Date of Award

1-1-2000

Thesis Type

Masters

Document Type

Thesis

Divisions

Institute for Advanced Studies

Department

-

Institution

Universiti Malaya

Abstract

Salmonella typhi S8 is resistant to ampicillin (Ac), chloramphenicol (Cm), cotrimoxazole (Ctm), streptomycin (Sm), and tetracycline (Tc). The Ac, Cm, Ctm. and Sm resistance traits have been shown to be mediated by a multiple antibiotic resistance transposon located on a large conjugative plasmid. Transposition of the transposon encoding Ac^R (ampicillin resistance), Cm^R Ctm^R and Sm^R designated TnX8, was done by Wong (1999) to a recipient replicon, plasmid pUB307. The Ac^R Cm^R, Ctm^R and Sm^R genes were subcloned into plasmid pKan by digesting the recombinant plasmid, des ignated pCL8 (pUB307::TnX8), with San. This subcloning strategy generated different recombinant pKans that harbour different antibiotic resistance genes. Plasmid pCLS55 is a recombinant pKan that harbours a Sa/l insert bearing Sm^R genes from the multiple antibiotic resistance transposon of S. typhi S8. The main objective of this study was to subclone and characterizes, by DNA sequencing, the Sm11 genes de rived from the transposon of S. typhi S8, in pCLS55. Successive subclonings into pUC19 and M13mp18 vectors, and construction of deletions localized the Sm11 genes on a 2.5 kb EcoRI DNA fragment. Southern hybridization, performed before DNA sequencing, confirmed that the 2.5 kb EcoRI Sm^R inserts carried by MI3pSMRI3 and M13pSMS16 originated from the plasmid in S. typhi S8. A total of 2,128 nucleotides was determined based on complete DNA sequencing of the 2.5 kb EcoRl Sm11 inse rt in recombinant M13mpl8. The nucleotide sequences were analyzed and the gene structure (containing str genes, strA and strB) within the 2.5 kb EcoRI Sm^R insert from pCLS55 was determined. These sequences were identical to the strA and strB genes encoding Sm resistance in plasmid RSF1010 (Scholz et al., 1989) and in Tn5393 of Erwinia amylovora (Chiou and Jones, I 993). The nucleotide sequences obtained in this study indicated that there are three open reading frames (ORF) in the 2.5 kb EcoRI Sm^R fragment. The first ORF of 267 amino acids was designated ORF A'. The deduced amino acid sequence corresponded to a molecular mass of 29,595 Daltons and has high similarity with the amino acid sequence of Sm phosphotransferase protein A, which is also encoded by RSF1010 and Tn5393 . This ORF translation starts at UUG (TTG in the DNA sequence) and this suggests that this gene did not originally come from members of the family Enterobacteriaceae. The second ORF, designated ORF B', encodes a polypeptide of 278 amino acids; which corresponded 10 a molecular mass of 30,824 Dallons and has high similarity with the amino acid sequence of Sm phosphotransferase protein B. The Sm^R genes were also observed to contain part of the transposase gene belonging to 1S26 and its variants. This was observed in the third ORF, ORF C', in the nucleotide sequences obtained. Partial DNA sequencing of the 5.5 kb Sa/l insert in pCLS55 showed that the two ends of this in se rt each contained part of the transposase gene of IS26 and its variants. The Sm^R genes in pCLS55 appeared to be borne on a composite element, flanked by 1S26 al its ends. The presence of the composite 1S26 element within TnXB supports the hypothesis that multiple antibiotic resistance transposons evolved by insertion of antibiotic resistance determinants, which are themselves transposable.

Initial

snms

Additional Information

Dissertation (M.A) -- Institute for Advanced Studies, Universiti Malaya, 2000.

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