Initial investigations of some gene transfer mechanisms in Rhodopseudomonas sphaeroides

Tucker, William Tinsley (1980). Initial investigations of some gene transfer mechanisms in Rhodopseudomonas sphaeroides PhD Thesis, School of Molecular and Microbial Sciences, The University of Queensland. doi:10.14264/uql.2014.515

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Author Tucker, William Tinsley
Thesis Title Initial investigations of some gene transfer mechanisms in Rhodopseudomonas sphaeroides
Formatted title
Initial investigations of some gene transfer mechanisms in Rhodopseudomonas sphaeroides
School, Centre or Institute School of Molecular and Microbial Sciences
Institution The University of Queensland
DOI 10.14264/uql.2014.515
Publication date 1980
Thesis type PhD Thesis
Supervisor Unknown
Total pages 201
Language eng
Subjects 0604 Genetics
Formatted abstract
In order to begin initial investigations of gene transfer systems in Rhodopseudomonas sphaeroides, strains of this photosynthetic bacterium were isolated from mud and stagnant water around Brisbane and used in preliminary genetic studies. Fifteen of seventeen strains were shown to be resistant to penicillin by means of a diffusible penicillinase. A slight variation in the level of resistance to other antibiotics was also noted. The seventeen strains were examined for the presence of spontaneously released temperate bacteriophages. Three distinct morphological types were observed. The most commonly isolated type had an octahedral head about 65 nm in diameter and a long flexuous noncontractile tail, 200-250 nm in length with 2-4 tail fibres. Another temperate bacteriophage had an icosahedral head with a thick tail and a distinct base plate, while the third type observed was an isometric, tailless particle.

A bacteriophage of the first, most common type was shown to encode the production of the diffusible penicillinase in its naturally occurring lysogen, R. sphaeroides RS 601. This bacteriophage had an unusual set of properties in that it possessed a circular supercoiled encapsidated genome of about 33 x 106 daltons in mass and also existed in a dimeric form as a plasmid in the prophage state. The physiological properties of this bacteriophage have been characterized. The prophage was readily induced by mitomycin C and cells cured of the prophage could be recovered from mitomycin C treated cultures, indicative of its extrachromosomal location. The prophage was not inducible by ultra-violet irradiation. No generalized transducing ability could be associated with preparations of this bacteriophage.

To investigate their potential for conjugation and chromosome mobilisation in R. sphaeroides, attempts were made to introduce a variety of R plasmids into this bacterium. Of the plasmids tested, only RP4, R68.45, R702 and R751 of the Inc P-1 incompatibility group, and R388 and S-a of the Inc W incompatibility group were shown to be transferrable to R. sphaeroides from E. coli at frequencies of between 1 x 10-3 and 1 x 10-5 transferants per donor cell. Once stably maintained in R. sphaeroides, these plasmids were able to transfer readily within the species at frequencies of between 1 x 10-1 and 1 x 10-3 per donor cell. The plasmids expressed most markers in these bacteria, but the expression of the β-lactamase encoded by transposon Tnl and the sensitivity to the male specific bacteriophages PR11 and PRR1 was never observed. All plasmids were demonstrated to mobilise chromosomal genes at low frequencies of about 1 X 10-8 per donor cell. Chromosome transfer did not appear to be polar.

The Inc P-1 plasmid RP4 was used to introduce the genome of the coliphage Mucts62 into the Mu-insensitive cells of R. sphaeroides. A hybrid RP4::Mucts62 plasmid, pJP20, was constructed in E. coli and transferred to R. sphaeroides. The bacteriophage Mu genome failed to express its thermosensitive phenotype when present in this organism, but a low level of spontaneously released Mu virus particles could be detected in culture supernatants. In R. sphaeroides the hybrid plasmid was shown to segregate, producing spontaneously cured cells. Plasmid pJP20 could be transferred intact back to E. coli where all markers were again expressed. No Mu-induced mutation of R. sphaeroides could be detected. The hybrid plasmid pSP601, bearing both an inserted Mu prophage and four translocatable drug resistance elements was introduced into R. sphaeroides in order to isolate insertions of the transposons into the chromosome following Mu-induced curing of the plasmid. The segregation of the plasmid-borne markers was observed but evidence for widespread transpositional insertion was not obtained.

R. sphaeroides was shown to be transformable with the extracted nucleic acid of the bacteriophage Rφ6P in the presence of a helper bacteriophage. This bacteriophage, Rφ9, was spontaneously released from the wild type isolate of R. sphaeroides RS901. It was morphologically indistinguishable from bacteriophage Rφ6P with an identical genome structure and similar physiological characteristics, yet was heteroimmune with Rφ6P and did not encode penicillinase production. The recovery of transformants was linearly related to the concentration of the transforming DNA, and destroyed by DNase but not by RNase or protease. It was dependent on the multiplicity of infection of the helper virus and the mechanism of the bacteriophage mediated transformation was apparently related to the adsorption of the helper bacteriophage to the recipient cells. Viable helper particles were not required as an ultra-violet inactivated preparation of Rφ9 could still mediate transformation. Transformants had the penicillin resistant phenotype and were lysogenic for both bacteriophage Rφ6P and bacteriophage Rφ9.

The implications of these results to the future of genetic studies of R. sphaeroides have been discussed, with regard to the development of recombinant DNA technology for the study of the photosynthetic mechanism.
Keyword Genetic transformation
Additional Notes Other Title: Gene transfer mechanisms in R. Sphaeroides.

Document type: Thesis
Collection: UQ Theses (RHD) - UQ staff and students only
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Created: Wed, 26 Nov 2014, 11:01:50 EST by Mary-Anne Marrington on behalf of Scholarly Communication and Digitisation Service