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Identification, sorting, and sequencing of individual cells directly from in situ samples have great potential for in-depth analysis of the structure and function of microbiomes. In this work, based on an artificial intelligence (AI)-assisted object detection model for cell phenotype screening and a cross-interface contact method for single-cell exporting, we developed an automatic and index-based system called EasySort AUTO, where individual microbial cells are sorted and then packaged in a microdroplet and automatically exported in a precisely indexed, "One-Cell-One-Tube" manner. The target cell is automatically identified based on an AI-assisted object detection model and then mobilized via an optical tweezer for sorting. Then, a cross-interface contact microfluidic printing method that we developed enables the automated transfer of cells from the chip to the tube, which leads to coupling with subsequent single-cell culture or sequencing. The efficiency of the system for single-cell printing is >93%. The throughput of the system for single-cell printing is ~120 cells/h. Moreover, >80% of single cells of both yeast and Escherichia coli are culturable, suggesting the superior preservation of cell viability during sorting. Finally, AI-assisted object detection supports automated sorting of target cells with high accuracy from mixed yeast samples, which was validated by downstream single-cell proliferation assays. The automation, index maintenance, and vitality preservation of EasySort AUTO suggest its excellent application potential for single-cell sorting.

We have developed a manually curated online reference database, DANMEL (http://124.239.252.254/danmel/), that addresses the lack of accurate dissection and annotation of the genetic structures of mobile genetic elements (MGEs) with genes for drug resistance. DANMEL contains accurately annotated and genetically dissected reference MGEs covering 5 categories and 135 subcategories/subfamilies of MGEs. Further, DANMEL provides a detailed guide on how to precisely annotate MGEs. DANMEL also provides SEARCH/BLAST functions to facilitate finding reference MGEs. Overall, DANMEL will aid researchers to conduct in-depth genetic analysis of sequenced bacterial MGEs with drug-resistance genes and further facilitate a better understanding of bacterial MGEs associated with drug resistance at a genomic level.

Thermus thermophilus is an attractive species in the bioindustry due to its valuable natural products, abundant thermophilic enzymes, and promising fermentation capacities. However, efficient and versatile genome editing tools are not available for this species. In this study, we developed an efficient genome editing tool for T. thermophilus HB27 based on its endogenous type I-B, I-C, and III-A/B CRISPR-Cas systems. First, we systematically characterized the DNA interference capabilities of the different types of the native CRISPR-Cas systems in T. thermophilus HB27. We found that genomic manipulations such as gene deletion, mutation, and in situ tagging could be easily implemented by a series of genome-editing plasmids carrying an artificial self-targeting mini-CRISPR and a donor DNA responsible for the recombinant recovery. We also compared the genome editing efficiency of different CRISPR-Cas systems and the editing plasmids with donor DNAs of different lengths. Additionally, we developed a reporter gene system for T. thermophilus based on a heat-stable β-galactosidase gene TTP0042, and constructed an engineered strain with a high production capacity of superoxide dismutases by genome modification.

Methane-producing microorganisms accelerate the corrosion of iron-containing metals. Previous studies have inferred that some methanogens might directly accept electrons from Fe(0), but when this possibility was more intensively investigated, H₂ was shown to be an intermediary electron carrier between Fe(0) and methanogens. Here, we report that Methanosarcina acetivorans catalyzes direct metal-to-microbe electron transfer to support methane production. Deletion of the gene for the multiheme, outer-surface c-type cytochrome MmcA eliminated methane production from Fe(0), consistent with the key role of MmcA in other forms of extracellular electron exchange. These findings, coupled with the previous demonstration that outer-surface c-type cytochromes are also electrical contacts for electron uptake from Fe(0) by Geobacter and Shewanella species, suggest that the presence of multiheme c-type cytochromes on corrosion surfaces might be diagnostic for direct metal-to-microbe electron transfer and that interfering with cytochrome function might be a strategy to mitigate corrosion.

