Biology subjects

Seshasayee, A. S. N.

Publications and source records attributed to Seshasayee, A. S. N..

At least 19 recordsLinked to original sources

bioRxiv2026

Phylogenetic evidence for the emergence of bacterial σ-factors by domain fusion

{sigma}-factors are dissociable subunits of RNA polymerase (RNAP), forming the basal regulatory machinery for bacterial transcription. The minimal {sigma}-factors contain two domains (called {sigma}2 or {sigma}4), both of which bind RNAP as well as the promoter DNA through their helix-turn-helix motif. Housekeeping {sigma}-factors contain four domains. We identify proteins containing at least one of the four {sigma}-factor domains in bacterial and bacteriophage genomes; we also show that proteins containing either {sigma}2 or {sigma}4, which are not defined as {sigma}-factors, are commonly found in both sets of organisms. Using phylogenetic analysis of these two domains, we suggest that the highly conserved major housekeeping {sigma}-factor, with its complex domain architecture, represents a late-evolving monophyletic clade, whereas single-domain proteins and minimal {sigma}-factor architectures comprising {sigma}2 and {sigma}4 might be ancestral. Thus, {sigma}-factors probably evolved from single-domain DNA-binding transcription factors by domain accretion.

evolutionary biology↗

bioRxiv2026

Early evolution of the prokaryotic transcription factor repertoire

Transcription initiation is regulated by proteins called transcription factors (TFs). Though TFs help determine phenotype across the tree of life, they are nonessential for minimal cellular life and are often absent in endosymbiotic and parasitic organisms. Given this and the idea that it is a certain level of organism complexity that calls for specific transcription regulation, we traced the evolutionary history of TF repertoire on a bacterio-archaeal tree of life using a dataset of [~]500,000 TFs, grouped into [~]1,700 orthologous groups (OGs) across [~]3,000 species. The most ancestral prokaryotes encoded multiple TFs. Going by known extant functions of these TFs, they possibly regulated sugar-fermentation metabolism, sensed overall metabolic state and redox, responded to DNA damage or bound metals; many of which are consistent with some reconstructions of ancestral gene pools and physiologies. The number of TFs as well as their superfamily-level diversity, through evolutionary history, matches expectations against genome size derived from extant bacteria, suggesting pre-LUCA diversification of TF sequence families. Emergence of new TFs along the phylogeny largely followed a smooth cumulative distribution curve, suggesting steady innovation, early in prokaryote evolution, in contrast to eukaryotes, in which a majority of TF families emerged in a burst manner at the ancestors of multicellular lineages. Gains of TFs late in prokaryotic evolution predominantly featured recycling of protein families discovered elsewhere in the prokaryotic tree, consistent with the dominance of horizontal gene transfer in these organisms. We speculate on the difference between the evolutionary trajectory of prokaryotic TF repertoire and compare it with the eukaryotic TF repertoire trajectory. This helps us in understanding the manner in which their TF repertoires have evolved in two different super-kingdoms. The difference between the evolutionary dynamics of TF-repertoires might be due to how complexity is envisioned in these two different kingdoms.

evolutionary biology↗

bioRxiv2025

Evolutionary analysis of Trehalose breakdown pathways

Trehalose is a widely prevalent, abundant disaccharide that acts as a cellular stress protectant, and functions as an energy source that enters central carbon metabolism when broken down. The evolution and distribution of trehalose breakdown pathways across kingdoms of life have not been studied, and therefore the ability of different organisms to consume trehalose as a carbon source is unknown. In this study, we build a comprehensive evolutionary analysis of the four known trehalose breakdown pathways - trehalase (acid, neutral, glycosyl hydrolase 15), trehalose phosphorylases (TP, treP), and trehalose specific phosphotransferases (PTS), by studying their distributions across ~4000 prokaryotic and eukaryotic genomes. Our study suggests the presence of trehalase in the Last Eukaryotic Common Ancestor (LECA), and reveals near-universal presence of trehalase in eukaryotes, except in all birds where trehalase was lost in the first bird ancestor. Fungi alone retain additional trehalose phosphorylases (TP) in addition to trehalase. In contrast, trehalose breakdown in prokaryotes is highly sporadic but can occur via multiple, independently evolved pathways, including trehalase, the trehalose-specific PTS and trehalose phosphorylase. Finally, we observe that a subset of fast-growing Gammaproteobacteria retain the trehalose specific PTS, the loss of which reduces growth in Escherichia coli. Overall, our findings uncover the evolutionary landscape of trehalose breakdown, and use of this versatile disaccharide as an energy reserve in different kingdoms of life.

