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This special issue on Advances in Kinase Drug Discovery provides a selection of research articles and topical reviews covering all aspects of drug discovery targeting the phosphotransferase enzyme family [...]

Choline phospholipids (PLs) such as phosphatidylcholine (PC) and 1-alkyl-2-acyl-sn-glycerophosphocholine are important components for cell membranes and also serve as a source of several lipid mediators. These lipids are biosynthesized in mammals in the final step of the CDP-choline pathway by the choline phosphotransferases choline phosphotransferase 1 (CPT1) and choline/ethanolamine phosphotransferase 1 (CEPT1). However, the contributions of these enzymes to the de novo biosynthesis of lipids remain unknown. Here, we established and characterized CPT1- and CEPT1-deficient human embryonic kidney 293 cells. Immunohistochemical analyses revealed that CPT1 localizes to the trans-Golgi network and CEPT1 to the endoplasmic reticulum. Enzyme assays and metabolic labeling with radiolabeled choline demonstrated that loss of CEPT1 dramatically decreases choline PL biosynthesis. Quantitative PCR and reintroduction of CPT1 and CEPT1 revealed that the specific activity of CEPT1 was much higher than that of CPT1. LC-MS/MS analysis of newly synthesized lipid molecular species from deuterium-labeled choline also showed that these enzymes have similar preference for the synthesis of PC molecular species, but that CPT1 had higher preference for 1-alkyl-2-acyl-sn-glycerophosphocholine with PUFA than did CEPT1. The endogenous level of PC was not reduced by the loss of these enzymes. However, several 1-alkyl-2-acyl-sn-glycerophosphocholine molecular species were reduced in CPT1-deficient cells and increased in CEPT1-deficient cells when cultured in 0.1% FBS medium. These results suggest that CEPT1 accounts for most choline PL biosynthesis activity, and that both enzymes are responsible for the production of different lipid molecular species in distinct organelles.

Adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3) (AK) is the enzyme that catalyzes the reversible conversion of MgATP + AMP to MgADP + ADP, a principal step in adenine nucleotide metabolism and cellular energy homeostasis. To enrich the information about fish AKs, we isolated this enzyme from the lateral muscle of bastard halibut Paralichthys olivaceus and biochemically characterized it. The halibut enzyme (PoAK) extracted from the lateral muscle was purified by column chromatographies on TOYOPEARL SP-650 M and Superdex 75 10/300. The purified PoAK showed a single protein band of ~ 22 kDa on SDS–polyacrylamide gel electrophoresis, and optimal temperature and pH at around 40 °C and 7, respectively. PoAK was appreciably heat stable, e.g., the temperature that caused 50% inactivation during 30-min incubation was 54 °C. The molar ratio for ATP:ADP:AMP in the equilibrium state of the reaction was ~ 1:1:1. Peptide mass fingerprinting indicated that PoAK is the product of adenylate kinase isoform 1 gene (GenBank, XP_019937160.1) encoded in the halibut genome. The deduced amino-acid sequence of the halibut AK comprised 194 residues and showed 92, 91 and 81% amino-acid identities to those of a putative rainbow trout AK, a carp AK and a chicken AK, respectively.

Proteins interact with one another across a broad spectrum of affinities. Our understanding of the low end of this spectrum, as characterized by millimolar dissociation constants, relies on a handful of cases in which weak encounters have experimentally been identified. These weak interactions away from the specific target binding site can lead toward a higher-affinity complex. Recently, we detected weak encounters between two paralogous phosphotransferase pathways of Escherichia coli, which regulate various metabolic processes and stress responses. In addition to encounters that are known to occur between cognate proteins, i.e., those that can exchange phosphate groups with each other, surprisingly, encounters involving noncognates were also observed. It is not clear whether these “futile” encounters have a cooperative or competitive role. Using agent-based simulations, we find that the encounter complexes can be cooperative or competitive so as to increase or lower the effective binding affinity of the specific complex under different circumstances. This finding invites further questions into how organisms might exploit such low affinities to connect their signaling components.

Fungal tRNA ligase (Trl1) rectifies RNA breaks with 2′,3′-cyclic-PO4 and 5′-OH termini. Trl1 consists of three catalytic modules: an N-terminal ligase (LIG) domain; a central polynucleotide kinase (KIN) domain; and a C-terminal cyclic phosphodiesterase (CPD) domain. Trl1 enzymes found in all human fungal pathogens are untapped targets for antifungal drug discovery. Here we report a 1.9 Å crystal structure of Trl1 KIN-CPD from the pathogenic fungus Candida albicans, which adopts an extended conformation in which separate KIN and CPD domains are connected by an unstructured linker. CPD belongs to the 2H phosphotransferase superfamily by dint of its conserved central concave β sheet and interactions of its dual HxT motif histidines and threonines with phosphate in the active site. Additional active site motifs conserved among the fungal CPD clade of 2H enzymes are identified. We present structures of the Candida Trl1 KIN domain at 1.5 to 2.0 Å resolution—as apoenzyme and in complexes with GTP•Mg2+, IDP•PO4, and dGDP•PO4—that highlight conformational switches in the G-loop (which recognizes the guanine base) and lid-loop (poised over the nucleotide phosphates) that accompany nucleotide binding.

Hygromycin B (HygB) is one of the aminoglycoside antibiotics, and it is widely used as a reagent in molecular-biology experiments. Two kinases are known to inactivate HygB through phosphorylation: aminoglycoside 7′′-phosphotransferase-Ia [APH(7′′)-Ia] from Streptomyces hygroscopicus and aminoglycoside 4-phosphotransferase-Ia [APH(4)-Ia] from Escherichia coli. They phosphorylate the hydroxyl groups at positions 7′′ and 4 of the HygB molecule, respectively. Previously, the crystal structure of APH(4)-Ia was reported as a ternary complex with HygB and 5′-adenylyl-β,γ-imidodiphosphate (AMP-PNP). To investigate the differences in the substrate-recognition mechanism between APH(7′′)-Ia and APH(4)-Ia, the crystal structure of APH(7′′)-Ia complexed with HygB is reported. The overall structure of APH(7′′)-Ia is similar to those of other aminoglycoside phosphotransferases, including APH(4)-Ia, and consists of an N-terminal lobe (N-lobe) and a C-terminal lobe (C-lobe). The latter also comprises a core and a helical domain. Accordingly, the APH(7′′)-Ia and APH(4)-Ia structures fit globally when the structures are superposed at three catalytically important conserved residues, His, Asp and Asn, in the Brenner motif, which is conserved in aminoglycoside phosphotransferases as well as in eukaryotic protein kinases. On the other hand, the phosphorylated hydroxyl groups of HygB in both structures come close to the Asp residue, and the HygB molecules in each structure lie in opposite directions. These molecules were held by the helical domain in the C-lobe, which exhibited structural differences between the two kinases. Furthermore, based on the crystal structures of APH(7′′)-Ia and APH(4)-Ia, some mutated residues in their thermostable mutants reported previously were located at the same positions in the two enzymes.

