Genes of the threonine operon of Escherichia coli were used for the construction of a Brevibacterium flavum strain excreting threonine. Using the shuttle vector pCEM300 and a newly constructed shuttle vector pEC71 (7.1 kb, Kmr/Nmr), various plasmids carrying E. coli thr genes were prepared. Mutants resistant to the threonine analog 2-amino-3-hydroxyvaleric acid (AHV) were isolated after the ethyl methanesulfonate treatment of B. flavum carrying these recombinant plasmids. A mutant of B. flavum CCM 351 carrying the cloned genes thrA and thrB accumulated 12 g/L of threonine after 48 h of cultivation.

• MeSH
• Genes, Bacterial genetics   MeSH
• Brevibacterium enzymology   genetics   metabolism   MeSH
• Escherichia coli genetics   MeSH
• Gene Expression physiology   MeSH
• Phosphotransferases (Alcohol Group Acceptor) metabolism   MeSH
• Genetic Vectors MeSH
• Homoserine Dehydrogenase metabolism   MeSH
• Lysine biosynthesis   MeSH
• Operon MeSH
• Plasmids MeSH
• Threonine biosynthesis   genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Phosphotransferases (Alcohol Group Acceptor) MeSH
• Homoserine Dehydrogenase MeSH
• homoserine kinase MeSH Browser
• Lysine MeSH
• Threonine MeSH

The plasmid vector pEM100 (13.5 kb) constructed from pGV1106, a miniderivative of the broad-host-range IncW pSa plasmid, and the pAM330 plasmid of Brevibacterium lactofermentum is not stably maintained in Escherichia coli host cells under nonselective growth conditions. By insertion of a 0.9 kb DNA fragment containing the parB locus (responsible for the maintenance of plasmid R1 in E. coli cells) to plasmid pEM100, plasmid pEM110 was prepared which is maintained in a population of E. coli cells growing without a selection pressure very stably.

• MeSH
• Drug Resistance, Microbial MeSH
• Brevibacterium genetics   MeSH
• Species Specificity MeSH
• Escherichia coli genetics   MeSH
• Genetic Markers MeSH
• R Factors genetics   MeSH
• Recombination, Genetic MeSH
• DNA Transposable Elements * MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Genetic Markers MeSH
• DNA Transposable Elements * MeSH

A new shuttle vector pCEM500 replicating in Escherichia coli and in Brevibacterium flavum was constructed. It carries two antibiotic resistance determinants (Kmr/Gmr from plasmid pSa of Gram-negative bacteria and Smr/Spr from plasmid pCG4 of Corynebacterium glutamicum) which are efficiently expressed in both hosts and can be inactivated by insertion of DNA fragments into the unique restriction endonuclease sites located within them. This vector was found to be stably maintained in B. flavum and can be used for transfer of the cloned genes into this amino-acid-producing coryneform bacterium.

• MeSH
• Drug Resistance, Microbial genetics   MeSH
• Transformation, Bacterial MeSH
• Brevibacterium genetics   MeSH
• Corynebacterium drug effects   genetics   MeSH
• DNA, Bacterial analysis   genetics   MeSH
• Escherichia coli genetics   MeSH
• Genetic Vectors * MeSH
• Cloning, Molecular MeSH
• R Factors * MeSH
• Gene Expression Regulation, Bacterial MeSH
• Restriction Mapping MeSH
• Spectinomycin pharmacology   MeSH
• Streptomycin pharmacology   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA, Bacterial MeSH
• Spectinomycin MeSH
• Streptomycin MeSH

Plasmid pIM138 which had been characterized by the higher resistance of its DNA replication to the action of clorobiocin in comparison with the progenitor plasmid, was tested for its stability in host cells in the absence of the antibiotic. Growing without selective pressure, pIM138 was better maintained in cells than pBR322. The stability in the presence and in the absence of clorobiocin can be unanimously assigned to the plasmid itself, but some influence of host cells cannot be excluded.

• MeSH
• DNA, Bacterial analysis   metabolism   MeSH
• Escherichia coli drug effects   metabolism   MeSH
• Molecular Sequence Data MeSH
• Novobiocin analogs & derivatives   pharmacology   MeSH
• Plasmids * drug effects   MeSH
• DNA Replication MeSH
• Base Sequence MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• clorobiocin MeSH Browser
• DNA, Bacterial MeSH
• Novobiocin MeSH