Biology subjects

Seshasayee, A. S. N.

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

23 records · Page 2Linked to original sources

bioRxiv2019

Laboratory Evolution Experiments Help Identify a Predominant Site of ΔrnhA-dnaA Constitutive Stable DNA Replication Initiation

The bacterium E. coli can initiate replication in the absence of the replication initiator protein DnaA and / or the canonical origin of replication oriC in a{Delta} rnhA 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 E. coli can find adaptations to loss of fitness caused by cSDR remain inadequately answered. We use laboratory evolution experiments of{Delta} rnhA-{Delta}dnaA followed by deep sequencing to show that DNA replication preferentially initiates within a broad region located [~]0.4-0.7 Mb clockwise of oriC. 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 favour 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 utilise only a miniscule 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. ImportanceThe bacterium E. coli 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 favours cSDR initiation predominantly at a region [~]0.6 Mb clockwise of oriC. 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 minimised. Understanding such evolutionary strategies in the context of cSDR can provide insights into the potential causes of resistance against antibiotics that target initiation of DNA replication.

genomics↗

bioRxiv2018

Modulation of rpoS fitness by loss of cpdA activity during stationary-phase in Escherichia coli

Experimental evolution of Escherichia coli in one month long stationary-phase in lysogeny broth batch cultures repeatedly selected mutations in the genes for the stationary-phase sigma factor RpoS and the cAMP phosphodiesterase CpdA. The founder strain carried a previously identified allele of rpoS, referred to as rpoS819, a partially functional variant that confers growth advantage in stationary-phase (GASP). The 46 base duplication at the 3 end of rpoS819 produces a longer protein present at very low levels compared to wild type RpoS. A new rpoS variant rpoS92, carrying a re-duplication of the original duplication in rpoS819, arose during the first week of our evolution experiment. In rpoS92, an in-frame stop codon truncated RpoS819 creating a shorter RpoS92 whose levels are restored to that of wild type RpoS. Transcription profiling of rpoS92 indicated a shift in gene-expression to that of wild-type rpoS, reversing some of the expression trends of rpoS819. {Delta}3cpdA, carrying an in-frame three base deletion, had arisen late in our evolution experiment. It is a loss of function mutation, which elevates cAMP levels. Using mixed culture competition experiments, we demonstrate that rpoS92 confers GASP, whereas {Delta}3cpdA confers relatively modest GASP in comparison to the ancestral rpoS819. {Delta}3cpdA mediates epistatic repression of rpoS92 GASP. The original survivor carrying both rpoS92 and {Delta}3cpdA besides other mutations displays robust GASP, highlighting the role of these additional mutations in reversing the epistatic interaction between {Delta}3cpdA and rpoS92. In 10- and 20-day old spent media, there is a reduction in the competitive fitness of rpoS92, which is arrested by {Delta}3cpdA. Thus the activity of RpoS fluctuates via genetic mutations in deep stationary phase, and additional mutations in CpdA helps modulate the competitive fitness of RpoS variants.

microbiology↗

bioRxiv2017

Molecular Noise Accelerates Cell Division And Death Under Antibiotic Treatment

It is well known that microbial cell populations can exhibit sustained exponential growth. More surprising is the fact that at high antibiotic levels, cell populations exhibit sustained exponential decay over several orders of magnitude. The boundary between growth and decay occurs at the Minimal Inhibitory Concentration (MIC) of the antibiotic, where the density of living cells remains constant over time. These observations suggest that positive (growth) or negative (decay) exponents arise as a difference of cell division and death rates obeying first-order kinetics. Thus for antibiotic concentrations below MIC, division dominates; for concentrations above MIC, death dominates; while MIC itself is a dynamic steady state of balanced division and death, rather than cell stasis. To measure these rates we separately tracked living and dead cells in Escherichia coli populations treated with the ribosome-targeting antibiotic kanamycin. We found that cells divide rapidly even at MIC: inferred division and death rates at MIC are 0.6 times the antibiotic-free division rate. A stochastic model of cells as collections of self-replicating units we term \"widgets\" reproduces both steady-state and transient features of our experiments, and explains first-order exponential kinetics. In this model cell division and death rates at MIC can be tuned from low to high values by amplifying molecular noise in the synthesis, degradation and partitioning of the widgets. At extremely low noise, cells approach the classic bacteriostatic limit at MIC: neither dividing nor dying. Noise-induced division and death of cells following antibiotic treatment could increase the likelihood of sepsis and antibiotic resistance.

microbiology↗

bioRxiv2017

Repression of YdaS Toxin is Mediated by Transcriptional Repressor RacR in the cryptic rac prophage of Escherichia coli K12

AbstractHorizontal gene transfer is a major driving force behind the genomic diversity seen in prokaryotes. The rac prophage in E.coli K12 encodes a putative transcription factor RacR, whose deletion is lethal. We have shown that the essentiality of racR in E.coli K12 is attributed to its role in transcriptionally repressing a toxin gene called ydaS, which is coded adjacent and divergently to racR.

genetics↗

bioRxiv2017

Genome-wide mutational diversity in Escherichia coli population evolving in prolonged stationary-phase

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 E. coli populations in lysogeny broth. A wide range of mutations accumulate over the course of one month in stationary-phase. SNPs constitute 64% of all mutations. A majority of these mutations are non-synonymous 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 obtained by mutations in our experimental populations can not 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.

genomics↗