Bo Yuan

The swnR gene was cloned in the endophytic fungus Alternaria oxytropis OW 7.8 isolated from Oxytropis glabra, and the gene knockout mutant ΔswnR was first constructed in this study. Compared with A. oxytropis OW 7.8, the ΔswnR exhibited distinct morphological alterations in both colony and mycelial structure, a slower growth rate, and significant reductions in swainsonine (SW) levels, indicating that the function of the swnR gene promoted SW biosynthesis. Six differentially expressed genes (DEGs) closely associated with SW synthesis were identified by transcriptomic analysis of A. oxytropis OW 7.8 and ΔswnR, with P5CR, swnR, swnK, swnH2, and swnH1 downregulating, and sac upregulating. The expression levels of the six genes were consistent with the transcriptomic analysis results. Five differential metabolites (DEMs) closely associated with SW synthesis were identified by metabolomic analysis, with L-Lys, L-Glutamic acid, Saccharopine, and L-Proline upregulating, and L-PA downregulating. The results lay the foundation for the in-depth elucidation of molecular mechanisms and SW synthesis pathways in fungi, and are also of importance for the prevention of locoism in livestock, the control and utilization of locoweeds, and the protection and sustainable development of grassland ecosystems.

Locoweeds, including Oxytropis and Astragalus species, are globally recognized as plants containing swainsonine (SW), a neurotoxic alkaloid that induces neurological dysfunction and growth inhibition in livestock. SW is produced by endophytic fungi in plants; the pyrroline-5-carboxylate reductase (P5CR) gene is critical in the fungal SW biosynthetic pathway. In this study, a P5CR gene knockout mutant (ΔP5CR) was constructed from the endophytic fungus Alternaria oxytropis OW7.8 isolated from Oxytropis glabra. Compared to the wild-type strain (A. oxytropis OW7.8), the SW content in the ΔP5CR mycelia was significantly reduced, indicating that the P5CR gene plays a crucial role in promoting SW biosynthesis. Compared to the wild-type strain A. oxytropis OW7.8, the ΔP5CR mutant exhibited distinct morphological alterations in both colony and mycelial structures. The transcriptomic analysis of A. oxytropis OW7.8 and ΔP5CR revealed the downregulation of six genes associated with SW biosynthesis. Metabolomic profiling further demonstrated altered levels of six metabolites linked to SW synthesis. These findings provide foundational insights into the molecular mechanisms and metabolic pathways underlying SW biosynthesis in fungi. They hold significant value for future strategies to control SW in Oxytropis glabra and contribute positively to the protection and sustainable development of grassland ecosystems.

The swnN gene in the endophytic fungus Alternaria oxytropis OW 7.8 isolated from Oxytropis glabra was identified, and the gene knockout mutant ΔswnN was first constructed in this study. Compared with A. oxytropis OW 7.8, the ΔswnN mutant exhibited altered colony and mycelia morphology, slower growth rate, and no swainsonine (SW) in mycelia. SW was detected in the gene function complementation strain ΔswnN/swnN, indicating that the function of the swnN gene promoted SW biosynthesis. Six differentially expressed genes (DEGs) closely associated with SW synthesis were identified by transcriptomic analysis of A. oxytropis OW 7.8 and ΔswnN, with P5CR, swnR, swnK, swnH2, and swnH1 down-regulating, and sac up-regulating. The expression levels of the six genes were consistent with the transcriptomic analysis results. Five differential metabolites (DEMs) closely associated with SW synthesis were identified by metabolomic analysis, with L-glutamate, α-ketoglutaric acid, and L-proline up-regulating, and phosphatidic acid (PA) and 2-aminoadipic acid down-regulating. The SW biosynthetic pathways in A. oxytropis OW 7.8 were predicted and refined. The results lay the foundation for in-depth elucidation of molecular mechanisms and the SW synthesis pathway in fungi. They are also of importance for the prevention of locoism in livestock, the control and utilization of locoweeds, and the protection and sustainable development of grassland ecosystems.

A novel Gram-stain-positive bacterium, designated NB10T, was isolated from the gut of Tenebrio molitor. The isolate was rod-shaped, aerobic, non-motile and non-spore-forming. Colonies of strain NB10T were light yellow, circular and smooth. Phylogenetic analysis based on 16S rRNA gene sequence comparisons indicated that the isolate was related to the genus Leucobacter. Its closest relatives were Leucobacter holotrichiae T14T (97.8 % 16S rRNA gene sequence similarity), Leucobacter zeae CC-MF41T (97.0%) and Leucobacter salsicius M1-8T (96.4%). The DNA G+C content of strain NB10T was 68.8 mol%. The average nucleotide identity (ANI) and digital DNA–DNA hybridization values among strain NB10T and the selected Leucobacter species were ≤83.8 % (ANI-blast), 87.6 % (ANI-MUMmer) and 29.6%, which were below the recommended cutoff values for species delineation. The predominant cellular fatty acids were anteiso-C15 : 0 (39.0%), anteiso-C17 : 0 (35.5%) and iso-C16 : 0 (17.0%). The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and glycolipids. The predominant respiratory quinones were MK-11 and MK-10. The cell wall amino acids were Gly, Glu, Tr, Ala, and DAB. Based on these phylogenetic and phenotypic results, strain NB10T can be clearly distinguished from all of the recognized species of the genus Leucobacter and is considered to represent a novel species of that genus. The name Leucobacter tenebrionis sp. nov. is proposed, with the type strain NB10T (=MCCC 1K07072T=KCTC 49728T).

Aerobic anoxygenic phototrophic bacteria (AAnPB) are photoheterotrophs, which use light as auxiliary energy and require organic carbon (OC) for growth. Herein, we report the unusual strain B3, which is a true AAnPB because it requires oxygen for growth, harbours genes for cbb3 - and bd -type cytochromes and acs F, and produces bacteriochlorophyll. The B3 genome encodes the complete metabolic pathways for AAnPB light utilization, CO ₂ fixation via Calvin-Benson-Bassham (CBB) cycle and oxidation of sulfite and H ₂ , and the transcriptome indicated that all components of these pathways were fully transcribed. Expression of the marker genes related to photosynthesis, including puf M for light harnessing and rbc L for CO ₂ fixation, and the activity of RubisCO, the key enzyme in the Calvin-Benson-Bassham (CBB) cycle, increased in response to decreased OC supply. Large amounts of cell biomass were obtained in liquid BG11 medium under illumination. The strain thus likely photoautotrophically grows using sulfite or H ₂ as an electron donor. Similar GC contents between photosynthesis, the CBB cycle and 16S rRNA genes and the consistency of their phylogenetic topologies implied that light harnessing and carbon fixation genes evolved vertically from an anaerobic phototrophic ancestor of Rhodospirillaceae in Alphaproteobacteria. In conclusion, strain B3 represents a novel AAnPB characterized by photoautotrophy using the CBB cycle. This kind of AAnPB may be ecologically significant in the global carbon cycle.