Studies on staphylococcal chloramphenicol-resistance plasmids.
thesisposted on 19.11.2015, 09:07 by Sarah Elizabeth. Skinner
Six staphylococcal chloramphenicol-resistance (CmR) plasmids were compared by incompatibility tests and restriction digest analysis: pCW8 was indistinguishable from pC194; pCW6 was very similar to pUB112. While interruption of the HindIII site of pCW6 failed to prevent the inducible synthesis of chloramphenicol acetyltransferase (CAT), the BstEII site was shown to lie in or near the CmR determinant. Four of the plasmids were compared by electronmicroscopy of heteroduplexes between derivatives of them, A 2 kilobase-pair (kb) region of homology which pCW6 and pCW7 shared with pC221 coincided with pCW41, a 2 kb derivative of pC221 carrying the functions for inducible synthesis of CAT and replication, maintenance and incompatibility in Staphylococcus aureus. While neither pC221 nor pCW6 showed any homology with pCW8, 0.28 kb of pCW7 and pCW8 hybridised. Detailed restriction maps were constructed for pC221 and pCW7 by Smith-Birnstiel mapping and double digests. These results are discussed in terms of the evolution of staphylococcal CmR plasmids. The organisation of the CmR determinant of pC221 was studied by induction experiments using S.aureus carrying derivatives of pC221, including pCW41. Interruption of the HpaII site failed to prevent the inducible synthesis of CAT; however, interruption of the BstEII site abolished detectable CAT synthesis. The induction of CAT was prevented by rifampicin. A small increase in cat RNA was observed on induction. Like the 3-deoxy analogue of Cm, 3-fluorochloramphenicol could be used as an inducer of CAT. Levels of expresson of the cat gene of pC221 in Escherichia coli were low and induction was generally poor unless transcription from an adjacent pBR322 promoter was possible. Sequences in pC221 could act as promoters for the tetracycline-resistance determinant of pBR322. These results are discussed in relation to various models for the regulation of Gram-positive cat genes.