Structural and biochemical characterization of the relaxosome auxiliary proteins encoded on the Bacillus subtilis plasmid pLS20

Bacterial conjugation is an important route for horizontal gene transfer. The initial step in this process involves a macromolecular protein-DNA complex called the relaxosome, which in plasmids consists of the origin of transfer (oriT) and several proteins that prepare the transfer. The relaxosome p...

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Autores: Crespo, Isidro, Bernardo, Nerea, Cuppari, Anna, Calisto, Barbara M., Val-Calvo, Jorge, Miguel-Arribas, Andrés, Meijer, Wilfried J.J., Carpena, Xavi, Gil-Ortiz, Fernando, Malfois, Marc, Boer, D. Roeland
Tipo de recurso: artículo
Estado:Versión publicada
Fecha de publicación:2022
País:España
Institución:Consejo Superior de Investigaciones Científicas (CSIC)
Repositorio:DIGITAL.CSIC. Repositorio Institucional del CSIC
OAI Identifier:oai:digital.csic.es:10261/415402
Acceso en línea:http://hdl.handle.net/10261/415402
Access Level:acceso abierto
Palabra clave:Structural biology
Bacterial conjugation
Relaxosome
Auxiliary protein
DNA binding protein
Ribbon-Helix-Helix
Antibiotic resistance
Firmicutes
Horizontal gene transfer
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spelling Structural and biochemical characterization of the relaxosome auxiliary proteins encoded on the Bacillus subtilis plasmid pLS20Crespo, IsidroBernardo, NereaCuppari, AnnaCalisto, Barbara M.Val-Calvo, JorgeMiguel-Arribas, AndrésMeijer, Wilfried J.J.Carpena, XaviGil-Ortiz, FernandoMalfois, MarcBoer, D. RoelandStructural biologyBacterial conjugationRelaxosomeAuxiliary proteinDNA binding proteinRibbon-Helix-HelixAntibiotic resistanceFirmicutesHorizontal gene transferBacterial conjugation is an important route for horizontal gene transfer. The initial step in this process involves a macromolecular protein-DNA complex called the relaxosome, which in plasmids consists of the origin of transfer (oriT) and several proteins that prepare the transfer. The relaxosome protein named relaxase introduces a nick in one of the strands of the oriT to initiate the process. Additional relaxosome proteins can exist. Recently, several relaxosome proteins encoded on the Bacillus subtilis plasmid pLS20 were identified, including the relaxase, named Rel, and two auxiliary DNA-binding factors, named Aux1 and Aux2. Here, we extend this characterization in order to define their function. We present the low-resolution SAXS envelope of the Aux1 and the atomic X-ray structure of the C-terminal domain of Aux2. We also study the interactions between the auxiliary proteins and the full-length Rel, as well as its separate domains. The results show that the quaternary structure of the auxiliary protein Aux1 involves a tetramer, as previously determined. The crystal structure of the C-terminal domain of Aux2 shows that it forms a tetramer and suggests that it is an analog of TraM of plasmid F. This is the first evidence of the existence of a TraM analog in gram positive conjugative systems, although, unlike other TraM analogs, Aux2 does not interact with the relaxase. Aux1 interacts with the C-terminal domain, but not the N-terminal domain, of the relaxase Rel. Thus, the pLS20 relaxosome exhibits some unique features despite the apparent similarity to some well-studied G- conjugation systems.This work was supported by Ministry of Economy and Competitiveness of the Spanish Government grants BIO2016-77883-C2-1-P and PID2019-108778GB-C21 to W.J.J.M, PID2020-117028 GB-I00 (AEI/FEDER, EU), BIO2016-77883-C2-2-P (AEI/FEDER, EU) and FIS2015-72574-EXP (AEI/FEDER, EU), which also supported N.B., to R.B. Funding for open access charge: Ministry of Economy and Competitiveness of the Spanish Government PID2020-117028 GB-I00. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Ministerio de Economía y Competitividad (España)Agencia Estatal de Investigación (España)European CommissionConsejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]2026202620222026info:eu-repo/semantics/articlehttp://purl.org/coar/resource_type/c_6501Publisher's versioninfo:eu-repo/semantics/publishedVersionhttp://hdl.handle.net/10261/415402reponame:DIGITAL.CSIC. Repositorio Institucional del CSICinstname:Consejo Superior de Investigaciones Científicas (CSIC)#PLACEHOLDER_PARENT_METADATA_VALUE##PLACEHOLDER_PARENT_METADATA_VALUE##PLACEHOLDER_PARENT_METADATA_VALUE##PLACEHOLDER_PARENT_METADATA_VALUE#info:eu-repo/grantAgreement/AEI//BES-2016-077883info:eu-repo/grantAgreement/AEI/Plan Estatal de Investigación Científica y Técnica y de Innovación 2017-2020/PID2019-108778GB-C21info:eu-repo/grantAgreement/AEI/Plan Estatal de Investigación Científica y Técnica y de Innovación 2017-2020/PID2020-117028GB-I00info:eu-repo/grantAgreement/MINECO//FIS2015-72574-EXPhttp://dx.doi.org/10.1016/j.csbj.2021.12.041Síinfo:eu-repo/semantics/openAccessoai:digital.csic.es:10261/4154022026-05-22T06:33:51Z
dc.title.none.fl_str_mv Structural and biochemical characterization of the relaxosome auxiliary proteins encoded on the Bacillus subtilis plasmid pLS20
title Structural and biochemical characterization of the relaxosome auxiliary proteins encoded on the Bacillus subtilis plasmid pLS20
spellingShingle Structural and biochemical characterization of the relaxosome auxiliary proteins encoded on the Bacillus subtilis plasmid pLS20
Crespo, Isidro
Structural biology
Bacterial conjugation
Relaxosome
Auxiliary protein
DNA binding protein
Ribbon-Helix-Helix
Antibiotic resistance
Firmicutes
Horizontal gene transfer
title_short Structural and biochemical characterization of the relaxosome auxiliary proteins encoded on the Bacillus subtilis plasmid pLS20
title_full Structural and biochemical characterization of the relaxosome auxiliary proteins encoded on the Bacillus subtilis plasmid pLS20
title_fullStr Structural and biochemical characterization of the relaxosome auxiliary proteins encoded on the Bacillus subtilis plasmid pLS20
title_full_unstemmed Structural and biochemical characterization of the relaxosome auxiliary proteins encoded on the Bacillus subtilis plasmid pLS20
title_sort Structural and biochemical characterization of the relaxosome auxiliary proteins encoded on the Bacillus subtilis plasmid pLS20
dc.creator.none.fl_str_mv Crespo, Isidro
Bernardo, Nerea
Cuppari, Anna
Calisto, Barbara M.
