A small molecule that inhibits the evolution of antibiotic resistance.

Antibiotic resistance rapidly develops against almost all available therapeutics. Therefore, searching for new antibiotics to overcome the problem of antibiotic resistance alone is insufficient. Given that antibiotic resistance can be driven by mutagenesis, an avenue for preventing it is the inhibition of mutagenic processes. We previously showed that the DNA translocase Mfd is mutagenic and accelerates antibiotic resistance development. Here, we present our discovery of a small molecule that inhibits Mfd-dependent mutagenesis, ARM-1 (anti-resistance molecule 1). We found ARM-1 using a high-throughput, small molecule, in vivo screen. Using biochemical assays, we characterized the mechanism by which ARM-1 inhibits Mfd. Critically, we found that ARM-1 reduces mutagenesis and significantly delays antibiotic resistance development across highly divergent bacterial pathogens. These results demonstrate that the mutagenic proteins accelerating evolution can be directly inhibited. Furthermore, our findings suggest that Mfd inhibition, alongside antibiotics, is a potentially effective approach for prevention of antibiotic resistance development during treatment of infections.

Replication-Transcription Conflicts: A Perpetual War on the Chromosome.

DNA replication and transcription occur in all living cells across all domains of life. Both essential processes occur simultaneously on the same template, leading to conflicts between the macromolecular machines that perform these functions. Numerous studies over the past few decades demonstrate that this is an inevitable problem in both prokaryotic and eukaryotic cells. We have learned that conflicts lead to replication fork reversal, breaks in the DNA, R-loop formation, topological stress, and mutagenesis, and they can ultimately impact evolution. Recent studies have also provided insight into the various mechanisms that mitigate, resolve, and allow tolerance of conflicts and how conflicts result in divergent pathological consequences across divergent species. In this review, we summarize current knowledge regarding the outcomes of encounters between replication and transcription machineries and explore how these clashes are dealt with across species.

Pathogenic bacteria experience pervasive RNA polymerase backtracking during infection.

IMPORTANCE: Eukaryotic hosts have defense mechanisms that may disrupt molecular transactions along the pathogen's chromosome through excessive DNA damage. Given that DNA damage stalls RNA polymerase (RNAP) thereby increasing mutagenesis, investigating how host defense mechanisms impact the movement of the transcription machinery on the pathogen chromosome is crucial. Using a new methodology we developed, we elucidated the dynamics of RNAP movement and association with the chromosome in the pathogenic bacterium Salmonella enterica during infection. We found that dynamics of RNAP movement on the chromosome change significantly during infection genome-wide, including at regions that encode for key virulence genes. In particular, we found that there is pervasive RNAP backtracking on the bacterial chromosome during infections and that anti-backtracking factors are critical for pathogenesis. Altogether, our results suggest that, interestingly, the host environment can promote the development of antimicrobial resistance and hypervirulence as stalled RNAPs can accelerate evolution through increased mutagenesis.

Oxidative stress drives mutagenesis through transcription-coupled repair in bacteria.

In bacteria, mutations lead to the evolution of antibiotic resistance, which is one of the main public health problems of the twenty-first century. Therefore, determining which cellular processes most frequently contribute to mutagenesis, especially in cells that have not been exposed to exogenous DNA damage, is critical. Here, we show that endogenous oxidative stress is a key driver of mutagenesis and the subsequent development of antibiotic resistance. This is the case for all classes of antibiotics and highly divergent species tested, including patient-derived strains. We show that the transcription-coupled repair pathway, which uses the nucleotide excision repair proteins (TC-NER), is responsible for endogenous oxidative stress-dependent mutagenesis and subsequent evolution. This suggests that a majority of mutations arise through transcription-associated processes rather than the replication fork. In addition to determining that the NER proteins play a critical role in mutagenesis and evolution, we also identify the DNA polymerases responsible for this process. Our data strongly suggest that cooperation between three different mutagenic DNA polymerases, likely at the last step of TC-NER, is responsible for mutagenesis and evolution. Overall, our work identifies a highly conserved pathway that drives mutagenesis due to endogenous oxidative stress, which has broad implications for all diseases of evolution, including antibiotic resistance development.

