Crystal structure of HPPD inhibitor sensitive protein from Oryza sativa.

4-hydroxyphenylpyruvate dioxygenase (HPPD) Inhibitor Sensitive 1 (HIS1) is an endogenous gene of rice, conferring broad-spectrum resistance to ß-triketone herbicides. Similar genes, known as HIS1-like genes (HSLs), exhibit analogous functions and can complement the herbicide-resistant characteristics endowed by HIS1. The identification of HIS1 and HSLs represents a valuable asset, as the intentional pairing of herbicides with resistance genes emerges as an effective strategy for crop breeding. Encoded by HIS1 is a Fe(II)/2-oxoglutarate-dependent oxygenase responsible for detoxifying ß-triketone herbicides through hydroxylation. However, the precise structure supporting this function remains unclear. This work, which determined the crystal structure of HIS1, reveals a conserved core motif of Fe(II)/2-oxoglutarate-dependent oxygenase and pinpoints the crucial residue dictating substrate preference between HIS1 and HSL.

Pyoluteorin regulates the biosynthesis of 2,4-DAPG through the TetR family transcription factor PhlH in Pseudomonas protegens Pf-5.

Soil and rhizosphere bacteria act as a rich source of secondary metabolites, effectively fighting against a diverse array of pathogens. Certain Pseudomonas species harbor biosynthetic gene clusters for producing both pyoluteorin and 2,4-diacetylphloroglucinol (2,4-DAPG), which are polyketides that exhibit highly similar antimicrobial spectrum against bacteria and fungi or oomycete. A complex cross talk exists between pyoluteorin and 2,4-DAPG biosynthesis, and production of 2,4-DAPG was strongly repressed by pyoluteorin, yet the underlying mechanism is still elusive. In this study, we find that the TetR family transcription factor PhlH is involved in the cross talk between pyoluteorin and 2,4-DAPG biosynthesis. PhlH binds to a palindromic sequence within the promoter of phlG (PphlG), which encodes a C-C bond hydrolase responsible for degrading 2,4-DAPG. As a signaling molecule, pyoluteorin disrupts the PhlH-PphlG complex by binding to PhlH, leading to decreased levels of 2,4-DAPG. Proteomics data suggest that pyoluteorin regulates multiple physiological processes including fatty acid biosynthesis and transportation of taurine, siderophore, and amino acids. Our work not only reveals a novel mechanism of cross talk between pyoluteorin and 2,4-DAPG biosynthesis, but also highlights pyoluteorin's role as a messenger in the complex communication network of Pseudomonas.IMPORTANCEAntibiosis serves as a crucial defense mechanism for microbes against invasive bacteria and resource competition. These bacteria typically orchestrate the production of multiple antibiotics in a coordinated fashion, wherein the synthesis of one antibiotic inhibits the generation of another. This strategic coordination allows the bacterium to focus its resources on producing the most advantageous antibiotic under specific circumstances. However, the underlying mechanisms of distinct antibiotic production in bacterial cells remain largely elusive. In this study, we reveal that the TetR family transcription factor PhlH detects the secondary metabolite pyoluteorin and mediates the cross talk between pyoluteorin and 2,4-DAPG biosynthesis in the biocontrol strain Pseudomonas protegens Pf-5. These findings hold promise for future research, potentially informing the manipulation of these systems to enhance the effectiveness of biocontrol agents.

Molecular basis for coordinating secondary metabolite production by bacterial and plant signaling molecules.

The production of secondary metabolites is a major mechanism used by beneficial rhizobacteria to antagonize plant pathogens. These bacteria have evolved to coordinate the production of different secondary metabolites due to the heavy metabolic burden imposed by secondary metabolism. However, for most secondary metabolites produced by bacteria, it is not known how their biosynthesis is coordinated. Here, we showed that PhlH from the rhizobacterium Pseudomonas fluorescens is a TetR-family regulator coordinating the expression of enzymes related to the biosynthesis of several secondary metabolites, including 2,4-diacetylphloroglucinol (2,4-DAPG), mupirocin, and pyoverdine. We present structures of PhlH in both its apo form and 2,4-DAPG-bound form and elucidate its ligand-recognizing and allosteric switching mechanisms. Moreover, we found that dissociation of 2,4-DAPG from the ligand-binding domain of PhlH was sufficient to allosterically trigger a pendulum-like movement of the DNA-binding domains within the PhlH dimer, leading to a closed-to-open conformational transition. Finally, molecular dynamics simulations confirmed that two distinct conformational states were stabilized by specific hydrogen bonding interactions and that disruption of these hydrogen bonds had profound effects on the conformational transition. Our findings not only reveal a well-conserved route of allosteric signal transduction in TetR-family regulators but also provide novel mechanistic insights into bacterial metabolic coregulation.

