@article {161133, title = {Bioenergetic reprogramming of macrophages reduces drug tolerance in Mycobacterium tuberculosis.}, journal = {Nat Commun}, volume = {16}, year = {2025}, month = {2025 Oct 23}, pages = {9370}, abstract = {

Effective clearance of Mycobacterium tuberculosis (Mtb) requires targeting drug-tolerant populations within host macrophages. Here, we show that macrophage metabolic states govern redox heterogeneity and drug response in intracellular Mtb. Using a redox-sensitive fluorescent reporter (Mrx1-roGFP2), flow cytometry, and transcriptomics, we found that macrophages with high oxidative phosphorylation (OXPHOS) and low glycolysis harbor reductive, drug-tolerant Mtb, whereas glycolytically active macrophages generate mitochondrial ROS via reverse electron transport, imposing oxidative stress on Mtb and enhancing drug efficacy. Computational and genetic analyses identified NRF2 as a key regulator linking host metabolism to bacterial redox state and drug tolerance. Pharmacological reprogramming of macrophages with the FDA-approved drug meclizine (MEC) shifted metabolism towards glycolysis, suppressed redox heterogeneity, and reduced Mtb drug tolerance in macrophages and mice. MEC exhibited no adverse interactions with frontline anti-TB drugs. These findings demonstrate the therapeutic potential of host metabolic reprogramming to overcome Mtb drug tolerance.

}, keywords = {Animals, Antitubercular Agents, Drug Tolerance, Energy Metabolism, Female, Glycolysis, Humans, Macrophages, Mice, Mice, Inbred C57BL, Mitochondria, Mycobacterium tuberculosis, NF-E2-Related Factor 2, Oxidation-Reduction, Oxidative Phosphorylation, Oxidative Stress, Reactive Oxygen Species, Tuberculosis}, issn = {2041-1723}, doi = {10.1038/s41467-025-64407-w}, author = {Yadav, Vikas and Sahoo, Sarthak and Malhotra, Nitish and Mishra, Richa and Sreedharan, Sreesa and Rajmani, Raju S and Shanmugam, Siva and Shandil, Radha K and Narayanan, Shridhar and Thacker, Vivek V and Laxman, Sunil and Jolly, Mohit Kumar and Seshasayee, Aswin Sai Narain and Singh, Amit} } @article {160481, title = {The evolution of function in the DNA binding domain of the CRP/FNR family.}, journal = {Genome Biol Evol}, year = {2025}, month = {2025 Sep 25}, abstract = {

Transcriptional regulation, facilitated by transcription factors (TFs), contributes to bacterial response to environmental and cellular perturbations. How have TFs diversified and gained their functions? We use the CRP/FNR family to study this question across \~{}6000 prokaryotic genomes. Characterized by homology to TFs CRP (cyclic AMP Receptor Protein) and FNR (Regulator of Fumarate and Nitrate Reductase) in Escherichia coli, the two functions of this family - sequence-specific DNA binding and transcriptional activation by direct contacts to RNA polymerase - are conferred by the helix-turn-helix containing DNA binding domain. We constructed a rooted phylogeny of this domain and performed residue conservation analysis on extant and in-silico reconstructed ancestral sequences of CRP/FNR family members. Residues that confer DNA binding specificity likely emerged in the overall ancestor of the sequence family. This ancestor does not fall into classes containing either E. coli CRP (Eco-CRP) or E. coli FNR (Eco-FNR) clades, but instead a large and diverse class that we call "CRP-like". Residues key for base-specific DNA binding remain largely conserved in the family. Residues (together called AR1) which make direct contacts with αCTD of RNA Polymerase are conserved only in the Eco-CRP class and restricted to Gammaproteobacteria. The corresponding {\textquoteright}287 determinant{\textquoteright} interface on αCTD is again fully conserved only in Gammaproteobacteria, which contain Eco-CRP members, following step-wise evolution initiated at the Proteobacterial common ancestor. These suggest suggesting co-emergence of the interface residues in Eco-CRP. Our analysis hence shows that Class I transcriptional activation through AR1 on CRP and the determinant of αCTD, as described in E. coli CRP, is phylogenetically restricted, while base-specific DNA binding has been present in the CRP/FNR family throughout its evolutionary history.