Glycosylphosphatidylinositol (GPI) is an important compound for the growth of fungi, because GPI-anchored proteins including glycosyltransferases and adhesins are involved in cell-wall integrity, adhesion, and nutrient uptake in this organism. In this study, we examined orf19.5244 in the genome database of the pathogenic fungus Candida albicans, a homologue of the Saccharomyces cerevisiae mannose-ethanolamine phosphotransferase gene, MCD4, which plays a role in GPI synthesis. Expression of this homologue, designated CaMCD4, restored cell growth in a defective conditional mcd4 mutant of S. cerevisiae, Scmcd4t, in which expression of native MCD4 was repressed in the presence of doxycycline (Dox). Analysis of radiolabeled lipids showed that the accumulation of abnormal GPI anchor precursors in Scmcd4t decreased markedly upon expression of CaMCD4. Moreover, we constructed a single mutant (Camcd4/CaMCD4) and a conditional double mutant (Camcd4/Camcd4t) at the MCD4 locus of C. albicans. Repression of CaMCD4 expression by Dox led to a decrease in growth and appearance of abnormal morphology in C. albicans, both in vitro and in a silkworm infection model. These results suggest that CaMcd4p is indispensable for growth of C. albicans both in vitro and in infected hosts and a candidate target for the development of new antifungals.

Protein phosphorylation regulates key processes in all organisms. In Gram-positive bacteria, protein arginine phosphorylation plays a central role in protein quality control by regulating transcription factors and marking aberrant proteins for degradation. Here, we report structural, biochemical, and in vivo data of the responsible kinase, McsB, the founding member of an arginine-specific class of protein kinases. McsB differs in structure and mechanism from protein kinases that act on serine, threonine, and tyrosine residues and instead has a catalytic domain related to that of phosphagen kinases (PhKs), metabolic enzymes that phosphorylate small guanidino compounds. In McsB, the PhK-like phosphotransferase domain is structurally adapted to target protein substrates and is accompanied by a novel phosphoarginine (pArg)-binding domain that allosterically controls protein kinase activity. The identification of distinct pArg reader domains in this study points to a remarkably complex signaling system, thus challenging simplistic views of bacterial protein phosphorylation.

Deactivation of aminoglycosides by their modifying enzymes, including a number of aminoglycoside O-phosphotransferases, is the most ubiquitous resistance mechanism in aminoglycoside-resistant pathogens. Nonetheless, in a couple of biosynthetic pathways for gentamicins, fortimicins, and istamycins, phosphorylation of aminoglycosides seems to be a unique and initial step for the creation of a natural defensive structural feature such as a 3',4'- dideoxy scaffold. Our aim was to elucidate the biochemical details on the beginning of these C3',4'-dideoxygenation biosynthetic steps for aminoglycosides. The biosynthesis of istamycins must surely involve these 3',4'-didehydroxylation steps, but much less has been reported in terms of characterization of istamycin biosynthetic genes, especially about the phosphotransferase-encoding gene. In the disruption and complementation experiments pointing to a putative gene, istP, in the genome of wild-type Streptomyces tenjimariensis, the function of the istP gene was proved here to be a phosphotransferase. Next, an in-frame deletion of a known phosphotransferase-encoding gene forP from the genome of wild-type Micromonospora olivasterospora resulted in the appearance of a hitherto unidentified fortimicin shunt product, namely 3-O-methyl-FOR-KK1, whereas complementation of forP restored the natural fortimicin metabolite profiles. The bilateral complementation of an istP gene (or forP) in the ΔforP mutant ( or ΔistP mutant strain) successfully restored the biosynthesis of 3',4'- dideoxy fortimicins and istamycins , thus clearly indicating that they are interchangeable launchers of the biosynthesis of 3',4'-dideoxy types of 1,4-diaminocyclitol antibiotics.

Summary Many bacteria take up carbohydrates by membrane‐integral sugar specific phosphoenolpyruvate‐dependent carbohydrate:phosphotransferase systems (PTS). Although the PTS is centrally involved in regulation of carbon metabolism in different bacteria, little is known about localization and putative oligomerization of the permease subunits (EII). Here, we analyzed localization of the fructose specific PtsF and the glucose specific PtsG transporters, as well as the general components EI and HPr from Corynebacterium glutamicum using widefield and single molecule localization microscopy. PtsF and PtsG form membrane embedded clusters that localize in a punctate pattern. Size, number and fluorescence of the membrane clusters change upon presence or absence of the transported substrate, and a direct influence of EI and HPr was not observed. In presence of the transport substrate, EII clusters significantly increased in size. Photo‐activated localization microscopy data revealed that, in presence of different carbon sources, the number of EII proteins per cluster remains the same, however, the density of these clusters reduces. Our work reveals a simple mechanism for efficient membrane occupancy regulation. Clusters of PTS EII transporters are densely packed in absence of a suitable substrate. In presence of a transported substrate, the EII proteins in individual clusters occupy larger membrane areas., PTS permeases cluster in substrate dependent manner in the membrane of Corynebacterium glutamicum. In absence of the transport substrate (here glucose) the PTS permease forms densely packed clusters. In presence of the correct sugar the permease proteins rearrange and cover a larger membrane area.

Systemic autoinflammatory diseases (sAIDs) are a heterogeneous group of disorders, having monogenic inherited forms with overlapping clinical manifestations. More than half of patients do not carry any pathogenic variant in formerly associated disease genes. Here, we report a cross-sectional study on targeted Next-Generation Sequencing (NGS) screening in patients with suspected sAIDs to determine the diagnostic utility of genetic screening. Fifteen autoinflammation/immune-related genes (ADA2-CARD14-IL10RA-LPIN2-MEFV-MVK-NLRC4-NLRP12-NLRP3-NOD2-PLCG2-PSTPIP1-SLC29A3-TMEM173-TNFRSF1A) were used to screen 196 subjects from adult/pediatric clinics, each with an initial clinical suspicion of one or more sAID diagnosis with the exclusion of typical familial Mediterranean fever (FMF) patients. Following the genetic screening, 140 patients (71.4%) were clinically followed-up and re-evaluated. Fifty rare variants in 41 patients (20.9%) were classified as pathogenic or likely pathogenic and 32 of those variants were located on the MEFV gene. We detected pathogenic or likely pathogenic variants compatible with the final diagnoses and inheritance patterns in 14/140 (10%) of patients for the following sAIDs: familial Mediterranean fever (n=7), deficiency of adenosine deaminase 2 (n=2), mevalonate kinase deficiency (n=2), Muckle-Wells syndrome (n=1), Majeed syndrome (n=1), and STING-associated vasculopathy with onset in infancy (n=1). Targeted NGS panels have impact on diagnosing rare monogenic sAIDs for a group of patients. We suggest that MEFV gene screening should be first-tier genetic testing especially in regions with high carrier rates. Clinical utility of multi-gene testing in sAIDs was as low as expected, but extensive genome-wide familial analyses in combination with exome screening would enlighten additional genetic factors causing disease. Istanbul University Scientific Research FundIstanbul University [49820, BYP-2017-22876] This study was funded by Istanbul University Scientific Research Fund (Grants: 49820 and BYP-2017-22876). WOS:000466048800017 30783801 Q3

This study aimed to develop a bile-responsive expression system for lactobacilli. The promoters of four genes, encoding phosphoenolpyruvate-dependent sugar phosphotransferase (mannose-specific), L-lactate dehydrogenase (LDH), HPr kinase, and D-alanine-D-alanine ligase, respectively, which were highly expressed by bile addition in Lactobacillus johnsonii PF01, were chosen. Each promoter was amplified by polymerase chain reaction and fused upstream of the β-glucuronidase gene as a reporter, respectively. Then, these constructs were cloned into E. coli-Lactobacillus shuttle vector pULP2, which was generated by the fusion of pUC19 with the L. plantarum plasmid pLP27. Finally, the constructed vectors were introduced into L. plantarum for a promoter activity assay. The LDH promoter showed the highest activity and its activity increased 1.8-fold by bile addition. The constructed vector maintained in L. plantarum until 80 generations without selection pressure. A bile-responsive expression vector, pULP3-PLDH, for Lactobacillus spp. can be an effective tool for the bile-inducible expression of bioactive proteins in intestine after intake in the form of fermented dairy foods.

