Identification and genetic dissection of convergent persister cell states.

Persister cells, rare phenotypic variants that survive normally lethal levels of antibiotics, present a major barrier to clearing bacterial infections. However, understanding the precise physiological state and genetic basis of persister formation has been a longstanding challenge. Here we generated a high-resolution single-cell RNA atlas of Escherichia coli growth transitions, which revealed that persisters from diverse genetic and physiological models converge to transcriptional states that are distinct from standard growth phases and instead exhibit a dominant signature of translational deficiency.

The genetic landscape of antibiotic sensitivity in Staphylococcus aureus.

Despite the critical importance of essential genes, systems-level investigations of their contribution to antibiotic sensitivity have been limited. Using CRISPR Adaptation-mediated Library Manufacturing (CALM), we generated ultra-dense CRISPR interference (CRISPRi) libraries in methicillin-sensitive and -resistant strains of Staphylococcus aureus, which allowed us to quantify gene fitness on a global scale across ten clinically relevant antibiotics. This led to the identification of a comprehensive set of known and novel biological processes modulating bacterial fitness in the antibiotics.

Multiscale regulation of nutrient stress responses in from chromatin structure to small regulatory RNAs.

Recent research has indicated the presence of heterochromatin-like regions of extended protein occupancy and transcriptional silencing of bacterial genomes. We utilized an integrative approach to track chromatin structure and transcription in K-12 across a wide range of nutrient conditions. In the process, we identified multiple loci which act similarly to facultative heterochromatin in eukaryotes, normally silenced but permitting expression of genes under specific conditions.

Systematic assessment of prognostic molecular features across cancers.

Precision oncology promises accurate prediction of disease trajectories by utilizing molecular features of tumors. We present a systematic analysis of the prognostic potential of diverse molecular features across large cancer cohorts. We find that the mRNA expression of biologically coherent sets of genes (modules) is substantially more predictive of patient survival than single-locus genomic and transcriptomic aberrations.

Arginine limitation causes a directed DNA sequence evolution response in colorectal cancer cells.

Utilization of specific codons varies significantly across organisms. Cancer represents a model for understanding DNA sequence evolution and could reveal causal factors underlying codon evolution. We found that across human cancer, arginine codons are frequently mutated to other codons.

OxPhos defects cause hypermetabolism and reduce lifespan in cells and in patients with mitochondrial diseases.

Patients with primary mitochondrial oxidative phosphorylation (OxPhos) defects present with fatigue and multi-system disorders, are often lean, and die prematurely, but the mechanistic basis for this clinical picture remains unclear. By integrating data from 17 cohorts of patients with mitochondrial diseases (n = 690) we find evidence that these disorders increase resting energy expenditure, a state termed hypermetabolism. We examine this phenomenon longitudinally in patient-derived fibroblasts from multiple donors.

Arginine limitation drives a directed codon-dependent DNA sequence evolution response in colorectal cancer cells.

Utilization of specific codons varies between organisms. Cancer represents a model for understanding DNA sequence evolution and could reveal causal factors underlying codon evolution. We found that across human cancer, arginine codons are frequently mutated to other codons.

Intracellular acidification is a hallmark of thymineless death in E. coli.

Thymidine starvation causes rapid cell death. This enigmatic process known as thymineless death (TLD) is the underlying killing mechanism of diverse antimicrobial and antineoplastic drugs. Despite decades of investigation, we still lack a mechanistic understanding of the causal sequence of events that culminate in TLD.

Functional genetic screen identifies ITPR3/calcium/RELB axis as a driver of colorectal cancer metastatic liver colonization.

Metastatic colonization is the primary cause of death from colorectal cancer (CRC). We employed genome-scale in vivo short hairpin RNA (shRNA) screening and validation to identify 26 promoters of CRC liver colonization. Among these genes, we identified a cluster that contains multiple targetable genes, including ITPR3, which promoted liver-metastatic colonization and elicited similar downstream gene expression programs.

Correction: Dynamic landscape of protein occupancy across the Escherichia coli chromosome.

[This corrects the article DOI: 10.1371/journal.pbio.

Molecular topography of an entire nervous system.

We have produced gene expression profiles of all 302 neurons of the C. elegans nervous system that match the single-cell resolution of its anatomy and wiring diagram. Our results suggest that individual neuron classes can be solely identified by combinatorial expression of specific gene families.

Dynamic landscape of protein occupancy across the Escherichia coli chromosome.

Free-living bacteria adapt to environmental change by reprogramming gene expression through precise interactions of hundreds of DNA-binding proteins. A predictive understanding of bacterial physiology requires us to globally monitor all such protein-DNA interactions across a range of environmental and genetic perturbations. Here, we show that such global observations are possible using an optimized version of in vivo protein occupancy display technology (in vivo protein occupancy display-high resolution, IPOD-HR) and present a pilot application to Escherichia coli.

An inducible CRISPR interference library for genetic interrogation of Saccharomyces cerevisiae biology.

Genome-scale CRISPR interference (CRISPRi) is widely utilized to study cellular processes in a variety of organisms. Despite the dominance of Saccharomyces cerevisiae as a model eukaryote, an inducible genome-wide CRISPRi library in yeast has not yet been presented. Here, we present a genome-wide, inducible CRISPRi library, based on spacer design rules optimized for S.

