Unique Plasmids Generated via pUC Replicon Mutagenesis in an Error-Prone Thermophile Derived from Geobacillus kaustophilus HTA426

<jats:title>ABSTRACT</jats:title> <jats:p> The plasmid pGKE75- <jats:italic> cat <jats:sub>A138T</jats:sub> </jats:italic> , which comprises pUC18 and the <jats:italic> cat <jats:sub>A138T</jats:sub> </jats:italic> gene encoding thermostable chloramphenicol acetyltransferase with an A138T amino acid replacement (CAT <jats:sub>A138T</jats:sub> ), serves as an <jats:named-content content-type="genus-species">Escherichia coli-Geobacillus kaustophilus</jats:named-content> shuttle plasmid that confers moderate chloramphenicol resistance on <jats:named-content content-type="genus-species">G. kaustophilus</jats:named-content> HTA426. The present study examined the thermoadaptation-directed mutagenesis of pGKE75- <jats:italic> cat <jats:sub>A138T</jats:sub> </jats:italic> in an error-prone thermophile, generating the mutant plasmid pGKE75 <jats:sup>αβ</jats:sup> - <jats:italic> cat <jats:sub>A138T</jats:sub> </jats:italic> responsible for substantial chloramphenicol resistance at 65°C. pGKE75 <jats:sup>αβ</jats:sup> - <jats:italic> cat <jats:sub>A138T</jats:sub> </jats:italic> contained no mutation in the <jats:italic> cat <jats:sub>A138T</jats:sub> </jats:italic> gene but had two mutations in the pUC replicon, even though the replicon has no apparent role in <jats:named-content content-type="genus-species">G. kaustophilus</jats:named-content> . Biochemical characterization suggested that the efficient chloramphenicol resistance conferred by pGKE75 <jats:sup>αβ</jats:sup> - <jats:italic> cat <jats:sub>A138T</jats:sub> </jats:italic> is attributable to increases in intracellular CAT <jats:sub>A138T</jats:sub> and acetyl-coenzyme A following a decrease in incomplete forms of pGKE75 <jats:sup>αβ</jats:sup> - <jats:italic> cat <jats:sub>A138T</jats:sub> </jats:italic> . The decrease in incomplete plasmids may be due to optimization of plasmid replication by RNA species transcribed from the mutant pUC replicon, which were actually produced in <jats:named-content content-type="genus-species">G. kaustophilus</jats:named-content> . It is noteworthy that <jats:named-content content-type="genus-species">G. kaustophilus</jats:named-content> was transformed with pGKE75 <jats:sup>αβ</jats:sup> - <jats:italic> cat <jats:sub>A138T</jats:sub> </jats:italic> using chloramphenicol selection at 60°C. In addition, a pUC18 derivative with the two mutations propagated in <jats:named-content content-type="genus-species">E. coli</jats:named-content> at a high copy number independently of the culture temperature and high plasmid stability. Since these properties have not been observed in known plasmids, the outcomes extend the genetic toolboxes for <jats:named-content content-type="genus-species">G. kaustophilus</jats:named-content> and <jats:named-content content-type="genus-species">E. coli</jats:named-content> . </jats:p>