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.

FIG 1

pBR322 and pUC replicons. The replication origin is indicated at position +1. RNA I and RNA II are indicated, with their transcription directions. Compared with the pBR322 replicon, the pUC replicon lacks a rom (also known as rop) gene and has a point mutation (C·G→T·A transition) at position −444.

FIG 2

Structures of pGKE75-cat (A), pUC18 (B), and pUC replicons of their derivatives (C). The replication origin is indicated at position +1. Mutation sites are indicated by open circles. pGKE75-cat and pGKE75-cat_A138T shared the usual pUC replicon with pUC18. pGKE75^αβ-cat_A138T and pUC18^αβ carry both a G·C→T·A transversion at +175 and a G·C→A·T transition at −252. pGKE75^α-cat_A138T and pUC18^α carry a G·C→T·A transversion at +175. pGKE75^β-cat_A138T and pUC18^β carry a G·C→A·T transition at −252. pUC18^γ carries a T·A→C·G transition at −444 and thus has a pBR322 replicon lacking the rom gene. P_gk704, a promoter functional in G. kaustophilus (18); bla, ampicillin resistance gene functional in E. coli; TK101, kanamycin resistance gene functional at high temperatures (40); pUC, pUC replicon functional in E. coli; pBST1, pBST1 replicon functional in Geobacillus spp. (38); and oriT, conjugative-transfer origin from pRK2013 (44).

FIG 3

Effects of pGKE75-cat derivatives on G. kaustophilus. G. kaustophilus MK242(pGKE75-cat derivative) was grown at 65°C on LK5 plates and analyzed for Cm resistance efficiency (A), intracellular CAT activity (B), plasmid copy number (C), and acetyl-CoA concentration (D). (A) Cells were incubated at 65°C on LK5 plates with and without Cm to determine the Cm resistance efficiency. The data are presented as means and SD (n = 4). (B) Cell extract was prepared and analyzed for CAT specific activity. The data are presented as means and SD (n = 5). (C) Total DNA was extracted from cells and used to analyze the plasmid copy number, which was determined using the ratio of bla (in pGKE75-cat derivatives) to rpoA (in the G. kaustophilus chromosome). The data are presented as means and SD (n = 3). (D) Cell extract was prepared and analyzed for acetyl-CoA concentration. The data were normalized by the protein concentration and are presented as means and SD (n = 4).

FIG 4

Plasmid copy number of pUC18 (top; solid bars), pUC18^αβ (top; open bars), pUC18^α (bottom; solid bars), pUC18^β (bottom; open bars), and pUC18^γ (bottom; gray bars) in E. coli. E. coli(pUC18 derivative) was cultured at the indicated temperatures. Total DNA was extracted from the cells and used to analyze the plasmid copy number, which is the ratio of bla (in pUC18 derivatives) to rpoA (in the E. coli chromosome). The data are presented as means and SD (n = 3).