Crystal structure of an aspartate aminotransferase Lpg0070 from Legionella pneumophila.

Legionella pneumophila aspartate aminotransferase (Lpg0070) is a member of the transaminase and belongs to the pyridoxal 5'-phosphate (PLP)-dependent superfamily. It is responsible for the transfer of α-amino between aspartate and α-ketoglutarate to form glutamate and oxaloacetate. Here, we report the crystal structure of Lpg0070 at the resolution of 2.14 Å and 1.7 Å, in apo-form and PLP-bound, respectively. Our structural analysis revealed the specific residues involved in the PLP binding and free form against PLP-bound supported conformational changes before substrate recognition. In vitro enzyme activity proves that the absence of the N-terminal arm reduces the enzyme activity of Lpg0070. These data provide further evidence to support the N-terminal arm plays a crucial role in catalytic activity.

Prenatal triclosan exposure impairs mammalian lung branching morphogenesis through activating Bmp4 signaling.

Triclosan (TCS) is a commonly used antibacterial agent present in personal care and household products. Recently, there have been increasing concerns about the association between children's health and TCS exposure during gestation, but the toxicological effects of TCS exposure on embryonic lung development remain undetermined. In this study, through using an ex vivo lung explant culture system, we found that prenatal exposure to TCS resulted in impaired lung branching morphogenesis and altered proximal-distal airway patterning. These TCS-induced dysplasias are accompanied by significantly reduced proliferation and increased apoptosis within the developing lung, as a consequence of activated Bmp4 signaling. Inhibition of Bmp4 signaling by Noggin partially rescues the lung branching morphogenesis and cellular defects in TCS-exposed lung explants. In addition, we provided in vivo evidence that administration of TCS during gestation leads to compromised branching formation and enlarged airspace in the lung of offspring. Thus, this study provides novel toxicological information on TCS and indicated a strong/possible association between TCS exposure during pregnancy and lung dysplasia in offspring.

Structural Characterization of an N-Acetyl Sugar Amidotransferase Involved in the Lipopolysaccharide Biosynthesis in Bacteria.

N-acetyl sugar amidotransferase (NASAT) is involved in the lipopolysaccharide (LPS) biosynthesis pathway that catalyzes the formation of the acetamido moiety (sugar-NC(=NH)CH3) on the O-chain. So far, little is known about its structural and functional properties. Here, we report the crystal structure of an N-acetyl sugar amidotransferase from Legionella pneumophila (LpNASAT) at 2.33 Å resolution. LpNASAT folds into a compact basin-shaped architecture with an unusually wide and open putative substrate-binding pocket and a conserved zinc ion-binding tetracysteine motif. The pocket contains a Rossmann-like fold with a PP-loop, suggesting that the NASAT-catalyzed amidotransfer reaction probably requires the conversion of ATP to AMP and PPi. Our data provide structural insights into the NASAT family of proteins, and allow us to possibly identify its functionally important regions.

Genetic mapping identifies a rice naringenin O-glucosyltransferase that influences insect resistance.

