Pseudomonas putida toksiin-antitoksiin süsteem GraTA: regulatsioon ja osalus stressitaluvuses

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Elu on stressirohke, eriti üheraksetel organismidel nagu bakterid. Sageli tundub, et parim viis stressiga toimetulekuks on rahulikult oodata tingimuste paranemist. Selline käitumismall on kasutust leidnud ka mikroobide maailmas. Bakteritel on palju erinevaid kasvu reguleerimise võimalusi, mille hulka on viimasel ajal arvatud ka toksiin-antitoksiin (TA) süsteemid. TA-süsteemid koosnevad kahest komponendist: rakule eluliselt olulisi protsesse või rakukesta kahjustavast toksiinist ja teda neutraliseerivast antitoksiinist. Selliste geenide olemasolu bakterite genoomis on esmapilgul mõistatuslik, sest miks peaks bakter tootma iseendale toksilist valku? Hiljutised uuringud mikroobide mudelorganismis Escherichia coli on näidanud, et toksiinid põhjustavad bakterite üleminekut uinuvasse olekusse, mida iseloomustab bakterite ainevahetuse aeglustumine ja peatunud kasv. Sellised mikroobid tekitavad suuri probleeme meditsiinis, kuna on väga paljude stressiolukordade, kaasa arvatud paljude antibiootikumide toime suhtes tundetumad ja võimelised üle elama tingimusi, mis kiirelt kasvavaid baktereid tapaks. Kui mudelorganismis E. coli on TA süsteemide osalus bakteri stressitaluvuses hästi kirjeldatud, siis teistes bakteriliikides ei ole neid potentsiaalselt toksilisi süsteeme nii süstemaatiliselt uuritud. Seetõttu ei ole ka selge, kas erinevates bakterites toimivad TA süsteemid erinevalt või mingi üldise mehhanismi alusel. Käesolev töö kirjeldab keskkonnabakteri Pseudomonas putida kasvukiirust mõjutavat GraTA süsteemi. Tavaliselt takistab antitoksiin GraA väga efektiivselt toksiini GraT aktiivsust, kuid antitoksiinist vabanenult suudab toksiin mõjutada selle bakteri stressitaluvust. Toksiini mõju on kahetine, sest olenevalt stressi tüübist võib toksiin nii suurendada kui ka vähendada bakteri stressitaluvust. Seetõttu on bakterile väga oluline, et potentsiaalselt kahjulik TA süsteem aktiveeruks vaid kindlatel stressitingimustel.Life is full of stress, especially for small unicellular organisms like bacteria. For bacteria, just like for us, the best option to survive harsh conditions is sometimes to just lie still and wait for things to get better. Bacteria have many mechanisms to regulate growth, among them also the intriguing toxin-antitoxin (TA) systems. These systems consist of two components: a toxic protein that can harm the vital functions or compartments of a cell, and an antitoxin that can inhibit the toxin’s action. The presence of the TA systems in bacterial chromosomes is puzzling at first sight: why should a bacterium waste energy and resources to produce a toxin against itself? Recent research in the model organism Escherichia coli has shown that the toxic proteins cause a dormant, hibernation-like state, which is characterized by reduced metabolism and ceased growth. These bacteria cause great medical concerns as they are highly persistent to different stresses, including antibiotics, and survive conditions that would kill rapidly growing bacteria. After the stress has passed, the antitoxins inactivate the toxins and bacteria can resume growth. So, TA systems contribute to stress survival of bacteria, at least of E. coli. The contribution of TA systems to stress tolerance has been studied less systematically in other bacteria and no universal mechanism for the TA-mediated stress management has emerged so far. The current work describes a growth-rate-affecting TA system GraTA in the environmental bacterium Pseudomonas putida and shows that the toxin is kept under strict regulation by the antitoxin. Yet, when toxin is freed from the antitoxin, it inhibits the protein production in a cold-sensitive manner. The GraT toxin plays a controversial role in stress tolerance as it can both increase and decrease the tolerance to certain chemicals. This vividly highlights both the benefits and costs that the TA systems can have for bacteria

The Rel stringent factor from Thermus thermophilus: Crystallization and X-ray analysis

