Mutagenesis of the Bacterial RNA Polymerase Alpha Subunit for Improvement of Complex Phenotypes

Klein‐Marcuschamer, Daniel; Santos, Christine Nicole S.; Yu, Huimin; Stephanopoulos, Gregory

doi:10.1128/aem.01888-08

Cited by 79 publications

(66 citation statements)

References 51 publications

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“…The use of global regulatory proteins, specifically RpoH, RpoE, and ArcA, may allow the modulation of entire stress regulons and more complex systems such as the NADH-ubiquinone oxidoreductase complex or the cytochrome complex. Such global approaches for trait selection have been conducted, including mutations in the E. coli rpoA gene for improved resistance to commercially important products including n-butanol (26). Recent studies also suggest a potential use of RNA chaperones such as Hfq to modulate the role of protein complexes such as the Opp system (15).…”

Section: Resultsmentioning

confidence: 99%

Functional Genomic Study of Exogenous n -Butanol Stress in Escherichia coli

Rutherford

Dahl

Price

et al. 2010

Appl Environ Microbiol

212

258

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n-Butanol has been proposed as an alternative biofuel to ethanol, and several industrially used microbes, including Escherichia coli, have been engineered to produce it. Unfortunately, n-butanol is more toxic than ethanol to these organisms. To understand the basis for its toxicity, cell-wide studies were conducted at the transcript, protein, and metabolite levels to obtain a global view of the n-butanol stress response. Analysis of the data indicates that n-butanol stress has components common to other stress responses, including perturbation of respiratory functions (nuo and cyo operons), oxidative stress (sodA, sodC, and yqhD), heat shock and cell envelope stress (rpoE, clpB, htpG, cpxR, and cpxP), and metabolite transport and biosynthesis (malE and opp operon). Assays using fluorescent dyes indicated a large increase in reactive oxygen species during n-butanol stress, confirming observations from the microarray and proteomics measurements. Mutant strains with mutations in several genes whose products changed most dramatically during n-butanol stress were examined for increased sensitivity to n-butanol. Results from these analyses allowed identification of key genes that were recruited to alleviate oxidative stress, protein misfolding, and other causes of growth defects. Cellular engineering based on these cues may assist in developing a high-titer, n-butanol-producing host.

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Section: Resultsmentioning

confidence: 99%

Functional Genomic Study of Exogenous n -Butanol Stress in Escherichia coli

Rutherford

Dahl

Price

et al. 2010

Appl Environ Microbiol

212

258

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“…Our proposal that ethanol exerts its toxic effects on E. coli in part through direct effects on the ribosome and RNAP contrasts with previous suggestions that ethanol tolerance mutations modifying proteins involved in transcription and translation function indirectly by "rewiring" gene-expression networks. For example, increased alcohol tolerance of strains with reduced Rho activity (14,20) or mutations in RNA polymerase subunits or the TATAbinding protein (4,22,63) have been attributed to altered expression of specific genes controlled by termination or initiation. Our results do not preclude the possibility that increased expression of some genes contributes to ethanol tolerance; both effects on the transcription/translation machinery and effects on gene expression may occur, and both may be important.…”

Section: Discussionmentioning

confidence: 99%

Correcting direct effects of ethanol on translation and transcription machinery confers ethanol tolerance in bacteria

Haft

Keating

Schwaegler

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

128

121

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The molecular mechanisms of ethanol toxicity and tolerance in bacteria, although important for biotechnology and bioenergy applications, remain incompletely understood. Genetic studies have identified potential cellular targets for ethanol and have revealed multiple mechanisms of tolerance, but it remains difficult to separate the direct and indirect effects of ethanol. We used adaptive evolution to generate spontaneous ethanol-tolerant strains of Escherichia coli, and then characterized mechanisms of toxicity and resistance using genome-scale DNAseq, RNAseq, and ribosome profiling coupled with specific assays of ribosome and RNA polymerase function. Evolved alleles of metJ, rho, and rpsQ recapitulated most of the observed ethanol tolerance, implicating translation and transcription as key processes affected by ethanol. Ethanol induced miscoding errors during protein synthesis, from which the evolved rpsQ allele protected cells by increasing ribosome accuracy. Ribosome profiling and RNAseq analyses established that ethanol negatively affects transcriptional and translational processivity. Ethanol-stressed cells exhibited ribosomal stalling at internal AUG codons, which may be ameliorated by the adaptive inactivation of the MetJ repressor of methionine biosynthesis genes. Ethanol also caused aberrant intragenic transcription termination for mRNAs with low ribosome density, which was reduced in a strain with the adaptive rho mutation. Furthermore, ethanol inhibited transcript elongation by RNA polymerase in vitro. We propose that ethanol-induced inhibition and uncoupling of mRNA and protein synthesis through direct effects on ribosomes and RNA polymerase conformations are major contributors to ethanol toxicity in E. coli, and that adaptive mutations in metJ, rho, and rpsQ help protect these central dogma processes in the presence of ethanol.

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“…Point mutation in rho has been shown to be a major contributor to ethanol tolerance in E. coli evolved under ethanol stress (21). Mutations in global regulators have also been shown to enhance ethanol tolerance and n-butanol tolerance (41)(42)(43). Thus, the observed mutation in nusA, in part, potentially explains the generalist behavior of this mutant toward different stressors by globally perturbing transcriptional regulation in the cell.…”

Section: Discussionmentioning

confidence: 99%

Genetic Determinants forn-Butanol Tolerance in Evolved Escherichia coli Mutants: Cross Adaptation and Antagonistic Pleiotropy betweenn-Butanol and Other Stressors

Reyes

Abdelaal

Kao

2013

Appl Environ Microbiol

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bCross-tolerance and antagonistic pleiotropy have been observed between different complex phenotypes in microbial systems. These relationships between adaptive landscapes are important for the design of industrially relevant strains, which are generally subjected to multiple stressors. In our previous work, we evolved Escherichia coli for enhanced tolerance to the biofuel n-butanol and discovered a molecular mechanism of n-butanol tolerance that also conferred tolerance to the cationic antimicrobial peptide polymyxin B in one specific lineage (green fluorescent protein [GFP] labeled) in the evolved population. In this work, we aim to identify additional mechanisms of n-butanol tolerance in an independent lineage (yellow fluorescent protein [YFP] labeled) from the same evolved population and to further explore potential cross-tolerance and antagonistic pleiotropy between n-butanol tolerance and other industrially relevant stressors. Analysis of the transcriptome data of the YFP-labeled mutants allowed us to discover additional membrane-related and osmotic stress-related genes that confer n-butanol tolerance in E. coli. Interestingly, the n-butanol resistance mechanisms conferred by the membrane-related genes appear to be specific to n-butanol and are in many cases antagonistic with isobutanol and ethanol. Furthermore, the YFP-labeled mutants showed cross-tolerance between n-butanol and osmotic stress, while the GFP-labeled mutants showed antagonistic pleiotropy between n-butanol and osmotic stress tolerance.

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Mutagenesis of the Bacterial RNA Polymerase Alpha Subunit for Improvement of Complex Phenotypes

Cited by 79 publications

References 51 publications

Functional Genomic Study of Exogenous n -Butanol Stress in Escherichia coli

Functional Genomic Study of Exogenous n -Butanol Stress in Escherichia coli

Correcting direct effects of ethanol on translation and transcription machinery confers ethanol tolerance in bacteria

Genetic Determinants forn-Butanol Tolerance in Evolved Escherichia coli Mutants: Cross Adaptation and Antagonistic Pleiotropy betweenn-Butanol and Other Stressors

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