ppk2 enzymes. During starvation, the specific activity of Pap in M. xanthus was increased by 2.3–2.4-fold at days 1 and 2 of incubation. In addition, recombinant Pap in combination with M. xanthus recombinant enzymes Ppk1 or adenylate kinase (AdkA) could generate ATP from AMP and polyP60-70. These results suggest a functional role of Pap during M. xanthus starvation, when it might act in cooperation with Ppk1 and/or AdkA to produce ATP from AMP, ADP, and polyP.

In this work, the effect of antimicrobial lipopeptide P34 on Listeria monocytogenes was evaluated for the first time through a proteomics approach. Bacteria were treated with sub-lethal doses of peptide P34 (F-P34) and P34 encapsulated into nanoliposomes (N-P34), while empty nanoliposomes (NE) and fresh buffer were used as controls. The proteomic analysis allowed the detection of one group of proteins commonly differentially represented in response to free and encapsulated P34 exposure. A second group of proteins was found to be exclusively differentially represented after exposure with encapsulated P34 only. The antimicrobial peptide P34 caused a significant downregulation of proteins associated with the transport of manganese and the over-representation of proteins related with iron transport in L. monocytogenes. In addition, reduction of stress tolerance proteins related to the σB and VirR regulons, together with the modulation of phosphoenolpyruvate phosphotransferase systems (PTS) for sugar transport were observed. The sugar and oligopeptide transporters regulated by antimicrobial action may influence the key virulence factor PrfA, reducing the pathogenicity of this microorganism.

Marine sponges often contain potent cytotoxic compounds, which in turn evokes the principle question of how marine sponges avoid self-toxicity. In a marine sponge Discodermia calyx, the highly toxic calyculin A is detoxified by the phosphorylation, which is catalyzed by the phosphotransferase CalQ of a producer symbiont, “Candidatus Entotheonella” sp. Here we show the activating mechanism to dephosphorylate the stored phosphocalyculin A protoxin. The phosphatase specific to phosphocalyculin A is CalL, which is also encoded in the calyculin biosynthetic gene cluster. CalL represents a new clade and unprecedently coordinates the heteronuclear metals Cu and Zn. CalL is localized in the periplasmic space of the sponge symbiont, where it is ready for the on-demand production of calyculin A in response to sponge tissue disruption., The phosphatase that activates calyculin biogenesis in the sponge Discodermia calyx turned out to originate from the bacterial symbiont Entotheonella.

Manure amendment generally bolsters soil organisms but not all bacteria equally. To understand why different taxa respond differently, we used shotgun metagenomic approaches to profile functional potentials and correlate them with taxon abundances. A soil originally unproductive was reclaimed using commercial manure and finally became productive. The abundance of Firmicutes in the soil decreased, whereas that of Bacteroidetes and Proteobacteria increased after manure addition. Thirty-nine KEGG modules were significantly different across fertilizer treatments. These modules were mainly associated with the phosphoenolpyruvate-dependent phosphotransferase system (PTS), ATP-binding cassette (ABC) transporters, and two-component signal transduction systems. The Proteobacteria and Firmicutes mainly contributed to these modules. Correlation between the abundances of phyla and orthologs showed two distinctive patterns. One linked the Firmicutes to cell wall biosynthesis, PTS, and ABC transporters, and the other linked the Betaproteobacteria, Bacteroidetes, and Verrucomicrobia to lipopolysaccharide biosynthesis, bacterial motility, and carbon metabolism. Correlation between the abundances of phyla and Carbohydrate-Active Enzyme Database families also showed two distinctive patterns, one of them linking the Betaproteobacteria, Bacteroidetes, and Verrucomicrobia to very high abundances of glycosyltransferases and glycoside hydrolases. Overall, the Proteobacteria and Firmicutes were main drivers of functional potential differences across fertilizer treatments. The Firmicutes were enriched with genes associated with cell wall biosynthesis and membrane transports, while Proteobacteria with lipopolysaccharide biosynthesis and carbohydrate metabolism, which supports our hypothesis that the Firmicutes have a lower potential for utilizing manure-derived carbohydrates, while Proteobacteria have a higher potential. This explains why the Proteobacteria and Firmicutes responded to manure differently.

