Conjugation of φBT1-derived integrative plasmid pDZL802 in <i>Amycolatopsis mediterranei</i> U32

Li, Chen; Zhou, Li; Wang, Ying; Zhao, Guoping; Ding, Xiaoming

doi:10.1080/21655979.2016.1270808

Cited by 8 publications

(11 citation statements)

References 20 publications

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“…Nocardia transformants allowed us to investigate individual insertion sites without any a priori knowledge of their location. Previous studies using ΦBT1 Int in Actinomycetes have determined insertion only by PCR around the known attB site (Gregory et al 2003;Li et al 2017), meaning that insertions at pseudo-attB sites would have been classed as "non-integrants" and most likely ignored. We sought to determine whether ΦBT1 pseudo-attB sites exist in various Streptomyces species and if these sites are utilised in strains with canonical attB sites.…”

Section: Identification Of Pseudo-attb Sites In Other Nocardia Speciesmentioning

confidence: 99%

“…has also been reported, although this appears to occur relatively infrequently (Baltz 2012;Combes et al 2002). While vectors utilizing ΦBT1 Int have also been shown to integrate in a range of Streptomyces species and Amycolatopsis mediterranei, their usage outside these organisms has been limited (Gregory et al 2003;Li et al 2017). Furthermore, the extent to which ΦBT1 Int can catalyse integration at pseudo-attB sites is unknown.…”

Section: Introductionmentioning

confidence: 99%

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Peer Review #1 of "The ΦBT1 large serine recombinase catalyzes DNA integration at pseudo-attB sites in the genus Nocardia (v0.1)"

2018

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Plasmid vectors based on bacteriophage integrases are important tools in molecular microbiology for the introduction of foreign DNA, especially into bacterial species where other systems for genetic manipulation are limited. Site specific integrases catalyze recombination between phage and bacterial attachment sites (attP and attB, respectively) and the best studied integrases in the Actinomycetes are the serine integrases from the Streptomyces bacteriophages ΦC31 and ΦBT1. As this reaction is unidirectional and highly stable, vectors containing phage integrase systems have been used in a number of genetic engineering applications. Plasmids bearing the ΦBT1 integrase have been used to introduce DNA into Streptomyces and Amycolatopsis strains, however, they have not been widely studied in other actinobacterial genera. Here, we show that vectors based on ΦBT1 integrase can stably integrate into the chromosomes of a range of Nocardia species, and that this integration occurs despite the absence of canonical attB sites in these genomes. Furthermore, we show that a ΦBT1 integrase-based vector can insert at multiple pseudo-attB sites within a single strain and we determine the sequence of a pseudo-attB motif. These data suggest that ΦBT1 integrase-based vectors can be used to readily and semi-randomly introduce foreign DNA into the genomes of a range of Nocardia species. However, the precise site of insertion will likely require empirical determination in each species to avoid unexpected off-target effects.

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Section: Identification Of Pseudo-attb Sites In Other Nocardia Speciesmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Peer Review #1 of "The ΦBT1 large serine recombinase catalyzes DNA integration at pseudo-attB sites in the genus Nocardia (v0.1)"

2018

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“…While ΦC31 attB sites are highly similar in a number of Streptomyces species, non-specific integration in sequences that have partial identity to attB (pseudo- attB sites) has also been reported, although this appears to occur relatively infrequently ( Baltz, 2012 ; Combes et al, 2002 ). While vectors utilizing ΦBT1 Int have also been shown to integrate in a range of Streptomyces species and A. mediterranei , their usage outside these organisms has been limited ( Gregory, Till & Smith, 2003 ; Li et al, 2017 ). Furthermore, the extent to which ΦBT1 Int can catalyze integration at pseudo- attB sites is unknown.…”

Section: Introductionmentioning

confidence: 99%

The ΦBT1 large serine recombinase catalyzes DNA integration at pseudo-attBsites in the genusNocardia

