Construction of a grpE-based plasmid addiction system in Escherichia coli and its application in phloroglucinol biosynthesis.

AIM: Biotechnical processes in Escherichia coli often operate with artificial plasmids. However, these bioprocesses frequently encounter plasmid loss. To ensure stable expression of heterologous genes in E. coli BL21(DE3), a novel plasmid addiction system (PAS) was developed. METHODS AND RESULTS: This PAS employed an essential gene grpE encoding a cochaperone in the DnaK-DnaJ-GrpE chaperone system as the selection marker, which represented a chromosomal ΔgrpE mutant harboring episomal expression plasmids that carry supplementary grpE alleles to restore the deficiency. To demonstrate the feasibility of this system, it was implemented in phloroglucinol (PG) biosynthesis, manifesting improved host tolerance to PG and increased PG production. Specifically, PG titer significantly improved from 0.78 ± 0.02 to 1.34 ± 0.04 g l-1, representing a 71.8% increase in shake-flask fermentation. In fed-batch fermentation, the titer increased from 3.71 ± 0.11 to 4.54 ± 0.10 g l-1, showing a 22.4% increase. RNA sequencing and transcriptome analysis revealed that the improvements were attributed to grpE overexpression and upregulation of various protective chaperones and the biotin acetyl-CoA carboxylase ligase coding gene birA. CONCLUSION: This novel PAS could be regarded as a typical example of nonanabolite- and nonmetabolite-related PAS. It effectively promoted plasmid maintenance in the host, improved tolerance to PG, and increased the titer of this compound.

Regiodivergent biosynthesis of bridged bicyclononanes.

Medicinal compounds from plants include bicyclo[3.3.1]nonane derivatives, the majority of which are polycyclic polyprenylated acylphloroglucinols (PPAPs). Prototype molecules are hyperforin, the antidepressant constituent of St. John's wort, and garcinol, a potential anticancer compound. Their complex structures have inspired innovative chemical syntheses, however, their biosynthesis in plants is still enigmatic. PPAPs are divided into two subclasses, named type A and B. Here we identify both types in Hypericum sampsonii plants and isolate two enzymes that regiodivergently convert a common precursor to pivotal type A and B products. Molecular modelling and substrate docking studies reveal inverted substrate binding modes in the two active site cavities. We identify amino acids that stabilize these alternative binding scenarios and use reciprocal mutagenesis to interconvert the enzymatic activities. Our studies elucidate the unique biochemistry that yields type A and B bicyclo[3.3.1]nonane cores in plants, thereby providing key building blocks for biotechnological efforts to sustainably produce these complex compounds for preclinical development.

Subinhibitory effects of 2,4-diacetylphloroglucinol on filamentous fungus Aspergillus fumigatus.

AIMS: Aspergillus fungi are common members of the soil microbiota. Some physiological and structural characteristics of Aspergillus species make them important participants in soil ecological processes. In this study, we aimed to evaluate the impact of 2,4-diacetylphloroglucinol (2,4-DAPG), a common metabolite of soil and rhizosphere bacteria, on the physiology of Aspergillus fumigatus. METHODS AND RESULTS: Integrated analysis using microscopy, spectrophotometry, and liquid chromatography showed the following effects of 2,4-DAPG on Aspergillus physiology. It was found that A. fumigatus in the biofilm state is resistant to high concentrations of 2,4-DAPG. However, experimental exposure led to a depletion of the extracellular polymeric substance, changes in the structure of the cell wall of the mycelium (increase in the content of α- and ß-glucans, chitin, and ergosterol), and conidia (decrease in the content of DHN-melanin). 2,4-DAPG significantly reduced the production of mycotoxins (gliotoxin and fumagillin) but increased the secretion of proteases and galactosaminogalactan. CONCLUSIONS: Overall, the data obtained suggest that 2,4-DAPG-producing Pseudomonas bacteria are unlikely to directly eliminate A. fumigatus fungi, as they exhibit a high level of resistance when in the biofilm state. However, at low concentrations, 2,4-DAPG significantly alters the physiology of aspergilli, potentially reducing the adaptive and competitive capabilities of these fungi.

Anti-Herpes Simplex Virus Type 1 Activity Evaluation of Natural Derived Phloroglucinol Derivatives and Their Molecular Mechanisms Study.

