Anushree Chatterjee

Convergent transcription confers a bistable switch in Enterococcus faecalis conjugation

Convergent gene pairs with head-to-head configurations are widespread in both eukaryotic and prokaryotic genomes and are speculated to be involved in gene regulation. Here we present a unique mechanism of gene regulation due to convergent transcription from the antagonistic prgX/prgQ operon in Enterococcus faecalis controlling conjugative transfer of the antibiotic resistance plasmid pCF10 from donor cells to recipient cells. Using mathematical modeling and experimentation, we demonstrate that convergent transcription in the prgX/prgQ operon endows the system with the properties of a robust genetic switch through premature termination of elongating transcripts due to collisions between RNA polymerases (RNAPs) transcribing from opposite directions and antisense regulation between complementary counter-transcripts. Evidence is provided for the presence of truncated RNAs resulting from convergent transcription from both the promoters that are capable of sense–antisense interactions. A mathematical model predicts that both RNAP collision and antisense regulation are essential for a robust bistable switch behavior in the control of conjugation initiation by prgX/prgQ operons. Moreover, given that convergent transcription is conserved across species, the mechanism of coupling RNAP collision and antisense interaction is likely to have a significant regulatory role in gene expression.

Mathematical modelling of HCV infection: what can it teach us in the era of direct-acting antiviral agents?

HCV infection is a major cause of chronic liver disease and affects nearly 170 million people worldwide. Whereas the previous standard of care with pegylated interferon and ribavirin had a modest effectiveness, the recent approval of two highly potent protease inhibitors and the ongoing development of dozens of direct-acting antiviral agents (DAAs) constitute a major milestone for HCV therapy. Mathematical modelling of viral kinetics under treatment has played an instrumental role in improving our understanding of virus pathogenesis and in guiding drug development. Here, we review the current state of HCV kinetic modelling, and challenges to the standard biphasic viral decline model that arise when fitting viral kinetic models to data obtained with DAAs.

Antagonistic self-sensing and mate-sensing signaling controls antibiotic-resistance transfer

Conjugation is one of the most common ways bacteria acquire antibiotic resistance, contributing to the emergence of multidrug-resistant “superbugs.” Bacteria of the genus Enterococcus faecalis are highly antibiotic-resistant nosocomial pathogens that use the mechanism of conjugation to spread antibiotic resistance between resistance-bearing donor cells and resistance-deficient recipient cells. Here, we report a unique quorum sensing-based communication system that uses two antagonistic signaling molecules to regulate conjugative transfer of tetracycline-resistance plasmid pCF10 in E. faecalis. A “mate-sensing” peptide sex pheromone produced by recipient cells is detected by donor cells to induce conjugative genetic transfer. Using mathematical modeling and experimentation, we show that a second antagonistic “self-sensing” signaling peptide, previously known to suppress self-induction of donor cells, also serves as a classic quorum-sensing signal for donors that functions to reduce antibiotic-resistance transfer at high donor density. This unique form of quorum sensing may provide a means of limiting the spread of the plasmid and present opportunities to control antibiotic-resistance transfer through manipulation of intercellular signaling, with implications in the clinical setting.

TWEAKING BIOLOGICAL SWITCHES THROUGH A BETTER UNDERSTANDING OF BISTABILITY BEHAVIOR

Many biological events are binary. The switch between mutually exclusive OFF to ON state in response to a stimulus is frequently mediated by a control circuit with a positive and/or a negative feedback. Such a system typically exhibits hysteresis with its switching ON and OFF stimulus levels dependent on the current state of the system. The system can be shown to be bistable both experimentally and mathematically. Work to synthesize such switches by combining natural or engineered components has begun to illustrate the potential of such control circuits in many areas of applications.