Naringenin, the biochemical precursor for predominant flavonoids in grasses, provides protection against UV damage, pathogen infection and insect feeding. To identify previously unknown loci influencing naringenin accumulation in rice (Oryza sativa), recombinant inbred lines derived from the Nipponbare and IR64 cultivars were used to map a quantitative trait locus (QTL) for naringenin abundance to a region of 50 genes on rice chromosome 7. Examination of candidate genes in the QTL confidence interval identified four predicted uridine diphosphate-dependent glucosyltransferases (Os07g31960, Os07g32010, Os07g32020 and Os07g32060). In vitro assays demonstrated that one of these genes, Os07g32020 (UGT707A3), encodes a glucosyltransferase that converts naringenin and uridine diphosphate-glucose to naringenin-7-O-ß-d-glucoside. The function of Os07g32020 was verified with CRISPR/Cas9 mutant lines, which accumulated more naringenin and less naringenin-7-O-ß-d-glucoside and apigenin-7-O-ß-d-glucoside than wild-type Nipponbare. Expression of Os12g13800, which encodes a naringenin 7-O-methyltransferase that produces sakuranetin, was elevated in the mutant lines after treatment with methyl jasmonate and insect pests, Spodoptera litura (cotton leafworm), Oxya hyla intricata (rice grasshopper) and Nilaparvata lugens (brown planthopper), leading to a higher accumulation of sakuranetin. Feeding damage from O. hyla intricata and N. lugens was reduced on the Os07g32020 mutant lines relative to Nipponbare. Modification of the Os07g32020 gene could be used to increase the production of naringenin and sakuranetin rice flavonoids in a more targeted manner. These findings may open up new opportunities for selective breeding of this important rice metabolic trait.

Structural insights into a new substrate binding mode of a histidine acid phosphatase from Legionella pneumophila.

MapA is a histidine acid phosphatase (HAP) from Legionella pneumophila that catalyzes the hydroxylation of a phosphoryl group from phosphomonoesters by an active-site histidine. Several structures of HAPs, including MapA, in complex with the inhibitor tartrate have been solved and the substrate binding tunnel identified; however, the substrate recognition mechanism remains unknown. To gain insight into the mechanism of substrate recognition, the crystal structures of apo-MapA and the MapAD281A mutant in complex with 5'-AMP were solved at 2.2 and 2.6 Å resolution, respectively. The structure of the MapAD281A/5'-AMP complex reveals that the 5'-AMP fits fully into the substrate binding tunnel, with the 2'-hydroxyl group of the ribose moiety stabilized by Glu201 and the adenine moiety sandwiched between His205 and Phe237. This is the second structure of a HAP/AMP complex solved with 5'-AMP binding in a unique manner in the active site. The structure presents a new substrate recognition mechanism of HAPs.

Effect of different disinfection treatments on the adhesion and separation of biofilm on stainless steel surface.

Attachment and separation of sulfate-reducing bacteria (SRB) biofilm on stainless steel (SS) in simulated cooling water with and without different sterilization treatments was investigated by calculation of surface energy, theoretical work of adhesion and analysis of Scanning Electron Microscope/Energy Dispersive Spectrometer. Two types of biocides, glutaraldehyde and Polyhexamethylene guanidine (PHMG), and electromagnetic treatment were used in this paper. The results show that PHMG had the best bactericidal performance, followed by glutaraldehyde, and electromagnetic treatment was the lowest one. The theoretical work of adhesion was used to quantitatively evaluate the adhesion of biofilm on the surface of the metal. Theoretical work of adhesion between biofilm and SS in simulated cooling water increased with time. The theoretical adhesion work and adhesive capacity of biofilm to SS surface increased after treating with glutaraldehyde while decreasing after treating with PHMG and electromagnetic field. As the theoretical adhesion work decreased, the biofilm was gradually removed from the stainless steel surface. On the contrary, the biofilm adhered more firmly. The results of SEM were also consistent with the calculation results of theoretical adhesion work. The results obtained indicated that electromagnetic treatment had the lowest effect in sterilization but the best in biofilm separation.

Crystal structure of a hypothetical T2SS effector Lpg0189 from Legionella pneumophila reveals a novel protein fold.

Lpg0189 is a type II secretion system-dependent extracellular protein with unknown function from Legionella pneumophila. Herein, we determined the crystal structure of Lpg0189 at 1.98 Šresolution by using single-wavelength anomalous diffraction (SAD). Lpg0189 folds into a novel chair-shaped architecture, with two sheets roughly perpendicular to each other. Bioinformatics analysis suggests Lpg0189 and its homologues are unique to Legionellales and evolved divergently. The interlinking structural and bioinformatics studies provide a better understanding of this hypothetical protein.

Identification of the Repressive Domain of the Negative Circadian Clock Component CHRONO.

