Transcriptome sequencing of Saccharina japonica sporophytes during whole developmental periods reveals regulatory networks underlying alginate and mannitol biosynthesis

Background: Alginate is an important cell wall component and mannitol is a soluble storage carbon substance in the brown seaweed Saccharina japonica. Their contents vary with kelp developmental periods and harvesting time. Alginate and mannitol regulatory networks and molecular mechanisms are largely unknown. Results: With WGCNA and trend analysis of 20,940 known genes and 4,264 new genes produced from transcriptome sequencing of 30 kelp samples from different stages and tissues, we deduced that ribosomal proteins, light harvesting complex proteins and "imm upregulated 3"gene family are closely associated with the meristematic growth and kelp maturity. Moreover, 134 and 6 genes directly involved in the alginate and mannitol metabolism were identified, respectively. Mannose-6-phosphate isomerase (MPI2), phosphomannomutase (PMM1), GDP-mannose 6-dehydrogenase (GMD3) and mannuronate C5-epimerase (MC5E70 and MC5E122) are closely related with the high content of alginate in the distal blade. Mannitol accumulation in the basal blade might be ascribed to high expression of mannitol-1-phosphate dehydrogenase (M1PDH1) and mannitol-1-phosphatase (M1Pase) (in biosynthesis direction) and low expression of mannitol-2-dehydrogenase (M2DH) and Fructokinase (FK) (in degradation direction). Oxidative phosphorylation and photosynthesis provide ATP and NADH for mannitol metabolism whereas glycosylated cycle and tricarboxylic acid (TCA) cycle produce GTP for alginate biosynthesis. RNA/protein synthesis and transportation might affect alginate complex polymerization and secretion processes. Cryptochrome (CRY-DASH), xanthophyll cycle, photosynthesis and carbon fixation influence the production of intermediate metabolite of fructose-6-phosphate, contributing to high content of mannitol in the basal blade. Conclusions: The network of co-responsive DNA synthesis, repair and proteolysis are presumed to be involved in alginate polymerization and secretion, while upstream light-responsive reactions are important for mannitol accumulation in meristem of kelp. Our transcriptome analysis provides new insights into the transcriptional regulatory networks underlying the biosynthesis of alginate and mannitol during S. japonica developments. Keywords: Alginate, Mannitol, Transcriptome, Regulatory networks, Growth, Development, Saccharina japonica

Transcriptome sequencing of Saccharina japonica sporophytes during whole developmental periods reveals regulatory networks underlying alginate and mannitol biosynthesis

Background Alginate is an important cell wall component and mannitol is a soluble storage carbon substance in the brown seaweed Saccharina japonica. Their contents vary with kelp developmental periods and harvesting time. Alginate and mannitol regulatory networks and molecular mechanisms are largely unknown. Results With WGCNA and trend analysis of 20,940 known genes and 4264 new genes produced from transcriptome sequencing of 30 kelp samples from different stages and tissues, we deduced that ribosomal proteins, light harvesting complex proteins and “imm upregulated 3” gene family are closely associated with the meristematic growth and kelp maturity. Moreover, 134 and 6 genes directly involved in the alginate and mannitol metabolism were identified, respectively. Mannose-6-phosphate isomerase (MPI2), phosphomannomutase (PMM1), GDP-mannose 6-dehydrogenase (GMD3) and mannuronate C5-epimerase (MC5E70 and MC5E122) are closely related with the high content of alginate in the distal blade. Mannitol accumulation in the basal blade might be ascribed to high expression of mannitol-1-phosphate dehydrogenase (M1PDH1) and mannitol-1-phosphatase (M1Pase) (in biosynthesis direction) and low expression of mannitol-2-dehydrogenase (M2DH) and Fructokinase (FK) (in degradation direction). Oxidative phosphorylation and photosynthesis provide ATP and NADH for mannitol metabolism whereas glycosylated cycle and tricarboxylic acid (TCA) cycle produce GTP for alginate biosynthesis. RNA/protein synthesis and transportation might affect alginate complex polymerization and secretion processes. Cryptochrome (CRY-DASH), xanthophyll cycle, photosynthesis and carbon fixation influence the production of intermediate metabolite of fructose-6-phosphate, contributing to high content of mannitol in the basal blade. Conclusions The network of co-responsive DNA synthesis, repair and proteolysis are presumed to be involved in alginate polymerization and secretion, while upstream light-responsive reactions are important for mannitol accumulation in meristem of kelp. Our transcriptome analysis provides new insights into the transcriptional regulatory networks underlying the biosynthesis of alginate and mannitol during S. japonica developments.

