Single-cell RNA sequencing reveals plasmid constrains bacterial population heterogeneity and identifies a non-conjugating subpopulation.

Transcriptional heterogeneity in isogenic bacterial populations can play various roles in bacterial evolution, but its detection remains technically challenging. Here, we use microbial split-pool ligation transcriptomics to study the relationship between bacterial subpopulation formation and plasmid-host interactions at the single-cell level. We find that single-cell transcript abundances are influenced by bacterial growth state and plasmid carriage. Moreover, plasmid carriage constrains the formation of bacterial subpopulations. Plasmid genes, including those with core functions such as replication and maintenance, exhibit transcriptional heterogeneity associated with cell activity. Notably, we identify a cell subpopulation that does not transcribe conjugal plasmid transfer genes, which may help reduce plasmid burden on a subset of cells. Our study advances the understanding of plasmid-mediated subpopulation dynamics and provides insights into the plasmid-bacteria interplay.

High-throughput single-cell transcriptomics of bacteria using combinatorial barcoding.

Microbial split-pool ligation transcriptomics (microSPLiT) is a high-throughput single-cell RNA sequencing method for bacteria. With four combinatorial barcoding rounds, microSPLiT can profile transcriptional states in hundreds of thousands of Gram-negative and Gram-positive bacteria in a single experiment without specialized equipment. As bacterial samples are fixed and permeabilized before barcoding, they can be collected and stored ahead of time. During the first barcoding round, the fixed and permeabilized bacteria are distributed into a 96-well plate, where their transcripts are reverse transcribed into cDNA and labeled with the first well-specific barcode inside the cells. The cells are mixed and redistributed two more times into new 96-well plates, where the second and third barcodes are appended to the cDNA via in-cell ligation reactions. Finally, the cells are mixed and divided into aliquot sub-libraries, which can be stored until future use or prepared for sequencing with the addition of a fourth barcode. It takes 4 days to generate sequencing-ready libraries, including 1 day for collection and overnight fixation of samples. The standard plate setup enables single-cell transcriptional profiling of up to 1 million bacterial cells and up to 96 samples in a single barcoding experiment, with the possibility of expansion by adding barcoding rounds. The protocol requires experience in basic molecular biology techniques, handling of bacterial samples and preparation of DNA libraries for next-generation sequencing. It can be performed by experienced undergraduate or graduate students. Data analysis requires access to computing resources, familiarity with Unix command line and basic experience with Python or R.

Pediatric Patients with Sitosterolemia: Next-Generation Sequencing and Biochemical Examination in Clinical Practice.

Here, we report the pediatric cases of sitosterolemia, a rare autosomal-recessive genetic disorder, characterized by high concentrations of plant sterols in blood and heterogeneity manifestations. All three patients (two girls aged 2 and 6 years old, and one boy aged 14 years old) were initially diagnosed with hypercholesterinemia. Next-generation sequencing (NGS) revealed homozygous (p.Leu572Pro/p.Leu572Pro) and compound (p.Leu572Pro/p.Gly512Arg and p.Leu572Pro/p.Trp361*) variants in the ABCG8 gene that allowed for the diagnosis of sitosterolemia. Two patients whose blood phytosterol levels were estimated before the diet demonstrated high levels of sitosterol/campesterol (69.6/29.2 and 28.3/12.4 µmol/L, respectively). Here, we demonstrate that NGS-testing led to the proper diagnosis that is essential for patients' management. The variant p.Leu572Pro might be prevalent among patients with sitosterolemia in Russia.

Expanding the Phenotype of Hereditary Congenital Facial Paresis Type 3.

The HOXB1 gene encodes a homeobox transcription factor pivotal in the development of rhombomere 4. Biallelic pathogenic variants in this gene are associated with congenital facial paresis type 3 (HCFP3). Only seven single nucleotide variants have been reported in the literature to date. Here, we report a 27-year-old female with a unique presentation of HCFP3 with two novel compound-heterozygous missense variants: c.763C>G, p.(Arg255Gly), which arose de novo and an inherited c.781C>T, p.(Arg261Cys) variant. The patient exhibited HCFP3 symptoms with mild upward esodeviation and lacked the documented ear malformations common in HCFP. For many years, she was misdiagnosed with facio-scapulo-humeral muscular dystrophy, due to complaints of shoulder girdle and neck muscle weakness. No alternative genetic or acquired causes of neck and shoulder girdle weakness were found, suggesting its potential inclusion in the phenotypic spectrum.

