Characterization of an Endogenous Plasmid and Development of Cloning Vectors and a Transformation System in Brevibacterium lactofermentum

Departamento de Microbiología, Facultad de Biología, Universidad de León, León, Spain ABSTRACT A cryptic plasmid, pBL1 of 4.3 kb, has been found in lysine-producing Brevibacterium lactofermentum strains BL0, BL70, BL74 and BL77. pBL1 had single restriction sites for Bal I, Bcl I, Hae II, Hind III an...

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Published inJournal of general microbiology Vol. 130; no. 9; pp. 2237 - 2246
Main Authors SANTAMARIA, RAMON, GIL, JOSE A, MESAS, JUAN M, MARTIN, JUAN F
Format Journal Article
LanguageEnglish
Published London Soc General Microbiol 1984
New York, NY Cambridge University Press
Subjects
Bacteriology
Biological and medical sciences
Brevibacterium lactofermentum
cloning vectors
Fundamental and applied biological sciences. Psychology
gene transfer
Genetics
host systems
Microbiology
plasmids
transformation
Plasmid
Transposition
Transformation
Brevibacterium lactofermentum
Brevibacteriaceae
Restriction map
Bacteria
Actinomycetes
Molecular cloning
Vector
Genetic transfer
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Summary:Departamento de Microbiología, Facultad de Biología, Universidad de León, León, Spain ABSTRACT A cryptic plasmid, pBL1 of 4.3 kb, has been found in lysine-producing Brevibacterium lactofermentum strains BL0, BL70, BL74 and BL77. pBL1 had single restriction sites for Bal I, Bcl I, Hae II, Hind III and Hpa I. It had four sites for Ava I, seven for Hae III, eight for Mbo I and a very large number for Alu I, but no sites were found for Pst I, Eco RI or Bam HI. The estimated copy number was 30. Three different pBL1-pBR322 hybrids named pUL1, pUL10 and pUL20 were constructed. Transposon Tn5 was inserted by transposition into either the pBR322 or the pBL1 components of plasmid pUL1, pUL10 and pUL20. A shuttle vector able to replicate in Escherichia coli, Streptomyces lividans and B. lactofermentum was constructed by cloning pBL1 into the plasmid pIJ860, a bifunctional E. coli-S. lividans vector carrying the tsr, bla and kan genes. A polyethylene glycol-assisted transformation system for B. lactofermentum protoplasts was developed. Transformation frequencies of 10 2 transformants (µg DNA) –1 were obtained. The kan resistance gene from Tn5 was expressed very efficiently in B. lactofermentum (up to 200 µg ml –1 ). A smaller plasmid, pUL62, was constructed in which the tsr (thiostrepton resistance) gene of pUL61 was deleted. Keywords: LA, Luria agar, LB, Luria broth
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ISSN:0022-1287
1350-0872
1465-2080
DOI:10.1099/00221287-130-9-2237