Transcriptome sequencing of Saccharina japonica sporophytes during whole developmental periods reveals regulatory networks underlying alginate and mannitol biosynthesis

Background: Alginate is an important cell wall component and mannitol is a soluble storage carbon substance in the brown seaweed Saccharina japonica. Their contents vary with kelp developmental periods and harvesting time. Alginate and mannitol regulatory networks and molecular mechanisms are largely unknown. Results: With WGCNA and trend analysis of 20,940 known genes and 4,264 new genes produced from transcriptome sequencing of 30 kelp samples from different stages and tissues, we deduced that ribosomal proteins, light harvesting complex proteins and “imm upregulated 3” gene family are closely associated with the meristematic growth and kelp maturity. Moreover, 134 and 6 genes directly involved in the alginate and mannitol metabolism were identified, respectively. Mannose-6-phosphate isomerase (MPI2), phosphomannomutase (PMM1), GDP-mannose 6-dehydrogenase (GMD3) and mannuronate C5-epimerase (MC5E70 and MC5E122) are closely related with the high content of alginate in the distal blade. Mannitol accumulation in the basal blade might be ascribed to high expression of mannitol-1-phosphate dehydrogenase (M1PDH1) and mannitol-1-phosphatase (M1Pase) (in biosynthesis direction) and low expression of mannitol-2-dehydrogenase (M2DH) and Fructokinase (FK) (in degradation direction). Oxidative phosphorylation and photosynthesis provide ATP and NADH for mannitol metabolism whereas glycosylated cycle and tricarboxylic acid (TCA) cycle produce GTP for alginate biosynthesis. RNA/protein synthesis and transportation might affect alginate complex polymerization and secretion processes. Cryptochrome (CRY-DASH), xanthophyll cycle, photosynthesis and carbon fixation influence the production of intermediate metabolite of fructose-6-phosphate, contributing to high content of mannitol in the basal blade. Conclusions: The network of co-responsive DNA synthesis, repair and proteolysis are presumed to be involved in alginate polymerization and secretion, while upstream light-responsive reactions are important for mannitol accumulation in meristem of kelp. Our transcriptome analysis provides new insights into the transcriptional regulatory networks underlying the biosynthesis of alginate and mannitol during S. japonica developments. Keywords: Alginate, Mannitol, Transcriptome, Regulatory networks, Growth, Development, Saccharina japonica

Transcriptome sequencing of Saccharina japonica sporophytes during whole developmental periods reveals regulatory networks underlying alginate and mannitol biosynthesis

Background: Alginate is an important cell wall component and mannitol is a soluble storage carbon substance in brown seaweed Saccharina japonica. Their contents vary with kelp developmental periods and harvesting time. Alginate and mannitol regulatory networks and molecular mechanisms are largely unknown.Results: With WGCNA and trend analysis of 20,940 known genes and 4,264 new genes produced from transcriptome sequencing of 30 kelp samples from different stages and tissues, we deduced that ribosomal proteins, light harvesting complex proteins and imm upregulated 3 gene family are closely associated with the meristematic growth and kelp maturity. Moreover, 134 and 6 genes directly involved in the alginate and mannitol metabolism were identified, respectively. Mannose-6-phosphate isomerase (MPI2), phosphomannomutase (PMM1), GDP-mannose 6-dehydrogenase (GMD3) and mannuronate C5-epimerase (MC5E70 and MC5E122) are closely related with the high content of alginate in the distal blade. Mannitol accumulation in the basal blade might be ascribed to high expression of mannitol-1-phosphate dehydrogenase (M1PDH1) and mannitol-1-phosphatase (M1Pase) (in biosynthesis direction) and low expression of mannitol-2-dehydrogenase (M2DH) and fructokinase (FK) (in degradation direction). Oxidative phosphorylation and photosynthesis provide ATP and NADH for mannitol metabolism whereas glycosylated cycle and tricarboxylic acid (TCA) cycle produce GTP for alginate biosynthesis. RNA/protein synthesis and transportation might affect alginate complex polymerization and secretion processes. Cryptochrome (CRY-DASH), xanthophyll cycle, photosynthesis and carbon fixation influence the production of intermediate metabolite of fructose-6-phosphate, contributing to high content of mannitol in the basal blade. Conclusions: The network of co-responsive DNA synthesis, repair and proteolysis are presumed to be involved in alginate polymerization and secretion, while upstream light-responsive reactions are important for mannitol accumulation in meristem of kelp. Our transcriptome analysis provides new insights into the transcriptional regulatory networks underlying the biosynthesis of alginate and mannitol during S. japonica developments.Keywords: Alginate, Mannitol, Transcriptome, Regulatory networks, Growth, Development, Saccharina japonica

Targeting Mannitol Metabolism as an Alternative Antimicrobial Strategy Based on the Structure-Function Study of Mannitol-1-Phosphate Dehydrogenase inStaphylococcus aureus

