Item – Theses Canada

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OCLC number
28217129
Author
Wang, Ying,1955-
Title
Transformation and antibiotic resistance in Campylobacter species.
Degree
Ph. D. -- University of Alberta, 1991
Publisher
Ottawa : National Library of Canada = Bibliothèque nationale du Canada, 1992.
Description
2 microfiches.
Notes
University Microfilms order no. UMI00322410.
Includes bibliographical references.
Abstract
Growing cells of Campylobacter coli and C. jejuni were naturally transformed by naked DNA without a requirement for any special treatment. Transformation frequencies for homologous chromosomal DNA were approximately 1 $\times$ 10$\sp{-3}$ transformants per viable cell in C. coli and 1 $\times$ 10$\sp{-4}$ in C. jejuni. C. coli UA585 is constitutively competent, and the competence level can be stimulated by DNA molecules. Maximum competence was found in the early exponential phase of growth. Campylobacters preferentially took up their own DNA in comparison with Escherichia coli chromosomal DNA which was taken up very poorly. Plasmid DNAs were taken up by campylobacters much less efficiently than homologous chromosomal DNA, and transformation into plasmid-free cells was very rare. However, with the use of recipients containing a homologous plasmid, approximately 1 $\times$ 10$\sp{-4}$ transformants per cell were obtained. A tet(M) determinant, originally obtained from Streptococcus, and a staphylococcal kanamycin resistance determinant were transformed and expressed in C. coli. A chloramphenicol resistance determinant, originally cloned from a C. coli plasmid pNR9589 in Japan, was isolated, and the nucleotide sequence determined. It contains an ORF of 621 bp, and the gene product was identified as a CAT (Cm acetyltransferase). The deduced amino acid sequence shows 43% to 57% identity with other CAT proteins of both Gram-positive and Gram-negative origin. Although expression of the cat gene was constitutive in both C. coli and E. coli, results of primer extension experiments indicated that transcription was initiated at different sites in these two species. A kanamycin resistance determinant, identified as the aphA-3 gene, was located downstream from the cat gene. The codon usage of the cat gene is very different from that used in E. coli; however, the CAT polypeptide was synthesized in large amounts in E. coli maxicells. Therefore, the codon usage bias is not one of the obstacles which affects Campylobacter spp. gene expression in E. coli. (Abstract shortened by UMI.)
ISBN
0315699264
9780315699267