genomics↗

bioRxiv2025

The evolution of function in the DNA binding domain of the CRP/FNR family

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 CTD of RNA Polymerase are conserved only in the Eco-CRP class and restricted to Gammaproteobacteria. The corresponding 287 determinant interface on CTD 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 CTD, 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.

bioinformatics↗

bioRxiv2025

Bioenergetic reprogramming of macrophages reduces drug tolerance in Mycobacterium tuberculosis

Eradication of Mycobacterium tuberculosis (Mtb) requires strategies targeting bacteria inside the host. Mtb exhibits heterogeneity in redox metabolism inside macrophages to evade killing by anti-TB drugs. If and how macrophage physiology correlates with bacterial redox heterogeneity and drug tolerance remains unclear. Using a fluorescent reporter of mycobacterial redox potential, flow sorting, and RNA sequencing of infected macrophages, we characterized transcriptional and metabolic responses of macrophages harboring redox-diverse Mtb populations. We found that macrophages with suppressed glycolysis and elevated oxidative phosphorylation (OXPHOS) correlated with Mtb populations exhibiting reductive stress and drug tolerance. Conversely, macrophages with elevated glycolysis and suppressed OXPHOS displayed higher mitochondrial reactive oxygen species through reverse electron transport, resulting in oxidative stress in Mtb and enhancing drug efficacy. Computational and genetic approaches identified Nrf2 as a key regulator of macrophage bioenergetics driving redox heterogeneity and drug tolerance in Mtb. Redirecting macrophage metabolism from OXPHOS to glycolysis using an FDA- approved antiemetic drug, meclizine, subverted redox heterogeneity and diminished drug tolerance in macrophages and mice. The pharmacological profile of meclizine (Cmax and AUClast) indicated no adverse interactions with first-line anti-TB drugs in mice. Our data demonstrate the feasibility of reprogramming macrophage metabolism to reduce drug tolerance in Mtb infection.

microbiology↗

bioRxiv2024

Hydrogen sulfide (H2S) coordinates redox balance, carbon metabolism, and mitochondrial bioenergetics to suppress SARS-CoV-2 infection.

Viruses exploit host metabolism for replication and immune-regulation. Understanding how SARS-CoV-2 alters the host metabolism may lead to treatments for COVID-19. We report that a ubiquitous gaseous molecule, hydrogen sulfide (H2S), regulates redox, metabolism, and mitochondrial bioenergetics to control SARS-CoV-2. Virus replication is associated with down-regulation of the H2S-producing enzymes cystathionine-{beta}-synthase (CBS), cystathionine-{gamma}-lyase (CTH), and 3-mercaptopyruvate sulfurtransferase (3-MST), resulting in diminished endogenous H2S levels. Inhibition of CTH resulted in SARS-CoV-2 proliferation. A slow-releasing H2S donor, GYY4137, diminished virus replication by inducing Nrf2/Keap1 pathway, restoring redox balance and mitochondrial bioenergetics. Treatment of SARS-CoV-2-infected animals with GYY4137 suppressed viral replication, ameliorated respiratory pathology, and restored antioxidant gene expression. Notably, whole-body plethysmography showed improved pulmonary function variables, including pulmonary obstruction and end-expiratory pause upon GYY4137 treatment in vivo. Data extend our understanding of H2S-mediated regulation of viral-infections, and open new avenues for investigating the pathogenic mechanisms and therapeutic opportunities for coronavirus-associated disorders.

microbiology↗

bioRxiv2024

Evolution of transcription factor-containing superfamilies in Eukaryotes

Regulation of gene expression helps determine various phenotypes in most cellular life forms. It is orchestrated at different levels and at the point of transcription initiation by transcription factors (TFs). TFs bind to DNA through domains that are evolutionarily related, by shared membership of the same superfamilies (TF-SFs), to those found in other nucleic acid binding and protein-binding functions (nTFs for non-TFs). Here we ask how TF DNA binding sequence families in eukaryotes have evolved in relation to their nTF relatives. TF numbers scale by power law with the total number of protein-coding genes differently in different clades, with fungi usually showing sub-linear powers whereas chordates show super-linear scaling. The LECA probably encoded a complex regulatory machinery with both TFs and nTFs, but with an excess of nTFs when compared to the relative distribution of TFs and nTFs in extant organisms. Losses drive the evolution of TFs and nTFs, with the possible exception of TFs in Animalia for some tree topologies. TFs are highly dynamic in evolution, showing higher gain and loss rates than nTFs though both are conserved to similar extents. Gains of TFs and nTFs are driven by the appearance of a large number of new sequence clusters in a small number of nodes, which determine the presence of as many as a third of extant TFs and nTFs as well as the relative presence of TFs and nTFs. Whereas nodes showing explosion of TF numbers belong to multicellular clades, those for nTFs lie among the fungi and the protists.