In mycobacteria, phosphatidylinositol (PI) acts as a common lipid anchor for key components of the cell wall, including the glycolipids phosphatidylinositol mannoside, lipomannan, and lipoarabinomannan. Glycolipids in Mycobacterium tuberculosis, the causative agent of tuberculosis, are important virulence factors that modulate the host immune response. The identity-defining step in PI biosynthesis in prokaryotes, unique to mycobacteria and few other bacterial species, is the reaction between cytidine diphosphate-diacylglycerol and inositol-phosphate to yield phosphatidylinositol-phosphate, the immediate precursor to PI. This reaction is catalyzed by the cytidine diphosphate-alcohol phosphotransferase phosphatidylinositol-phosphate synthase (PIPS), an essential enzyme for mycobacterial viability. Here we present structures of PIPS from Mycobacterium kansasii with and without evidence of donor and acceptor substrate binding obtained using a crystal engineering approach. PIPS from Mycobacterium kansasii is 86% identical to the ortholog from M. tuberculosis and catalytically active. Functional experiments guided by our structural results allowed us to further characterize the molecular determinants of substrate specificity and catalysis in a new mycobacterial species. This work provides a framework to strengthen our understanding of phosphatidylinositol-phosphate biosynthesis in the context of mycobacterial pathogens.

The final step of the CDP-ethanolamine pathway is catalyzed by ethanolamine phosphotransferase 1 (EPT1) and choline/EPT1 (CEPT1). These enzymes are likely involved in the transfer of ethanolamine phosphate from CDP-ethanolamine to lipid acceptors such as 1,2-diacylglycerol (DAG) for PE production and 1-alkyl-2-acyl-glycerol (AAG) for the generation of 1-alkyl-2-acyl-glycerophosphoethanolamine. Here, we investigated the intracellular location and contribution to ethanolamine phospholipid (EP) biosynthesis of EPT1 and CEPT1 in HEK293 cells. Immunohistochemical analyses revealed that EPT1 localizes to the Golgi apparatus and CEPT1 to the ER. We created EPT1-, CEPT1-, and EPTI-CEPT1-deficient cells, and labeling of these cells with radio- or deuterium-labeled ethanolamine disclosed that EPT1 is more important for the de novo biosynthesis of 1-alkenyl-2-acyl-glycerophosphoethanolamine than is CEPT1. EPT1 also contributed to the synthesis of PE species containing the fatty acids 36:1, 36:4, 38:5, 38:4, 38:3, 40:6, 40:5, and 40:4. In contrast, CEPT1 was important for PE formation from shorter fatty acids such as 32:2, 32:1, 34:2, and 34:1. Brefeldin A treatment did not significantly affect the levels of the different PE species, indicating that the subcellular localization of the two enzymes is not responsible for their substrate preferences. In vitro enzymatic analysis revealed that EPT1 prefers AAG 16-20:4 > DAG 18:0-20:4 > DAG 16:0-18:1 = AAG 16-18:1 as lipid acceptors and that CEPT1 greatly prefers DAG 16:0-18:1 to other acceptors. These results suggest that EPT1 and CEPT1 differ in organelle location and are responsible for the biosynthesis of distinct EP species.

We developed a self-priming compartmentalization (SPC) micro-device made of polymethyl methacrylate (PMMA) and integrated a loop-mediated isothermal amplification (LAMP) system for performing multiplex visual detection. The approach had a high throughput of identification of selectable marker gene (SMG) (phosphomannose isomerase (PMI) gene, hygromycin B phosphotransferase (HPT) gene, noglycoside phosphotransferase II (NPTII), β-glucuronidase (GUS), and CP4-5-enolpyruvylshikimate-3-phosphate synthase (CP4-EPSPS)) and was able to perform five SMG analyses simultaneously within 1 h. This micro-device can detect five SMGs in one injection, each gene is repeated three times, and each micro-reactor arrays without cross-contamination. The method of extracting genomic DNA from GM rice grains can be very simple and the detection method only requires cell homogenization. In addition, the limit of detections of the method for PMI, HPT, and NPTII were 10−4. The limit of detections of the method for GUS and CP4-EPSPS were 10−5. The results demonstrated that our method could specifically recognize in genetically modified crops.

Forward genetics in model organisms has boosted our knowledge of the genetic bases of development, aging, and human diseases. In this experimental pipeline, it is crucial to start by inducing a large number of random mutations in the genome of the model organism to search for phenotypes of interest. Many chemical mutagens are used to this end because most of them display particular reactivity properties and act differently over DNA. Here we report the use of N-ethyl-N-nitrosourea (ENU) as a mutagen in the fission yeast Schizosaccharomyces pombe. As opposed to many other alkylating agents, ENU only induces an SN1-type reaction with a low s constant (s = 0.26), attacking preferentially O2 and O4 in thymine and O6 deoxyguanosine, leading to base substitutions rather than indels, which are extremely rare in its resulting mutagenic repertoire. Using ENU, we gathered a collection of 13 temperature-sensitive mutants and 80 auxotrophic mutants including two deleterious alleles of the human ortholog ATIC. Defective alleles of this gene cause AICA-ribosiduria, a severe genetic disease. In this screen, we also identified 13 aminoglycoside-resistance inactivating mutations in APH genes. Mutations reported here may be of interest for metabolism related diseases and antibiotic resistance research fields., This work was supported by Grupos Emergentes UPO grant APP2D1011 to V.A.T. and BFU2016-77297 grant from the Spanish MINECO to J.J. R.H.M. is supported by FPU grant FPU16/04283 from the Spanish MECD. S.V.C is supported by FPU grant FPU18/04507 from the Spanish MEFP.

