Targeted accurate RNA consensus sequencing (tARC-seq) reveals mechanisms of replication error affecting SARS-CoV-2 divergence.

RNA viruses, like SARS-CoV-2, depend on their RNA-dependent RNA polymerases (RdRp) for replication, which is error prone. Monitoring replication errors is crucial for understanding the virus's evolution. Current methods lack the precision to detect rare de novo RNA mutations, particularly in low-input samples such as those from patients. Here we introduce a targeted accurate RNA consensus sequencing method (tARC-seq) to accurately determine the mutation frequency and types in SARS-CoV-2, both in cell culture and clinical samples. Our findings show an average of 2.68 × 10-5 de novo errors per cycle with a C > T bias that cannot be solely attributed to APOBEC editing. We identified hotspots and cold spots throughout the genome, correlating with high or low GC content, and pinpointed transcription regulatory sites as regions more susceptible to errors. tARC-seq captured template switching events including insertions, deletions and complex mutations. These insights shed light on the genetic diversity generation and evolutionary dynamics of SARS-CoV-2.

Drugging evolution of antibiotic resistance at a regulatory network hub.

Evolution of antibiotic resistance is a world health crisis, fueled by new mutations. Drugs to slow mutagenesis could, as cotherapies, prolong the shelf-life of antibiotics, yet evolution-slowing drugs and drug targets have been underexplored and ineffective. Here, we used a network-based strategy to identify drugs that block hubs of fluoroquinolone antibiotic-induced mutagenesis. We identify a U.S. Food and Drug Administration- and European Medicines Agency-approved drug, dequalinium chloride (DEQ), that inhibits activation of the Escherichia coli general stress response, which promotes ciprofloxacin-induced (stress-induced) mutagenic DNA break repair. We uncover the step in the pathway inhibited: activation of the upstream "stringent" starvation stress response, and find that DEQ slows evolution without favoring proliferation of DEQ-resistant mutants. Furthermore, we demonstrate stress-induced mutagenesis during mouse infections and its inhibition by DEQ. Our work provides a proof-of-concept strategy for drugs to slow evolution in bacteria and generally.

ppGpp and RNA-polymerase backtracking guide antibiotic-induced mutable gambler cells.

Antibiotic resistance is a global health threat and often results from new mutations. Antibiotics can induce mutations via mechanisms activated by stress responses, which both reveal environmental cues of mutagenesis and are weak links in mutagenesis networks. Network inhibition could slow the evolution of resistance during antibiotic therapies. Despite its pivotal importance, few identities and fewer functions of stress responses in mutagenesis are clear. Here, we identify the Escherichia coli stringent starvation response in fluoroquinolone-antibiotic ciprofloxacin-induced mutagenesis. Binding of response-activator ppGpp to RNA polymerase (RNAP) at two sites leads to an antibiotic-induced mutable gambler-cell subpopulation. Each activates a stress response required for mutagenic DNA-break repair: surprisingly, ppGpp-site-1-RNAP triggers the DNA-damage response, and ppGpp-site-2-RNAP induces σS-response activity. We propose that RNAP regulates DNA-damage processing in transcribed regions. The data demonstrate a critical node in ciprofloxacin-induced mutagenesis, imply RNAP-regulation of DNA-break repair, and identify promising targets for resistance-resisting drugs.

Conjugation's Toolkit: the Roles of Nonstructural Proteins in Bacterial Sex.

Bacterial conjugation, a form of horizontal gene transfer, relies on a type 4 secretion system (T4SS) and a set of nonstructural genes that are closely linked. These nonstructural genes aid in the mobile lifestyle of conjugative elements but are not part of the T4SS apparatus for conjugative transfer, such as the membrane pore and relaxosome, or the plasmid maintenance and replication machineries. While these nonstructural genes are not essential for conjugation, they assist in core conjugative functions and mitigate the cellular burden on the host. This review compiles and categorizes known functions of nonstructural genes by the stage of conjugation they modulate: dormancy, transfer, and new host establishment. Themes include establishing a commensalistic relationship with the host, manipulating the host for efficient T4SS assembly and function and assisting in conjugative evasion of recipient cell immune functions. These genes, taken in a broad ecological context, play important roles in ensuring proper propagation of the conjugation system in a natural environment.

