IS<italic toggle="yes">26</italic>-Mediated Formation of Transposons Carrying Antibiotic Resistance Genes

ABSTRACT The IS26 transposase, Tnp26, catalyzes IS26 movement to a new site and deletion or inversion of adjacent DNA via a replicative route. The intramolecular deletion reaction produces a circular molecule consisting of a DNA segment and a single IS26, which we call a translocatable unit or TU. R...

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Autores principales:	Christopher J. Harmer, Ruth M. Hall
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2016
Materias:	IS26 translocatable unit transposition transposons Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/6cdce6083da348978e213d1a2e49f930
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spelling	oai:doaj.org-article:6cdce6083da348978e213d1a2e49f9302021-11-15T15:21:21ZIS<italic toggle="yes">26</italic>-Mediated Formation of Transposons Carrying Antibiotic Resistance Genes10.1128/mSphere.00038-162379-5042https://doaj.org/article/6cdce6083da348978e213d1a2e49f9302016-04-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSphere.00038-16https://doaj.org/toc/2379-5042ABSTRACT The IS26 transposase, Tnp26, catalyzes IS26 movement to a new site and deletion or inversion of adjacent DNA via a replicative route. The intramolecular deletion reaction produces a circular molecule consisting of a DNA segment and a single IS26, which we call a translocatable unit or TU. Recently, Tnp26 was shown to catalyze an additional intermolecular, conservative reaction between two preexisting copies of IS26 in different plasmids. Here, we have investigated the relative contributions of homologous recombination and Tnp26-catalyzed reactions to the generation of a transposon from a TU. Circular TUs containing the aphA1a kanamycin and neomycin resistance gene or the tet(D) tetracycline resistance determinant were generated in vitro and transformed into Escherichia coli recA cells carrying R388::IS26. The TU incorporated next to the IS26 in R388::IS26 forms a transposon with the insertion sequence (IS) in direct orientation. Introduction of a second TU produced regions containing both the aphA1a gene and the tet(D) determinant in either order but with only three copies of IS26. The integration reaction, which required a preexisting IS26, was precise and conservative and was 50-fold more efficient when both IS26 copies could produce an active Tnp26. When both ISs were inactivated by a frameshift in tnp26, TU incorporation was not detected in E. coli recA cells, but it did occur in E. coli recA+ cells. However, the Tnp-catalyzed reaction was 100-fold more efficient than RecA-dependent homologous recombination. The ability of Tnp26 to function in either a replicative or conservative mode is likely to explain the prominence of IS26-bounded transposons in the resistance regions found in Gram-negative bacteria. IMPORTANCE In Gram-negative bacteria, IS26 recruits antibiotic resistance genes into the mobile gene pool by forming transposons carrying many different resistance genes. In addition to replicative transposition, IS26 was recently shown to use a novel conservative movement mechanism in which an incoming IS26 targets a preexisting one. Here, we have demonstrated how IS26-bounded class I transposons can be produced from translocatable units (TUs) containing only an IS26 and a resistance gene via the conservative reaction. TUs were incorporated next to an existing IS26, creating a class I transposon, and if the targeted IS26 is in a transposon, the product resembles two transposons sharing a central IS26, a configuration observed in some resistance regions and when a transposon is tandemly duplicated. Though homologous recombination could also incorporate a TU, Tnp26 is far more efficient. This provides insight into how IS26 builds transposons and brings additional transposons into resistance regions.Christopher J. HarmerRuth M. HallAmerican Society for MicrobiologyarticleIS26translocatable unittranspositiontransposonsMicrobiologyQR1-502ENmSphere, Vol 1, Iss 2 (2016)
institution	DOAJ
collection	DOAJ
language	EN
topic	IS26 translocatable unit transposition transposons Microbiology QR1-502
