Codon usage influences fitness through RNA toxicity

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.

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Codon usage influences fitness through RNA toxicity

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1810022115 ◽

2018 ◽

Vol 115 (34) ◽

pp. 8639-8644 ◽

Cited By ~ 28

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.

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Pathovar Transcriptomes

mSystems ◽

10.1128/msystems.00049-17 ◽

2017 ◽

Vol 2 (4) ◽

Cited By ~ 1

Author(s):

Amy Platenkamp ◽

Jay L. Mellies

Keyword(s):

Gene Expression ◽

Escherichia Coli ◽

Regulatory Networks ◽

Virulence Gene ◽

Model System ◽

Genomic Sequences ◽

Bacterial Strains ◽

Content Type ◽

Virulence Gene Expression ◽

Link Type

ABSTRACT Archetypal pathogenic bacterial strains are often used to elucidate regulatory networks of an entire pathovar, which encompasses multiple lineages and phylogroups. With enteropathogenic Escherichia coli (EPEC) as a model system, Hazen and colleagues (mSystems 6:e00024-17, 2017, https://doi.org/10.1128/mSystems.00024-17 ) used 9 isolates representing 8 lineages and 3 phylogroups to find that isolates with similar genomic sequences exhibit similarities in global transcriptomes under conditions of growth in medium that induces virulence gene expression, and they found variation among individual isolates. Archetypal pathogenic bacterial strains are often used to elucidate regulatory networks of an entire pathovar, which encompasses multiple lineages and phylogroups. With enteropathogenic Escherichia coli (EPEC) as a model system, Hazen and colleagues (mSystems 6:e00024-17, 2017, https://doi.org/10.1128/mSystems.00024-17 ) used 9 isolates representing 8 lineages and 3 phylogroups to find that isolates with similar genomic sequences exhibit similarities in global transcriptomes under conditions of growth in medium that induces virulence gene expression. They also found variation among individual isolates. Their work illustrates the importance of moving beyond observing regulatory phenomena of a limited number of regulons in a few archetypal strains, with the possibility of correlating clinical symptoms to key transcriptional pathways across lineages and phylogroups.

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The identification of bacterial gene expression differences using mRNA-based isothermal subtractive hybridization

Canadian Journal of Microbiology ◽

10.1139/m95-020 ◽

1995 ◽

Vol 41 (2) ◽

pp. 152-156 ◽

Cited By ~ 16

Author(s):

Eric A. Utt ◽

Jeffery P. Brousal ◽

Lynne C. Kikuta-Oshima ◽

Frederick D. Quinn

Keyword(s):

Gene Expression ◽

Listeria Monocytogenes ◽

Subtractive Hybridization ◽

Listeriolysin O ◽

Bacterial Strains ◽

Bacterial Gene ◽

Total Rna ◽

Bacterial Gene Expression ◽

Hybridization Probes ◽

The Difference

We describe a method for isolating and determining differences in gene expression between related bacterial strains. The method is based upon differences in mRNA expression. To demonstrate this procedure, cDNA generated from total RNA of Listeria monocytogenes serotype 1/2a was hybridized to total RNA from a Tn916 mutant of serogroup 1/2a (M3) that was deficient in the production of listeriolysin O, the product of the hly gene. The single-stranded cDNA fragments remaining after hybridization represent the difference in expressed genes between the two strains. These subtraction products were used as hybridization probes to identify the corresponding hly gene in a Southern hybridization.Key words: subtractive hybridization, Listeria monocytogenes, hemolysin, gene expression, isogenic.

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The nrfA and nirB Nitrite Reductase Operons in Escherichia coli Are Expressed Differently in Response to Nitrate than to Nitrite

Journal of Bacteriology ◽

10.1128/jb.182.20.5813-5822.2000 ◽

2000 ◽

Vol 182 (20) ◽

pp. 5813-5822 ◽

Cited By ~ 66

Author(s):

Henian Wang ◽

Robert P. Gunsalus

Keyword(s):

