High-Throughput Cultivation for the Selective Isolation of Acidobacteria From Termite Nests.
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ABSTRACT:
SUBMITTER: Oberpaul M
PROVIDER: S-EPMC7677567 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The termite nest is one of the architectural wonders of the living world, built by the collective action of workers in a colony. Each nest has several characteristic structural motifs that allow for efficient ventilation, cooling, and traversal. We use tomography to quantify the nest architecture of the African termite Apicotermes lamani, consisting of regularly spaced floors connected by scattered linear and helicoidal ramps. To understand how these elaborate structures are built and arranged, we formulate a minimal model for the spatiotemporal evolution of three hydrodynamic fields-mud, termites, and pheromones-linking environmental physics to collective building behavior using simple local rules based on experimental observations. We find that floors and ramps emerge as solutions of the governing equations, with statistics consistent with observations of A. lamani nests. Our study demonstrates how a local self-reinforcing biotectonic scheme is capable of generating an architecture that is simultaneously adaptable and functional, and likely to be relevant for a range of other animal-built structures.
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Project description:BackgroundMiniaturization of biochemical reaction volumes within artificial microcompartments has been the key driver for directed evolution of several catalysts in the past two decades. Typically, single cells are co-compartmentalized within water-in-oil emulsion droplets with a fluorogenic substrate whose conversion allows identification of catalysts with improved performance. However, emulsion droplet-based technologies prevent cell proliferation to high density and preclude the feasibility of biochemical reactions that require the exchange of small molecule substrates. Here, we report on the development of a high-throughput screening method that addresses these shortcomings and that relies on a novel selective permeable polymer hydrogel microcapsule.ResultsHollow-core polyelectrolyte-coated chitosan alginate microcapsules (HC-PCAMs) with selective permeability were successfully constructed by jet break-up and layer-by-layer (LBL) technology. We showed that HC-PCAMs serve as miniaturized vessels for single cell encapsulation, enabling cell growth to high density and cell lysis to generate monoclonal cell lysate compartments suitable for high-throughput analysis using a large particle sorter (COPAS). The feasibility of using HC-PCAMs as reaction compartments which exchange small molecule substrates was demonstrated using the transpeptidation reaction catalyzed by the bond-forming enzyme sortase F from P. acnes. The polyelectrolyte shell surrounding microcapsules allowed a fluorescently labelled peptide substrate to enter the microcapsule and take part in the transpeptidation reaction catalyzed by the intracellularly expressed sortase enzyme retained within the capsule upon cell lysis. The specific retention of fluorescent transpeptidation products inside microcapsules enabled the sortase activity to be linked with a fluorescent readout and allowed clear separation of microcapsules expressing the wild type SrtF from those expressing the inactive variant.ConclusionA novel polymer hydrogel microcapsule-based method, which allows for high-throughput analysis based on encapsulation of single cells has been developed. The method has been validated for the transpeptidation activity of sortase enzymes and represents a powerful tool for screening of libraries of sortases, other bond-forming enzymes, as well as of binding affinities in directed evolution experiments. Moreover, selective permeable microcapsules encapsulating microcolonies provide a new and efficient means for preparing novel caged biocatalyst and biosensor agents.
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Project description:Fungus-growing termite (FGT) nests possess an oxalate pool derived from termite input and fungal oxalogenesis. The effect of oxalate biotransformation in the termite nest on the symbiotic association between FGTs and Termitomyces fungi is poorly understood. Here, we measured the pH value, mineral composition, oxalate and carbonate contents, along with the abundance and composition of oxalotrophic bacteria (OxB) in termite nests. The results showed the community structures of OxB in different parts of the termite nest across fungus comb, termite nest wall and surface soil, were significantly different. The diversity of OxB in the fungus comb was significantly lower than that in the termite nest wall and surface soil. Results also showed the abundance of OxB in the fungus comb was higher than that in the termite nest wall and significantly lower than that in the surface soil. In addition, we isolated and screened an oxalotrophic bacterium Methylobacterium sp. TA1 from the fungus comb, which can degrade calcium oxalate and convert it into calcite. Our results from the perspective of oxalate biodegradation and transformation show that the oxalate-carbonate pathway driven by OxB in active termite nests can maintain stable microecological environments in termite nests and is beneficial to the symbiotic association between FGTs and Termitomyces.
