scholarly journals Stochastic Analysis Demonstrates the Dual Role of Hfq in Chaperoning E. coli Sugar Shock Response

2020 ◽  
Author(s):  
David M. Bianchi ◽  
Troy A. Brier ◽  
Anustup Poddar ◽  
Muhammad S. Azam ◽  
Carin K. Vanderpool ◽  
...  
Keyword(s):  
Single Molecule ◽  
Super Resolution ◽  
Population Level ◽  
Cellular Heterogeneity ◽  
Bacterial Gene ◽  
E Coli ◽  
Gene Replication ◽  
Bacterial Gene Expression ◽  
Glucose Phosphate ◽  
Cell Variation

AbstractSmall RNAs (sRNAs) play a crucial role in the regulation of bacterial gene expression by silencing the translation of target mRNAs. SgrS is an sRNA that relieves glucose-phosphate stress, or “sugar shock” in E. coli. The power of single cell measurements is their ability to obtain population level statistics that illustrate cell-to-cell variation. Here, we utilize single molecule super-resolution microscopy in single E. coli cells coupled with stochastic modeling to analyze glucose-phosphate stress regulation by SgrS. We present a kinetic model that captures the combined effects of transcriptional regulation, gene replication and chaperone mediated RNA silencing in the SgrS regulatory network. This more complete kinetic description, simulated stochastically, recapitulates experimentally observed cellular heterogeneity and characterizes the binding of SgrS to the chaperone protein Hfq as a slow process that not only stabilizes SgrS but also may be critical in restructuring the sRNA to facilitate association with its target ptsG mRNA.

scholarly journals Stochastic Analysis Demonstrates the Dual Role of Hfq in Chaperoning E. coli Sugar Shock Response

2020 ◽  
Vol 7 ◽  
Author(s):  
David M. Bianchi ◽  
Troy A. Brier ◽  
Anustup Poddar ◽  
Muhammad S. Azam ◽  
Carin K. Vanderpool ◽  
...  
Keyword(s):  
Single Molecule ◽  
Super Resolution ◽  
Population Level ◽  
Cellular Heterogeneity ◽  
Bacterial Gene ◽  
E Coli ◽  
Gene Replication ◽  
Bacterial Gene Expression ◽  
Glucose Phosphate ◽  
Cell Variation

Small RNAs (sRNAs) play a crucial role in the regulation of bacterial gene expression by silencing the translation of target mRNAs. SgrS is an sRNA that relieves glucose-phosphate stress, or “sugar shock” in E. coli. The power of single cell measurements is their ability to obtain population level statistics that illustrate cell-to-cell variation. Here, we utilize single molecule super-resolution microscopy in single E. coli cells coupled with stochastic modeling to analyze glucose-phosphate stress regulation by SgrS. We present a kinetic model that captures the combined effects of transcriptional regulation, gene replication and chaperone mediated RNA silencing in the SgrS regulatory network. This more complete kinetic description, simulated stochastically, recapitulates experimentally observed cellular heterogeneity and characterizes the binding of SgrS to the chaperone protein Hfq as a slow process that not only stabilizes SgrS but also may be critical in restructuring the sRNA to facilitate association with its target ptsG mRNA.

scholarly journals Evaluation of Live Bacterial Prophylactics to Decrease IncF Plasmid Transfer and Association With Intestinal Small RNAs

2021 ◽  
Vol 11 ◽  
Author(s):  
Graham A. J. Redweik ◽  
Mary Kate Horak ◽  
Ryley Hoven ◽  
Logan Ott ◽  
Melha Mellata
Keyword(s):  
Virulence Genes ◽  
Positive Association ◽  
Multidrug Resistant ◽  
Plasmid Transfer ◽  
Siderophore Production ◽  
Causal Mechanism ◽  
Bacterial Gene ◽  
E Coli ◽  
Bacterial Gene Expression