Glycyrrhizic acid (GL) and its deglycosylated metabolite glycyrrhetinic acid (GA) have been recognized as effective drugs for liver diseases. However, the underlying molecular mechanism of GL and GA remains unclear. Here, CCl4 was used to induce the liver cirrhosis in rat, GL and GA both significantly alleviate the liver fibrosis by decreasing the ALT, AST level in serum. While, only GL was observed to significantly down-regulate the TLR4 and LBP gene expression, which indicated that GL could have direct impact on the intestinal flora. Additionally, 16S rRNA gene sequencing showed that GL especially restore the abundance of Lactobacillus compared with the model group, however, GA has no effect on the cirrhosis-induced gut dysbiosis. Furthermore, PICRUSt analysis showed that GL could improve the relative abundance of phosphotransferase systems and phosphor-glucosidases which may promote the bioavailability of GL. These results may provide novel strategies for prevention and treatment of liver cirrhosis.

Despite their comparatively low abundance, phosphoinositides play a central role in membrane traffic and signalling. PtdIns(3,4,5)P3 and PtdIns(3,4)P2 are uniquely important, as they promote cell growth, survival, and migration. Pathogenic organisms have developed means to subvert phosphoinositide metabolism to promote successful infection and their survival within host organisms. We demonstrate that PtdIns(3,4)P2 is generated in host cells by effectors of the enteropathogenic bacteria Salmonella and Shigella. Pharmacological, gene silencing and heterologous expression experiments revealed that, remarkably, the biosynthesis of PtdIns(3,4)P2 occurs independently of phosphoinositide 3-kinases. Instead, we found that the Salmonella effector SopB, heretofore believed to be a phosphatase, generates PtdIns(3,4)P2 de novo via a phosphotransferase/phosphoisomerase mechanism. Recombinant SopB is capable of generating PtdIns(3,4)P2 from PtdIns(4,5)P2 in a cell-free system. Through a remarkable instance of convergent evolution, bacterial effectors acquired the ability to synthesize 3-phosphorylated phosphoinositides by an ATP- and kinase-independent mechanism, thereby subverting host signaling to gain entry and even provoke oncogenic transformation.

Enzyme I (EI) is a phosphotransferase enzyme responsible for converting phosphoenolpyruvate (PEP) into pyruvate. This reaction initiates a five-step phosphorylation cascade in the bacterial phosphotransferase (PTS) transduction pathway. Under physiological conditions, EI exists in an equilibrium between a functional dimer and an inactive monomer. The monomer–dimer equilibrium is a crucial factor regulating EI activity and the phosphorylation state of the overall PTS. Experimental studies of EI’s monomeric state have yet been hampered by the dimer’s high thermodynamic stability, which prevents its characterization by standard structural techniques. In this study, we modified the dimerization domain of EI (EIC) by mutating three amino acids involved in the formation of intersubunit salt bridges. The engineered variant forms an active dimer in solution that can bind and hydrolyze PEP. Using hydrostatic pressure as an additional perturbation, we were then able to study the complete dissociation of the variant from 1 bar to 2.5 kbar in the absence and the presence of EI natural ligands. Backbone residual dipolar couplings collected under high-pressure conditions allowed us to determine the conformational ensemble of the isolated EIC monomeric state in solution. Our calculations reveal that three catalytic loops near the dimerization interface become unstructured upon monomerization, preventing the monomeric enzyme from binding its natural substrate. This study provides an atomic-level characterization of EI’s monomeric state and highlights the role of the catalytic loops as allosteric connectors controlling both the activity and oligomerization of the enzyme.

Different external stimuli are perceived by multiple sensor histidine kinases and transmitted by phosphorylation via the phosphotransfer protein Ypd1p in the multistep phosphorelay system of the high osmolarity glycerol signaling pathway of filamentous fungi. How the signal propagation takes place is still not known in detail since multiple sensor histidine kinase genes in most filamentous fungi are coded in the genome, whereas only one gene for Ypd1p exists. That raises the hypothesis that various Ypd1p isoforms are produced from a single gene sequence, perhaps by alternative splicing, facilitating a higher variability in signal transduction. We found that the mRNA of MoYPD1 in the rice blast fungus Magnaporthe oryzae is subjected to an increased structural variation and amplified putative isoforms on a cDNA level. We then generated mutant strains overexpressing these isoforms, purified the products, and present here one previously unknown MoYpd1p isoform on a proteome level. Alternative splicing was found to be a valid molecular mechanism to increase the signal diversity in eukaryotic multistep phosphorelay systems.

Tpt1, an essential component of the fungal and plant tRNA splicing machinery, catalyzes transfer of an internal RNA 2′-PO4 to NAD+ yielding RNA 2′-OH and ADP-ribose-1′,2′-cyclic phosphate products. Here, we report NMR structures of the Tpt1 ortholog from the bacterium Runella slithyformis (RslTpt1), as apoenzyme and bound to NAD+. RslTpt1 consists of N- and C-terminal lobes with substantial inter-lobe dynamics in the free and NAD+-bound states. ITC measurements of RslTpt1 binding to NAD+ (KD ∼31 μM), ADP-ribose (∼96 μM) and ADP (∼123 μM) indicate that substrate affinity is determined primarily by the ADP moiety; no binding of NMN or nicotinamide is observed by ITC. NAD+-induced chemical shift perturbations (CSPs) localize exclusively to the RslTpt1 C-lobe. NADP+, which contains an adenylate 2′-PO4 (mimicking the substrate RNA 2′-PO4), binds with lower affinity (KD ∼1 mM) and elicits only N-lobe CSPs. The RslTpt1·NAD+ binary complex reveals C-lobe contacts to adenosine ribose hydroxyls (His99, Thr101), the adenine nucleobase (Asn105, Asp112, Gly113, Met117) and the nicotinamide riboside (Ser125, Gln126, Asn163, Val165), several of which are essential for RslTpt1 activity in vivo. Proximity of the NAD+ β-phosphate to ribose-C1″ suggests that it may stabilize an oxocarbenium transition-state during the first step of the Tpt1-catalyzed reaction.