The stringent response, controlled by (p)ppGpp, enables bacteria to trigger a strong phenotypic resetting that is crucial to cope with adverse environmental changes and is required for stress survival and virulence. In the bacterial cell, (p)ppGpp levels are regulated by the concerted opposing activities of RSH (RelA/SpoT homologue) enzymes that can transfer a pyrophosphate group of ATP to the 3′ position of GDP (or GTP) or remove the 3′ pyrophosphate moiety from (p)ppGpp. Bifunctional Rel enzymes are notoriously difficult to crystallize owing to poor stability and a propensity for aggregation, usually leading to a loss of biological activity after purification. Here, the production, biochemical analysis and crystallization of the bifunctional catalytic region of the Rel stringent factor from Thermus thermophilus (Rel Tt NTD) in the resting state and bound to nucleotides are described. Rel Tt and Rel Tt NTD are monomers in solution that are stabilized by the binding of Mn2+ and mellitic acid. Rel Tt NTD crystallizes in space group P4122, with unit-cell parameters a = b = 88.4, c = 182.7 Å, at 4°C and in space group P41212, with unit-cell parameters a = b = 105.7, c = 241.4 Å, at 20°C.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Production, biophysical characterization and crystallization of Pseudomonas putida GraA and its complexes with GraT and the graTA operator

The graTA operon from Pseudomonas putida encodes a toxin-antitoxin module with an unusually moderate toxin. Here, the production, SAXS analysis and crystallization of the antitoxin GraA, the GraTA complex and the complex of GraA with a 33 bp operator fragment are reported. GraA forms a homodimer in solution and crystallizes in space group P21, with unit-cell parameters a = 66.9, b = 48.9, c = 62.7 Å, β = 92.6°. The crystals are likely to contain two GraA dimers in the asymmetric unit and diffract to 1.9 Å resolution. The GraTA complex forms a heterotetramer in solution. Crystals of the GraTA complex diffracted to 2.2 Å resolution and are most likely to contain a single heterotetrameric GraTA complex in the asymmetric unit. They belong to space group P41 or P43, with unit-cell parameters a = b = 56.0, c = 128.2 Å. The GraA-operator complex consists of a 33 bp operator region that binds two GraA dimers. It crystallizes in space group P31 or P32, with unit-cell parameters a = b = 105.6, c = 149.9 Å. These crystals diffract to 3.8 Å resolution.The antitoxin GraA from P. putida and its complexes with the toxin GraT and with the 33 bp operator of the graTA operon were crystallized.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

(p)ppGpp controls stringent factors by exploiting antagonistic allosteric coupling between catalytic domains

Amino acid starvation is sensed by Escherichia coli RelA and Bacillus subtilis Rel through monitoring the aminoacylation status of ribosomal A-site tRNA. These enzymes are positively regulated by their product—the alarmone nucleotide (p)ppGpp—through an unknown mechanism. The (p)ppGpp-synthetic activity of Rel/RelA is controlled via auto-inhibition by the hydrolase/pseudo-hydrolase (HD/pseudo-HD) domain within the enzymatic N-terminal domain region (NTD). We localize the allosteric pppGpp site to the interface between the SYNTH and pseudo-HD/HD domains, with the alarmone stimulating Rel/RelA by exploiting intra-NTD autoinhibition dynamics. We show that without stimulation by pppGpp, starved ribosomes cannot efficiently activate Rel/RelA. Compromised activation by pppGpp ablates Rel/RelA function in vivo, suggesting that regulation by the second messenger (p)ppGpp is necessary for mounting an acute starvation response via coordinated enzymatic activity of individual Rel/RelA molecules. Control by (p)ppGpp is lacking in the E. coli (p)ppGpp synthetase SpoT, thus explaining its weak synthetase activity

Nonhydrolysable Analogues of (p)ppGpp and (p)ppApp Alarmone Nucleotides as Novel Molecular Tools