Pathogenic bacteria experience pervasive RNA polymerase backtracking during infection.

Pathogenic bacteria and their eukaryotic hosts are in a constant arms race. Hosts have numerous defense mechanisms at their disposal that not only challenge the bacterial invaders, but have the potential to disrupt molecular transactions along the bacterial chromosome. However, it is unclear how the host impacts association of proteins with the bacterial chromosome at the molecular level during infection. This is partially due to the lack of a method that could detect these events in pathogens while they are within host cells. We developed and optimized a system capable of mapping and measuring levels of bacterial proteins associated with the chromosome while they are actively infecting the host (referred to as PIC-seq). Here, we focused on the dynamics of RNA polymerase (RNAP) movement and association with the chromosome in the pathogenic bacterium Salmonella enterica as a model system during infection. Using PIC-seq, we found that RNAP association patterns with the chromosome change during infection genome-wide, including at regions that encode for key virulence genes. Importantly, we found that infection of a host significantly increases RNAP backtracking on the bacterial chromosome. RNAP backtracking is the most common form of disruption to RNAP progress on the chromosome. Interestingly, we found that the resolution of backtracked RNAPs via the anti-backtracking factors GreA and GreB is critical for pathogenesis, revealing a new class of virulence genes. Altogether, our results strongly suggest that infection of a host significantly impacts transcription by disrupting RNAP movement on the chromosome within the bacterial pathogen. The increased backtracking events have important implications not only for efficient transcription, but also for mutation rates as stalled RNAPs increase the levels of mutagenesis.

Direct visualization of transcription-replication conflicts reveals post-replicative DNA:RNA hybrids.

Transcription-replication collisions (TRCs) are crucial determinants of genome instability. R-loops were linked to head-on TRCs and proposed to obstruct replication fork progression. The underlying mechanisms, however, remained elusive due to the lack of direct visualization and of non-ambiguous research tools. Here, we ascertained the stability of estrogen-induced R-loops on the human genome, visualized them directly by electron microscopy (EM), and measured R-loop frequency and size at the single-molecule level. Combining EM and immuno-labeling on locus-specific head-on TRCs in bacteria, we observed the frequent accumulation of DNA:RNA hybrids behind replication forks. These post-replicative structures are linked to fork slowing and reversal across conflict regions and are distinct from physiological DNA:RNA hybrids at Okazaki fragments. Comet assays on nascent DNA revealed a marked delay in nascent DNA maturation in multiple conditions previously linked to R-loop accumulation. Altogether, our findings suggest that TRC-associated replication interference entails transactions that follow initial R-loop bypass by the replication fork.

The in vivo measurement of replication fork velocity and pausing by lag-time analysis.

An important step towards understanding the mechanistic basis of the central dogma is the quantitative characterization of the dynamics of nucleic-acid-bound molecular motors in the context of the living cell. To capture these dynamics, we develop lag-time analysis, a method for measuring in vivo dynamics. Using this approach, we provide quantitative locus-specific measurements of fork velocity, in units of kilobases per second, as well as replisome pause durations, some with the precision of seconds. The measured fork velocity is observed to be both locus and time dependent, even in wild-type cells. In this work, we quantitatively characterize known phenomena, detect brief, locus-specific pauses at ribosomal DNA loci in wild-type cells, and observe temporal fork velocity oscillations in three highly-divergent bacterial species.

Locus-Specific Analysis of Replication Dynamics and Detection of DNA-RNA Hybrids by Immuno Electron Microscopy.

DNA-RNA hybrids can interfere with DNA replication, but the underlying intermediates and molecular mechanisms have remained elusive. Here, we describe a single molecule approach that allows to monitor DNA-RNA hybrids locus-specifically in the context of ongoing replication. Using restriction digestion, gel electrophoresis and gel elution, this workflow allows to efficiently isolate replication intermediates and to study replication dynamics across a specific genomic locus. Here, we applied this procedure to isolate a bacterial genomic locus carrying an inducible transcription-replication conflict. Moreover, we combined electron microscopy with S9.6-Gold immuno-labeling to detect DNA-RNA hybrids on the isolated replication intermediates. With some limitations, this approach may be adapted to locus-specific replication analyses in different organisms.

