A Multiplexed Single-Cell CRISPR Screening Platform Enables Systematic Dissection of the Unfolded Protein Response.

Functional genomics efforts face tradeoffs between number of perturbations examined and complexity of phenotypes measured. We bridge this gap with Perturb-seq, which combines droplet-based single-cell RNA-seq with a strategy for barcoding CRISPR-mediated perturbations, allowing many perturbations to be profiled in pooled format. We applied Perturb-seq to dissect the mammalian unfolded protein response (UPR) using single and combinatorial CRISPR perturbations. Two genome-scale CRISPR interference (CRISPRi) screens identified genes whose repression perturbs ER homeostasis. Subjecting ∼100 hits to Perturb-seq enabled high-precision functional clustering of genes. Single-cell analyses decoupled the three UPR branches, revealed bifurcated UPR branch activation among cells subject to the same perturbation, and uncovered differential activation of the branches across hits, including an isolated feedback loop between the translocon and IRE1α. These studies provide insight into how the three sensors of ER homeostasis monitor distinct types of stress and highlight the ability of Perturb-seq to dissect complex cellular responses.

A Comprehensive, CRISPR-based Functional Analysis of Essential Genes in Bacteria.

Essential gene functions underpin the core reactions required for cell viability, but their contributions and relationships are poorly studied in vivo. Using CRISPR interference, we created knockdowns of every essential gene in Bacillus subtilis and probed their phenotypes. Our high-confidence essential gene network, established using chemical genomics, showed extensive interconnections among distantly related processes and identified modes of action for uncharacterized antibiotics. Importantly, mild knockdown of essential gene functions significantly reduced stationary-phase survival without affecting maximal growth rate, suggesting that essential protein levels are set to maximize outgrowth from stationary phase. Finally, high-throughput microscopy indicated that cell morphology is relatively insensitive to mild knockdown but profoundly affected by depletion of gene function, revealing intimate connections between cell growth and shape. Our results provide a framework for systematic investigation of essential gene functions in vivo broadly applicable to diverse microorganisms and amenable to comparative analysis.

Cold Shock Response in Bacteria.

Bacteria often encounter temperature fluctuations in their natural habitats and must adapt to survive. The molecular response of bacteria to sudden temperature upshift or downshift is termed the heat shock response (HSR) or the cold shock response (CSR), respectively. Unlike the HSR, which activates a dedicated transcription factor that predominantly copes with heat-induced protein folding stress, the CSR is mediated by a diverse set of inputs. This review provides a picture of our current understanding of the CSR across bacteria. The fundamental aspects of CSR involved in sensing and adapting to temperature drop, including regulation of membrane fluidity, protein folding, DNA topology, RNA metabolism, and protein translation, are discussed. Special emphasis is placed on recent findings of a CSR circuitry in Escherichia coli mediated by cold shock family proteins and RNase R that monitors and modulates messenger RNA structure to facilitate global translation recovery during acclimation.

Chemical-genetic interrogation of RNA polymerase mutants reveals structure-function relationships and physiological tradeoffs.

The multi-subunit bacterial RNA polymerase (RNAP) and its associated regulators carry out transcription and integrate myriad regulatory signals. Numerous studies have interrogated RNAP mechanism, and RNAP mutations drive Escherichia coli adaptation to many health- and industry-relevant environments, yet a paucity of systematic analyses hampers our understanding of the fitness trade-offs from altering RNAP function. Here, we conduct a chemical-genetic analysis of a library of RNAP mutants. We discover phenotypes for non-essential insertions, show that clustering mutant phenotypes increases their predictive power for drawing functional inferences, and demonstrate that some RNA polymerase mutants both decrease average cell length and prevent killing by cell-wall targeting antibiotics. Our findings demonstrate that RNAP chemical-genetic interactions provide a general platform for interrogating structure-function relationships in vivo and for identifying physiological trade-offs of mutations, including those relevant for disease and biotechnology. This strategy should have broad utility for illuminating the role of other important protein complexes.