HSV-1 is a common infection that can cause cold sores. In this study, the anti-HSV-1 virus activity of three series compounds A1-A9, B1-B12, C1-C22 was screened by MTT assay, qRT-PCR assay, Western blot assay and viruses' plaque assays. The results of MTT assay disclosed that phloroglucinol derivatives C2 and C3 effectively inhibited the death of HSV-1 infected vero cells with the CC50 values of C2 and C3 were 72.64 µmol/L and 32.62 µmol/L in HaCaT cells, 137.6 µmol/L and 48.55 µmol/L in Hela cells. The IC50 values of C3 in vero cells and Hela cells were 19.26 µmol/L and 22.98 µmol/L, respectively. In the qRT-PCR experiments, it showed that C2 and C3 effectively reduced the synthesis of HSV-1 early viral gene VP16 and late viral gene gD. The Western blot results showed that both C2 and C3 inhibited the expression of HSV-1 gD protein in a concentration-dependent manner. Lastly, viruses' plaque assay results showed that C2 and C3 inhibited the production of HSV-1 progeny virus in Hela cells and HaCaT cells in a concentration-dependent manner. Taken together, these results suggest that C2 and C3 are promising candidate that warrants further attention in the development of anti-HSV-1 drugs.

Soil bacterium manipulates antifungal weapons by sensing intracellular type IVA secretion system effectors of a competitor.

Soil beneficial bacteria can effectively inhibit bacterial pathogens by assembling contact-dependent killing weapons, such as the type IVA secretion system (T4ASS). It's not clear whether these antibacterial weapons are involved in biotrophic microbial interactions in soil. Here we showed that an antifungal antibiotic 2,4-DAPG production of the soil bacterium, Pseudomonas protegens can be triggered by another soil bacterium, Lysobacter enzymogenes, via T4ASS by co-culturing on agar plates to mimic cell-to-cell contact. We demonstrated that the induced 2,4-DAPG production of P. protegens is achieved by intracellular detection of the T4ASS effector protein Le1519 translocated from L. enzymogenes. We defined Le1519 as LtaE (Lysobacter T4E triggering antifungal effects), which specifically stimulates the expression of 2,4-DAPG biosynthesis genes in P. protegens, thereby protecting soybean seedlings from infection by the fungus Rhizoctonia solani. We further found that LtaE directly bound to PhlF, a pathway-specific transcriptional repressor of the 2,4-DAPG biosynthesis, then activated the 2,4-DAPG production. Our results highlight a novel pattern of microbial interspecies and interkingdom interactions, providing a unique case for expanding the diversity of soil microbial interactions.

Biochemical investigations of polyphenol degradation enzymes in the phototrophic bacterium Rubrivivax gelatinosus.

Phloroglucinol (1,3,5-trihydroxybenzene) is an important intermediate in the degradation of flavonoids and tannins by anaerobic bacteria. Recent studies have shed light on the enzymatic mechanism of phloroglucinol degradation in butyrate-forming anaerobic bacteria, including environmental and intestinal bacteria such as Clostridium and Flavonifractor sp. Phloroglucinol degradation gene clusters have also been identified in other metabolically diverse bacteria, although the polyphenol metabolism of these microorganisms remain largely unexplored. Here, we describe biochemical studies of polyphenol degradation enzymes found in the purple non-sulfur bacterium Rubrivivax gelatinosus IL144, an anaerobic photoheterotroph reported to utilize diverse organic compounds as carbon sources for growth. In addition to the phloroglucinol reductase and dihydrophloroglucinol cyclohydrolase that catalyze phloroglucinol degradation, we characterize a Mn2+-dependent phloretin hydrolase that catalyzes the cleavage of phloretin into phloroglucinol and phloretic acid. We also report a Mn2+-dependent decarboxylase (DeC) that catalyzes the reversible decarboxylation of 2,4,6-trihydroxybenzoate to form phloroglucinol. A bioinformatics search led to the identification of DeC homologs in diverse soil and gut bacteria, and biochemical studies of a DeC homolog from the human gut bacterium Flavonifractor plautii demonstrated that it is also a 2,4,6-trihydroxybenzoate decarboxylase. Our study expands the range of enzymatic mechanisms for phloroglucinol formation, and provides further biochemical insight into polyphenol metabolism in the anaerobic biosphere.

