Biology subjects

Kannoly, S.

Publications and source records attributed to Kannoly, S..

7 recordsLinked to original sources

bioRxiv2026

A Data-Analysis Pipeline for High-Throughput Systematic Evolution of Ligands by Exponential Enrichment (HT-SELEX) in the Characterization of Telomeric Proteins

Telomeres are nucleoprotein structures at the ends of eukaryotic chromosomes that safeguard them from triggering inappropriate DNA damage signaling. POT1, a member of the mammalian shelterin complex, binds single-stranded (ss) telomeric DNA and blocks the activation of the ATR kinase-mediated DNA damage response at telomeres. Yet until recently, it was poorly understood how the double-stranded (ds)-ss telomeric junction was protected from DNA damage response factors. An initial study of the DNA-binding activity of human POT1 (hPOT1) using systematic evolution of ligands by exponential enrichment (SELEX) and subsequent investigation revealed that POT1 contains a binding pocket, known as the POT-hole, that binds the 5 phosphorylated dC of the telomeric ds-ss junction. The amino acid residues composing the POT-hole show full sequence identity with telomeric proteins from diverse eukaryotes, including Caenorhabditis elegans POT-1. The current study builds on this SELEX method, developing an extensive analysis pipeline for SELEX datasets sequenced by next-generation sequencing and achieving a deeper analysis of the resulting sequences. We validated our approach by applying it to the DNA-binding domain of hPOT1, yielding results consistent with a previous SELEX study. Furthermore, we employ our pipeline to characterize the DNA-binding activity of C. elegans proteins that are considered homologs of hPOT1: POT-1, POT-2, POT-3, and MRT-1. Our analysis suggests that all four proteins show a binding preference for G-enriched DNA sequences, with POT-1 additionally binding secondary structural elements. Overall, we present a bioinformatics pipeline that is accessible and applicable for determining the nucleic acid-binding properties of a variety of proteins.

biochemistry↗

bioRxiv2025

A Chemostat-Based Model for Growing Bacterial Biofilms

Biofilms are groups of microbes that live together in dense communities, often attached to a surface. They play an outsized role in all aspects of microbial life, from chronic infections to biofouling to dental decay. In recent decades, appreciation for the diversity of roles that biofilms play in the environment has grown. Yet, most bacterial studies still rely upon approaches developed in the 19th century and center on planktonic populations alone. Here we present a chemostat-based experimental platform to investigate not only biofilms themselves, but how they interact with their surrounding environments. Our results show that biofilms grow to larger sizes in chemostats as opposed to flasks. In addition, we show that biofilms may be a consistent source of migrants into planktonic populations. We also show that secondary biofilms rapidly develop, although these may be more susceptible to environmental conditions. Taken together, our data suggest that chemostats may be a flexible and insightful platform for the study of biofilms in vitro. IMPORTANCEBiofilms are the predominant way that bacteria live in natural environments and are characterized by three emergent properties: ubiquity, resilience, and impact. They can be found across all environments, both natural and human-made, and across all of recorded time, dating back at least 3.5 billion years. Biofilms also represent a major economic impact of over $5 trillion annually. Yet, most of what is known about bacteria is the result of studies using planktonically growing liquid cultures under laboratory conditions. Here, we propose a comprehensive experimental platform that allows for study of biofilms on the molecular, organismal, and community levels using chemostats.

microbiology↗

bioRxiv2025

Translation Efficiency Impacts Phage Lysis Timing and Its Precision in Single Cells

The timing of host cell lysis is a fundamental life history parameter in bacteriophages as it presents an evolutionary trade-off between maximizing intracellular phage replication and optimizing transmission to new host cells, ultimately determining viral fitness in dynamic bacterial populations. In the bacteriophage {lambda}, lysis time is dependent on the expression of a timekeeper protein, holin, which accumulates in the Escherichia coli inner membrane. Cell lysis is triggered when the membrane concentration of holin crosses a critical threshold level. In the present study, we investigated the effects of the rate of holin translation on lysis timing and its precision. We show that modulating holin translation efficiency through genetic modifications and antibiotic treatment alters bacteriophage lysis timing and its precision, providing insight into how phages optimize the evolutionary trade-off between replication and transmission. Reducing ribosomal binding affinity decreased holin expression and delayed lysis, while optimizing the Shine-Dalgarno sequence enhanced translation and accelerated lysis. Surprisingly, very low tetracycline concentrations may have improved translation efficiency and hastened lysis, whereas higher doses predictably delayed it. In all cases, longer lysis times corresponded with decreased timing variability. A model that incorporates stochastic gene expression with additional stochasticity in the initiation of holin expression was sufficient to explain the data. Thus, we demonstrated that modifying the rate of holin translation can be used as an evolutionary strategy to achieve optimum lysis timing. ImportanceOur prior work demonstrated that phage lysis timing is calibrated to optimize fitness based on environmental conditions affecting host availability and phage replication. We identified an optimal threshold--the critical holin concentration--that minimizes lysis time variability. Genetic manipulation of the holin gene altered the lysis threshold and generated phages with non-optimal early and late lysis times. The current study reveals that adjusting holin translation rates offers another mechanism for modifying lysis timing. Phages therefore have multiple means to adjust the timing of host lysis in order to maximize fitness under different selective pressures.