Selective ribosome profiling reveals the cotranslational chaperone action of trigger factor in vivo.

As nascent polypeptides exit ribosomes, they are engaged by a series of processing, targeting, and folding factors. Here, we present a selective ribosome profiling strategy that enables global monitoring of when these factors engage polypeptides in the complex cellular environment. Studies of the Escherichia coli chaperone trigger factor (TF) reveal that, though TF can interact with many polypeptides, ß-barrel outer-membrane proteins are the most prominent substrates. Loss of TF leads to broad outer-membrane defects and premature, cotranslational protein translocation. Whereas in vitro studies suggested that TF is prebound to ribosomes waiting for polypeptides to emerge from the exit channel, we find that in vivo TF engages ribosomes only after ~100 amino acids are translated. Moreover, excess TF interferes with cotranslational removal of the N-terminal formyl methionine. Our studies support a triaging model in which proper protein biogenesis relies on the fine-tuned, sequential engagement of processing, targeting, and folding factors.

Phenotypic landscape of a bacterial cell.

The explosion of sequence information in bacteria makes developing high-throughput, cost-effective approaches to matching genes with phenotypes imperative. Using E. coli as proof of principle, we show that combining large-scale chemical genomics with quantitative fitness measurements provides a high-quality data set rich in discovery. Probing growth profiles of a mutant library in hundreds of conditions in parallel yielded > 10,000 phenotypes that allowed us to study gene essentiality, discover leads for gene function and drug action, and understand higher-order organization of the bacterial chromosome. We highlight new information derived from the study, including insights into a gene involved in multiple antibiotic resistance and the synergy between a broadly used combinatory antibiotic therapy, trimethoprim and sulfonamides. This data set, publicly available at http://ecoliwiki.net/tools/chemgen/, is a valuable resource for both the microbiological and bioinformatic communities, as it provides high-confidence associations between hundreds of annotated and uncharacterized genes as well as inferences about the mode of action of several poorly understood drugs.

A Stress Response that Monitors and Regulates mRNA Structure Is Central to Cold Shock Adaptation.

Temperature influences the structural and functional properties of cellular components, necessitating stress responses to restore homeostasis following temperature shift. Whereas the circuitry controlling the heat shock response is well understood, that controlling the E. coli cold shock adaptation program is not. We found that during the growth arrest phase (acclimation) that follows shift to low temperature, protein synthesis increases, and open reading frame (ORF)-wide mRNA secondary structure decreases. To identify the regulatory system controlling this process, we screened for players required for increased translation. We identified a two-member mRNA surveillance system that enables recovery of translation during acclimation: RNase R assures appropriate mRNA degradation and the Csps dynamically adjust mRNA secondary structure to globally modulate protein expression level. An autoregulatory switch in which Csps tune their own expression to cellular demand enables dynamic control of global translation. The universality of Csps in bacteria suggests broad utilization of this control mechanism.

Correction of a widespread bias in pooled chemical genomics screens improves their interpretability.

Chemical genomics is a powerful and increasingly accessible technique to probe gene function, gene-gene interactions, and antibiotic synergies and antagonisms. Indeed, multiple large-scale pooled datasets in diverse organisms have been published. Here, we identify an artifact arising from uncorrected differences in the number of cell doublings between experiments within such datasets. We demonstrate that this artifact is widespread, show how it causes spurious gene-gene and drug-drug correlations, and present a simple but effective post hoc method for removing its effects. Using several published datasets, we demonstrate that this correction removes spurious correlations between genes and conditions, improving data interpretability and revealing new biological insights. Finally, we determine experimental factors that predispose a dataset for this artifact and suggest a set of experimental and computational guidelines for performing pooled chemical genomics experiments that will maximize the potential of this powerful technique.

Regulation of peptidoglycan synthesis by outer-membrane proteins.