Characterizing stochastic cell-cycle dynamics in exponential growth.

Two powerful and complementary experimental approaches are commonly used to study the cell cycle and cell biology: One class of experiments characterizes the statistics (or demographics) of an unsynchronized exponentially growing population, while the other captures cell-cycle dynamics, either by time-lapse imaging of full cell cycles or in bulk experiments on synchronized populations. In this paper, we study the subtle relationship between observations in these two distinct experimental approaches. We begin with an existing model: A single-cell deterministic description of cell-cycle dynamics where cell states (i.e., periods or phases) have precise lifetimes. We then generalize this description to a stochastic model in which the states have stochastic lifetimes, as described by arbitrary probability distribution functions. Our analyses of the demographics of an exponential culture reveal a simple and exact correspondence between the deterministic and stochastic models: The corresponding state ages in the deterministic model are equal to the exponential mean of the age in the stochastic model. An important implication is therefore that the demographics of an exponential culture will be well fit by a deterministic model even if the state timing is stochastic. Although we explore the implications of the models in the context of the Escherichia coli cell cycle, we expect both the models as well as the significance of the exponential-mean lifetimes to find many applications in the quantitative analysis of cell-cycle dynamics in other biological systems.

Topological stress is responsible for the detrimental outcomes of head-on replication-transcription conflicts.

Conflicts between the replication and transcription machineries have profound effects on chromosome duplication, genome organization, and evolution across species. Head-on conflicts (lagging-strand genes) are significantly more detrimental than codirectional conflicts (leading-strand genes). The fundamental reason for this difference is unknown. Here, we report that topological stress significantly contributes to this difference. We find that head-on, but not codirectional, conflict resolution requires the relaxation of positive supercoils by the type II topoisomerases DNA gyrase and Topo IV, at least in the Gram-positive model bacterium Bacillus subtilis. Interestingly, our data suggest that after positive supercoil resolution, gyrase introduces excessive negative supercoils at head-on conflict regions, driving pervasive R-loop formation. Altogether, our results reveal a fundamental mechanistic difference between the two types of encounters, addressing a long-standing question in the field of replication-transcription conflicts.

Mfd regulates RNA polymerase association with hard-to-transcribe regions in vivo, especially those with structured RNAs.

RNA polymerase (RNAP) encounters various roadblocks during transcription. These obstacles can impede RNAP movement and influence transcription, ultimately necessitating the activity of RNAP-associated factors. One such factor is the bacterial protein Mfd, a highly conserved DNA translocase and evolvability factor that interacts with RNAP. Although Mfd is thought to function primarily in the repair of DNA lesions that stall RNAP, increasing evidence suggests that it may also be important for transcription regulation. However, this is yet to be fully characterized. To shed light on Mfd's in vivo functions, we identified the chromosomal regions where it associates. We analyzed Mfd's impact on RNAP association and transcription regulation genome-wide. We found that Mfd represses RNAP association at many chromosomal regions. We found that these regions show increased RNAP pausing, suggesting that they are hard to transcribe. Interestingly, we noticed that the majority of the regions where Mfd regulates transcription contain highly structured regulatory RNAs. The RNAs identified regulate a myriad of biological processes, ranging from metabolism to transfer RNA regulation to toxin-antitoxin (TA) functions. We found that cells lacking Mfd are highly sensitive to toxin overexpression. Finally, we found that Mfd promotes mutagenesis in at least one toxin gene, suggesting that its function in regulating transcription may promote evolution of certain TA systems and other regions containing strong RNA secondary structures. We conclude that Mfd is an RNAP cofactor that is important, and at times critical, for transcription regulation at hard-to-transcribe regions, especially those that express structured regulatory RNAs.

Targeting evolution to inhibit antibiotic resistance.