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Project description:The emergence of bacterial pathogens that are resistant to clinical antibiotics poses an increasing risk to human health. An important reservoir from which bacterial pathogens can acquire resistance is the human gut microbiota. However, thus far, a substantial fraction of the gut microbiota remains uncultivated and has been little-studied with respect to its resistance reservoir-function. Here, we aimed to isolate yet uncultivated resistant gut bacteria by a targeted approach. Therefore, faecal samples from 20 intensive care patients who had received the prophylactic antibiotic treatment selective digestive decontamination (SDD), i.e. tobramycin, polymyxin E, amphotericin B and cefotaxime, were inoculated anaerobically on porous aluminium oxide chips placed on top of poor and rich agar media, including media supplemented with the SDD antibiotics. Biomass growing on the chips was analysed by 16S rRNA gene amplicon sequencing, showing large inter-individual differences in bacterial cultivability, and enrichment of a range of taxonomically diverse operational taxonomic units (OTUs). Furthermore, growth of Ruminococcaceae (2 OTUs), Enterobacteriaceae (6 OTUs) and Lachnospiraceae (4 OTUs) was significantly inhibited by the SDD antibiotics. Strains belonging to 16 OTUs were candidates for cultivation to pure culture as they shared ?95% sequence identity with the closest type strain and had a relative abundance of ?2%. Six of these OTUs were detected on media containing SDD antibiotics, and as such were prime candidates to be studied regarding antibiotic resistance. One of these six OTUs was obtained in pure culture using targeted isolation. This novel strain was resistant to the antibiotics metrodinazole and imipenem. It was initially classified as member of the Ruminococcaceae, though later it was found to share 99% nucleotide identity with the recently published Sellimonas intestinalis BR72T. In conclusion, we show that high-throughput cultivation-based screening of microbial communities can guide targeted isolation of bacteria that serve as reservoirs of antibiotic resistance.
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Project description:This paper describes the microfluidic streak plate (MSP), a facile method for high-throughput microbial cell separation and cultivation in nanoliter sessile droplets. The MSP method builds upon the conventional streak plate technique by using microfluidic devices to generate nanoliter droplets that can be streaked manually or robotically onto petri dishes prefilled with carrier oil for cultivation of single cells. In addition, chemical gradients could be encoded in the droplet array for comprehensive dose-response analysis. The MSP method was validated by using single-cell isolation of Escherichia coli and antimicrobial susceptibility testing of Pseudomonas aeruginosa PAO1. The robustness of the MSP work flow was demonstrated by cultivating a soil community that degrades polycyclic aromatic hydrocarbons. Cultivation in droplets enabled detection of the richest species diversity with better coverage of rare species. Moreover, isolation and cultivation of bacterial strains by MSP led to the discovery of several species with high degradation efficiency, including four Mycobacterium isolates and a previously unknown fluoranthene-degrading Blastococcus species.
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Project description:A total of 118 actinobacterial isolates were collected from the three types of termite nests (mound, carton, and subterranean nests) to evaluate their potential as a source of bioactive actinobacteria with antimicrobial activity. The highest number (67 isolates) and generic abundance (7 known genera) of actinobacterial isolates were obtained from carton nests. Streptomyces was the dominant genus in each type of termite nest. In the non-Streptomyces group, Nocardia was the dominant genus detected in mound and carton nests, while Pseudonocardia was the dominant genus in subterranean nests. A discovery trend of novel species (<99% similarity in the 16S rRNA gene sequence) was also observed in the termite nests examined. Each type of termite nest housed >20% of bioactive actinobacteria that could inhibit the growth of at least one test organism, while 12 isolates, belonging to the genera Streptomyces, Amycolatopsis, Pseudonocardia, Micromonospora and Nocardia, exhibited distinct antimicrobial activities. Streptomyces sp. CMU-NKS-3 was the most distinct bioactive isolate. It was closely related to S. padanus MITKK-103T, which was confirmed by 99% similarities in their 16S rRNA gene sequences. The highest level of extracellular antimicrobial substances was produced by the isolate CMU-NKS-3, which was grown in potato dextrose broth and exhibited a wide range (6.10×10(-4)-1.25 mg mL(-1)) of minimum inhibitory concentrations against diverse pathogens. We concluded that termite nests are an abundant source of bioactive strains of cultivable actinobacteria for future biotechnological needs.
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Project description:Termite nests have been widely studied as effective examples for ventilation and thermoregulation. However, the mechanisms by which these properties are controlled by the microstructure of the outer walls remain unclear. Here, we combine multiscale X-ray imaging with three-dimensional flow field simulations to investigate the impact of the architectural design of nest walls on CO2 exchange, heat transport and water drainage. We show that termites build outer walls that contain both small and percolating large pores at the microscale. The network of larger microscale pores enhances permeability by one to two orders of magnitude compared to the smaller pores alone, and it increases CO2 diffusivity up to eight times. In addition, the pore network offers enhanced thermal insulation and allows quick drainage of rainwater, thereby restoring the ventilation and providing structural stability to the wet nest.
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Project description:Cell-based therapies are emerging as the next frontier of medicine, offering a plausible path forward in the treatment of many devastating diseases. Critically, current methods for antigen positive cell sorting lack a high throughput method for delivering ultrahigh purity populations, prohibiting the application of some cell-based therapies to widespread diseases. Here we show the first use of targeted, protective polymer coatings on cells for the high speed enrichment of cells. Individual, antigen-positive cells are coated with a biocompatible hydrogel which protects the cells from a surfactant solution, while uncoated cells are immediately lysed. After lysis, the polymer coating is removed through orthogonal photochemistry, and the isolate has >50% yield of viable cells and these cells proliferate at rates comparable to control cells. Minority cell populations are enriched from erythrocyte-depleted blood to >99% purity, whereas the entire batch process requires 1 h and <$2000 in equipment. Batch scale-up is only contingent on irradiation area for the coating photopolymerization, as surfactant-based lysis can be easily achieved on any scale.
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