Although the accomplishments of microbiome engineering highlight its significance for the targeted manipulation of microbial communities, knowledge and technical gaps still limit the applications of microbiome engineering in biotechnology, especially for environmental use. Addressing the environmental challenges of refractory pollutants and fluctuating environmental conditions requires an adequate understanding of the theoretical achievements and practical applications of microbiome engineering. Here, we review recent cutting-edge studies on microbiome engineering strategies and their classical applications in bioremediation. Moreover, a framework is summarized for combining both top-down and bottom-up approaches in microbiome engineering toward improved applications. A strategy to engineer microbiomes for environmental use, which avoids the build-up of toxic intermediates that pose a risk to human health, is suggested. We anticipate that the highlighted framework and strategy will be beneficial for engineering microbiomes to address difficult environmental challenges such as degrading multiple refractory pollutants and sustain the performance of engineered microbiomes in situ with indigenous microorganisms under fluctuating conditions.

Higher biodiversity is often assumed to be a more desirable scenario for maintaining the functioning of ecosystems, but whether species-richer communities are also more disturbance-tolerant remains controversial. In this study, we investigated the bacterial communities based on 472 soil samples from 28 forests across China with associated edaphic and climatic properties. We developed two indexes (i.e., community mean tolerance breadth [CMTB] and community mean response asynchrony [CMRA]) to explore the relationship between diversity and community resistance potential. Moreover, we examined this resistance potential along the climatic and latitudinal gradients. We revealed that CMTB was significantly and negatively related to species richness, resulting from the changes in balance between relative abundances of putative specialists and generalists. In comparison, we found a unimodal relationship between CMRA and richness, suggesting that higher biodiversity might not always lead to higher community resistance. Moreover, our results showed differential local patterns along latitude. In particular, local patterns in the northern region mainly followed general relationships rather than those for the southern forests, which may be attributed to the differences in annual means and annual variations of climate conditions. Our findings highlight that the community resistance potential depends on the composition of diverse species with differential environmental tolerance and responses. This study provides a new, testable evaluation by considering tolerance breadth and response asynchrony at the community level, which will be helpful in assessing the influence of disturbance under rapid shifts in biodiversity and species composition as a result of global environmental change.

Despite the fast progress in our understanding of the complex functions of gut microbiota, it is still challenging to directly investigate the in vivo microbial activities and processes on an individual cell basis. To gain knowledge of the indigenous growth/division patterns of the diverse mouse gut bacteria with a relatively high throughput, here, we propose an integrative strategy, which combines the use of fluorescent probe labeling, confocal imaging with single-cell sorting, and sequencing. Mouse gut bacteria sequentially labeled by two fluorescent d-amino acid probes in vivo were first imaged by confocal microscopy to visualize their growth patterns, which can be unveiled by the distribution of the two fluorescence signals on each bacterium. Bacterial cells of interest on the imaging slide were then sorted using a laser ejection equipment, and the collected cells were then sequenced individually to identify their taxa. Our strategy allows integrated acquirement of the growth pattern knowledge of a variety of gut bacteria and their genomic information on a single-cell basis, which should also have great potential in studying many other complex bacterial systems.

Streptomyces is a model bacterium to study multicellular differentiation and the major reservoir for antibiotics discovery. However, the cellular-level lifecycle of Streptomyces has not been well studied due to its complexity and lack of research tools that can mimic their natural conditions. In this study, we developed a simple microfluidic chip for the cultivation and observation of the entire lifecycle of Streptomyces development from the single-cell perspective. The chip consists of channels for loading samples and supplying nutrients, microwell arrays for the seeding and growth of single spores, and air chambers beside the microwells that facilitate the development of aerial hyphae and spores. A unique feature of this chip is that each microwell is surrounded by a 1.5 µm nanogap connected to an air chamber, which provides a stabilized water-air interface. We used this chip to observe the lifecycle development of Streptomyces coelicolor and Streptomyces griseus germinated from single spores, which revealed differentiation of aerial hyphae with progeny spores at micron-scale water-air interfaces and air chambers. Finally, we demonstrated the applicability of this chip in phenotypic assays by showing that the microbial hormone A-Factor is involved in the regulatory pathways of aerial hyphae and spore formation. The microfluidic chip could become a robust tool for studying multicellular differentiation, single-spore heterogeneity, and secondary metabolism of single-spore germinated Streptomyces.