Chicken intestinal Escherichia coli are a reservoir for virulence and antimicrobial resistance (AMR) genes that are often carried on incompatibility group F (IncF) plasmids. The rapid transfer of these plasmids between bacteria in the gut contributes to the emergence of new multidrug-resistant and virulent bacteria that threaten animal agriculture and human health. Thus, the aim of the present study was to determine whether live bacterial prophylactics could affect the distribution of large virulence plasmids and AMR in the intestinal tract and the potential role of smRNA in this process. In this study, we tested ∼100 randomly selected E. coli from pullet feces (n = 3 per group) given no treatment (CON), probiotics (PRO), a live Salmonella vaccine (VAX), or both (P + V). E. coli isolates were evaluated via plasmid profiles and several phenotypic (siderophore production and AMR), and genotypic (PCR for virulence genes and plasmid typing) screens. P + V isolates exhibited markedly attenuated siderophore production, lack of AMR and virulence genes, which are all related to the loss of IncF and ColV plasmids (P < 0.0001). To identify a causal mechanism, we evaluated smRNA levels in the ceca mucus and found a positive association between smRNA concentrations and plasmid content, with both being significantly reduced in P + V birds compared to other groups (P < 0.01). To test this positive association between IncF plasmid transfer and host smRNA concentration, we evenly pooled smRNA per group and treated E. coli mating pairs with serial concentrations of smRNA in vitro. Higher smRNA concentrations resulted in greater rates of IncF plasmid transfer between E. coli donors (APEC O2 or VAX isolate IA-EC-001) and recipient (HS-4) (all groups; P < 0.05). Finally, RNAHybrid predictive analyses detected several chicken miRNAs that hybridize with pilus assembly and plasmid transfer genes on the IncF plasmid pAPEC-O2-R. Overall, we demonstrated P + V treatment reduced smRNA levels in the chicken ceca, which was associated with a reduction in potentially virulent E. coli. Furthermore, we propose a novel mechanism in which intestinal smRNAs signal plasmid exchange between E. coli. Investigations to understand the changes in bacterial gene expression as well as smRNAs responsible for this phenomenon are currently underway.

scholarly journals Subpopulations of sensorless bacteria drive fitness in fluctuating environments

2020 ◽  
Author(s):  
Thomas Julou ◽  
Ludovit Zweifel ◽  
Diana Blank ◽  
Athos Fiori ◽  
Erik van Nimwegen
Keyword(s):  
Gene Expression ◽  
Single Molecule ◽  
Population Level ◽  
General Mechanism ◽  
Gene Expression Noise ◽  
E Coli ◽  
Expression Noise ◽  
Basal Expression ◽  
Fluctuating Environments ◽  
Operon Expression

AbstractPopulations of bacteria often undergo a lag in growth when switching conditions. Because growth lags can be large compared to typical doubling times, variations in growth lag are an important but often overlooked component of bacterial fitness in fluctuating environments. We here explore how growth lag variation is determined for the archetypical switch from glucose to lactose as a carbon source in E. coli. First, we show that single-cell lags are bimodally distributed and controlled by a single-molecule trigger. That is, gene expression noise causes the population before the switch to divide cells with zero pre-existing into subpopulations with zero and nonzero lac operon expression. While ’sensorless’ lac expression at the switch have long lags because they are unable to sense the lactose signal, any nonzero lac operon expression suffices to ensure a short lag. Second, we show that the growth lag at the population level depends crucially on the fraction of sensorless cells, and that this fraction in turn depends sensitively on the growth condition before the switch. Consequently, even small changes in basal expression affecting the fraction of sensorless cells can significantly affect population lags and fitness under switching conditions, and may thus be subject to significant natural selection. Indeed, we show that condition-dependent population lags vary across wild E. coli isolates. Since many sensory genes are naturally low expressed in conditions where their inducer is not present, bimodal responses due to subpopulations of sensorless cells may be a general mechanism inducing phenotypic heterogeneity and controlling population lags in switching environments. This mechanism also illustrates how gene expression noise can turn even simple sensory gene circuits into a bet-hedging module, and underlines the profound role of gene expression noise in regulatory responses.

scholarly journals Role of Hfq in Genome Evolution: Instability of G-Quadruplex Sequences in E. coli