}, issn = {1759-6653}, doi = {10.1093/gbe/evaf182}, author = {Krishnaswamy, Madhumitha and Seshasayee, Aswin Sai Narain} } @article {157490, title = {The Evolution of Sequence Specificity in a DNA Binding Protein Family.}, journal = {J Mol Biol}, volume = {437}, year = {2025}, month = {2025 Jul 15}, pages = {169177}, abstract = {

Transcriptional regulation enables bacteria to adjust to its environment. This is driven by transcription factors (TFs), which display DNA site recognition specificity with some flexibility built in. TFs, however, are not considered essential to a minimal cellular life. How did they evolve? It has been hypothesized that TFs evolve by gaining specificity (and other functions) on a background of non-specific chromosome structuring proteins. We used the IHF/HU family of DNA binding proteins, in which IHF binds DNA in a sequence-specific manner, whereas HU binds more indiscriminately, to test this hypothesis. We show that HUβ has been present from the bacterial root, while both IHF subunits emerged much later and diversified in Proteobacteria, with HUα having possibly arisen from transfer events in Gammaproteobacteria. By reconstructing ancestral sequences in-silico on a rooted phylogeny of IHF/HU we show that the common ancestor of this family was probably HU-like and therefore non-specific in binding DNA. IHF evolved from a branch of HU after HU had substantially diverged. Various residues characteristic of IHFα and shown to be involved in specific sequence recognition (at least in E. coli) have likely been co-opted from preexisting residues in HU, while those residues of IHFβ have likely evolved independently, suggesting that each of the IHF subunits has undergone different trajectories to acquire their DNA binding properties.

}, keywords = {HU; IHF; bacteria; evolution; transcription factor.}, issn = {1089-8638}, doi = {10.1016/j.jmb.2025.169177}, url = {https://www.sciencedirect.com/science/article/pii/S0022283625002438?via\%3Dihub}, author = {Nandy, Meghna and Krishnaswamy, Madhumitha and Sharda, Mohak and Seshasayee, Aswin Sai Narain} } @article {147303, title = {Intracellular peroxynitrite perturbs redox balance, bioenergetics, and Fe-S cluster homeostasis in Mycobacterium tuberculosis.}, journal = {Redox Biol}, volume = {75}, year = {2024}, month = {2024 Sep}, pages = {103285}, abstract = {

The ability of Mycobacterium tuberculosis (Mtb) to tolerate nitric oxide (NO) and superoxide (O) produced by phagocytes contributes to its success as a human pathogen. Recombination of NO and O generates peroxynitrite (ONOO), a potent oxidant produced inside activated macrophages causing lethality in diverse organisms. While the response of Mtb toward NO and O is well established, how Mtb responds to ONOO remains unclear. Filling this knowledge gap is important to understand the persistence mechanisms of Mtb during infection. We synthesized a series of compounds that generate both NO and O, which should combine to produce ONOO. From this library, we identified CJ067 that permeates Mtb to reliably enhance intracellular ONOO levels. CJ067-exposed Mtb strains, including multidrug-resistant (MDR) and extensively drug-resistant (XDR) clinical isolates, exhibited dose-dependent, long-lasting oxidative stress and growth inhibition. In contrast, Mycobacterium smegmatis (Msm), a fast-growing, non-pathogenic mycobacterial species, maintained redox balance and growth in response to intracellular ONOO. RNA-sequencing with Mtb revealed that CJ067 induces antioxidant machinery, sulphur metabolism, metal homeostasis, and a 4Fe-4S cluster repair pathway (suf operon). CJ067 impaired the activity of the 4Fe-4S cluster-containing TCA cycle enzyme, aconitase, and diminished bioenergetics of Mtb. Work with Mtb strains defective in SUF and IscS involved in Fe-S cluster biogenesis pathways showed that both systems cooperatively protect Mtb from intracellular ONOO in vitro and inducible nitric oxide synthase (iNOS)-dependent growth inhibition during macrophage infection. Thus, Mtb is uniquely sensitive to intracellular ONOO and targeting Fe-S cluster homeostasis is expected to promote iNOS-dependent host immunity against tuberculosis (TB).

}, keywords = {Energy Metabolism, Homeostasis, Humans, Iron-Sulfur Proteins, Macrophages, Mycobacterium smegmatis, Mycobacterium tuberculosis, Nitric Oxide, Oxidation-Reduction, Oxidative Stress, Peroxynitrous Acid, Superoxides, Tuberculosis}, issn = {2213-2317}, doi = {10.1016/j.redox.2024.103285}, author = {Dewan, Arshiya and Jain, Charu and Das, Mayashree and Tripathi, Ashutosh and Sharma, Ajay Kumar and Singh, Harshit and Malhotra, Nitish and Seshasayee, Aswin Sai Narain and Chakrapani, Harinath and Singh, Amit} } @article {137347, title = {Cysteine desulfurase (IscS)-mediated fine-tuning of bioenergetics and SUF expression prevents hypervirulence.}, journal = {Sci Adv}, volume = {9}, year = {2023}, month = {2023 Dec 15}, pages = {eadh2858}, abstract = {

Iron-sulfur (Fe-S) biogenesis requires multiprotein assembly systems, SUF and ISC, in most prokaryotes. () encodes a complete SUF system, the depletion of which was bactericidal. The ISC operon is truncated to a single gene (cysteine desulfurase), whose function remains uncertain. Here, we show that Δ is bioenergetically deficient and hypersensitive to oxidative stress, antibiotics, and hypoxia. Δ resisted killing by nitric oxide (NO). RNA sequencing indicates that IscS is important for expressing regulons of DosR and Fe-S-containing transcription factors, WhiB3 and SufR. Unlike wild-type , Δ could not enter a stable persistent state, continued replicating in mice, and showed hypervirulence. The operon was overexpressed in Δ during infection in a NO-dependent manner. Suppressing expression in Δ either by CRISPR interference or upon infection in inducible NO-deficient mice arrests hypervirulence. Together, redesigned the ISC system to "fine-tune" the expression of SUF machinery for establishing persistence without causing detrimental disease in the host.

