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Functional Characterization of Small Alarmone Synthetase and Small Alarmone Hydrolase Proteins from Treponema denticola

The stringent response enables bacteria to survive nutrient starvation, antibiotic challenge, and other threats to cellular survival. Two alarmone (magic spot) second messengers, guanosine pentaphosphate (pppGpp) and guanosine tetraphosphate (ppGpp), which are synthesized by RelA/SpoT homologue (RSH...

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Autores principales: Wang, Miao, Tang, Nga-Yeung, Xie, Shujie, Watt, Rory M.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society for Microbiology 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10434055/
https://www.ncbi.nlm.nih.gov/pubmed/37289081
http://dx.doi.org/10.1128/spectrum.05100-22
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author Wang, Miao
Tang, Nga-Yeung
Xie, Shujie
Watt, Rory M.
author_facet Wang, Miao
Tang, Nga-Yeung
Xie, Shujie
Watt, Rory M.
author_sort Wang, Miao
collection PubMed
description The stringent response enables bacteria to survive nutrient starvation, antibiotic challenge, and other threats to cellular survival. Two alarmone (magic spot) second messengers, guanosine pentaphosphate (pppGpp) and guanosine tetraphosphate (ppGpp), which are synthesized by RelA/SpoT homologue (RSH) proteins, play central roles in the stringent response. The pathogenic oral spirochete bacterium Treponema denticola lacks a long-RSH homologue but encodes putative small alarmone synthetase (Tde-SAS, TDE1711) and small alarmone hydrolase (Tde-SAH, TDE1690) proteins. Here, we characterize the respective in vitro and in vivo activities of Tde-SAS and Tde-SAH, which respectively belong to the previously uncharacterized RSH families DsRel and ActSpo2. The tetrameric 410-amino acid (aa) Tde-SAS protein preferentially synthesizes ppGpp over pppGpp and a third alarmone, pGpp. Unlike RelQ homologues, alarmones do not allosterically stimulate the synthetic activities of Tde-SAS. The ~180 aa C-terminal tetratricopeptide repeat (TPR) domain of Tde-SAS acts as a brake on the alarmone synthesis activities of the ~220-aa N-terminal catalytic domain. Tde-SAS also synthesizes “alarmone-like” nucleotides such as adenosine tetraphosphate (ppApp), albeit at considerably lower rates. The 210-aa Tde-SAH protein efficiently hydrolyzes all guanosine and adenosine-based alarmones in a Mn(II) ion-dependent manner. Using a growth assays with a ΔrelAΔspoT strain of Escherichia coli that is deficient in pppGpp/ppGpp synthesis, we demonstrate that Tde-SAS can synthesize alarmones in vivo to restore growth in minimal media. Taken together, our results add to our holistic understanding of alarmone metabolism across diverse bacterial species. IMPORTANCE The spirochete bacterium Treponema denticola is a common component of the oral microbiota. However, it may play important pathological roles in multispecies oral infectious diseases such as periodontitis: a severe and destructive form of gum disease, which is a major cause of tooth loss in adults. The operation of the stringent response, a highly conserved survival mechanism, is known to help many bacterial species cause persistent or virulent infections. By characterizing the biochemical functions of the proteins putatively responsible for the stringent response in T. denticola, we may gain molecular insight into how this bacterium can survive within harsh oral environments and promote infection. Our results also expand our general understanding of proteins that synthesize nucleotide-based intracellular signaling molecules in bacteria.
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spelling pubmed-104340552023-08-18 Functional Characterization of Small Alarmone Synthetase and Small Alarmone Hydrolase Proteins from Treponema denticola Wang, Miao Tang, Nga-Yeung Xie, Shujie Watt, Rory M. Microbiol Spectr Research Article The stringent response enables bacteria to survive nutrient starvation, antibiotic challenge, and other threats to cellular survival. Two alarmone (magic spot) second messengers, guanosine pentaphosphate (pppGpp) and guanosine tetraphosphate (ppGpp), which are synthesized by RelA/SpoT homologue (RSH) proteins, play central roles in the stringent response. The pathogenic oral spirochete bacterium Treponema denticola lacks a long-RSH homologue but encodes putative small alarmone synthetase (Tde-SAS, TDE1711) and small alarmone hydrolase (Tde-SAH, TDE1690) proteins. Here, we characterize the respective in vitro and in vivo activities of Tde-SAS and Tde-SAH, which respectively belong to the previously uncharacterized RSH families DsRel and ActSpo2. The tetrameric 410-amino acid (aa) Tde-SAS protein preferentially synthesizes ppGpp over pppGpp and a third alarmone, pGpp. Unlike RelQ homologues, alarmones do not allosterically stimulate the synthetic activities of Tde-SAS. The ~180 aa C-terminal tetratricopeptide repeat (TPR) domain of Tde-SAS acts as a brake on the alarmone synthesis activities of the ~220-aa N-terminal catalytic domain. Tde-SAS also synthesizes “alarmone-like” nucleotides such as adenosine tetraphosphate (ppApp), albeit at considerably lower rates. The 210-aa Tde-SAH protein efficiently hydrolyzes all guanosine and adenosine-based alarmones in a Mn(II) ion-dependent manner. Using a growth assays with a ΔrelAΔspoT strain of Escherichia coli that is deficient in pppGpp/ppGpp synthesis, we demonstrate that Tde-SAS can synthesize alarmones in vivo to restore growth in minimal media. Taken together, our results add to our holistic understanding of alarmone metabolism across diverse bacterial species. IMPORTANCE The spirochete bacterium Treponema denticola is a common component of the oral microbiota. However, it may play important pathological roles in multispecies oral infectious diseases such as periodontitis: a severe and destructive form of gum disease, which is a major cause of tooth loss in adults. The operation of the stringent response, a highly conserved survival mechanism, is known to help many bacterial species cause persistent or virulent infections. By characterizing the biochemical functions of the proteins putatively responsible for the stringent response in T. denticola, we may gain molecular insight into how this bacterium can survive within harsh oral environments and promote infection. Our results also expand our general understanding of proteins that synthesize nucleotide-based intracellular signaling molecules in bacteria. American Society for Microbiology 2023-06-08 /pmc/articles/PMC10434055/ /pubmed/37289081 http://dx.doi.org/10.1128/spectrum.05100-22 Text en Copyright © 2023 Wang et al. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Research Article
Wang, Miao
Tang, Nga-Yeung
Xie, Shujie
Watt, Rory M.
Functional Characterization of Small Alarmone Synthetase and Small Alarmone Hydrolase Proteins from Treponema denticola
title Functional Characterization of Small Alarmone Synthetase and Small Alarmone Hydrolase Proteins from Treponema denticola
title_full Functional Characterization of Small Alarmone Synthetase and Small Alarmone Hydrolase Proteins from Treponema denticola
title_fullStr Functional Characterization of Small Alarmone Synthetase and Small Alarmone Hydrolase Proteins from Treponema denticola
title_full_unstemmed Functional Characterization of Small Alarmone Synthetase and Small Alarmone Hydrolase Proteins from Treponema denticola
title_short Functional Characterization of Small Alarmone Synthetase and Small Alarmone Hydrolase Proteins from Treponema denticola
title_sort functional characterization of small alarmone synthetase and small alarmone hydrolase proteins from treponema denticola
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10434055/
https://www.ncbi.nlm.nih.gov/pubmed/37289081
http://dx.doi.org/10.1128/spectrum.05100-22
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