Negative allosteric regulation of
            <i>Enterococcus faecalis</i>
            small alarmone synthetase RelQ by single-stranded RNA

Beljantseva, Jelena; Kudrin, Pavel; Andresen, Liis; Shingler, Victoria; Atkinson, Gemma C.; Tenson, Tanel; Hauryliuk, Vasili

doi:10.1073/pnas.1617868114

Cited by 48 publications

(63 citation statements)

References 33 publications

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“…Mycobacterium phage Squirty PhRel(18) did not display significant toxicity even when the expression was driven with a strong Shine-Dalgarno sequence (Supplementary Figure S1A). Additionally, we tested well-studied bacterial SASs that are not encoded in bicistronic arrangements (Staphylococcus aureus RelP (34,35) and Enterococcus faecalis RelQ (36,37)). We detected no toxicity, even with strong Shine-Dalgarno motifs (Supplementary Figure S1B).…”

Section: Bicistronic Toxsas Operons Encode Bona Fide Type II and Typementioning

confidence: 99%

“…We then investigated whether toxSAS antitoxins inhibit toxSASs on the level of RNA (as in type I and III TA systems) or protein (as in type II and IV TA systems). The former scenario is theoretically possible, since, as we have shown earlier, E. faecalis SAS RelQ binds single-strand RNA and is inhibited in a sequence-specific manner (36). To discriminate between the two alternatives, we mutated the start codon of the aTphRel2, aTfaRel2 and aTphRel antitoxin ORFs to a stop codon, TAA.…”

Section: Bicistronic Toxsas Operons Encode Bona Fide Type II and Typementioning

confidence: 99%

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A widespread toxin-antitoxin system exploiting growth control via alarmone signalling

Jimmy

Saha

Stavropoulos

et al. 2019

Preprint

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22Under stressful conditions, bacterial RelA-SpoT Homologue (RSH) enzymes synthesise the alarmone 23 (p)ppGpp, a nucleotide messenger. (p)ppGpp rewires bacterial transcription and metabolism to cope 24 with stress, and at high concentrations inhibits the process of protein synthesis and bacterial growth 25 to save and redirect resources until conditions improve. Single domain Small Alarmone Synthetases 26 (SASs) are RSH family members that contain the (p)ppGpp synthesis (SYNTH) domain, but lack the 27 hydrolysis (HD) domain and regulatory C-terminal domains of the long RSHs such as Rel, RelA and 28SpoT. We have discovered that multiple SAS subfamilies can be encoded in broadly distributed 29 conserved bicistronic operon architectures in bacteria and bacteriophages that are reminiscent of 30 those typically seen in toxin-antitoxin (TA) operons. We have validated five of these SASs as being 31 toxic (toxSASs), with neutralisation by the protein products of six neighbouring antitoxin genes. The 32 toxicity of Cellulomonas marina ToxSAS FaRel is mediated by alarmone accumulation combined with 33 depletion of cellular ATP and GTP pools, and this is counteracted by its HD domain-containing 34 antitoxin. Thus, the ToxSAS-antiToxSAS system is a novel TA paradigm comprising multiple different 35 antitoxins that exemplifies how ancient nucleotide-based signalling mechanisms can be repurposed 36 as TA modules during evolution, potentially multiple times independently. 37 1 Bacteria encounter a variety of different environmental conditions during their life cycles, to which 2 they need to respond and adapt in order to survive. This can include slowing down their growth and 3 redirecting their metabolic resources during nutritional stress, until conditions improve and the 4 growth rate can increase. One of the main signals that bacteria use for signalling stress are the 5 alarmone nucleotides ppGpp and pppGpp, collectively referred to as (p)ppGpp 1 . At high 6 concentrations (p)ppGpp is a potent inhibitor of bacterial growth 2 , targeting transcription, 7 translation and ribosome assembly 1 . (p)ppGpp is produced and degraded by proteins of the 8 RelA/SpoT homologue (RSH) superfamily, named after the two Escherichia coli representatives -9 multi-domain 'long' RSH factors RelA and SpoT 3 . In addition to long RSHs, bacteria can encode single-10 domain RSHs: Small Alarmone Synthetases (SAS) and Small Alarmone Hydrolases (SAH). 11It is currently unknown why some bacteria carry multiple SASs and SAHs, which can belong to many 12 different subfamilies. Conservation of gene order through evolution can reveal potentially interacting 13 proteins and shed light on the cellular role of proteins 4 . Therefore, we developed a computational 14 tool -FlaGs, standing for Flanking Genes 5 -for analysing the conservation of genomic 15 neighbourhoods, and applied it to our updated database of RSH sequences classified into 16 subfamilies. Surprisingly, we find that some subfamilies of SAS can be encoded in apparently bi-(and 17 sometimes tri-) ...