Regulatory and evolutionary adaptation of yeast to acute lethal ethanol stress.

The yeast Saccharomyces cerevisiae has been the subject of many studies aimed at understanding mechanisms of adaptation to environmental stresses. Most of these studies have focused on adaptation to sub-lethal stresses, upon which a stereotypic transcriptional program called the environmental stress response (ESR) is activated. However, the genetic and regulatory factors that underlie the adaptation and survival of yeast cells to stresses that cross the lethality threshold have not been systematically studied.

In vivo mRNA display enables large-scale proteomics by next generation sequencing.

Large-scale proteomic methods are essential for the functional characterization of proteins in their native cellular context. However, proteomics has lagged far behind genomic approaches in scalability, standardization, and cost. Here, we introduce in vivo mRNA display, a technology that converts a variety of proteomics applications into a DNA sequencing problem.

Prokaryotic single-cell RNA sequencing by in situ combinatorial indexing.

Despite longstanding appreciation of gene expression heterogeneity in isogenic bacterial populations, affordable and scalable technologies for studying single bacterial cells have been limited. Although single-cell RNA sequencing (scRNA-seq) has revolutionized studies of transcriptional heterogeneity in diverse eukaryotic systems, the application of scRNA-seq to prokaryotes has been hindered by their extremely low mRNA abundance, lack of mRNA polyadenylation and thick cell walls. Here, we present prokaryotic expression profiling by tagging RNA in situ and sequencing (PETRI-seq)-a low-cost, high-throughput prokaryotic scRNA-seq pipeline that overcomes these technical obstacles.

Comprehensive Genome-wide Perturbations via CRISPR Adaptation Reveal Complex Genetics of Antibiotic Sensitivity.

Genome-wide CRISPR screens enable systematic interrogation of gene function. However, guide RNA libraries are costly to synthesize, and their limited diversity compromises the sensitivity of CRISPR screens. Using the Streptococcus pyogenes CRISPR-Cas adaptation machinery, we developed CRISPR adaptation-mediated library manufacturing (CALM), which turns bacterial cells into "factories" for generating hundreds of thousands of crRNAs covering 95% of all targetable genomic sites.

Extreme Antibiotic Persistence via Heterogeneity-Generating Mutations Targeting Translation.

Antibiotic persistence, the noninherited tolerance of a subpopulation of bacteria to high levels of antibiotics, is a bet-hedging phenomenon with broad clinical implications. Indeed, the isolation of bacteria with substantially increased persistence rates from chronic infections suggests that evolution of hyperpersistence is a significant factor in clinical therapy resistance. However, the pathways that lead to hyperpersistence and the underlying cellular states have yet to be systematically studied.

Genome wide analysis of 3' UTR sequence elements and proteins regulating mRNA stability during maternal-to-zygotic transition in zebrafish.

Posttranscriptional regulation plays a crucial role in shaping gene expression. During the maternal-to-zygotic transition (MZT), thousands of maternal transcripts are regulated. However, how different -elements and -factors are integrated to determine mRNA stability remains poorly understood.

Unconventional function of an Achaete-Scute homolog as a terminal selector of nociceptive neuron identity.

Proneural genes are among the most early-acting genes in nervous system development, instructing blast cells to commit to a neuronal fate. Drosophila Atonal and Achaete-Scute complex (AS-C) genes, as well as their vertebrate orthologs, are basic helix-loop-helix (bHLH) transcription factors with such proneural activity. We show here that a C.

Stochastic tuning of gene expression enables cellular adaptation in the absence of pre-existing regulatory circuitry.

Cells adapt to familiar changes in their environment by activating predefined regulatory programs that establish adaptive gene expression states. These hard-wired pathways, however, may be inadequate for adaptation to environments never encountered before. Here, we reveal evidence for an alternative mode of gene regulation that enables adaptation to adverse conditions without relying on external sensory information or genetically predetermined -regulation.

Multifactorial Competition and Resistance in a Two-Species Bacterial System.

Microorganisms exist almost exclusively in interactive multispecies communities, but genetic determinants of the fitness of interacting bacteria, and accessible adaptive pathways, remain uncharacterized. Here, using a two-species system, we studied the antagonism of Pseudomonas aeruginosa against Escherichia coli. Our unbiased genome-scale approach enabled us to identify multiple factors that explained the entire antagonism observed.

HNRNPA2B1 Is a Mediator of m(6)A-Dependent Nuclear RNA Processing Events.

N(6)-methyladenosine (m(6)A) is the most abundant internal modification of messenger RNA. While the presence of m(6)A on transcripts can impact nuclear RNA fates, a reader of this mark that mediates processing of nuclear transcripts has not been identified. We find that the RNA-binding protein HNRNPA2B1 binds m(6)A-bearing RNAs in vivo and in vitro and its biochemical footprint matches the m(6)A consensus motif.

Metastasis-suppressor transcript destabilization through TARBP2 binding of mRNA hairpins.

Aberrant regulation of RNA stability has an important role in many disease states. Deregulated post-transcriptional modulation, such as that governed by microRNAs targeting linear sequence elements in messenger RNAs, has been implicated in the progression of many cancer types. A defining feature of RNA is its ability to fold into structures.