Extracellular Alkaline Phosphatase Research Articles

Combination of Cinnamaldehyde/β-cyclodextrin inclusion complex and L-phenylalanine effectively reduces the postharvest green mold in citrus fruit

The essential oil and β-cyclodextrin inclusion complex was able to inhibit the growth of Penicillium digitatum, a damaging pathogen that causes green mold in citrus fruit. In this study, cinnamaldehyde-β-cyclodextrin inclusion complex (β-CDCA) for controlling citrus green mold was synthesized by the co-precipitation method. Characterization of β-CDCA revealed that the aromatic ring skeleton of cinnamaldehyde (CA) was successfully embedded into the cavity of β-CD to form the inclusion complex. β-CDCA inhibited P. digitatum at a minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of 4.0 g/L. FT-IR spectroscopy analysis, calcofluor white staining, extracellular alkaline phosphatase (AKP) activity and propidium iodide (PI) staining of hyphae morphology showed that β-CDCA may damage the cell ultrastructure and membrane permeability of P. digitatum. The study further demonstrated that hydrogen peroxide (H2O2), malondialdehyde (MDA), and reactive oxygen species (ROS) markedly accumulated in 1/2 MIC β-CDCA treated hyphae. This implied that β-CDCA inhibited growth of P. digitatum by the triggering oxidative stress, which may have caused cell death by altering cell membrane permeability. In addition, in vivo results showed that β-CDCA alone or combined with L-phenylalanine (L-PHe) displayed a comparable level to that of prochloraz. Therefore, β-CDCA combined with L-PHe can thus be used as an eco-friendly preservative for the control green mold in postharvest citrus fruit.

Enzyme-responsive oncolytic polypeptide for tumor therapy

Host defense peptide-mimicking cationic oncolytic polymers have attracted increasing attention for cancer treatment in recent years. However, polymers with large amounts of positive charge may cause rapid clearance and severe off-target toxicity. To facilitate in vivo application, an alkaline phosphatase (ALP)-responsive oncolytic polypeptide precursor (C12-PLL/PA) has been reported in this work. C12-PLL/PA could be hydrolyzed into the active form of the oncolytic polypeptide (C12-PLL) by the extracellular alkaline phosphatase within solid tumors, thereby resulting in the conversion of the negative charge to positive charge and restoring its membrane-lytic activity. Detailed mechanistic studies showed that C12-PLL/PA could effectively destroy cancer cell membranes and subsequently result in rapid necrosis of cancer cells. More importantly, C12-PLL/PA significantly inhibited the tumor growth in the 4T1 orthotopic breast tumor model with negligible side effects. In summary, these findings demonstrated that the shielding of the amino groups with phosphate groups represents a secure and effective strategy to develop cationic oncolytic polypeptide, which represents a valuable reference for the design of enzyme-activated oncolytic polymers. Statement of significanceRecently, there has been a growing interest in fabricating host defense peptide-mimicking cationic oncolytic polymers for cancer therapy. However, there remain concerns about the tumor selectivity and off-target toxicity of these cationic polymers. In this study, an alkaline phosphatase-responsive oncolytic polypeptide precursor (C12-PLL/PA) has been developed to selectively target cancer cells while sparing normal cells. Mechanistic investigations demonstrated that C12-PLL/PA effectively disrupted cancer cell membranes, leading to rapid necrosis. Both in vitro and in vivo experiments showed promising anticancer activity and reliable safety of C12-PLL/PA. The findings suggest that this synthetic enzyme-responsive polypeptide holds potential as a tumor-specific oncolytic polymer, paving the way for future applications in cancer therapy.