spellingShingle	IS26 translocatable unit transposition transposons Microbiology QR1-502 Christopher J. Harmer Ruth M. Hall IS<italic toggle="yes">26</italic>-Mediated Formation of Transposons Carrying Antibiotic Resistance Genes
description	ABSTRACT The IS26 transposase, Tnp26, catalyzes IS26 movement to a new site and deletion or inversion of adjacent DNA via a replicative route. The intramolecular deletion reaction produces a circular molecule consisting of a DNA segment and a single IS26, which we call a translocatable unit or TU. Recently, Tnp26 was shown to catalyze an additional intermolecular, conservative reaction between two preexisting copies of IS26 in different plasmids. Here, we have investigated the relative contributions of homologous recombination and Tnp26-catalyzed reactions to the generation of a transposon from a TU. Circular TUs containing the aphA1a kanamycin and neomycin resistance gene or the tet(D) tetracycline resistance determinant were generated in vitro and transformed into Escherichia coli recA cells carrying R388::IS26. The TU incorporated next to the IS26 in R388::IS26 forms a transposon with the insertion sequence (IS) in direct orientation. Introduction of a second TU produced regions containing both the aphA1a gene and the tet(D) determinant in either order but with only three copies of IS26. The integration reaction, which required a preexisting IS26, was precise and conservative and was 50-fold more efficient when both IS26 copies could produce an active Tnp26. When both ISs were inactivated by a frameshift in tnp26, TU incorporation was not detected in E. coli recA cells, but it did occur in E. coli recA+ cells. However, the Tnp-catalyzed reaction was 100-fold more efficient than RecA-dependent homologous recombination. The ability of Tnp26 to function in either a replicative or conservative mode is likely to explain the prominence of IS26-bounded transposons in the resistance regions found in Gram-negative bacteria. IMPORTANCE In Gram-negative bacteria, IS26 recruits antibiotic resistance genes into the mobile gene pool by forming transposons carrying many different resistance genes. In addition to replicative transposition, IS26 was recently shown to use a novel conservative movement mechanism in which an incoming IS26 targets a preexisting one. Here, we have demonstrated how IS26-bounded class I transposons can be produced from translocatable units (TUs) containing only an IS26 and a resistance gene via the conservative reaction. TUs were incorporated next to an existing IS26, creating a class I transposon, and if the targeted IS26 is in a transposon, the product resembles two transposons sharing a central IS26, a configuration observed in some resistance regions and when a transposon is tandemly duplicated. Though homologous recombination could also incorporate a TU, Tnp26 is far more efficient. This provides insight into how IS26 builds transposons and brings additional transposons into resistance regions.
format	article
author	Christopher J. Harmer Ruth M. Hall
author_facet	Christopher J. Harmer Ruth M. Hall
author_sort	Christopher J. Harmer
title	IS<italic toggle="yes">26</italic>-Mediated Formation of Transposons Carrying Antibiotic Resistance Genes
title_short	IS<italic toggle="yes">26</italic>-Mediated Formation of Transposons Carrying Antibiotic Resistance Genes
title_full	IS<italic toggle="yes">26</italic>-Mediated Formation of Transposons Carrying Antibiotic Resistance Genes
title_fullStr	IS<italic toggle="yes">26</italic>-Mediated Formation of Transposons Carrying Antibiotic Resistance Genes
title_full_unstemmed	IS<italic toggle="yes">26</italic>-Mediated Formation of Transposons Carrying Antibiotic Resistance Genes
title_sort	is<italic toggle="yes">26</italic>-mediated formation of transposons carrying antibiotic resistance genes
publisher	American Society for Microbiology
publishDate	2016
url	https://doaj.org/article/6cdce6083da348978e213d1a2e49f930
work_keys_str_mv	AT christopherjharmer isitalictoggleyes26italicmediatedformationoftransposonscarryingantibioticresistancegenes AT ruthmhall isitalictoggleyes26italicmediatedformationoftransposonscarryingantibioticresistancegenes
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IS<italic toggle="yes">26</italic>-Mediated Formation of Transposons Carrying Antibiotic Resistance Genes

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