How can systems approaches help us understand and treat infectious disease?

Leading researchers at the intersection of infectious disease and systems biology speak about how systems approaches have influenced modern infectious disease research and what these tools can offer for the future of the field.

Microbial single-cell RNA sequencing by split-pool barcoding.

Single-cell RNA sequencing (scRNA-seq) has become an essential tool for characterizing gene expression in eukaryotes, but current methods are incompatible with bacteria. Here, we introduce microSPLiT (microbial split-pool ligation transcriptomics), a high-throughput scRNA-seq method for Gram-negative and Gram-positive bacteria that can resolve heterogeneous transcriptional states. We applied microSPLiT to >25,000 Bacillus subtilis cells sampled at different growth stages, creating an atlas of changes in metabolism and lifestyle. We retrieved detailed gene expression profiles associated with known, but rare, states such as competence and prophage induction and also identified unexpected gene expression states, including the heterogeneous activation of a niche metabolic pathway in a subpopulation of cells. MicroSPLiT paves the way to high-throughput analysis of gene expression in bacterial communities that are otherwise not amenable to single-cell analysis, such as natural microbiota.

Single-cell profiling of the developing mouse brain and spinal cord with split-pool barcoding.

To facilitate scalable profiling of single cells, we developed split-pool ligation-based transcriptome sequencing (SPLiT-seq), a single-cell RNA-seq (scRNA-seq) method that labels the cellular origin of RNA through combinatorial barcoding. SPLiT-seq is compatible with fixed cells or nuclei, allows efficient sample multiplexing, and requires no customized equipment. We used SPLiT-seq to analyze 156,049 single-nucleus transcriptomes from postnatal day 2 and 11 mouse brains and spinal cords. More than 100 cell types were identified, with gene expression patterns corresponding to cellular function, regional specificity, and stage of differentiation. Pseudotime analysis revealed transcriptional programs driving four developmental lineages, providing a snapshot of early postnatal development in the murine central nervous system. SPLiT-seq provides a path toward comprehensive single-cell transcriptomic analysis of other similarly complex multicellular systems.

Deep learning of the regulatory grammar of yeast 5' untranslated regions from 500,000 random sequences.

Our ability to predict protein expression from DNA sequence alone remains poor, reflecting our limited understanding of cis-regulatory grammar and hampering the design of engineered genes for synthetic biology applications. Here, we generate a model that predicts the protein expression of the 5' untranslated region (UTR) of mRNAs in the yeast Saccharomyces cerevisiae. We constructed a library of half a million 50-nucleotide-long random 5' UTRs and assayed their activity in a massively parallel growth selection experiment. The resulting data allow us to quantify the impact on protein expression of Kozak sequence composition, upstream open reading frames (uORFs), and secondary structure. We trained a convolutional neural network (CNN) on the random library and showed that it performs well at predicting the protein expression of both a held-out set of the random 5' UTRs as well as native S. cerevisiae 5' UTRs. The model additionally was used to computationally evolve highly active 5' UTRs. We confirmed experimentally that the great majority of the evolved sequences led to higher protein expression rates than the starting sequences, demonstrating the predictive power of this model.

Slowdown of growth controls cellular differentiation.

How can changes in growth rate affect the regulatory networks behavior and the outcomes of cellular differentiation? We address this question by focusing on starvation response in sporulating Bacillus subtilis We show that the activity of sporulation master regulator Spo0A increases with decreasing cellular growth rate. Using a mathematical model of the phosphorelay-the network controlling Spo0A-we predict that this increase in Spo0A activity can be explained by the phosphorelay protein accumulation and lengthening of the period between chromosomal replication events caused by growth slowdown. As a result, only cells growing slower than a certain rate reach threshold Spo0A activity necessary for sporulation. This growth threshold model accurately predicts cell fates and explains the distribution of sporulation deferral times. We confirm our predictions experimentally and show that the concentration rather than activity of phosphorelay proteins is affected by the growth slowdown. We conclude that sensing the growth rates enables cells to indirectly detect starvation without the need for evaluating specific stress signals.