Drug-resistant bacterial infections have led to a global health crisis. Although much effort is placed on the development of new antibiotics or variants that are less subject to existing resistance mechanisms, history shows that this strategy by itself is unlikely to solve the problem of drug resistance. Here, we discuss inhibiting evolution as a strategy that, in combination with antibiotics, may resolve the problem. Although mutagenesis is the main driver of drug resistance development, attacking the drivers of genetic diversification in pathogens has not been well explored. Bacteria possess active mechanisms that increase the rate of mutagenesis, especially at times of stress, such as during replication within eukaryotic host cells, or exposure to antibiotics. We highlight how the existence of these promutagenic proteins (evolvability factors) presents an opportunity that can be capitalized upon for the effective inhibition of drug resistance development. To help move this idea from concept to execution, we first describe a set of criteria that an 'optimal' evolvability factor would likely have to meet to be a viable therapeutic target. We then discuss the intricacies of some of the known mutagenic mechanisms and evaluate their potential as drug targets to inhibit evolution. In principle, and as suggested by recent studies, we argue that the inhibition of these and other evolvability factors should reduce resistance development. Finally, we discuss the challenges of transitioning anti-evolution drugs from the laboratory to the clinic.

The enigmatic role of Mfd in replication-transcription conflicts in bacteria.

Conflicts between replication and transcription can have life-threatening consequences. RNA polymerase (RNAP) is the major impediment to replication progression, and its efficient removal from DNA should mitigate the consequences of collisions with replication. Cells have various proteins that can resolve conflicts by removing stalled (or actively translocating) RNAP from DNA. It would therefore seem logical that RNAP-associated factors, such as the bacterial DNA translocase Mfd, would minimize the effects of conflicts. Despite seemingly conclusive statements in most textbooks, the role of Mfd in conflicts remains an enigma. In this review, we will discuss the different physical states of RNAP during transcription, and how each distinct state can influence conflict severity and potentially trigger the involvement of Mfd. We propose models to explain the contradictory conclusions from published studies on the potential role of Mfd in resolving conflicts.

Inhibiting the Evolution of Antibiotic Resistance.

Efforts to battle antimicrobial resistance (AMR) are generally focused on developing novel antibiotics. However, history shows that resistance arises regardless of the nature or potency of new drugs. Here, we propose and provide evidence for an alternate strategy to resolve this problem: inhibiting evolution. We determined that the DNA translocase Mfd is an "evolvability factor" that promotes mutagenesis and is required for rapid resistance development to all antibiotics tested across highly divergent bacterial species. Importantly, hypermutator alleles that accelerate AMR development did not arise without Mfd, at least during evolution of trimethoprim resistance. We also show that Mfd's role in AMR development depends on its interactions with the RNA polymerase subunit RpoB and the nucleotide excision repair protein UvrA. Our findings suggest that AMR development can be inhibited through inactivation of evolvability factors (potentially with "anti-evolution" drugs)-in particular, Mfd-providing an unexplored route toward battling the AMR crisis.

Gene inversion potentiates bacterial evolvability and virulence.

Most bacterial genes are encoded on the leading strand, co-orienting the movement of the replication machinery with RNA polymerases. This bias reduces the frequency of detrimental head-on collisions between the two machineries. The negative outcomes of these collisions should lead to selection against head-on alleles, maximizing genome co-orientation. Our findings challenge this model. Using the GC skew calculation, we reveal the evolutionary inversion record of all chromosomally encoded genes in multiple divergent bacterial pathogens. Against expectations, we find that a large number of co-oriented genes have inverted to the head-on orientation, presumably increasing the frequency of head-on replication-transcription conflicts. Furthermore, we find that head-on genes, (including key antibiotic resistance and virulence genes) have higher rates of non-synonymous mutations and are more frequently under positive selection (dN/dS > 1). Based on these results, we propose that spontaneous gene inversions can increase the evolvability and pathogenic capacity of bacteria through head-on replication-transcription collisions.

Crystal structure of a membrane-bound O-acyltransferase.