}, keywords = {Animals, Energy Metabolism, Escherichia coli, Iron-Sulfur Proteins, Mice, Mycobacterium tuberculosis}, issn = {2375-2548}, doi = {10.1126/sciadv.adh2858}, author = {Das, Mayashree and Sreedharan, Sreesa and Shee, Somnath and Malhotra, Nitish and Nandy, Meghna and Banerjee, Ushashi and Kohli, Sakshi and Rajmani, Raju S and Chandra, Nagasuma and Seshasayee, Aswin Sai Narain and Laxman, Sunil and Singh, Amit} } @article {113538, title = {Replication-dependent organisation constrains positioning of long DNA repeats in bacterial genomes.}, journal = {Genome Biol Evol}, year = {2022}, month = {2022 Jul 01}, abstract = {

Bacterial genome organisation is primarily driven by chromosomal replication from a single origin of replication. However chromosomal rearrangements, which can disrupt such organisation, are inevitable in nature. Long DNA repeats are major players mediating rearrangements, large and small, via homologous recombination. Since changes to genome organisation affect bacterial fitness - and more so in fast-growing than slow-growing bacteria - and are under selection, it is reasonable to expect that genomic positioning of long DNA repeats is also under selection. To test this, we identified identical DNA repeats of at least 100 base pairs across \~{}6,000 bacterial genomes and compared their distribution in fast and slow-growing bacteria. We found that long identical DNA repeats are distributed in a non-random manner across bacterial genomes. Their distribution differs in their overall number, orientation and proximity to the origin of replication, between fast and slow-growing bacteria. We show that their positioning - which might arise from a combination of the processes that produce repeats and selection on rearrangements that recombination between repeat elements might cause - permits less disruption to the replication-dependent genome organisation of bacteria compared to random suggesting it as a major constraint to positioning of long DNA repeats.

}, issn = {1759-6653}, doi = {10.1093/gbe/evac102}, author = {Malhotra, Nitish and Seshasayee, Aswin Sai Narain} } @article {120170, title = {Understanding the Genome-Wide Transcription Response To Various cAMP Levels in Bacteria Using Phenomenological Models.}, journal = {mSystems}, year = {2022}, month = {2022 Nov 21}, pages = {e0090022}, abstract = {

Attempts to understand gene regulation by global transcription factors have largely been limited to expression studies under binary conditions of presence and absence of the transcription factor. Studies addressing genome-wide transcriptional responses to changing transcription factor concentration at high resolution are lacking. Here, we create a data set containing the entire Escherichia coli transcriptome in Luria-Bertani (LB) broth as it responds to 10 different cAMP concentrations spanning the biological range. We use the Hill{\textquoteright}s model to accurately summarize individual gene responses into three intuitively understandable parameters, , , and , reflecting the sensitivity, nonlinearity, and midpoint of the dynamic range. Our data show that most cAMP-regulated genes have an of \>2, with their values centered around the wild-type concentration of cAMP. Additionally, cAMP receptor protein (CRP) affinity to a promoter is correlated with but not , hinting that a high-affinity CRP promoter need not ensure transcriptional activation at lower cAMP concentrations and instead affects the magnitude of the response. Finally, genes belonging to different functional classes are tuned to have different , , and values. We demonstrate that phenomenological models are a better alternative for studying gene expression trends than classical clustering methods, with the phenomenological constants providing greater insights into how genes are tuned in a regulatory network. Different genes may follow different trends in response to various transcription factor concentrations. In this study, we ask two questions: (i) what are the trends that different genes follow in response to changing transcription factor concentrations and (ii) what methods can be used to extract information from the gene trends so obtained. We demonstrate a method to analyze transcription factor concentration-dependent genome-wide expression data using phenomenological models. Conventional clustering methods and principal-component analysis (PCA) can be used to summarize trends in data but have limited interpretability. The use of phenomenological models greatly enhances the interpretability and thus utility of conventional clustering. Transformation of dose-response data into phenomenological constants opens up avenues to ask and answer many different kinds of question. We show that the phenomenological constants obtained from the model fits can be used to generate insights about network topology and allows integration of other experimental data such as chromatin immunoprecipitation sequencing (ChIP-seq) to understand the system in greater detail.

