Biology subjects

Chib, S.

Publications and source records attributed to Chib, S..

5 recordsLinked to original sources

bioRxiv2026

Unwinding of an RNA duplex by the Orthoflavivirus NS3 helicase requires translocation beyond the displaced strand and is stimulated by the NS5 RdRp

The NS3 helicases from the Flaviviridae family of viruses exhibit nucleotide-hydrolysis-dependent, nucleic-acid-unwinding activity. The RNA unwinding activity for NS3 helicases from the Orthoflavivirus genus has not been fully explored and contrasts with NS3 helicase from Hepatitis C virus (HCV) of the Hepacivirus genus, which has thus far served as the prototypical model enzyme from this family of viruses. To begin to understand the functional differences between flavivirus NS3 helicases, we first developed an expression and purification system for full-length untagged NS3 protein from West Nile virus (WNV) and Zika virus (ZIKV). Both enzymes exhibit RNA-stimulated ATPase activity and are dependent on the nucleoside triphosphatase active site of the enzyme. Unlike HCV NS3, orthoflavivirus NS3s do not efficiently pre-assemble on a 3-ssRNA-tailed dsRNA substrate in the absence of ATP-Mg which is a prerequisite for formation of a productive HCV NS3-RNA complex that can exhibit a rapid burst of RNA unwinding. Instead, to observe RNA unwinding by WNV and ZIKV NS3s, low Mg-ATP concentrations are required at a time coincident when NS3 encounters the RNA substrate. In addition, we find that orthoflavivirus NS3s require translocation beyond the displaced strand to completely unwind a dsRNA substrate. Last, we find that orthoflavivirus NS5 stimulates the ability of NS3 to unwind dsRNA. These results suggest that functional differences exist between the flavivirus NS3 helicases and illuminate that orthoflavivirus NS3s require a functional interaction with the NS5 protein for coordination of its activity, as it is believed these two proteins constitute the viral replicase.

biochemistry↗

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↗

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

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↗