A carrier-free injectable hydrogel self-assembled using natural thymol and glycyrrhizin for MRSA-infected wound healing in rats

A carrier-free, self-assembled, injectable hydrogel was developed as wound dressing to deal with skin wound bleeding, bacterial infections, and wound healing. Natural thymol (THY) and glycyrrhizin were used to construct THY-loaded glycymicelles (THY@glycymicelles), which greatly improved the aqueous solubility of THY. Optimal THY@glycymicelles had a high encapsulation efficiency of THY and a uniform nanoparticle size. Interestingly, THY@glycymicelles could reversibly transform into THY@glycyrrhizin hydrogel (THY@glycygel) by adjusting neutral pH to skin pH (pH ∼ 4.9), and vice versa. THY@glycygel had a dense porous sponge microstructure and showed many excellent features, including injectable, hemostatic, antibacterial, and anti-inflammatory properties. In vitro antibacterial evaluation, THY@glycygel showed lower minimum inhibitory concentration to three tested bacteria than free THY. In terms of antibacterial mechanism, THY@glycygel could destroy the bacterial cell wall and membrane, increasing the extracellular alkaline phosphatase release, and inhibiting biofilm. THY@glycygel had strong hemostatic ability and exhibited great potential for antimicrobial activity and improving wound healing infected by methicillin-resistant Staphylococcus aureus (MRSA). Mechanisms including antimicrobial, promoting hair follicle growth, improving epidermis remolding and collagen deposition, decreasing inflammatory cytokine levels, and increasing growth factor levels promoted the MRSA-infected skin wound healing process of THY@glycygel. Totally, this novel hydrogel has great potential as wound dressing.

Antibacterial activity and mechanism of X33 antimicrobial oligopeptide against Acinetobacter baumannii

Acinetobacter baumannii is a pathogenic bacterium widespread in human environments, especially in intensive care units, and is associated with high morbidity and infection rates. Multiple drug resistance in A. baumannii frequently leads to the death of patients, making the development of multi-effect antibacterial agents against this bacterium a research hotspot. We have previously found that the X33 antimicrobial oligopeptide can effectively inhibit the growth of Penicillium digitatum and Candida albicans. Herein, we evaluated the antibacterial activity of X33 antimicrobial oligopeptide against A. baumannii by determining the minimum inhibitory concentration, inhibition zone, and growth curve. The increase in extracellular alkaline phosphatase and the leakage of intracellular compounds confirmed the effect of X33 antimicrobial oligopeptide on the cell wall and membrane. Changes in reactive oxygen species, malondialdehyde, ATP, reducing sugar, soluble protein, and pyruvate content demonstrated that the incubation with X33 antimicrobial oligopeptide affected energy metabolism and oxidative stress. Consistent with the physiological characteristics, transcriptomics analysis indicated that incubation with X33 antimicrobial oligopeptide significantly induced changes in the expression of 2339 genes, including 1262 upregulated and 1077 downregulated genes, which participate in oxidative phosphorylation, ribosome, quorum sensing, fatty acid degradation, glycolysis/gluconeogenesis, and citrate cycle pathways. These results provide a fundamental basis for investigating the mechanism of X33 antimicrobial oligopeptide as a potential drug against A. baumannii.

Presence of pharmaceutical contaminants of emerging concerns in two rivers of western Cuba and their relationship with the extracellular enzymatic activity of microbial communities

In recent years, the concern derived from the presence of emerging contaminants in the environment and the possible effects on the One Health trilogy has increased. This study determined the concentration of pharmaceutical contaminants of emerging concern and their relationship with the extracellular enzymatic activity of microbial communities from two rivers in western Cuba. Two sampling stations were analyzed; one in the Almendares River (urban) and the other in the San Juan River (rural), taking into account the pollution sources that arrive at these stations and previous physicochemical characterizations. Extracellular protease, acid phosphatase, alkaline phosphatase, lipase, and catalase activities in water and sediments were determined and correlated with contaminants of emerging concern determined by liquid chromatography with mass spectrometry. This study evidenced the presence of different pharmaceutical contaminants found in the categories of antihypertensives, stimulants, anti-inflammatories, and antibiotics in both rivers. Concentrations of contaminants of emerging concern were greater in the Almendares River compared to the San Juan River. In addition, through the canonical redundancy analysis, the influence of these contaminants on the extracellular enzymatic activities of microbial communities was documented, where in most cases they inhibit protease, phosphatase, and lipase activities and enhance catalase activity in response to oxidative stress. The present investigation constitutes the first report in Cuba of the presence of pharmaceutical contaminants of emerging concern and one of the few works that exist in the Latin American region.