Circadian rhythm is an endogenous, self-sustainable oscillation that participates in regulating organisms' physiological activities. Key to this oscillation is a negative feedback by the main clock components Periods and Cryptochromes that repress the transcriptional activity of BMAL1/CLOCK (defined in the Abbreviations) complexes. In addition, a novel repressor, CHRONO, has been identified recently, but details of CHRONO's function during repressing the circadian cycle remain unclear. Here we report that a domain of CHRONO mainly composed of α-helixes is critical to repression through the exploitation of protein-protein interactions according to luciferase reporter assays, co-immunoprecipitation, immunofluorescence, genome editing, and structural information analysis via circular dichroism spectroscopy. This repression is fulfilled by interactions between CHRONO and a region on the C-terminus of BMAL1 where Cryptochrome and CBP (defined in the Abbreviations) bind. Our resultsindicate that CHRONO and PER differentially function as BMAL1/CLOCK-dependent repressors. Besides, the N-terminus of CHRONO is important for its nuclear localization. We further develop a repression model of how PER, CRY, and CHRONO proteins associate with BMAL1, respectively.

Effects of Al2O3 Nanoparticles on the Crystallization of Calcium Carbonate in Aqueous Solution.

The effects of Al2O3 nanoparticles on the precipitation behavior of CaCO3 and on the anti-scale performance of 2-phosphonobutane-1,2,4-tricarboxylic acid (PBTCA) in CaCO3 growth solution were studied by means of solution analysis, gravimetric methods, scanning electron microscopy, Fourier transform infrared spectroscopy and X-ray diffraction. The results illustrate that Al2O3 nanoparticles had little effect on the concentration of calcium ions in the test solution without PBTCA, but significantly changed the form and morphology of calcium carbonate crystals, which were transformed from calcite to aragonite. As a commonly used and effective scale inhibitor, PBTCA showed good Ca2+ retention ability in the test solution, distorting the calcite crystal lattice and promoting the formation of vaterite. When Al2O3 nanoparticles co-existed with PBTCA in the test solution, calcium carbonate was more likely to precipitate, and the Ca2+ retention ability of PBTCA reduced. A newly designed gravimetric method was used to evaluate the scale inhibition performance of Al2O3 nanoparticles on the heat exchange surface. When the concentration of Al2O3 nanoparticles reached 1 g/L, the surface scale inhibition efficiency of Al2O3 nanoparticles exceeded 80%.

Comparison on treatment strategy for chemical cleaning wastewater: Pollutants removal, process design and techno-economic analysis.

Chemical cleaning wastewater (CCW) usually consists of pickling wastewater (PW) and alkaline cleaning wastewater (ACW), and the strategy of separate treatment or combined treatment affects pollutant removal efficiency and cost. In this study, separate and combined treatment of real PW and ACW generated from an on-site cleaning campaign were investigated. A neutralization - fluoride removal - coagulation - oxidation process was constructed for PW and mixed wastewater (MW) treatment, and operational conditions of each process were optimized. The optimal mixing ratio of PW and ACW in the primary neutralization tank was 3:7, which obtained a near neutral pH, efficient chromaticity and turbidity removal and good settling performance. The neutralized MW and PW were both adjusted pH to 9.5 to precipitate metal ions as hydroxides. After fluoride precipitated as CaF2, the fluoride removal rates of MW and PW were both 99.9%, respectively, and polyaluminum chloride was dosed to improve the settleability of CaF2. Then sodium hypochlorite oxidization was employed to remove NH3-N and soluble COD. Techno-economic analysis based on pilot-scale tests showed that separate treatment of PW and ACW obtained better effluent quality than combined treatment. The total cost of combined treatment (37.44 $/m3) was greatly higher than that of separate treatment of PW and ACW (18.20 $/m3). This study proposed a cost-effective strategy for CCW treatment, and suggested that neutralization with acidic or alkaline wastewater should be systematically considered for technical and economic feasibility.

The C-terminus of ubiquitin plays a critical role in deamidase Lpg2148 recognition.

