Two CRISPR/Cas9 Systems Developed in Thermomyces dupontii and Characterization of Key Gene Functions in Thermolide Biosynthesis and Fungal Adaptation.

Thermomyces dupontii, a widely distributed thermophilic fungus, is an ideal organism for investigating the mechanism of thermophilic fungal adaptation to diverse environments. However, genetic analysis of this fungus is hindered by a lack of available and efficient gene-manipulating tools. In this study, two different Cas9 proteins from mesophilic and thermophilic bacteria, with in vivo expression of a single guide RNA (sgRNA) under the control of tRNAGly, were successfully adapted for genome editing in T. dupontii We demonstrated the feasibility of applying these two gene editing systems to edit one or two genes in T. dupontii The mesophilic CRISPR/Cas9 system displayed higher editing efficiency (50 to 86%) than the thermophilic CRISPR/Cas9 system (40 to 67%). However, the thermophilic CRISPR/Cas9 system was much less time-consuming than the mesophilic CRISPR/Cas9 system. Combining the CRISPR/Cas9 systems with homologous recombination, a constitutive promoter was precisely knocked in to activate a silent polyketide synthase-nonribosomal peptide synthase (PKS-NRPS) biosynthetic gene, leading to the production of extra metabolites that did not exist in the parental strains. Metabolic analysis of the generated biosynthetic gene mutants suggested that a key biosynthetic pathway existed for the biosynthesis of thermolides in T. dupontii, with the last two steps being different from those in the heterologous host Aspergillus Further analysis suggested that these biosynthetic genes might be involved in fungal mycelial growth, conidiation, and spore germination, as well as in fungal adaptation to osmotic, oxidative, and cell wall-perturbing agents.IMPORTANCEThermomyces represents a unique ecological taxon in fungi, but a lack of flexible genetic tools has greatly hampered the study of gene function in this taxon. The biosynthesis of potent nematicidal thermolides in T. dupontii remains largely unknown. In this study, mesophilic and thermophilic CRISPR/Cas9 gene editing systems were successfully established for both disrupting and activating genes in T. dupontii In this study, a usable thermophilic CRISPR/Cas9 gene editing system derived from bacteria was constructed in thermophilic fungi. Chemical analysis of the mutants generated by these two gene editing systems identified the key biosynthetic genes and pathway for the biosynthesis of nematocidal thermolides in T. dupontii Phenotype analysis and chemical stress experiments revealed potential roles of secondary metabolites or their biosynthetic genes in fungal development and adaption to chemical stress conditions. These two genomic editing systems will not only accelerate investigations into the biosynthetic mechanisms of unique natural products and functions of cryptic genes in T. dupontii but also offer an example for setting up CRISPR/Cas9 systems in other thermophilic fungi.

Integrated Metabolomics and Morphogenesis Reveal Volatile Signaling of the Nematode-Trapping Fungus Arthrobotrys oligospora.

The adjustment of metabolic patterns is fundamental to fungal biology and plays vital roles in adaptation to diverse ecological challenges. Nematode-trapping fungi can switch their lifestyle from saprophytic to pathogenic by developing specific trapping devices induced by nematodes to infect their prey as a response to nutrient depletion in nature. However, the chemical identity of the specific fungal metabolites used during the switch remains poorly understood. We hypothesized that these important signal molecules might be volatile in nature. Gas chromatography-mass spectrometry was used to carry out comparative analysis of fungal metabolomics during the saprophytic and pathogenic lifestyles of the model species Arthrobotrys oligospora Two media commonly used in research on this species, cornmeal agar (CMA) and potato dextrose agar (PDA), were chosen for use in this study. The fungus produced a small group of volatile furanone and pyrone metabolites that were associated with the switch from the saprophytic to the pathogenic stage. A. oligospora fungi grown on CMA tended to produce more traps and employ attractive furanones to improve the utilization of traps, while fungi grown on PDA developed fewer traps and used nematode-toxic furanone metabolites to compensate for insufficient traps. Another volatile pyrone metabolite, maltol, was identified as a morphological regulator for enhancing trap formation. Deletion of the gene AOL_s00079g496 in A. oligospora led to increased amounts of the furanone attractant (2-fold) in mutants and enhanced the attractive activity (1.5-fold) of the fungus, while it resulted in decreased trap formation. This investigation provides new insights regarding the comprehensive tactics of fungal adaptation to environmental stress, integrating both morphological and metabolomic mechanisms.IMPORTANCE Nematode-trapping fungi are a unique group of soil-living fungi that can switch from the saprophytic to the pathogenic lifestyle once they come into contact with nematodes as a response to nutrient depletion. In this study, we investigated the metabolic response during the switch and the key types of metabolites involved in the interaction between fungi and nematodes. Our findings indicate that A. oligospora develops multiple and flexible metabolic tactics corresponding to different morphological responses to nematodes. A. oligospora can use similar volatile furanone and pyrone metabolites with different ecological functions to help capture nematodes in the fungal switch from the saprophytic to the pathogenic lifestyle. Furthermore, studies with A. oligospora mutants with increased furanone and pyrone metabolites confirmed the results. This investigation reveals the importance of volatile signaling in the comprehensive tactics used by nematode-trapping fungi, integrating both morphological and metabolomic mechanisms.

