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.

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.

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.

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.

The transcription factor DksA prevents conflicts between DNA replication and transcription machinery.

Actively dividing cells perform robust and accurate DNA replication during fluctuating nutrient availability, yet factors that prevent disruption of replication remain largely unknown. Here we report that DksA, a nutrient-responsive transcription factor, ensures replication completion in Escherichia coli by removing transcription roadblocks. In the absence of DksA, replication is rapidly arrested upon amino acid starvation. This arrest requires active transcription and is alleviated by RNA polymerase mutants that compensate for DksA activity. This replication arrest occurs independently of exogenous DNA damage, yet it induces the DNA-damage response and recruits the main recombination protein RecA. This function of DksA is independent of its transcription initiation activity but requires its less-studied transcription elongation activity. Finally, GreA/B elongation factors also prevent replication arrest during nutrient stress. We conclude that transcription elongation factors alleviate fundamental conflicts between replication and transcription, thereby protecting replication fork progression and DNA integrity.

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.

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.

DksA guards elongating RNA polymerase against ribosome-stalling-induced arrest.

In bacteria, translation-transcription coupling inhibits RNA polymerase (RNAP) stalling. We present evidence suggesting that, upon amino acid starvation, inactive ribosomes promote rather than inhibit RNAP stalling. We developed an algorithm to evaluate genome-wide polymerase progression independently of local noise and used it to reveal that the transcription factor DksA inhibits promoter-proximal pausing and increases RNAP elongation when uncoupled from translation by depletion of charged tRNAs. DksA has minimal effect on RNAP elongation in vitro and on untranslated RNAs in vivo. In these cases, transcripts can form RNA structures that prevent backtracking. Thus, the effect of DksA on transcript elongation may occur primarily upon ribosome slowing/stalling or at promoter-proximal locations that limit the potential for RNA structure. We propose that inactive ribosomes prevent formation of backtrack-blocking mRNA structures and that, in this circumstance, DksA acts as a transcription elongation factor in vivo.

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.

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.

DksA involvement in transcription fidelity buffers stochastic epigenetic change.

DksA is an auxiliary transcription factor that interacts with RNA polymerase and influences gene expression. Depending on the promoter, DksA can be a positive or negative regulator of transcription initiation. Moreover, DksA has a substantial effect on transcription elongation where it prevents the collision of transcription and replication machineries, plays a key role in maintaining transcription elongation when translation and transcription are uncoupled and has been shown to be involved in transcription fidelity. Here, we assessed the role of DksA in transcription fidelity by monitoring stochastic epigenetic switching in the lac operon (with and without an error-prone transcription slippage sequence), partial phenotypic suppression of a lacZ nonsense allele, as well as monitoring the number of lacI mRNA transcripts produced in the presence and absence of DksA via an operon fusion and single molecule fluorescent in situ hybridization studies. We present data showing that DksA acts to maintain transcription fidelity in vivo and the role of DksA seems to be distinct from that of the GreA and GreB transcription fidelity factors.

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.

Removal of 8-oxo-GTP by MutT hydrolase is not a major contributor to transcriptional fidelity.

Living in an oxygen-rich environment is dangerous for a cell. Reactive oxygen species can damage DNA, RNA, protein and lipids. The MutT protein in Escherichia coli removes 8-oxo-deoxyguanosine triphosphate (8-oxo-dGTP) and 8-oxo-guanosine triphosphate (8-oxo-GTP) from the nucleotide pools precluding incorporation into DNA and RNA. While 8-oxo-dGTP incorporation into DNA is mutagenic, it is not clear if 8-oxo-GTP incorporation into RNA can have phenotypic consequences for the cell. We use a bistable epigenetic switch sensitive to transcription errors in the Escherichia coli lacI transcript to monitor transient RNA errors. We do not observe any increase in epigenetic switching in mutT cells. We revisit the original observation of partial phenotypic suppression of a lacZamber allele in a mutT background that was attributed to RNA errors. We find that Lac+ revertants can completely account for the increase in ß-galactosidase levels in mutT lacZamber cultures, without invoking participation of transient transcription errors. Moreover, we observe a fluctuation type of distribution of ß-galactosidase appearance in a growing culture, consistent with Lac+ DNA revertant events. We conclude that the absence of MutT produces a DNA mutator but does not equally create an RNA mutator.

Heritable change caused by transient transcription errors.

