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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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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., (Published by Elsevier Inc.)

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, β-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., (Copyright © 2011 Elsevier Inc. All rights reserved.)

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., (Copyright © 2011 Elsevier Inc. All rights reserved.)

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., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Transmembrane signaling between intracellular compartments is often controlled by regulated proteolysis. Escherichia coli respond to misfolded or unfolded outer-membrane porins (OMPs) in the periplasm by inducing sigma(E)-dependent transcription of stress genes in the cytoplasm. This process requires a proteolytic cascade initiated by the DegS protease, which destroys a transmembrane protein (RseA) that normally binds to and inhibits sigma(E). Here, we show that peptides ending with OMP-like C-terminal sequences bind the DegS PDZ domain, activate DegS cleavage of RseA, and induce sigma(E)-dependent transcription. These results suggest that DegS acts as a sensor of envelope stress by binding unassembled OMPs. DegS activation involves relief of inhibitory interactions between its PDZ and protease domains. Peptide binding to inhibitory PDZ domains in proteases related to DegS, including DegP/HtrA, may also regulate the degradation of specific substrates by these enzymes.

GroEL/S chaperonin ring complexes fold many unrelated proteins. To understand the basis and extent of the chaperonin substrate spectrum, we used rounds of selection and DNA shuffling to obtain GroEL/S variants that dramatically enhanced folding of a single substrate-green fluorescent protein (GFP). Changes in the substrate-optimized chaperonins increase the polarity of the folding cavity and alter the ATPase cycle. These findings reveal a surprising plasticity of GroEL/S, which can be exploited to aid folding of recombinant proteins. Our studies also reveal a conflict between specialization and generalization of chaperonins as increased GFP folding comes at the expense of the ability of GroEL/S to fold its natural substrates. This conflict and the nature of the ring structure may help explain the evolution of cellular chaperone systems.

Initiation of transcription is the first step in gene expression and a major point of regulation. Recent structural studies reveal the nature of the initiating complex and suggest new ways of accomplishing the processes required for initiation.

For transcription to initiate, RNA polymerase must recognize and melt promoters. Selective binding to the nontemplate strand of the -10 region of the promoter is central to this process. We show that a 48 amino acid (aa) coiled-coil from the beta' subunit (aa 262--309) induces sigma(70) to perform this function almost as efficiently as core RNA polymerase itself. We provide evidence that interaction between the beta' coiled-coil and region 2.2 of sigma(70) promotes an allosteric transition that allows sigma(70) to selectively recognize the nontemplate strand. As the beta' 262--309 peptide can function with the previously crystallized portion of sigma(70), nontemplate recognition can be reconstituted with only 47 kDa, or 1/10 of holoenzyme.

The sigma 70 subunit of E. coli RNA polymerase is required for sequence-specific recognition of promoter DNA. Genetic studies and sequence analysis have indicated that sigma 70 contains two specific DNA-binding domains that recognize the two conserved portions of the prokaryotic promoter. However, intact sigma 70 does not bind to DNA. Using C-terminal and internal polypeptides of sigma 70, carrying one or both putative DNA-binding domains, we demonstrate that sigma 70 does contain two DNA-binding domains, but that N-terminal sequences inhibit the ability of intact sigma 70 to bind to DNA. Thus, we propose that sigma 70 is a sequence-specific DNA-binding protein that normally functions through an allosteric interaction with the core subunits of RNA polymerase.

The rpoD gene encoding the sigma subunit of E. coli RNA polymerase is cotranscribed with rpsU and dnaG, encoding ribosomal protein S21 and DNA primase, respectively. After temperature upshift, a heat shock promoter (Phs) located within dnaG is transiently induced, causing increased transcription of rpoD. The extent of induction is sufficient to account for the heat shock response of sigma synthesis. The initiation site of this promoter was located about 360 bp upstream of rpoD by promoter cloning and S1 nuclease mapping. Plasmid deletions generated with Bal 31 nuclease show that the DNA sequence CTGCCACCC in the -44 to -36 region of this promoter is necessary for its heat shock activity. Heat induction of transcription from Phs is under the control of HtpR, a positive regulator of the heat shock response.

The htpR gene of E. coli encodes a positive regulator of the heat-shock response. We have fused the htpR gene to the inducible PL promoter of phage lambda. Overproduction of HtpR following a temperature upshift resulted in the overexpression of heat-shock proteins. We describe the purification and initial in vitro characterization of the factor controlling expression of heat-shock genes. The factor was the 32 kd htpR gene product. In vitro, a mixture of HtpR and core RNA polymerase initiated transcription at heat-shock promoters. The sigma factor encoded by rpoD was not required for this reaction. Therefore, HtpR is a sigma factor that promotes transcription initiation at heat-shock promoters. We propose that htpR be renamed rpoH and that the gene product be called sigma-32.

The sigma subunit of E. coli RNA polymerase is encoded by the rpoD gene. Within the sequence upstream from rpoD, we have identified the structural genes rpsU and dnaG, which encode the 30S ribosomal protein S21 and DNA primase, respectively. The three genes are in the order rpsU, dnaG rpoD, and are all encoded by the same DNA strand. Analysis of in vivo transcripts from this region shows that these genes are all within the same operon. By correlating the 5' and 3' ends of in vivo transcripts with our DNA sequence, we have identified several regulatory features of the operon. These features include tandem promoters upstream from rpsU, a terminator between rpsU and dnaG, an RNA processing site separating dnaG and rpoD, and the operon terminator just downstream from rpoD. Immediately upstream of the operon promoters is an active promoter for an unidentified gene. We discuss the regulatory significance of the operon features and the biological significance of an operon encoding proteins essential for translation, replication and transcription.

We have isolated spontaneous temperature-resistant revertants of a temperature-sensitive mutation (rpoD800) in the sigma subunit of E. coli K12 RNA polymerase. These revertants still contained the rpoD800 allele. They were mucoid, and sensitive to ultraviolet light and the radiomimetic agent nitrofurantoin, which are characteristics of lon mutants. One revertant, Tr29, was mapped to the lon region of the chromosome. Lon- rpoD800 double mutants were constructed, and were phenotypically indistinguishable from the spontaneous temperature-resistant revertant. It is the degradation-deficient property of lon mutants that is responsible for the suppression of the temperature-sensitive phenotype. We show that the rpoD800 sigma polypeptide is a substrate for the ion proteolytic system, and that mutations in lon decrease the rate of mutant sigma degradation. The rate of synthesis of mutant sigma was also affected in lon- strains. The net effect of lon-mutations was to increase the concentration of mutant sigma. We conclude that the temperature-sensitive phenotype results from insufficient concentration, rather than altered function, of the mutant protein.