[Distribution of pathogenic bacteria in patients of injuries combined with infection after May 12 Wenchuan Earthquake and drug resistance thereof].

OBJECTIVE: To investigate the distribution of pathogenic bacteria in the patients of injuries combined with infection after May 12 Wenchuan Earthquake and the drug resistance thereof. METHODS: Bacterial culture and identification were conducted on 139 clinical specimens from 108 patients with open injuries combined with infection. Drug-sensitive test was carried out by Kirby-Bauer method on the isolated strains. RESULTS: Ninety-six stains were isolated, 79.86% of them were from the infected wound secretion and 58.33% were from three department of orthopedics. The bacteria with high isolation rates included Acinetobacter spp. (20 strains), Enterobacter cloacae (20 strains), Escherichia coli (17 strains), and Staphylococcus spp. (13 strains). The isolated Gram negative bacteria showed lower resistance to imipenem, amikacin, and cefoperazone/sulbactam. CONCLUSION: The Gram negative bacteria isolated from the wounds caused by earthquake show lower resistance to imipenem, amikacin, and cefoperazone/sulbactam. No Staphylococcus spp. is found resistant to glycopeptides.

Craniometrics Reveal "Two Layers" of Prehistoric Human Dispersal in Eastern Eurasia.

This cranio-morphometric study emphasizes a "two-layer model" for eastern Eurasian anatomically modern human (AMH) populations, based on large datasets of 89 population samples including findings directly from ancient archaeological contexts. Results suggest that an initial "first layer" of AMH had related closely to ancestral Andaman, Australian, Papuan, and Jomon groups who likely entered this region via the Southeast Asian landmass, prior to 65-50 kya. A later "second layer" shared strong cranial affinities with Siberians, implying a Northeast Asian source, evidenced by 9 kya in central China and then followed by expansions of descendant groups into Southeast Asia after 4 kya. These two populations shared limited initial exchange, and the second layer grew at a faster rate and in greater numbers, linked with contexts of farming that may have supported increased population densities. Clear dichotomization between the two layers implies a temporally deep divergence of distinct migration routes for AMH through both southern and northern Eurasia.

Author Correction: Craniometrics Reveal "Two Layers" of Prehistoric Human Dispersal in Eastern Eurasia.

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[Study of the Raman spectrum of CCl4 Fermi resonance].

Fermi resonance is a very common phenomenon in molecule vibration spectra, especially in polyatomic molecule with complex structure. Fermi resonance appears when a fundamental vibration frequency lies closely to an overtone or combination frequencies. One can observe two peaks coming from Fermi resonance and the energy transfer also occurs between the two peaks. Fermi resonance phenomenon appears in both infrared spectrum and Raman spectrum. The Raman spectrum of CCl4 was measured. The frequency separation between the two peaks in a Fermi resonance doublet was observed and the integrated intensities were calculated with the software of Origin Pro 7.5. Fermi resonance interaction was caused by the CCl4 C-Cl f-symmetrical stretching fundamental nu3 coupling. The combination frequency (nu1 + nu4) of C-Cl a1-symmetrical stretching nu1 and band C-Cl2 f-symmetrical bending nu4 was calculated. Based on Bertran's theory, Fermi coupling coefficient W was calculated and the theoretical value nu3(0) of nu3 was estimated. The present article provided good reference for better understanding of the relationship between molecular vibration frequency and molecular structure.