Biofilm growth alters regulation of conjugation by a bacterial pheromone

Conjugation is an important mode of horizontal gene transfer in bacteria, enhancing the spread of antibiotic resistance. In clinical settings, biofilms are likely locations for antibiotic resistance transfer events involving nosocomial pathogens such as Enterococcus faecalis. Here we demonstrate that growth in biofilms alters the induction of conjugation by a sex pheromone in E. faecalis. Mathematical modelling suggested that a higher plasmid copy number in biofilm cells would enhance a switch‐like behaviour in the pheromone response of donor cells with a delayed, but increased response to the mating signal. Alterations in plasmid copy number, and a bimodal response to induction of conjugation in populations of plasmid‐containing donor cells were both observed in biofilms, consistent with the predictions of the model. The pheromone system may have evolved such that donor cells in biofilms are only induced to transfer when they are in extremely close proximity to potential recipients in the biofilm community. These results may have important implications for development of chemotherapeutic agents to block resistance transfer and treat biofilm‐related clinical infections.

Bistability versus Bimodal Distributions in Gene Regulatory Processes from Population Balance

In recent times, stochastic treatments of gene regulatory processes have appeared in the literature in which a cell exposed to a signaling molecule in its environment triggers the synthesis of a specific protein through a network of intracellular reactions. The stochastic nature of this process leads to a distribution of protein levels in a population of cells as determined by a Fokker-Planck equation. Often instability occurs as a consequence of two (stable) steady state protein levels, one at the low end representing the “off” state, and the other at the high end representing the “on” state for a given concentration of the signaling molecule within a suitable range. A consequence of such bistability has been the appearance of bimodal distributions indicating two different populations, one in the “off” state and the other in the “on” state. The bimodal distribution can come about from stochastic analysis of a single cell. However, the concerted action of the population altering the extracellular concentration in the environment of individual cells and hence their behavior can only be accomplished by an appropriate population balance model which accounts for the reciprocal effects of interaction between the population and its environment. In this study, we show how to formulate a population balance model in which stochastic gene expression in individual cells is incorporated. Interestingly, the simulation of the model shows that bistability is neither sufficient nor necessary for bimodal distributions in a population. The original notion of linking bistability with bimodal distribution from single cell stochastic model is therefore only a special consequence of a population balance model.

Convergent Transcription in the Butyrolactone Regulon in Streptomyces coelicolor Confers a Bistable Genetic Switch for Antibiotic Biosynthesis

cis-encoded antisense RNAs (cis asRNA) have been reported to participate in gene expression regulation in both eukaryotic and prokaryotic organisms. Its presence in Streptomyces coelicolor has also been reported recently; however, its role has yet to be fully investigated. Using mathematical modeling we explore the role of cis asRNA produced as a result of convergent transcription in scbA-scbR genetic switch. scbA and scbR gene pair, encoding repressor–amplifier proteins respectively, mediates the synthesis of a signaling molecule, the γ-butyrolactone SCB1 and controls the onset of antibiotic production. Our model considers that transcriptional interference caused by convergent transcription of two opposing RNA polymerases results in fatal collision and transcriptional termination, which suppresses transcription efficiency. Additionally, convergent transcription causes sense and antisense interactions between complementary sequences from opposing strands, rendering the full length transcript inaccessible for translation. We evaluated the role of transcriptional interference and the antisense effect conferred by convergent transcription on the behavior of scbA-scbR system. Stability analysis showed that while transcriptional interference affects the system, it is asRNA that confers scbA-scbR system the characteristics of a bistable switch in response to the signaling molecule SCB1. With its critical role of regulating the onset of antibiotic synthesis the bistable behavior offers this two gene system the needed robustness to be a genetic switch. The convergent two gene system with potential of transcriptional interference is a frequent feature in various genomes. The possibility of asRNA regulation in other such gene-pairs is yet to be examined.

Hepatitis C Viral Kinetics: The Past, Present, and Future

Mathematical modeling of hepatitis C viral kinetics has been an important tool in understanding hepatitis C virus (HCV) infection dynamics and in estimating crucial in vivo parameters characterizing the effectiveness of HCV therapy. Because of the introduction of direct-acting antiviral agents, there is a need to extend previous models so as to understand, characterize, and compare various new HCV treatment regimens. Here we review recent modeling efforts in this direction.