Protein phosphorylation is a universal mechanism for transducing cellular signals in prokaryotes and eukaryotes. The histidine kinase CckA, the histidine phosphotransferase ChpT, and the response regulator CtrA are conserved throughout the alphaproteobacteria. In Rhodobacter capsulatus, these proteins are key regulators of the gene transfer agent (RcGTA), which is present in several alphaproteobacteria. Using purified recombinant R. capsulatus proteins, we show in vitro autophosphorylation of CckA protein, and phosphotransfer to ChpT and thence to CtrA, to demonstrate biochemically that they form a phosphorelay. The secondary messenger cyclic di-GMP changed CckA from a kinase to a phosphatase, resulting in reversal of the phosphotransfer flow in the relay. The substitutions of two residues in CckA greatly affected the kinase or phosphatase activity of the protein in vitro, and production of mutant CckA proteins in vivo confirmed the importance of kinase but not phosphatase activity for the lytic release of RcGTA. However, phosphatase activity was needed to produce functional RcGTA particles. The binding of cyclic di-GMP to the wild-type and mutant CckA proteins was evaluated directly using a pulldown assay based on biotinylated cyclic di-GMP and streptavidin-linked beads. IMPORTANCE The CckA, ChpT, and CtrA phosphorelay proteins are widespread in the alphaproteobacteria, and there are two groups of organisms that differ in terms of whether this pathway is essential for cell viability. Little is known about the biochemical function of these proteins in organisms where the pathway is not essential, a group that includes Rhodobacter capsulatus. This work demonstrates biochemically that CckA, ChpT, and CtrA also form a functional phosphorelay in the latter group and that the direction of phosphotransfer is reversed by cyclic di-GMP. It is important to improve understanding of more representatives of this pathway in order to obtain deeper insight into the function, composition, and evolutionary significance of a wider range of bacterial regulatory networks.

Capsular polysaccharide (CPS) is a crucial virulence factor for Klebsiella pneumoniae infection. We demonstrated an association of CPS production with two phosphoenolpyruvate:carbohydrate phosphotransferase systems (PTSs). Deficiency of crr, encoding enzyme IIA of PTS, in K. pneumoniae enhanced the transcriptional activities of galF, wzi and gnd, which are in the cps gene cluster, leading to high CPS production. A crr mutant exhibited a higher survival rate in 1% hydrogen peroxide than the wild-type. The crr mutant showed less sensitivity to engulfment by macrophage (RAW 264.7) than the wild-type by observing the intracellular bacteria using confocal laser scanning microscopy (CLSM) and by calculating the colony-forming units (CFU) of intracellular bacteria. After long-term incubation, the survival rate of the intracellular crr mutant was higher than that of the wild-type. Deficiency of crr enhanced the transcriptional activities of etcABC which encodes another putative enzyme II complex of a PTS. Deletion of etcABC in the crr mutant reduced CPS production and the transcriptional activities of galF compared to those of the crr mutant. These results indicated that one PTS component, Crr, represses CPS production by repressing another PTS component, EtcABC, in K. pneumoniae. In addition, PTS plays a role in bacterial resistance to macrophage phagocytosis.

Phosphorous-NMR is scarcely employed to evaluate enzyme kinetics of kinase driven monophosphorylations, despite of being a powerful and reliable tool to undoubtedly detect the actual phosphoryl transfer to the targeted substrate. Another advantage is that an external supplementation source of the NMR active isotope is not required, since 31P is highly abundant in nature. Glycerol kinase (GlpK) from E. coli is an exemplary ATP-dependent kinase/phosphotransferase model to illustrate the value and usefulness of a 31P-NMR-based approach to assess the enzymatically driven monophosphorylation of glycerol. Moreover, the described approach offers an alternative to the indirect coupled glycerol kinase enzyme assays. Herein, we provided a real time 31P-NMR-based method customized for the direct assessment of the glycerol kinase enzyme activity.•Real-time detection for phosphoryl group dynamics in the GlpK driven reaction•Direct assessment of product formation (glycerol-monophosphate)•Parallel determination of cosubstrate (ATP) consumption and coproduct (ADP) generation•Method validation was performed via 31P-NMR for each phosphorylated molecule involved in the reaction in order to assist in the molecular assignments, Graphical abstract Image, graphical abstract

10 Pág. Centro de Biotecnología y Genómica de Plantas (CBGP), The poverty of disease resistance gene reservoirs limits the breeding of crops for durable resistance against evolutionary dynamic pathogens. Zymoseptoria tritici which causes Septoria tritici blotch (STB), represents one of the most genetically diverse and devastating wheat pathogens worldwide. No fully virulent Z. tritici isolates against synthetic wheats carrying the major resistant gene Stb16q have been identified. Here, we use comparative genomics, mutagenesis and complementation to identify Stb16q, which confers broad-spectrum resistance against Z. tritici. The Stb16q gene encodes a plasma membrane cysteine-rich receptor-like kinase that was recently introduced into cultivated wheat and which considerably slows penetration and intercellular growth of the pathogen., Our works was supported by the “Fond de Soutien à l’Obtention Vegetale” (FSOV), the French National Institute for Agricultural Research (INRA), and the ETH Zurich Career Seed Grant (SEED-58 16-1). We thank Sreedhar Kilaru and Gero Steinberg for providing the strain 3D7 expressing eGFP. Confocal laser scanning microscopy experiments were performed in the Scientific Center for Optical and Electron Microscopy (ScopeM), ETH Zurich and at the SPIBOC imaging facility of the Institut Sophia Agrobiotech. B.F. was supported by INRA and the French Government (National Research Agency, ANR) through the ‘Investments for the Future’ LabEx SIGNALIFE: program reference #ANR-11-LABX-0028-01.

Objectives To describe a novel chromosomal aminoglycoside phosphotransferase named APH(3′)-IId identified in an MDR Brucella intermedia ZJ499 isolate from a cancer patient. Methods Species identity was determined by PCR and MALDI-TOF MS analysis. WGS was performed to determine the genetic elements conferring antimicrobial resistance. Gene cloning, transcriptional analysis and targeted gene deletion, as well as protein purification and kinetic analysis, were performed to investigate the mechanism of resistance. Results APH(3′)-IId consists of 266 amino acids and shares the highest identity (48.25%) with the previously known APH(3′)-IIb. Expression of aph(3′)-IId in Escherichia coli decreased susceptibility to kanamycin, neomycin, paromomycin and ribostamycin. The aph(3′)-IId gene in ZJ499 was transcriptionally active under laboratory conditions and the relative abundance of this transcript was unaffected by treatment with the above four antibiotics. However, deletion of aph(3′)-IId in ZJ499 results in decreased MICs of these drugs. The purified APH(3′)-IId showed phosphotransferase activity against kanamycin, neomycin, paromomycin and ribostamycin, with catalytic efficiencies (kcat/Km) ranging from ∼105 to 107 M−1 s−1. Genetic environment and comparative genomic analyses suggested that aph(3′)-IId is probably a ubiquitous gene in Brucella, with no mobile genetic elements detected in its surrounding region. Conclusions APH(3′)-IId is a novel chromosomal aminoglycoside phosphotransferase and plays an important role in the resistance of B. intermedia ZJ499 to kanamycin, neomycin, paromomycin and ribostamycin. To the best of our knowledge, APH(3′)-IId represents the fourth characterized example of an APH(3′)-II enzyme.