microbiology↗

bioRxiv2021

A single-cell approach reveals variation in cellular phage-producing capacities

Virus burst size, a component of viral fitness, is the average number of viral particles released from a single infected cell. In this study, we estimated bacteriophage lambda ({lambda}) burst size mean and distribution at different lysis times. To estimate phage {lambda} burst sizes at single-cell level, we employed a lysis-deficient E. coli lysogen, which allowed chemical lysis at desired times after the induction of lytic cycle. Induced cultures of E. coli lysogen were diluted and aliquoted into the wells of 96-well plates. A high dilution rate results in mostly empty wells and minimizes the probability of having multiple cells in wells that do receive cells. Burst size was estimated by titering single-cell lysates obtained after chemical lysis at desired times. Our data shows that the viral burst size initially increases exponentially with the lysis time, and then saturates at longer lysis times. We also demonstrate that cell-to-cell variation or "noise" in lysis timing does not significantly contribute to the burst size noise. The burst size noise remains constant with increasing mean burst size. The most likely explanation for the experimentally observed constant burst size noise is that cell-to-cell differences in burst size originate from differences in cellular capacity to produce phages. The mean burst size measured at different lysis times is positively correlated to cell volume, which may determine the cellular phage production capacity. However, experiments controlling for cell size indicates that there are other factors in addition to cell size that determine this cellular capacity. ARTICLE IMPORTANCEPhages produce offspring by hijacking a cells replicative machinery. Previously, it was noted that the variation in the number of phages produced by single infected cells far exceeded cell size variation. It has been hypothesized that this variation is a consequence of variation in the timing of host cell lysis. Here we show that cell-to-cell variation in lysis timing does not significantly contribute to the burst size variation. We suggest that the constant burst size variation across different host lysis times results from cell-to-cell differences in capacity to produce phages. We find that the mean burst size measured at different lysis times is positively correlated to cell volume, which may determine the cellular phage production capacity. However, experiments controlling for cell size indicates that there are other factors in addition to cell size that determine this cellular capacity.

microbiology↗

bioRxiv2020

The role of incoherent feedforward circuits in regulating precision of event timing

Triggering of cellular events often relies on the level of a key gene product crossing a critical threshold. Achieving precision in event timing in spite of noisy gene expression facilitates high-fidelity functioning of diverse processes from biomolecular clocks, apoptosis and cellular differentiation. Here we investigate the role of an incoherent feedforward circuit in regulating the time taken by a bacterial virus (bacteriophage lambda) to lyse an infected Escherichia coli cell. Lysis timing is the result of expression and accumulation of a single lambda protein (holin) in the E. coli cell membrane up to a critical threshold level, which triggers the formation of membrane lesions. This easily visualized process provides a simple model system for characterizing event-timing stochasticity in single cells. Intriguingly, lambdas lytic pathway synthesizes two functionally opposite proteins: holin and antiholin from the same mRNA in a 2:1 ratio. Antiholin sequesters holin and inhibits the formation of lethal membrane lesions, thus creating an incoherent feedforward circuit. We develop and analyze a stochastic model for this feedforward circuit that considers correlated bursty expression of holin/antiholin, and their concentrations are diluted from cellular growth. Interestingly, our analysis shows the noise in timing is minimized when both proteins are expressed at an optimal ratio, hence revealing an important regulatory role for antiholin. These results are in agreement with single cell data, where removal of antiholin results in enhanced stochasticity in lysis timing.

systems biology↗

bioRxiv2020

An optimal lysis time maximizes bacteriophage fitness in quasi-continuous culture

Optimality models have a checkered history in evolutionary biology. While optimality models have been successful in providing valuable insight into the evolution of a wide variety of biological traits, a common objection is that optimality models are overly simplistic and ignore organismal genetics. We revisit evolutionary optimization in the context of a major bacteriophage life history trait, lysis time. Lysis time refers to the period spanning phage infection of a host cell and its lysis, whereupon phage progeny are released. Lysis time, therefore, directly determines phage fecundity assuming progeny assembly rate is maximized. Noting that previous tests of lysis time optimality rely on batch culture, we implemented a quasi-continuous culture system to observe productivity of a panel of isogenic phage {lambda} mutants differing in lysis time. We report that {lambda} phage productivity in our experiments is maximized around an optimal lysis time of 63 min, which is the lysis time of the {lambda} "wildtype" strain. We discuss this finding in light of recent results that lysis time variation is also minimized in the {lambda} "wildtype" strain.

evolutionary biology↗

bioRxiv2020

Optimum Threshold Minimizes Noise in Timing of Intracellular Events

How the noisy expression of regulatory proteins affects timing of intracellular events is an intriguing fundamental problem that influences diverse cellular processes. Here we use the bacteriophage {lambda} to study event timing in individual cells where cell lysis is the result of expression and accumulation of a single protein (holin) in the Escherchia coli cell membrane up to a critical threshold level. Site-directed mutagenesis of the holin gene was used to generate phage variants that vary in their timing of lysis from 30 to 190 min. Observation of the lysis times of single cells reveals an intriguing finding - the noise in lysis timing first decreases with increasing lysis time to reach a minimum, and then sharply increases at longer longer lysis times. A mathematical model with stochastic expression of holin together with dilution from cell growth was sufficient to explain the non-monotonic noise profile, and identify holin accumulation thresholds that generate precision in lysis timing.

biophysics↗