Graduate Thesis Or Dissertation

 

Studies on transformation of Bacillus licheniformis Public Deposited

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  • The original purpose of this research was to study the metabolic pathway to the synthesis of glutamyl polypeptide in B. licheniformis. Colonies of B. licheniformis 9945A, when grown on an appropriate medium appear very smooth as a result of glutamyl polypeptide accumulation. The plan of approach was to obtain mutants blocked at various steps in the route of synthesis and to attempt to identify the blocked reactions by searching for accumulation of precursors by certain classes of mutants and utilization of the accumulated products by other classes of mutants. Two transducing phages for B. licheniformis were available and the plan of approach included the use of these phages to distinguish classes of mutants by transduction. However, with the low frequencies of transduction which were characteristic of this sytem and without a specific method for selecting cells that were transduced for the ability to synthesize peptide, it soon became apparent that a different procedure would have to be used. Since frequencies of transformation are, in general, much higher than frequencies of transduction, it seemed logical to use transformation as the system of genetic transfer. Transformation had not been reported in B. licheniformis, but it was expected that techniques which gave good results with B. subtilis would work with B. licheniformis as well. It was soon learned, however, that B. licheniformis behaved very differently from E. subtilis, and because of this, the problem became one of how to get transformation to occur in B. licheniformis. Auxotrophic mutants and rough mutants of B. licheniformis 9945A were isolated and a number of experiments were run in unsuccessful attempts to transform them. The conditions for growth of the recipient cells, the media, and the mutants were manipulated in trying to obtain a transforming system. In most of the later experiments efforts were concentrated on attempts to transfer nutritional markers since selection methods for these markers presented no problem and the probability of detecting transformants occurring at low frequencies would be increased. All of these attempts to transform B. licheniformis failed. The next approach was based on the assumption that it should be possible to isolate transformable mutants. The plan was to isolate numerous mutants and to screen them for transformation. The screening method consisted of spreading 0.1 ml of a culture and 0.05 ml of DNA, prepared from wild type 9945A, on a minimal agar pIate. Control plates included one with cells alone and one with cells and DNA plus DNase. The plates were incubated and observed daily for four or five days. This approach turned out to be very fuitful within a short period of time. Three auxotrophs, 9945A-M28 (glycine⁻), -M30 (uncharacterized), and -M33 (purine⁻), produced transformants. M28 transformed at a much higher frequency than M30 or M33 and for that reason, it was studied in greater detail. The transformants of 9945A-M28 were of two colonial types on minimal agar; a few of them synthesized peptide and were smooth and a larger number did not synthesize peptide and were rough. DNA was prepared from the two types of transformants and from 9945A-M28. No transformants were produced with the DNA isolated from the mutant. Transformants were produced with the DNA isolated from the two types of transformants. The transformants that were produced with the DNA from the rough type were all of the rough colony type, whereas the transformants produced with the DNA from the smooth type included both rough and smooth colony types. Cells of 9945A-M28 when spread on minimal agar plates became competent after a period of incubation and transformed well when 9945A DNA was present. The most competent M28 cells for transformation in liquid medium were from 22-hour cultures grown in minimal medium salts plus nutrient broth and glycerol and the highest frequency of transformation occurred when the DNA-cell mixture was incubated in a serum bottle on a rotary shaker for 1 hour. Transformation was obtained with five doubly-marked auxotrophs of 9945A-M28. Glycine⁺ transformants were obtained with each of the five mutants and transformants for three of the other markers, serine, histidine, and leucine, were also obtained. However, transformants for adenine or tryptophan were not detected. From the above experiment, glycine⁺serine⁻, glycine⁺ leucine⁻, and glycine⁺histidine⁻ transformants were isolated and tested for transformability. Each of the three isolates, although glycine independent, was transformed to prototrophy. These results, which show that transformation was not specific for the glycine marker, suggested that M28 carries, in addition to the mutation responsible for glycine dependence, a second unidentified mutation which renders it amenable to transformation.
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