Evolutionary action of mutations reveals antimicrobial resistance genes in Escherichia coli.

Since antibiotic development lags, we search for potential drug targets through directed evolution experiments. A challenge is that many resistance genes hide in a noisy mutational background as mutator clones emerge in the adaptive population. Here, to overcome this noise, we quantify the impact of mutations through evolutionary action (EA). After sequencing ciprofloxacin or colistin resistance strains grown under different mutational regimes, we find that an elevated sum of the evolutionary action of mutations in a gene identifies known resistance drivers. This EA integration approach also suggests new antibiotic resistance genes which are then shown to provide a fitness advantage in competition experiments. Moreover, EA integration analysis of clinical and environmental isolates of antibiotic resistant of E. coli identifies gene drivers of resistance where a standard approach fails. Together these results inform the genetic basis of de novo colistin resistance and support the robust discovery of phenotype-driving genes via the evolutionary action of genetic perturbations in fitness landscapes.

RNA polymerase inaccuracy underlies SARS-CoV-2 variants and vaccine heterogeneity.

Both the SARS-CoV-2 virus and its mRNA vaccines depend on RNA polymerases (RNAP)1,2; however, these enzymes are inherently error-prone and can introduce variants into the RNA3. To understand SARS-CoV-2 evolution and vaccine efficacy, it is critical to identify the extent and distribution of errors introduced by the RNAPs involved in each process. Current methods lack the sensitivity and specificity to measure de novo RNA variants in low input samples like viral isolates3. Here, we determine the frequency and nature of RNA errors in both SARS-CoV-2 and its vaccine using a targeted Accurate RNA Consensus sequencing method (tARC-seq). We found that the viral RNA-dependent RNAP (RdRp) makes ~1 error every 10,000 nucleotides - higher than previous estimates4. We also observed that RNA variants are not randomly distributed across the genome but are associated with certain genomic features and genes, such as S (Spike). tARC-seq captured a number of large insertions, deletions and complex mutations that can be modeled through non-programmed RdRp template switching. This template switching feature of RdRp explains many key genetic changes observed during the evolution of different lineages worldwide, including Omicron. Further sequencing of the Pfizer-BioNTech COVID-19 vaccine revealed an RNA variant frequency of ~1 in 5,000, meaning most of the vaccine transcripts produced in vitro by T7 phage RNAP harbor a variant. These results demonstrate the extraordinary genetic diversity of viral populations and the heterogeneous nature of an mRNA vaccine fueled by RNAP inaccuracy. Along with functional studies and pandemic data, tARC-seq variant spectra can inform models to predict how SARS-CoV-2 may evolve. Finally, our results may help improve future vaccine development and study design as mRNA therapies continue to gain traction.

Escherichia coli Metabolite Profiling Leads to the Development of an RNA Interference Strain for Caenorhabditis elegans.

The relationship of genotypes to phenotypes can be modified by environmental inputs. Such crucial environmental inputs include metabolic cues derived from microbes living together with animals. Thus, the analysis of genetic effects on animals' physiology can be confounded by variations in the metabolic profile of microbes. Caenorhabditis elegans exposed to distinct bacterial strains and species exhibit phenotypes different at cellular, developmental, and behavioral levels. Here we reported metabolomic profiles of three Escherichia coli strains, B strain OP50, K-12 strain MG1655, and B-K-12 hybrid strain HB101, as well as different mitochondrial and fat storage phenotypes of C. elegans exposed to MG1655 and HB101 vs. OP50. We found that these metabolic phenotypes of C. elegans are not correlated with overall metabolic patterning of bacterial strains, but their specific metabolites. In particular, the fat storage phenotype is traced to the betaine level in different bacterial strains. HT115 is another K-12 E. coli strain that is commonly utilized to elicit an RNA interference response, and we showed that C. elegans exposed to OP50 and HT115 exhibit differences in mitochondrial morphology and fat storage levels. We thus generated an RNA interference competent OP50 (iOP50) strain that can robustly and consistently knockdown endogenous C. elegans genes in different tissues. Together, these studies suggest the importance of specific bacterial metabolites in regulating the host's physiology and provide a tool to prevent confounding effects when analyzing genotype-phenotype interactions under different bacterial backgrounds.