Clostridioides difficile (C. difficile) is the leading cause of antibiotic-induced bacterial colitis and life-threatening diarrhea worldwide. The commonly existing anionic polysaccharide II (PSII) ...

The spectrum of kinase alterations displays distinct functional characteristics and requires kinase mutation‐oriented strategies for therapeutic interference. Besides phosphotransferase activity, protein abundance, and intermolecular interactions, particular patient‐mutations promote pathological kinase conformations. Despite major advances in identifying lead molecules targeting clinically relevant oncokinase functions, still many kinases are neglected and not part of drug discovery efforts. One explanation is attributed to challenges in tracking kinase activities. Chemical probes are needed to functionally annotate kinase functions, whose activities may not always depend on catalyzing phospho‐transfer. Such non‐catalytic kinase functions are related to transitions of full‐length kinase conformations. Recent findings underline that cell‐based reporter systems can be adapted to record conformation changes of kinases. Here, we discuss the possible applications of an extendable kinase conformation (KinCon) reporter toolbox for live‐cell recording of kinase states. KinCon is a genetically encoded bioluminescence‐based biosensor platform, which can be subjected for measurements of conformation dynamics of mutated kinases upon small molecule inhibitor exposure. We hypothesize that such biosensors can be utilized to delineate the molecular modus operandi for kinase and pseudokinase regulation. This should pave the path for full‐length kinase‐targeted drug discovery efforts aiming to identify single and combinatory kinase inhibitor therapies with increased specificity and efficacy.

Understanding the molecular mechanisms governing antifungal resistance is crucial for identifying new cellular targets for developing new antifungal therapeutics. In this study, we performed a transposon-mediated genome-wide genetic screen in haploid Candida albicans to identify mutants resistant to caspofungin, the first member of the echinocandin class of antifungal drugs. A mutant exhibiting the highest resistance possessed a transposon insertion that inactivates GPI7, a gene encoding the mannose-ethanolamine phosphotransferase. Deleting GPI7 in diploid C. albicans caused similar caspofungin resistance. gpi7Δ/Δ cells showed significantly elevated cell wall chitin content and enhanced phosphorylation of Mkc1, a core component of the PKC-MAPK cell-wall integrity pathway. Deleting MKC1 suppressed the chitin elevation and caspofungin resistance of gpi7Δ/Δ cells, but overexpressing the dominant inactive form of RHO1, an upstream activator of PKC-MAPK signaling, did not. Transcriptome analysis uncovered 406 differentially expressed genes in gpi7Δ/Δ cells, many related to cell wall construction. Our results suggest that GPI7 deletion impairs cell wall integrity, which triggers the cell-wall salvage mechanism via the PKC-MAPK pathway independently of Rho1, resulting in the compensatory chitin synthesis to confer caspofungin resistance.

Cellular signaling systems transmit information over long distances using allosteric transitions and/or post-translational modifications. In two-component systems the sensor histidine kinase and response regulator are wired through phosphoryl-transfer reactions, using either a uni- or bi-directional transmission mode, allowing to build rich regulatory networks. Using the thermosensor DesK-DesR two-component system from Bacillus subtilis and combining crystal structures, QM/MM calculations and integrative kinetic modeling, we uncover that: i) longer or shorter distances between the phosphoryl-acceptor and -donor residues can shift the phosphoryl-transfer equilibrium; ii) the phosphorylation-dependent dimerization of the regulator acts as a sequestering mechanism by preventing the interaction with the histidine kinase; and iii) the kinase's intrinsic conformational equilibrium makes the phosphotransferase state unlikely in the absence of histidine phosphorylation, minimizing backwards transmission. These mechanisms allow the system to control the direction of signal transmission in a very efficient way, showcasing the key role that structure-encoded allostery plays in signaling proteins to store and transmit information.

Probiotics containing functional food confer health benefits in addition to their nutritional properties. In this study, we have evaluated the differential proteomic responses of a potential novel probiotic Pediococcus pentosaceus M41 under heat, cold, acid, and bile stress conditions. We identified stress response proteins that could provide tolerances against these stresses and could be used as probiotic markers for evaluating stress tolerance. Pediococcus pentosaceus M41 was exposed for 2 h to each condition: 50°C (heat stress), 4°C (cold stress), pH 3.0 (acid stress) and 0.05% bile (bile stress). Proteomic analysis was carried out using 2D-IEF SDS PAGE and LC-MS/MS. Out of 60 identified proteins, 14 upregulated and 6 downregulated proteins were common among all the stress conditions. These proteins were involved in different biological functions such as translation-related proteins, carbohydrate metabolism (phosphoenolpyruvate phosphotransferase), histidine biosynthesis (imidazole glycerol phosphate synthase) and cell wall synthesis (tyrosine-protein kinase CapB). Proteins such as polysaccharide deacetylase, lactate oxidase, transcription repressor NrdR, dihydroxyacetone kinase were upregulated under three out of the four stress conditions. The differential expression of these proteins might be responsible for tolerance and protection of P. pentosaceus M41 against different stress conditions.