Assessment of degradation mechanism of imidacloprid residues in grape rhizosphere soil by UHPLC-Orbitrap™-MS and its residual impact on soil enzyme activity.

Imidacloprid (IM) is a systemic insecticide persistent in the environment and possesses a negative impact on the non-targeted ecosystem. The objective of the present study was to evaluate the dissipation and degradation mechanism of IM residues in grape rhizosphere soil and to investigate its residual effect on soil enzyme activity at different IM spiking levels. The half-life of IM residue in soil was 27, 36, and 43.5days at a spiking level of 1, 10, and 50mgkg-1, respectively following a bi-phasic first + first-order dissipation kinetics. UHPLC-Orbitrap™-MS analysis by targeted metabolomics approach revealed that IM metabolites such as IM-amine analogue, guanidine (reduction), 5-hydroxy IM (hydroxylation), IM-Urea (oxidation), reduced NO analogue of IM (oxidation), and olefin of guanidine IM (dehydrogenation) were identified and proposed the degradation mechanism in grape rhizosphere soil. Toxicity of IM residues on five extracellular enzymes, viz., dehydrogenase, acid phosphatase, alkaline phosphatase, β-glucosidase, and urease revealed that activity of dehydrogenase, acid phosphatase, and alkaline phosphatase remained unaffected at 60th day of sampling. The β-glucosidase and urease were negatively affected throughout the incubation period indicating the influence of IM residues on carbon and nitrogen mineralization in soil. Thus, long-term exposure of IM to grape rhizosphere through soil drenching could affect soil enzyme activity which has a negative effect on the soil nutrient cycle and soil microbiome.

Coordinated phosphate uptake by extracellular alkaline phosphatase and solute carrier transporters in marine diatoms.

Marine diatoms express genes encoding potential phosphate transporter and alkaline phosphatase (APase) under phosphate-limited (-P) condition. This indicates that diatoms use high-affinity phosphate uptake system with organic phosphate hydration. The function of molecules playing roles for Pi uptake was determined in this study. Pi uptake and APase activity of two marine diatoms, Phaeodactylum tricornutum and Thalassiosira pseudonana, were monitored during acclimation to -P condition. The transcript levels of Pi transporter were analyzed, and Pi transporters were localized with GFP tagging in diatom cells. KO mutants of plasma membrane solute carrier proteins (SLC34s) or APase were established, and their phenotype was evaluated. Some Na+ /Pi transporter candidates, SLC34s in P. tricornutum and T. pseudonana, increased transcript under -P condition. Whole-cell Pi transport was specifically stimulated by sodium ion but independent of potassium, lithium, or proton. Genome-editing KO of PtSLC34-5 and APase (Pt49678) in P. tricornutum was highly inhibitory for Pi uptake, and KO of TpSLC34-2 was also highly inhibitory for Pi uptake in T. pseudonana. SLC34s and APase were co-expressed under -P conditions in marine diatoms. SLC34s play a major role in the initial acclimation stage of diatom cells to -P condition and APase plays an increasing role in the prolonged Pi-starved condition.

Fluorimetric microplate assay for the determination of extracellular alkaline phosphatase kinetics and inhibition kinetics in activated sludge

