Bakterite toksiin-antitoksiin süsteemid: transkriptsiooniline rist-aktivatsioon ja uue mqsRA süsteemi iseloomustamine

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Paljud bakterid kinnituvad pindadele ja moodustavad enda ümber limase kaitsekihi. Sellist kasvuviisi kutsutakse biokileks ja see tagab bakterile parema kaitse kahjulike mõjurite eest nagu näiteks antibiootikumid või peremehe immuunsüsteem. Biokilede tõttu tekivad korduvad infektsioonid, mis on tõsiseks meditsiiniliseks probleemiks. Biokiledes on suurenenud ajutiselt mitte-kasvavate bakterite hulk, kes ei oma eriomaseid mehhanisme antibiootikumide vastu võitlemiseks, kuid kes oma uinunud oleku tõttu ei allu ravile. Neis uinunud rakkudes on tugevalt avaldunud toksiin-antitoksiini (TA) süsteeme kodeerivad geenid. Toksiin on valk, mis takistab mõnda bakteri enda olulist rakulist protsessi. Paljud toksiinid lõikavad mRNA-d, takistades nii valkude sünteesi ja bakterite kasvu. Antitoksiin on valk, mis toksiiniga seondudes võimaldab bakteril jälle kasvada. Seega aktiveerunud toksiinid võivad põhjustada nende uinunud bakterite teket biofilmis. Käesoleva doktoritöö eesmärgiks oli kirjeldada TA süsteemide omavahelist rist-aktiveerimist ja testida, kas soolebakteri Escherichia coli geenid mqsR ja mqsA moodustavad uue TA süsteemi. Uurimustulemustest selgus, et MqsR pidurdab tugevalt bakterite kasvu, samas MqsA pärsib MqsR toksilist mõju ja reguleerib mõlema geeni avaldumist. Seega tõestasime, et MqsRA on uus TA süsteem. Teiseks näitasime, et TA süsteemide vahel esineb rist-aktivatsioon. Täiendavalt selgus, et see rist-aktivatsioon võib olla mõjutatud TA mRNAde lõikamisest, mille tulemusel toodab bakter rohkem toksiine kui antitoksiine. Kokkuvõtteks, töö tulemusel selgitati TA geenide üheaegse avaldumise tagamaid, mis toetavad TA süsteemide seost uinunud bakterite tekkel.Many bacteria attach to surfaces and enclose into slime. This type of growth is called biofilm and it provides better protection for bacteria to fight against harmful factors such as antibiotics and immune system. It has been proposed that most chronic infections are caused by bacteria growing in biofilms, which is a serious medical problem. Biofilm is enriched with temporarily non-dividing bacteria that have no specific antibiotic resistance mechanisms. These bacteria are not killed by the drug due to their dormant state. After the treatment, these cells wake up and start growing again. In these dormant cells, the toxin-antitoxin (TA) system genes are highly expressed. Toxin is a protein that inhibits some important process inside the bacterium. Many toxins cleave mRNA, thus, inhibiting protein synthesis and bacterial growth. Antitoxin is a protein that, by binding to the toxin, allows a cell to grow again. Therefore, activated toxins may result in formation of dormant cells in biofilms. The aim of the current dissertation was to examine the cross-activation between TA systems and to test whether mqsR and mqsA genes encode a new TA system in Eschericha coli. Firstly, we demonstrated that MqsR arrests cell growth and MqsA neutralizes the toxic effect of MqsR. In addition, MqsA regulates expression of the both genes, which is a characteristic for the antitoxins. Hence, we characterized and validated a new MqsRA TA system. Secondly, we proved cross-activation between different TA systems. The results indicate that TA cross-activation may be influenced by TA mRNA cleavage, through which bacteria produce more toxin compared to its antitoxin. In conclusion, this study provides new knowledge about simultaneous expression of TA systems and supports their possible role in dormancy

Transcriptional cross-activation between toxin-antitoxin systems of Escherichia coli

BACKGROUND: Bacterial toxin-antitoxin (TA) systems are formed by potent regulatory or suicide factors (toxins) and their short-lived inhibitors (antitoxins). Antitoxins are DNA-binding proteins and auto-repress transcription of TA operons. Transcription of multiple TA operons is activated in temporarily non-growing persister cells that can resist killing by antibiotics. Consequently, the antitoxin levels of persisters must have been dropped and toxins are released of inhibition. RESULTS: Here, we describe transcriptional cross-activation between different TA systems of Escherichia coli. We find that the chromosomal relBEF operon is activated in response to production of the toxins MazF, MqsR, HicA, and HipA. Expression of the RelE toxin in turn induces transcription of several TA operons. We show that induction of mazEF during amino acid starvation depends on relBE and does not occur in a relBEF deletion mutant. Induction of TA operons has been previously shown to depend on Lon protease which is activated by polyphospate accumulation. We show that transcriptional cross-activation occurs also in strains deficient for Lon, ClpP, and HslV proteases and polyphosphate kinase. Furthermore, we find that toxins cleave the TA mRNA in vivo, which is followed by degradation of the antitoxin-encoding fragments and selective accumulation of the toxin-encoding regions. We show that these accumulating fragments can be translated to produce more toxin. CONCLUSION: Transcriptional activation followed by cleavage of the mRNA and disproportionate production of the toxin constitutes a possible positive feedback loop, which can fire other TA systems and cause bistable growth heterogeneity. Cross-interacting TA systems have a potential to form a complex network of mutually activating regulators in bacteria