By bearing a papain-like core structure and a cysteine-based catalytic triad, deamidase can convert glutamine to glutamic acid or asparagine to aspartic acid to modify the functions of host target proteins resulting in the blocking of eukaryotic host cell function. Legionella pneumophila effector Lpg2148 (MvcA) is a deamidase, a structural homolog of cycle inhibiting factor (Cif) effectors. Lpg2148 and Cif effectors are functionally diverse, with Lpg2148 only catalyzing ubiquitin but not NEDD8. However, a detailed understanding of substrate specificity is still missing. Here, we resolved the crystal structure of Lpg2148 at 2.5 Šresolution and obtained rigid-body modeling of Lpg2148 with C-terminus deleted ubiquitin (1-68) (ubΔc) complex using HADDOCK, which shows that the C-terminus of ubiquitin is flexible in recognition. We also conducted the truncated analysis to demonstrate that Leu71 of ubiquitin is necessary for its interaction with Lpg2148. Moreover, Val33 of Lpg2148 at the edge of a channel plays a vital role in the interaction and is limited by the length of the C-terminus of ubiquitin, which may help to explain the selectivity of ubiquitin over NEDD8. In summary, these results enrich our knowledge of substrate recognition of deamidase.

Effects of TiO2 Nanofluid on the Surface State of Brass.

The surface states of brass in simulated cooling water (SCW) containing or free of sodium dodecyl benzene sulfonate (SDBS) and TiO2 nanofluid were analyzed by means of scanning electron microscope (SEM), energy spectrum analysis (EDS) and X-ray diffraction (XRD). The concentrations of Cu and Zn ions in the solution after brass immersion were analyzed using a plasma emission spectrometer. The relationship between the surface states and corrosion resistance of brass was investigated by electrochemical impedance spectroscopy (EIS). The results showed that the brass surface was mainly covered with zinc compound Zn5(OH)6(CO3)2 as corrosion product in SCW. In SCW containing SDBS, a large amount of SDBS was adsorbed on the brass surface. In TiO2 nanofluid, the brass surface was relatively bare and mainly contained cuprous oxide. There was no obvious adhesion of SDBS aggregates and no accumulation of zinc compound on brass surface in TiO2 nanofluid. TiO2 nanoparticles inhibit the adsorption of SDBS on brass surface. Solution analysis results showed that the concentrations of Cu and Zn ions in TiO2 nanofluid was obviously higher than that in SCW and SCW containing SDBS, indicating that most of corrosion products of brass dissolved into the nanofluid. The EIS results illustrated the brass electrode had a larger reaction resistance in SCW containing SDBS, indicating the good protective performance of the adsorbed SDBS film on brass surface. The reaction resistance of the brass electrode was the smallest in TiO2 nanofluid, which illustrated that TiO2 nanoparticles in solution promoted the corrosion of brass.

Influence of Al2O3 Nanoparticles on Corrosion Inhibition Performance of Benzotriazole to Brass.

The influence of Al2O3 nanoparticles on corrosion inhibition of benzotriazole (BTA) in brass/ simulated water system was studied by potentiodynamic polarization curve and electrochemical impedance spectroscopy (EIS). The results show that BTA has good corrosion inhibition effect on brass. Al2O3 nanoparticles could reduce the corrosion inhibition performance of BTA. The higher the concentration of Al2O3 nanoparticles in simulated water, the lower corrosion inhibition performance of BTA. The isothermal adsorption of BTA on brass surface in simulated water and Al2O3 nanofluids was analyzed. The results indicated that the adsorption of BTA on brass surface followed the Langmuirs' adsorption isotherm, the adsorption Gibbs free energy ΔG was less than -40 kJ/mol, corresponding to chemical adsorption, in both simulated water and Al2O3 nanofluids. The -ΔG value of BTA on brass surface decreased in Al2O3 nanofluids, indicating the weakening of the BTA adsorption on the brass surface. Surface analysis of brass samples by optical microscope and X-ray diffraction confirmed the above results.

Treatment of chemical cleaning wastewater and cost optimization by response surface methodology coupled nonlinear programming.