The microbial enzyme alkaline phosphatase contributes to the removal of organic phosphorus compounds from wastewaters. To cope with regulatory threshold values for permitted maximum phosphor concentrations in treated wastewaters, a high activity of this enzyme in the biological treatment stage, e.g., the activated sludge process, is required. To investigate the reaction dynamics of this enzyme, to analyze substrate selectivities, and to identify potential inhibitors, the determination of enzyme kinetics is necessary. A method based on the application of the synthetic fluorogenic substrate 4-methylumbelliferyl phosphate is proven for soils, but not for activated sludges. Here, we adapt this procedure to the latter. The adapted method offers the additional benefit to determine inhibition kinetics. In contrast to conventional photometric assays, no particle removal, e.g., of sludge pellets, is required enabling the analysis of the whole sludge suspension as well as of specific sludge fractions. The high sensitivity of fluorescence detection allows the selection of a wide substrate concentration range for sound modeling of kinetic functions.•Fluorescence array technique for fast and sensitive analysis of high sample numbers•No need for particle separation – analysis of the whole (diluted) sludge suspension•Simultaneous determination of standard and inhibition kinetics

An O-GlcNAc transferase pathogenic variant linked to intellectual disability affects pluripotent stem cell self-renewal.

O-linked β-N-acetylglucosamine (O-GlcNAc) transferase (OGT) is an essential enzyme that modifies proteins with O-GlcNAc. Inborn OGT genetic variants were recently shown to mediate a novel type of congenital disorder of glycosylation (OGT-CDG), which is characterised by X-linked intellectual disability (XLID) and developmental delay. Here, we report an OGTC921Y variant that co-segregates with XLID and epileptic seizures, and results in loss of catalytic activity. Colonies formed by mouse embryonic stem cells carrying OGTC921Y showed decreased levels of protein O-GlcNAcylation accompanied by decreased levels of Oct4 (encoded by Pou5f1), Sox2 and extracellular alkaline phosphatase (ALP), implying reduced self-renewal capacity. These data establish a link between OGT-CDG and embryonic stem cell self-renewal, providing a foundation for examining the developmental aetiology of this syndrome.

Transcriptomics and physiological analyses unveil the distinct mechanisms of ATP and glucose-6-phosphate utilization in Phaeodactylum tricornutum

Phosphoesters are a dominant component of marine dissolved organic phosphorus (DOP) and an important source of the phosphorus nutrient for phytoplankton, but the molecular mechanisms of their utilization by phytoplankton are divergent and poorly understood. In this study, we used the model diatom Phaeodactylum tricornutum to investigate and compare the utilization mechanisms of two different phosphoesters, adenosine triphosphate (ATP) and glucose-6-phosphate (G6P). We found that ATP and G6P can both be efficiently used to support the growth of P. tricornutum. Cells grown on ATP or G6P showed lower pigment contents and photosynthetic rates but higher cellular lipids relative to those grown on NaH2PO4 (DIP). Surprisingly, in neither the ATP nor the G6P group were significant increases in whole-cell alkaline phosphatase (AP) activity detected, suggesting that utilization of both DOPs was not reliant on extracellular AP. Yet, ATP-grown cultures released DIP into the medium (i.e., ATP hydrolyzed extracellularly) whereas G6P-grown cultures did not. Furthermore, transcriptomic and RT-qPCR results showed that the gene encoding 5’ nucleotidase (5NT) in the ATP group and PhoD in the G6P group was upregulated. These results indicated that different pathways are involved in the use of these two DOPs, with ATP being hydrolyzed extracellularly likely by 5NT (PHATRDRAFT_44177) to release DIP for uptake, and G6P being directly absorbed and hydrolyzed intracellularly likely by PhoD (PHATRDRAFT_45757). Nevertheless, P. tricornutum under ATP and G6P conditions showed more similar transcriptomic profiles to each other than either compared to DIP-grown cultures, indicating similar metabolic functions of these two DOPs. These findings demonstrate that despite the high similarity in transcriptomic response to ATP and G6P conditions, the utilization mechanisms of these phosphoesters in the same species can be totally different, and the lack of AP activity does not necessarily signal the absence of DIP deficiency or the absence of DOP utilization.