A Functionalizable Analog of the Yariv Reagent for AGP Imaging using Fluorescence Microscopy.

Small molecule fluorescent probes that bind selectively to plant cell wall polysaccharides have been instrumental in elucidating the localization and function of these glycans. Arabinogalactan proteins (AGPs) are cell wall proteoglycans implicated in essential functions such as cell signaling, plant growth, and programmed cell death. There is currently no small molecule probe capable of fluorescently labeling AGPs. The Yariv reagents are the only small molecules that bind AGPs, and have been used to study AGP function and isolate AGPs via precipitation of an AGP-Yariv complex. However, the Yariv reagents are not fluorescent, rendering them ineffective for localization studies using fluorescence microscopy. A fluorescent version of a Yariv reagent that is capable of both binding as well as imaging AGPs would provide a powerful tool for studying AGPs in planta. Herein, we describe the synthesis of an azido analog of the Yariv reagent that can be further functionalized with a fluorophore to provide a glycoconjugate that binds AGPs and is fluorescent. We show that the modified reagent binds gum arabic in in vitro binding assays when used in conjunction with the ßGlcYariv reagent. Fluorescent imaging of AGPs in fixed maize leaf tissue enables localization of AGPs to cell walls in the leaf. Significantly, imaging can also be carried out using fresh tissue. This represents the first small molecule probe that can be used to visualize AGPs using fluorescence microscopy.

Anaerobic phloroglucinol degradation by Clostridium scatologenes.

Polyphenols are abundant in nature, and their anaerobic biodegradation by gut and soil bacteria is a topic of great interest. The O2 requirement of phenol oxidases is thought to explain the microbial inertness of phenolic compounds in anoxic environments, such as peatlands, termed the enzyme latch hypothesis. A caveat of this model is that certain phenols are known to be degraded by strict anaerobic bacteria, although the biochemical basis for this process is incompletely understood. Here, we report the discovery and characterization of a gene cluster in the environmental bacterium Clostridium scatologenes for the degradation phloroglucinol (1,3,5-trihydroxybenzene), a key intermediate in the anaerobic degradation of flavonoids and tannins, which constitute the most abundant polyphenols in nature. The gene cluster encodes the key C-C cleavage enzyme dihydrophloroglucinol cyclohydrolase, as well as (S)-3-hydroxy-5-oxo-hexanoate dehydrogenase and triacetate acetoacetate-lyase, which enable phloroglucinol to be utilized as a carbon and energy source. Bioinformatics studies revealed the presence of this gene cluster in phylogenetically and metabolically diverse gut and environmental bacteria, with potential impacts on human health and carbon preservation in peat soils and other anaerobic environmental niches. IMPORTANCE This study provides novel insights into the microbiota's anaerobic metabolism of phloroglucinol, a critical intermediate in the degradation of polyphenols in plants. Elucidation of this anaerobic pathway reveals enzymatic mechanisms for the degradation of phloroglucinol into short-chain fatty acids and acetyl-CoA, which are used as a carbon and energy source for bacterium growth. Bioinformatics studies suggested the prevalence of this pathway in phylogenetically and metabolically diverse gut and environmental bacteria, with potential impacts on carbon preservation in peat soils and human gut health.

Evaluating the effect of phenolic compounds as hydrogen acceptors when ruminal methanogenesis is inhibited in vitro - Part 1. Dairy cows.

Some antimethanogenic feed additives for ruminants promote rumen dihydrogen (H2) accumulation potentially affecting the optimal fermentation of diets. We hypothesised that combining an H2 acceptor with a methanogenesis inhibitor can decrease rumen H2 build-up and improve the production of metabolites that can be useful for the host ruminant. We performed three in vitro incubation experiments using rumen fluid from lactating Holstein cows: Experiment 1 examined the effect of phenolic compounds (phenol, catechol, resorcinol, hydroquinone, pyrogallol, phloroglucinol, and gallic acid) at 0, 2, 4, and 6 mM on ruminal fermentation for 24 h; Experiment 2 examined the combined effect of each phenolic compound from Experiment 1 at 6 mM with two different methanogenesis inhibitors (Asparagopsis taxiformis or 2-bromoethanesulfonate (BES)) for 24 h incubation; Experiment 3 examined the effect of a selected phenolic compound, phloroglucinol, with or without BES over a longer term using sequential incubations for seven days. Results from Experiment 1 showed that phenolic compounds, independently of the dose, did not negatively affect rumen fermentation, whereas results from Experiment 2 showed that phenolic compounds did not decrease H2 accumulation or modify CH4 production when methanogenesis was decreased by up to 75% by inhibitors. In Experiment 3, after three sequential incubations, phloroglucinol combined with BES decreased H2 accumulation by 72% and further inhibited CH4 production, compared to BES alone. Interestingly, supplementation with phloroglucinol (alone or in combination with the CH4 inhibitor) decreased CH4 production by 99% and the abundance of methanogenic archaea, with just a nominal increase in H2 accumulation. Supplementation of phloroglucinol also increased total volatile fatty acid (VFA), acetate, butyrate, and total gas production, and decreased ammonia concentration. This study indicates that some phenolic compounds, particularly phloroglucinol, which are naturally found in plants, could improve VFA production, decrease H2 accumulation and synergistically decrease CH4 production in the presence of antimethanogenic compounds.

