Nucleotide sequence and functional map of pC194, a plasmid that specifies inducible chloramphenicol resistance
- 1 May 1982
- journal article
- research article
- Published by American Society for Microbiology in Journal of Bacteriology
- Vol. 150 (2), 815-825
- https://doi.org/10.1128/jb.150.2.815-825.1982
Abstract
The nucleotide sequence of pC194, a small plasmid from Staphylococcus aureus which is capable of replication in Bacillus subtilis, was determined. The genetic determinant of chloramphenicol (CAM) resistance, which includes the chloramphenicol acetyl transferase (CAT) structural gene, the putative promoter and controlling element of this determinant, were mapped functionally by subcloning a 1035-nucleotide fragment which specifies the resistance phenotype using plasmid pBR322 as vector. Expression of CAM resistance is autogenously regulated since the 1035-nucleotide fragment containing the CAT gene sequence and its promoter cloned into pBR322 expresses resistance inducibly in the Escherichia coli host. A presumed controlling element of CAT expression consists of a 37-nucleotide inverted complementary repeat sequence that is located between the -10 and ribosome-loading sequences of the CAT structural gene. Whereas the composite plasmid containing the minimal CAT determinant cloned in pBR322 could not replicate in B. subtilis, ability to replicate in B. subtilis was seen if the fragment cloned included an extension consisting of an additional 300 nucleotides beyond the 5'' end of the single pC194 MspI site associated with replication. This 5'' extension contained a 120-nucleotide inverted complementary repeat sequence similar to that found in pE194 TaqI fragment B which contains replication sequences of the plasmid. pC194 was found to contain 4 open reading frames theoretically capable of coding for proteins with maximum molecular masses, as follows: A, 27,800 daltons; B, 26,200 daltons; C, 15,000 daltons; and D, 9600 daltons. Interruption or deletion of either frame A or D does not entail loss of ability to replicate or to express CAM resistance; frame B contains the CAT structural gene; and frame C contains sequences associated with plasmid replication.This publication has 27 references indexed in Scilit:
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