Polysaccharide Biosynthesis Research Articles

Candidate genes involved in biosynthesis and degradation of the main extracellular matrix polysaccharides of brown algae and their probable evolutionary history

BackgroundBrown algae belong to the Stramenopiles phylum and are phylogenetically distant from plants and other multicellular organisms. This independent evolutionary history has shaped brown algae with numerous metabolic characteristics specific to this group, including the synthesis of peculiar polysaccharides contained in their extracellular matrix (ECM). Alginates and fucose-containing sulphated polysaccharides (FCSPs), the latter including fucans, are the main components of ECMs. However, the metabolic pathways of these polysaccharides remain poorly described due to a lack of genomic data.ResultsAn extensive genomic dataset has been recently released for brown algae and their close sister species, for which we previously performed an expert annotation of key genes involved in ECM-carbohydrate metabolisms. Here we provide a deeper analysis of this set of genes using comparative genomics, phylogenetics analyses, and protein modelling. Two key gene families involved in both the synthesis and degradation of alginate were suggested to have been acquired by the common ancestor of brown algae and their closest sister species Schizocladia ischiensis. Our analysis indicates that this assumption can be extended to additional metabolic steps, and thus to the whole alginate metabolic pathway. The pathway for the biosynthesis of fucans still remains biochemically unresolved and we also investigate putative fucosyltransferase genes that may harbour a fucan synthase activity in brown algae.ConclusionsOur analysis is the first extensive survey of carbohydrate-related enzymes in brown algae, and provides a valuable resource for future research into the glycome and ECM of brown algae. The expansion of specific families related to alginate metabolism may have represented an important prerequisite for the evolution of developmental complexity in brown algae. Our analysis questions the possible occurrence of FCSPs outside brown algae, notably within their closest sister taxon and in other Stramenopiles such as diatoms. Filling this knowledge gap in the future will help determine the origin and evolutionary history of fucan synthesis in eukaryotes.

Light-Regulated Growth, Anatomical, Metabolites Biosynthesis and Transcriptional Changes in Angelica sinensis

Angelica sinensis is an alpine medicinal plant that has been widely used as a general blood tonic and gynecological indications over 2000 years, which depend on the bioactive metabolites (e.g., volatile oils, organic acids, and flavonoids). Although the accumulation of these metabolites is significantly affected by the environmental factors (e.g., altitude, temperature, and sunshine) as found in previous studies, the regulatory mechanism of different lights has not been clearly revealed. Here, growth parameters, contents of bioactive metabolites, and expression levels of related genes were examined when A. sinensis was exposed to different white-light (WL) and UV-B radiation treatments. The results showed that the differences in growth parameters (e.g., plant height, root length, and plant biomass) and leaf tissue characteristics (e.g., leaf thickness, stomatal density and shape, and chloroplast density) were observed under different light treatments. The contents of Z-ligustilide and ferulic acid elevated with the increase of WL (50 to 150 µmol·m2/s) and maximized under the combination of WL-100 and UV-B (107 µW/m2, UV-107) radiation, while the total flavonoids and polysaccharides contents, as well as in vitro antioxidant capacity, elevated with the increasing of WL and UV-B. mRNA transcripts encoding for the biosynthesis of volatile oils, ferulic acid, flavonoids, and polysaccharides were found to be differentially regulated under the different WL and UV-B treatments. These morphological, anatomical, and transcriptional changes are consistent with the elevated bioactive metabolites in A. sinensis under the combination of WL and UV-B. These findings will provide useful references for improving bioactive metabolite production via the cultivation and bioengineering of A. sinensis.

Metabolomic and transcriptomic analyses reveal the mechanism of polysaccharide and secondary metabolite biosynthesis in Bletilla striata tubers in response to shading

