title: Unique plasmids generated via pUC replicon mutagenesis in an error-prone thermophile derived from Geobacillus kaustophilus HTA426.
abstract: The plasmid pGKE75-catA138T, which comprises pUC18 and the catA138T gene encoding thermostable chloramphenicol acetyltransferase with an A138T amino acid replacement (CATA138T), serves as an Escherichia coli-Geobacillus kaustophilus shuttle plasmid that confers moderate chloramphenicol resistance on G. kaustophilus HTA426. The present study examined the thermoadaptation-directed mutagenesis of pGKE75-catA138T in an error-prone thermophile, generating the mutant plasmid pGKE75(αβ)-catA138T responsible for substantial chloramphenicol resistance at 65°C. pGKE75(αβ)-catA138T contained no mutation in the catA138T gene but had two mutations in the pUC replicon, even though the replicon has no apparent role in G. kaustophilus. Biochemical characterization suggested that the efficient chloramphenicol resistance conferred by pGKE75(αβ)-catA138T is attributable to increases in intracellular CATA138T and acetyl-coenzyme A following a decrease in incomplete forms of pGKE75(αβ)-catA138T. 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 G. kaustophilus. It is noteworthy that G. kaustophilus was transformed with pGKE75(αβ)-catA138T using chloramphenicol selection at 60°C. In addition, a pUC18 derivative with the two mutations propagated in E. coli 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 G. kaustophilus and E. coli.
authors: Department of Chemistry and Biotechnology, Graduate School of Engineering, Tottori University, Tottori, Japan Functional Genomics of Extremophiles, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka, Japan.
authors: Jyumpei
authors: J
authors: Kobayashi
authors: Department of Chemistry and Biotechnology, Graduate School of Engineering, Tottori University, Tottori, Japan.
authors: Misaki
authors: M
authors: Tanabiki
authors: Department of Biotechnology, Faculty of Engineering, Tottori University, Tottori, Japan.
authors: Shohei
authors: S
authors: Doi
authors: Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, Kobe, Hyogo, Japan.
authors: Akihiko
authors: A
authors: Kondo
authors: Department of Chemistry and Biotechnology, Graduate School of Engineering, Tottori University, Tottori, Japan.
authors: Takashi
authors: T
authors: Ohshiro
authors: Department of Chemistry and Biotechnology, Graduate School of Engineering, Tottori University, Tottori, Japan Functional Genomics of Extremophiles, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka, Japan hirokap@xpost.plala.or.jp.
authors: Hirokazu
authors: H
authors: Suzuki
mesh_terms: D000105:Acetyl Coenzyme A / Q000378:metabolism; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D002702:Chloramphenicol Resistance; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors; D056506:Geobacillus / Q000235:genetics*; D006358:Hot Temperature; D016296:Mutagenesis; D009154:Mutation; D010957:Plasmids
chemical_list: D000105:Acetyl Coenzyme A; D015500:Chloramphenicol O-Acetyltransferase