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

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.

Discovery and Biosynthesis of the Cytotoxic Polyene Terpenomycin in Human Pathogenic Nocardia

“…Mining of the complete N. terpenica AUSMDU00012715 genome (GenBank accession CP046173.1) using antiSMASH v6.0 revealed 49 predicted BGCs, with one BGC showing 59% similarity to the concanamycin BGC. Concanamycin is a polyene macrolide related to the bafilomycins, yet it has an 18-membered macrolactone core (as opposed to the 16-membered core of terpenomycin) and lacks the pentaene side chain of terpenomycin.…”

“…As we were unable to find any previous studies that had utilized transposon mutagenesis in the genus Nocardia, we looked to systems that had been used in the closely related and better studied Streptomyces. Previous work showed that a codon-optimized hyperactive variant of the transposase Tn 5 could be used to successfully mutagenize Streptomyces species. − Due to our previous work adapting Streptomyces genetic elements to function in Nocardia, , Tn5’s preference for high-GC regions of DNA, and similarity in codon usage and GC content between Streptomyces and Nocardia, we hypothesized that this Streptomyces codon-optimized Tn 5 may also function in the Nocardia. Utilizing the vector pHL734, we performed multiple rounds of transformation of N.…”

Identification and Mobilization of a Cryptic Antibiotic Biosynthesis Gene Locus from a Human-Pathogenic Nocardia Isolate

“…Genome sequences for Nocardia terpenica AUSMDU00012715 and Nocardia arthritidis AUSMDU00012717 have been published previously. 41,42 All genomes were analyzed using the AntiSMASH 5.0 Web server (antismash. secondarymetabolites.org).…”

“…DNA Extraction. DNA was isolated from N. brasiliensis AUSMDU00024985 according to Herisse et al 41 Transformation Associated Recombination (TAR). TAR capture was performed according to the "Direct Cloning and Heterologous Expression of Biosynthetic Gene Cluster" protocol provided with pCAP03-aac(3)IV (https://media.addgene.org/data/ plasmids/69/69862/69862-attachment_VaRga0ZsUaHw.pdf; see also Tang et al 26 ).…”

“…The genome of each Nocardia strain was obtained from Genbank (corresponding accession numbers are shown in Supplementary Table 3), except for N. brasiliensis AUSMDU00024985, which was sequenced and assembled in-house (see below). Genome sequences for Nocardia terpenica AUSMDU00012715 and Nocardia arthritidis AUSMDU00012717 have been published previously. , All genomes were analyzed using the AntiSMASH 5.0 Web server () . The AntiSMASH output for each genome was then used as the input for BiG-SCAPE .…”