Polysaccharides and various secondary metabolites are the major bioactive ingredients in Bletilla striata tubers and their biosynthesis and accumulation are influenced by light intensity. However, the mechanisms underlying shading effects remain largely unknown. In the present study, we used a combined analysis of the physiology, metabolome, and transcriptome to investigate the physiological activities and bioactive component accumulation of B. striata under different shading treatments (S0, S50, S70, and S90). The dry weight of shoots and tubers, net photosynthetic rate, and polysaccharide content were highest in S50 and lowest in S90. The content of precursors (sucrose, Glucose-6P, and Mannose-6P) for polysaccharide synthesis significantly increased in S50. However, the expression levels of genes involved in starch biosynthesis decreased in S50. Several structural genes involved in secondary metabolism, including cinnamic acid 4-hydroxylase (C4H), chalcone synthase (CHS), and 1-Deoxy-D-xylulose-5-phosphate synthase (DXS), showed decreased expression in S50. However, the shading effect on the biosynthesis of secondary metabolites (phenylpropanoids, flavonoids, and terpenoids) was inconsistent. Our study provides the molecular mechanisms underlying the effects of shading on the biosynthesis of polysaccharides and secondary metabolites in B. striata and offers a theoretical basis for the artificial cultivation and industrial production of bioactive ingredients.

Pathogenic and Comparative Genomic Analysis of Ralstonia pseudosolanacearum Isolated from Casuarina.

Casuarina equisetifolia is crucial in protecting coastal regions of China against typhoon attacks but has faced a substantial challenge due to wilt disease caused by pathogens of the Ralstonia solanacearum species complex (RSSC). Although the initial outbreak of Casuarina wilt in the 1970s was effectively controlled by disease-resistant C. equisetifolia varieties, the disease has recently re-emerged in coastal regions of Guangdong. In this study, we report the isolation, characterization, and comparative genomic analysis of 11 RSSC strains from diseased C. equisetifolia at various locations along the coast of Guangdong. Phylogenomic analysis showed that the strains were closely related and clustered with phylotype I strains previously isolated from peanuts. Single-gene-based analysis further suggested these strains could be derived from strains present in Guangdong since the 1980s, indicating a historical context to their current pathogenicity. Casuarina-isolated strains exhibited notably higher virulence against C. equisetifolia and peanuts than the representative RSSC strains GMI1000 and EP1, suggesting host-specific adaptations that possibly contributed to the recent outbreak. Comparative genomic analysis among RSSC strains revealed a largely conserved genome structure and high levels of conservation in gene clusters encoding extracellular polysaccharide biosynthesis, secretion systems, and quorum sensing regulatory systems. However, we also found a number of unique genes in the Casuarina-isolated strains that were absent in GMI1000 and EP1, and vice versa, pointing to potential genetic factors underpinning their differential virulence. These unique genes offer promising targets for future functional studies. Overall, our findings provide crucial insights into the RSSC pathogens causing Casuarina wilt in Guangdong, guiding future efforts in disease control and prevention.

Insights into the Differences in Polysaccharide and Alkaloid Biosynthesis in the Medicinal Orchids Dendrobium nobile and D. officinale.

Both Dendrobium nobile and D. officinale are widely used medicinal plants in China and their major medicinal components are alkaloids and polysaccharides, respectively. It is still unclear why these two closely related orchids synthesize and accumulate different chemical components. Here, we investigated the molecular mechanisms underlying polysaccharide and alkaloid biosynthesis in D. nobile and D. officinale through transcriptome and metabolomic analysis at different growth stages. A total of 1267 metabolites were identified in the juvenile and mature stages of the two species. D. nobile accumulated a large number of alkaloids, benzenoids/phenylpropanoids, flavonoids, and terpenoids during the transition from juvenile to mature plants. In contrast, D. officinale accumulated a small number of those metabolites and an absence of flavonoids. The correlation analysis of polysaccharide contents with the differentially expressed genes suggested that the differential expression of GH1, GH3, and GH9 might be related to the difference in polysaccharide contents between the two Dendrobium species. Meanwhile, the difference in the biosynthesis of dendrobine, the main component of alkaloids in D. nobile, was involved in the differential expression of HMGCR, DXR, DXS, ISPH and eight CYP450s. These findings provided new insights into understanding the biosynthetic mechanisms of the main medicinal components in Dendrobium species.

