Biology subjects

Ross, T. D.

Publications and source records attributed to Ross, T. D..

5 recordsLinked to original sources

bioRxiv2022

Massively parallel single-cell sequencing of genetic loci in diverse microbial populations

Single cell genetic heterogeneity is ubiquitous in microbial populations and an important aspect of microbial biology. However, we lack a broadly applicable and accessible method to study this heterogeneity at the single cell level. Here, we introduce a simple, robust, and generalizable platform for quantitative and massively parallel single cell sequencing of target genetic loci in microbes using ultrahigh-throughput droplet microfluidics (Droplet Targeted Amplicon Sequencing or DoTA-seq). Using DoTA-seq, we elucidate the highly diverse single cell ON/OFF states of the phase-variable capsule synthesis operons in the prevalent human gut species Bacteroides fragilis. In addition, we quantify the shifts in antibiotic resistance gene abundances in different species in a 25 member human gut microbial community in response to antibiotics. By sequencing tens of thousands of single-cells derived from a human fecal sample, we identify links between plasmid replicons and the taxonomic lineages of their associated hosts. In sum, DoTA-seq is an accessible and broadly applicable tool for profiling single-cell genetic variation in microbiomes.

microbiology↗

bioRxiv2022

Single-cell bacterial sequencing of invertible loci reveals promoter inversion rates as a strong determinant of population heterogeneity

Population heterogeneity can promote bacterial fitness in response to unpredictable environmental conditions. Human gut symbiont Bacteroides spp., displays variability in single cell surface architectures by combinatorial regulation of promoter inversions that drive expression of capsular polysaccharides (CPS). Using high-throughput single-cell sequencing, we reveal population heterogeneity generated through combinatorial promoter inversion and show that B. fragilis populations can access diverse CPS promoter states. Combining our data with stochastic computational modeling, we demonstrate that the rates of promoter inversion regulated by a broadly conserved serine recombinase are a major mechanism shaping population heterogeneity. Exploiting control over the expression of the recombinase, we devise a method for creating phase-locked variants and show that populations with different initial compositions converge to a similar steady-state composition over time. Our approach can be used to interrogate single-cell phase variable states of diverse microbes including bacterial pathogens. Summary sentenceHigh-throughput single cell sequencing of phase variation reveals regulation as a major driver of population diversification

systems biology↗

bioRxiv2022

Programming bacteria to sense environmental DNA for multiplexed pathogen detection

DNA is a universal and programmable signal of living organisms. Here we developed cell-based DNA sensors by engineering the naturally competent bacterium Bacillus subtilis (B. subtilis) to detect specific DNA sequences in the environment. The DNA sensor strains can identify diverse bacterial species including major human pathogens with high specificity and sensitivity. Multiplexed detection of genomic DNA from different species in complex samples can be achieved by coupling the sensing mechanism to orthogonal fluorescent reporters. We also demonstrate that the DNA sensors can detect the presence of species in the complex samples without requiring DNA extraction. The modularity of the living cell-based DNA sensing mechanism and simple detection procedure could enable programmable DNA sensing for broad applications.

synthetic biology↗

bioRxiv2021

Motor processivity and speed determine structure and dynamics of microtubule-motor assemblies

1Active matter systems can generate highly ordered structures, avoiding equilibrium through the consumption of energy by individual constituents. How the microscopic parameters that characterize the active agents are translated to the observed mesoscopic properties of the assembly has remained an open question. These active systems are prevalent in living matter; for example, in cells, the cytoskeleton is organized into structures such as the mitotic spindle through the coordinated activity of many motor proteins walking along microtubules. Here, we investigate how the microscopic motor-microtubule interactions affect the coherent structures formed in a reconstituted motor-microtubule system. This question is of deeper evolutionary significance as we suspect motor and microtubule type contribute to the shape and size of resulting structures. We explore key parameters experimentally and theoretically, using a variety of motors with different speeds, proces-sivities, and directionalities. We demonstrate that aster size depends on the motor used to create the aster, and develop a model for the distribution of motors and microtubules in steady-state asters that depends on parameters related to motor speed and processivity. Further, we show that network contraction rates scale linearly with the single-motor speed in quasi one-dimensional contraction experiments. In all, this theoretical and experimental work helps elucidate how microscopic motor properties are translated to the much larger scale of collective motor-microtubule assemblies.

biophysics↗

bioRxiv2020

Investigating the dynamics of microbial consortia in spatially structured environments

The spatial organization of microbial communities arises from a complex interplay of biotic and abiotic interactions and is a major determinant of ecosystem functions. We design a microfluidic platform to investigate how the spatial arrangement of microbes impacts gene expression and growth. We elucidate key biochemical parameters that dictate the mapping between spatial positioning and gene expression patterns. We show that distance can establish a low-pass filter to periodic inputs, and can enhance the fidelity of information processing. Positive and negative feedback can play disparate roles in the synchronization and robustness of a genetic oscillator distributed between two strains to spatial separation. Quantification of growth and metabolite release in an amino-acid auxotroph community demonstrates that the interaction network and stability of the community are highly sensitive to temporal perturbations and spatial arrangements. In sum, our microfluidic platform can quantify spatiotemporal parameters influencing diffusion-mediated interactions in microbial consortia.

systems biology↗