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Identification and Functional Characterization of Small Alarmone Synthetases in Corynebacterium glutamicum

The hyperphosphorylated guanosine derivatives ppGpp and pppGpp represent global regulators of the bacterial stress response, as they act as central elements of the stringent response system. Although it was assumed that both, (p)ppGpp synthesis and hydrolysis, are catalyzed by one bifunctional RSH-p...

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Autores principales: Ruwe, Matthias, Kalinowski, Jörn, Persicke, Marcus
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5566576/
https://www.ncbi.nlm.nih.gov/pubmed/28871248
http://dx.doi.org/10.3389/fmicb.2017.01601
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author Ruwe, Matthias
Kalinowski, Jörn
Persicke, Marcus
author_facet Ruwe, Matthias
Kalinowski, Jörn
Persicke, Marcus
author_sort Ruwe, Matthias
collection PubMed
description The hyperphosphorylated guanosine derivatives ppGpp and pppGpp represent global regulators of the bacterial stress response, as they act as central elements of the stringent response system. Although it was assumed that both, (p)ppGpp synthesis and hydrolysis, are catalyzed by one bifunctional RSH-protein in the actinobacterial model organism Corynebacterium glutamicum ATCC 13032, two putative short alarmone synthetases (SASs) were identified by bioinformatic analyses. The predicted sequences of both enzymes, designated as RelP(*)(Cg) and RelS(Cg), exhibit high similarities to the conserved (p)ppGpp synthetase catalytic domain. In the context of sequence analysis, significant differences were found between the RelP variants of different C. glutamicum isolates. In contrast to the bifunctional RelA/SpoT homolog (RSH) protein Rel(Cg), whose gene deletion results in a reduced growth rate, no change in growth characteristics were observed for deletion mutants of the putative SAS proteins under standard growth conditions. The growth deficit of the Δrel strain could be restored by the additional deletion of the gene encoding RelS(Cg), which clearly indicates a functional relationship between both enzymes. The predicted pyrophosphokinase activity of RelS(Cg) was demonstrated by means of genetic complementation of an Escherichia coli ΔrelAΔspoT strain. For the expression of RelP(*)(Cg), as well as the slightly differing variant RelP(Cg) from C. glutamicum AS1.542, no complementation was observed, concluding that both RelP versions possess no significant pyrophosphokinase activity in vivo. The results were confirmed by in vitro characterization of the corresponding proteins. In the course of this investigation, the additional conversion of GMP to pGpp was determined for the enzyme RelS(Cg). Since the SAS species analyzed extend both the network of stringent response related enzymes and the number of substances involved, the study of this class of enzymes is an important component in understanding the bacterial stress response. In addition to the comprehension of important biological processes, such as growth rate regulation and the survival of pathogenic species in the host organism, SAS enzymes can be used to produce novel hyperphosphorylated nucleotide species, such as pGpp.
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spelling pubmed-55665762017-09-04 Identification and Functional Characterization of Small Alarmone Synthetases in Corynebacterium glutamicum Ruwe, Matthias Kalinowski, Jörn Persicke, Marcus Front Microbiol Microbiology The hyperphosphorylated guanosine derivatives ppGpp and pppGpp represent global regulators of the bacterial stress response, as they act as central elements of the stringent response system. Although it was assumed that both, (p)ppGpp synthesis and hydrolysis, are catalyzed by one bifunctional RSH-protein in the actinobacterial model organism Corynebacterium glutamicum ATCC 13032, two putative short alarmone synthetases (SASs) were identified by bioinformatic analyses. The predicted sequences of both enzymes, designated as RelP(*)(Cg) and RelS(Cg), exhibit high similarities to the conserved (p)ppGpp synthetase catalytic domain. In the context of sequence analysis, significant differences were found between the RelP variants of different C. glutamicum isolates. In contrast to the bifunctional RelA/SpoT homolog (RSH) protein Rel(Cg), whose gene deletion results in a reduced growth rate, no change in growth characteristics were observed for deletion mutants of the putative SAS proteins under standard growth conditions. The growth deficit of the Δrel strain could be restored by the additional deletion of the gene encoding RelS(Cg), which clearly indicates a functional relationship between both enzymes. The predicted pyrophosphokinase activity of RelS(Cg) was demonstrated by means of genetic complementation of an Escherichia coli ΔrelAΔspoT strain. For the expression of RelP(*)(Cg), as well as the slightly differing variant RelP(Cg) from C. glutamicum AS1.542, no complementation was observed, concluding that both RelP versions possess no significant pyrophosphokinase activity in vivo. The results were confirmed by in vitro characterization of the corresponding proteins. In the course of this investigation, the additional conversion of GMP to pGpp was determined for the enzyme RelS(Cg). Since the SAS species analyzed extend both the network of stringent response related enzymes and the number of substances involved, the study of this class of enzymes is an important component in understanding the bacterial stress response. In addition to the comprehension of important biological processes, such as growth rate regulation and the survival of pathogenic species in the host organism, SAS enzymes can be used to produce novel hyperphosphorylated nucleotide species, such as pGpp. Frontiers Media S.A. 2017-08-21 /pmc/articles/PMC5566576/ /pubmed/28871248 http://dx.doi.org/10.3389/fmicb.2017.01601 Text en Copyright © 2017 Ruwe, Kalinowski and Persicke. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Microbiology
Ruwe, Matthias
Kalinowski, Jörn
Persicke, Marcus
Identification and Functional Characterization of Small Alarmone Synthetases in Corynebacterium glutamicum
title Identification and Functional Characterization of Small Alarmone Synthetases in Corynebacterium glutamicum
title_full Identification and Functional Characterization of Small Alarmone Synthetases in Corynebacterium glutamicum
title_fullStr Identification and Functional Characterization of Small Alarmone Synthetases in Corynebacterium glutamicum
title_full_unstemmed Identification and Functional Characterization of Small Alarmone Synthetases in Corynebacterium glutamicum
title_short Identification and Functional Characterization of Small Alarmone Synthetases in Corynebacterium glutamicum
title_sort identification and functional characterization of small alarmone synthetases in corynebacterium glutamicum
topic Microbiology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5566576/
https://www.ncbi.nlm.nih.gov/pubmed/28871248
http://dx.doi.org/10.3389/fmicb.2017.01601
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