How bacteria grow and divide while retaining a defined shape is a fundamental question in microbiology, but technological advances are now driving a new understanding of how the shape-maintaining ...bacterial peptidoglycan sacculus grows. In this Review, we highlight the relationship between peptidoglycan synthesis complexes and cytoskeletal elements, as well as recent evidence that peptidoglycan growth is regulated from outside the sacculus in Gram-negative bacteria. We also discuss how growth of the sacculus is sensitive to mechanical force and nutritional status, and describe the roles of peptidoglycan hydrolases in generating cell shape and of D-amino acids in sacculus remodelling.
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.
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► Ribosome profiling broadly enables quantitative analysis of translation in bacteria ► Selective ribosome profiling reveals cotranslational chaperone action of trigger factor ► Trigger factor engages nascent chains only after ∼100 residues have been translated ► Outer-membrane porins are highly enriched among trigger factor substrates
Ribosome profiling offers a dynamic view of a chaperone's engagement with nascent polypeptides, yielding insights into its selective activity that have been opaque to more traditional biochemical and genetic analyses.
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.
This document is a focused update to the 2017 colorectal cancer (CRC) screening recommendations from the U.S. Multi-Society Task Force on Colorectal Cancer, which represents the American College of ...Gastroenterology, the American Gastroenterological Association, and the American Society for Gastrointestinal Endoscopy. This update is restricted to addressing the age to start and stop CRC screening in average-risk individuals and the recommended screening modalities. Although there is no literature demonstrating that CRC screening in individuals under age 50 improves health outcomes such as CRC incidence or CRC-related mortality, sufficient data support the U.S. Multi-Society Task Force to suggest average-risk CRC screening begin at age 45. This recommendation is based on the increasing disease burden among individuals under age 50, emerging data that the prevalence of advanced colorectal neoplasia in individuals ages 45 to 49 approaches rates in individuals 50 to 59, and modeling studies that demonstrate the benefits of screening outweigh the potential harms and costs. For individuals ages 76 to 85, the decision to start or continue screening should be individualized and based on prior screening history, life expectancy, CRC risk, and personal preference. Screening is not recommended after age 85.
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.
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•Perturb-seq allows parallel screening with rich phenotypic output from single cells•Simultaneous delivery and identification of up to three CRISPR perturbations•Genome-scale screens dissect the mammalian unfolded protein response•Analytical methods separate perturbation responses from confounding effects
A strategy for barcoding CRISPR-mediated perturbations allows pooled expression profiling via single-cell RNA sequencing. Application to the mammalian unfolded protein response then enabled systematic delineation of the transcriptional arms of the response and functional clustering of genes affecting ER homeostasis.
Microorganisms live in fluctuating environments, requiring stress response pathways to resist environmental insults and stress. These pathways dynamically monitor cellular status, and mediate ...adaptive changes by remodeling the proteome, largely accomplished by remodeling transcriptional networks and protein degradation. The complementarity of fast, specific proteolytic degradation and slower, broad transcriptomic changes gives cells the mechanistic repertoire to dynamically adjust cellular processes and optimize response behavior. Together, this enables cells to minimize the ‘cost’ of the response while maximizing the ability to survive environmental stress. Here we highlight recent progress in our understanding of transcriptional networks and proteolysis that illustrates the design principles used by bacteria to generate the complex behaviors required to resist stress.
Microorganisms live in fluctuating environments, requiring stress response pathways to resist environmental insults and stress. Guo and Gross highlight recent progress in our understanding of transcriptional networks and proteolysis that illustrates the design principles used by bacteria to generate the complex behaviors required to resist stress.
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.
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•mRNA unfolding drives translation recovery during acclimation after cold shock•Csps and RNase R are the major players in the response•Csps promote mRNA unfolding and RNase R degrades mRNA during translation recovery•CspA modulates its 5′ UTR structure to tune expression to demand
Zhang et al. identified an mRNA structure surveillance system mediated by Csps and RNase R that facilitates translation recovery after cold shock in E. coli. Their work proves insights into a post-transcriptionally regulated bacterial stress response and suggests broad utilization of this control mechanism across all bacteria.
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.
•Bacterial CRISPR approaches are used for gene editing, repression, and activation.•CRISPRi, which blocks transcription of targeted genes, is the main bacterial modality.•Chemical genomics and ...titratable CRISPRi enable studies of essential gene functions.•Pooled, genome-wide CRISPRi screens identify novel gene functions.•CRISPRi has been demonstrated in many non-model bacteria.
In this review we describe the application of CRISPR tools for functional genomics screens in bacteria, with a focus on the use of interference (CRISPRi) approaches. We review recent developments in CRISPRi titration, which has enabled essential gene functional screens, and genome-scale pooled CRISPRi screens. We summarize progress toward enabling CRISPRi screens in non-model and pathogenic bacteria, including the development of new dCas9 variants. Taking into account the current state of the field, we provide a forward-looking analysis of CRISPRi strategies for determining gene function in bacteria.
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