}, issn = {2379-5077}, doi = {10.1128/msystems.00900-22}, author = {Chakraborty, Shweta and Singh, Parul and Seshasayee, Aswin Sai Narain} } @article {72074, title = {Early fate of exogenous promoters in E.~coli.}, journal = {Nucleic Acids Res}, year = {2020}, month = {2020 Jan 21}, abstract = {

Gene gain by horizontal gene transfer is a major pathway of genome innovation in bacteria. The current view posits that acquired genes initially need to be silenced and that a bacterial chromatin protein, H-NS, plays a role in this silencing. However, we lack direct observation of the early fate of a horizontally transferred gene to prove this theory. We combine sequencing, flow cytometry and sorting, followed by microscopy to monitor gene expression and its variability after large-scale random insertions of a reporter gene in a population of Escherichia coli bacteria. We find that inserted promoters have a wide range of gene-expression variability related to their location. We find that high-expression clones carry insertions that are not correlated with H-NS binding. Conversely, binding of H-NS correlates with silencing. Finally, while most promoters show a common level of extrinsic noise, some insertions show higher noise levels. Analysis of these high-noise clones supports a scenario of switching due to transcriptional interference from divergent ribosomal promoters. Altogether, our findings point to evolutionary pathways where newly-acquired genes are not necessarily silenced, but may immediately explore a wide range of expression levels to probe the optimal ones.

}, issn = {1362-4962}, doi = {10.1093/nar/gkz1196}, author = {Yousuf, Malikmohamed and Iuliani, Ilaria and Veetil, Reshma T and Seshasayee, Aswin Sai Narain and Sclavi, Bianca and Cosentino Lagomarsino, Marco} } @article {84079, title = {Evolutionary and Comparative analysis of bacterial Non-Homologous End Joining Repair.}, journal = {Genome Biol Evol}, year = {2020}, month = {2020 Oct 20}, abstract = {

DNA double-strand breaks (DSBs) are a threat to genome stability. In all domains of life, DSBs are faithfully fixed via homologous recombination. Recombination requires the presence of an uncut copy of duplex DNA which is used as a template for repair. Alternatively, in the absence of a template, cells utilize error-prone Non-homologous end joining (NHEJ). Although ubiquitously found in eukaryotes, NHEJ is not universally present in bacteria. It is unclear as to why many prokaryotes lack this pathway. Towards understanding what could have led to the current distribution of bacterial NHEJ, we carried out comparative genomics and phylogenetic analysis across \~{}6000 genomes. Our results show that this pathway is sporadically distributed across the phylogeny. Ancestral reconstruction further suggests that NHEJ was absent in the eubacterial ancestor, and can be acquired via specific routes. Integrating NHEJ occurrence data for archaea, we also find evidence for extensive horizontal exchange of NHEJ genes between the two kingdoms as well as across bacterial clades. The pattern of occurrence in bacteria is consistent with correlated evolution of NHEJ with key genome characteristics of genome size and growth rate; NHEJ presence is associated with large genome sizes and/or slow growth rates, with the former being the dominant correlate. Given the central role these traits play in determining the ability to carry out recombination, it is possible that the evolutionary history of bacterial NHEJ may have been shaped by requirement for efficient DSB repair.

}, issn = {1759-6653}, doi = {10.1093/gbe/evaa223}, author = {Sharda, Mohak and Badrinarayanan, Anjana and Seshasayee, Aswin Sai Narain} } @article {73644, title = {Laboratory Evolution Experiments Help Identify a Predominant Region of Constitutive Stable DNA Replication Initiation.}, journal = {mSphere}, volume = {5}, year = {2020}, month = {2020 Feb 26}, abstract = {

The bacterium can initiate replication in the absence of the replication initiator protein DnaA and/or the canonical origin of replication in a background. This phenomenon, which can be primed by R-loops, is called constitutive stable DNA replication (cSDR). Whether DNA replication during cSDR initiates in a stochastic manner through the length of the chromosome or at specific sites and how can find adaptations to loss of fitness caused by cSDR remain inadequately answered. We use laboratory evolution experiments of strains followed by deep sequencing to show that DNA replication preferentially initiates within a broad region located \~{}0.4 to 0.7 Mb clockwise of This region includes many bisulfite-sensitive sites, which have been previously defined as R-loop-forming regions, and includes a site containing sequence motifs that favor R-loop formation. Initiation from this region would result in head-on replication-transcription conflicts at rRNA loci. Inversions of these rRNA loci, which can partly resolve these conflicts, help the bacterium suppress the fitness defects of cSDR. These inversions partially restore the gene expression changes brought about by cSDR. The inversion, however, increases the possibility of conflicts at essential mRNA genes, which would utilize only a minuscule fraction of RNA polymerase molecules, most of which transcribe rRNA genes. Whether subsequent adaptive strategies would attempt to resolve these conflicts remains an open question. The bacterium can replicate its DNA even in the absence of the molecules that are required for canonical replication initiation. This often requires the formation of RNA-DNA hybrid structures and is referred to as constitutive stable DNA replication (cSDR). Where on the chromosome does cSDR initiate? We answer this question using laboratory evolution experiments and genomics and show that selection favors cSDR initiation predominantly at a region \~{}0.6 Mb clockwise of Initiation from this site will result in more head-on collisions of DNA polymerase with RNA polymerase operating on rRNA loci. The bacterium adapts to this problem by inverting a region of the genome including several rRNA loci such that head-on collisions between the two polymerases are minimized. Understanding such evolutionary strategies in the context of cSDR can provide insights into the potential causes of resistance to antibiotics that target initiation of DNA replication.