Gene Expression ◽

Escherichia Coli ◽

Nitrite Reductase ◽

Transcriptional Activation ◽

Response Regulator ◽

Physiological Role ◽

Growth Conditions ◽

Potent Inducer ◽

Culture Techniques ◽

Nitrite Reductase Gene

ABSTRACT Escherichia coli possesses two distinct nitrite reductase enzymes encoded by the nrfA and nirBoperons. The expression of each operon is induced during anaerobic cell growth conditions and is further modulated by the presence of either nitrite or nitrate in the cells' environment. To examine how each operon is expressed at low, intermediate, and high levels of either nitrate or nitrite, anaerobic chemostat culture techniques were employed using nrfA-lacZ and nirB-lacZ reporter fusions. Steady-state gene expression studies revealed a differential pattern of nitrite reductase gene expression where optimalnrfA-lacZ expression occurred only at low to intermediate levels of nitrate and where nirB-lacZ expression was induced only by high nitrate conditions. Under these conditions, the presence of high levels of nitrate suppressed nrfA gene expression. While either NarL or NarP was able to inducenrfA-lacZ expression in response to low levels of nitrate, only NarL could repress at high nitrate levels. The different expression profile for the alternative nitrite reductase operon encoded by nirBDC under high-nitrate conditions was due to transcriptional activation by either NarL or NarP. Neither response regulator could repress nirB expression. Nitrite was also an inducer of nirB and nrfA gene expression, but nitrate was always the more potent inducer by >100-fold. Lastly, since nrfA operon expression is only induced under low-nitrate concentrations, the NrfA enzyme is predicted to have a physiological role only where nitrate (or nitrite) is limiting in the cell environment. In contrast, the nirB nitrite reductase is optimally synthesized only when nitrate or nitrite is in excess of the cell's capacity to consume it. Revised regulatory schemes are presented for NarL and NarP in control of the two operons.

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Metal homeostasis in Escherichia coli: The regulation of bacterial gene expression by cupric and mercuric ions.

Journal of Inorganic Biochemistry ◽

10.1016/0162-0134(91)84481-n ◽

1991 ◽

Vol 43 (2-3) ◽

pp. 504

Author(s):

Nigel L. Brown ◽

Siobhán R. Barrett ◽

Barry T.O. Lee ◽

Julian Parkhill ◽

Duncan A. Rouch

Keyword(s):

Gene Expression ◽

Escherichia Coli ◽

Metal Homeostasis ◽

Bacterial Gene ◽

Bacterial Gene Expression ◽

Mercuric Ions

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Genome-Wide Expression Analysis Indicates that FNR of Escherichia coli K-12 Regulates a Large Number of Genes of Unknown Function

Journal of Bacteriology ◽

10.1128/jb.187.3.1135-1160.2005 ◽

2005 ◽

Vol 187 (3) ◽

pp. 1135-1160 ◽

Cited By ~ 190

Author(s):

Yisheng Kang ◽

K. Derek Weber ◽

Yu Qiu ◽

Patricia J. Kiley ◽

Frederick R. Blattner

Keyword(s):

Gene Expression ◽

Escherichia Coli ◽

Dna Binding ◽

Growth Conditions ◽

Anaerobic Growth ◽

Computer Search ◽

Anaerobic Conditions ◽

Metabolic Potential ◽

Fermentation Products ◽

K 12

ABSTRACT The major regulator controlling the physiological switch between aerobic and anaerobic growth conditions in Escherichia coli is the DNA binding protein FNR. To identify genes controlled by FNR, we used Affymetrix Antisense GeneChips to compare global gene expression profiles from isogenic MG1655 wild-type and Δfnr strains grown in glucose minimal media under aerobic or anaerobic conditions. We found that 297 genes contained within 184 operons were regulated by FNR and/or by O2 levels. The expression of many genes known to be involved in anaerobic respiration and fermentation was increased under anaerobic growth conditions, while that of genes involved in aerobic respiration and the tricarboxylic acid cycle were repressed as expected. The expression of nine operons associated with acid resistance was also increased under anaerobic growth conditions, which may reflect the production of acidic fermentation products. Ninety-one genes with no presently defined function were also altered in expression, including seven of the most highly anaerobically induced genes, six of which we found to be directly regulated by FNR. Classification of the 297 genes into eight groups by k-means clustering analysis indicated that genes with common gene expression patterns also had a strong functional relationship, providing clues for studying the function of unknown genes in each group. Six of the eight groups showed regulation by FNR; while some expression groups represent genes that are simply activated or repressed by FNR, others, such as those encoding functions for chemotaxis and motility, showed a more complex pattern of regulation. A computer search for FNR DNA binding sites within predicted promoter regions identified 63 new sites for 54 genes. We suggest that E. coli MG1655 has a larger metabolic potential under anaerobic conditions than has been previously recognized.