2019 ◽  
Vol 8 (1) ◽  
pp. 28 ◽  
Author(s):  
Virali J. Parekh ◽  
Brittany A. Niccum ◽  
Rachna Shah ◽  
Marisa A. Rivera ◽  
Mark J. Novak ◽  
...  
Keyword(s):  
Genomic Instability ◽  
Dna Repeats ◽  
Loop Formation ◽  
Bacterial Gene ◽  
Quadruplex Dna ◽  
E Coli ◽  
Alternative Structures ◽  
Bacterial Gene Expression ◽  
G Quadruplex

Certain G-rich DNA repeats can form quadruplex in bacterial chromatin that can present blocks to DNA replication and, if not properly resolved, may lead to mutations. To understand the participation of quadruplex DNA in genomic instability in Escherichia coli (E. coli), mutation rates were measured for quadruplex-forming DNA repeats, including (G3T)4, (G3T)8, and a RET oncogene sequence, cloned as the template or nontemplate strand. We evidence that these alternative structures strongly influence mutagenesis rates. Precisely, our results suggest that G-quadruplexes form in E. coli cells, especially during transcription when the G-rich strand can be displaced by R-loop formation. Structure formation may then facilitate replication misalignment, presumably associated with replication fork blockage, promoting genomic instability. Furthermore, our results also evidence that the nucleoid-associated protein Hfq is involved in the genetic instability associated with these sequences. Hfq binds and stabilizes G-quadruplex structure in vitro and likely in cells. Collectively, our results thus implicate quadruplexes structures and Hfq nucleoid protein in the potential for genetic change that may drive evolution or alterations of bacterial gene expression.

Pulses and delays, anticipation and memory: seeing bacterial stress responses from a single-cell perspective

2020 ◽  
Vol 44 (5) ◽  
pp. 565-571
Author(s):  
Valentine Lagage ◽  
Stephan Uphoff
Keyword(s):  
Stress Response ◽  
Single Cell ◽  
Single Molecule ◽  
Stress Responses ◽  
Phenotypic Diversity ◽  
Single Cells ◽  
Super Resolution ◽  
Population Level ◽  
Protective Mechanisms ◽  
Single Cell Imaging

ABSTRACT Stress responses are crucial for bacteria to survive harmful conditions that they encounter in the environment. Although gene regulatory mechanisms underlying stress responses in bacteria have been thoroughly characterised for decades, recent advances in imaging technologies helped to uncover previously hidden dynamics and heterogeneity that become visible at the single-cell level. Despite the diversity of stress response mechanisms, certain dynamic regulatory features are frequently seen in single cells, such as pulses, delays, stress anticipation and memory effects. Often, these dynamics are highly variable across cells. While any individual cell may not achieve an optimal stress response, phenotypic diversity can provide a benefit at the population level. In this review, we highlight microscopy studies that offer novel insights into how bacteria sense stress, regulate protective mechanisms, cope with response delays and prepare for future environmental challenges. These studies showcase developments in the single-cell imaging toolbox including gene expression reporters, FRET, super-resolution microscopy and single-molecule tracking, as well as microfluidic techniques to manipulate cells and create defined stress conditions.

scholarly journals In situ reprogramming of gut bacteria by oral delivery

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Bryan B. Hsu ◽  
Isaac N. Plant ◽  
Lorena Lyon ◽  
Frances M. Anastassacos ◽  
Jeffrey C. Way ◽  
...  
Keyword(s):  
Oral Delivery ◽  
Multidisciplinary Approach ◽  
Gut Bacteria ◽  
Release Mechanism ◽  
Bacterial Gene ◽  
E Coli ◽  
Bacterial Gene Expression ◽  
Bacterial Function

Abstract Abundant links between the gut microbiota and human health indicate that modification of bacterial function could be a powerful therapeutic strategy. The inaccessibility of the gut and inter-connections between gut bacteria and the host make it difficult to precisely target bacterial functions without disrupting the microbiota and/or host physiology. Herein we describe a multidisciplinary approach to modulate the expression of a specific bacterial gene within the gut by oral administration. We demonstrate that an engineered temperate phage λ expressing a programmable dCas9 represses a targeted E. coli gene in the mammalian gut. To facilitate phage administration while minimizing disruption to host processes, we develop an aqueous-based encapsulation formulation with a microbiota-based release mechanism and show that it facilitates oral delivery of phage in vivo. Finally we combine these technologies and show that bacterial gene expression in the mammalian gut can be precisely modified in situ with a single oral dose.