}, issn = {2379-5042}, doi = {10.1128/mSphere.00939-19}, author = {Veetil, Reshma T and Malhotra, Nitish and Dubey, Akshara and Seshasayee, Aswin Sai Narain} } @article {84075, title = {Whole-Genome Sequencing of sp. Strain RSMS, a Highly Efficient Tributyl Phosphate-Degrading Bacterium.}, journal = {Microbiol Resour Announc}, volume = {9}, year = {2020}, month = {2020 Oct 15}, abstract = {

sp. strain RSMS was described earlier as an efficient degrader of tributyl phosphate, an organic pollutant. This report describes the generation and annotation of the genome sequence of sp. strain RSMS, which will facilitate future studies to identify genetic elements responsible for the degradation of tributyl phosphate.

}, issn = {2576-098X}, doi = {10.1128/MRA.00600-20}, author = {Rangu, Shyam Sunder and Beck, Ashish and Sharda, Mohak and Mukhopadhyaya, Rita and Seshasayee, Aswin Sai Narain and Rath, Devashish} } @article {67836, title = {Targeting redox heterogeneity to counteract drug tolerance in replicating .}, journal = {Sci Transl Med}, volume = {11}, year = {2019}, month = {2019 Nov 13}, abstract = {

The capacity of () to tolerate multiple antibiotics represents a major problem in tuberculosis (TB) management. Heterogeneity in populations is one of the factors that drives antibiotic tolerance during infection. However, the mechanisms underpinning this variation in bacterial population remain poorly understood. Here, we show that phagosomal acidification alters the redox physiology of to generate a population of replicating bacteria that display drug tolerance during infection. RNA sequencing of this redox-altered population revealed the involvement of iron-sulfur (Fe-S) cluster biogenesis, hydrogen sulfide (HS) gas, and drug efflux pumps in antibiotic tolerance. The fraction of the pH- and redox-dependent tolerant population increased when infected macrophages with actively replicating HIV-1, suggesting that redox heterogeneity could contribute to high rates of TB therapy failure during HIV-TB coinfection. Pharmacological inhibition of phagosomal acidification by the antimalarial drug chloroquine (CQ) eradicated drug-tolerant , ameliorated lung pathology, and reduced postchemotherapeutic relapse in in vivo models. The pharmacological profile of CQ ( and AUC) exhibited no major drug-drug interaction when coadministered with first line anti-TB drugs in mice. Our data establish a link between phagosomal pH, redox metabolism, and drug tolerance in replicating and suggest repositioning of CQ to shorten TB therapy and achieve a relapse-free cure.

}, issn = {1946-6242}, doi = {10.1126/scitranslmed.aaw6635}, author = {Mishra, Richa and Kohli, Sakshi and Malhotra, Nitish and Bandyopadhyay, Parijat and Mehta, Mansi and Munshi, MohamedHusen and Adiga, Vasista and Ahuja, Vijay Kamal and Shandil, Radha K and Rajmani, Raju S and Seshasayee, Aswin Sai Narain and Singh, Amit} } @article {33715, title = {Regulation of Global Transcription in by Rsd and 6S RNA.}, journal = {G3 (Bethesda)}, year = {2018}, month = {2018 Apr 23}, abstract = {

In , the sigma factor σ directs RNA polymerase to transcribe growth-related genes, while σ directs transcription of stress response genes during stationary phase. Two molecules hypothesized to regulate RNA polymerase are the protein Rsd, which binds to σ, and the non-coding 6S RNA which binds to the RNA polymerase-σ holoenzyme. Despite multiple studies, the functions of Rsd and 6S RNA remain controversial. Here we use RNA-Seq in five phases of growth to elucidate their function on a genome-wide scale. We show for the first time that Rsd and 6S RNA facilitate σ activity throughout bacterial growth, while 6S RNA also regulates widely different genes depending upon growth phase. We discover novel interactions between 6S RNA and Rsd and show widespread expression changes in a strain lacking both regulators. Finally, we present a mathematical model of transcription which highlights the crosstalk between Rsd and 6S RNA as a crucial factor in controlling sigma factor competition and global gene expression.

