Temperature-sensitive cloning vector for Corynebacterium glutamicum

• Published in: Plasmid Vol. 56; no. 3; pp. 179 - 186
• Main Authors: Nakamura, Jun, Kanno, Sohei, Kimura, Eiichiro, Matsui, Kazuhiko, Nakamatsu, Tsuyoshi, Wachi, Masaaki
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01-11-2006
• Subjects: Amino Acid Sequence; Base Sequence; Corynebacterium glutamicum; Corynebacterium glutamicum - genetics; DNA Primers; DNA Replication - genetics; Escherichia coli; Gene Components; Genetic Vectors - genetics; Hydroxylamine; Molecular Sequence Data; Mutagenesis, Site-Directed; pBL1; Sequence Analysis, DNA; Temperature; Temperature-sensitive plasmid; pBL1; Temperature-sensitive plasmid; Corynebacterium glutamicum
• ISSN: 0147-619X; 1095-9890
• DOI: 10.1016/j.plasmid.2006.05.003

We constructed a temperature-sensitive form of the
Corynebacterium glutamicum ATCC13869 cryptic plasmid, pBL1. The
C. glutamicum/
Escherichia coli shuttle vector pSFK6, which is composed of pBL1 and the
E. coli cloning vector pK1, was mutagenized
in vitro by treatment with hydroxylamine, and introduced into
C. glutamicum cells. A mutant plasmid, which was stably maintained at 25
°C but not at 34
°C, was isolated from the cells. Sequencing the plasmid, which was named p48K, revealed four substitutions in the Rep protein coding region. Moreover, site-directed single-nucleotide substitutions showed that a G to A transition at position 2,920, which resulted in a Pro-47 to Ser substitution in the Rep protein, was responsible for its temperature-sensitive replication. Pro-47 is conserved among the Rep proteins of the pIJ101/pJV1 family of plasmids. This temperature-sensitive cloning vector will be useful for disrupting genes in this industrially important bacterium.