Nitric oxide mediates positive regulation of Nostoc flagelliforme polysaccharide yield via potential S-nitrosylation of G6PDH and UGDH

Based on our previous findings that salicylic acid and jasmonic acid increased Nostoc flagelliforme polysaccharide yield by regulating intracellular nitric oxide (NO) levels, the mechanism through which NO affects polysaccharide biosynthesis in Nostoc flagelliforme was explored from the perspective of S-nitrosylation (SNO). The addition of NO donor and scavenger showed that intracellular NO had a significant positive effect on the polysaccharide yield of N. flagelliforme. To explore the mechanism, we investigated the relationship between NO levels and the activity of several key enzymes involved in polysaccharide biosynthesis, including fructose 1,6-bisphosphate aldolase (FBA), glucokinase (GK), glucose 6-phosphate dehydrogenase (G6PDH), mitochondrial isocitrate dehydrogenase (ICDH), and UDP-glucose dehydrogenase (UGDH). The enzymatic activities of G6PDH, ICDH, and UGDH were shown to be significantly correlated with the shifts in intracellular NO levels. For further validation, G6PDH, ICDH, and UGDH were heterologously expressed in Escherichia coli and purified via Ni+-NAT affinity chromatography, and subjected to a biotin switch assay and western blot analysis, which revealed that UGDH and G6PDH were susceptible to SNO. Furthermore, mass spectrometry analysis of proteins treated with S-nitrosoglutathione (GSNO) identified the SNO modification sites for UGDH and G6PDH as cysteine 423 and cysteine 249, respectively. These findings suggest that NO modulates polysaccharide biosynthesis in N. flagelliforme through SNO of UGDH and G6PDH. This reveals a potential mechanism through which NO promotes polysaccharide synthesis in N. flagelliforme, while also providing a new strategy for improving the industrial production of polysaccharides.

Co-Expression Network Analysis and Introgressive Gene Identification for Fiber Length and Strength Reveal Transcriptional Differences in 15 Cotton Chromosome Substitution Segment Lines and Their Upland and Sea Island Parents.

Fiber length (FL) and strength (FS) are the core indicators for evaluating cotton fiber quality. The corresponding stages of fiber elongation and secondary wall thickening are of great significance in determining FL and FS formation, respectively. QTL mapping and high-throughput sequencing technology have been applied to dissect the molecular mechanism of fiber development. In this study, 15 cotton chromosome segment substitution lines (CSSLs) with significant differences in FL and FS, together with their recurrent parental Gossypium hirsutum line CCRI45 and donor parent G. barbadense line Hai1, were chosen to conduct RNA-seq on developing fiber samples at 10 days post anthesis (DPA) and 20 DPA. Differentially expressed genes (DEGs) were obtained via pairwise comparisons among all 24 samples (each one with three biological repeats). A total of 969 DEGs related to FL-high, 1285 DEGs to FS-high, and 997 DEGs to FQ-high were identified. The functional enrichment analyses of them indicated that the GO terms of cell wall structure and ROS, carbohydrate, and phenylpropanoid metabolism were significantly enriched, while the GO terms of glucose and polysaccharide biosynthesis, and brassinosteroid and glycosylphosphatidylinositol metabolism could make great contributions to FL and FS formation, respectively. Weighted gene co-expressed network analyses (WGCNA) were separately conducted for analyzing FL and FS traits, and their corresponding hub DEGs were screened in significantly correlated expression modules, such as EXPA8, XTH, and HMA in the fiber elongation and WRKY, TDT, and RAC-like 2 during secondary wall thickening. An integrated analysis of these hub DEGs with previous QTL identification results successfully identified a total of 33 candidate introgressive DEGs with non-synonymous mutations between the Gh and Gb species. A common DEG encoding receptor-like protein kinase 1 was reported to likely participate in fiber secondary cell thickening regulation by brassionsteroid signaling. Such valuable information was conducive to enlightening the developing mechanism of cotton fiber and also provided an abundant gene pool for further molecular breeding.

Significant response of coral-associated bacteria and their carbohydrate-active enzymes diversity to coral bleaching

Analysis of bacterial carbohydrate-active enzymes (CAZymes) contributes significantly to comprehending the response exhibited by coral symbionts to the external environment. This study explored the impact of bleaching on the bacteria and their CAZymes in coral Favites sp. through metagenomic sequencing. Notably, principal coordinates analysis (PCoA) unveiles substantial difference in bacterial communities between bleached and unbleached corals. Proteobacteria, Actinobacteria, Acidobacteria, Bacteroidota, and Chloroflexi, exhibit noteworthy alterations during coral bleaching. CAZymes profiles in bleached coral disclosed a significant increase in Glycosyltransferases (GTs) abundance, suggesting an intensified biosynthesis of polysaccharides. Conversely, there is a marked reduction in other CAZymes abundance in bleached coral. Proteobacteria, Bacteroidota, Chlorobi, and Planctomycetota exhibit greater contributions to CAZymes in bleached corals, with Rhodobacterales, Cytophagales, Burkholderiales, Caulobacterales, and Hyphomicrobiales being the main contributors. While Acidobacteria, Actinobacteria, and Chloroflexi demonstrate higher contributions to CAZymes in unbleached corals. The changes in bacteria and their CAZymes reflect the ecological adaptability of coral holobionts when facing environmental stress. The alterations in CAZymes composition caused by bleaching events may have profound impacts on coral nutrient absorption and ecosystem stability. Therefore, understanding the dynamic changes in CAZymes is crucial for assessing the health and recovery potential of coral ecosystems.

