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Molecular genetics of antibiotic resistance determinants from Clostridium perfringens

Abraham, Lawrence Joseph (1986) Molecular genetics of antibiotic resistance determinants from Clostridium perfringens. PhD thesis, Murdoch University.

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Abstract

Clostridium perfringens is a sporulating, Gram positive, anaerobic bacterium that is a normal inhabitant of the gastrointestinal tract and is known to cause a number of diseases in both man and animals. Conjugative tetracycline resistance plasmids from C. perfringens strains isolated in Australia, the United States, France, Belgium, and Japan were analysed by comparison with the reference plasmid, pCW3. This conjugative 47 kilobasepair (kb) plasmid was isolated from a human C. perfringens strain and confers inducible tetracycline resistance. Fifteen of the plasmids encoded tetracycline resistance whereas three carried both tetracycline and chloramphenicol resistance. Nine of the tetracycline resistance plasmids had restriction profiles that were identical to those of pCW3. The remaining nine R-plasmids were different to pCW3. Comparison of partial restriction maps of these plasmids with a complete map of pCW3 indicated that they contain at least 17 kb of DNA that was present in pCW3. Hybridization analysis confirmed that these plasmids shared substantial homology with pCW3. The results showed that a pCW3-like element forms the basis of all transferable R-plasmids found in C. perfringens and that this element was widely disseminated in nature.

To compare regions of homology between the conjugative plasmids it was necessary to construct a detailed restriction map of pCW3. The five Cla I fragments that together comprise the entire pCW3 molecule were cloned in Escherichia coli. The recombinant plasmids were mapped and the restriction data were used to construct an unambiguous pCW3 map. The C. perfringens tetracycline resistance determinant was expressed in E. coli and was shown to be located on two juxtaposed Eco RI fragments that together encompass a 4 kb region of pCW3. Deletion and transposon mutagenesis experiments showed that the tetracycline resistance gene was contained within a 1. 4 kb segment of the 4 kb region. This gene, designated tetP, was compared with those of other bacteria by Southern hybridization. The results indicated that there was no homology between tetP and any of the other classes of tetracycline resistance determinant. Consequently, tetP represents a new class of tetracycline resistance determinant that is distinct from those of other bacteria.

Three of the conjugative tetracycline resistance plasmids that were characterised as being related to pCW3, also coded for chloramphenicol resistance. The homologous 6. 2 kb chloramphenicol resistance regions delete with high frequency upon transfer of these plasmids. The chloramphenicol resistance regions from two of these plasmids, pIP401 and pJIR27, were cloned in E. coli. The recombinant plasmids, pJIR45 and pJIR97 respectively, confer chloramphenicol resistance upon E. coli. However, cultures carrying these plasmids rapidly became chloramphenicol sensitive when grown in the absence of chloramphenicol. Loss of resistance was associated with the loss of the 6. 2 kb segments from both plasmids. Transposition of these segments to different sites on the E. coli chromosome was demonstrated after cloning the 6. 2 kb region onto a temperature sensitive replicon. Heteroduplex analysis and restriction mapping indicated that the transposons, Tn4451 (pIP401) and Tn4452 (pJIR27), were closely related and did not contain large inverted or directly repeated sequences.

Comparison of the end sequences of Tn4451 with those of the Tn3 family of transposons indicated some similarity. In contrast to the Tn3- like transposons which contain ca. 30 basepair (bp) inverted repeat sequences at their ends and duplicate 5 bp of the target sequence, TN4451 contained 3 bp inverted repeat sequences at its ends, and appeared to generate 2 bp direct repeats of the target DNA upon insertion. The regions surrounding the site of deletion of TN4451 also were sequenced. Deletion fragments derived from excision of TN4451 in both C. perfringens and E. coli were sequenced and it was shown that the deletion of TN4451was precise. The chloramphenicol resistance transposons TN4451 and TN4452 represent the first transposons to be identified from C. perfringens.

Item Type: Thesis (PhD)
Murdoch Affiliation: School of Veterinary Studies
Notes: Note to the author: If you would like to make your thesis openly available on Murdoch University Library's Research Repository, please contact: repository@murdoch.edu.au. Thank you.
Supervisor(s): Rood, Julian
URI: http://researchrepository.murdoch.edu.au/id/eprint/53043
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