@article {133186, title = {Biosensor-integrated transposon mutagenesis reveals as a coordinator of redox homeostasis in .}, journal = {Elife}, volume = {12}, year = {2023}, month = {2023 Aug 29}, abstract = {

() is evolutionarily equipped to resist exogenous reactive oxygen species but shows vulnerability to an increase in endogenous ROS (eROS). Since eROS is an unavoidable consequence of aerobic metabolism, understanding how manages eROS levels is essential yet needs to be characterized. By combining the Mrx1-roGFP2 redox biosensor with transposon mutagenesis, we identified 368 genes (redoxosome) responsible for maintaining homeostatic levels of eROS in . Integrating redoxosome with a global network of transcriptional regulators revealed a hypothetical protein (Rv0158) as a critical node managing eROS in Disruption of ( KO) impaired growth, redox balance, respiration, and metabolism of on glucose but not on fatty acids. Importantly, KO exhibited enhanced growth on propionate, and the Rv0158 protein directly binds to methylmalonyl-CoA, a key intermediate in propionate catabolism. Metabolite profiling, ChIP-Seq, and gene-expression analyses indicate that Rv0158 manages metabolic neutralization of propionate toxicity by regulating the methylcitrate cycle. Disruption of enhanced the sensitivity of to oxidative stress, nitric oxide, and anti-TB drugs. Lastly, KO showed poor survival in macrophages and persistence defect in mice. Our results suggest that Rv0158 is a metabolic integrator for carbon metabolism and redox balance in .

}, issn = {2050-084X}, doi = {10.7554/eLife.80218}, author = {Shee, Somnath and Veetil, Reshma T and Mohanraj, Karthikeyan and Das, Mayashree and Malhotra, Nitish and Bandopadhyay, Devleena and Beig, Hussain and Birua, Shalini and Niphadkar, Shreyas and Nagarajan, Sathya Narayanan and Sinha, Vikrant Kumar and Thakur, Chandrani and Rajmani, Raju S and Chandra, Nagasuma and Laxman, Sunil and Singh, Mahavir and Samal, Areejit and Seshasayee, Aswin N and Singh, Amit} } @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 {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 {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} }