Combined metabolomics and transcriptomics reveal the secondary metabolite networks in different growth stages of Bletilla striata (Thunb.) Reichb.f.

Bletilla striata (Thunb.) Reichb.f. (B. striata) is a traditional Chinese medicinal herb. B. striata polysaccharides (BSP), stilbenes and 2-isobutyl malic acid glucosoxy-benzyl ester compounds are the main active ingredients in B. striata. However, there is limited report on the changes of medicinal components and their biosynthesis regulation mechanisms in the tubers of B. striata at different stages. The tubers of B. striata were collected during the flowering period, fruiting period, and harvest period to determine the total polysaccharide content using the phenol sulfuric acid method. The changes in secondary metabolites in the tubers at these stages were analyzed by ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS), and transcriptomics was conducted for further exploration of their biosynthetic pathways. The BSP content gradually increases from the flowering period to the fruiting period as the tubers develop, reaching its peak, but subsequently decreases at harvest time, which may be associated with the germination of B. striata buds in later stage. A total of 294 compounds were identified in this study. Among them, a majority of the compounds, such as 2-isobutyl malate gluconoxy-benzyl ester, exhibited high content during the fruit stage, while stilbenes like coelonin, 3'-O-methylbatatasin III, and blestriarene A accumulated during the harvesting period. The transcriptome data also revealed a substantial number of differentially expressed genes at various stages, providing a partial explanation for the complex changes in metabolites. We observed a correspondence between the expression pattern of GDP-Man biosynthesis-related enzyme genes and cumulative changes in BSP. And identified a positive correlation between 9 transcription factors and genes associated with polysaccharide biosynthesis, while 5 transcription factors were positively correlated with accumulation of 2-isobutyl malate gluconoxy-benzyl ester compounds and 5 transcription factors exhibited negative correlated with stilbene accumulation. It is imperative to determine the appropriate harvesting period based on the specific requirements of different active ingredients and the accumulation patterns of their metabolites. Considering the involvement of multiple transcription factors in the biosynthesis and accumulation of its active ingredients, a comprehensive investigation into the specific regulatory mechanisms that facilitate high-quality cultivation of B. striata is imperative.

Integrative analyses of metabolome and transcriptome reveal the dynamic accumulation and regulatory network in rhizomes and fruits of Polygonatum cyrtonema Hua

BackgroundAccording to Chinese ancient books, both fruits and rhizomes of Polygonatum cyrtonema Hua have medicinal and edible values. Up to now, there is no report about the metabolite profiles and regulatory network in fruits and different year-old rhizomes of P. cyrtonema.ResultsIn this study, we performed integrative analyses of metabolome and transcriptome to reveal the dynamic accumulation and regulatory network of fruits and different year-old rhizomes in P. cyrtonema. The relative content of phenolic acids, lignans and coumarins, flavonoids and alkaloids increased with growth years, while steroids and lipids decreased with it. In addition, the relative content of nucleotides and derivatives, flavonoids, organic acids, steroids and lipids in fruits were higher than rhizomes. Genes that might relate to the biosynthesis of polysaccharides, flavonoids, triterpene saponins and alkaloids biosynthesis were further analyzed by transcriptome analysis, including sacA, GMPP, PMM, CCoAOMT, CHI, ANR, CHS, DXS, GGPS, ZEP, CYP72A219 and so on, for their expressions were positively correlated with the relative content of the metabolites. Additionally, the correlation network in sugar and aromatic amino acids metabolites were constructed to further illustrate the biosynthesis of polysaccharides, flavonoids and alkaloids in P. cyrtonema, and some transcription factors (TFs) were screened, such as C2C2, MYB, bZIP, GRAS and NAC.ConclusionsThis study can deepen our understanding of the accumulation patterns and molecular mechanism of the main compounds in P. cyrtonema, and provide reference for the standardize production of P. cyrtonema.