While alarmone nucleotides guanosine-3',5'-bisdiphosphate (ppGpp) and guanosine-5'-triphosphate-3'-diphosphate (pppGpp) are archetypical bacterial second messengers, their adenosine analogues ppApp (adenosine-3',5'-bisdiphosphate) and pppApp (adenosine-5'-triphosphate-3'-diphosphate) are toxic effectors that abrogate bacterial growth. The alarmones are both synthesized and degraded by the members of the RelA-SpoT Homologue (RSH) enzyme family. Because of the chemical and enzymatic liability of (p)ppGpp and (p)ppApp, these alarmones are prone to degradation during structural biology experiments. To overcome this limitation, we have established an efficient and straightforward procedure for synthesizing nonhydrolysable (p)ppNuNpp analogues starting from 3'-azido-3'-deoxyribonucleotides as key intermediates. To demonstrate the utility of (p)ppGNpp as a molecular tool, we show that (i) as an HD substrate mimic, ppGNpp competes with ppGpp to inhibit the enzymatic activity of human MESH1 Small Alarmone Hyrolase, SAH; and (ii) mimicking the allosteric effects of (p)ppGpp, (p)ppGNpp acts as a positive regulator of the synthetase activity of long ribosome-associated RSHs Rel and RelA. Finally, by solving the structure of the N-terminal domain region (NTD) of T. thermophilus Rel complexed with pppGNpp, we show that as an HD substrate mimic, the analogue serves as a bona fide orthosteric regulator that promotes the same intra-NTD structural rearrangements as the native substrate

Direct activation of a bacterial innate immune system by a viral capsid protein

Bacteria have evolved diverse immunity mechanisms to protect themselves against the constant onslaught of bacteriophages1-3. Similar to how eukaryotic innate immune systems sense foreign invaders through pathogen-associated molecular patterns4 (PAMPs), many bacterial immune systems that respond to bacteriophage infection require phage-specific triggers to be activated. However, the identities of such triggers and the sensing mechanisms remain largely unknown. Here we identify and investigate the anti-phage function of CapRelSJ46, a fused toxin-antitoxin system that protects Escherichia coli against diverse phages. Using genetic, biochemical and structural analyses, we demonstrate that the C-terminal domain of CapRelSJ46 regulates the toxic N-terminal region, serving as both antitoxin and phage infection sensor. Following infection by certain phages, newly synthesized major capsid protein binds directly to the C-terminal domain of CapRelSJ46 to relieve autoinhibition, enabling the toxin domain to pyrophosphorylate tRNAs, which blocks translation to restrict viral infection. Collectively, our results reveal the molecular mechanism by which a bacterial immune system directly senses a conserved, essential component of phages, suggesting a PAMP-like sensing model for toxin-antitoxin-mediated innate immunity in bacteria. We provide evidence that CapRels and their phage-encoded triggers are engaged in a 'Red Queen conflict'5, revealing a new front in the intense coevolutionary battle between phages and bacteria. Given that capsid proteins of some eukaryotic viruses are known to stimulate innate immune signalling in mammalian hosts6-10, our results reveal a deeply conserved facet of immunity

Nonhydrolysable Analogues of (p)ppGpp and (p)ppApp Alarmone Nucleotides as Novel Molecular Tools

While alarmone nucleotides guanosine-3′,5′-bisdiphosphate (ppGpp) and guanosine-5′-triphosphate-3′-diphosphate (pppGpp) are archetypical bacterial second messengers, their adenosine analogues ppApp (adenosine-3′,5′-bisdiphosphate) and pppApp (adenosine-5′-triphosphate-3′-diphosphate) are toxic effectors that abrogate bacterial growth. The alarmones are both synthesized and degraded by the members of the RelA-SpoT Homologue (RSH) enzyme family. Because of the chemical and enzymatic liability of (p)ppGpp and (p)ppApp, these alarmones are prone to degradation during structural biology experiments. To overcome this limitation, we have established an efficient and straightforward procedure for synthesizing nonhydrolysable (p)ppNuNpp analogues starting from 3′-azido-3′-deoxyribonucleotides as key intermediates. To demonstrate the utility of (p)ppGNpp as a molecular tool, we show that (i) as an HD substrate mimic, ppGNpp competes with ppGpp to inhibit the enzymatic activity of human MESH1 Small Alarmone Hyrolase, SAH; and (ii) mimicking the allosteric effects of (p)ppGpp, (p)ppGNpp acts as a positive regulator of the synthetase activity of long ribosome-associated RSHs Rel and RelA. Finally, by solving the structure of the N-terminal domain region (NTD) of T. thermophilus Rel complexed with pppGNpp, we show that as an HD substrate mimic, the analogue serves as a bona fide orthosteric regulator that promotes the same intra-NTD structural rearrangements as the native substrate