Evaluating the effect of phenolic compounds as hydrogen acceptors when ruminal methanogenesis is inhibited in vitro - Part 2. Dairy goats.

Most mitigation strategies to reduce enteric methane (CH4) production in the rumen induce an excess of rumen dihydrogen (H2) that is expelled and consequently not redirected to the synthesis of metabolites that can be utilised by the ruminant. We hypothesised that phenolic compounds can be potential H2 acceptors when added to the diet, as they can be degraded to compounds that may be beneficial for the animal, using part of the H2 available when ruminal methanogenesis is inhibited. We performed four in vitro incubation experiments using rumen inoculum from Murciano-Granadina adult goats: Experiment 1 examined the inhibitory potential of Asparagopsis taxiformis (AT) at different concentrations (0, 1, 2, 3, 4 and 5% of the substrate on a DM basis) in 24 h incubations; Experiment 2 investigated the effect of a wide range of phenolic compounds (phenol, catechol, resorcinol, hydroquinone, pyrogallol, phloroglucinol, gallic acid and formic acid) at different doses (0, 2, 4, and 6 mM) on rumen fermentation for 24 h; Experiment 3 evaluated the combined effect of each phenolic compound at 6 mM with AT at 2% DM in sequential batch cultures for 5 days; and Experiment 4 examined the dose-response effect of phloroglucinol at different concentrations (0, 6, 16, 26 and 36 mM) combined with AT in sequential batch cultures for 5 days. Results from Experiment 1 confirmed that AT at 2% DM substantially inhibited CH4 production while significantly increasing H2 accumulation and decreasing the acetate:propionate ratio. Results from Experiment 2 showed that phenolic compounds did not negatively affect rumen fermentation at any dose. In Experiment 3, each phenolic compound at 6 mM combined with AT at 2% DM inhibited CH4 production. Phloroglucinol numerically decreased H2 accumulation and significantly increased total gas production (TGP), volatile fatty acid (VFA) production and the acetate:propionate ratio. In Experiment 4, phloroglucinol at increasing doses supplemented with AT at 2% DM significantly decreased H2 accumulation and the abundances of archaea, protozoa and fungi abundances, and increased TGP, total VFA production and the acetate:propionate ratio in a dose-dependent way. In conclusion, combined treatment with AT and phloroglucinol was successful to mitigate CH4 production while preventing the accumulation of H2, leading to an increase in acetate and total VFA production and therefore an improvement in rumen fermentation in goats.

Sequential interspecies interactions affect production of antimicrobial secondary metabolites in Pseudomonas protegens DTU9.1.

Soil and rhizosphere microbiomes play important roles in suppression of plant pathogens through production of antagonistic secondary metabolites, yet mechanisms that determine the strength of pathogen control are not well understood. Many Pseudomonas species are associated with soil and rhizosphere microbiomes, and their ability to suppress pathogens is well documented. Here, we investigate how interactions within the Pseudomonas genus affect their production of antimicrobial metabolites. From a biosensor-based screen, we identify P. capeferrum species as capable of modulating secondary metabolite production in P. protegens. We show that P. capeferrum alters production of pyoluteorin and 2,4-diacetylphloroglucinol (DAPG) in P. protegens via two distinct and sequential mechanisms that depends on spatial proximity of the two species. Specifically, P. capeferrum secretes a diffusible signal that induce pyoluteorin production up to 100-fold in neighboring P. protegens colonies. In contrast, the interaction results in reduced DAPG production, but only within mixed-species colonies. Additionally, we found that increased pyoluteorin production and cell lysis of P. capeferrum is required for inhibition of DAPG production, suggesting that pyoluteorin-facilitated antibiosis of P. protegens on P. capeferrum leads to release of cell-associated metabolites and subsequent inhibition of DAPG production in P. protegens. As the interaction modulates in vitro bioactivity of the species, genus-specific interactions may assist in improving efficacy of biocontrol strains and consortia.

