The split G-quadruplex DNAzyme has emerged as a valuable tool for visual DNA detection. Here, we successfully integrated colorimetric split G-quadruplex DNAzyme assay with nucleic acid sequence-based amplification to generate a novel detection approach, allowing visual and rapid detection for the RNA of Shimen and HCLV strains of Classic Swine Fever Virus (CSFV). CSFV is a RNA virus that causes a highly contagious disease in domestic pigs and wild boar. With this method, we were able to detect as little as 10 copies/ml of CSF viral RNA within 3 h in serum samples taken from the field. No interference was encountered in the amplification and detection of Classic Swine Fever Virus in the presence of non-target RNA or DNA. Moreover, Shimen and HCLV strains of Classic Swine Fever Virus could be easily differentiated using the NASBA-DNAzyme system. These findings indicate the NASBA-DNAzyme system is a rapid and practical technique for detecting and discriminating CSFV strains and may be applied to the detection of other RNA viruses.
The geological role of microorganisms has been widely studied in the karst cave ecosystem. However, microbial interactions and ecological functions in such a dark, humid, and oligotrophic habitat have received far less attention, which is crucial to understanding cave biogeochemistry. Herein, microorganisms from weathered rock and sediment along the Heshang Cave depth were analyzed by random matrix theory-based network and Tax4Fun functional prediction. The results showed that although the cave microbial communities have spatial heterogeneity, differential habitats drove the community structure and diversity. Actinobacteria were predominant in weathered rock, whereas Proteobacteria dominated the sediment. The sediment communities presented significantly higher alpha diversities due to the relatively abundant nutrition from the outside by the intermittent stream. Consistently, microbial interactions in sediment were more complex, as visualized by more nodes and links. The abundant taxa presented more positive correlations with other community members in both of the two networks, indicating that they relied on promotion effects to adapt to the extreme environment. The keystones in weathered rock were mainly involved in the biodegradation of organic compounds, whereas the keystone Nitrospira in sediment contributed to carbon/nitrogen fixation. Collectively, these findings suggest that microbial interactions may lead to distinct taxonomic and functional communities in weathered rock and sediment in the subsurface Heshang Cave. IMPORTANCE In general, the constant physicochemical conditions and limited nutrient sources over long periods in the subsurface support a stable ecosystem in karst cave. Previous studies on cave microbial ecology were mostly focused on community composition, diversity, and the relationship with local environmental factors. There are still many unknowns about the microbial interactions and functions in such a dark environment with little human interference. Two representative habitats, including weathered rock and sediment in Heshang Cave, were selected to give an integrated insight into microbial interactions and potential functions. The cooccurrence network, especially the subnetwork, was used to characterize the cave microbial interactions in detail. We demonstrated that abundant taxa primarily relied on promotion effects rather than inhibition effects to survive in Heshang Cave. Keystone species may play important metabolic roles in sustaining ecological functions. Our study provides improved understanding of microbial interaction patterns and community ecological functions in the karst cave ecosystem.
Karst caves are usually considered as natural laboratories to study pristine microbiomes in subsurface biosphere. However, effects of the increasingly detected nitrate in underground karst ecosystem due to the acid rain impact on microbiota and their functions in subsurface karst caves have remained largely unknown. In this study, samples of weathered rocks and sediments were collected from the Chang Cave, Hubei province and subjected to high-throughput sequencing of 16S rRNA genes. The results showed that nitrate significantly impacted bacterial compositions, interactions, and functions in different habitats. Bacterial communities clustered according to their habitats with distinguished indicator groups identified for each individual habitat. Nitrate shaped the overall bacterial communities across two habitats with a contribution of 27.2%, whereas the pH and TOC, respectively, structured bacterial communities in weathered rocks and sediments. Alpha and beta diversities of bacterial communities increased with nitrate concentration in both habitats, with nitrate directly affecting alpha diversity in sediments, but indirectly on weathered rocks by lowering pH. Nitrate impacted more on bacterial communities in weathered rocks at the genus level than in sediments because more genera significantly correlated with nitrate concentration in weathered rocks. Diverse keystone taxa involved in nitrogen cycling were identified in the co-occurrence networks such as nitrate reducers, ammonium-oxidizers, and N
Bio-production of cadaverine from cheap carbon sources for synthesizing bio-based polyamides is becoming more commom. Here, a novel fermentation process for cadaverine bio-production from glucose was implemented by using a microbial consortium of two engineered Escherichia coli strains to relieve the toxic effect of cadaverine on fermentation efficiency. To achieve controllable growth of strains in the microbial consortium, two engineered E. coli strains grown separately on different carbon sources were first constructed. The strains were, an L-lysine-producing E. coli NT1004 with glucose as carbon source, and a cadaverine-producing E. coli CAD03 with glucose metabolism deficiency generated by modifying the PTSGlc system with CRISPR-Cas9 technology and inactivating cadaverine degradation pathways. Co-culturing these two engineered E. coli strains with a mixture of glucose and glycerol led to successful production of cadaverine. After optimizing cultivation conditions, a cadaverine titer of 28.5 g/L was achieved with a multi-stage constant-speed feeding strategy.
Microbial communities composed of few abundant and many rare species are widely involved in the biogeochemical cycles of elements. Yet little is known about the ecological roles of rare taxa in antimony (Sb) contaminated groundwater. Groundwater samples were collected along a Sb concentration gradient in the Xikuangshan antimony mine area and subjected to high through-put sequencing of 16S rRNA to investigate the bacterial communities. Bacterial communities were dominated by Betaproteobacteria , Gammaproteobacteria , and Alphaproteobacteria for both abundant and rare sub-communities , while rare taxa was much more complex in diversity. Multivariate analysis showed that both the abundant and rare taxa were under the stress of Sb, but the impact on rare taxa was greater. Nitrate explained a large part for the variation of the abundant sub-communities, indicating the critical role of nitrate for their activities under anoxic conditions. In contrast, bicarbonate significantly impacted rare sub-communities, suggesting their potential autotrophic characteristics. To further explore the role of rare taxa in the communities and the mechanism of affecting the community composition, a network was constructed to display the co-occurrence pattern of bacterial communities. The rare taxa contributed most of the network nodes and served as keystone species to maintain the stability of community. Abiotic factors (mainly Sb and pH) and bacterial interspecific interactions (interactions between keystone species and other bacterial groups) jointly affect the community dynamics. Functional prediction was performed to further reveal the ecological function of rare taxa in the Sb-disturbed groundwater environment. The results indicated that the rare taxa harbored much more diverse functions than the abundant counterparts. Notably, elevated Sb concentration promoted some potential autotrophic functions in rare taxa such as the oxidation of S-, N-, and Fe(II)-compounds. These results offer new insights into the roles of rare species in elemental cycles in the Sb-impacted groundwater.
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