Presentation Information
[P03-359]Development of new Thermus thermophilus—Escherichia coli shuttle vectors
○Haruki Omichi1, Sora Murayama1, Kentaro Kentaro2, Hiroya Tomita2,3, Kohsuke Honda2,3 (1. Eng. Osaka Univ. (Japan), 2. Biotech Osaka Univ. (Japan), 3. OTRI Osaka Univ. (Japan))
Keywords:
Thermus thermophilus,compatibility,persistence,plasmid,replication protein,shuttle vector,synthetic biology
Thermus thermophilus is a model thermophilic bacterium. However, Genetic engineering in this organism has primarily relied on the pTT8-type replication origin, and alternative replication modules remain scarce.Genomic analysis of T. thermophilus strains isolated from the Senami Hot Spring in Japan revealed numerous plasmids (Miyazaki, Microbiol Resour Announc. 2022), including five plasmids containing putative replication proteins (REP proteins) distinct from the pTT8-type. Among them, two plasmids contained toxin–antitoxin-like tandemly aligned short open reading frames (ORFs) located downstream of the putative REP protein.
To evaluate these replication modules, a series of shuttle vectors, designated pIOK, was constructed by cloning the putative REP protein gene and its flanking regions into an E. coli plasmid (ColE1, hygromycin-resistant). All five vectors were stably maintained in T. thermophilus HB27 in the presence of hygromycin. Among them, two vectors 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 plasmids in T. thermophilus 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. To assess functional applicability, the xylan utilization pathway from Thermus brockianus (13 kbp) was divided into two parts and cloned separately into pIOK and pSN2. Wild-type HB27 could not utilize xylan as the sole carbon source, whereas the double transformant carrying the two plasmids acquired this ability, indicating successful reconstitution of the complete xylan utilization pathway from two compatible plasmids.
The pIOK vectors and the toxin–antitoxin-like module provide a system for stable maintenance of multiple plasmids in T. thermophilus and will facilitate synthetic biology in this organism.
To evaluate these replication modules, a series of shuttle vectors, designated pIOK, was constructed by cloning the putative REP protein gene and its flanking regions into an E. coli plasmid (ColE1, hygromycin-resistant). All five vectors were stably maintained in T. thermophilus HB27 in the presence of hygromycin. Among them, two vectors 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 plasmids in T. thermophilus 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. To assess functional applicability, the xylan utilization pathway from Thermus brockianus (13 kbp) was divided into two parts and cloned separately into pIOK and pSN2. Wild-type HB27 could not utilize xylan as the sole carbon source, whereas the double transformant carrying the two plasmids acquired this ability, indicating successful reconstitution of the complete xylan utilization pathway from two compatible plasmids.
The pIOK vectors and the toxin–antitoxin-like module provide a system for stable maintenance of multiple plasmids in T. thermophilus and will facilitate synthetic biology in this organism.
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