Giant linear plasmids in Mycobacterium avium harbour a tRNA array unit

Nontuberculous mycobacteria occasionally harbour clustered tRNA genes, referred to as a tRNA array unit, which is considered a putative antidefense system within their genomes. However, the precise genomic location of these tRNA array units remains unclear. To address this, we sequenced the complete genomes of 5 Mycobacterium avium strains carrying a tRNA array unit using a hybrid assembly of long and short reads followed by manual curation. The assemblies indicated that each strain harbours 3 to 5 extrachromosomal elements. In all genomes, the tRNA array unit was found on a linear contig exceeding 300 kb. Pulse-field gel electrophoresis (PFGE) and sodium dodecyl sulphate-PFGE revealed that the strains harbour linear plasmids corresponding to these large contigs with protein-capped termini. These linear plasmids encode a hybrid type VII/type IV secretion system but lack relaxase genes, which are typically present in mycobacterial circular plasmids. Additionally, they contain approximately 415 bp inverted repeats at the termini. Sequences of related plasmids were identified exclusively in the genomes of M. avium isolates from Japan available in public databases, suggesting a possible Asian origin. This study provides the first experimental evidence that M. avium harbours giant invertron-type linear plasmids carrying a tRNA array unit.