Self-Regulation of Soil Enzyme Activity and Stoichiometry under Nitrogen Addition and Plastic Film Mulching in the Loess Plateau Area, Northwest China

Soil extracellular enzyme activity (EA) and its eco-enzyme stoichiometric ratio (ES) are extremely sensitive to environmental change. This study aimed to clarify the change law of EA and ES in soil with different nitrogen addition levels under plastic film mulching, and to optimize the application amount of nitrogen fertilizer that was used. Based on the location experiment of plastic film mulching fertilization that has been ongoing since 2015, soil samples were collected from different depths (0–10 cm, 10–20 cm and 20–30 cm) during the harvest period of spring maize in October 2021. Four soil extracellular enzyme activities (β-1,4 glucosidase (βG), β-1, 4-N-acetylglucosidase (NAG), leucine aminopeptidase (LAP) and alkaline phosphatase (AP)) involved in soil carbon (C), nitrogen (N) and phosphorus (P) cycling at different nitrogen application levels (0, 90, 150, 225 and 300 kg·hm−2) were studied under two planting patterns of no plastic film mulching (LD) and plastic film mulching (PM). The latest discovery of this study is that the activities of soil EA involved in the cycling of soil carbon C, N and P are similar in different soil depths (0–10 cm, 10–20 cm and 20–30 cm). Both EC:P and EC:P in the soil in this area are less than 1:1, indicating that the soil is limited by N and P. Comprehensive analysis showed that a nitrogen application level of 225 kg·hm−2 was beneficial to the balance of soil nutrients and the improvement of soil EA at harvest. At the same time, PM can effectively improve the soil EA and is more conducive to the balance of soil nutrients. Redundancy analysis (RDA) showed that EA and ES were strongly correlated with pH, soil organic carbon (SOC), total nitrogen (TN) and total phosphorus (TP). Most importantly, this study revealed that the activity of extracellular enzymes in arid and semi-arid areas was constantly self-regulated with the addition of nitrogen, which provided theoretical and technical support for the efficient use of nitrogen under the condition of plastic film mulching.

Antibacterial activity and mechanism of sanguinarine against Staphylococcus aureus by interfering with the permeability of the cell wall and membrane and inducing bacterial ROS production.

Staphylococcus aureus (SA) is representative of gram-positive bacteria. Sanguinarine chloride hydrate (SGCH) is the hydrochloride form of sanguinarine (SG), one of the main extracts of Macleaya cordata (M. cordata). There are few reports on its antibacterial mechanism against SA. Therefore, in this study, we investigated the in vitro antibacterial activity and mechanism of SGCH against SA. The inhibitory zone, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) were measured, and the bactericidal activity curve was plotted. In addition, the micromorphology, alkaline phosphatase (AKP) activity, Na+K+, Ca2+Mg2+-adenosine triphosphate (ATP) activity, intracellular reactive oxygen species (ROS), and fluorescein diacetate (FDA) were observed and detected. The results showed that the inhibitory zone of SGCH against SA was judged as medium-sensitive; the MIC and MBC were 128 and 256 μg/mL, respectively; in the bactericidal activity curve, SGCH with 8 × MIC could completely kill SA within 24 h. SGCH was able to interfere with the integrity and permeability of the SA cell wall and membrane, as confirmed by the scanning electron microscopy (SEM) images, the increase in extracellular AKP and Na+ K+, Ca2+ Mg2+-ATP activities as well as the fluorescein diacetate (FDA) staining experiment results. Moreover, a high concentration of SGCH could induce SA to produce large amounts of ROS. In summary, these findings revealed that SGCH has a preferable antibacterial effect on SA, providing an experimental and theoretical basis for using SG as an antibiotic substitute in animal husbandry and for the clinical control and treatment of diseases caused by SA.