scholarly journals A Prophage-Encoded Small RNA Controls Metabolism and Cell Division in Escherichia coli

mSystems ◽  
2016 ◽  
Vol 1 (1) ◽  
Author(s):  
Divya Balasubramanian ◽  
Preethi T. Ragunathan ◽  
Jingyi Fei ◽  
Carin K. Vanderpool
Keyword(s):  
Gene Expression ◽  
Cell Division ◽  
Posttranscriptional Control ◽  
Bacterial Gene ◽  
Content Type ◽  
E Coli ◽  
Bacterial Gene Expression ◽  
Metabolic Gene Expression ◽  
Defective Prophage ◽  
Ectopic Production

ABSTRACT sRNAs are ubiquitous and versatile regulators of bacterial gene expression. A number of well-characterized examples in E. coli are highly conserved and present in the E. coli core genome. In contrast, the sRNA DicF (identified over 20 years ago but remaining poorly characterized) is encoded by a gene carried on a defective prophage element in many E. coli genomes. Here, we characterize DicF in order to better understand how horizontally acquired sRNA regulators impact bacterial gene expression and physiology. Our data confirm the long-hypothesized DicF-mediated regulation of ftsZ, encoding the bacterial tubulin homolog required for cell division. We further uncover DicF-mediated posttranscriptional control of metabolic gene expression. Ectopic production of DicF is highly toxic to E. coli cells, but the toxicity is not attributable to DicF regulation of ftsZ. Further work is needed to reveal the biological roles of and benefits for the host conferred by DicF and other products encoded by defective prophages. Hundreds of small RNAs (sRNAs) have been identified in diverse bacterial species, and while the functions of most remain unknown, some regulate key processes, particularly stress responses. The sRNA DicF was identified over 25 years ago as an inhibitor of cell division but since then has remained uncharacterized. DicF consists of 53 nucleotides and is encoded by a gene carried on a prophage (Qin) in the genomes of many Escherichia coli strains. We demonstrated that DicF inhibits cell division via direct base pairing with ftsZ mRNA to repress translation and prevent new synthesis of the bacterial tubulin homolog FtsZ. Systems analysis using computational and experimental methods identified additional mRNA targets of DicF: xylR and pykA mRNAs, encoding the xylose uptake and catabolism regulator and pyruvate kinase, respectively. Genetic analyses showed that DicF directly base pairs with and represses translation of these targets. Phenotypes of cells expressing DicF variants demonstrated that DicF-associated growth inhibition is not solely due to repression of ftsZ, indicating that the physiological consequences of DicF-mediated regulation extend beyond effects on cell division caused by reduced FtsZ synthesis. IMPORTANCE sRNAs are ubiquitous and versatile regulators of bacterial gene expression. A number of well-characterized examples in E. coli are highly conserved and present in the E. coli core genome. In contrast, the sRNA DicF (identified over 20 years ago but remaining poorly characterized) is encoded by a gene carried on a defective prophage element in many E. coli genomes. Here, we characterize DicF in order to better understand how horizontally acquired sRNA regulators impact bacterial gene expression and physiology. Our data confirm the long-hypothesized DicF-mediated regulation of ftsZ, encoding the bacterial tubulin homolog required for cell division. We further uncover DicF-mediated posttranscriptional control of metabolic gene expression. Ectopic production of DicF is highly toxic to E. coli cells, but the toxicity is not attributable to DicF regulation of ftsZ. Further work is needed to reveal the biological roles of and benefits for the host conferred by DicF and other products encoded by defective prophages.

scholarly journals In situ reprogramming of gut bacteria by oral delivery

2020 ◽  
Author(s):  
Bryan B. Hsu ◽  
Isaac N. Plant ◽  
Lorena Lyon ◽  
Frances M. Anastassacos ◽  
Jeffrey C. Way ◽  
...  
Keyword(s):  
Oral Delivery ◽  
Multidisciplinary Approach ◽  
Gut Bacteria ◽  
Release Mechanism ◽  
Bacterial Gene ◽  
E Coli ◽  
Bacterial Gene Expression ◽  
Bacterial Function