Roles of α-1,3-glucosyltransferase in growth and polysaccharides biosynthesis of Ganoderma lucidum

Ganoderma lucidum polysaccharides are valuable natural compounds possessing significant biological activity, with glycosyltransferases playing a crucial role in their biosynthesis. Although the function of β-1,3-glucosyltransferase in polysaccharides production is well understood, the role of α-1,3-glucosyltransferase in edible fungi remains unclear. In this study, over-expression of the α-1,3-glucosyltransferase gene in G. lucidum (glagt) was found to suppress the growth, with the maximum biomass and mycelial growth rate decreasing by 21.78 % and 79.61 %, respectively, a behavior distinct from β-1,3-glucosyltransferase. The fungal pellet diameter decreased by 38 % and the cell-wall thickness by 32.44 %, whereas intracellular and extracellular polysaccharides production increased by 27.58 % and 66.08 %, respectively. In the transcription level, overexpressing the glagt gene i) downregulated the citrate synthase and isocitrate dehydrogenase gene in the TCA cycle, disrupting energy metabolism and fungal growth; ii) upregulated key enzymes involved in UDP-glucose synthesis and glycosyltransferases (gl24465, gl24971, and gl22535); and iii) universally increased the transcriptional level of glucosidases gl21451, gl30087, and gl24581 by 22 %–397 %, contributing to cell-wall thinning to facilitate polysaccharides export. Conversely, the glagt gene downregulation promoted G. lucidum growth and decreased polysaccharides production. The results elucidate the roles of GLAGT and are expected to inspire in-depth exploration of polysaccharides biosynthesis pathways.

In Vivo Proximity Cross-Linking and Immunoprecipitation of Cell Wall Epitopes Identify Proteins Associated with the Biosynthesis of Matrix Polysaccharides.

Identification of proteins involved in cell wall matrix polysaccharide biosynthesis is crucial to understand plant cell wall biology. We utilized in vivo cross-linking and immunoprecipitation with cell wall antibodies that recognized xyloglucan, xylan, mannan, and homogalacturonan to capture proteins associated with matrix polysaccharides in Arabidopsis protoplasts. The use of cross-linkers allowed us to capture proteins actively associated with cell wall polymers, including those directly interacting with glycans via glycan-protein (GP) cross-linkers and those associated with proteins linked to glycans via a protein-protein (PP) cross-linker. Immunoprecipitations led to the identification of 65 Arabidopsis protein IDs localized in the Golgi, ER, plasma membrane, and others without subcellular localization data. Among these, we found several glycosyltransferases directly involved in polysaccharide synthesis, along with proteins related to cell wall modification and vesicle trafficking. Protein interaction networks from DeepAraPPI and AtMAD databases showed interactions between various IDs, including those related to cell-wall-associated proteins and membrane/vesicle trafficking proteins. Gene expression and coexpression analyses supported the presence and relevance of the proteins to the cell wall processes. Reverse genetic studies using T-DNA insertion mutants of selected proteins revealed changes in cell wall composition and saccharification, further supporting their potential roles in cell wall biosynthesis. Overall, our approach represents a novel approach for studying cell wall polysaccharide biosynthesis and associated proteins, providing advantages over traditional immunoprecipitation techniques. This study provides a list of putative proteins associated with different matrix polysaccharides for further investigation and highlights the complexity of cell wall biosynthesis and trafficking within plant cells.

The GE296_RS03820 and GE296_RS03830 genes are involved in capsular polysaccharide biosynthesis in Riemerella anatipestifer.