Transcription fidelity: New paradigms in epigenetic inheritance, genome instability and disease.

RNA transcription errors are transient, yet frequent, events that do have consequences for the cell. However, until recently we lacked the tools to empirically measure and study these errors. Advances in RNA library preparation and next generation sequencing (NGS) have allowed the spectrum of transcription errors to be empirically measured over the entire transcriptome and in nascent transcripts. Combining these powerful methods with forward and reverse genetic strategies has refined our understanding of transcription factors known to enhance RNA accuracy and will enable the discovery of new candidates. Furthermore, these approaches will shed additional light on the complex interplay between transcription fidelity and other DNA transactions, such as replication and repair, and explore a role for transcription errors in cellular evolution and disease.

Gamblers: An Antibiotic-Induced Evolvable Cell Subpopulation Differentiated by Reactive-Oxygen-Induced General Stress Response.

Antibiotics can induce mutations that cause antibiotic resistance. Yet, despite their importance, mechanisms of antibiotic-promoted mutagenesis remain elusive. We report that the fluoroquinolone antibiotic ciprofloxacin (cipro) induces mutations by triggering transient differentiation of a mutant-generating cell subpopulation, using reactive oxygen species (ROS). Cipro-induced DNA breaks activate the Escherichia coli SOS DNA-damage response and error-prone DNA polymerases in all cells. However, mutagenesis is limited to a cell subpopulation in which electron transfer together with SOS induce ROS, which activate the sigma-S (σS) general-stress response, which allows mutagenic DNA-break repair. When sorted, this small σS-response-"on" subpopulation produces most antibiotic cross-resistant mutants. A U.S. Food and Drug Administration (FDA)-approved drug prevents σS induction, specifically inhibiting antibiotic-promoted mutagenesis. Further, SOS-inhibited cell division, which causes multi-chromosome cells, promotes mutagenesis. The data support a model in which within-cell chromosome cooperation together with development of a "gambler" cell subpopulation promote resistance evolution without risking most cells.

Bacteria-to-Human Protein Networks Reveal Origins of Endogenous DNA Damage.

DNA damage provokes mutations and cancer and results from external carcinogens or endogenous cellular processes. However, the intrinsic instigators of endogenous DNA damage are poorly understood. Here, we identify proteins that promote endogenous DNA damage when overproduced: the DNA "damage-up" proteins (DDPs). We discover a large network of DDPs in Escherichia coli and deconvolute them into six function clusters, demonstrating DDP mechanisms in three: reactive oxygen increase by transmembrane transporters, chromosome loss by replisome binding, and replication stalling by transcription factors. Their 284 human homologs are over-represented among known cancer drivers, and their RNAs in tumors predict heavy mutagenesis and a poor prognosis. Half of the tested human homologs promote DNA damage and mutation when overproduced in human cells, with DNA damage-elevating mechanisms like those in E. coli. Our work identifies networks of DDPs that provoke endogenous DNA damage and may reveal DNA damage-associated functions of many human known and newly implicated cancer-promoting proteins.

Fluorescent fusions of the N protein of phage Mu label DNA damage in living cells.

The N protein of phage Mu was indicated from studies in Escherichia coli to hold linear Mu chromosomes in a circular conformation by non-covalent association, and thus suggested potentially to bind DNA double-stranded ends. Because of its role in association with linear Mu DNA, we tested whether fluorescent-protein fusions to N might provide a useful tool for labeling DNA damage including double-strand break (DSB) ends in single cells. We compared N-GFP with a biochemically well documented DSB-end binding protein, the Gam protein of phage Mu, also fused to GFP. We find that N-GFP produced in live E. coli forms foci in response to DNA damage induced by radiomimetic drug phleomycin, indicating that it labels damaged DNA. N-GFP also labels specific DSBs created enzymatically by I-SceI double-strand endonuclease, and by X-rays, with the numbers of foci corresponding with the numbers of DSBs generated, indicating DSB labeling. However, whereas N-GFP forms about half as many foci as GamGFP with phleomycin, its labeling of I-SceI- and X-ray-induced DSBs is far less efficient than that of GamGFP. The data imply that N-GFP binds and labels DNA damage including DSBs, but may additionally label phleomycin-induced non-DSB damage, with which DSB-specific GamGFP does not interact. The data indicate that N-GFP labels DNA damage, and may be useful for general, not DSB-specific, DNA-damage detection.