Hérisse

Porter

Guérillot

et al. 2018

PeerJ

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Plasmid vectors based on bacteriophage integrases are important tools in molecular microbiology for the introduction of foreign DNA, especially into bacterial species where other systems for genetic manipulation are limited. Site specific integrases catalyze recombination between phage and bacterial attachment sites (attP and attB, respectively) and the best studied integrases in the actinomycetes are the serine integrases from the Streptomyces bacteriophages ΦC31 and ΦBT1. As this reaction is unidirectional and highly stable, vectors containing phage integrase systems have been used in a number of genetic engineering applications. Plasmids bearing the ΦBT1 integrase have been used to introduce DNA into Streptomyces and Amycolatopsis strains; however, they have not been widely studied in other actinobacterial genera. Here, we show that vectors based on ΦBT1 integrase can stably integrate into the chromosomes of a range of Nocardia species, and that this integration occurs despite the absence of canonical attB sites in these genomes. Furthermore, we show that a ΦBT1 integrase-based vector can insert at multiple pseudo-attB sites within a single strain and we determine the sequence of a pseudo-attB motif. These data suggest that ΦBT1 integrase-based vectors can be used to readily and semi-randomly introduce foreign DNA into the genomes of a range of Nocardia species. However, the precise site of insertion will likely require empirical determination in each species to avoid unexpected off-target effects.

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“…The ϕBT1 attB sites have been more commonly identified in Amycolatopsis , and there has been successful conjugative transfer of vectors based on ϕBT1 int/attP in A. orientalis(20) and A. mediterranei (21). Furthermore, electroporation remains the most widely applied method for transfer of integrative plasmids into this genus, rather than conjugation(20, 21).…”

Section: Introductionmentioning

confidence: 99%

“…There is only one reported example of a conjugation system based on ϕC31 integrase in Amycolatopsis Japonicum MG4I7-CFI7(19), and it has been reported that other Amycolatopsis species lack ϕC31 attB sites in their chromosomes(20). The ϕBT1 attB sites have been more commonly identified in Amycolatopsis , and there has been successful conjugative transfer of vectors based on ϕBT1 int/attP in A. orientalis(20) and A. mediterranei (21). Furthermore, electroporation remains the most widely applied method for transfer of integrative plasmids into this genus, rather than conjugation(20, 21).…”

Section: Introductionmentioning

confidence: 99%

Integrating Vectors for Genetic Studies in the Rare ActinomyceteAmycolatopsis marina

Gao

Murugesan

Hoßbach

et al. 2018

Preprint

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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 integrating vectors, derived using phage int/attP loci, that would efficiently integrate site-specifically in the rare Actinomycete, Amycolatopsis marina DSM45569. 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 E. coli to A. marina DSM45569. Site-specific recombination of the plasmids into the host TG1 or R4 attB sites was confirmed by sequencing. The presence of homologous TG1 and R4 attB sites in other species of this genus indicates that vectors based on TG1 and R4 integrases could be widely applicable.ImportanceRare Actinomycetes have the same potential of natural product discovery as Streptomyces, but the potential has not been fully explored due to the lack of efficient molecular biology tools. In this study, we identified two serine integrases, TG1 and R4, which could be used in the rare Actinomycetes species, Amycolatopsis marina, as tools for genome integration. The high level of conservation between the attB sites for TG1 and R4 in a number of Amycolatopsis species suggested that plasmids with the integration systems from these phages should be widely useful in this genus.

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Conjugation of φBT1-derived integrative plasmid pDZL802 in Amycolatopsis mediterranei U32

Cited by 8 publications

References 20 publications

Peer Review #1 of "The ΦBT1 large serine recombinase catalyzes DNA integration at pseudo-attB sites in the genus Nocardia (v0.1)"

Peer Review #1 of "The ΦBT1 large serine recombinase catalyzes DNA integration at pseudo-attB sites in the genus Nocardia (v0.1)"

The ΦBT1 large serine recombinase catalyzes DNA integration at pseudo-attBsites in the genusNocardia

Integrating Vectors for Genetic Studies in the Rare ActinomyceteAmycolatopsis marina

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