The phosphorus limitation in the post-fire forest soils increases soil CO2 emission via declining cellular carbon use efficiency and increasing extracellular phosphatase

Wildfires cause severe disruptions to forest soils and modify the carbon (C) cycle in the post-fire soils. Yet, the contribution of abiotic and biotic drivers to CO2 emission in the post-fire forest soils is not well understood. This study investigated soil CO2 emission from the burned and corresponding unburned forest soils across a large latitudinal gradient of mean average precipitation (from 422 to 1640 mm) and temperature (from 0.20 to 25.8 °C). We compared the soil chemical properties (e.g., dissolved organic carbon, nutrients) and microbial properties (biomass, metabolic quotient (qCO2), and community diversity and composition) in the burned and unburned soils. Microbial metabolism was also addressed through extracellular enzyme activity and intracellular C use efficiency (CUE) using the stoichiometric modeling approach. Here, we showed that the wildfires: i) increased phosphorous (P) pressure to soil microbes (indicated by increased qCO2), ii) decreased microbial biomass, community diversity and abundance of some microbial groups (e.g., Phylum Actinobacteria), and iii) resulted in larger basal respiration due to lower cellular CUE and higher extracellular alkaline phosphatase (AP) ratio to other enzymes like β-glucosidase, Cellobiohydrolase and N-Acetyl-glucosaminidase. The latter indicated the wildfires-induced P:N imbalance (P-limitation) caused significant C loss via microbial exploitation of P from soil organic matter. The study highlights the ecological significance of intracellular and extracellular metabolisms in soil respiration and consequent C sequestration in post-fire forest soils.

Antibacterial activity and mechanism of flavonoids from Chimonanthus salicifolius S. Y. Hu. and its transcriptome analysis against Staphylococcus aureus.

Chimonanthus salicifolius S. Y. Hu. (FCS) possess many biological activities, but the antibacterial activity and underlying mechanisms of flavonoids from Chimonanthus salicifolius S. Y. Hu. (FCS) is still unknown. Maximum diameter of inhibition zone (DIZ), maximum diameter of inhibition zone (DIZ), the lowest minimum inhibition concentration (MIC), and the lowest minimum bactericide concentration (MBC) were used to detect the antibacterial activity. Meanwhile, related enzyme activities, the transcriptome analysis and quantitative RT-PCR were used to investigate the antibacterial activity mechanisms. The results showed that FCS (with a purity of 84.2 ± 2.0%) has potential effects on tested strains with the maximum diameter of inhibition zone (DIZ) was 15.93 ± 2.63 mm, the lowest minimum inhibition concentration (MIC) was 1.56 mg/ml and the lowest minimum bactericide concentration (MBC) was 6.25 mg/ml. In addition, the bacterial growth curve test, release of extracellular alkaline phosphatase (AKP), loss of intracellular components, DNA damage and transmission electron microscope (TEM) suggested that FCS could destroy the cell wall and membrane, cause the loss of intracellular substance, cause DNA damage and even lead to cell death. Moreover, the antibacterial mechanism of FCS against Staphylococcus aureus (S. aureus, Gram-positive bacteria) was further confirmed by the transcriptome analysis and quantitative RT-PCR at the molecular level for the first time. A total of 671 differentially expressed genes (DEGs) were identified after treated with FCS (1/2 MIC), with 338 and 333 genes showing up-regulation and down-regulation, respectively. The highlighted changes were those related to the biosynthesis of bacteria wall and membrane, DNA replication and repair, and energy metabolism. Overall, our research provides theoretical guidance for the application of FCS, which is expected to be potentially used as a natural antimicrobial agent in food safety.

Purification of Polysaccharides from Orostachys cartilaginous Cell Cultures by Macroporous Resin Absorption and Bioactivities of the Purified Polysaccharides.

