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Stress-Induced MazF-Mediated Proteins in Escherichia coli.

ABSTRACT: Escherichia coli mazEF is an extensively studied stress-induced toxin-antitoxin (TA) system. The toxin MazF is an endoribonuclease that cleaves RNAs at ACA sites. By that means, under stress, the induced MazF generates a stress-induced translation machinery (STM) composed of MazF-processed mRNAs and selective ribosomes that specifically translate the processed mRNAs. Here, we performed a proteomic analysis of all the E. coli stress-induced proteins that are mediated through the chromosomally borne mazF gene. We show that the mRNAs of almost all of them are characterized by the presence of an ACA site up to 100 nucleotides upstream of the AUG initiator. Therefore, under stressful conditions, induced MazF processes mRNAs that are translated by STM. Furthermore, the presence of the ACA sites far upstream (up to 100 nucleotides) of the AUG initiator may still permit translation by the canonical translation machinery. Thus, such dual-translation mechanisms enable the bacterium under stress also to prepare proteins for immediate functions while coming back to normal growth conditions.IMPORTANCE The stress response, the strategy that bacteria have developed in order to cope up with all kinds of adverse conditions, is so far understood at the level of transcription. Our previous findings of a uniquely modified stress-induced translation machinery (STM) generated in E. coli under stress by the endoribonucleolytic activity of the toxin MazF opens a new chapter in understanding microbial physiology under stress at the translational level. Here, we performed a proteomic analysis of all the E. coli stress-induced proteins that are mediated by chromosomally borne MazF through STM.

SUBMITTER: Nigam A

PROVIDER: S-EPMC6437054 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Stress-induced MazF-mediated proteins in Escherichia coli

Project description:Escherichia coli (E. coli) mazEF is an extensively studied stress-induced toxin-antitoxin (TA) system. The toxin MazF is an endoribonuclease that cleaves RNAs at ACA sites. Thereby, under stress the induced MazF generates a Stress induced Translation Machinery (STM), composed of MazF processed mRNAs and selective ribosomes that specifically translate the processed mRNAs. Here we performed a proteomic analysis of all the E. coli stress-induced proteins that are mediated through chromosomally borne mazF gene . We show that the mRNAs of almost all of them is characterized by the presence of an ACA site up to 100 nucleotides upstream to the AUG initiator. Thereby, under stressful conditions the induced MazF is processing mRNAs that are translated by STM. Furthermore, the presence of the ACA site far enough upstream (up to 100 nucleotides) to the AUG initiator may still permit translation by the canonical translation machinery. Thus, such dual translation mechanisms, enable under stress also to prepare proteins for immediate functions while coming back to normal growth conditions.

2019-02-25 | PXD010101 | Pride

A Bias in the Reading of the Genetic Code of <i>Escherichia coli</i> is a Characteristic for Genes that Specify Stress-induced MazF-mediated Proteins.

Project description:BackgroundEscherichia coli (E. coli) mazEF, a stress-induced toxin-antitoxin (TA) system, has been studied extensively. The MazF toxin is an endoribonuclease that cleaves RNAs at ACA sites. Thereby, under stress, the induced MazF generates a Stress-induced Translation Machinery (STM), composed of MazF processed mRNAs and selective ribosomes that specifically translate the processed mRNAs.Materials and methodsBased on the data from the EcoCyc website of the National Center for Biotechnology Information (NCBI), the sequence of all E. coli MG1655 genes were scanned for ACA sites upstream from the initiation codons. Among these sequences, the fuzznuc program of the "European Molecular Biology Open Software Suite" (EMBOSS) was used to find the "ACA" pattern. The distribution of the ACA threonine codon, both in-frame and out-of-frame, was determined by using the HTML Script Program (Supplementary Material).ResultsHere it is reported that for most of the E. coli proteins mediated by stress-induced MazF, the ACA threonine codon in their mRNAs is not in-frame but rather out-of-frame; in these same RNAs, the three synonymous threonine codons, ACG, ACU, and ACC, are in-frame. In contrast, for proteins translated by the canonical translation system, in the majority of mRNAs, the ACA codon is located in-frame.ConclusionThe described bias in the genetic code is a characteristic of E. coli genes specifying for stress-induced MazF-mediated proteins.

| S-EPMC7521043 | biostudies-literature

The RNA ligase RtcB reverses MazF-induced ribosome heterogeneity in Escherichia coli.

