Isolation and in silico analysis of a new subclass of parasporin 4 from Bacillus thuringiensis coreanensis

Background: Bacillus thuringiensis (Bt) is a Gram-positive bacterium whose strains have been studied mainly for the control of insect pests, due to the insecticidal capacity of its Cry and Vip proteins. However, recent studies indicate the presence of other proteins with no known insecticidal action...

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Authors: Santos, Thais N.F. [UNESP], Moreira, Raquel O. [UNESP], Rodrigues, Jardel D.B. [UNESP], Rojas, Luis A.C. [UNESP], Souza, Jackson A.M. [UNESP], Desidério, Janete A. [UNESP]
Format: article
Status:Published version
Publication Date:2025
Country:Brasil
Institution:Universidade Estadual Paulista (UNESP)
Repository:Repositório Institucional da UNESP
Language:English
OAI Identifier:oai:repositorio.unesp.br:11449/298581
Online Access:http://dx.doi.org/10.7717/peerj.19061
https://hdl.handle.net/11449/298581
Access Level:Open access
Keyword:Antitumor activity
Bacillus thuringiensis
Bioinformatics
Cancer
Cry
Cytotoxicity
Modeling
Parasporin
Phylogenetics
Pore former
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spelling Isolation and in silico analysis of a new subclass of parasporin 4 from Bacillus thuringiensis coreanensisAntitumor activityBacillus thuringiensisBioinformaticsCancerCryCytotoxicityModelingParasporinPhylogeneticsPore formerBackground: Bacillus thuringiensis (Bt) is a Gram-positive bacterium whose strains have been studied mainly for the control of insect pests, due to the insecticidal capacity of its Cry and Vip proteins. However, recent studies indicate the presence of other proteins with no known insecticidal action. These proteins denominated “parasporins” (PS) have cytotoxic activity and are divided into six classes, namely PS1, PS2, PS3, PS4, PS5, and PS6. Among these, parasporins 4 (PS4) has only one described subclass, present in the Bacillus thuringiensis shandongiensis strain. Given the importance and limited knowledge about the actions of PS4 proteins and the existence of only one described subclass, the present work aimed to characterize the Bacillus thuringiensis coreanensis strain as a potential source of PS4 protein. Methods: A preliminary screening to detect the ps4 gene was conducted in a bank of standard strains and isolates of Bacillus thuringiensis from the Laboratory of Bacterial Genetics and Applied Biotechnology, FCAV/UNESP. The positive strain for this gene had its genomic DNA extracted, the ps4 gene was isolated, cloned and in silico analyses of its sequence were performed. Tools such as Bioedit, BLAST, Clustal Omega, Geneious, IQ-Tree, and iTOL were used in these analyses. For the structural analysis of the PS4 detected, in comparison to the database PS4 (BAD22577), the tools Alphafold2, Pymol, and InterPro were used. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel analyses allowed the visualization of the inactive and active PS4 protein from the positive strain, after solubilization and activation with Proteinase K. Results: Previous screening of Bt standard strains revealed the presence of a partial ps4 gene in Bacillus thuringiensis coreanensis strain. The alignment obtained by the BLAST tool revealed 100% identity between the fragment detected in this work with a hypothetical protein (ANN35810.1) from the genome of that same strain. Considering this, the isolation of the complete gene present in this strain was performed by applying the polymer chain reaction (PCR) technique, using the hypothetical sequence as a basis for the primers elaboration. The in silico analysis of the obtained sequence revealed 92.03% similarity with the ps4 sequence presented in the database (AB180980). Protein modeling studies and comparison of their structures revealed that the B. thuringiensis coreanensis has a new subclass of PS4, denominated PS4Ab1, being an important source of parasporin to be explored in biotechnological applications.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Biology Department São Paulo State University, JaboticabalDepartment of Agricultural and Environmental Biotechnology São Paulo State University, JaboticabalBiology Department São Paulo State University, JaboticabalDepartment of Agricultural and Environmental Biotechnology São Paulo State University, JaboticabalFAPESP: 2019/21621-0Universidade Estadual Paulista (UNESP)2025-04-29T18:37:33Z2025-01-01info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articlehttp://dx.doi.org/10.7717/peerj.19061PeerJ, v. 13, n. 3, 2025.2167-8359https://hdl.handle.net/11449/29858110.7717/peerj.190612-s2.0-105001014457Scopusreponame:Repositório Institucional da UNESPinstname:Universidade Estadual Paulista (UNESP)instacron:UNESPengPeerJinfo:eu-repo/semantics/openAccessSantos, Thais N.F. [UNESP]Moreira, Raquel O. [UNESP]Rodrigues, Jardel D.B. [UNESP]Rojas, Luis A.C. [UNESP]Souza, Jackson A.M. [UNESP]Desidério, Janete A. [UNESP]2025-10-22T13:37:45Zoai:repositorio.unesp.br:11449/298581Repositório InstitucionalPUBhttp://repositorio.unesp.br/oai/requestrepositoriounesp@unesp.bropendoar:29462025-10-22T13:37:45Repositório Institucional da UNESP - Universidade Estadual Paulista (UNESP)false
dc.title.none.fl_str_mv Isolation and in silico analysis of a new subclass of parasporin 4 from Bacillus thuringiensis coreanensis
title Isolation and in silico analysis of a new subclass of parasporin 4 from Bacillus thuringiensis coreanensis
spellingShingle Isolation and in silico analysis of a new subclass of parasporin 4 from Bacillus thuringiensis coreanensis