Membrane-bound O-acyltransferases (MBOATs) are a superfamily of integral transmembrane enzymes that are found in all kingdoms of life1. In bacteria, MBOATs modify protective cell-surface polymers. In vertebrates, some MBOAT enzymes-such as acyl-coenzyme A:cholesterol acyltransferase and diacylglycerol acyltransferase 1-are responsible for lipid biosynthesis or phospholipid remodelling2,3. Other MBOATs, including porcupine, hedgehog acyltransferase and ghrelin acyltransferase, catalyse essential lipid modifications of secreted proteins such as Wnt, hedgehog and ghrelin, respectively4-10. Although many MBOAT proteins are important drug targets, little is known about their molecular architecture and functional mechanisms. Here we present crystal structures of DltB, an MBOAT responsible for the D-alanylation of cell-wall teichoic acid in Gram-positive bacteria11-16, both alone and in complex with the D-alanyl donor protein DltC. DltB contains a ring of 11 peripheral transmembrane helices, which shield a highly conserved extracellular structural funnel extending into the middle of the lipid bilayer. The conserved catalytic histidine residue is located at the bottom of this funnel and is connected to the intracellular DltC through a narrow tunnel. Mutation of either the catalytic histidine or the DltC-binding site of DltB abolishes the D-alanylation of lipoteichoic acid and sensitizes the Gram-positive bacterium Bacillus subtilis to cell-wall stress, which suggests cross-membrane catalysis involving the tunnel. Structure-guided sequence comparison among DltB and vertebrate MBOATs reveals a conserved structural core and suggests that MBOATs from different organisms have similar catalytic mechanisms. Our structures provide a template for understanding structure-function relationships in MBOATs and for developing therapeutic MBOAT inhibitors.

The Clash of Macromolecular Titans: Replication-Transcription Conflicts in Bacteria.

Within the last decade, it has become clear that DNA replication and transcription are routinely in conflict with each other in growing cells. Much of the seminal work on this topic has been carried out in bacteria, specifically, Escherichia coli and Bacillus subtilis; therefore, studies of conflicts in these species deserve special attention. Collectively, the recent findings on conflicts have fundamentally changed the way we think about DNA replication in vivo. Furthermore, new insights on this topic have revealed that the conflicts between replication and transcription significantly influence many key parameters of cellular function, including genome organization, mutagenesis, and evolution of stress response and virulence genes. In this review, we discuss the consequences of replication-transcription conflicts on the life of bacteria and describe some key strategies cells use to resolve them. We put special emphasis on two critical aspects of these encounters: ( a) the consequences of conflicts on replisome stability and dynamics, and ( b) the resulting increase in spontaneous mutagenesis.

The bacterial replisome has factory-like localization.

DNA replication is essential to cellular proliferation. The cellular-scale organization of the replication machinery (replisome) and the replicating chromosome has remained controversial. Two competing models describe the replication process: In the track model, the replisomes translocate along the DNA like a train on a track. Alternately, in the factory model, the replisomes form a stationary complex through which the DNA is pulled. We summarize the evidence for each model and discuss a number of confounding aspects that complicate interpretation of the observations. We advocate a factory-like model for bacterial replication where the replisomes form a relatively stationary and weakly associated complex that can transiently separate.

Compensatory mutations improve general permissiveness to antibiotic resistance plasmids.

Horizontal gene transfer mediated by broad-host-range plasmids is an important mechanism of antibiotic resistance spread. While not all bacteria maintain plasmids equally well, plasmid persistence can improve over time, yet no general evolutionary mechanisms have emerged. Our goal was to identify these mechanisms and to assess if adaptation to one plasmid affects the permissiveness to others. We experimentally evolved Pseudomonas sp. H2 containing multidrug resistance plasmid RP4, determined plasmid persistence and cost using a joint experimental-modelling approach, resequenced evolved clones, and reconstructed key mutations. Plasmid persistence improved in fewer than 600 generations because the fitness cost turned into a benefit. Improved retention of naive plasmids indicated that the host evolved towards increased plasmid permissiveness. Key chromosomal mutations affected two accessory helicases and the RNA polymerase ß-subunit. Our and other findings suggest that poor plasmid persistence can be caused by a high cost involving helicase-plasmid interactions that can be rapidly ameliorated.