A role for the Saccharomyces cerevisiae ABCF protein New1 in translation termination/recycling

Translation is controlled by numerous accessory proteins and translation factors. In the yeast Saccharomyces cerevisiae, translation elongation requires an essential elongation factor, the ABCF ATPase eEF3. A closely related protein, New1, is encoded by a non-essential gene with cold sensitivity and ribosome assembly defect knock-out phenotypes. Since the exact molecular function of New1 is unknown, it is unclear if the ribosome assembly defect is direct, i.e. New1 is a bona fide assembly factor, or indirect, for instance due to a defect in protein synthesis. To investigate this, we employed yeast genetics, cryo-electron microscopy (cryo-EM) and ribosome profiling (Ribo-Seq) to interrogate the molecular function of New1. Overexpression of New1 rescues the inviability of a yeast strain lacking the otherwise strictly essential translation factor eEF3. The structure of the ATPase-deficient (EQ2) New1 mutant locked on the 80S ribosome reveals that New1 binds analogously to the ribosome as eEF3. Finally, Ribo-Seq analysis revealed that loss of New1 leads to ribosome queuing upstream of 3'-terminal lysine and arginine codons, including those genes encoding proteins of the cytoplasmic translational machinery. Our results suggest that New1 is a translation factor that fine-tunes the efficiency of translation termination or ribosome recycling

The Escherichia coli mqsR and ygiT Genes Encode a New Toxin-Antitoxin Pair▿ †

Toxin-antitoxin (TA) systems are plasmid- or chromosome-encoded protein complexes composed of a stable toxin and a short-lived inhibitor of the toxin. In cultures of Escherichia coli, transcription of toxin-antitoxin genes was induced in a nondividing subpopulation of bacteria that was tolerant to bactericidal antibiotics. Along with transcription of known toxin-antitoxin operons, transcription of mqsR and ygiT, two adjacent genes with multiple TA-like features, was induced in this cell population. Here we show that mqsR and ygiT encode a toxin-antitoxin system belonging to a completely new family which is represented in several groups of bacteria. The mqsR gene encodes a toxin, and ectopic expression of this gene inhibits growth and induces rapid shutdown of protein synthesis in vivo. ygiT encodes an antitoxin, which protects cells from the effects of MqsR. These two genes constitute a single operon which is transcriptionally repressed by the product of ygiT. We confirmed that transcription of this operon is induced in the ampicillin-tolerant fraction of a growing population of E. coli and in response to activation of the HipA toxin. Expression of the MqsR toxin does not kill bacteria but causes reversible growth inhibition and elongation of cells

Ribosome profiling analysis of eEF3-depleted Saccharomyces cerevisiae

In addition to the standard set of translation factors common in eukaryotic organisms, protein synthesis in the yeast Saccharomyces cerevisiae requires an ABCF ATPase factor eEF3, eukaryotic Elongation Factor 3. eEF3 is an E-site binder that was originally identified as an essential factor involved in the elongation stage of protein synthesis. Recent biochemical experiments suggest an additional function of eEF3 in ribosome recycling. We have characterised the global effects of eEF3 depletion on translation using ribosome profiling. Depletion of eEF3 results in decreased ribosome density at the stop codon, indicating that ribosome recycling does not become rate limiting when eEF3 levels are low. Consistent with a defect in translation elongation, eEF3 depletion causes a moderate redistribution of ribosomes towards the 5' part of the open reading frames. We observed no E-site codon-or amino acid-specific ribosome stalling upon eEF3 depletion, supporting its role as a general elongation factor. Surprisingly, depletion of eEF3 leads to a relative decrease in P-site proline stalling, which we hypothesise is a secondary effect of generally decreased translation and/or decreased competition for the E-site with eIF5A

Toxins MazF and MqsR cleave <i>Escherichia coli</i> rRNA precursors at multiple sites

<p>The endoribonuclease toxins of the <i>E. coli</i> toxin-antitoxin systems arrest bacterial growth and protein synthesis by targeting cellular mRNAs. As an exception, <i>E. coli</i> 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.</p