Riemerella anatipestifer is a pathogenic bacterium that causes duck serositis and meningitis, leading to significant harm to the duck industry. To escape from the host immune system, the meningitis-causing bacteria must survive and multiply in the bloodstream, relying on specific virulence factors such as capsules. Therefore, it is essential to study the genes involved in capsule biosynthesis in R. anatipestifer. In this study, we successfully constructed gene deletion mutants Δ3820 and Δ3830, targeting the GE296_RS03820 and GE296_RS03830 genes, respectively, using the RA-LZ01 strain as the parental strain. The growth kinetics analysis revealed that these two genes contribute to bacterial growth. Transmission and scanning electron microscopy (TEM and SEM) and silver staining showed that Δ3820 and Δ3830 produced the altered capsules and compounds of capsular polysaccharides (CPSs). Serum resistance test showed the mutants also exhibited reduced C3b deposition and decreased resistance serum killing. Invivo, Δ3820 and Δ3830 exhibited markedly declining capacity to cross the blood-brain barrier, compared to RA-LZ01. These findings indicate that the GE296_RS03820 and GE296_RS03830 genes are involved in CPSs biosynthesis and play a key role in the pathogenicity of R. anatipestifer. Furthermore, Δ3820 and Δ3830 mutants presented a tendency toward higher survival rates from RA-LZ01 challenge invivo. Additionally, sera from ducklings immunized with the mutants showed cross-immunoreactivity with different serotypes of R. anatipestifer, including 1, 2, 7 and 10. Western blot and SDS-PAGE assays revealed that the altered CPSs of Δ3820 and Δ3830 resulted in the exposure of some conserved proteins playing the key role in the cross-immunoreactivity. Our study clearly demonstrated that the GE296_RS03820 and GE296_RS03830 genes are involved in CPS biosynthesis in R. anatipestifer and the capsule is a target for attenuation in vaccine development.

Clonality Analysis of Streptococcus pneumoniae in Clinical Specimens

Pneumococcal pneumonia is a significant cause of illness and death globally, particularly among young children and the elderly. The cpsB gene is involved in the biosynthesis of the capsule polysaccharide, and polymorphisms in the cpsB gene are the basis for sequetyping, a molecular biology-based approach to serotyping. In this study, we attempted the sequetyping of pneumococci directly from clinical sputum specimens collected from adult patients diagnosed with community-acquired pneumonia (CAP). We performed conventional PCR for the cpsB gene, followed by TA cloning and Sanger sequencing of the amplicon. The results showed the status of clonality of pneumococci in each specimen. We also performed real-time PCR targeting pneumococci for each specimen. It revealed a significant association between the Ct value of the real-time PCR and the clonality status of pneumococci among the specimens (p-value 0.0007 by Fisher’s exact test analysis). Specifically, when the Ct value was below 22, there was a high probability that pneumococcus existed as a single clone. Thus, this study demonstrates the possible correlation between pneumococcal clonality and bacterial load in clinical specimens, which might indicate the infection status.

PHOTOREGULATION OF THE BIOSYNTHETIC ACTIVITY OF LARICIFOMES OFFICINALIS USING COLLOIDAL SOLUTIONS OF METAL NANOPARTICLES AND LASER IRRADIATION

The aim of the work was to study the influence of biogenic metal nanoparticles on the growth characteristics and biosynthetic activity of the fungus Laricifomes officinalis, as well as the effects of photocatalytic activity of NPs after exposure to low-intensity laser radiation under deep cultivation conditions. Material and Methods. Traditional mycological methods, colloidal solutions of nanoparticles biogenic metals, and unique photobiological methods were used. Results. Treatment of the inoculum with colloidal solutions of nanoparticles of all used metals ncreased the growth of L. officinalis by 31–54%, while irradiation of the fungal inoculum with laser light in a medium with nanoparticles reduced the growth activity of the L. officinalis mycelium by 14.4–22.6%. All nanoparticles suppressed the biosynthesis of extracellular polysaccharides, whereas treatment of the inoculum with colloidal solutions of FeNPs and MgNPs stimulated the synthesis of endopolysaccharides. At the same time, laser light irradiation in the presence of AgNPs increased the amount of endopolysaccharides, while FeNPs and MgNPs slightly inhibited their synthesis. Treatment of the inoculum with colloidal metal solutions and laser light affected the total phenolic content (TPC) in the mycelial mass. The highest TPC values in ethanol extracts with AgNPs and laser light irradiation were 97.31Ѓ}3.7 mg of GAEs/g of dry mass. Conclusions. The research results gave ground to consider nanoparticles of biogenic metals (AgNPs, FeNPs, MgNPs) and low-intensity laser light as a promising regulators of the biosynthetic activity of L. officinalis in the biotechnology of its cultivation.

