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

Abstract

The plasmid pGKE75-cat A138T , which comprises pUC18 and the cat A138T gene encoding thermostable chloramphenicol acetyltransferase with an A138T amino acid replacement (CAT A138T ), 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-cat A138T in an error-prone thermophile, generating the mutant plasmid pGKE75 ␣␤ -cat A138T responsible for substantial chloramphenicol resistance at 65°C. pGKE75 ␣␤ -cat A138T contained no mutation in the cat A138T 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␣␤ -cat A138T is attributable to increases in intracellular CAT A138T and acetyl-coenzyme A following a decrease in incomplete forms of pGKE75 ␣␤ -cat A138T . 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 ␣␤ -cat A138T 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. C olE1-type plasmids, such as pBR322 and pUC, replicate in Escherichia coli autonomously with substantial copy numbers and are extensively utilized in microbial genetic engineering. As shown by pBR322 (Fig. 1), the replicon of ColE1-type plasmids generally contains genes for a precursor of RNA primer (RNA II), a replication-regulatory RNA (RNA I), and a replication-regulatory protein (Rom). The precursor RNA II is transcribed from 555 bp upstream of the replication origin and adopts a stem-loop structure that forms a persistent hybrid at the origin. This structure is subsequently cleaved by RNase H to serve as a primer for plasmid replication (1). RNA I consists of 108 nucleotides transcribed from 445 bp upstream of the origin to near the initiation site for RNA II synthesis (2-4). Because RNA I synthesis proceeds in the direction opposite to that of RNA II synthesis, RNA I can hybridize to RNA II and trigger conformational changes in RNA II. This event, which prevents RNA II from hybridization at the replication origin, inhibits plasmid replication and decreases the plasmid copy number. Plasmid replication is also negatively regulated by the Rom protein (2, 5-8), which facilitates the initial interaction between RNA I and RNA II. Thus, the copy number of ColE1-type plasmids is under the tripartite control of RNA I, RNA II, and the Rom protein.Genetic alterations in or near the replicon are known to affect the copy numbe...

Small Klebsiella pneumoniae Plasmids: Neglected Contributors to Antibiotic Resistance

“…However, it should be noted that some of these latter studies were carried out with plasmids with non-natural replicons such as pUC19. This is a derivative of the ColE1-type pMB1 replicon but in which the rom gene has been deleted, and a nucleotide substitution within RNA II alters its spatial structure in a temperature-dependent manner affecting the interaction between RNA II and the negative regulator RNA I (Minton et al, 1988; Lin-Chao et al, 1992). As a consequence, plasmids with this origin of replication have a copy number abnormally higher.…”

Plasmid Curing is a Promising Approach to Improve Thermophiles for Biotechnological Applications: Perspectives in Archaea

“…Genetic tools are available for this strain [10][11][12][13][14][15][16]. The whole genome sequence has been determined [17], showing that G. kaustophilus HTA426 harbors the circular plasmid pHTA426 (Figure 1).…”