The production and isolation of mutants of Acinetobacter calcoaceticus NCIB 8250

Ahlquist, Eric Fredolin (1974) The production and isolation of mutants of Acinetobacter calcoaceticus NCIB 8250. MSc(R) thesis, University of Glasgow.

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The work described in this thesis was undertaken in an attempt to establish systems for the induction, selection and isolation of mutants for the bacterium Acinetobacter calcoaceticus NCIB 8250. This study formed part of a general biochemical and genetic investigation of the control of aromatic carbon compound catabolism and involved a systematic analysis of mutagenesis, and the development of methods for the enrichment and isolation of both nonspecific mutants (e.g. amino acid auxotrophs) and specific mutants affecting enzymes catalysing particular reactions in the catabolism of aromatic carbon compounds. A study was made of the mutagenic and lethal effects of N-methyl-N'-nitro-N-nitrosoguanidine, ultraviolet light irradiation, ethyl methanesulphonate and near ultraviolet light irradiation with 8-methoxypsoralen. In each case the percentage of auxotrophs obtained following exposure to these treatments was used as the measure of their mutagenic efficiencies. All four agents were found to be mutagenic, however, ethyl methanesulphonate and N-methyl-N'-nitro-N-nitrosoguanidine, although more effective than irradiation, did not cause such a high frequency of mutation as has been observed with other bacteria, A range of antibiotics was screened for their suitability for use in the selection of mutants. Vancomycin and penicillin V, which both inhibit bacterial wall synthesis, proved most suitable particularly when used together. Vancomycin causes a much greater inactivation of growing bacteria than non-growing bacteria, and penicillin V was found to protect non-growing bacteria from even the small effects of vancomycin. The various parameters of a selection system, such as substrate and antibiotic concentration, length of antibiotic treatment and ratio of live to inactivated bacteria during antibiotic treatment, were tested. Using reconstruction experiments the vancomycin and vancomycin/penicillin V selection systems were further developed for the isolation of auxotrophs and for mutants blocked in the catabolism of aromatic compounds. Cysteine, tyrosine, isoleucine and isoleucine/valine requiring auxotrophs and mutants blocked at "benzoate oxidase", between catechol 1,2-oxygenase and beta-ketoadipate enol-lactone hydrolase and at or below beta-ketoadipate succinyl-CoA transferase were isolated and partially characterised by growth experiments. Streptomycin-resistant mutants were obtained by a positive selection method. Positive selection of blocked aromatic mutants with p-fluoro-analogues of substrates was, however, not successful. A continuous-culture method was used for isolating a mutant strain mesoconstitutive for L-mandelate dehydrogenase but failed to select for magnoconstitutive mutants. Multiple mutants were constructed from existing single mutants by genetic transformation. One class of these grew on D-mandelate but not on L-mandelate: this gave proof that the selection of mutant strains of A. calcoaceticus able to grow on D-mandelate is due to the presence of a D-mandelate dehydrogenase and not to the presence of a mandelate racemase. The results of this work provide general methods suitable for the isolation of various types of mutant for use in the genetic analysis of this organism.

Item Type: Thesis (MSc(R))
Qualification Level: Masters
Additional Information: Adviser: C A Fewson
Keywords: Microbiology
Date of Award: 1974
Depositing User: Enlighten Team
Unique ID: glathesis:1974-72099
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 17 May 2019 13:01
Last Modified: 17 May 2019 13:01

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