Plasmid pSA5700 from Staphylococcus aureus coding for erythromycin (Em(R)) and chloramphenicol (Cm(R)) resistance was transformed into Streptococcus pneumoniae. High-copy-number and Em(R) constitutive mutants of this plasmid were isolated. Transformation frequencies in S. pneumoniae as high as 70% were obtained with a constitutive plasmid as donor DNA, into a recipient cell containing a resident, inducible, high-copy-number plasmid. With the aid of these high frequencies, the site of constitutive mutations could be mapped via a simple marker rescue technique that uses purified restriction endonuclease-generated fragments. One of the Em(R) constitutive mutants, pFB9, a plasmid originating from a Gram-positive host, was shown to replicate and express Em(R) and Cm(R) in a Gram-negative organism, Escherichia coli. Four derivatives of pFB9 containing large (0.6-0.9 megadalton) insertion sequences that arose spontaneously in E. coli demonstrated unusual tranforming activity, as well as enhanced Em(R), in E. coli. The inserted elements mapped to the region in front of the Em(R) gene. Three of these inserted elements had the size and restriction patterns of insertion sequence IS1, IS2, and IS5. Plasmid pFB9 and derivatives are useful for isolation of new insertion sequences and for comparison of gene expression and illegitimate recombination between Gram-positive and Gram-negative species.
|Original language||English (US)|
|Number of pages||5|
|Journal||Proceedings of the National Academy of Sciences of the United States of America|
|Issue number||9 I|
|State||Published - 1982|
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