GlcNAc-1-phosphotransferase (GNPT) catalyzes the initial step in the formation of the mannose-6-phosphate tag that labels ∼60 lysosomal proteins for transport. Mutations in GNPT cause lysosomal storage disorders such as mucolipidoses. However, the molecular mechanism of GNPT remains unclear. Mammalian GNPTs are α2β2γ2 hexamers in which the core catalytic α- and β-subunits are derived from GNPTAB. Here, we present the cryo-electron microscopy structure of the Drosophila melanogaster GNPTAB homolog (DmGNPTAB). Four conserved regions located far apart in the sequence fold into the catalytic domain, which exhibits structural similarity to that of the UDP-glucose glycoprotein glucosyltransferase (UGGT). Comparison with UGGT revealed a putative donor substrate-binding site, and the functional requirements of critical residues in human GNPTAB were validated using GNPTAB-knockout cells. DmGNPTAB forms an evolutionarily conserved homodimer, and perturbing the dimer interface undermines the maturation and activity of human GNPTAB. These results provide important insights into GNPT function and related diseases.

Mucopolysaccharidoses (MPS) and mucolipidoses (ML) are disorders that alter lysosome function. While MPS are caused by mutation in enzymes that degrade glycosaminoglycans, the ML are disorders characterized by reduced function in the phosphotransferase enzyme. Multiple clinical features are associated with these diseases and the exact mechanisms that could explain such different clinical manifestations in patients are still unknown. Furthermore, there are no curative treatment for any of MPS and ML conditions so far. Gene editing holds promise as a tool for the creation of cell and animal models to help explain disease pathogenesis, as well as a platform for gene therapy. In this chapter, we discuss the main studies involving genome editing for MPS and the prospect applications for ML.

2-Aminovinyl-cysteine (AviCys) is a thioether amino acid shared by a variety of ribosomally synthesized and posttranslationally modified peptides (RiPPs). Based on investigations into the biosynthesis of thioviridamide RiPPs in Streptomyces sp. NRRL S-87, we here report a path for the formation of this unusual thioether residue. This path relies on four dedicated proteins: phosphotransferase TvaCS-87, Lyase TvaDS-87, kinase homolog TvaES-87, and LanD-like flavoprotein TvaFS-87. TvaES-87 plays a critical role in effective AviCys formation. During the posttranslational modifications of the precursor peptide, it works with TvaFS-87 to form a minimum AviCys synthetase complex, which follows the combined activity of TvaCDS-87 for Thr dehydration and catalyzes Cys oxidative decarboxylation and subsequent Michael addition of the resulting enethiol nucleophile onto the newly formed dehydroamino acid residue for cyclization. With TvaES-87, TvaFS-87 activity for Cys processing can be coordinated with TvaCDS-87 activity for minimizing competitive or unexpected spontaneous reactions and forming AviCys effectively.

2-Aminovinyl-cysteine (AviCys) is an unusual thioether amino acid shared by a variety of ribosomally synthesized and posttranslationally modified peptides (RiPPs), as part of a macrocyclic ring system that contains theC-terminal 4 or 6 residues of a precursor peptide. This amino acid is nonproteinogenic and arises from processing theC-terminal Cys residue and an internal Ser/Thr residue to form an unsaturated thioether linkage. Enzyme activities for forming lanthionine (Lan), a distinct saturated thioether residue characteristic of lanthipeptide-related RiPPs, has long been speculated to be necessary for AviCys formation. Based on investigations into the biosynthesis of thioviridamide non-lanthipeptdes inStreptomyces sp. NRRL S-87, we here report an alternative path for AviCys formation that is independent of known Lan synthetase activity. This path relies on four dedicated enzymes for posttranslational modifications of the precursor peptide, in which TvaES-87, a phosphotransferase homolog, plays a critical role. It works with LanD-like flavoprotein TvaFS-87to form a minimum AviCys synthetase complex that follows the combined activity of TvaCDS-87for Thr dehydration and catalyzes Cys oxidative decarboxylation and subsequent Michael addition of the resulting enethiol nucleophile onto the newly formed dehydrobutyrine residue for cyclization. With TvaES-87, TvaFS-87activity for Cys processing can be coordinated with TvaCDS-87activity for minimizing competitive or unexpected spontaneous reactions and forming AviCys effectively.

Autophagy is a process in which a myriad membrane structures called autophagosomes are formed de novo in a single cell, which deliver the engulfed substrates into lysosomes for degradation. The size of the autophagosomes is relatively uniform in non-selective autophagy and variable in selective autophagy. It has been recently established that autophagosome formation occurs near the endoplasmic reticulum (ER). In this review, we have discussed recent advances in the relationship between autophagosome formation and endoplasmic reticulum. Autophagosome formation occurs near the ER subdomain enriched with phospholipid synthesizing enzymes like phosphatidylinositol synthase (PIS)/CDP-diacylglycerol-inositol 3-phosphatidyltransferase (CDIPT) and choline/ethanolamine phosphotransferase 1 (CEPT1). Autophagy-related protein 2 (Atg2), which is involved in autophagosome formation has a lipid transfer capacity and is proposed to directly transfer the lipid molecules from the ER to form autophagosomes. Vacuole membrane protein 1 (VMP1) and transmembrane protein 41b (TMEM41b) are ER membrane proteins that are associated with the formation of the subdomain. Recently, we have reported that an uncharacterized ER membrane protein possessing the DNAJ domain, called ERdj8/DNAJC16, is associated with the regulation of the size of autophagosomes. The localization of ERdj8/DNAJC16 partially overlaps with the PIS-enriched ER subdomain, thereby implying its association with autophagosome size determination.

Hariharan and Guan find that binding of either melibiose or Na+ to their transporter, MelB, may favor MelB open states, while cooperative binding may trigger dehydration and cavity closure, thus enforcing cotransport. Cooperative binding is the core mechanism for their cotransport., MelB catalyzes the obligatory cotransport of melibiose with Na+, Li+, or H+. Crystal structure determination of the Salmonella typhimurium MelB (MelBSt) has revealed a typical major facilitator superfamily (MFS) fold at a periplasmic open conformation. Cooperative binding of Na+ and melibiose has been previously established. To determine why cotranslocation of sugar solute and cation is obligatory, we analyzed each binding in the thermodynamic cycle using three independent methods, including the determination of melting temperature by circular dichroism spectroscopy, heat capacity change (ΔCp), and regulatory phosphotransferase EIIAGlc binding with isothermal titration calorimetry (ITC). We found that MelBSt thermostability is increased by either substrate (Na+ or melibiose) and observed a cooperative effect of both substrates. ITC measurements showed that either binary formation yields a positive sign in the ΔCp, suggesting MelBSt hydration and a likely widening of the periplasmic cavity. Conversely, formation of a ternary complex yields negative values in ΔCp, suggesting MelBSt dehydration and cavity closure. Lastly, we observed that EIIAGlc, which has been suggested to trap MelBSt at an outward-open state, readily binds to the MelBSt apo state at an affinity similar to MelBSt/Na+. However, it has a suboptimal binding to the ternary state, implying that MelBSt in the ternary complex may be conformationally distant from the EIIAGlc-preferred outward-facing conformation. Our results consistently support the notion that binding of one substrate (Na+ or melibiose) favors MelBSt at open states, whereas the cooperative binding of both substrates triggers the alternating-access process, thus suggesting this conformational regulation could ensure the obligatory cotransport.

