The C-Terminal RRM/ACT Domain Is Crucial for Fine-Tuning the Activation of 'Long' RelA-SpoT Homolog Enzymes by Ribosomal Complexes.

Hiraku Takada,Vasili Hauryliuk,Abel Garcia-Pino,Rikinori Murayama,Mohammad Roghanian,Gemma C Atkinson,Victoriia Murina,Genki Akanuma,Ievgen Dzhygyr

doi:10.3389/fmicb.2020.00277

Abstract

The (p)ppGpp-mediated stringent response is a bacterial stress response implicated in virulence and antibiotic tolerance. Both synthesis and degradation of the (p)ppGpp alarmone nucleotide are mediated by RelA-SpoT Homolog (RSH) enzymes which can be broadly divided in two classes: single-domain ‘short’ and multi-domain ‘long’ RSH. The regulatory ACT (Aspartokinase, Chorismate mutase and TyrA)/RRM (RNA Recognition Motif) domain is a near-universal C-terminal domain of long RSHs. Deletion of RRM in both monofunctional (synthesis-only) RelA as well as bifunctional (i.e., capable of both degrading and synthesizing the alarmone) Rel renders the long RSH cytotoxic due to overproduction of (p)ppGpp. To probe the molecular mechanism underlying this effect we characterized Escherichia coli RelA and Bacillus subtilis Rel RSHs lacking RRM. We demonstrate that, first, the cytotoxicity caused by the removal of RRM is counteracted by secondary mutations that disrupt the interaction of the RSH with the starved ribosomal complex – the ultimate inducer of (p)ppGpp production by RelA and Rel – and, second, that the hydrolytic activity of Rel is not abrogated in the truncated mutant. Therefore, we conclude that the overproduction of (p)ppGpp by RSHs lacking the RRM domain is not explained by a lack of auto-inhibition in the absence of RRM or/and a defect in (p)ppGpp hydrolysis. Instead, we argue that it is driven by misregulation of the RSH activation by the ribosome.

Highlights

Bacteria employ diverse mechanisms to sense and respond to stress
By characterizing truncated versions of E. coli RelA and B. subtilis, we demonstrate that the cytotoxicity of mutant RelA/SpoT Homolog (RSH) variants is strictly dependent on the interaction with the ribosome and deacylated tRNA, and, cannot be explained by defects in intra-molecular regulation alone
Our results demonstrate that (i) Rel RNA Recognition Motif domain (RRM) is toxic in ppGpp0 B. subtilis, (ii) this toxicity requires intact (p)ppGpp synthesis activity of the enzyme (iii) it is abrogated by mutations disrupting the interaction with tRNA and starved ribosomes and (iv) deletion of the RRM domain does not abrogate the hydrolysis activity of B. subtilis Rel

Summary

Introduction

Bacteria employ diverse mechanisms to sense and respond to stress. One such mechanism is the stringent response – a near-universal stress response orchestrated by hyper-phosphorylated derivatives of housekeeping nucleotides GDP and GTP: guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), collectively referred to as (p)ppGpp (Hauryliuk et al, 2015; Liu et al, 2015; Steinchen and Bange, 2016). Rel/RelA Regulation by the RRM/ACT Domain signaling are implicated in virulence, antibiotic resistance and tolerance (Dalebroux et al, 2010; Dalebroux and Swanson, 2012; Hauryliuk et al, 2015), this stress signaling system has been recently targeted for development of new anti-infective compounds (Kushwaha et al, 2019). Both synthesis and degradation of (p)ppGpp is mediated by RelA/SpoT Homolog (RSH) enzymes. In addition to long RSHs, bacteria often encode single domain RSH enzymes: Small Alarmone Synthetases (SAS) and Small Alarmone Hydrolases (SAH) (Atkinson et al, 2011; Jimmy et al, 2019), such as RelQ and RelP in the Firmicute bacterium B. subtilis (Nanamiya et al, 2008)

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Conclusion