}, issn = {2160-1836}, doi = {10.1534/g3.118.200265}, author = {Lal, Avantika and Krishna, Sandeep and Seshasayee, Aswin Sai Narain} } @article {33713, title = {Reply to Jobling, "Ectopic Expression of the and Genes of the Cryptic Prophage Rac of K-12 May Be Toxic but Do They Really Encode Toxins?: a Case for Using Genetic Context To Understand Function".}, journal = {mSphere}, volume = {3}, year = {2018}, month = {2018 Apr 25}, issn = {2379-5042}, doi = {10.1128/mSphere.00177-18}, author = {Seshasayee, Aswin Sai Narain} } @article {53990, title = {Using stochastic cell division and death to probe minimal units of cellular replication.}, journal = {New J Phys}, volume = {20}, year = {2018}, month = {2018 Mar}, pages = {035004}, abstract = {

The invariant cell initiation mass measured in bacterial growth experiments has been interpreted as a minimal unit of cellular replication. Here we argue that the existence of such minimal units induces a coupling between the rates of stochastic cell division and death. To probe this coupling we tracked live and dead cells in populations treated with a ribosome-targeting antibiotic. We find that the growth exponent from macroscopic cell growth or decay measurements can be represented as the difference of microscopic first-order cell division and death rates. The boundary between cell growth and decay, at which the number of live cells remains constant over time, occurs at the minimal inhibitory concentration (MIC) of the antibiotic. This state appears macroscopically static but is microscopically dynamic: division and death rates exactly cancel at MIC but each is remarkably high, reaching 60\% of the antibiotic-free division rate. A stochastic model of cells as collections of minimal replicating units we term {\textquoteright}widgets{\textquoteright} reproduces both steady-state and transient features of our experiments. Sub-cellular fluctuations of widget numbers stochastically drive each new daughter cell to one of two alternate fates, division or death. First-order division or death rates emerge as eigenvalues of a stationary Markov process, and can be expressed in terms of the widget{\textquoteright}s molecular properties. High division and death rates at MIC arise due to low mean and high relative fluctuations of widget number. Isolating cells at the threshold of irreversible death might allow molecular characterization of this minimal replication unit.

}, issn = {1367-2630}, doi = {10.1088/1367-2630/aab197}, author = {Chib, Savita and Das, Suman and Venkatesan, Soumya and Seshasayee, Aswin Sai Narain and Thattai, Mukund} } @article {27275, title = {CRISPR-Cas-Mediated Gene Silencing Reveals RacR To Be a Negative Regulator of YdaS and YdaT Toxins in Escherichia coli K-12.}, journal = {mSphere}, volume = {2}, year = {2017}, month = {2017 Nov-Dec}, abstract = {

Bacterial genomes are rich in horizontally acquired prophages. racR is an essential gene located in the rac prophage that is resident in many Escherichia coli genomes. Employing a clustered regularly interspaced short palindromic repeat (CRISPR)-Cas-based gene silencing approach, we show that RacR is a negative regulator of the divergently transcribed and adjacent ydaS-ydaT operon in Escherichia coli K-12. Overexpression of YdaS and YdaT due to RacR depletion leads to cell division defects and decrease in survival. We further show that both YdaS and YdaT can act independently as toxins and that RacR serves to counteract the toxicity by tightly downregulating the expression of these toxins. IMPORTANCEracR is an essential gene and one of the many poorly studied genes found on the rac prophage element that is present in many Escherichia coli genomes. Employing a CRISPR-based approach, we have silenced racR expression to various levels and elucidated its physiological consequences. We show that the downregulation of racR leads to upregulation of the adjacent ydaS-ydaT operon. Both YdaS and YdaT act as toxins by perturbing the cell division resulting in enhanced cell killing. This work establishes a physiological role for RacR, which is to keep the toxic effects of YdaS and YdaT in check and promote cell survival. We, thus, provide a rationale for the essentiality of racR in Escherichia coli K-12 strains.

}, issn = {2379-5042}, doi = {10.1128/mSphere.00483-17}, author = {Bindal, Gargi and Krishnamurthi, Revathy and Seshasayee, Aswin Sai Narain and Rath, Devashish} } @article {18677, title = {Genomewide Mutational Diversity in Escherichia coli Population Evolving in Prolonged Stationary Phase.}, journal = {mSphere}, volume = {2}, year = {2017}, month = {2017 May-Jun}, abstract = {