Apart from the canonical fingers, palm and thumb domains, the RNA dependent RNA polymerases (RdRp) from the viral order Nidovirales possess two additional domains. Of these, the function of the Nidovirus RdRp associated nucleotidyl transferase domain (NiRAN) remains unanswered. The elucidation of the 3D structure of RdRp from the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), provided the first ever insights into the domain organisation and possible functional characteristics of the NiRAN domain. Using in silico tools, we predict that the NiRAN domain assumes a kinase or phosphotransferase like fold and binds nucleoside triphosphates at its proposed active site. Additionally, using molecular docking we have predicted the binding of three widely used kinase inhibitors and five well characterized anti-microbial compounds at the NiRAN domain active site along with their drug-likeliness. For the first time ever, using basic biochemical tools, this study shows the presence of a kinase like activity exhibited by the SARS-CoV-2 RdRp. Interestingly, a well-known kinase inhibitor- Sorafenib showed a significant inhibition and dampened viral load in SARS-CoV-2 infected cells. In line with the current global COVID-19 pandemic urgency and the emergence of newer strains with significantly higher infectivity, this study provides a new anti-SARS-CoV-2 drug target and potential lead compounds for drug repurposing against SARS-CoV-2., Author summary The on-going coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is significantly affecting the world health. Unfortunately, over 180 million cases of COVID-19 resulting in nearly 4 million deaths have been reported till June, 2021. In this study, using a combination of bioinformatics, biochemical and mass spectrometry methods, we show that the Nidovirus RdRp associated Nucleotidyl transferase (NiRAN) domain of the RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 exhibits a kinase like activity. Additionally, we also show that few broad spectrum anti-cancer and anti-microbial drugs dampen this kinase like activity. Of note, Sorafenib, an FDA approved anti-cancer kinase inhibiting drug significantly reduces the SARS-CoV-2 load in cell lines. Our study suggests that NiRAN domain of the SARS-CoV-2 RdRp is indispensible for the successful viral life cycle and shows that abolishing this enzymatic function of RdRp by small molecule inhibitors may open novel avenues for COVID-19 therapeutics.

BackgroundGlycolytic pathway is common in all plant organs, especially in oxygen-deficient tissues. Phosphofructokinase (PFK) is a rate-limiting enzyme in the glycolytic pathway and catalyses the phosphorylation of fructose-6-phosphate to fructose-1,6-bisphosphate. Cassava (M. esculenta) root is a huge storage organ with low amount of oxygen. However, less is known about the functions of PFK fromM. esculenta(MePFK). We conducted a systematic analysis ofMePFKgenes to explore the function of theMePFKgene family under hypoxic stress.ResultsWe identified 13MePFKgenes and characterised their sequence structure. The phylogenetic tree divided the 13 genes into two groups: nine wereMePFKs and four were pyrophosphate-fructose-6-phosphate phosphotransferase (MePFPs). We confirmed by green fluorescent protein fusion protein expression that MePFK03 and MePFPA1 were localised in the chloroplast and cytoplasm, respectively. The expression profiles of the 13MePFKs detected by quantitative reverse transcription polymerase chain reaction revealed thatMePFK02,MePFK03,MePFPA1,MePFPB1displayed higher expression in leaves, root and flower. The expression ofMePFK03,MePFPA1andMePFPB1in tuber root increased gradually with plant growth. We confirmed that hypoxia occurred in the cassava root, and the concentration of oxygen was sharply decreasing from the outside to the inside root. The expression ofMePFK03,MePFPA1andMePFPB1decreased with the decrease in the oxygen concentration in cassava root. Waterlogging stress treatment showed that the transcript level of PPi-dependentMePFPandMeSuSywere up-regulated remarkably and PPi-dependent glycolysis bypass was promoted.ConclusionA systematic survey of phylogenetic relation, molecular characterisation, chromosomal and subcellular localisation and cis-element prediction of MePFKs were performed in cassava. The expression profiles of MePFKs in different development stages, organs and under waterlogging stress showed that MePFPA1 plays an important role during the growth and development of cassava. Combined with the transcriptional level ofMeSuSy, we found that pyrophosphate (PPi)-dependent glycolysis bypass was promoted when cassava was under waterlogging stress. The results would provide insights for further studying the function of MePFKs under hypoxic stress.