RNA-Mediated Reciprocal Regulation between Two Bacterial Operons Is RNase III Dependent

ABSTRACT In bacteria, RNAs regulate gene expression and function via several mechanisms. An RNA may pair with complementary sequences in a target RNA to impact transcription, translation, or degradation of the target. Control of conjugation of pCF10, a pheromone response plasmid of Enterococcus faecalis, is a well-characterized system that serves as a model for the regulation of gene expression in bacteria by intercellular signaling. The prgQ operon, whose products mediate conjugation, is negatively regulated by two products of the prgX operon, Anti-Q, a small RNA, and PrgX, the transcriptional repressor of the prgQ promoter. Here we show that Qs, an RNA from the 5′ end of the prgQ operon, represses expression of PrgX by targeting prgX mRNA for cleavage by RNase III. Our results demonstrate that the prgQ and prgX operons each use RNAs to negatively regulate gene expression from the opposing operon by different mechanisms. Such reciprocal regulation between two operons using RNAs has not been previously demonstrated. Furthermore, these results show that Qs is an unusually versatile RNA, serving three separate functions in the regulation of conjugation. Understanding the potential versatility of RNAs and their various roles in gene regulatory networks will allow us to better understand how cells regulate complex behavior. IMPORTANCE Bacteria use RNA to regulate gene expression by a variety of mechanisms. The prgQ and prgX operons of pCF10, a conjugative plasmid of Enterococcus faecalis, have been shown to negatively regulate one another by a variety of mechanisms. One of these mechanisms involves Anti-Q, a small RNA from the prgX operon that prevents gene expression from the prgQ operon. In this work, we find that Qs, an RNA from the prgQ operon, negatively regulates gene expression from the prgX operon. These findings have a number of implications. (i) The Anti-Q and Qs RNAs act by different mechanisms, highlighting the variety of ways in which bacteria can regulate gene expression using RNAs. (ii) Reciprocal regulation between operons mediated by small RNAs has not been previously described, deepening our understanding of how bacteria regulate complex behavior. (iii) Additional roles for Qs have been described, demonstrating the versatility of this RNA. Bacteria use RNA to regulate gene expression by a variety of mechanisms. The prgQ and prgX operons of pCF10, a conjugative plasmid of Enterococcus faecalis, have been shown to negatively regulate one another by a variety of mechanisms. One of these mechanisms involves Anti-Q, a small RNA from the prgX operon that prevents gene expression from the prgQ operon. In this work, we find that Qs, an RNA from the prgQ operon, negatively regulates gene expression from the prgX operon. These findings have a number of implications. (i) The Anti-Q and Qs RNAs act by different mechanisms, highlighting the variety of ways in which bacteria can regulate gene expression using RNAs. (ii) Reciprocal regulation between operons mediated by small RNAs has not been previously described, deepening our understanding of how bacteria regulate complex behavior. (iii) Additional roles for Qs have been described, demonstrating the versatility of this RNA.

CRISPR Perturbation of Gene Expression Alters Bacterial Fitness under Stress and Reveals Underlying Epistatic Constraints

The evolution of antibiotic resistance has engendered an impending global health crisis that necessitates a greater understanding of how resistance emerges. The impact of nongenetic factors and how they influence the evolution of resistance is a largely unexplored area of research. Here we present a novel application of CRISPR-Cas9 technology for investigating how gene expression governs the adaptive pathways available to bacteria during the evolution of resistance. We examine the impact of gene expression changes on bacterial adaptation by constructing a library of deactivated CRISPR-Cas9 synthetic devices to tune the expression of a set of stress-response genes in Escherichia coli. We show that artificially inducing perturbations in gene expression imparts significant synthetic control over fitness and growth during stress exposure. We present evidence that these impacts are reversible; strains with synthetically perturbed gene expression regained wild-type growth phenotypes upon stress removal, while maintaining divergent growth characteristics under stress. Furthermore, we demonstrate a prevailing trend toward negative epistatic interactions when multiple gene perturbations are combined simultaneously, thereby posing an intrinsic constraint on gene expression underlying adaptive trajectories. Together, these results emphasize how CRISPR-Cas9 can be employed to engineer gene expression changes that shape bacterial adaptation, and present a novel approach to synthetically control the evolution of antimicrobial resistance.