The real alkaline cleaning wastewater (ACW) was treated by a process consisting of neutralization, NaClO oxidation and aluminum sulfate (AS) coagulation, and a novel response surface methodology coupled nonlinear programming (RSM-NLP) approach was developed and used to optimize the oxidation-coagulation process under constraints of relevant discharge standards. Sulfuric acid neutralization effectively removed chemical oxygen demand (COD), surfactant alkylphenol ethoxylates (OP-10) and silicate at the optimum pH of 7.0, with efficiencies of 62.3%, >82.7% and 94.2%, respectively. Coagulation and adsorption by colloidal hydrated silica formed during neutralization were the major removal mechanisms. NaClO oxidation achieved almost complete removal of COD, but was ineffective for the removal of surfactant OP-10. AS coagulation followed by oxidation can efficiently remove OP-10 with the formation of Si-O-Al compounds. The optimum conditions for COD ≤100 mg/L were obtained at hypochlorite to COD molar ratio of 2.25, pH of 10.0 and AS dosage of 0.65 g Al/L, with minimum cost of 9.58 $/m3 ACW. This study shows that the integrative RSM-NLP approach could effectively optimize the oxidation-coagulation process, and is attractive for techno-economic optimization of systems with multiple factors and threshold requirements for response variables.

Crystal structures of Apo and GMP bound hypoxanthine-guanine phosphoribosyltransferase from Legionella pneumophila and the implications in gouty arthritis.

Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) (EC 2.4.2.8) reversibly catalyzes the transfer of the 5-phophoribosyl group from 5-phosphoribosyl-alpha-1-pyrophosphate (PRPP) to hypoxanthine or guanine to form inosine monophosphate (IMP) or guanosine monophosphate (GMP) in the purine salvage pathway. To investigate the catalytic mechanism of this enzyme in the intracellular pathogen Legionella pneumophila, we determined the crystal structures of the L. pneumophila HGPRT (LpHGPRT) both in its apo-form and in complex with GMP. The structures reveal that LpHGPRT comprises a core domain and a hood domain which are packed together to create a cavity for GMP-binding and the enzymatic catalysis. The binding of GMP induces conformational changes of the stable loop II. This new binding site is closely related to the Gout arthritis-linked human HGPRT mutation site (Ser103Arg). Finally, these structures of LpHGPRT provide insights into the catalytic mechanism of HGPRT.

Crystal structure of Legionella pneumophila dephospho-CoA kinase reveals a non-canonical conformation of P-loop.

Dephospho-CoA kinase (DPCK; EC 2.7.1.24) catalyzes the final step in the coenzyme A biosynthetic pathway. DPCK transfers a phosphate group from ATP to the 3-hydroxyl group of the ribose of dephosphocoenzyme A (dCoA) to yield CoA and ADP. Upon the binding of ligands, large conformational changes is induced in DPCKs, as well as in many other kinases, to shield the bound ATP in their catalytic site from the futile hydrolysis by bulk water molecules. To investigate the molecular mechanisms underlying the phosphoryl transfer during DPCK catalytic cycle, we determined the crystal structures of the Legionellapneumophila DPCK (LpDPCK) both in its apo-form and in complex with ATP. The structures reveal that LpDPCK comprises of three domains, the classical core domain, the CoA domain, and the LID domain, which are packed together to create a central cavity for substrate-binding and enzymatic catalysis. The binding of ATP induces large conformational changes, including a hinge-bending motion of the CoA binding domain and the "helix to loop" conformational change of the P-loop. Finally, modeling of a dCoA molecule to the enzyme provides insights into the catalytic mechanism of DPCK.

Silver Mineralized Protein Hydrogel with Intrinsic Cell Proliferation Promotion and Broad-Spectrum Antimicrobial Properties for Accelerated Infected Wound Healing.

The presence of multidrug-resistant bacteria has challenged the clinical treatment of bacterial infection. There is a real need for the development of novel biocompatible materials with broad-spectrum antimicrobial activities. Antimicrobial hydrogels show great potential in infected wound healing but are still being challenged. Herein, broad-spectrum antibacterial and mechanically tunable amyloid-based hydrogels based on self-assembly and local mineralization of silver nanoparticles are reported. The mineralized hydrogels are biocompatible and have the advantages of sustained release of silver, prolonged antimicrobial effect, and improved adhesion capacity. Moreover, the mineralized hydrogels display a significant antimicrobial effect against both Gram-positive and Gram-negative bacteria in cells and mice by inducing membrane damage and reactive oxygen species toxicity in bacteria. In addition, the mineralized hydrogels can rapidly accelerate wound healing by the synergy between their antibacterial activity and intrinsic improvement for cell proliferation and migration. This study provides a modular approach to developing a multifunctional protein hydrogel platform based on biomolecule-coordinated self-assembly for a wide range of biomedical applications.