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Section: Bicistronic Toxsas Operons Encode Bona Fide Type II and Typementioning

confidence: 99%

Section: Bicistronic Toxsas Operons Encode Bona Fide Type II and Typementioning

confidence: 99%

A widespread toxin-antitoxin system exploiting growth control via alarmone signalling

Jimmy

Saha

Stavropoulos

et al. 2019

Preprint

Self Cite

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“…The alarmones pppGpp and ppGpp have been shown to (allosterically) stimulate the synthetic activities of the SAS proteins from E. faecalis (RelQ Ef ) [40,42] and B. subtilis (SAS1, YjbM) [58], but to (allosterically) inhibit the activities of the Sa-RelP protein [41]. However, the modulatory effects of alarmones on the Sa-RelQ protein remain to be determined.…”

Section: Kinetic Analysis Of Sa-relp and Sa-relq Alarmone Synthesizinmentioning

confidence: 99%

“…210 aa and ca. 230 aa in length, and share high levels of sequence similarity and structural homology [14,[39][40][41][42]. It was recently shown that the RelQ protein from E. faecalis could efficiently synthesize a third alarmone molecule: guanosine-5'monophosphate 3'-diphosphate (pGpp) from GMP + ATP, in addition to its previously established pppGpp and ppGpp-synthesizing activities [42].…”

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The Ps and Qs of alarmone synthesis inStaphylococcus aureus

Yang

Xie

Choi

et al. 2019

Preprint

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During the stringent response, bacteria synthesize guanosine-3',5'-bis(diphosphate) (ppGpp) and guanosine-5'-triphosphate 3'-diphosphate (pppGpp), which act as secondary messengers to promote cellular survival and adaptation. (p)ppGpp 'alarmones' are synthesized and/or hydrolyzed by proteins belonging to the RelA/SpoT Homologue (RSH) family. Many bacteria also encode 'small alarmone synthetase' (SAS) proteins (e.g. RelP, RelQ) which may also be capable of synthesizing a third alarmone: guanosine-5'-phosphate 3'-diphosphate (pGpp). Here, we report the biochemical properties of the Rel (RSH), RelP and RelQ proteins from Staphylococcus aureus (Sa-Rel, Sa-RelP, Sa-RelQ, respectively). Sa-Rel synthesized pppGpp more efficiently than ppGpp, but lacked the ability to produce pGpp. However, Sa-Rel efficiently hydrolyzed all three alarmones in a Mn(II) ion-dependent manner. The removal of the C-terminal regulatory domain of Sa-Rel increased its rate of (p)ppGpp synthesis ca. 10fold, but had negligible effects on its rate of (pp)pGpp hydrolysis. Sa-RelP and Sa-RelQ efficiently synthesized pGpp in addition to pppGpp and ppGpp. The alarmone-synthesizing abilities of Sa-RelQ, but not Sa-RelP, were allosterically-stimulated by the addition of pppGpp, ppGpp or pGpp. The respective (pp)pGpp-synthesizing activities of Sa-RelP/Sa-RelQ were compared and contrasted with SAS homologues from Enterococcus faecalis (Ef-RelQ) andStreptococcus mutans (Sm-RelQ, Sm-RelP). Results indicated that EF-RelQ, Sm-RelQ and Sa-RelQ were functionally-equivalent; but exhibited considerable variations in their respective biochemical properties, and the degrees to which alarmones and single-stranded RNA molecules allosterically stimulated their respective alarmone-synthesizing activities. The respective (pp)pGpp-synthesizing capabilities of Sa-RelP and Sm-RelP proteins were inhibited by pGpp, ppGpp and pppGpp. Our results support the premise that RelP and RelQ proteins may synthesize pGpp in addition to (p)ppGpp within S. aureus and other Gram-positive bacterial species. intracellular messengers that directly or indirectly up-regulate cellular processes that promote the conservation, recycling and biosynthesis of important molecular building-blocks; initiate physiological mechanisms that preserve cellular integrity and tolerance in response to certain cytotoxic exogenous agents or harsh extracellular conditions; and down-regulate processes that are superfluous or non-essential for short-term viability (reviewed in [4][5][6][7][8][9][10][11][12]).Alarmones are synthesized and/or hydrolyzed by proteins belonging to the RelA/SpoT-Homologue (RSH) family [13,14] RelA proteins, such as the Escherichia coli RelA protein, are termed monofunctional or 'synthase-only', in that they lack a catalytic Histidine-Aspartate (HD) domain responsible for hydrolytic activities, and only catalyze (p)ppGpp production [15,16]. This synthase activity involves the transfer of a diphosphate unit from ATP to the 3'hydroxyl group of GTP or GDP in a Mg 2+ -dependent manner to pr...

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“…Ao passo que as proteínas RSHs parecem responder a estímulos de carência nutricional, as sintetases minoritárias de bactérias Gram-positivas, ainda pouco exploradas, parecem ter papel importante em danos relacionados ao envelope celular e choque alcalino (Nanamiya et al, 2008). Recentemente, se propôs um mecanismo regulatório de uma das sintetases minoritárias dependente da interação com RNAs, no qual a ligação de um RNAm inibiria a atividade de síntese da proteína (Beljantseva et al, 2017a).…”

Section: Figura 1 Representação Das Reações De Síntese E Degradação unclassified

Estudos da resposta estringente de <i>Bacillus subtilis</i> e busca por pequenas moléculas moduladoras de RelA

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Negative allosteric regulation of Enterococcus faecalis small alarmone synthetase RelQ by single-stranded RNA

Cited by 48 publications

References 33 publications

A widespread toxin-antitoxin system exploiting growth control via alarmone signalling

A widespread toxin-antitoxin system exploiting growth control via alarmone signalling

The Ps and Qs of alarmone synthesis inStaphylococcus aureus

Estudos da resposta estringente de <i>Bacillus subtilis</i> e busca por pequenas moléculas moduladoras de RelA

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