The ΦBT1 large serine recombinase catalyzes DNA integration at pseudo-attB sites in the genus Nocardia.

Abstract:

: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.

journal_name

PeerJ

journal_title

PeerJ

authors

Herisse M,Porter JL,Guerillot R,Tomita T,Goncalves Da Silva A,Seemann T,Howden BP,Stinear TP,Pidot SJ

doi

10.7717/peerj.4784

subject

Has Abstract

pub_date

2018-05-04 00:00:00

pages

e4784

issn

2167-8359

pii

4784

journal_volume

6

pub_type

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