genomics↗

bioRxiv2024

The evolution of sequence specificity in a DNA binding protein family

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 do 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 HU{beta} has been present from the bacterial root, while both IHF subunits emerged much later and diversified in Proteobacteria, with HU 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 IHF 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 IHF{beta} have likely evolved independently, suggesting that each of the IHF subunits has undergone different trajectories to acquire their DNA binding properties.

evolutionary biology↗

bioRxiv2022

Understanding the genome-wide transcription response to varying cAMP levels using phenomenological models in bacteria

Attempts to understand gene regulation by global transcription factors (TF) have largely been limited to expression studies under binary conditions of presence and absence of the TF. Studies addressing genome-wide transcriptional responses to changing TF concentration at high resolution are lacking. Here, we create a dataset containing the entire E.coli transcriptome as it responds to 10 different cAMP concentrations spanning the biological range. We use the Hills model to accurately summarise individual gene responses into 3 intuitively understandable parameters - k, n and Emax reflecting the midpoint of dynamic range, non-linearity and sensitivity of a gene. cAMP-regulated genes show a small dynamic range with midpoints centred around wild-type cAMP concentrations, with genes activating in a switch-like fashion. Using this approach we show that cAMP-CRP affinity at promoters is well correlated to the sensitivity(Emax) of genes but not to the midpoints of dynamic range(k). Finally, genes belonging to different functional classes are tuned to different k, n and Emax. We show phenomenological models to be a better alternative for studying gene expression trends compared to classical clustering methods with the phenomenological constants providing greater insights into how genes are tuned in a regulatory network.

bioinformatics↗

bioRxiv2022

Replication-dependent organisation constrains positioning of long DNA repeats in bacterial genomes

Bacterial genome organisation is primarily driven by chromosome 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 find 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, in 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 minimum disruption to the replication-dependent genome organisation of bacteria, thus acting as a major constraint.

genomics↗

bioRxiv2021

Emergence of networks of shared restriction-modification systems in phage-bacteria ecosystems

1Restriction-modification (RM) systems are the most ubiquitous bacterial defense system against bacteriophages and an important part of controlling phage predation. Using genomic sequence data, we show that RM systems are often shared among bacterial strains in a structured way. Examining the network of interconnections between bacterial strains within each genus, we find that in many genera strains share more RM systems than expected from a random network. We also find that many genera have a larger than expected number of bacterial strains with unique RM systems. We use population dynamics models of closed and open phage-bacteria ecosystems to qualitatively understand the selection pressures that could lead to these non-random network structures with enhanced overlap or uniqueness. In our models we find that the phages impose a pressure that favours bacteria with more RM systems, and more overlap of RM systems with other strains, but in bacteria dominated states this is opposed by the increased cost to the growth rate of these bacteria. Similar to what we observe in the genome data, we find that two distinct bacterial strategies emerge - strains either have a larger overlap than expected, or they have more unique RM systems than one expects from a null model. The former strategy appears to dominate when the repertoire of available RM systems is smaller but the average number of RM systems per strain is larger.

microbiology↗

bioRxiv2020

PAN-INDIA 1000 SARS-CoV-2 RNA Genome Sequencing Reveals Important Insights into the Outbreak