Integrated multi-omic analysis reveals the carbon metabolism-mediated regulation of polysaccharide biosynthesis by suitable light intensity in Bletilla striata leaves

Bletilla striata, valued for its medicinal and ornamental properties, remains largely unexplored in terms of how light intensity affects its physiology, biochemistry, and polysaccharide formation. In this 5-month study, B. striata plants were exposed to three different light intensities: low light (LL) (5–20 μmol m−2·s−1), middle light (ML) (200 μmol m−2·s−1), and high light (HL) (400 μmol m−2·s−1). The comprehensive assessment included growth, photosynthetic apparatus, chlorophyll fluorescence electron transport, and analysis of differential metabolites based on the transcriptome and metabolome data. The results indicated that ML resulted in the highest plant height and total polysaccharide content, enhanced photosynthetic apparatus performance and light energy utilization, and stimulated carbon metabolism and carbohydrate accumulation. HL reduced Chl content and photosynthetic apparatus functionality, disrupted OEC activity and electron transfer, stimulated carbon metabolism and starch and glucose accumulation, and hindered energy metabolism related to carbohydrate degradation and oxidation. In contrast, LL facilitated leaf growth and increased chlorophyll content but decreased plant height and total polysaccharide content, compromised the photosynthetic apparatus, hampered light energy utilization, stimulated energy metabolism related to carbohydrate degradation and oxidation, and inhibited carbon metabolism and carbohydrate synthesis. Numerous genes in carbon metabolism were strongly related to polysaccharide metabolites. The katE and cysK genes in carbon metabolism were strongly related not only to polysaccharide metabolites, but also to genes involved in polysaccharide biosynthesis. Our results highlight that light intensity plays a crucial role in affecting polysaccharide biosynthesis in B. striata, with carbon metabolism acting as a mediator under suitable light intensity conditions.

Integration of transcriptomics and metabolomics to reveal the mechanism of allyl isothiocyanate delaying postharvest quality deterioration of Coprinus comatus

The regulatory mechanism underlying allyl isothiocyanate (AITC)-mediated quality maintenance in postharvest Coprinus comatus fruiting bodies remains unclear. Herein, transcriptomics and metabolomics were applied to investigate the potential protective mechanism of AITC. Metabolomics analysis revealed that 189 differentially accumulated metabolites were identified and AITC significantly regulated the metabolic pathways (ko01100). Transcriptomics data demonstrated that a total of 2484, 2990 and 437 differentially expressed gens (DEGs) were determined in CON12 vs CON0, AITC12 vs CON0 and AITC12 vs CON12, respectively. The expression of 66 DEGs was markedly reversed by AITC. KEGG analysis indicated that these DEGs were mainly involved in galactose metabolism (ko00052) and sphingolipid metabolism (ko00600), which was also highlighted by the omics integrated analysis. In detail, AITC enhanced the biosynthesis of cell wall polysaccharides via up-regulation of aldose 1-epimerase and UTP-glucose-1-phosphate uridylyltransferase, as well as suppressed the degradation of glucan via down-regulation of alpha-galactosidase. Meanwhile, AITC evidently hindered the excessive accumulation of cytotoxic intermediates in sphingolipid metabolism and reduced the generation of ROS mainly through down-regulating the key rate-limiting enzyme, serine palmitoyltransferase. Overall, AITC effectively maintained cell wall integrity and mitigated oxidative damage on mushroom cells. In addition, the expression of 24 transcription factors was significantly regulated by AITC, and they were mainly from GATA, GRAS, NAC and C2H2 families. These findings indicated that AITC is a promising preservative for alleviating quality deterioration of postharvest edible mushrooms.

Nano-sized polystyrene and magnetite collectively promote biofilm stability and resistance due to enhanced oxidative stress response

