Breadon, Robert Leslie (1988) Development of a system of genetic analyses for Rhodomicrobium vannielii. PhD thesis, University of Warwick.

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Abstract

Preliminary experiments, to find phage or bacteriocin activity which might be plasmid-encoded amongst available strains of the Rhodopirillaceae were unsuccessful, and no phage were isolated from the natural environment. The technique for phototrophic plate growth of Rm. vannielii was found to be unsuitable for basic genetic experiments and a method using microaerophilic growth was developed. The latter was found to yield better results in both mutagenesis and conjugation experiments despite an increased incubation time relative to phototrophic growth (i.e. 14 instead of 3-5 days to obtain colonies). The growth rate under microaerophilic conditions was increased by adding more yeast extract to the media and workable colonies were obtained after 10 days.

NTG and UV-mutagenesis were successfully used to isolate a variety of mutant types including pigment mutants, motility mutants and temperature sensitive motility mutants.

The pigment mutants were classified into four groups according to colour and spectroscopic analysis. A number of them were selected for further analysis. PAGE showed that in general these mutants were very similar to pigment mutants of other purple non-sulphur bacteria.

Transposon mutagenesis was successfully carried out using pSUP2021 and pJB4JI. Tn5-insertion pigment mutants, were isolated and Southern hybridization demonstrated that the transposon was inserted at single non-specific sites on the chromosome. This was verified by analysis of revertants obtained using a * light-lethal’ selection technique on a photosynthetically incompetent mutant. An EcoRI fragment containing Tn5 plus flanking sequences was subsequently cloned from this mutant into pBR322.

Conjugation experiments showed that broad-host-range plasmids from incompatability groups P, Q and W could be transferred to and maintained in Rm.vannielii.

Item Type:	Thesis or Dissertation (PhD)
Subjects:	Q Science > QR Microbiology
Library of Congress Subject Headings (LCSH):	Bacteria -- Genetics, Bacteria -- Motility, Bacteriophages, Transposons, Mutagenesis
Official Date:	1988
Dates:	Date Event 1988 UNSPECIFIED
Institution:	University of Warwick
Theses Department:	School of Life Sciences
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Salmond, George ; Dow, Crawford S.
Sponsors:	Northern Ireland. Department of Education
Extent:	xx, 284 leaves : charts
Language:	eng

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