The PAN-INDIA 1000 SARS-CoV-2 RNA Genome Sequencing Consortium has achieved its initial goal of completing the sequencing of 1000 SARS-CoV-2 genomes from nasopharyngeal and oropharyngeal swabs collected from individuals testing positive for COVID-19 by Real Time PCR. The samples were collected across 10 states covering different zones within India. Given the importance of this information for public health response initiatives investigating transmission of COVID-19, the sequence data is being released in GISAID database. This information will improve our understanding on how the virus is spreading, ultimately helping to interrupt the transmission chains, prevent new cases of infection, and provide impetus to research on intervention measures. This will also provide us with information on evolution of the virus, genetic predisposition (if any) and adaptation to human hosts. One thousand and fifty two sequences were used for phylodynamic, temporal and geographic mutation patterns and haplotype network analyses. Initial results indicate that multiple lineages of SARS-CoV-2 are circulating in India, probably introduced by travel from Europe, USA and East Asia. A2a (20A/B/C) was found to be predominant, along with few parental haplotypes 19A/B. In particular, there is a predominance of the D614G mutation, which is found to be emerging in almost all regions of the country. Additionally, mutations in important regions of the viral genome with significant geographical clustering have also been observed. The temporal haplotype diversities landscape in each region appears to be similar pan India, with haplotype diversities peaking between March-May, while by June A2a (20A/B/C) emerged as the predominant one. Within haplotypes, different states appear to have different proportions. Temporal and geographic patterns in the sequences obtained reveal interesting clustering of mutations. Some mutations are present at particularly high frequencies in one state as compared to others. The negative estimate Tajimas D (D = -2.26817) is consistent with the rapid expansion of SARS-CoV-2 population in India. Detailed mutational analysis across India to understand the gradual emergence of mutants at different regions of the country and its possible implication will help in better disease management.

genomics↗

bioRxiv2020

Elucidating the regulatory role of CRP in coordinating protein biosynthesis machinery with metabolism that defines growth optimality in Escherichia coli

Evolution entails the orchestration of cellular resources together with mutations to achieve fitter phenotypes. Here, we determined the system-wide pleiotropic effects that redress the significant perturbations caused by the deletion of global transcriptional regulator CRP in Escherichia coli when evolved in the presence of glucose. We elucidated that absence of CRP results in alterations in key metabolic pathways instrumental for the precise functioning of protein biosynthesis machinery that subsequently corroborated with intracellular metabolite profiles. Apart from acquiring mutations in the promoter of glucose transporter ptsG, the evolved populations recovered the metabolic pathways to their pre-perturbed state with amelioration of protein biosynthesis machinery coupled with fine-tuned proteome re-allocation that enabled growth recovery. However, ineffective utilization of carbon towards biomass as perceived from ATP maintenance flux and costly amino acid accumulations poses a limitation. Overall, we comprehensively illustrate the genetic and metabolic adjustments underlying adaptive evolvability, fundamental for understanding the growth physiology.

systems biology↗

bioRxiv2020

SARS-CoV-2 sequence typing, evolution and signatures of selection using CoVa, a Python-based command-line utility

The current global pandemic COVID-19, caused by SARS-CoV-2, has resulted in millions of infections worldwide in a few months. Global efforts to tackle this situation have produced a tremendous body of genomic data, which can be used for tracing transmission routes, characterization of isolates, and monitoring variants with potential for unusual virulence. Several groups have analyzed these genomes using different approaches. However, as new data become available, the research community needs a pipeline to perform a set of routine analyses, that can quickly incorporate new genome sequences and update the analysis reports. We developed a programmatic tool, CoVa, with this objective. It is a fast, accurate and user-friendly utility to perform a variety of genome analyses on hundreds of SARS-CoV-2 sequences. Using CoVa, we define a modified sequence typing nomenclature and identify sites under positive selection. Further analysis identified some peptides and sites showing geographical patterns of selection. Specifically, we show differences in sequence type distribution between sequences from India and those from the rest of the world. We also show that several sites show signatures of positive selection uniquely in sequences from India. Preliminary evolutionary analysis, using features that will be incorporated into CoVa in the near future, show a mutation rate of 7.4 x 10-4 substitutions/site/year, confirm a temporal signal with a November 2019 origin of SARS-CoV-2, and a heterogeneity in the geographical distribution of Indian samples.

bioinformatics↗

bioRxiv2020

Gcn4 (ATF4) drives a methionine-dependent anabolic program by enabling metabolic precursor supply.