Orostachys cartilaginous is a traditional herbal medicine and its cell cultures contain large amounts of polysaccharides. To utilize the cultured O. cartilaginous cells, this study purified the crude polysaccharides of O. cartilaginous cells by macroporous resin absorption and optimized the purification process in the experiment of orthogonal design with four factors (sample concentration and volume, and eluent concentration and volume) and three levels; the antibacterial and anti-cancer effects of the purified polysaccharides (OTP) were further examined. The results showed that polysaccharide purity reached 95 % in the optimized group, i. e., 1.6 mg/mL of sample (crude polysaccharides) concentration, 3.0 bed volume (BV) of sample volume, 65 % eluent (ethanol) concentration, and 3.0 BV of eluent volume. In the antibacterial experiment, the growth of three bacterial species, i. e., Pseudomonas aeruginosa, Staphylococcus aureus, and Bacillus subtilis was inhibited by OTP, whereas that of Escherichia coli was not affected; the largest diameter of the inhibitory zone was found on B. subtilis; the extracellular alkaline phosphatase activity and the electrical conductivity, nucleic acid, and protein levels of B. subtilis increased after OTP treatment, indicating that the inhibition of B. subtilis growth was caused by the leakage of cell contents. In the anti-cancer experiment, OTP decreased the cell viabilities of the tested human cancer cells, i. e., AGS (gastric cancer), HCT116 (colon cancer), HepG2 (liver cancer), and HeLa (cervical cancer), and the highest inhibitory effect was on HCT116. OTP promoted HCT116 apoptosis and affected the expression of apoptosis-related proteins, i. e., the expression of B-cell lymphoma-2 decreased and that of bcl-2 associated X protein, cytochrome c, caspase 9 and caspase 3 increased. The findings of the present study suggest that O. cartilaginous cell cultures have a potential application in food or drug production.

Underlying mechanism of menthol on controlling postharvest citrus sour rot caused by Geotrichum citri-aurantii

Sour rot, caused by Geotrichum citri-aurantii, has affected fruit quality and caused severe economic losses. Menthol is widely applied to medicine and food industries due to its antiseptic and antimicrobial effects, whereas its application in postharvest fruit management is rarely reported. The present study investigated the antifungal activity of menthol and its underlying mechanism involved. The in vitro experiments showed that the minimum inhibitory concentration (MIC) of menthol was 6.4 mmol L-1. Menthol induced the formation and accumulation of reactive oxygen species (ROS) in G. citri-aurantii and triggered severe membrane lipid peroxidation, resulting in the destruction of plasma membrane integrity. The activity of extracellular alkaline phosphatase (AKP) was increased after menthol treatment. Otherwise, the decrease of blue fluorescence in the cell wall and the change of cell wall morphology were also observed, indicating that the cell wall structure was disrupted. Notably, in vivo, the disease incidence of citrus fruit decreased by 43% after the application of 128 mmol L-1 menthol. Furthermore, menthol treatment maintained the quality of citrus fruit and enhanced defense-related enzyme activities in citrus. To further explore the potential pathogenicity of G. citri-aurantii, we de novo assembled the genome of G. citri-aurantii G50 based on Illumina NovaSeq PE150 and PacBio Sequel. The revised genome size was 25.41 Mb, assembled onto 27 scaffolds with a total length of 27,548,716 bp, 38.49% GC content and 4181 protein-coding genes. Herein, we identified four CWI-MAPK-related genes (GciPKC1, GciBCK1, GciMKK1 and GciSLT2). The expressions of these genes were down-regulated after menthol treatment, influencing on downstream chitin and glucan synthesis. Collectively, these results demonstrated that menthol might be beneficial as a potential alternative for decay management of citrus sour rot. Our research provided new perspectives for postharvest disease control and management control of this pathogen and elucidated its pathogenic mechanism.

Cyanobacterial extracellular alkaline phosphatase: detection and ecological function

Cyanobacterial extracellular alkaline phosphatase: detection and ecological function

Dissolved Organic Matter and Activities of Extracellular Enzymes in Two Lagoons (South China Sea) Affected by Aquaculture

