RelA-SpoT homolog enzymes as effectors of Toxin-Antitoxin systems
Date
2022-10-25
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Abstract
Nagu kõik elusorganismid, tunnetavad bakterid keskkonda ja reageerivad suurele hulgale erinevatele stressidele, kohandades vastavalt oma füsioloogiat.
Üks peamisi stressivastuseid on poomisvastus. Poomisvastus vahendab bakterite kohanemist toitainete vähesusega, samuti vastust abiootilisele keskkonnastressidele nagu näiteks kuumašokk. Rohkem kui kuus aastakümmet tagasi avastati, et häirenukleotiidid ppGpp ja pppGpp – ühiselt viidatud kui (p)ppGpp – ehk maagilised laigud tekivad Escherichia coli rakkudes vastusena aminohapete vähesusele. Poomisvastuse esimene füsioloogiline roll, mis tuvastati, oli stabiilse RNA (rRNA ja tRNA) sünteesi pärssimine, mis on kooskõlastatud aminohapete biosünteesi ja stressitaluvusega seotud geenide ekspressiooni indutseerimisega. Aastakümneid kestnud uuringud on aga näidanud, et lisaks transkriptsioonile on (p)ppGpp sihtmärkideks ka mitmed muud rakus toimuvad protsessid, nagu translatsioon, ribosoomide kokkupanek, antibiootikumiresistentsus ja virulentsus.
Veel üks oluline bakterite regulatsioonisüsteem põhineb toksiini – antitoksiin (TA) süsteemidel. Esimesed toksiini-antitoksiin (TA) süsteemide esindajad avastati 80ndate alguses. Klassikalised TA süsteemid on bitsistroonilised – st koosnevad kahest geenist – operonist, milles üks geen kodeerib valgulist toksiini ja teine antitoksiini, valku või RNAd, mis toksiini kas otseselt või kaudselt neutraliseerib. TA-süsteemide uuringud on viimastel aastatel plahvatuslikult kasvanud, avastatud on arvukalt uusi TA perekondi, iseloomustatud nende toimemehhanisme, iseloomustatud bioloogilisi funktsioone ja pakutud välja võimalikke rakendusi biotehnoloogias. Enim iseloomustatud funktsioonid hõlmavad plasmiidi säilitamist, kaitset bakteriofaagide vastu ja rakufüsioloogia reguleerimist.
Käesolevas uuringus kirjeldati RSH perekonna ensüümide uusi aktiivsusi ja toksiinide neutraliseerimise spetsiifilisust PanA antitoksiini perekonna liikmete poolt.
Lisaks eelpool kirjeldatud protsessidele toimub stressi ajal ribosoomide dimerisatsioon. See stressivastus on kasulik rakkudele ellujäämiseks, kuid võib lüsaatide kasutamise korral biotehnoloogias olla probleemiks kuna vähendab rakuvabade translatsioonisüsteemide aktiivsust. Seetõttu uuriti ribosoomi dimeriseerumise eest vastutavate valkude eemaldamise mõju rakulüsaatide aktiivsusele.
Leiti, et RSH ensüümide ensümaatiline aktiivsus ei piirdu (p)ppGpp tootmise ja lagunemisega. ToxSAS RSH PhRel2, FaRel2, PhRel ja CapRel alamperekondade liikmed katalüüsivad tRNA 3'CCA otsa pürofosforüülimist ja FaRel perekonna liikmed katalüüsivad (pp)pApp sünteesi. SAH alamperekonna liikmed MESH1 ja ATfaRel katalüüsivad pürofosfaadi eemaldamist PP-tRNA-st ja (pp)pApp lagunemist.
Ühist PanA domeeni sisaldavad antitoksiinid neutraliseerivad erinevaid toksiine. PanA-vahendatud toksiinide neutraliseerimine on toksiini osas siiski spetsiifiline.
Ribosoomi dimerisatsioonifaktorite geneetiline elimineerimine bakteri B. subtilis (hfp) ja pärmi S. cerevisiae (stm1) tüvedes on paljulubav strateegia aktiivsemate rakuvabade translatsioonilüsaatide tootmiseks. Reaktsiooni optimeerimisel on oluline panna tähele Mg2+ ja muude komponentide kontsentratsioone ja omavahelisi suhteid.