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.

Convergent gene clusters underpin hyperforin biosynthesis in St John's wort.

The meroterpenoid hyperforin is responsible for the antidepressant activity of St John's wort extracts, but the genes controlling its biosynthesis are unknown. Using genome mining and biochemical work, we characterize two biosynthetic gene clusters (BGCs) that encode the first three steps in the biosynthesis of hyperforin precursors. The findings of syntenic and phylogenetic analyses reveal the parallel assembly of the two BGCs. The syntenous BGC in Mesua ferrea indicates that the first cluster was assembled before the divergence of the Hypericaceae and Calophyllaceae families. The assembly of the second cluster is the result of a coalescence of genomic fragments after a major duplication event. The differences between the two BGCs - in terms of gene expression, response to methyl jasmonate, substrate specificity and subcellular localization of key enzymes - suggest that the presence of the two clusters could serve to generate separate pools of precursors. The parallel assembly of two BGCs with similar compositions in a single plant species is uncommon, and our work provides insights into how and when these gene clusters form. Our discovery helps to advance our understanding of the evolution of plant specialized metabolism and its genomic organization. Additionally, our results offer a foundation from which hyperforin biosynthesis can be more fully understood, and which can be used in future metabolic engineering applications.

Phloroglucinol Promotes Fucoxanthin Synthesis by Activating the cis-Zeatin and Brassinolide Pathways in Thalassiosira pseudonana.

Phloroglucinol improves shoot formation and somatic embryogenesis in several horticultural and grain crops, but its function in microalgae remains unclear. Here, we found that sufficiently high concentrations of phloroglucinol significantly increased fucoxanthin synthesis, growth, and photosynthetic efficiency in the microalga Thalassiosira pseudonana. These results suggested that the role of phloroglucinol is conserved across higher plants and microalgae. Further analysis showed that, after phloroglucinol treatment, the contents of cis-zeatin and brassinolide in T. pseudonana increased significantly, while the contents of trans-zeatin, N6-isopentenyladenine (iP), auxin, and gibberellin were unaffected. Indeed, functional studies showed that the effects of cis-zeatin and brassinolide in T. pseudonana were similar to those of phloroglucinol. Knockout of key enzyme genes in the cis-zeatin synthesis pathway of T. pseudonana or treatment of T. pseudonana with a brassinolide synthesis inhibitor (brassinazole) significantly reduced growth and fucoxanthin content in T. pseudonana, and phloroglucinol treatment partially alleviated these inhibitory effects. However, phloroglucinol treatment was ineffective when the cis-zeatin and brassinolide pathways were simultaneously inhibited. These results suggested that the cis-zeatin and brassinolide signaling pathways are independent regulators of fucoxanthin synthesis in T. pseudonana and that phloroglucinol affects both pathways. Thus, this study not only characterizes the mechanism by which phloroglucinol promotes fucoxanthin synthesis but also demonstrates the roles of cis-zeatin and brassinolide in T. pseudonana. IMPORTANCE Here, we demonstrate that phloroglucinol, a growth promoter in higher plants, also increases growth and fucoxanthin synthesis in the microalga Thalassiosira pseudonana and therefore may have substantial practical application for industrial fucoxanthin production. Phloroglucinol treatment also induced the synthesis of cis-zeatin and brassinolide in T. pseudonana, and the cis-zeatin and brassinolide signaling pathways were implicated in the phloroglucinol-driven increases in T. pseudonana growth and fucoxanthin synthesis. Thus, our work clarified the molecular mechanism of phloroglucinol promoting the growth and fucoxanthin synthesis of Thalassiosira pseudonana and suggested that cis-zeatin and brassinolide, in addition to phloroglucinol, have potential utility as inducers of increased microalgal fucoxanthin production.