Santos, Thais N.F. [UNESP]
Antitumor activity
Bacillus thuringiensis
Bioinformatics
Cancer
Cry
Cytotoxicity
Modeling
Parasporin
Phylogenetics
Pore former
title_short Isolation and in silico analysis of a new subclass of parasporin 4 from Bacillus thuringiensis coreanensis
title_full Isolation and in silico analysis of a new subclass of parasporin 4 from Bacillus thuringiensis coreanensis
title_fullStr Isolation and in silico analysis of a new subclass of parasporin 4 from Bacillus thuringiensis coreanensis
title_full_unstemmed Isolation and in silico analysis of a new subclass of parasporin 4 from Bacillus thuringiensis coreanensis
title_sort Isolation and in silico analysis of a new subclass of parasporin 4 from Bacillus thuringiensis coreanensis
dc.creator.none.fl_str_mv Santos, Thais N.F. [UNESP]
Moreira, Raquel O. [UNESP]
Rodrigues, Jardel D.B. [UNESP]
Rojas, Luis A.C. [UNESP]
Souza, Jackson A.M. [UNESP]
Desidério, Janete A. [UNESP]
author Santos, Thais N.F. [UNESP]
author_facet Santos, Thais N.F. [UNESP]
Moreira, Raquel O. [UNESP]
Rodrigues, Jardel D.B. [UNESP]
Rojas, Luis A.C. [UNESP]
Souza, Jackson A.M. [UNESP]
Desidério, Janete A. [UNESP]
author_role author
author2 Moreira, Raquel O. [UNESP]
Rodrigues, Jardel D.B. [UNESP]
Rojas, Luis A.C. [UNESP]
Souza, Jackson A.M. [UNESP]
Desidério, Janete A. [UNESP]
author2_role author
author
author
author
author
dc.contributor.none.fl_str_mv Universidade Estadual Paulista (UNESP)
dc.subject.por.fl_str_mv Antitumor activity
Bacillus thuringiensis
Bioinformatics
Cancer
Cry
Cytotoxicity
Modeling
Parasporin
Phylogenetics
Pore former
topic Antitumor activity
Bacillus thuringiensis
Bioinformatics
Cancer
Cry
Cytotoxicity
Modeling
Parasporin
Phylogenetics
Pore former
description Background: Bacillus thuringiensis (Bt) is a Gram-positive bacterium whose strains have been studied mainly for the control of insect pests, due to the insecticidal capacity of its Cry and Vip proteins. However, recent studies indicate the presence of other proteins with no known insecticidal action. These proteins denominated “parasporins” (PS) have cytotoxic activity and are divided into six classes, namely PS1, PS2, PS3, PS4, PS5, and PS6. Among these, parasporins 4 (PS4) has only one described subclass, present in the Bacillus thuringiensis shandongiensis strain. Given the importance and limited knowledge about the actions of PS4 proteins and the existence of only one described subclass, the present work aimed to characterize the Bacillus thuringiensis coreanensis strain as a potential source of PS4 protein. Methods: A preliminary screening to detect the ps4 gene was conducted in a bank of standard strains and isolates of Bacillus thuringiensis from the Laboratory of Bacterial Genetics and Applied Biotechnology, FCAV/UNESP. The positive strain for this gene had its genomic DNA extracted, the ps4 gene was isolated, cloned and in silico analyses of its sequence were performed. Tools such as Bioedit, BLAST, Clustal Omega, Geneious, IQ-Tree, and iTOL were used in these analyses. For the structural analysis of the PS4 detected, in comparison to the database PS4 (BAD22577), the tools Alphafold2, Pymol, and InterPro were used. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel analyses allowed the visualization of the inactive and active PS4 protein from the positive strain, after solubilization and activation with Proteinase K. Results: Previous screening of Bt standard strains revealed the presence of a partial ps4 gene in Bacillus thuringiensis coreanensis strain. The alignment obtained by the BLAST tool revealed 100% identity between the fragment detected in this work with a hypothetical protein (ANN35810.1) from the genome of that same strain. Considering this, the isolation of the complete gene present in this strain was performed by applying the polymer chain reaction (PCR) technique, using the hypothetical sequence as a basis for the primers elaboration. The in silico analysis of the obtained sequence revealed 92.03% similarity with the ps4 sequence presented in the database (AB180980). Protein modeling studies and comparison of their structures revealed that the B. thuringiensis coreanensis has a new subclass of PS4, denominated PS4Ab1, being an important source of parasporin to be explored in biotechnological applications.
publishDate 2025
dc.date.none.fl_str_mv 2025-04-29T18:37:33Z
2025-01-01
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
dc.type.driver.fl_str_mv info:eu-repo/semantics/article
format article
status_str publishedVersion
dc.identifier.uri.fl_str_mv http://dx.doi.org/10.7717/peerj.19061
PeerJ, v. 13, n. 3, 2025.
2167-8359
https://hdl.handle.net/11449/298581
10.7717/peerj.19061
2-s2.0-105001014457
url http://dx.doi.org/10.7717/peerj.19061
https://hdl.handle.net/11449/298581
identifier_str_mv PeerJ, v. 13, n. 3, 2025.
2167-8359
10.7717/peerj.19061
2-s2.0-105001014457
dc.language.iso.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv PeerJ
dc.rights.driver.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.source.none.fl_str_mv Scopus
reponame:Repositório Institucional da UNESP
instname:Universidade Estadual Paulista (UNESP)
instacron:UNESP
instname_str Universidade Estadual Paulista (UNESP)
instacron_str UNESP
institution UNESP
reponame_str Repositório Institucional da UNESP
collection Repositório Institucional da UNESP
repository.name.fl_str_mv Repositório Institucional da UNESP - Universidade Estadual Paulista (UNESP)
repository.mail.fl_str_mv repositoriounesp@unesp.br
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