Transmission of cellular identity relies on the faithful transfer of information from the mother to the daughter cell. This process includes accurate replication of the DNA, but also the correct propagation of regulatory programs responsible for cellular identity. Errors in DNA replication (mutations) and protein conformation (prions) can trigger stable phenotypic changes and cause human disease, yet the ability of transient transcriptional errors to produce heritable phenotypic change ('epimutations') remains an open question. Here, we demonstrate that transcriptional errors made specifically in the mRNA encoding a transcription factor can promote heritable phenotypic change by reprogramming a transcriptional network, without altering DNA. We have harnessed the classical bistable switch in the lac operon, a memory-module, to capture the consequences of transient transcription errors in living Escherichia coli cells. We engineered an error-prone transcription sequence (A9 run) in the gene encoding the lac repressor and show that this 'slippery' sequence directly increases epigenetic switching, not mutation in the cell population. Therefore, one altered transcript within a multi-generational series of many error-free transcripts can cause long-term phenotypic consequences. Thus, like DNA mutations, transcriptional epimutations can instigate heritable changes that increase phenotypic diversity, which drives both evolution and disease.

Activation of the σE-dependent stress pathway by conjugative TraR may anticipate conjugational stress.

Horizontal gene transfer by conjugation plays a major role in bacterial evolution, allowing the acquisition of new traits, such as virulence and resistance to antibacterial agents. With the increased antibiotic resistance in bacterial pathogens, a better understanding of how bacteria modulate conjugation under changing environments and the genetic factors involved is needed. Despite the evolutionary advantages conjugation may confer, the process can be quite stressful for the donor cell. Here, we characterize the ability of TraR, encoded on the episomal F' plasmid, to upregulate the σ(E) extracytoplasmic stress pathway in Escherichia coli. TraR, a DksA homolog, modulates transcription initiation through the secondary channel of RNA polymerase. We show here that TraR activates transcription directly; however, unlike DksA, it does so without using ppGpp as a cofactor. TraR expression can stimulate the σ(E) extracytoplasmic stress response independently of the DegS/RseA signal transduction cascade. In the absence of TraR, bacteria carrying conjugative plasmids become more susceptible to external stress. We propose that TraR increases the concentrations of periplasmic chaperones and proteases by directly activating the transcription of σ(E)-dependent promoters; this increased protein folding capacity may prepare the bacterium to endure the periplasmic stress of sex pilus biosynthesis during mating.

Separation of recombination and SOS response in Escherichia coli RecA suggests LexA interaction sites.

RecA plays a key role in homologous recombination, the induction of the DNA damage response through LexA cleavage and the activity of error-prone polymerase in Escherichia coli. RecA interacts with multiple partners to achieve this pleiotropic role, but the structural location and sequence determinants involved in these multiple interactions remain mostly unknown. Here, in a first application to prokaryotes, Evolutionary Trace (ET) analysis identifies clusters of evolutionarily important surface amino acids involved in RecA functions. Some of these clusters match the known ATP binding, DNA binding, and RecA-RecA homo-dimerization sites, but others are novel. Mutation analysis at these sites disrupted either recombination or LexA cleavage. This highlights distinct functional sites specific for recombination and DNA damage response induction. Finally, our analysis reveals a composite site for LexA binding and cleavage, which is formed only on the active RecA filament. These new sites can provide new drug targets to modulate one or more RecA functions, with the potential to address the problem of evolution of antibiotic resistance at its root.

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.

Characterization of a novel RNA polymerase mutant that alters DksA activity.

The auxiliary factor DksA is a global transcription regulator and, with the help of ppGpp, controls the nutritional stress response in Escherichia coli. Although the consequences of its modulation of RNA polymerase (RNAP) are becoming better explained, it is still not fully understood how the two proteins interact. We employed a series of genetic suppressor selections to find residues in RNAP that alter its sensitivity to DksA. Our approach allowed us to identify and genetically characterize in vivo three single amino acid substitutions: ß' E677G, ß V146F, and ß G534D. We demonstrate that the mutation ß' E677G affects the activity of both DksA and its homolog, TraR, but does not affect the action of other secondary interactors, such as GreA or GreB. Our mutants provide insight into how different auxiliary transcription factors interact with RNAP and contribute to our understanding of how different stages of transcription are regulated through the secondary channel of RNAP in vivo.

Super DksAs: substitutions in DksA enhancing its effects on transcription initiation.

At specific times during bacterial growth, the transcription factor DksA and the unusual nucleotide regulator ppGpp work synergistically to inhibit some Escherichia coli promoters (e.g. rRNA promoters) and to stimulate others (e.g. promoters for amino-acid synthesis and transport). However, the mechanism of DksA action remains uncertain, in part because DksA does not function like conventional transcription factors. To gain insights into DksA function, we identified mutations in dksA that bypassed the requirement for ppGpp by selecting for growth of cells lacking ppGpp on minimal medium without amino acids. We show here that two substitutions in DksA, L15F and N88I, result in higher DksA activity both in vivo and in vitro, primarily by increasing the apparent affinity of DksA for RNA polymerase (RNAP). The mutant DksA proteins suggest potential roles for ppGpp in DksA function, identify potential surfaces on DksA crucial for RNAP binding, and provide tools for future studies to elucidate the mechanism of DksA action.