AbstractAbundant links between the gut microbiota and human health indicate that the modification of bacterial function could be a powerful therapeutic strategy. The inaccessibility of the gut and inter-connections between gut bacteria and the host make it difficult to precisely target bacterial functions without disrupting the microbiota and/or host physiology. Herein we describe a multidisciplinary approach to modulate the expression of a specific bacterial gene within the gut by oral administration. We first demonstrate that an engineered temperate phage λ expressing a programmable dCas9 represses a targeted E. coli gene in the mammalian gut. To facilitate phage administration while minimizing disruption to host processes, we develop an aqueous-based encapsulation formulation with a microbiota-based release mechanism and show that it facilitates the oral delivery of phage in vivo. Finally we combine these technologies and show that bacterial gene expression in the mammalian gut can be precisely modified in situ with a single oral dose.

scholarly journals Unprecedented tunability of riboswitch structure and regulatory function by sub-millimolar variations in physiological Mg2+

2019 ◽  
Vol 47 (12) ◽  
pp. 6478-6487 ◽  
Author(s):  
Kaley McCluskey ◽  
Julien Boudreault ◽  
Patrick St-Pierre ◽  
Cibran Perez-Gonzalez ◽  
Adrien Chauvier ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Molecule ◽  
Fine Tuning ◽  
Regulatory Function ◽  
Divalent Metal Ions ◽  
Ligand Complex ◽  
Physiological Concentration ◽  
Bacterial Gene ◽  
Bacterial Gene Expression ◽  
Amino Acid Sensing

Abstract Riboswitches are cis-acting regulatory RNA biosensors that rival the efficiency of those found in proteins. At the heart of their regulatory function is the formation of a highly specific aptamer–ligand complex. Understanding how these RNAs recognize the ligand to regulate gene expression at physiological concentrations of Mg2+ ions and ligand is critical given their broad impact on bacterial gene expression and their potential as antibiotic targets. In this work, we used single-molecule FRET and biochemical techniques to demonstrate that Mg2+ ions act as fine-tuning elements of the amino acid-sensing lysC aptamer's ligand-free structure in the mesophile Bacillus subtilis. Mg2+ interactions with the aptamer produce encounter complexes with strikingly different sensitivities to the ligand in different, yet equally accessible, physiological ionic conditions. Our results demonstrate that the aptamer adapts its structure and folding landscape on a Mg2+-tunable scale to efficiently respond to changes in intracellular lysine of more than two orders of magnitude. The remarkable tunability of the lysC aptamer by sub-millimolar variations in the physiological concentration of Mg2+ ions suggests that some single-aptamer riboswitches have exploited the coupling of cellular levels of ligand and divalent metal ions to tightly control gene expression.

Noise in bacterial gene expression

2018 ◽  
Vol 47 (1) ◽  
pp. 209-217 ◽  
Author(s):  
Christoph Engl
Keyword(s):  
Gene Expression ◽  
Single Molecule ◽  
Detailed Knowledge ◽  
Extrinsic Factors ◽  
Bacterial Gene ◽  
Bacterial Gene Expression ◽  
Genome Wide ◽  
A Genome ◽  
Single Molecule Studies ◽  
Low Activity

AbstractThe expression level of a gene can fluctuate significantly between individuals within a population of genetically identical cells. The resultant phenotypic heterogeneity could be exploited by bacteria to adapt to changing environmental conditions. Noise is hence a genome-wide phenomenon that arises from the stochastic nature of the biochemical reactions that take place during gene expression and the relatively low abundance of the molecules involved. The production of mRNA and proteins therefore occurs in bursts, with alternating episodes of high and low activity during transcription and translation. Single-cell and single-molecule studies demonstrated that noise within gene expression is influenced by a combination of both intrinsic and extrinsic factors. However, our mechanistic understanding of this process at the molecular level is still rather limited. Further investigation is necessary that takes into account the detailed knowledge of gene regulation gained from biochemical studies.

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