Project description:When Escherichia coli encounters stress, the endoribonuclease MazF initiates a post-transcriptional response that results in the reprogramming of protein synthesis. By removing the 3΄-terminus of the 16S rRNA, MazF generates specialized ribosomes that selectively translate mRNAs likewise processed by MazF. Given the energy required for de novo ribosome biosynthesis, we considered the existence of a repair mechanism operating upon stress relief to recycle the modified ribosomes. Here, we show that the stress-ribosomes and the 3΄-terminal 16S rRNA fragment are stable during adverse conditions. Moreover, employing in vitro and in vivo approaches we demonstrate that the RNA ligase RtcB catalyzes the re-ligation of the truncated 16S rRNA present in specialized ribosomes Thereby their ability to translate canonical mRNAs is fully restored. Together, our findings not only provide a physiological function for the RNA ligase RtcB in bacteria but highlight the reversibility of ribosome heterogeneity, a crucial but hitherto undescribed concept for translational regulation.

| S-EPMC5416887 | biostudies-literature

Escherichia coli MazF leads to the simultaneous selective synthesis of both "death proteins" and "survival proteins".

Project description:The Escherichia coli mazEF module is one of the most thoroughly studied toxin-antitoxin systems. mazF encodes a stable toxin, MazF, and mazE encodes a labile antitoxin, MazE, which prevents the lethal effect of MazF. MazF is an endoribonuclease that leads to the inhibition of protein synthesis by cleaving mRNAs at ACA sequences. Here, using 2D-gels, we show that in E. coli, although MazF induction leads to the inhibition of the synthesis of most proteins, the synthesis of an exclusive group of proteins, mostly smaller than about 20 kDa, is still permitted. We identified some of those small proteins by mass spectrometry. By deleting the genes encoding those proteins from the E. coli chromosome, we showed that they were required for the death of most of the cellular population. Under the same experimental conditions, which induce mazEF-mediated cell death, other such proteins were found to be required for the survival of a small sub-population of cells. Thus, MazF appears to be a regulator that induces downstream pathways leading to death of most of the population and the continued survival of a small sub-population, which will likely become the nucleus of a new population when growth conditions become less stressful.

| S-EPMC2646832 | biostudies-literature

The MazF-regulon: a toolbox for the post-transcriptional stress response in Escherichia coli.

Project description:Flexible adaptation to environmental stress is vital for bacteria. An energy-efficient post-transcriptional stress response mechanism in Escherichia coli is governed by the toxin MazF. After stress-induced activation the endoribonuclease MazF processes a distinct subset of transcripts as well as the 16S ribosomal RNA in the context of mature ribosomes. As these 'stress-ribosomes' are specific for the MazF-processed mRNAs, the translational program is changed. To identify this 'MazF-regulon' we employed Poly-seq (polysome fractionation coupled with RNA-seq analysis) and analyzed alterations introduced into the transcriptome and translatome after mazF overexpression. Unexpectedly, our results reveal that the corresponding protein products are involved in all cellular processes and do not particularly contribute to the general stress response. Moreover, our findings suggest that translational reprogramming serves as a fast-track reaction to harsh stress and highlight the so far underestimated significance of selective translation as a global regulatory mechanism in gene expression. Considering the reported implication of toxin-antitoxin (TA) systems in persistence, our results indicate that MazF acts as a prime effector during harsh stress that potentially introduces translational heterogeneity within a bacterial population thereby stimulating persister cell formation.

| S-EPMC5001579 | biostudies-literature

MazF-mediated cell death in Escherichia coli: a point of no return.

Project description:mazEF is a stress-induced toxin-antitoxin module, located on the chromosome of Escherichia coli, that we have previously described to be responsible for programmed cell death in E. coli. mazF specifies a stable toxin, and mazE specifies a labile antitoxin. Recently, it was reported that inhibition of translation and cell growth by ectopic overexpression of the toxin MazF can be reversed by the action of the antitoxin MazE ectopically overexpressed at a later time. Based on these results, it was suggested that rather than inducing cell death, mazF induces a state of reversible bacteriostasis (K. Pederson, S. K. Christensen, and K. Gerdes, Mol. Microbiol. 45:501-510, 2002). Using a similar ectopic overexpression system, we show here that overexpression of MazE could reverse MazF lethality only over a short window of time. The size of that window depended on the nature of the medium in which MazF was overexpressed. Thus, we found "a point of no return," which occurred sooner in minimal M9 medium than it did in the rich Luria-Bertani medium. We also describe a state in which the effect of MazF on translation could be separated from its effect on cell death: MazE overproduction could completely reverse the inhibitory effect of MazF on translation, while not affecting the bacteriocidic effect of MazF at all. Our results reported here support our view that the mazEF module mediates cell death and is part of a programmed cell death network.

| S-EPMC532418 | biostudies-other

Universal stress proteins in Escherichia coli.