In plants, phosphate (Pi) homeostasis is regulated by the interaction of Pi starvation response transcription factors (PHRs) with stand-alone SPX proteins, which act as sensors for inositol pyrophosphates (PP-InsPs). Recently, ITPK1 was shown to generate the PP-InsP InsP7 from InsP6in vitro, but the importance of this activity in Pi signaling remained unknown. Here, we show that uncontrolled Pi accumulation in ITPK1-deficient plants is accompanied by impaired Pi-dependent InsP7 and InsP8 synthesis. Reciprocal grafting demonstrates that Pi starvation responses are mainly controlled by ITPK1 activity in shoots. Nuclear magnetic resonance assays and PAGE analyses with recombinant protein reveal that besides InsP6 phosphorylation, ITPK1 is also able to catalyze ATP synthesis using 5-InsP7 but not any other InsP7 isomer as a P-donor when ATP is low. Additionally, we show that the dynamic changes in InsP7 and InsP8 to cellular Pi are conserved from land plant species to human cells, suggesting that Pi-dependent PP-InsP synthesis is a common component of Pi signaling across kingdoms. Together, our study demonstrates how Pi-dependent changes in nutritional and energetic states modulate ITPK1 activities to fine-tune the synthesis of PP-InsPs.

Selenoprotein I (SELENOI) is an ethanolamine phosphotransferase that catalyzes the third reaction of the Kennedy pathway for the synthesis of phosphatidylethanolamine. Since the role of SELENOI in murine embryogenesis has not been investigated, SELENOI−/+ mating pairs were used to generate global KO offspring. Of 323 weanling pups, no homozygous KO genotypes were found. E6.5-E18.5 embryos (165 total) were genotyped, and only two E18.5 KO embryos were detected with no discernable anatomical defects. To screen embryos prior to uterine implantation that occurs ~ E6, blastocyst embryos (E3.5-E4.4) were flushed from uteruses of pregnant females and analyzed for morphology and genotype. KO embryos were detected in 5 of 6 pregnant females, and 7 of the 32 genotyped embryos were found to be SELENOI KO that exhibited no overt pathological features. Overall, these results demonstrate that, except for rare cases (2/490 = 0.4%), global SELENOI deletion leads to early embryonic lethality.

Abstract Due to a high unresponsiveness to chemotherapy, biofilm formation is an important medical problem that frequently occurs during infection with many bacterial pathogens. In this study, the marine sponge-derived natural compounds 4,6-dibromo-2-(2′,4′-dibromophenoxy)phenol and 3,4,6-tribromo-2-(2′,4′-dibromophenoxy)phenol were found to exhibit broad antibacterial activity against medically relevant gram-positive and gram-negative pathogens. The compounds were not only bactericidal against both replicating and stationary phase–persistent planktonic cells of methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa; they also killed biofilm-incorporated cells of both species while not affecting biofilm structural integrity. Moreover, these compounds were active against carbapenemase-producing Enterobacter sp. This simultaneous activity of compounds against different growth forms of both gram-positive and gram-negative bacteria is rare. Genome sequencing of spontaneous resistant mutants and proteome analysis suggest that resistance is mediated by downregulation of the bacterial EIIBC phosphotransferase components scrA and mtlA in MRSA likely leading to a lower uptake of the molecules. Due to their only moderate cytotoxicity against human cell lines, phenoxyphenols provide an interesting new scaffold for development of antimicrobial agents with activity against planktonic cells, persisters and biofilm-incoporated cells of ESKAPE pathogens. Key points • Brominated phenoxyphenols kill actively replicating and biofilm-incorporated bacteria. • Phosphotransferase systems mediate uptake of brominated phenoxyphenols. • Downregulation of phosphotransferase systems mediate resistance.

Prebiotics are increasingly used as food supplements, especially in infant formulas, to modify the functioning and composition of the microbiota. However, little is currently known about the mechanisms of prebiotic recognition and transport by gut bacteria, while these steps are crucial in their metabolism. In this study, we established a new strategy to profile the specificity of oligosaccharide transporters, combining microbiomics, genetic locus and strain engineering, and state-of-the art metabolomics. We revisited the transporter classification database and proposed a new way to classify these membrane proteins based on their structural and mechanistic similarities. Based on these developments, we identified and characterized, at the molecular level, a fructooligosaccharide transporting phosphotransferase system, which constitutes a biomarker of diet and gut pathology. The deciphering of this prebiotic metabolization mechanism by a nonbeneficial bacterium highlights the controversial use of prebiotics, especially in the context of chronic gut diseases., Prebiotic oligosaccharides, such as fructooligosaccharides, are increasingly being used to modulate the composition and activity of the gut microbiota. However, carbohydrate utilization analyses and metagenomic studies recently revealed the ability of deleterious and uncultured human gut bacterial species to metabolize these functional foods. Moreover, because of the difficulties of functionally profiling transmembrane proteins, only a few prebiotic transporters have been biochemically characterized to date, while carbohydrate binding and transport are the first and thus crucial steps in their metabolization. Here, we describe the molecular mechanism of a phosphotransferase system, highlighted as a dietary and pathology biomarker in the human gut microbiome. This transporter is encoded by a metagenomic locus that is highly conserved in several human gut Firmicutes, including Dorea species. We developed a generic strategy to deeply analyze, in vitro and in cellulo, the specificity and functionality of recombinant transporters in Escherichia coli, combining carbohydrate utilization locus and host genome engineering and quantification of the binding, transport, and growth rates with analysis of phosphorylated carbohydrates by mass spectrometry. We demonstrated that the Dorea fructooligosaccharide transporter is specific for kestose, whether for binding, transport, or phosphorylation. This constitutes the biochemical proof of effective phosphorylation of glycosides with a degree of polymerization of more than 2, extending the known functional diversity of phosphotransferase systems. Based on these new findings, we revisited the classification of these carbohydrate transporters. IMPORTANCE Prebiotics are increasingly used as food supplements, especially in infant formulas, to modify the functioning and composition of the microbiota. However, little is currently known about the mechanisms of prebiotic recognition and transport by gut bacteria, while these steps are crucial in their metabolism. In this study, we established a new strategy to profile the specificity of oligosaccharide transporters, combining microbiomics, genetic locus and strain engineering, and state-of-the art metabolomics. We revisited the transporter classification database and proposed a new way to classify these membrane proteins based on their structural and mechanistic similarities. Based on these developments, we identified and characterized, at the molecular level, a fructooligosaccharide transporting phosphotransferase system, which constitutes a biomarker of diet and gut pathology. The deciphering of this prebiotic metabolization mechanism by a nonbeneficial bacterium highlights the controversial use of prebiotics, especially in the context of chronic gut diseases.