ABSTRACTMannitol-1-phosphate dehydrogenase (M1PDH) is a key enzyme inStaphylococcus aureusmannitol metabolism, but its roles in pathophysiological settings have not been established. We performed comprehensive structure-function analysis of M1PDH fromS. aureusUSA300, a strain of community-associated methicillin-resistantS. aureus, to evaluate its roles in cell viability and virulence under pathophysiological conditions. On the basis of our results, we propose M1PDH as a potential antibacterial target.In vitrocell viability assessment of ΔmtlDknockout and complemented strains confirmed that M1PDH is essential to endure pH, high-salt, and oxidative stress and thus that M1PDH is required for preventing osmotic burst by regulating pressure potential imposed by mannitol. The mouse infection model also verified that M1PDH is essential for bacterial survival during infection. To further support the use of M1PDH as an antibacterial target, we identified dihydrocelastrol (DHCL) as a competitive inhibitor ofS. aureusM1PDH (SaM1PDH) and confirmed that DHCL effectively reduces bacterial cell viability during host infection. To explain physiological functions ofSaM1PDH at the atomic level, the crystal structure ofSaM1PDH was determined at 1.7-Å resolution. Structure-based mutation analyses and DHCL molecular docking to theSaM1PDH active site followed by functional assay identified key residues in the active site and provided the action mechanism of DHCL. Collectively, we proposeSaM1PDH as a target for antibiotic development based on its physiological roles with the goals of expanding the repertory of antibiotic targets to fight antimicrobial resistance and providing essential knowledge for developing potent inhibitors ofSaM1PDH based on structure-function studies.IMPORTANCEDue to the shortage of effective antibiotics against drug-resistantStaphylococcus aureus, new targets are urgently required to develop next-generation antibiotics. We investigated mannitol-1-phosphate dehydrogenase ofS. aureusUSA300 (SaM1PDH), a key enzyme regulating intracellular mannitol levels, and explored the possibility of usingSaM1PDH as a target for developing antibiotic. Since mannitol is necessary for maintaining the cellular redox and osmotic potential, the homeostatic imbalance caused by treatment with aSaM1PDH inhibitor or knockout of the gene encodingSaM1PDH results in bacterial cell death through oxidative and/or mannitol-dependent cytolysis. We elucidated the molecular mechanism ofSaM1PDH and the structural basis of substrate and inhibitor recognition by enzymatic and structural analyses ofSaM1PDH. Our results strongly support the concept that targeting ofSaM1PDH represents an alternative strategy for developing a new class of antibiotics that cause bacterial cell death not by blocking key cellular machinery but by inducing cytolysis and reducing stress tolerance through inhibition of the mannitol pathway.

Changes in Mannitol Content, Regulation of Genes Involved in Mannitol Metabolism, and the Protective Effect of Mannitol onVolvariella volvaceaat Low Temperature

The mechanism of autolysis ofVolvariella volvacea(V. volvacea) at low temperature has not been fully explained. As mannitol is among the most important osmotic adjustment substances in fungal resistance, this study sampled mycelia of strains V23 and VH3 treated at 0°C for 0, 2, 4, 8, and 10 h to analyze changes in intracellular mannitol content by high-performance anion chromatography with pulsed amperometric detection (HAPEC-PAD). Reverse transcription quantitative PCR (RT-qPCR) analysis was applied to assess differences in the transcript levels of genes associated with mannitol metabolism under low-temperature stress. A mannitol solution was added to cultures ofV. volvaceafruiting bodies, and effects on the hypothermic resistance of these organs were explored by evaluating variations in sensory properties during cryogenic storage after harvest. The results suggested that in the initial stage of low-temperature treatment, intracellular mannitol was largely catabolized as an energy storage material and the expression of genes encoding enzymes involved in synthetic reactions was inhibited. However, low-temperature resistance was induced with further treatment, with activation of mannitol synthesis and inhibition of degradation; the cells accumulated mannitol, leading to osmoregulation. No significant elongation ofV. volvaceafruiting bodies during storage at 4°C was observed, and these organs tended to shrink and collapse. The sensory quality of mannitol-treated fruiting bodies was much better than that of control fruiting bodies. Application of a mannitol solution at the cultivation stage ofV. volvaceasomewhat improved the low-temperature resistance of the fruiting bodies, verifying the correlation between mannitol and resistance to this stress inV. volvacea. The results of this study lay a foundation for a deeper understanding of the autolysis mechanism ofV. volvacea, providing technical support for increasing the cryopreservation time of this species and extending the postharvest shelf life of its fruiting bodies. In addition, the mechanism underlying the low-temperature tolerance of the VH3 strain should be further explained at the molecular level.