Biology subjects

Rinehart, J.

Publications and source records attributed to Rinehart, J..

4 recordsLinked to original sources

bioRxiv2024

Genomically recoded Escherichia coli with optimized functional phenotypes

Genomically recoded organisms hold promise for many biotechnological applications, but they may exhibit substantial fitness defects relative to their non-recoded counterparts. We used targeted metabolic screens, genetic analysis, and proteomics to identify the origins of fitness impairment in a model recoded organism, Escherichia coli C321.{Delta}A. We found that defects in isoleucine biosynthesis and release factor activity, caused by mutations extant in all K-12 lineage strains, elicited profound fitness impairments in C321.{Delta}A, suggesting that genome recoding exacerbates suboptimal traits present in precursor strains. By correcting these and other C321.{Delta}A-specific mutations, we engineered C321.{Delta}A strains with doubling time reductions of 17% and 42% in rich and minimal medium, respectively, compared to ancestral C321. Strains with improved growth kinetics also demonstrated enhanced ribosomal non-standard amino acid incorporation capabilities. Proteomic analysis indicated that C321.{Delta}A lacks the ability to regulate essential amino acid and nucleotide biosynthesis pathways, and that targeted mutation reversion restored regulatory capabilities. Our work outlines a strategy for the rapid and precise phenotypic optimization of genomically recoded organisms and other engineered microbes.

synthetic biology↗

bioRxiv2022

Cell type-independent profiling of interactions between intracellular pathogens and the human phosphoproteome

Interactions between proteins from intracellular pathogens and host proteins in an infected cell are often mediated by post-translational modifications encoded in the host proteome. Identifying protein modifications, such as phosphorylation, that dictate these interactions remains a defining challenge in unraveling the molecular mechanisms of pathogenesis. We have developed a platform in engineered bacteria that displays over 110,000 phosphorylated human proteins coupled to a fluorescent reporter system capable of identifying the host-pathogen interactome of phosphoproteins (H-PIP). This resource broadly enables cell-type independent interrogation and discovery of proteins from intracellular pathogens capable of binding phosphorylated human proteins. As an example of the H-PIP platform, we generated a unique, high-resolution SARS-CoV-2 interaction network which expanded our knowledge of viral protein function and identified understudied areas of host pathology.

synthetic biology↗

bioRxiv2021

Principles for Systematic Optimization of an Orthogonal Translation System with Enhanced Biological Tolerance

Over the past twenty years, the development of orthogonal biological systems has sparked a revolution in our ability to study cellular physiology. Orthogonal translation systems (OTSs) enable site-specific incorporation of hundreds of non-standard amino acids, offering unprecedented access to the study of cellular mechanisms modulated by post-translational modifications (e.g. protein phosphorylation). Although development of phosphoserine-OTSs (pSerOTS) has been significant, little work has focused on the biology of OTS development and utilization. To better understand the impact of OTSs on host physiology, we utilize pSerOTS as a model to systematically explore the extent to which OTS components interact with Escherichia coli. Using this information, we constructed pSerOTS variants designed to enhance OTS orthogonality by minimizing interactions with host processes and decreasing stress response activation. Our expanded understanding of OTS:host interactions enables informed OTS design practices which minimize the negative impact of OTSs while improving OTS performance across a range of experimental settings.

synthetic biology↗

bioRxiv2020

Hyperosmotic stress induces downstream-of-gene transcription and alters the RNA Polymerase II interactome despite widespread transcriptional repression

SummaryStress-induced readthrough transcription results in the synthesis of thousands of downstream-of-gene (DoG) containing transcripts. The mechanisms underlying DoG formation during cellular stress remain unknown. Nascent transcription profiles during DoG induction in human cell lines using TT-TimeLapse-seq revealed that hyperosmotic stress induces widespread transcriptional repression. Yet, DoGs are produced regardless of the transcriptional level of their upstream genes. ChIP-seq confirmed that the stress-induced redistribution of RNA Polymerase (Pol) II correlates with the transcriptional output of genes. Stress-induced alterations in the Pol II interactome are observed by mass spectrometry. While subunits of the cleavage and polyadenylation machinery remained Pol II-associated, Integrator complex subunits dissociated from Pol II under stress conditions. Depleting the catalytic subunit of the Integrator complex, Int11, using siRNAs induces hundreds of readthrough transcripts, whose parental genes partially overlap those of stress-induced DoGs. Our results provide insights into the mechanisms underlying DoG production and how Integrator activity influences DoG transcription.In brief Rosa-Mercado et al. report that hyperosmotic stress causes widespread transcriptional repression in human cells, yet DoGs arise regardless of the transcriptional response of their upstream genes. They find that the interaction between Pol II and Integrator is disrupted by hypertonicity and that knocking down the Integrator nuclease leads to DoG production.HighlightsHyperosmotic stress triggers transcriptional repression of many genes.DoG RNAs arise independent of the transcriptional level of their upstream gene.The interaction between Pol II and Integrator subunits decreases after salt stress.Depletion of the Int11 nuclease subunit induces the production of hundreds of DoGs.Competing Interest StatementThe authors have declared no competing interest.View Full Text

molecular biology↗