Temperature-sensitive cloning vector for Corynebacterium glutamicum.
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 degrees C but not at 34 degrees 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.
Nakamura
Jun
J
Fermentation and Biotechnology Laboratories, Ajinomoto CO., Inc., Kawasaki-shi, Kanagawa 210-8681, Japan. jun_nakamura@ajinomoto.com
Kanno
Sohei
S
Kimura
Eiichiro
E
Matsui
Kazuhiko
K
Nakamatsu
Tsuyoshi
T
Wachi
Masaaki
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D048230:Corynebacterium glutamicum / Q000235:genetics*; D017931:DNA Primers; D004261:DNA Replication / Q000235:genetics*; D040461:Gene Components; D005822:Genetic Vectors / Q000235:genetics*; D019811:Hydroxylamine; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017422:Sequence Analysis, DNA; D013696:Temperature
D017931:DNA Primers; D019811:Hydroxylamine