Genetic studies of Thermus

Abstract

The work presented in this thesis examines some of the requirements for the establishment of a gene transfer system in Thermus and the transcription, organisation and control of the leucine operon in Thermus HB8. The major findings are: 1) No correlation was found between the plasmid content of Thermus strains and the antibiotic and heavy metal ion resistances expressed by the strains. 2) Two plasmids isolated from Thermus were unable to replicate and be maintained in E.coli. Several potential shuttle plasmids were constructed and used in attempts to transform Thermus cells. The potential shuttle plasmids were unable to replicate in the absence of the E.coli vector replicon in E.coli. In addition, no polypeptides produced by the potential shuttle plasmids were observed in E.coli. 3) A bacteriophage capable of infecting one strain of Thermus was characterised. The phage has a similar appearance to that of lambda and contains double-stranded DNA. Transfection of spheroplasts or cells of Thermus with the phage DNA did not result in plaque production. 4) Linear or supercoiled Thermus DNA can be taken up by naturally competent Thermus cells. Thermus DNA passaged through E.coli is unable to transform Thermus. 5) The leuB genes of Thermus HB8, T351 and T.sp. share DNA homology throughout a 1.62Kb fragment and are identical for 180 bases. The leuA and leuB genes of Thermus HB8 and E.coli share no significant DNA sequence homology. 6) The sequence of Thermus DNA capable of functioning as a promoter in E.coli was determined. This promoter is responsible for the expression of the leuB, leuC and leuD genes of Thermus HB8 in E.coli. 7) The order of the structural genes in the leucine operon of Thermus HB8 is either D, C-B or C, D-B where the latter two genes overlap. This arrangement differs from that in E.coli and S.typhimurium.