Prolonged stationary phase is an approximation of natural environments presenting a range of stresses. Survival in prolonged stationary phase requires alternative metabolic pathways for survival. This study describes the repertoire of mutations accumulating in starving Escherichia\ coli populations in lysogeny broth. A wide range of mutations accumulates over the course of 1\ month in stationary phase. Single nucleotide polymorphisms (SNPs) constitute 64\% of all mutations. A majority of these mutations are nonsynonymous and are located at conserved loci. There is an increase in genetic diversity in the evolving populations over time. Computer simulations of evolution in stationary phase suggest that the maximum frequency of mutations observed in our experimental populations cannot be explained by neutral drift. Moreover, there is frequent genetic parallelism across populations, suggesting that these mutations are under positive selection. Finally, functional analysis of mutations suggests that regulatory mutations are frequent targets of selection. IMPORTANCE Prolonged stationary phase in bacteria, contrary to its name, is highly dynamic, with extreme nutrient limitation as a predominant stress. Stationary-phase cultures adapt by rapidly selecting a mutation(s) that confers a growth advantage in stationary phase (GASP). The phenotypic diversity of starving E.\ coli populations has been studied in detail; however, only a few mutations that accumulate in prolonged stationary phase have been described. This study documented the spectrum of mutations appearing in Escherichia coli during 28\ days of prolonged starvation. The genetic diversity of the population increases over time in stationary phase to an extent that cannot be explained by random, neutral drift. This suggests that prolonged stationary phase offers a great model system to study adaptive evolution by natural selection.

}, doi = {10.1128/mSphere.00059-17}, author = {Chib, Savita and Ali, Farhan and Seshasayee, Aswin Sai Narain} } @article {28248, title = {Modulation of Global Transcriptional Regulatory Networks as a Strategy for Increasing Kanamycin Resistance of the Translational Elongation Factor-G Mutants in Escherichia coli.}, journal = {G3 (Bethesda)}, volume = {7}, year = {2017}, month = {2017 Dec 04}, pages = {3955-3966}, abstract = {

Evolve and resequence experiments have provided us a tool to understand bacterial adaptation to antibiotics. In our previous work, we used short-term evolution to isolate mutants resistant to the ribosome targeting antibiotic kanamycin, and reported that Escherichia coli develops low cost resistance to kanamycin via different point mutations in the translation Elongation Factor-G (EF-G). Furthermore, we had shown that the resistance of EF-G mutants could be increased by second site mutations in the genes rpoD/cpxA/topA/cyaA Mutations in three of these genes had been discovered in earlier screens for aminoglycoside resistance. In this work, we expand our understanding of these second site mutations, the goal being to understand how these mutations affect the activities of the mutated gene products to confer resistance. We show that the mutation in cpxA most likely results in an active Cpx stress response. Further evolution of an EF-G mutant in a higher concentration of kanamycin than what was used in our previous experiments identified the cpxA locus as a primary target for a significant increase in resistance. The mutation in cyaA results in a loss of catalytic activity and probably results in resistance via altered CRP function. Despite a reduction in cAMP levels, the CyaAN600Y mutant has a transcriptome indicative of increased CRP activity, pointing to an unknown role for CyaA and / or cAMP in gene expression. From the transcriptomes of double and single mutants, we describe the epistasis between the mutation in EF-G and these second site mutations. We show that the large scale transcriptomic changes in the topoisomerase I (FusAA608E-TopAS180L) mutant likely result from increased negative supercoiling in the cell. Finally, genes with known roles in aminoglycoside resistance were present among the misregulated genes in the mutants.

}, issn = {2160-1836}, doi = {10.1534/g3.117.300284}, author = {Mogre, Aalap and Veetil, Reshma T and Seshasayee, Aswin Sai Narain} } @article {22811, title = {Nucleoid-Associated Proteins: Genome Level Occupancy and Expression Analysis.}, journal = {Methods Mol Biol}, volume = {1624}, year = {2017}, month = {2017}, pages = {85-97}, abstract = {

The advent of Chromatin Immunoprecipitation sequencing (ChIP-Seq) has allowed the identification of genomic regions bound by a DNA binding protein in-vivo on a genome-wide scale. The impact of the DNA binding protein on gene expression can be addressed using transcriptome experiments in appropriate genetic settings. Overlaying the above two sources of data enables us to dissect the direct and indirect effects of a DNA binding protein on gene expression. Application of these techniques to Nucleoid Associated Proteins (NAPs) and Global Transcription Factors (GTFs) has underscored the complex relationship between DNA-protein interactions and gene expression change, highlighting the role of combinatorial control. Here, we demonstrate the usage of ChIP-Seq to infer binding properties and transcriptional effects of NAPs such as Fis and HNS, and the GTF CRP in the model organism Escherichia coli K12 MG1655 (E. coli).