Chromosomal Arrangement of Phosphorelay Genes Couples Sporulation and DNA Replication.

Genes encoding proteins in a common regulatory network are frequently located close to one another on the chromosome to facilitate co-regulation or couple gene expression to growth rate. Contrasting with these observations, here, we demonstrate a functional role for the arrangement of Bacillus subtilis sporulation network genes on opposite sides of the chromosome. We show that the arrangement of two sporulation network genes, one located close to the origin and the other close to the terminus, leads to a transient gene dosage imbalance during chromosome replication. This imbalance is detected by the sporulation network to produce cell-cycle coordinated pulses of the sporulation master regulator Spo0A∼P. This pulsed response allows cells to decide between sporulation and continued vegetative growth during each cell cycle spent in starvation. The simplicity of this coordination mechanism suggests that it may be widely applicable in a variety of gene regulatory and stress-response settings. VIDEO ABSTRACT.

Inverse Gillespie for inferring stochastic reaction mechanisms from intermittent samples.

Gillespie stochastic simulation is used extensively to investigate stochastic phenomena in many fields, ranging from chemistry to biology to ecology. The inverse problem, however, has remained largely unsolved: How to reconstruct the underlying reactions de novo from sparse observations. A key challenge is that often only aggregate concentrations, proportional to the population numbers, are observable intermittently. We discovered that under specific assumptions, the set of relative population updates in phase space forms a convex polytope whose vertices are indicative of the dominant underlying reactions. We demonstrate the validity of this simple principle by reconstructing stochastic models (reaction structure plus propensities) from a variety of simulated and experimental systems, where hundreds and even thousands of reactions may be occurring in between observations. In some cases, the inferred models provide mechanistic insight. This principle can lead to the understanding of a broad range of phenomena, from molecular biology to population ecology.

Temporal competition between differentiation programs determines cell fate choice.

Multipotent differentiation, where cells adopt one of several possible fates, occurs in diverse systems ranging from bacteria to mammals. This decision-making process is driven by multiple differentiation programs that operate simultaneously in the cell. How these programs interact to govern cell fate choice is poorly understood. To investigate this issue, we simultaneously measured activities of the competing sporulation and competence programs in single Bacillus subtilis cells. This approach revealed that these competing differentiation programs progress independently without cross-regulation before the decision point. Cells seem to arrive at a fate choice through differences in the relative timing between the two programs. To test this proposed dynamic mechanism, we altered the relative timing by engineering artificial cross-regulation between the sporulation and competence circuits. Results suggest a simple model that does not require a checkpoint or intricate cross-regulation before cellular decision-making. Rather, cell fate choice appears to be the outcome of a 'molecular race' between differentiation programs that compete in time, providing a simple dynamic mechanism for decision-making.

Reversible and noisy progression towards a commitment point enables adaptable and reliable cellular decision-making.

Cells must make reliable decisions under fluctuating extracellular conditions, but also be flexible enough to adapt to such changes. How cells reconcile these seemingly contradictory requirements through the dynamics of cellular decision-making is poorly understood. To study this issue we quantitatively measured gene expression and protein localization in single cells of the model organism Bacillus subtilis during the progression to spore formation. We found that sporulation proceeded through noisy and reversible steps towards an irreversible, all-or-none commitment point. Specifically, we observed cell-autonomous and spontaneous bursts of gene expression and transient protein localization events during sporulation. Based on these measurements we developed mathematical population models to investigate how the degree of reversibility affects cellular decision-making. In particular, we evaluated the effect of reversibility on the 1) reliability in the progression to sporulation, and 2) adaptability under changing extracellular stress conditions. Results show that reversible progression allows cells to remain responsive to long-term environmental fluctuations. In contrast, the irreversible commitment point supports reliable execution of cell fate choice that is robust against short-term reductions in stress. This combination of opposite dynamic behaviors (reversible and irreversible) thus maximizes both adaptable and reliable decision-making over a broad range of changes in environmental conditions. These results suggest that decision-making systems might employ a general hybrid strategy to cope with unpredictably fluctuating environmental conditions.