Summary Persistence, the stress-tolerant state, is arguably the most vital phenotype since nearly all cells experience nutrient stress, which causes a sub-population to become dormant. However, how persister cells wake to reconstitute infections is not understood well. Here, using single-cell observations, we determined that Escherichia coli persister cells resuscitate primarily when presented with specific carbon sources, rather than spontaneously. In addition, we found that the mechanism of persister cell waking is through sensing nutrients by chemotaxis and phosphotransferase membrane proteins. Furthermore, nutrient transport reduces the level of secondary messenger cAMP through enzyme IIA; this reduction in cAMP levels leads to ribosome resuscitation and rescue. Resuscitating cells also immediately commence chemotaxis toward nutrients, although flagellar motion is not required for waking. Hence, persister cells wake by perceiving nutrients via membrane receptors that relay the signal to ribosomes via the secondary messenger cAMP, and persisters wake and utilize chemotaxis to acquire nutrients., Graphical Abstract, Highlights • Persister cells wake primarily by sensing nutrients rather than spontaneously • Persisters wake using chemotaxis sensors and phosphotransferase membrane proteins • Persisters wake by reducing cAMP and by activating stalled/hibernating ribosomes • Persister cells undergo chemotaxis as they resuscitate, Biological Sciences; Microbiology; Molecular Microbiology; Cell Biology

Aims: Nondiabetic patients were studied to determine whether modest elevations in plasma mannose may be associated with a greater incidence of coronary artery disease (CAD). Materials and Methods: Plasma insulin, mannose, glucose, hexokinase 1-2, GLUT1-GLUT4 levels, and serum mannose phosphate isomerase enzyme levels were evaluated with respect to subsequent CAD using records from 120 nondiabetic CAD patients and 120 healthy volunteers. CAD was identified from myocardial infarction and new diagnoses of angina. Results: Of 120 nondiabetic CAD patients studied, their plasma GLUT4 and HK1 levels were significantly lower than those of the control group. In addition, a significant increase in plasma mannose levels was found in the patient group compared to the control group. Conclusion: Our findings showed that elevated baseline mannose levels in plasma are associated with an increased risk of CAD over time. © Copyright 2020, Mary Ann Liebert, Inc., publishers 2020.

RNA 2′-phosphotransferase Tpt1 converts an internal RNA 2′-monophosphate to a 2′-OH via a two-step NAD+-dependent mechanism in which: (i) the 2′-phosphate attacks the C1″ of NAD+ to expel nicotinamide and form a 2′-phospho-ADP-ribosylated RNA intermediate; and (ii) the ADP-ribose O2″ attacks the phosphate of the RNA 2′-phospho-ADPR intermediate to expel the RNA 2′-OH and generate ADP-ribose 1″–2″ cyclic phosphate. Tpt1 is an essential component of the fungal tRNA splicing pathway that generates a unique 2′-PO4, 3′-5′ phosphodiester splice junction during tRNA ligation. The wide distribution of Tpt1 enzymes in taxa that have no fungal-type RNA ligase raises the prospect that Tpt1 might catalyze reactions other than RNA 2′-phosphate removal. A survey of Tpt1 enzymes from diverse sources reveals that whereas all of the Tpt1 enzymes are capable of NAD+-dependent conversion of an internal RNA 2′-PO4 to a 2′-OH (the canonical Tpt1 reaction), a subset of Tpt1 enzymes also catalyzed NAD+-dependent ADP-ribosylation of an RNA or DNA 5′-monophosphate terminus. Aeropyrum pernix Tpt1 (ApeTpt1) is particularly adept in this respect. One-step synthesis of a 5′-phospho-ADP-ribosylated cap structure by ApeTpt1 (with no subsequent 5′-phosphotransferase step) extends the repertoire of the Tpt1 enzyme family and the catalogue of ADP-ribosylation reactions involving nucleic acid acceptors.

Introduction Aminoglycoside resistance (AR) is common in health care-associated methicillin-resistant Staphylococcus aureus (HA-MRSA). AR is most often associated with the production of antibiotic modifying enzymes: bidomain AAC(6′)-Ie/APH(2″)-Ia acetyltransferase and phosphotransferase, ANT(4′)-Ia nucleotidyltransferase, and APH(3″)-IIIa phosphotransferase. Aim Determination of aminoglycoside sensitivity, presence of genes encoding enzymes, and molecular typing of HA-MRSA strains derived from patients hospitalized in surgical and transplantation wards. Materials and methods Fifty-four HA-MRSA strains, isolated from various materials from patients in the surgical and transplantation wards of Warsaw's clinical hospital, hospitalized between 1991 and 2007. The MIC values of gentamicin-GEN/tobramycin-TOB/amikacin-AK/netilmicin-NET were determined by the E-test (CLSI/EUCAST). Genes mecA/aacA-aphD/aadD/aph(3″)-IIIa were detected using PCR. SCCmec types were determined according to the Oliveira method and the sequence type (ST)/clonal complex (CC) by the MLST method. Results Of the isolates tested, 36 (66.7%) showed resistance to at least one aminoglycoside: TOB (57.4%), GEN (53.7%), AK (55.6%), NET (24.1%). The aacA-aphD gene was present in 29 MRSA-GEN-R (most often in combination with aadD, 15/29 or aph(3″)-IIIa, 10/29); the aacA-aphD gene was the only determinant of resistance in 1 isolate. The AR variants mainly belonged to the CC8 clonal complex (ST239/247/241/254/8) and most frequently contained SCCmec type III (3A) cassettes. Conclusions Resistance to at least one aminoglycoside was present in 66.7% of HA-MRSA and in more than 22% to all of them. The presence of the aacA-aphD gene was sufficient to express the resistance phenotype to GEN/TOB/AK/NET. Resistant isolates were closely related to each other.

Combinatorial metabolic engineering enabled the development of efficient microbial cell factories for modulating gene expression to produce desired products. Here, we report the combinatorial metabolic engineering of Corynebacterium glutamicum to produce butyrate by introducing a synthetic butyrate pathway including phosphotransferase and butyrate kinase reactions and repressing the essential acn gene-encoding aconitase, which has been targeted for downregulation in a genome-scale model. An all-in-one clustered regularly interspaced short palindromic repeats interference system for C. glutamicum was used for tunable downregulation of acn in an engineered strain, where by-product-forming reactions were deleted and the synthetic butyrate pathway was inserted, resulting in butyrate production (0.52 ± 0.02 g/L). Subsequently, biotin limitation enabled the engineered strain to produce butyrate (0.58 ± 0.01 g/L) without acetate formation for the entire duration of the culture. These results demonstrate the potential homo-production of butyrate using engineered C. glutamicum. This method can also be applied to other industrial microorganisms.

This work was supported by Instituto Evandro Chagas/Secretaria de Vigil?ncia em Sa?de/Minist?rio da Sa?de (IEC/SVS/MS) and Funda??o Amaz?nia Paraense de Amparo ? Pesquisa (FAPESPA) Universidade Federal do Par?. N?cleo de Medicina Tropical. Programa de P?s-Gradua??o Stricto Sensu em Patologia Tropical. Bel?m, PA, Brasil / Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Laborat?rio de Virologia e Biologia Molecular. Ananindeua, PA, Brasil. Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Centro de Inova??es Tecnol?gicas. Ananindeua, PA, Brasil. Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Centro de Inova??es Tecnol?gicas. Ananindeua, PA, Brasil. Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Laborat?rio de Virologia e Biologia Molecular. Ananindeua, PA, Brasil. Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Laborat?rio de Virologia e Biologia Molecular. Ananindeua, PA, Brasil. Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Laborat?rio de Virologia e Biologia Molecular. Ananindeua, PA, Brasil. Universidade Federal do Par?. N?cleo de Medicina Tropical. Programa de P?s-Gradua??o Stricto Sensu em Patologia Tropical. Bel?m, PA, Brasil. Introduction: Human cytomegalovirus is one of the causes of opportunist infections in immunocompromised patients, and is triggered by factors such as state of viral latency, weakened immune responses, and development of antiviral resistance to ganciclovir, the only drug offered by the public health system in Brazil to treat the infection. The goal of this study was to identify mutations that may be associated with antiviral resistance in immunocompromised patients. Methods: Molecular analysis was performed in 82 blood samples and subjected to genomic DNA extraction by a silica-based method. Three sequences of the HCMV UL97 gene, which encodes a phosphotransferase protein required for activation of ganciclovir, were amplified by polymerase chain reaction. Pyrosequencing methods were applied to one external 2096-bp segment DNA and two internal sequences between nucleotides 1087 to 1828 to detect mutations in this gene. Results: Approximately 10% of sequences contained mutations between nucleotides 377 and 594, in conserved regions of the UL97 gene, leading to amino acid changes. Eleven coding mutations were identified, including changes leading to amino acid substitutions, E596K and S604F, which were observed in 100% of samples and are described for the first time in Brazil. In addition, one mutation (A594V) that is associated with ganciclovir resistance was detected in a kidney transplant patient. Conclusions: Further studies to detect mutations associated with HCMV resistance to antiviral drugs are required to demonstrate the need to increase the variety and availability of drugs used to treat viral infections in the public health care system in Brazil

