Integrating vectors for genetic studies in the rare Actinomycete Amycolatopsis marina
Abstract Background Few natural product pathways from rare Actinomycetes have been studied due to the difficulty in applying molecular approaches in these genetically intractable organisms. In this study, we sought to identify more integrating vectors, using phage int/attP loci, that would efficient...
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doaj-718f25bbf7474f3f9867bb7fa0824f002020-11-25T03:43:22ZengBMCBMC Biotechnology1472-67502019-06-0119111010.1186/s12896-019-0521-yIntegrating vectors for genetic studies in the rare Actinomycete Amycolatopsis marinaHong Gao0Buvani Murugesan1Janina Hoßbach2Stephanie K. Evans3W. Marshall Stark4Margaret C. M. Smith5Department of Biology, University of YorkDepartment of Biology, University of YorkDepartment of Biology, University of YorkDepartment of Biology, University of YorkInstitute of Molecular, Cell and Systems Biology, University of GlasgowDepartment of Biology, University of YorkAbstract Background Few natural product pathways from rare Actinomycetes have been studied due to the difficulty in applying molecular approaches in these genetically intractable organisms. In this study, we sought to identify more integrating vectors, using phage int/attP loci, that would efficiently integrate site-specifically in the rare Actinomycete, Amycolatopsis marina DSM45569. Results Analysis of the genome of A. marina DSM45569 indicated the presence of attB-like sequences for TG1 and R4 integrases. The TG1 and R4 attBs were active in in vitro recombination assays with their cognate purified integrases and attP loci. Integrating vectors containing either the TG1 or R4 int/attP loci yielded exconjugants in conjugation assays from Escherichia coli to A. marina DSM45569. Site-specific recombination of the plasmids into the host TG1 or R4 attB sites was confirmed by sequencing. Conclusions The homologous TG1 and R4 attB sites within the genus Amycolatopsis have been identified. The results indicate that vectors based on TG1 and R4 integrases could be widely applicable in this genus.http://link.springer.com/article/10.1186/s12896-019-0521-yRare ActinomycetesAmycolatopsisIntegrating vectorsTG1 integraseR4 integrase |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Hong Gao Buvani Murugesan Janina Hoßbach Stephanie K. Evans W. Marshall Stark Margaret C. M. Smith |
spellingShingle |
Hong Gao Buvani Murugesan Janina Hoßbach Stephanie K. Evans W. Marshall Stark Margaret C. M. Smith Integrating vectors for genetic studies in the rare Actinomycete Amycolatopsis marina BMC Biotechnology Rare Actinomycetes Amycolatopsis Integrating vectors TG1 integrase R4 integrase |
author_facet |
Hong Gao Buvani Murugesan Janina Hoßbach Stephanie K. Evans W. Marshall Stark Margaret C. M. Smith |
author_sort |
Hong Gao |
title |
Integrating vectors for genetic studies in the rare Actinomycete Amycolatopsis marina |
title_short |
Integrating vectors for genetic studies in the rare Actinomycete Amycolatopsis marina |
title_full |
Integrating vectors for genetic studies in the rare Actinomycete Amycolatopsis marina |
title_fullStr |
Integrating vectors for genetic studies in the rare Actinomycete Amycolatopsis marina |
title_full_unstemmed |
Integrating vectors for genetic studies in the rare Actinomycete Amycolatopsis marina |
title_sort |
integrating vectors for genetic studies in the rare actinomycete amycolatopsis marina |
publisher |
BMC |
series |
BMC Biotechnology |
issn |
1472-6750 |
publishDate |
2019-06-01 |
description |
Abstract Background Few natural product pathways from rare Actinomycetes have been studied due to the difficulty in applying molecular approaches in these genetically intractable organisms. In this study, we sought to identify more integrating vectors, using phage int/attP loci, that would efficiently integrate site-specifically in the rare Actinomycete, Amycolatopsis marina DSM45569. Results Analysis of the genome of A. marina DSM45569 indicated the presence of attB-like sequences for TG1 and R4 integrases. The TG1 and R4 attBs were active in in vitro recombination assays with their cognate purified integrases and attP loci. Integrating vectors containing either the TG1 or R4 int/attP loci yielded exconjugants in conjugation assays from Escherichia coli to A. marina DSM45569. Site-specific recombination of the plasmids into the host TG1 or R4 attB sites was confirmed by sequencing. Conclusions The homologous TG1 and R4 attB sites within the genus Amycolatopsis have been identified. The results indicate that vectors based on TG1 and R4 integrases could be widely applicable in this genus. |
topic |
Rare Actinomycetes Amycolatopsis Integrating vectors TG1 integrase R4 integrase |
url |
http://link.springer.com/article/10.1186/s12896-019-0521-y |
work_keys_str_mv |
AT honggao integratingvectorsforgeneticstudiesintherareactinomyceteamycolatopsismarina AT buvanimurugesan integratingvectorsforgeneticstudiesintherareactinomyceteamycolatopsismarina AT janinahoßbach integratingvectorsforgeneticstudiesintherareactinomyceteamycolatopsismarina AT stephaniekevans integratingvectorsforgeneticstudiesintherareactinomyceteamycolatopsismarina AT wmarshallstark integratingvectorsforgeneticstudiesintherareactinomyceteamycolatopsismarina AT margaretcmsmith integratingvectorsforgeneticstudiesintherareactinomyceteamycolatopsismarina |
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