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Localized Gene Expression in Pseudomonas aeruginosa Biofilms

Applied and Environmental Microbiology ◽

10.1128/aem.00710-08 ◽

2008 ◽

Vol 74 (14) ◽

pp. 4463-4471 ◽

Cited By ~ 109

Author(s):

Ailyn P. Lenz ◽

Kerry S. Williamson ◽

Betsey Pitts ◽

Philip S. Stewart ◽

Michael J. Franklin

Keyword(s):

Gene Expression ◽

Pseudomonas Aeruginosa ◽

Green Fluorescent Protein ◽

16S Rrna ◽

Fluorescent Protein ◽

Mrna Levels ◽

Protein Fluorescence ◽

Bacterial Gene ◽

Qrt Pcr ◽

Green Fluorescent

ABSTRACT Gene expression in biofilms is dependent on bacterial responses to the local environmental conditions. Most techniques for studying bacterial gene expression in biofilms characterize average values across the entire population. Here, we describe the use of laser capture microdissection microscopy (LCMM) combined with multiplex quantitative real-time reverse transcriptase PCR (qRT-PCR) to isolate and quantify RNA transcripts from small groups of cells at spatially resolved sites within biofilms. The approach was first tested and analytical parameters were determined for Pseudomonas aeruginosa containing an isopropyl-β-d-thiogalactopyranoside-inducible gene for the green fluorescent protein (gfp). The results show that the amounts of gfp mRNA were greatest in the top zones of the biofilms, and that gfp mRNA levels correlated with the zone of active green fluorescent protein fluorescence. The method then was used to quantify transcripts from wild-type P. aeruginosa biofilms for a housekeeping gene, acpP; the 16S rRNA; and two genes regulated by quorum sensing, phzA1 and aprA. The results demonstrated that the amount of acpP mRNA was greatest in the top 30 μm of the biofilm, with little or no mRNA for this gene at the base of the biofilms. In contrast, 16S rRNA amounts were relatively uniform throughout biofilm strata. Using this strategy, the RNA amounts of individual genes were determined, and therefore the results are dependent on both gene expression and the half-life of the transcripts. Therefore, the uniform amount of rRNA throughout the biofilms likely is due to the stability of the rRNA within ribosomes. The levels of aprA mRNA showed stratification, with the largest amounts in the upper 30-μm zone of these biofilms. The results demonstrate that mRNA levels for individual genes are not uniformly distributed throughout biofilms but may vary by orders of magnitude over small distances. The LCMM/qRT-PCR technique can be used to resolve and quantify this RNA variability at high spatial resolution.

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Genome-wide gene expression noise in Escherichia coli is condition-dependent and determined by propagation of noise through the regulatory network

PLoS Biology ◽

10.1371/journal.pbio.3001491 ◽

2021 ◽

Vol 19 (12) ◽

pp. e3001491

Author(s):

Arantxa Urchueguía ◽

Luca Galbusera ◽

Dany Chauvin ◽

Gwendoline Bellement ◽

Thomas Julou ◽

...

Keyword(s):

Gene Expression ◽

Escherichia Coli ◽

Growth Rate ◽

Regulatory Network ◽

Growth Conditions ◽

Noise Levels ◽

Transcriptional Noise ◽

Expression Noise ◽

Genome Wide ◽

Absolute Expression

Although it is well appreciated that gene expression is inherently noisy and that transcriptional noise is encoded in a promoter’s sequence, little is known about the extent to which noise levels of individual promoters vary across growth conditions. Using flow cytometry, we here quantify transcriptional noise in Escherichia coli genome-wide across 8 growth conditions and find that noise levels systematically decrease with growth rate, with a condition-dependent lower bound on noise. Whereas constitutive promoters consistently exhibit low noise in all conditions, regulated promoters are both more noisy on average and more variable in noise across conditions. Moreover, individual promoters show highly distinct variation in noise across conditions. We show that a simple model of noise propagation from regulators to their targets can explain a significant fraction of the variation in relative noise levels and identifies TFs that most contribute to both condition-specific and condition-independent noise propagation. In addition, analysis of the genome-wide correlation structure of various gene properties shows that gene regulation, expression noise, and noise plasticity are all positively correlated genome-wide and vary independently of variations in absolute expression, codon bias, and evolutionary rate. Together, our results show that while absolute expression noise tends to decrease with growth rate, relative noise levels of genes are highly condition-dependent and determined by the propagation of noise through the gene regulatory network.