How Acts of Infidelity Promote DNA Break Repair: Collision and Collusion Between DNA Repair and Transcription.

Transcription is a fundamental cellular process and the first step in gene regulation. Although RNA polymerase (RNAP) is highly processive, in growing cells the progression of transcription can be hindered by obstacles on the DNA template, such as damaged DNA. The authors recent findings highlight a trade-off between transcription fidelity and DNA break repair. While a lot of work has focused on the interaction between transcription and nucleotide excision repair, less is known about how transcription influences the repair of DNA breaks. The authors suggest that when the cell experiences stress from DNA breaks, the control of RNAP processivity affects the balance between preserving transcription integrity and DNA repair. Here, how the conflict between transcription and DNA double-strand break (DSB) repair threatens the integrity of both RNA and DNA are discussed. In reviewing this field, the authors speculate on cellular paradigms where this equilibrium is well sustained, and instances where the maintenance of transcription fidelity is favored over genome stability.

CRISPR-FRT targets shared sites in a knock-out collection for off-the-shelf genome editing.

CRISPR advances genome engineering by directing endonuclease sequence specificity with a guide RNA molecule (gRNA). For precisely targeting a gene for modification, each genetic construct requires a unique gRNA. By generating a gRNA against the flippase recognition target (FRT) site, a common genetic element shared by multiple genetic collections, CRISPR-FRT circumvents this design constraint to provide a broad platform for fast, scarless, off-the-shelf genome engineering.

Transcription infidelity and genome integrity: the parallax view.

It was recently shown that removal of GreA, a transcription fidelity factor, enhances DNA break repair. This counterintuitive result, arising from unresolved backtracked RNA polymerase impeding DNA resection and thereby facilitating RecA-loading, leads to an interesting corollary: error-free full-length transcripts and broken chromosomes. Therefore, transcription fidelity may compromise genomic integrity.

DksA and ppGpp Regulate the σS Stress Response by Activating Promoters for the Small RNA DsrA and the Anti-Adapter Protein IraP.

σS is an alternative sigma factor, encoded by the rpoS gene, that redirects cellular transcription to a large family of genes in response to stressful environmental signals. This so-called σS general stress response is necessary for survival in many bacterial species and is controlled by a complex, multifactorial pathway that regulates σS levels transcriptionally, translationally, and posttranslationally in Escherichia coli It was shown previously that the transcription factor DksA and its cofactor, ppGpp, are among the many factors governing σS synthesis, thus playing an important role in activation of the σS stress response. However, the mechanisms responsible for the effects of DksA and ppGpp have not been elucidated fully. We describe here how DksA and ppGpp directly activate the promoters for the anti-adaptor protein IraP and the small regulatory RNA DsrA, thereby indirectly influencing σS levels. In addition, based on effects of DksAN88I, a previously identified DksA variant with increased affinity for RNA polymerase (RNAP), we show that DksA can increase σS activity by another indirect mechanism. We propose that by reducing rRNA transcription, DksA and ppGpp increase the availability of core RNAP for binding to σS and also increase transcription from other promoters, including PdsrA and PiraP By improving the translation and stabilization of σS, as well as the ability of other promoters to compete for RNAP, DksA and ppGpp contribute to the switch in the transcription program needed for stress adaptation.IMPORTANCE Bacteria spend relatively little time in log phase outside the optimized environment found in a laboratory. They have evolved to make the most of alternating feast and famine conditions by seamlessly transitioning between rapid growth and stationary phase, a lower metabolic mode that is crucial for long-term survival. One of the key regulators of the switch in gene expression that characterizes stationary phase is the alternative sigma factor σS Understanding the factors governing σS activity is central to unraveling the complexities of growth, adaptation to stress, and pathogenesis. Here, we describe three mechanisms by which the RNA polymerase binding factor DksA and the second messenger ppGpp regulate σS levels.

The transcription fidelity factor GreA impedes DNA break repair.