Growth of the mesh-like peptidoglycan (PG) sacculus located between the bacterial inner and outer membranes (OM) is tightly regulated to ensure cellular integrity, maintain cell shape, and orchestrate division. Cytoskeletal elements direct placement and activity of PG synthases from inside the cell, but precise spatiotemporal control over this process is poorly understood. We demonstrate that PG synthases are also controlled from outside of the sacculus. Two OM lipoproteins, LpoA and LpoB, are essential for the function, respectively, of PBP1A and PBP1B, the major E. coli bifunctional PG synthases. Each Lpo protein binds specifically to its cognate PBP and stimulates its transpeptidase activity, thereby facilitating attachment of new PG to the sacculus. LpoB shows partial septal localization, and our data suggest that the LpoB-PBP1B complex contributes to OM constriction during cell division. LpoA/LpoB and their PBP-docking regions are restricted to γ-proteobacteria, providing models for niche-specific regulation of sacculus growth.

The context of the ribosome binding site in mRNAs defines specificity of action of kasugamycin, an inhibitor of translation initiation.

Kasugamycin (KSG) is an aminoglycoside antibiotic widely used in agriculture and exhibits considerable medical potential. Previous studies suggested that KSG interferes with translation by blocking binding of canonical messenger RNA (mRNA) and initiator transfer tRNA (tRNA) to the small ribosomal subunit, thereby preventing initiation of protein synthesis. Here, by using genome-wide approaches, we show that KSG can interfere with translation even after the formation of the 70S initiation complex on mRNA, as the extent of KSG-mediated translation inhibition correlates with increased occupancy of start codons by 70S ribosomes. Even at saturating concentrations, KSG does not completely abolish translation, allowing for continuing expression of some Escherichia coli proteins. Differential action of KSG significantly depends on the nature of the mRNA residue immediately preceding the start codon, with guanine in this position being the most conducive to inhibition by the drug. In addition, the activity of KSG is attenuated by translational coupling as genes whose start codons overlap with the coding regions or the stop codons of the upstream cistrons tend to be less susceptible to drug-mediated inhibition. Altogether, our findings reveal KSG as an example of a small ribosomal subunit-targeting antibiotic with a well-pronounced context specificity of action.

Expansion and re-classification of the extracytoplasmic function (ECF) σ factor family.

Extracytoplasmic function σ factors (ECFs) represent one of the major bacterial signal transduction mechanisms in terms of abundance, diversity and importance, particularly in mediating stress responses. Here, we performed a comprehensive phylogenetic analysis of this protein family by scrutinizing all proteins in the NCBI database. As a result, we identified an average of ∼10 ECFs per bacterial genome and 157 phylogenetic ECF groups that feature a conserved genetic neighborhood and a similar regulation mechanism. Our analysis expands previous classification efforts ∼50-fold, enriches many original ECF groups with previously unclassified proteins and identifies 22 entirely new ECF groups. The ECF groups are hierarchically related to each other and are further composed of subgroups with closely related sequences. This two-tiered classification allows for the accurate prediction of common promoter motifs and the inference of putative regulatory mechanisms across subgroups composing an ECF group. This comprehensive, high-resolution description of the phylogenetic distribution of the ECF family, together with the massive expansion of classified ECF sequences and an openly accessible data repository called 'ECF Hub' (https://www.computational.bio.uni-giessen.de/ecfhub), will serve as a powerful hypothesis-generator to guide future research in the field.

Rewiring the specificity of extracytoplasmic function sigma factors.