The Chirality of Aggregated Yariv Reagents Correlates with Their AGP-Binding Ability.

Yariv reagents are glycosylated triphenylazo dyes that bind to arabinogalactan proteins (AGPs), proteoglycans found in plant cell walls that are integral for plant growth and development. Yariv reagents are widely utilized as imaging, purification, and quantification tools for AGPs and represent the only small molecule probe for interrogating AGP function. The ability of Yariv reagents to bind to AGPs is dependent on the structure of the terminal glycoside on the dye. The reason for this selectivity has not been understood until the present work. Using circular dichroism spectroscopy, we show that the Yariv reagents form supramolecular aggregates with helical chirality. More significantly, the ability of the Yariv reagent to bind AGPs is correlated with this helical chirality. This finding paves the way towards developing a more detailed understanding of the nature of the Yariv-AGP complex, and the design of AGP-binding reagents with higher affinities and selectivities.

The phytochemical hyperforin triggers thermogenesis in adipose tissue via a Dlat-AMPK signaling axis to curb obesity.

Stimulation of adipose tissue thermogenesis is regarded as a promising avenue in the treatment of obesity. However, pharmacologic engagement of this process has proven difficult. Using the Connectivity Map (CMap) approach, we identified the phytochemical hyperforin (HPF) as an anti-obesity agent. We found that HPF efficiently promoted thermogenesis by stimulating AMPK and PGC-1α via a Ucp1-dependent pathway. Using LiP-SMap (limited proteolysis-mass spectrometry) combined with a microscale thermophoresis assay and molecular docking analysis, we confirmed dihydrolipoamide S-acetyltransferase (Dlat) as a direct molecular target of HPF. Ablation of Dlat significantly attenuated HPF-mediated adipose tissue browning both in vitro and in vivo. Furthermore, genome-wide association study analysis indicated that a variation in DLAT is significantly associated with obesity in humans. These findings suggest that HPF is a promising lead compound in the pursuit of a pharmacological approach to promote energy expenditure in the treatment of obesity.

A Rationale for Drug Design Provided by Co-Crystal Structure of IC261 in Complex with Tubulin.

Microtubules composed of α/ß tubulin heterodimers are an essential part of the cytoskeleton of eukaryotic cells and are widely regarded as targets for cancer chemotherapy. IC261, which is discovered as an ATP-competitive inhibitor of serine/threonine-specific casein kinase 1 (CK1), has shown its inhibitory activity on microtubule polymerization in recent studies. However, the structural information of the interaction between tubulin and IC261 is still unclear. Here, we provided a high-resolution (2.85 Å) crystal structure of tubulin and IC261 complex, revealed the intermolecular interaction between tubulin and IC261, and analyzed the structure-activity relationship (SAR). Subsequently, the structure of tubulin-IC261 complex was compared with tubulin-colchicine complex to further elucidate the novelty of IC261. Furthermore, eight optimal candidate compounds of new IC261-based microtubule inhibitors were obtained through molecular docking studies. In conclusion, the co-crystal structure of tubulin-IC261 complex paves a way for the design and development of microtubule inhibitor drugs.

Polymethylated acylphloroglucinols from Rhodomyrtus tomentosa exert acetylcholinesterase inhibitory effects.

Chemical investigation of the twigs and leaves of Rhodomyrtus tomentosa led to the isolation and structural identification of a novel polymethylated phloroglucinol meroterpenoid (PPM) featuring a 6/6/6/6 tetracyclic system, rhotomentodione F (1), five new polymethylated polycyclic phloroglucinols (PPPs) with a rare bis-furan framework, rhotomentosones A-E (2-6), and one new adduct composed of an acylphloroglucinol and two ß-triketone units, rhotomentosone F (7), as well as five known analogues (8-12). Their structures and absolute configurations were unambiguously determined by comprehensive spectroscopic data and electronic circular dichroism (ECD) calculations. All isolates were evaluated for their anti-inflammatory and acetylcholinesterase (AChE) inhibitory activities. Compound 6 displayed significant AChE inhibitory effect with an IC50 value of 8.68 µM. Further molecular docking studies of 6 revealed that the interactions with AChE residues Ser125, Glu202, and Tyr133 are crucial for AChE inhibitory activity. The current study not only enriches the chemical diversity of phloroglucinols in Myrtaceae species, but also provides potential lead compounds for the further design and development of new AChE inhibitors to treat Alzheimer's disease.

