Type IV pili trigger episymbiotic association of Saccharibacteria with its bacterial host.

Recent characterization of the obligate episymbiont Saccharibacteria (TM7) belonging to the candidate phyla radiation (CPR) has expanded the extent of microbial diversity. However, the episymbiotic lifestyle of TM7 is still underexploited due to the deficiency of cultivated representatives. Here, we describe gene-targeted TM7 cultivation guided by repurposing epicPCR (emulsion, paired isolation, and concatenation PCR) to capture in situ TM7‒host associations. Using this method, we obtained a novel Saccharibacteria isolate TM7i and its host Leucobacter aridicollis J1 from Cicadae Periostracum, the castoff shell of cicada. Genomic analyses and microscopic characterizations revealed that TM7i could bind to J1 through twitching-like motility mediated by type IV pili (T4P). We further showed that the inhibition of T4P extrusion suppressed the motility and host adherence of TM7i, resulting in its reduced growth. However, the inactivation of T4P had little effect on the growth of TM7i that had already adhered to J1, suggesting the essential role of T4P in host recognition by TM7i. By capturing CPR‒host association and elaborating the T4P-dependent episymbiotic association mechanism, our studies shed light on the distinct yet widespread lifestyle of CPR bacteria.

µMET: A Novel Reusable Microfluidic Chip for Precision Microbial Enumeration Tests.

This work describes µMET, a novel microfluidic device for precise microbial enumeration tests (MET), essential in pharmaceutical, cosmetic, and food industries for ensuring microbiological safety standards. The µMET chip, comprising two hydrophobic glass plates, features a 15-µm deep µMET chamber enhanced by nanopillars and air supply units, facilitating both immediate and growth-dependent MET. Experimental results, with E. coli as a model bacterium, demonstrate that µMET provides counting linearity that outperforms traditional hemocytometers. The chip's design mitigates challenges like evaporation and ensures high-resolution imaging, making it a cost-effective and reusable alternative to conventional methods. Notably, bright-field µMET eliminates the need for fluorescent staining, streamlining operations with deep-learning algorithms for bacterial counts. Furthermore, we have developed a high-parallel µMET chip featuring 16 counting chambers, enhancing throughput and accommodating immediate and growth-dependent MET approaches. Its innovative design and adaptability render the µMET chip as a valuable tool for microbiology, medicine, and industry applications.

Tunable and Contamination-Free Injection with Microfluidics by Stepinjection.

Droplet microfluidics with picoinjection provides significant advantages to multistep reactions and screenings. The T-junction design for picoinjection is convenient in adding picoliter reagents into passing droplets to initiate reactions. However, conventional picoinjectors face difficulties in eliminating cross-contamination between droplets, preventing them from widespread use in sensitive biological and molecular assays. Here, we introduce stepinjection, which uses a T-junction with a stepped channel design to elevate the diffusional buffer zone into the main channel and consequently increases the pressure difference between droplets and the inlet of the injection channel. To demonstrate the stepinjector's ability to perform contamination-sensitive enzymatic assays, we inject casein fluorescein isothiocyanate (FITC-casein) into a mixture of savinase and savinase-free (labeled with a red fluorescent dye) droplets. We observe no cross-contamination using stepinjection but find a severe cross-talk using an optimal picoinjection design. We envision that the simple, tunable, and reliable stepinjector can be easily integrated in various droplet processing devices, and facilitate various biomedical and biochemical applications including multiplex digital PCR, single-cell sequencing, and enzymatic screening.

High-throughput single-cell cultivation reveals the underexplored rare biosphere in deep-sea sediments along the Southwest Indian Ridge.

Microorganisms in the deep sea play vital roles in marine ecosystems. However, despite great advances brought by high throughput sequencing and metagenomics, only a small portion of microorganisms living in the environment can be cultivated in the laboratory and systematically studied. In this study, an improved high-throughput microfluidic streak plate (MSP) platform was developed to speed up the isolation of microorganisms from deep-sea sediments and evaluated with deep-sea sediments collected from the Southwest Indian Ridge (SWIR). Based on our previously reported MSP method, we improved its isolation efficiency with a semi-automated droplet picker and improved humidity control to enable long-term cultivation with a low-nutrient medium for up to five months according to the slow-growing nature of most deep-sea species. The improved MSP method allows the isolation of microbes by selection and investigation of microbial diversity by high throughput sequencing of the pooled sample cultures. By picking individual droplets and scale-up cultivation, a total of 772 strains that were taxonomically assigned to 70 species were isolated from the deep-sea sediments in the SWIR, including 15 potential novel species. On the other hand, based on 16S rRNA gene amplicon sequencing analysis, the microbial diversity of the SWIR was studied and documented with culture-dependent and independent methods in this study. The superiority of the MSP platform in revealing the rare biosphere was also evaluated based on amplicon sequencing. The results show that droplet-based single-cell cultivation of the MSP has a much higher ability than traditional agar plate cultivation in obtaining microbial species and more than 90% of operational taxonomic units (OTUs) detected in the MSP pool belong to the rare biosphere. Our results indicate the high robustness and efficiency of the improved MSP platform in revealing the environmentally rare biosphere, especially for slow-growing species. Overall, the MSP platform has a superior ability to recover microbial diversity than conventional agar plates and it was found to hold great potential for recovering rare microbial resources from various environments.