Like all living organisms, bacteria sense the environment and respond to plethora of stresses by adjusting their physiology accordingly. One of the central bacterial stress pathways is the stringent response (SR). The stringent response mediates the bacterial adaptation to nutrient limitation as well as to in response to abiotic environmental stresses like heat shock. More than six decades ago alarmone nucleotides ppGpp and pppGpp – collectively referred to as (p)ppGpp – or the “magic spots” were discovered to be produced in Escherichia coli cells as a response to amino acids limitation. The first physiologial role of the SR to be characterised was inhibition of stable RNA (rRNA and tRNA) synthesis, coordinated with induction of expression of genes involved in amino acid biosynthesis and stress tolerance. However, decades of research have established that in addition to transcription, (p)ppGpp targets multiple other processes in the cell, such as translation, ribosome assembly, metabolism, and impact all the aspects of cell physiology including adaptation to nutrient limitation, antibiotic resistance and virulence. Another regulatory system in bacteria is based on the toxin – antitoxin (TA) systems. First representatives of toxin -antitoxin (TA) systems were discovered in the early 80s. The classical TA systems are bicistronic – i.e. comprised of two gene – operons, in which one gene encodes a protein toxin and the other encodes a protein or RNA antitoxin which neutralises the toxin, either directly or indirectly. Studies of TA systems have exploded in the last years, with numerous new TA families being discovered, their mechanisms of action being characterised, biological functions established and possible applications for biotechnology put forward. The most established functions include plasmid maintenance, defence against bacteriophages and regulation of cell physiology. In the current study new activities of the RSH family enzymes were described and the specificity of toxin neutralization by an PanA antitoxin family members described. Additionally, dimerization of ribosomes occurs during stress. This activity is useful for the cell survival but might decrease the activity of cell free translation systems in case lysates are used in biotechnology. Therefore, the effect of removing the proteins responsible for ribosome dimerization was investigated. It was found that enzymatic activities of RSH enzymes are not limited to production and degradation of (p)ppGpp. Members of toxSAS RSH PhRel2, FaRel2, PhRel and CapRel subfamilies catalyse pyrophosphorylation of tRNA 3'CCA end, and members of FaRel family catalyse synthesis of (pp)pApp. Members of SAH subfamily MESH1 and ATfaRel catalyse removal of the pyrophosphate from PP-tRNA and degradation of (pp)pApp. Antitoxins containing common PanA domain neutralize diverse toxin families. PanA-mediated toxin neutralisation is highly specific for the cognate toxin-antitoxin pair. Genetic elimination of ribosome dimerization factors in Firmicute bacterium B. subtilis (hfp) and yeast S. cerevisiae (stm1) strains is a promising strategy for producing more active in vitro translation lysates. Titration of Mg2+ and different reaction components are essential for achieving the optimal activity of the lysate.
Like all living organisms, bacteria sense the environment and respond to plethora of stresses by adjusting their physiology accordingly. One of the central bacterial stress pathways is the stringent response (SR). The stringent response mediates the bacterial adaptation to nutrient limitation as well as to in response to abiotic environmental stresses like heat shock. More than six decades ago alarmone nucleotides ppGpp and pppGpp – collectively referred to as (p)ppGpp – or the “magic spots” were discovered to be produced in Escherichia coli cells as a response to amino acids limitation. The first physiologial role of the SR to be characterised was inhibition of stable RNA (rRNA and tRNA) synthesis, coordinated with induction of expression of genes involved in amino acid biosynthesis and stress tolerance. However, decades of research have established that in addition to transcription, (p)ppGpp targets multiple other processes in the cell, such as translation, ribosome assembly, metabolism, and impact all the aspects of cell physiology including adaptation to nutrient limitation, antibiotic resistance and virulence. Another regulatory system in bacteria is based on the toxin – antitoxin (TA) systems. First representatives of toxin -antitoxin (TA) systems were discovered in the early 80s. The classical TA systems are bicistronic – i.e. comprised of two gene – operons, in which one gene encodes a protein toxin and the other encodes a protein or RNA antitoxin which neutralises the toxin, either directly or indirectly. Studies of TA systems have exploded in the last years, with numerous new TA families being discovered, their mechanisms of action being characterised, biological functions established and possible applications for biotechnology put forward. The most established functions include plasmid maintenance, defence against bacteriophages and regulation of cell physiology. In the current study new activities of the RSH family enzymes were described and the specificity of toxin neutralization by an PanA antitoxin family members described. Additionally, dimerization of ribosomes occurs during stress. This activity is useful for the cell survival but might decrease the activity of cell free translation systems in case lysates are used in biotechnology. Therefore, the effect of removing the proteins responsible for ribosome dimerization was investigated. It was found that enzymatic activities of RSH enzymes are not limited to production and degradation of (p)ppGpp. Members of toxSAS RSH PhRel2, FaRel2, PhRel and CapRel subfamilies catalyse pyrophosphorylation of tRNA 3'CCA end, and members of FaRel family catalyse synthesis of (pp)pApp. Members of SAH subfamily MESH1 and ATfaRel catalyse removal of the pyrophosphate from PP-tRNA and degradation of (pp)pApp. Antitoxins containing common PanA domain neutralize diverse toxin families. PanA-mediated toxin neutralisation is highly specific for the cognate toxin-antitoxin pair. Genetic elimination of ribosome dimerization factors in Firmicute bacterium B. subtilis (hfp) and yeast S. cerevisiae (stm1) strains is a promising strategy for producing more active in vitro translation lysates. Titration of Mg2+ and different reaction components are essential for achieving the optimal activity of the lysate.
Description
Väitekirja elektrooniline versioon ei sisalda publikatsioone
Keywords
bacteria, stringent response, toxin-antitoxin systems