Xincun Lagoon and Li’an Lagoon are typical tropical lagoons containing unique seagrass meadow and mangrove forest ecosystems. However, nutrient pollution from human activities threatens ecosystem health in these areas. A year-long bimonthly survey was carried out to study the bioavailability of organic nutrients through hydrolysis of the extracellular enzymes β-glucosidase (GLU), leucine aminopeptidase (LAP), and alkaline phosphatase (AP) in the two lagoons. The results showed that the nutrient statuses in Xincun Lagoon and Li’an Lagoon were highly variable and enriched with organic nutrients, especially nitrogen. Organic nitrogen and phosphorus were actively recycled in the lagoons through active LAP and AP hydrolysis. The enzyme activities were mostly produced by particulate-associated bacteria and were positively regulated by the existence of organic nutrients. The enzymatic stoichiometry results indicated the importance of dissolved proteins and aminopolysaccharides as sources of both carbon and nitrogen and a potential phosphorus limitation on microbial growth in the lagoons. Our results emphasized the importance of EEAs in the recycling of organic nutrients and the potential influence of enriched organic nutrients on both microorganisms and plants in Xincun Lagoon and Li’an Lagoon.

Alkaline Phosphatase Activity of Plant Growth-Promoting Actinomycetes and Their Genetic Diversity Based on the phoD Gene

Actinomycete is one of the beneficial bacteria groups inhabiting rhizosphere soil. They can promote plant growth through various mechanisms. In the previous study we have isolated rhizosphere actinomycetes from maize rhizosphere with direct plant growth promotion characters. The aims of the present study were to analyze the ability of maize rhizosphere actinomycetes to solubilize phosphate, determine alkaline phosphatase activity, and study their genetic diversity based on phoD gene. Thirteen rhizosphere actinomycete isolates were able to solubilize phosphate at concertation range 55.84±2.27 mg/L to 144.48±5.71 mg/L. The activity of extracellular alkaline phosphatase was exhibited by all maize rhizosphere actinomycetes isolates in various level ranging from 0.08 mU/mL to 0.51 mU/mL. The phoD gene, one of the three homologous genes which encode alkaline phosphatases, was successfully detected in all isolates and identified as alkaline phosphatase D of Streptomyces spp. The partial phoD sequences of the isolates were located within metallophosphatase domain of alkaline phosphatase D. Alignment analysis showed that the deduced amino acid sequences of PhoD were mostly conserved in the isolates and Streptomyces spp. Essential residues involved in the active core arrangement of PhoD which binds metal ion cofactors were conserved. Constructed phylogenetic tree showed that the isolates were divided into two groups within PhoD cluster. PhoD of the isolates and Streptomyces spp. had closer relationship to purple acid phosphatase compared to other homologous PhoA and PhoX which form separate cluster. Generated three-dimensional structure model of partial PhoD had high similarity to alkaline phosphatase D of Bacillus subtilis (2YEQ) and showed overlapping structure based on super-positioning analysis.

Synergistic effect of sulfidated nanoscale zerovalent iron in donor and recipient bacterial inactivation and gene conjugative transfer inhibition

Antibiotic resistance genes (ARGs) and antibiotic resistant bacteria (ARB) are widespread in urban wastewater treatment plants (UWTPs). In this research, a horizontal transfer model of recipient (Pseudomonas. HLS-6) and donor (Escherichia coli DH5α carries RP4 plasmid) was constructed to explore the effect of sulfidated nanoscale zerovalent iron (S-nZVI) on the efficiency of plasmid-mediated horizontal transfer. When the S/Fe was 0.1, the inactivation efficiency of 1120 mg/L S-nZVI on the donor and recipient bacteria were 2.36 ± 0.03 log and 3.50 ± 0.17 log after 30 min, respectively (initial ARB concentration ≈ 5 ×107 CFU/mL). Effects of treatment time, S/Fe molar ratio, S-nZVI dosage and initial bacterial concentration were systemically studied. S-nZVI treatment could increase the extracellular alkaline phosphatase and malondialdehyde content of the ARB, cause oxidative stress in the bacteria, destroy the cell structure and damage the intracellular DNA. This study provided evidence and insights into possible underlying mechanisms for reducing conjugative transfer, such as hindering cell membrane repair, inducing the overproduction of reactive oxygen species, inhibiting the SOS response, reducing the expression of ARGs and related transfer genes. S-nZVI could inhibit the gene conjugative transfer while inactivating the ARB. The findings provided an alternative method for controlling antibiotic resistance.