Bacterial genomes are being sequenced at an exponentially increasing rate, but our inability to decipher their transcriptional wiring limits our ability to derive new biology from these sequences. De novo determination of regulatory interactions requires accurate prediction of regulators' DNA binding and precise determination of biologically significant binding sites. Here we address these challenges by solving the DNA-specificity code of extracytoplasmic function sigma factors (ECF σs), a major family of bacterial regulators, and determining their putative regulons. We generated an aligned collection of ECF σs and their promoters by leveraging the autoregulatory nature of ECF σs as a means of promoter discovery and analyzed it to identify and characterize the conserved amino acid-nucleotide interactions that determine promoter specificity. This enabled de novo prediction of ECF σ specificity, which we combined with a statistically rigorous phylogenetic footprinting pipeline based on precomputed orthologs to predict the direct targets of ∼67% of ECF σs. This global survey indicated that some ECF σs are conserved global regulators controlling many genes throughout the genome, which are important under many conditions, while others are local regulators, controlling a few closely linked genes in response to specific stimuli in select species. This analysis reveals important organizing principles of bacterial gene regulation and presents a conceptual and computational framework for deciphering gene regulatory networks.

MicL, a new σE-dependent sRNA, combats envelope stress by repressing synthesis of Lpp, the major outer membrane lipoprotein.

In enteric bacteria, the transcription factor σ(E) maintains membrane homeostasis by inducing synthesis of proteins involved in membrane repair and two small regulatory RNAs (sRNAs) that down-regulate synthesis of abundant membrane porins. Here, we describe the discovery of a third σ(E)-dependent sRNA, MicL (mRNA-interfering complementary RNA regulator of Lpp), transcribed from a promoter located within the coding sequence of the cutC gene. MicL is synthesized as a 308-nucleotide (nt) primary transcript that is processed to an 80-nt form. Both forms possess features typical of Hfq-binding sRNAs but surprisingly target only a single mRNA, which encodes the outer membrane lipoprotein Lpp, the most abundant protein of the cell. We show that the copper sensitivity phenotype previously ascribed to inactivation of the cutC gene is actually derived from the loss of MicL and elevated Lpp levels. This observation raises the possibility that other phenotypes currently attributed to protein defects are due to deficiencies in unappreciated regulatory RNAs. We also report that σ(E) activity is sensitive to Lpp abundance and that MicL and Lpp comprise a new σ(E) regulatory loop that opposes membrane stress. Together MicA, RybB, and MicL allow σ(E) to repress the synthesis of all abundant outer membrane proteins in response to stress.

Topoisomerase IV can functionally replace all type 1A topoisomerases in Bacillus subtilis.

DNA topoisomerases play essential roles in chromosome organization and replication. Most bacteria possess multiple topoisomerases which have specialized functions in the control of DNA supercoiling or in DNA catenation/decatenation during recombination and chromosome segregation. DNA topoisomerase I is required for the relaxation of negatively supercoiled DNA behind the transcribing RNA polymerase. Conflicting results have been reported on the essentiality of the topA gene encoding topoisomerase I in the model bacterium Bacillus subtilis. In this work, we have studied the requirement for topoisomerase I in B. subtilis. All stable topA mutants carried different chromosomal amplifications of the genomic region encompassing the parEC operon encoding topoisomerase IV. Using a fluorescent amplification reporter system we observed that each individual topA mutant had acquired such an amplification. Eventually, the amplifications were replaced by a point mutation in the parEC promoter region which resulted in a fivefold increase of parEC expression. In this strain both type I topoisomerases, encoded by topA and topB, were dispensable. Our results demonstrate that topoisomerase IV at increased expression is necessary and sufficient to take over the function of type 1A topoisomerases.

Discovery of the extracytoplasmic function σ factors.

This special issue of Molecular Microbiology marks the 25th anniversary of the discovery of the extracytoplasmic function (ECF) σ factors, proteins that subsequently emerged as the largest group of alternative σ factors and one of the three major pillars of signal transduction in bacteria, alongside one- and two-component systems. A single bacterial genome can encode > 100 ECF σ factors, and combined with their cognate anti-σ factors, they represent a modular design that primarily functions in transmembrane signal transduction. Here, we first describe the immediate events that led to the 1994 publication in the Proceeding of the National Academy of Sciences USA, and then set them in the broader context of key events in the history of σ biology research.

