Biology subjects

Kralj, J. M.

Publications and source records attributed to Kralj, J. M..

6 recordsLinked to original sources

bioRxiv2026

Context-dependent peptide recognition shapes tyrosine kinase substrate specificity beyond consensus motifs

Protein tyrosine kinases (PTKs) regulate cellular biochemistry by phosphorylating tyrosine residues that alter protein function; their substrate preferences define the topology of signaling cascades. Previous studies of PTKs have mapped their average preferences for amino acids surrounding phosphorylation sites, but their sensitivity to sequence variation remains poorly understood. Here, we used microbial biosensors for PTK activity to examine the influence of local sequence context on substrate specificity. Across five well-studied PTKs, we identified amino acid substitutions within consensus substrates that could confer sensitivity to substrate length or enhance selectivity for one PTK over others. Using a secondary decoy screen, we found sequence-diverse substrates with unexpectedly orthogonal PTK compatibilities. Our findings show how context-specific sequence features alter PTK substrate specificity far beyond what might be expected from classical consensus models and establish an experimental framework for defining the limits of substrate overlap between closely related kinases.

biochemistry↗

bioRxiv2023

High Throughput Viability Assay for Microbiology

Counting viable cells is a universal practice in microbiology. The colony forming unit (CFU) assay has remained the gold standard to measure viability across disciplines; however, it is time-intensive and resource-consuming. Herein, we describe the Geometric Viability Assay (GVA) that replicates CFU measurements over 6-orders of magnitude while reducing over 10-fold the time and consumables. GVA computes a samples viable cell count based on the distribution of embedded colonies growing inside a pipette tip. GVA is compatible with gram-positive and -negative planktonic bacteria, biofilms, and yeast. Laborious CFU experiments such as checkerboard assays, treatment time-courses, and drug screens against slow-growing cells are simplified by GVA. We therefore screened a drug library against exponential and stationary phase E. coli leading to the discovery of the ROS-mediated, bactericidal mechanism of diphenyliodonium. The ease and low cost of GVA evinces it can accelerate existing viability assays and enable measurements at previously impractical scales.

microbiology↗

bioRxiv2021

Aminoglycosides induce a bacterial senescent state that increases antibiotic tolerance in treatment-naïve cells

1.Bacterial evolution of antibiotic resistance is facilitated by non-genetic resistance that increases drug tolerance, buying time for evolutionary innovation. Escherichia coli treated with aminoglycosides permanently lose the ability to divide within four hours, yet we discovered a majority of cells maintain membrane integrity and metabolic activity greater than two days post treatment - a bacterial senescent-like state. These cells, which we term zombies, exhibit dynamic gene expression and metabolomic profiles, even after irreversible exit from the cell cycle. Our data reveal zombies upregulate the phage shock protein pathway to maintain membrane integrity. Remarkably, though unable to form new colonies, zombies increase the antibiotic tolerance of treatment-naive cells, implying chemical communication. Chemical supplementation and genetic knockouts show that zombies communicate with treatment-naive cells by secreting indole. In summary, our study revealed a bacterial senescent-like state, induced by aminoglycosides, that decreases the antibiotic susceptibility of multiple bacterial species. Thus, E. coli zombies utilize paracrine signaling to promote non-genetic antibiotic tolerance.

microbiology↗

bioRxiv2020

Genome wide functional screen for calcium transients in E. coli identifies decreased membrane potential adaptation to persistent DNA damage

1.Calcium plays numerous critical roles in signaling and homeostasis in eukaryotic cells. Unlike eukaryotic cells, far less is known about calcium signaling in bacteria, and few genes controlling influx and efflux have been identified. Previous work in Escherichia coli showed calcium influx is induced by voltage depolarization, which were enhanced by mechanical stimulation, suggesting a role in bacterial mechanosensation. To identify proteins and pathways affecting calcium handling in bacteria, we designed a live cell screen to monitor calcium dynamics in single cells across a genome wide knockout panel in E. coli. The screen measured cells from the Keio collection of knockouts and quantified calcium transients across the population. Overall, we found 143 gene knockouts that decreased calcium transients, and 32 genes knockouts that increased transients. Knockouts involved in energy production and regulation appeared, as expected, as well as knockouts of the voltage sink, the F1Fo-ATPase. Knockouts in exopolysaccharide and outer membrane synthesis showed reduced transients and refined our model of electrophysiology mediated mechanosensation in E. coli. Additionally, knockouts annotated in DNA repair had reduced calcium transients and voltage. However, acute DNA damage did not affect voltage, and suggested that only long term adaptation to DNA damage decreased membrane potential and calcium transients. Our work showed a distinct separation between the acute and long term DNA damage responses in bacteria, which has implications for mitochondrial DNA damage in eukaryotes. ImportanceAll eukaryotic cells use calcium as a critical signaling molecule. There is tantalizing evidence that bacteria also use calcium for cellular signaling, but much less is known about the molecular actors and physiological roles. To identify genes regulating cytoplasmic calcium in Escherichia coli, we created a single cell screen for modulators of calcium dynamics. The genes uncovered in this screen helped refine a model for voltage mediated bacterial mechanosensation. Additionally, we were able to more carefully dissect the mechanisms of adaptation to long term DNA damage, which has implications for both bacteria and mitochondria in the face of unrepaired DNA.

microbiology↗

bioRxiv2020

Membrane voltage dysregulation driven by metabolic dysfunction underlies bactericidal activity of aminoglycosides

Aminoglycosides are broad-spectrum antibiotics whose mechanism of bactericidal activity has been under debate. It is widely accepted, however, that membrane voltage potentiates aminoglycoside activity, which is ascribed to voltage dependent drug uptake. In this paper, we measured the single cell response of Escherichia coli treated with aminoglycosides and discovered that the bactericidal action arises not from the downstream effects of voltage dependent drug uptake, but rather directly from dysregulated membrane potential. In the absence of voltage, aminoglycosides are taken into cells and exert bacteriostatic effects by inhibiting translation. However, cell killing was immediate upon re-polarization. The hyperpolarization arose from altered ATP flux, which induced a reversal of the F1Fo-ATPase to hydrolyze ATP and generated the deleterious voltage. Heterologous expression of an ATPase inhibitor from Salmonella completely eliminated bactericidal activity, while loss of the F-ATPase significantly reduced the electrophysiological response to aminoglycosides. Our data support a model of voltage induced death, which could be resolved in real-time at the single cell level, and separates the mechanisms of aminoglycoside bacteriostasis and bactericide in E. coli.

microbiology↗

bioRxiv2018

All-optical electrophysiology for high-throughput functional characterization of human iPSC-derived motor neuron model of ALS

Human induced pluripotent stem cell (iPSC)-derived neurons are an attractive substrate for modeling disease, yet the heterogeneity of these cultures presents a challenge for functional characterization by manual patch clamp electrophysiology. Here we describe an optimized all-optical electrophysiology, \"Optopatch\", pipeline for high-throughput functional characterization of human iPSC-derived neuronal cultures. We demonstrate the method in a human iPSC-derived motor neuron model of ALS. In a comparison of neurons with an ALS-causing mutation (SOD1 A4V) with their genome-corrected controls, the mutants showed elevated spike rates under weak or no stimulus, and greater likelihood of entering depolarization block under strong optogenetic stimulus. We compared these results to numerical simulations of simple conductance-based neuronal models and to literature results in this and other iPSC-based models of ALS. Our data and simulations suggest that deficits in slowly activating potassium channels may underlie the changes in electrophysiology in the SOD1 A4V mutation.

neuroscience↗