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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.

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