Resistance to serine in Bacillus subtilis: identification of the serine transporter YbeC and of a metabolic network that links serine and threonine metabolism.

The Gram-positive bacterium Bacillus subtilis uses serine not only as a building block for proteins but also as an important precursor in many anabolic reactions. Moreover, a lack of serine results in the initiation of biofilm formation. However, excess serine inhibits the growth of B. subtilis. To unravel the underlying mechanisms, we isolated suppressor mutants that can tolerate toxic serine concentrations by three targeted and non-targeted genome-wide screens. All screens as well as genetic complementation in Escherichia coli identified the so far uncharacterized permease YbeC as the major serine transporter of B. subtilis. In addition to YbeC, the threonine transporters BcaP and YbxG make minor contributions to serine uptake. A strain lacking these three transporters was able to tolerate 100 mM serine whereas the wild type strain was already inhibited by 1 mM of the amino acid. The screen for serine-resistant mutants also identified mutations that result in increased serine degradation and in increased expression of threonine biosynthetic enzymes suggesting that serine toxicity results from interference with threonine biosynthesis.

Global analysis of translation termination in E. coli.

Terminating protein translation accurately and efficiently is critical for both protein fidelity and ribosome recycling for continued translation. The three bacterial release factors (RFs) play key roles: RF1 and 2 recognize stop codons and terminate translation; and RF3 promotes disassociation of bound release factors. Probing release factors mutations with reporter constructs containing programmed frameshifting sequences or premature stop codons had revealed a propensity for readthrough or frameshifting at these specific sites, but their effects on translation genome-wide have not been examined. We performed ribosome profiling on a set of isogenic strains with well-characterized release factor mutations to determine how they alter translation globally. Consistent with their known defects, strains with increasingly severe release factor defects exhibit increasingly severe accumulation of ribosomes over stop codons, indicative of an increased duration of the termination/release phase of translation. Release factor mutant strains also exhibit increased occupancy in the region following the stop codon at a significant number of genes. Our global analysis revealed that, as expected, translation termination is generally efficient and accurate, but that at a significant number of genes (≥ 50) the ribosome signature after the stop codon is suggestive of translation past the stop codon. Even native E. coli K-12 exhibits the ribosome signature suggestive of protein extension, especially at UGA codons, which rely exclusively on the reduced function RF2 variant of the K-12 strain for termination. Deletion of RF3 increases the severity of the defect. We unambiguously demonstrate readthrough and frameshifting protein extensions and their further accumulation in mutant strains for a few select cases. In addition to enhancing recoding, ribosome accumulation over stop codons disrupts attenuation control of biosynthetic operons, and may alter expression of some overlapping genes. Together, these functional alterations may either augment the protein repertoire or produce deleterious proteins.

Correction: A Chemical-Genomic Screen of Neglected Antibiotics Reveals Illicit Transport of Kasugamycin and Blasticidin S.

[This corrects the article DOI: 10.1371/journal.pgen.1006124.].

Bacterial sigma factors: a historical, structural, and genomic perspective.

Transcription initiation is the crucial focal point of gene expression in prokaryotes. The key players in this process, sigma factors (σs), associate with the catalytic core RNA polymerase to guide it through the essential steps of initiation: promoter recognition and opening, and synthesis of the first few nucleotides of the transcript. Here we recount the key advances in σ biology, from their discovery 45 years ago to the most recent progress in understanding their structure and function at the atomic level. Recent data provide important structural insights into the mechanisms whereby σs initiate promoter opening. We discuss both the housekeeping σs, which govern transcription of the majority of cellular genes, and the alternative σs, which direct RNA polymerase to specialized operons in response to environmental and physiological cues. The review concludes with a genome-scale view of the extracytoplasmic function σs, the most abundant group of alternative σs.