Showing papers by "Suparna Sanyal published in 2016"

Molecular mechanism of viomycin inhibition of peptide elongation in bacteria

Abstract: Viomycin is a tuberactinomycin antibiotic essential for treating multidrug-resistant tuberculosis. It inhibits bacterial protein synthesis by blocking elongation factor G (EF-G) catalyzed translocation of messenger RNA on the ribosome. Here we have clarified the molecular aspects of viomycin inhibition of the elongating ribosome using pre-steady-state kinetics. We found that the probability of ribosome inhibition by viomycin depends on competition between viomycin and EF-G for binding to the pretranslocation ribosome, and that stable viomycin binding requires an A-site bound tRNA. Once bound, viomycin stalls the ribosome in a pretranslocation state for a minimum of ∼45 s. This stalling time increases linearly with viomycin concentration. Viomycin inhibition also promotes futile cycles of GTP hydrolysis by EF-G. Finally, we have constructed a kinetic model for viomycin inhibition of EF-G catalyzed translocation, allowing for testable predictions of tuberactinomycin action in vivo and facilitating in-depth understanding of resistance development against this important class of antibiotics.

Experimental evolution of Escherichia coli harboring an ancient translation protein

Abstract: The ability to design synthetic genes and engineer biological systems at the genome scale opens new means by which to characterize phenotypic states and the responses of biological systems to perturbations. One emerging method involves inserting artificial genes into bacterial genomes, and examining how the genome and its new genes adapt to each other. Here we report the development and implementation of a modified approach to this method, in which phylogenetically inferred genes are inserted into a microbial genome, and laboratory evolution is then used to examine the adaptive potential of the resulting hybrid genome. Specifically, we engineered an approximately 700-million-year old inferred ancestral variant of tufB, an essential gene encoding Elongation Factor Tu, and inserted it in a modern Escherichia coli genome in place of the native tufB gene. While the ancient homolog was not lethal to the cell, it did cause a two-fold decrease in organismal fitness, mainly due to reduced protein dosage. We subsequently evolved replicate hybrid bacterial populations for 2,000 generations in the laboratory, and examined the adaptive response via fitness assays, whole-genome sequencing, proteomics and biochemical assays. We found that the hybrid lineages exhibit a general adaptive strategy in which the fitness cost of the ancient gene was ameliorated in part by up-regulation of protein production. We expect that this ancient-modern recombinant method may pave the way for the synthesis of organisms that exhibit ancient phenotypes, and that laboratory evolution of these organisms may prove useful in elucidating insights into historical adaptive processes.

Experimental evolution of modern Escherichia coli harboring an ancient Elongation Factor gene

Abstract: Species achieve evolutionary innovations through two major genetic mechanisms, namely regulatory- and structural-level mutations. The ability of populations to evolve involves a balance between selection, genetic drift, epistasis, biochemical and biophysical requirements, thermodynamic properties and other factors. This adaptive diversity begs the question as to whether a restricted pathway governs adaptations or whether multiple pathways are possible to achieve an adaptive response. By combining a unique set of tools drawn from synthetic biology, evolutionary biology and genomics, we experimentally evolved and then characterized the adaptive properties of a modern E. coli strain containing a 700 million-year-old reconstructed ancestral Elongation Factor Tu (EF-Tu) gene inserted into its genome for the first time. We then tracked the evolutionary steps taken by the ancient-modern hybrid microorganism through laboratory evolution by monitoring genomic mutations. This study reveals that lineages respond to the ancient gene by increasing the expression levels of the maladapted protein, rather than through direct accumulation of mutations in the open reading frame. In particular, these findings show that the general strategy for the bacteria to adapt to the ancient protein is to accumulate mutations in the cis-regulatory region; gene-coding mutations appear to preclude rapid adaptation upon integration of the ancient gene for our system.