Project description: Not available

| S-EPMC1236659 | biostudies-literature

Quantifying heterologous gene expression during ectopic MazF production in Escherichia coli.

Project description:ObjectiveMazF is a sequence-specific endoribonuclease-toxin of the MazEF toxin-antitoxin system. MazF cleaves single-stranded ribonucleic acid (RNA) regions at adenine-cytosine-adenine (ACA) sequences in the bacterium Escherichia coli. The MazEF system has been used in various biotechnology and synthetic biology applications. In this study, we infer how ectopic mazF overexpression affects production of heterologous proteins. To this end, we quantified the levels of fluorescent proteins expressed in E. coli from reporters translated from the ACA-containing or ACA-less messenger RNAs (mRNAs). Additionally, we addressed the impact of the 5'-untranslated region of these reporter mRNAs under the same conditions by comparing expression from mRNAs that comprise (canonical mRNA) or lack this region (leaderless mRNA).ResultsFlow cytometry analysis indicates that during mazF overexpression, fluorescent proteins are translated from the canonical as well as leaderless mRNAs. Our analysis further indicates that longer mazF overexpression generally increases the concentration of fluorescent proteins translated from ACA-less mRNAs, however it also substantially increases bacterial population heterogeneity. Finally, our results suggest that the strength and duration of mazF overexpression should be optimized for each experimental setup, to maximize the heterologous protein production and minimize the amount of phenotypic heterogeneity in bacterial populations, which is unfavorable in biotechnological processes.

| S-EPMC9102682 | biostudies-literature

Substrate Recognition and Activity Regulation of the Escherichia coli mRNA Endonuclease MazF.

Project description:Escherichia coli MazF (EcMazF) is the archetype of a large family of ribonucleases involved in bacterial stress response. The crystal structure of EcMazF in complex with a 7-nucleotide substrate mimic explains the relaxed substrate specificity of the E. coli enzyme relative to its Bacillus subtilis counterpart and provides a framework for rationalizing specificity in this enzyme family. In contrast to a conserved mode of substrate recognition and a conserved active site, regulation of enzymatic activity by the antitoxin EcMazE diverges from its B. subtilis homolog. Central in this regulation is an EcMazE-induced double conformational change as follows: a rearrangement of a crucial active site loop and a relative rotation of the two monomers in the EcMazF dimer. Both are induced by the C-terminal residues Asp-78-Trp-82 of EcMazE, which are also responsible for strong negative cooperativity in EcMazE-EcMazF binding. This situation shows unexpected parallels to the regulation of the F-plasmid CcdB activity by CcdA and further supports a common ancestor despite the different activities of the MazF and CcdB toxins. In addition, we pinpoint the origin of the lack of activity of the E24A point mutant of EcMazF in its inability to support the substrate binding-competent conformation of EcMazF.

| S-EPMC4900246 | biostudies-literature

Toxins MazF and MqsR cleave Escherichia coli rRNA precursors at multiple sites.

Project description:The endoribonuclease toxins of the E. coli toxin-antitoxin systems arrest bacterial growth and protein synthesis by targeting cellular mRNAs. As an exception, E. coli MazF was reported to cleave also 16S rRNA at a single site and separate an anti-Shine-Dalgarno sequence-containing RNA fragment from the ribosome. We noticed extensive rRNA fragmentation in response to induction of the toxins MazF and MqsR, which suggested that these toxins can cleave rRNA at multiple sites. We adapted differential RNA-sequencing to map the toxin-cleaved 5'- and 3'-ends. Our results show that the MazF and MqsR cleavage sites are located within structured rRNA regions and, therefore, are not accessible in assembled ribosomes. Most of the rRNA fragments are located in the aberrant ribosomal subunits that accumulate in response to toxin induction and contain unprocessed rRNA precursors. We did not detect MazF- or MqsR-cleaved rRNA in stationary phase bacteria and in assembled ribosomes. Thus, we conclude that MazF and MqsR cleave rRNA precursors before the ribosomes are assembled and potentially facilitate the decay of surplus rRNA transcripts during stress.

| S-EPMC5270532 | biostudies-literature

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