Despite the growing prevalence of nanoplastics in drinking water distribution systems, the collective influence of nanoplastics and background nanoparticles on biofilm formation and microbial risks remains largely unexplored. Here, we demonstrate that nano-sized polystyrene modified with carboxyl groups (nPS) and background magnetite (nFe3O4) nanoparticles at environmentally relevant concentrations can collectively stimulate biofilm formation and prompt antibiotic resistance. Combined exposure of nPS and nFe3O4 by P. aeruginosa biofilm cells stimulated intracellular reactive oxidative species (ROS) production more significantly compared with individual exposure. The resultant upregulation of quorum sensing (QS) and c-di-GMP signaling pathways enhanced the biosynthesis of polysaccharides by 50 %− 66 % and increased biofilm biomass by 36 %− 40 % relative to unexposed control. Consistently, biofilm mechanical stability (measured as Young’s modulus) increased by 7.2–9.1 folds, and chemical stress resistance (measured with chlorine disinfection) increased by 1.4–2.0 folds. For P. aeruginosa, the minimal inhibitory concentration of different antibiotics also increased by 1.1–2.5 folds after combined exposure. Moreover, at a microbial community-wide level, metagenomic analysis revealed that the combined exposure enhanced the multi-species biofilm’s resistance to chlorine, enriched the opportunistic pathogenic bacteria, and promoted their virulence and antibiotic resistance. Overall, the enhanced formation of biofilms (that may harbor opportunistic pathogens) by nanoplastics and background nanoparticles is an overlooked phenomenon, which may jeopardize the microbial safety of drinking water distribution systems.

Antibiofilm activity of d-limonene against spoilage Bacillus amyloliquefaciens

This study aimed to determine the efficacy of d-limonene in preventing biofilm formation by Bacillus amyloliquefaciens, a soy food spoilage bacterium. The minimal biofilm inhibitory concentrations (MBICs) of d-limonene against two strains of B. amyloliquefaciens were determined using a twofold dilution method. Furthermore, the ability of d-limonene to eradicate preformed biofilms was evaluated using the TTC reduction method. Subsequent analyses investigated the effects of d-limonene on biofilm morphology, cell motility, cell surface characteristics, and the composition of the biofilm matrix. The MBICs of d-limonene for B. amyloliquefaciens strains DY1a and DY1b were determined to be 24 and 36 μL/mL, respectively. At 2 MBIC, d-limonene eradicated 63.67% of the DY1a biofilms and 52.63% of the DY1b biofilms. Significantly, d-limonene markedly reduced swimming and swarming motility at concentrations ranging from 1/2 to 1 MBIC. In particular, at 1/2 MBIC, d-limonene decreased swimming and swarming abilities by 85.88% and 37.84%, respectively, for strain DY1a and by 75.00% and 95.07%, respectively, for strain DY1b. Furthermore, d-limonene suppressed the biosynthesis of polysaccharides and proteins in the extracellular biofilm matrix and reduced cell surface hydrophobicity, surface charge, and autoaggregation. These findings suggest that d-limonene could be an effective antibiofilm agent for the control of B. amyloliquefaciens in the soybean food industry.

Connection between protein-tyrosine kinase inhibition and coping with oxidative stress in Bacillus subtilis

In bacteria, attenuation of protein-tyrosine phosphorylation occurs during oxidative stress. The main described mechanism behind this effect is the H2O2-triggered conversion of bacterial phospho-tyrosines to protein-bound 3,4-dihydroxyphenylalanine. This disrupts the bacterial tyrosine phosphorylation-based signaling network, which alters the bacterial polysaccharide biosynthesis. Herein, we report an alternative mechanism, in which oxidative stress leads to a direct inhibition of bacterial protein-tyrosine kinases (BY-kinases). We show that DefA, a minor peptide deformylase, inhibits the activity of BY-kinase PtkA when Bacillus subtilis is exposed to oxidative stress. High levels of PtkA activity are known to destabilize B. subtilis pellicle formation, which leads to higher sensitivity to oxidative stress. Interaction with DefA inhibits both PtkA autophosphorylation and phosphorylation of its substrate Ugd, which is involved in exopolysaccharide formation. Inactivation of defA drastically reduces the capacity of B. subtilis to cope with oxidative stress, but it does not affect the major oxidative stress regulons PerR, OhrR, and Spx, indicating that PtkA inhibition is the main pathway for DefA involvement in this stress response. Structural analysis identified DefA residues Asn95, Tyr150, and Glu152 as essential for interaction with PtkA. Inhibition of PtkA depends also on the presence of a C-terminal α-helix of DefA, which resembles PtkA-interacting motifs from known PtkA activators, TkmA, SalA, and MinD. Loss of either the key interacting residues or the inhibitory helix of DefA abolishes inhibition of PtkA in vitro and impairs postoxidative stress recovery in vivo, confirming the involvement of these structural features in the proposed mechanism.