Christophe Herman

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.

Transcriptional Infidelity Promotes Heritable Phenotypic Change in a Bistable Gene Network

Bistable epigenetic switches are fundamental for cell fate determination in unicellular and multicellular organisms. Regulatory proteins associated with bistable switches are often present in low numbers and subject to molecular noise. It is becoming clear that noise in gene expression can influence cell fate. Although the origins and consequences of noise have been studied, the stochastic and transient nature of RNA errors during transcription has not been considered in the origin or modeling of noise nor has the capacity for such transient errors in information transfer to generate heritable phenotypic change been discussed. We used a classic bistable memory module to monitor and capture transient RNA errors: the lac operon of Escherichia coli comprises an autocatalytic positive feedback loop producing a heritable all-or-none epigenetic switch that is sensitive to molecular noise. Using single-cell analysis, we show that the frequency of epigenetic switching from one expression state to the other is increased when the fidelity of RNA transcription is decreased due to error-prone RNA polymerases or to the absence of auxiliary RNA fidelity factors GreA and GreB (functional analogues of eukaryotic TFIIS). Therefore, transcription infidelity contributes to molecular noise and can effect heritable phenotypic change in genetically identical cells in the same environment. Whereas DNA errors allow genetic space to be explored, RNA errors may allow epigenetic or expression space to be sampled. Thus, RNA infidelity should also be considered in the heritable origin of altered or aberrant cell behaviour.

ATP-dependent Proteases Differ Substantially in Their Ability to Unfold Globular Proteins

ATP-dependent proteases control the concentrations of hundreds of regulatory proteins and remove damaged or misfolded proteins from cells. They select their substrates primarily by recognizing sequence motifs or covalent modifications. Once a substrate is bound to the protease, it has to be unfolded and translocated into the proteolytic chamber to be degraded. Some proteases appear to be promiscuous, degrading substrates with poorly defined targeting signals, which suggests that selectivity may be controlled at additional levels. Here we compare the abilities of representatives from all classes of ATP-dependent proteases to unfold a model substrate protein and find that the unfolding abilities range over more than 2 orders of magnitude. We propose that these differences in unfolding abilities contribute to the fates of substrate proteins and may act as a further layer of selectivity during protein destruction.

The σE stress response is required for stress-induced mutation and amplification in Escherichia coli

Pathways of mutagenesis are induced in microbes under adverse conditions controlled by stress responses. Control of mutagenesis by stress responses may accelerate evolution specifically when cells are maladapted to their environments, i.e. are stressed. Stress‐induced mutagenesis in the Escherichia coli Lac assay occurs either by ‘point’ mutation or gene amplification. Point mutagenesis is associated with DNA double‐strand‐break (DSB) repair and requires DinB error‐prone DNA polymerase and the SOS DNA‐damage‐ and RpoS general‐stress responses. We report that the RpoE envelope‐protein‐stress response is also required. In a screen for mutagenesis‐defective mutants, we isolated a transposon insertion in the rpoE P2 promoter. The insertion prevents rpoE induction during stress, but leaves constitutive expression intact, and allows cell viability. rpoE insertion and suppressed null mutants display reduced point mutagenesis and maintenance of amplified DNA. Furthermore, σE acts independently of stress responses previously implicated: SOS/DinB and RpoS, and of σ32, which was postulated to affect mutagenesis. I‐SceI‐induced DSBs alleviated much of the rpoE phenotype, implying that σE promoted DSB formation. Thus, a third stress response and stress input regulate DSB‐repair‐associated stress‐induced mutagenesis. This provides the first report of mutagenesis promoted by σE, and implies that extracytoplasmic stressors may affect genome integrity and, potentially, the ability to evolve.

Engineered proteins detect spontaneous DNA breakage in human and bacterial cells

Spontaneous DNA breaks instigate genomic changes that fuel cancer and evolution, yet direct quantification of double-strand breaks (DSBs) has been limited. Predominant sources of spontaneous DSBs remain elusive. We report synthetic technology for quantifying DSBs using fluorescent-protein fusions of double-strand DNA end-binding protein, Gam of bacteriophage Mu. In Escherichia coli GamGFP forms foci at chromosomal DSBs and pinpoints their subgenomic locations. Spontaneous DSBs occur mostly one per cell, and correspond with generations, supporting replicative models for spontaneous breakage, and providing the first true breakage rates. In mammalian cells GamGFP—labels laser-induced DSBs antagonized by end-binding protein Ku; co-localizes incompletely with DSB marker 53BP1 suggesting superior DSB-specificity; blocks resection; and demonstrates DNA breakage via APOBEC3A cytosine deaminase. We demonstrate directly that some spontaneous DSBs occur outside of S phase. The data illuminate spontaneous DNA breakage in E. coli and human cells and illustrate the versatility of fluorescent-Gam for interrogation of DSBs in living cells. DOI: http://dx.doi.org/10.7554/eLife.01222.001

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.

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.

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.

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.

TraR, a Homolog of a RNAP Secondary Channel Interactor, Modulates Transcription

Recent structural and biochemical studies have identified a novel control mechanism of gene expression mediated through the secondary channel of RNA Polymerase (RNAP) during transcription initiation. Specifically, the small nucleotide ppGpp, along with DksA, a RNAP secondary channel interacting factor, modifies the kinetics of transcription initiation, resulting in, among other events, down-regulation of ribosomal RNA synthesis and up-regulation of several amino acid biosynthetic and transport genes during nutritional stress. Until now, this mode of regulation of RNAP was primarily associated with ppGpp. Here, we identify TraR, a DksA homolog that mimics ppGpp/DksA effects on RNAP. First, expression of TraR compensates for dksA transcriptional repression and activation activities in vivo. Second, mutagenesis of a conserved amino acid of TraR known to be critical for DksA function abolishes its activity, implying both structural and functional similarity to DksA. Third, unlike DksA, TraR does not require ppGpp for repression of the rrnB P1 promoter in vivo and in vitro or activation of amino acid biosynthesis/transport genes in vivo. Implications for DksA/ppGpp mechanism and roles of TraR in horizontal gene transfer and virulence are discussed.