}, issn = {1940-6029}, doi = {10.1007/978-1-4939-7098-8_8}, author = {Singh, Parul and Seshasayee, Aswin Sai Narain} } @article {27276, title = {Repression of YdaS Toxin Is Mediated by Transcriptional Repressor RacR in the Cryptic rac Prophage of Escherichia coli K-12.}, journal = {mSphere}, volume = {2}, year = {2017}, month = {2017 Nov-Dec}, abstract = {

Horizontal gene transfer is a major driving force behind the genomic diversity seen in prokaryotes. The cryptic rac prophage in Escherichia\ coli K-12 carries the gene for a putative transcription factor RacR, whose deletion is lethal. We have shown that the essentiality of racR in E.\ coli K-12 is attributed to its role in transcriptionally repressing toxin gene(s) called ydaS and ydaT, which are adjacent to and coded divergently to racR. IMPORTANCE Transcription factors in the bacterium E.\ coli are rarely essential, and when they are essential, they are largely toxin-antitoxin systems. While studying transcription factors encoded in horizontally acquired regions in E.\ coli, we realized that the protein RacR, a putative transcription factor encoded by a gene on the rac prophage, is an essential protein. Here, using genetics, biochemistry, and bioinformatics, we show that its essentiality derives from its role as a transcriptional repressor of the ydaS and ydaT genes, whose products are toxic to the cell. Unlike type II toxin-antitoxin systems in which transcriptional regulation involves complexes of the toxin and antitoxin, repression by RacR is sufficient to keep ydaS transcriptionally silent.

}, issn = {2379-5042}, doi = {10.1128/mSphere.00392-17}, author = {Krishnamurthi, Revathy and Ghosh, Swagatha and Khedkar, Supriya and Seshasayee, Aswin Sai Narain} } @article {5304, title = {Comparative Genomics of Interreplichore Translocations in Bacteria: A Measure of Chromosome Topology?}, journal = {G3 (Bethesda)}, volume = {6}, year = {2016}, month = {2016}, pages = {1597-606}, abstract = {

Genomes evolve not only in base sequence but also in terms of their architecture, defined by gene organization and chromosome topology. Whereas genome sequence data inform us about the changes in base sequences for a large variety of organisms, the study of chromosome topology is restricted to a few model organisms studied using microscopy and chromosome conformation capture techniques. Here, we exploit whole genome sequence data to study the link between gene organization and chromosome topology in bacteria. Using comparative genomics across \~{}250 pairs of closely related bacteria we show that: (a) many organisms show a high degree of interreplichore translocations throughout the chromosome and not limited to the inversion-prone terminus (ter) or the origin of replication (oriC); (b) translocation maps may reflect chromosome topologies; and (c) symmetric interreplichore translocations do not disrupt the distance of a gene from oriC or affect gene expression states or strand biases in gene densities. In summary, we suggest that translocation maps might be a first line in defining a gross chromosome topology given a pair of closely related genome sequences.

}, issn = {2160-1836}, doi = {10.1534/g3.116.028274}, author = {Khedkar, Supriya and Seshasayee, Aswin Sai Narain} } @article {905, title = {The genome-scale interplay amongst xenogene silencing, stress response and chromosome architecture in Escherichia coli.}, journal = {Nucleic Acids Res}, volume = {43}, year = {2015}, month = {2015 Jan}, pages = {295-308}, abstract = {

The gene expression state of exponentially growing Escherichia coli cells is manifested by high expression of essential and growth-associated genes and low levels of stress-related and horizontally acquired genes. An important player in maintaining this homeostasis is the H-NS-StpA gene silencing system. A Δhns-stpA deletion mutant results in high expression of otherwise-silent horizontally acquired genes, many located in the terminus-half of the chromosome, and an indirect downregulation of many highly expressed genes. The Δhns-stpA double mutant displays slow growth. Using laboratory evolution we address the evolutionary strategies that E. coli would adopt to redress this gene expression imbalance. We show that two global gene regulatory mutations-(i) point mutations inactivating the stress-responsive sigma factor RpoS or σ38 and (ii) an amplification of \~{}40\% of the chromosome centred around the origin of replication-converge in partially reversing the global gene expression imbalance caused by Δhns-stpA. Transcriptome data of these mutants further show a three-way link amongst the global gene regulatory networks of H-NS and σ38, as well as chromosome architecture. Increasing gene expression around the terminus of replication results in a decrease in the expression of genes around the origin and vice versa; this appears to be a persistent phenomenon observed as an association across \~{}300 publicly-available gene expression data sets for E. coli. These global suppressor effects are transient and rapidly give way to more specific mutations, whose roles in reversing the growth defect of H-NS mutations remain to be understood.

}, keywords = {Chromosomes, Bacterial, Directed Molecular Evolution, DNA-Binding Proteins, DNA-Directed RNA Polymerases, Escherichia coli, Escherichia coli Proteins, Gene Deletion, Gene Expression Regulation, Bacterial, Gene Silencing, Genome, Bacterial, Molecular Chaperones, Sigma Factor, Stress, Physiological, Transcription, Genetic}, issn = {1362-4962}, doi = {10.1093/nar/gku1229}, author = {Srinivasan, Rajalakshmi and Scolari, Vittore Ferdinando and Lagomarsino, Marco Cosentino and Seshasayee, Aswin Sai Narain} }