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Synonymous but not Silent: The Codon Usage Code for Gene Expression and Protein Folding

Annual Review of Biochemistry ◽

10.1146/annurev-biochem-071320-112701 ◽

2021 ◽

Vol 90 (1) ◽

Author(s):

Yi Liu ◽

Qian Yang ◽

Fangzhou Zhao

Keyword(s):

Gene Expression ◽

Protein Folding ◽

Codon Usage ◽

Protein Structures ◽

Large Body ◽

Annual Review ◽

Publication Date ◽

Translation Efficiency ◽

Mrna Levels ◽

Synonymous Mutations

Codon usage bias, the preference for certain synonymous codons, is found in all genomes. Although synonymous mutations were previously thought to be silent, a large body of evidence has demonstrated that codon usage can play major roles in determining gene expression levels and protein structures. Codon usage influences translation elongation speed and regulates translation efficiency and accuracy. Adaptation of codon usage to tRNA expression determines the proteome landscape. In addition, codon usage biases result in nonuniform ribosome decoding rates on mRNAs, which in turn influence the cotranslational protein folding process that is critical for protein function in diverse biological processes. Conserved genome-wide correlations have also been found between codon usage and protein structures. Furthermore, codon usage is a major determinant of mRNA levels through translation-dependent effects on mRNA decay and translation-independent effects on transcriptional and posttranscriptional processes. Here, we discuss the multifaceted roles and mechanisms of codon usage in different gene regulatory processes. Expected final online publication date for the Annual Review of Biochemistry, Volume 90 is June 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

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Differential expression of antioxidant genes during clinical mastitis of cow caused by Staphylococcus aureus and Escherichia coli

Veterinarski arhiv ◽

10.24099/vet.arhiv.1000 ◽

2021 ◽

Vol 91 (5) ◽

pp. 451-458

Author(s):

Reza Asadpour ◽

◽

Pedram Zangiband ◽

Katayoon Nofouzi ◽

Adel Saberivand

Keyword(s):

Gene Expression ◽

Escherichia Coli ◽

Staphylococcus Aureus ◽

Expression Profiles ◽

Clinical Mastitis ◽

Mrna Levels ◽

Antioxidant Genes ◽

E Coli ◽

Antioxidant Gene Expression ◽

The Mean

There is considerable interest in the hypothesis that oxidative stress is enhanced in the pathophysiology of clinical mastitis. The main goal of this research was to establish profiles of antioxidant gene expression in the milk of cows with clinical mastitis. Standard bacteriology was conducted on pretreatment milk specimens from 77 cows with clinical mastitis between 15 and 70 days in milk (DIM). Examinations were performed on mRNA expression of antioxidant genes, such as catalase (CAT), glutathione peroxidase (GPx), and superoxide dismutase (SOD). Additionally, levels of lipid peroxidation were measured in milk samples from healthy cows and those with clinical mastitis. The isolated bacteria consisted of Staphylococcus aureus (S. aureus, 10.48%), Streptococcus agalactiae (7.69%), Streptococcus dysagalactiae (6.29%), and Escherichia coli (E. coli, 29.37%). E. coli was the most prevalent pathogen found in the milk of cows with clinical mastitis in early lactation. The mean level of malondialdehyde (MDA) in clinical mastitis samples was significantly higher (P<0.05) than that of healthy cows. The results revealed that the expression profiles of SOD in mastitis milk induced by S. aureus were significantly (P<0.0001) up-regulated compared with E.coli. In addition, the mRNA levels of GPx in mastitis milk due to E.coli were significantly (P<0.0001) over expressed compared to S. aureus. CAT gene expression had a tendency to be enhanced in mastitis milk induced by S. aureus compared with mastitis in cows due to E.coli. These results showed that the mRNA levels of antioxidant genes may differ depending on the type of bacteria, and diminished expression of antioxidant genes might increase susceptibility to mastitis.

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