Growth and starvation are considered opposite ends of a spectrum. To sustain growth, cells must manage biomolecule supply to balance constructive metabolism with high translation, through coordinated gene expression programs. Global growth programs couple increased ribosomal biogenesis with sufficient carbon metabolism, amino acid and nucleotide biosynthesis, and how this is collectively managed is a fundamental question. Conventionally, the role of the Gcn4/ATF4 transcription factor has been studied only in the context of amino acid starvation. However, high Gcn4/ATF4 has been observed in contexts of rapid cell proliferation, and the specific role of Gcn4 in growth contexts are unclear. Here, using a methionine-induced growth program in yeast, we show that Gcn4/ATF4 is the fulcrum through which metabolic supply dependent sustenance of translation outputs is maintained. Integrating time-matched transcriptome and ChIP-Seq analysis, we decipher genome-wide direct and indirect roles for Gcn4 in this growth program. Genes that enable metabolic precursor biosynthesis indispensably require Gcn4; contrastingly ribosomal genes are partly repressed by Gcn4. Gcn4 directly binds promoter-regions and transcribes a subset of metabolic genes, particularly driving lysine and arginine biosynthesis. Gcn4 also globally represses lys/arg enriched transcripts, which include the translation machinery. The sustained Gcn4 dependent lys/arg supply is required to maintain sufficient translation capacity, by allowing the synthesis of the translation machinery itself. Gcn4 thereby enables metabolic-precursor supply to bolster protein synthesis, and drive a growth program. Thus, we illustrate how growth and starvation outcomes are both controled using the same Gcn4 transcriptional outputs, in entirely distinct contexts.Competing Interest StatementThe authors have declared no competing interest.View Full Text

systems biology↗

bioRxiv2020

A mutant RNA polymerase activates the general stress response, enabling E. coli adaptation to late prolonged stationary phase

Escherichia coli populations undergo repeated replacement of parental genotypes with fitter variants deep in stationary phase. We isolated one such variant, which emerged after three weeks o of maintaining an E. coli K12 population in stationary phase. This variant displayed a small colony phenotype, slow growth and was able to outcompete its ancestor over a narrow time window in stationary phase. The variant also shows tolerance to beta-lactam antibiotics, though not previously exposed to the antibiotic. We show that an RpoC (A494V) mutation confers the slow growth and small colony phenotype to this variant. The ability of this mutation to confer a growth advantage in stationary phase depends on the availability of the stationary phase sigma factor {sigma}S. The RpoC (A494V) mutation up-regulates the {sigma}S regulon. As shown over 20 years ago, early in prolonged stationary phase, {sigma}S attenuation, but not complete loss of activity, confers a fitness advantage. Our study shows that later mutations enhance {sigma}S activity, either by mutating the gene for {sigma}S directly, or via mutations such as RpoC (A494V). The balance between the activities of the housekeeping major sigma factor and {sigma}S sets up a trade-off between growth and stress tolerance, which is tuned repeatedly during prolonged stationary phase. ImportanceAn important general mechanism of bacterial adaptation to its environment involves adjusting the balance between growing fast, and tolerating stresses. One paradigm where this plays out is in prolonged stationary phase: early studies showed that attenuation, but not complete elimination, of the general stress response enables early adaptation of the bacterium E. coli to the conditions established about 10 days into stationary phase. We show here that this balance is not static and that it is tilted back in favour of the general stress response about two weeks later. This can be established by direct mutations in the master regulator of the general stress response, or by mutations in the core RNA polymerase enzyme itself. These conditions can support the development of antibiotic tolerance though the bacterium is not exposed to the antibiotic. Further exploration of the growth-stress balance over the course of stationary phase will necessarily require a deeper understanding of the events in the extracellular milieu.

microbiology↗

bioRxiv2019

Evolutionary analysis of bacterial Non-Homologous End Joining Repair

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 that 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. To understand 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 rates; 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.

bioinformatics↗

bioRxiv2019

The Evolution and Diversity of Bacterial Transcription Factors across Time-scales

The evolution of bacterial regulatory networks has largely been explained at macroevolutionary scales through lateral gene transfer and gene duplication. Transcription factors (TF) have been found to be less conserved across species than their target genes (TG). This would be expected if TFs accumulate mutations faster than TGs. This hypothesis is supported by several lab evolution studies which found TFs, especially global regulators, to be frequently mutated. Despite these studies, the contribution of point mutations in TFs to the evolution of regulatory network is poorly understood. We tested if TFs show greater genetic variation than their TGs using whole-genome sequencing data from a large collection of E coli isolates. We found TFs to be less diverse, across natural isolates, due to their regulatory roles. TFs were enriched in mutations in multiple adaptive lab evolution studies but not in mutation accumulation. However, over long-term evolution, relative frequency of mutations in TFs showed a gradual decay after a rapid initial burst. Our results suggest that point mutations, conferring large-scale expression changes, may drive the early stages of adaptation but gene regulation is subjected to stronger purifying selection post adaptation.

evolutionary biology↗