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The LspC3–41I restriction-modification system is the major determinant for genetic manipulations of Lysinibacillus sphaericus C3–41

BACKGROUND: Lysinibacillus sphaericus has been widely used in integrated mosquito control program and it is one of the minority bacterial species unable to metabolize carbohydrates. In consideration of the high genetic conservation at genomic level and difficulty of genetic horizontal transfer, it i...

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Autores principales: Fu, Pan, Ge, Yong, Wu, Yiming, Zhao, Ni, Yuan, Zhiming, Hu, Xiaomin
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5437673/
https://www.ncbi.nlm.nih.gov/pubmed/28525986
http://dx.doi.org/10.1186/s12866-017-1014-6
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author Fu, Pan
Ge, Yong
Wu, Yiming
Zhao, Ni
Yuan, Zhiming
Hu, Xiaomin
author_facet Fu, Pan
Ge, Yong
Wu, Yiming
Zhao, Ni
Yuan, Zhiming
Hu, Xiaomin
author_sort Fu, Pan
collection PubMed
description BACKGROUND: Lysinibacillus sphaericus has been widely used in integrated mosquito control program and it is one of the minority bacterial species unable to metabolize carbohydrates. In consideration of the high genetic conservation at genomic level and difficulty of genetic horizontal transfer, it is hypothesized that effective restriction-modification (R-M) systems existed in mosquitocidal L. sphaericus. RESULTS: In this study, six type II R-M systems including LspC3–41I were predicted in L. sphaericus C3–41 genome. It was found that the cell free extracts (CFE) from this strain shown similar restriction and methylation activity on exogenous Bacillus/Escherichia coli shuttle vector pBU4 as the HaeIII, which is an isoschizomer of BspRI. The Bsph_0498 (encoding the predicted LspC3–41IR) knockout mutant Δ0498 and the complement strain RC0498 were constructed. It was found that the unmethylated pBU4 can be digested by the CFE of C3–41 and RC0498, but not by that of Δ0498. Furthermore, the exogenous plasmid pBU4 can be transformed at very high efficacy into Δ0498, low efficacy into RC0498, but no transformation into C3–41, indicating that LspC3–41I might be a major determinant for the genetic restriction barrier of strain C3–41. Besides, lspC3–41IR and lspC3–41IM genes are detected in other two strains besides C3–41 of the tested 16 L. sphaericus strains, which all belonging to serotype H5 and MLST sequence type (ST) 1. Furthermore, the three strains are not horizontal transferred, and this restriction could be overcome by in vitro methylation either by the host CFE or by commercial methytransferase M. HaeIII. The results provide an insight to further study the genetic restriction, modification and evolution of mosquitocidal L. sphaericus, also a theoretical basis and a method for the genetic manipulations of L. sphaericus. CONCLUSIONS: LspC3–41I is identified as the major determinant for the restriction barrier of L. sphaericus C3–41. Only three strains of the tested 16 L. sphaericus strains, which all belonging to serotype H5 and ST1 by MLST scheme, contain LspC3–41I system. Two different methods can be used to overcome the restriction barrier of the three isolates to get transformants efficiently: 1) to methylate plasmid DNA prior to the electroporation; and 2) to delete the major restriction endonuclease encoding gene lspC3–41IR.
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spelling pubmed-54376732017-05-22 The LspC3–41I restriction-modification system is the major determinant for genetic manipulations of Lysinibacillus sphaericus C3–41 Fu, Pan Ge, Yong Wu, Yiming Zhao, Ni Yuan, Zhiming Hu, Xiaomin BMC Microbiol Research Article BACKGROUND: Lysinibacillus sphaericus has been widely used in integrated mosquito control program and it is one of the minority bacterial species unable to metabolize carbohydrates. In consideration of the high genetic conservation at genomic level and difficulty of genetic horizontal transfer, it is hypothesized that effective restriction-modification (R-M) systems existed in mosquitocidal L. sphaericus. RESULTS: In this study, six type II R-M systems including LspC3–41I were predicted in L. sphaericus C3–41 genome. It was found that the cell free extracts (CFE) from this strain shown similar restriction and methylation activity on exogenous Bacillus/Escherichia coli shuttle vector pBU4 as the HaeIII, which is an isoschizomer of BspRI. The Bsph_0498 (encoding the predicted LspC3–41IR) knockout mutant Δ0498 and the complement strain RC0498 were constructed. It was found that the unmethylated pBU4 can be digested by the CFE of C3–41 and RC0498, but not by that of Δ0498. Furthermore, the exogenous plasmid pBU4 can be transformed at very high efficacy into Δ0498, low efficacy into RC0498, but no transformation into C3–41, indicating that LspC3–41I might be a major determinant for the genetic restriction barrier of strain C3–41. Besides, lspC3–41IR and lspC3–41IM genes are detected in other two strains besides C3–41 of the tested 16 L. sphaericus strains, which all belonging to serotype H5 and MLST sequence type (ST) 1. Furthermore, the three strains are not horizontal transferred, and this restriction could be overcome by in vitro methylation either by the host CFE or by commercial methytransferase M. HaeIII. The results provide an insight to further study the genetic restriction, modification and evolution of mosquitocidal L. sphaericus, also a theoretical basis and a method for the genetic manipulations of L. sphaericus. CONCLUSIONS: LspC3–41I is identified as the major determinant for the restriction barrier of L. sphaericus C3–41. Only three strains of the tested 16 L. sphaericus strains, which all belonging to serotype H5 and ST1 by MLST scheme, contain LspC3–41I system. Two different methods can be used to overcome the restriction barrier of the three isolates to get transformants efficiently: 1) to methylate plasmid DNA prior to the electroporation; and 2) to delete the major restriction endonuclease encoding gene lspC3–41IR. BioMed Central 2017-05-19 /pmc/articles/PMC5437673/ /pubmed/28525986 http://dx.doi.org/10.1186/s12866-017-1014-6 Text en © The Author(s). 2017 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research Article
Fu, Pan
Ge, Yong
Wu, Yiming
Zhao, Ni
Yuan, Zhiming
Hu, Xiaomin
The LspC3–41I restriction-modification system is the major determinant for genetic manipulations of Lysinibacillus sphaericus C3–41
title The LspC3–41I restriction-modification system is the major determinant for genetic manipulations of Lysinibacillus sphaericus C3–41
title_full The LspC3–41I restriction-modification system is the major determinant for genetic manipulations of Lysinibacillus sphaericus C3–41
title_fullStr The LspC3–41I restriction-modification system is the major determinant for genetic manipulations of Lysinibacillus sphaericus C3–41
title_full_unstemmed The LspC3–41I restriction-modification system is the major determinant for genetic manipulations of Lysinibacillus sphaericus C3–41
title_short The LspC3–41I restriction-modification system is the major determinant for genetic manipulations of Lysinibacillus sphaericus C3–41
title_sort lspc3–41i restriction-modification system is the major determinant for genetic manipulations of lysinibacillus sphaericus c3–41
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5437673/
https://www.ncbi.nlm.nih.gov/pubmed/28525986
http://dx.doi.org/10.1186/s12866-017-1014-6
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