Development of new <i>Thermus thermophilus—Escherichia coli</i> shuttle vectors

Genomic analysis revealed numerous plasmids in <i>Thermus thermophilus</i> strains isolated from the Senami Hot Spring in Japan. Five plasmids contained putative replication proteins (REP proteins) distinct from the pTT8-type commonly used in <i>T. thermophilus-Escherichia coli</i> shuttle vectors. Among them, two plasmids contained toxin-antitoxin-like tandemly aligned short open reading frames (ORFs) downstream of the putative REP protein. A series of shuttle vectors, pIOK, was constructed by cloning the putative REP protein gene and its flanking regions into an <i>E. coli</i> plasmid (ColE1, hygromycin-resistant). All five vectors were stably maintained in <i>T. thermophilus</i> HB27 in the presence of hygromycin. Among them, two containing the toxin-antitoxin-like module demonstrated greater persistence, exhibiting no plasmid loss after 168 generations of subcultivation without hygromycin. This module also enhanced the persistence of the pTT8-type shuttle vector pSN2, suggesting its broad applicability to the <i>Thermus</i> plasmids for stable maintenance. Quantitative PCR analysis demonstrated that the copy numbers of the pIOK vectors were equal to or greater than threefold those of the chromosomes. The pIOK vectors were compatible with one another and with pSN2. The xylan utilization pathway from <i>Thermus brockianus</i> (13 kbp) was split into two parts, which were cloned into pIOK and pSN2. Wild-type HB27 could not utilize xylan as the sole carbon source, while the double transformant carrying the two plasmids could, indicating the successful reconstitution of the complete xylan utilization pathway from two compatible plasmids. The pIOK vectors and toxin-antitoxin-like module will be invaluable for advancing synthetic biology research in <i>T. thermophilus</i>.IMPORTANCEThe rapid accumulation of genomic data from <i>Thermus thermophilus</i>, which we recently isolated from hot springs in Japan, has revealed the presence of plasmids with novel replication origins. We have developed a series of shuttle vectors, called pIOK, that are compatible with existing pTT8-based shuttle vectors. The effective use of multiple plasmid systems in <i>T. thermophilus</i> was demonstrated by dividing a relatively large (approximately 13 kbp) xylan assimilation pathway and reconstituting it using two compatible plasmids. The toxin-antitoxin-like module, located downstream of the newly identified replication proteins, significantly enhanced persistence, enabling the cultivation of the recombinant strain without the use of antibiotics. The pIOK vectors and the toxin-antitoxin-like module are expected to be valuable tools in the synthetic biology of <i>T. thermophilus</i>.