A Whole-Cell Biosensor for Detection of 2,4-Diacetylphloroglucinol (DAPG)-Producing Bacteria from Grassland Soil.

Fluorescent Pseudomonas spp. producing the antibiotic 2,4-diacetylphloroglucinol (DAPG) are ecologically important in the rhizosphere, as they can control phytopathogens and contribute to disease suppression. DAPG can also trigger a systemic resistance response in plants and stimulate root exudation and branching as well as induce plant-beneficial activities in other rhizobacteria. While studies of DAPG-producing Pseudomonas have predominantly focused on rhizosphere niches, the ecological role of DAPG as well as the distribution and dynamics of DAPG-producing bacteria remains less well understood for other environments, such as bulk soil and grassland, where the level of DAPG producers are predicted to be low. In this study, we constructed a whole-cell biosensor for detection of DAPG and DAPG-producing bacteria from environmental samples. The constructed biosensor contains a phlF response module and either lacZ or lux genes as output modules assembled on a pSEVA plasmid backbone for easy transfer to different host species and to enable easy future genetic modifications. We show that the sensor is highly specific toward DAPG, with a sensitivity in the low nanomolar range (>20 nM). This sensitivity is comparable to the DAPG levels identified in rhizosphere samples by chemical analysis. The biosensor enables guided isolation of DAPG-producing Pseudomonas Using the biosensor, we probed the same grassland soil sampling site to isolate genetically related DAPG-producing Pseudomonas kilonensis strains over a period of 12 months. Next, we used the biosensor to determine the frequency of DAPG-producing pseudomonads within three different grassland soil sites and showed that DAPG producers can constitute part of the Pseudomonas population in the range of 0.35 to 17% at these sites. Finally, we showed that the biosensor enables detection of DAPG produced by non-Pseudomonas species. Our study shows that a whole-cell biosensor for DAPG detection can facilitate isolation of bacteria that produce this important secondary metabolite and provide insight into the population dynamics of DAPG producers in natural grassland soil.IMPORTANCE The interest in bacterial biocontrol agents as biosustainable alternatives to pesticides to increase crop yields has grown. To date, we have a broad knowledge of antimicrobial compounds, such as DAPG, produced by bacteria growing in the rhizosphere surrounding plant roots. However, compared to the rhizosphere niches, the ecological role of DAPG as well as the distribution and dynamics of DAPG-producing bacteria remains less well understood for other environments, such as bulk and grassland soil. Currently, we are restricted to chemical methods with detection limits and time-consuming PCR-based and probe hybridization approaches to detect DAPG and its respective producer. In this study, we developed a whole-cell biosensor, which can circumvent the labor-intensive screening process as well as increase the sensitivity at which DAPG can be detected. This enables quantification of relative amounts of DAPG producers, which, in turn, increases our understanding of the dynamics and ecology of these producers in natural soil environments.

Investigations of changes in the arabinogalactan proteins (AGPs) structure, size and composition during the fruit ripening process.

Arabinogalactan proteins (AGPs) are ubiquitous cell wall and plasma membrane components and are characterised by extensive glycosylation and heterogeneity of their carbohydrate and protein units. The aim of the study was to evaluate the structural features of AGPs present in apple fruits at different stages of the ripening process. AGPs were extracted using the Yariv reagent and examined using SDS-PAGE, immunoblotting, FT-IR, and AFM. In situ analysis, immunofluorescence (CLSM) and immunogold-labelling (TEM), were performed. We demonstrated that AGPs were indeed present in apple fruits at the different stages of the ripening process. The changes in the amount (1.52-2.08 mg g-1), diameter (152.73-75.05 nm), molecular mass (50-250 kDa), and distribution in the cell of AGPs demonstrate their variable presence and changeable structure during the ripening process. We propose specific wavenumbers, i.e. 1265 cm-1, 1117 cm-1, and 960 cm-1, which could be assigned to AGPs. The immunofluorescence and immunogold-labelling results indicate that the JIM13 antibody is the most characteristic for AGPs in apple fruits. This study quantitatively demonstrated for the first time that AGP accumulation occurs in ripe fruits, which is supported by the highest AGPs content, the highest molecular mass, and the appearance of a specific distribution pattern at the cellular level.