scholarly journals Codon usage influences fitness through RNA toxicity

2018 ◽  
Vol 115 (34) ◽  
pp. 8639-8644 ◽  
Author(s):  
Pragya Mittal ◽  
James Brindle ◽  
Julie Stephen ◽  
Joshua B. Plotkin ◽  
Grzegorz Kudla
Keyword(s):  
Mrna Levels ◽  
Bacterial Gene ◽  
E Coli ◽  
Rna Toxicity ◽  
Synonymous Mutations ◽  
Bacterial Gene Expression ◽  
Synonymous Codons ◽  
Synonymous Sites ◽  
Bacterial Fitness ◽  
Promoter Mutations

Many organisms are subject to selective pressure that gives rise to unequal usage of synonymous codons, known as codon bias. To experimentally dissect the mechanisms of selection on synonymous sites, we expressed several hundred synonymous variants of the GFP gene inEscherichia coli, and used quantitative growth and viability assays to estimate bacterial fitness. Unexpectedly, we found many synonymous variants whose expression was toxic toE. coli. Unlike previously studied effects of synonymous mutations, the effect that we discovered is independent of translation, but it depends on the production of toxic mRNA molecules. We identified RNA sequence determinants of toxicity and evolved suppressor strains that can tolerate the expression of toxic GFP variants. Genome sequencing of these suppressor strains revealed a cluster of promoter mutations that prevented toxicity by reducing mRNA levels. We conclude that translation-independent RNA toxicity is a previously unrecognized obstacle in bacterial gene expression.

scholarly journals Codon usage influences fitness through RNA toxicity

2018 ◽  
Author(s):  
Pragya Mittal ◽  
James Brindle ◽  
Julie Stephen ◽  
Joshua B. Plotkin ◽  
Grzegorz Kudla
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Growth Conditions ◽  
Mrna Levels ◽  
Heterologous Proteins ◽  
Bacterial Strains ◽  
Bacterial Gene ◽  
Bacterial Physiology ◽  
Rna Toxicity ◽  
Synonymous Mutations

AbstractMany organisms are subject to selective pressure that gives rise to unequal usage of synonymous codons, known as codon bias. To experimentally dissect the mechanisms of selection on synonymous sites, we expressed several hundred synonymous variants of the GFP gene inEscherichia coli, and used quantitative growth and viability assays to estimate bacterial fitness. Unexpectedly, we found many synonymous variants whose expression was toxic toE. coli. Unlike previously studied effects of synonymous mutations, the effect that we discovered is independent of translation, but it depends on the production of toxic mRNA molecules. We identified RNA sequence determinants of toxicity, and evolved suppressor strains that can tolerate the expression of toxic GFP variants. Genome sequencing of these suppressor strains revealed a cluster of promoter mutations that prevented toxicity by reducing mRNA levels. We conclude that translation-independent RNA toxicity is a previously unrecognized obstacle in bacterial gene expression.Significance statementSynonymous mutations in genes do not change protein sequence, but they may affect gene expression and cellular function. Here we describe an unexpected toxic effect of synonymous mutations inEscherichia coli, with potentially large implications for bacterial physiology and evolution. Unlike previously studied effects of synonymous mutations, the effect that we discovered is independent of translation, but it depends on the production of toxic mRNA molecules. We hypothesize that the mechanism we identified influences the evolution of endogenous genes in bacteria, by imposing selective constraints on synonymous mutations that arise in the genome. Of interest for biotechnology and synthetic biology, we identify bacterial strains and growth conditions that alleviate RNA toxicity, thus allowing efficient overexpression of heterologous proteins.

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 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.

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 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.

In Vivo Introduction of Unpreferred Synonymous Codons Into the Drosophila Adh Gene Results in Reduced Levels of ADH Protein

Genetics ◽  
2003 ◽  
Vol 163 (1) ◽  
pp. 239-243 ◽  
Author(s):  
David B Carlini ◽  
Wolfgang Stephan
Keyword(s):  
Natural Selection ◽  
Codon Bias ◽  
Protein Production ◽  
Empirical Relationship ◽  
Abundant Species ◽  
Translational Efficiency ◽  
Synonymous Mutations ◽  
Synonymous Codons ◽  
Synonymous Sites

Abstract The evolution of codon bias, the unequal usage of synonymous codons, is thought to be due to natural selection for the use of preferred codons that match the most abundant species of isoaccepting tRNA, resulting in increased translational efficiency and accuracy. We examined this hypothesis by introducing 1, 6, and 10 unpreferred codons into the Drosophila alcohol dehydrogenase gene (Adh). We observed a significant decrease in ADH protein production with number of unpreferred codons, confirming the importance of natural selection as a mechanism leading to codon bias. We then used this empirical relationship to estimate the selection coefficient (s) against unpreferred synonymous mutations and found the value (s ≥ 10-5) to be approximately one order of magnitude greater than previous estimates from population genetics theory. The observed differences in protein production appear to be too large to be consistent with current estimates of the strength of selection on synonymous sites in D. melanogaster.

scholarly journals The association between Hfq and RNase E in long-term nitrogen starved Escherichia coli

2021 ◽  
Author(s):  
Josh McQuail ◽  
Agamemnon J. Carpousis ◽  
Sivaramesh Wigneshweraraj
Keyword(s):  
Escherichia Coli ◽  
Rna Degradation ◽  
Rnase E ◽  
Bacterial Gene ◽  
E Coli ◽  
Bacterial Gene Expression ◽  
Live Bacteria ◽  
Specific Mrnas ◽  
Rna Degradosome

AbstractThe regulation of bacterial gene expression is underpinned by the synthesis and degradation of mRNA. In Escherichia coli, RNase E is the central enzyme involved in RNA degradation and serves as a scaffold for the assembly of the multiprotein complex known as the RNA degradosome. The activity of RNase E against specific mRNAs can also be regulated by the action of small RNAs (sRNA). The ubiquitous bacterial chaperone Hfq bound to sRNAs interacts with the RNA degradosome for the sRNA guided degradation of target mRNAs. The association between RNase E and Hfq has never been observed in live bacteria. We now show that in long-term nitrogen starved E. coli, both RNase E and Hfq co-localise in a single, large focus. This subcellular assembly, which we refer to as the H-body, also includes components of the RNA degradosome, namely, the helicase RhlB and the exoribonuclease polynucleotide phosphorylase. We further show that H-bodies are important for E. coli to optimally survive sustained nitrogen starvation. Collectively, the properties and features of the H-body suggests that it represents a hitherto unreported example of subcellular compartmentalisation of a process(s) associated with RNA management in stressed bacteria.

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.

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