Val-Calvo, Jorge
Miguel-Arribas, Andrés
Meijer, Wilfried J.J.
Carpena, Xavi
Gil-Ortiz, Fernando
Malfois, Marc
Boer, D. Roeland
author Crespo, Isidro
author_facet Crespo, Isidro
Bernardo, Nerea
Cuppari, Anna
Calisto, Barbara M.
Val-Calvo, Jorge
Miguel-Arribas, Andrés
Meijer, Wilfried J.J.
Carpena, Xavi
Gil-Ortiz, Fernando
Malfois, Marc
Boer, D. Roeland
author_role author
author2 Bernardo, Nerea
Cuppari, Anna
Calisto, Barbara M.
Val-Calvo, Jorge
Miguel-Arribas, Andrés
Meijer, Wilfried J.J.
Carpena, Xavi
Gil-Ortiz, Fernando
Malfois, Marc
Boer, D. Roeland
author2_role author
author
author
author
author
author
author
author
author
author
dc.contributor.none.fl_str_mv Ministerio de Economía y Competitividad (España)
Agencia Estatal de Investigación (España)
European Commission
Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]
dc.subject.none.fl_str_mv Structural biology
Bacterial conjugation
Relaxosome
Auxiliary protein
DNA binding protein
Ribbon-Helix-Helix
Antibiotic resistance
Firmicutes
Horizontal gene transfer
topic Structural biology
Bacterial conjugation
Relaxosome
Auxiliary protein
DNA binding protein
Ribbon-Helix-Helix
Antibiotic resistance
Firmicutes
Horizontal gene transfer
description Bacterial conjugation is an important route for horizontal gene transfer. The initial step in this process involves a macromolecular protein-DNA complex called the relaxosome, which in plasmids consists of the origin of transfer (oriT) and several proteins that prepare the transfer. The relaxosome protein named relaxase introduces a nick in one of the strands of the oriT to initiate the process. Additional relaxosome proteins can exist. Recently, several relaxosome proteins encoded on the Bacillus subtilis plasmid pLS20 were identified, including the relaxase, named Rel, and two auxiliary DNA-binding factors, named Aux1 and Aux2. Here, we extend this characterization in order to define their function. We present the low-resolution SAXS envelope of the Aux1 and the atomic X-ray structure of the C-terminal domain of Aux2. We also study the interactions between the auxiliary proteins and the full-length Rel, as well as its separate domains. The results show that the quaternary structure of the auxiliary protein Aux1 involves a tetramer, as previously determined. The crystal structure of the C-terminal domain of Aux2 shows that it forms a tetramer and suggests that it is an analog of TraM of plasmid F. This is the first evidence of the existence of a TraM analog in gram positive conjugative systems, although, unlike other TraM analogs, Aux2 does not interact with the relaxase. Aux1 interacts with the C-terminal domain, but not the N-terminal domain, of the relaxase Rel. Thus, the pLS20 relaxosome exhibits some unique features despite the apparent similarity to some well-studied G- conjugation systems.
publishDate 2022
dc.date.none.fl_str_mv 2022
2026
2026
2026
dc.type.none.fl_str_mv info:eu-repo/semantics/article
http://purl.org/coar/resource_type/c_6501
Publisher's version
info:eu-repo/semantics/publishedVersion
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/10261/415402
url http://hdl.handle.net/10261/415402
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#PLACEHOLDER_PARENT_METADATA_VALUE#
#PLACEHOLDER_PARENT_METADATA_VALUE#
#PLACEHOLDER_PARENT_METADATA_VALUE#
info:eu-repo/grantAgreement/AEI//BES-2016-077883
info:eu-repo/grantAgreement/AEI/Plan Estatal de Investigación Científica y Técnica y de Innovación 2017-2020/PID2019-108778GB-C21
info:eu-repo/grantAgreement/AEI/Plan Estatal de Investigación Científica y Técnica y de Innovación 2017-2020/PID2020-117028GB-I00
info:eu-repo/grantAgreement/MINECO//FIS2015-72574-EXP
http://dx.doi.org/10.1016/j.csbj.2021.12.041
Sí
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
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instname:Consejo Superior de Investigaciones Científicas (CSIC)
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reponame_str DIGITAL.CSIC. Repositorio Institucional del CSIC
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