Dibutyl phthalate (DBP) is well known as a high-priority pollutant. This study explored the impacts of DBP on the metabolic pathways of microbes in black soils in the short term (20 days). The results showed that the microbial communities were changed in black soils with DBP. In nitrogen cycling, the abundances of the genes were elevated by DBP. DBP contamination facilitated 3′-phosphoadenosine-5′-phosphosulfate (PAPS) formation, and the gene flux of sulfate metabolism was increased. The total abundances of ABC transporters and the gene abundances of the monosaccharide-transporting ATPases MalK and MsmK were increased by DBP. The total abundance of two-component system (TCS) genes and the gene abundances of malate dehydrogenase, histidine kinase and citryl-CoA lyase were increased after DBP contamination. The total abundance of phosphotransferase system (PTS) genes and the gene abundances of phosphotransferase, Crr and BglF were raised by DBP. The increased gene abundances of ABC transporters, TCS and PTS could be the reasons for the acceleration of nitrogen, carbon and sulfate metabolism. The degrading-genes of DBP were increased markedly in soil exposed to DBP. In summary, DBP contamination altered the microbial community and enhanced the gene abundances of the carbon, nitrogen and sulfur metabolism in black soils in the short term.

Background: The phosphoenolpyruvate (PEP):lactose phosphotransferase system of Staphylococcus aureus transports and phosphorylates lactose and various phenylgalactosides. Their phosphorylation is catalyzed by the Cys476-phosphorylated EIIB domain of the lactose-specific permease enzyme IICB (EIICBLac). Phosphorylation causes the release of galactosides bound to the EIIC domain into the cytoplasm by a mechanism not yet understood. Results: Irradiation of a reaction mixture containing the photoactivatable p-azidophenyl-β-D-galactopyranoside and EIICBLac with UV light caused a loss of EIICBLac activity. Nevertheless, photoinactivated EIICBLac could still be phosphorylated with [32P]PEP. Proteolysis of photoinactivated [32P]P-EIICBLac with subtilisin provided an 11-kDa radioactive peptide. Only the sequence of its first three amino acids (-H-G-P-, position 245–247) could be determined. They are part of the substrate binding pocket in EIICs of the lactose/cellobiose PTS family. Surprisingly, while acid treatment caused hydrolysis of the phosphoryl group in active [32P]P∼EIICBLac, photoinactivated [32P]P-EIICBLac remained strongly phosphorylated. Conclusion: Phosphorylation of the –OH group at C6 of p-nitrenephenyl-β-D-galactopyranoside covalently bound to EIICLac by the histidyl-phosphorylated [32P]P∼EIIBLac domain is a likely explanation for the observed acid resistance. Placing p-nitrenephenyl-β-D-galactopyranoside into the active site of modelled EIICLac suggested that the nitrene binds to the -NH- group of Ser248, which would explain why no sequence data beyond Pro247could be obtained.

In this study, the effect of epinephrine on the biofilm formation of Micrococcus luteus C01 isolated from human skin was investigated in depth for the first time. This hormone has a complex effect on biofilms in various systems. In a system with polytetrafluoroethylene (PTFE) cubes, treatment with epinephrine at a physiological concentration of 4.9 × 10−9 M increased the total amount of 72-h biofilm biomass stained with crystal violet and increased the metabolic activity of biofilms, but at higher and lower concentrations, the treatment had no significant effect. On glass fiber filters, treatment with the hormone decreased the number of colony forming units (CFUs) and changed the aggregation but did not affect the metabolic activity of biofilm cells. In glass bottom plates examined by confocal microscopy, epinephrine notably inhibited the growth of biofilms. RNA-seq analysis and RT–PCR demonstrated reproducible upregulation of genes encoding Fe–S cluster assembly factors and cyanide detoxification sulfurtransferase, whereas genes encoding the co-chaperone GroES, the LysE superfamily of lysine exporters, short-chain alcohol dehydrogenase and the potential c-di-GMP phosphotransferase were downregulated. Our results suggest that epinephrine may stimulate matrix synthesis in M. luteus biofilms, thereby increasing the activity of NAD(H) oxidoreductases. Potential c-di-GMP pathway proteins are essential in these processes.

Protein phosphorylation is a post-translational modification that affects protein activity through the addition of a phosphate moiety by protein kinases or phosphotransferases. It occurs in all life forms. In addition to Hanks kinases found also in eukaryotes, bacteria encode membrane histidine kinases that, with their cognate response regulator, constitute two-component systems and phosphotransferases that phosphorylate proteins involved in sugar utilization on histidine and cysteine residues. In addition, they encode BY-kinases and arginine kinases that phosphorylate protein specifically on tyrosine and arginine residues respectively. They also possess unusual bacterial protein kinases illustrated here by examples from Bacillus subtilis.

The selenoprotein family includes 25 members, many of which are antioxidant or redox regulating enzymes. A unique member of this family is Selenoprotein I (SELENOI), which does not catalyze redox reactions, but instead is an ethanolamine phosphotransferase (Ept). In fact, the characteristic selenocysteine residue that defines selenoproteins lies far outside of the catalytic domain of SELENOI. Furthermore, data using recombinant SELENOI lacking the selenocysteine residue have suggested that the selenocysteine amino acid is not directly involved in the Ept reaction. SELENOI is involved in two different pathways for the synthesis of phosphatidylethanolamine (PE) and plasmenyl PE, which are constituents of cellular membranes. Ethanolamine phospholipid synthesis has emerged as an important process for metabolic reprogramming that occurs in pluripotent stem cells and proliferating tumor cells, and this review discusses roles for upregulation of SELENOI during T cell activation, proliferation, and differentiation. SELENOI deficiency lowers but does not completely diminish de novo synthesis of PE and plasmenyl PE during T cell activation. Interestingly, metabolic reprogramming in activated SELENOI deficient T cells is impaired and this reduces proliferative capacity while favoring tolerogenic to pathogenic phenotypes that arise from differentiation. The implications of these findings are discussed related to vaccine responses, autoimmunity, and cell-based therapeutic approaches.