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

Abstract

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.

Increased production of L-serine in Escherichia coli through Adaptive Laboratory Evolution

“…The Rho R87L mutant showed the second highest enrichment (37-fold) in the presence of L-serine. Mutation in rho gene (L270M) has previously been observed in an ethanol tolerant strain (Haft et al, 2014), and Rho L270M was shown to have reduced activity and reduced the premature transcription terminations in the cell. It is possible that the R87L mutation and the insertion of transposon in the promoter region may also reduce the expression and activity of Rho.…”

The reduction in small ribosomal subunit abundance in ethanol-stressed cells of Bacillus subtilis is mediated by a SigB-dependent antisense RNA

“…During their lifetime, all organisms are challenged with a wide range 42 of stresses. In bacteria these stresses can range from oxidative stress, 43 heat or cold stress, hypoxia, starvation, osmotic stress, and antibiotic ex-44 posure, to ethanol stress (Refs.…”

Yeast Interspecies Comparative Proteomics Reveals Divergence in Expression Profiles and Provides Insights into Proteome Resource Allocation and Evolutionary Roles of Gene Duplication

“…The sequences and synonymous and nonsynonymous mutation rates of abundant proteins are reported to be constrained to contribute to mRNA stability, translation fidelity, and robust protein folding (62,77). Considered together with the recent report that in bacteria, ethanol stress increases the frequency of misreading at protein translation (78), the existence of a compensatory counterpart gene might enhance the probability that proteins containing fewer errors are synthesized from at least one of the duplicate genes having a constrained DNA sequence more compatible with a high production rate, even when exposed to higher amount of self-generated ethanol.…”