Homologous recombination repairs DNA double-strand breaks and must function even on actively transcribed DNA. Because break repair prevents chromosome loss, the completion of repair is expected to outweigh the transcription of broken templates. However, the interplay between DNA break repair and transcription processivity is unclear. Here we show that the transcription factor GreA inhibits break repair in Escherichia coli. GreA restarts backtracked RNA polymerase and hence promotes transcription fidelity. We report that removal of GreA results in markedly enhanced break repair via the classic RecBCD-RecA pathway. Using a deep-sequencing method to measure chromosomal exonucleolytic degradation, we demonstrate that the absence of GreA limits RecBCD-mediated resection. Our findings suggest that increased RNA polymerase backtracking promotes break repair by instigating RecA loading by RecBCD, without the influence of canonical Chi signals. The idea that backtracked RNA polymerase can stimulate recombination presents a DNA transaction conundrum: a transcription fidelity factor that compromises genomic integrity.

Microbial Genetic Composition Tunes Host Longevity.

Homeostasis of the gut microbiota critically influences host health and aging. Developing genetically engineered probiotics holds great promise as a new therapeutic paradigm to promote healthy aging. Here, through screening 3,983 Escherichia coli mutants, we discovered that 29 bacterial genes, when deleted, increase longevity in the host Caenorhabditis elegans. A dozen of these bacterial mutants also protect the host from age-related progression of tumor growth and amyloid-beta accumulation. Mechanistically, we discovered that five bacterial mutants promote longevity through increased secretion of the polysaccharide colanic acid (CA), which regulates mitochondrial dynamics and unfolded protein response (UPRmt) in the host. Purified CA polymers are sufficient to promote longevity via ATFS-1, the host UPRmt-responsive transcription factor. Furthermore, the mitochondrial changes and longevity effects induced by CA are conserved across different species. Together, our results identified molecular targets for developing pro-longevity microbes and a bacterial metabolite acting on host mitochondria to promote longevity.

Cooperativity of Negative Autoregulation Confers Increased Mutational Robustness.

Negative autoregulation is universally found across organisms. In the bacterium Escherichia coli, transcription factors often repress their own expression to form a negative feedback network motif that enables robustness to changes in biochemical parameters. Here we present a simple phenomenological model of a negative feedback transcription factor repressing both itself and another target gene. The strength of the negative feedback is characterized by three parameters: the cooperativity in self-repression, the maximal expression rate of the transcription factor, and the apparent dissociation constant of the transcription factor binding to its own promoter. Analysis of the model shows that the target gene levels are robust to mutations in the transcription factor, and that the robustness improves as the degree of cooperativity in self-repression increases. The prediction is tested in the LexA transcriptional network of E. coli by altering cooperativity in self-repression and promoter strength. Indeed, we find robustness is correlated with the former. Considering the proposed importance of gene regulation in speciation, parameters governing a transcription factor's robustness to mutation may have significant influence on a cell or organism's capacity to evolve.

Holliday junction trap shows how cells use recombination and a junction-guardian role of RecQ helicase.

DNA repair by homologous recombination (HR) underpins cell survival and fuels genome instability, cancer, and evolution. However, the main kinds and sources of DNA damage repaired by HR in somatic cells and the roles of important HR proteins remain elusive. We present engineered proteins that trap, map, and quantify Holliday junctions (HJs), a central DNA intermediate in HR, based on catalytically deficient mutant RuvC protein of Escherichia coli. We use RuvCDefGFP (RDG) to map genomic footprints of HR at defined DNA breaks in E. coli and demonstrate genome-scale directionality of double-strand break (DSB) repair along the chromosome. Unexpectedly, most spontaneous HR-HJ foci are instigated, not by DSBs, but rather by single-stranded DNA damage generated by replication. We show that RecQ, the E. coli ortholog of five human cancer proteins, nonredundantly promotes HR-HJ formation in single cells and, in a novel junction-guardian role, also prevents apparent non-HR-HJs promoted by RecA overproduction. We propose that one or more human RecQ orthologs may act similarly in human cancers overexpressing the RecA ortholog RAD51 and find that cancer genome expression data implicate the orthologs BLM and RECQL4 in conjunction with EME1 and GEN1 as probable HJ reducers in such cancers. Our results support RecA-overproducing E. coli as a model of the many human tumors with up-regulated RAD51 and provide the first glimpses of important, previously elusive reaction intermediates in DNA replication and repair in single living cells.