## Doctoral Dissertations

Fall 1991

Dissertation

Microbiology

#### Degree Name

Doctor of Philosophy

Robert M Zsigray

#### Abstract

The relationship between growth rate and competence for genetic transformation in B. subtilis 168 was investigated. B. subtilis grown in chemostat cultures expressed competence at a relatively rapid growth rate of 2.5 h (mass doubling time, t$\sb{\rm d}$). Since the literature suggested competence normally occurred at slower growth rates in batch cultures, B. subtilis was grown in a biomass recycling fermentor and extremely slow growth rates were examined for the expression of competence under carbon/energy limitation. No additional peaks on the number of competent cells were detected. However, major metabolic manifestations were observed at the growth rate where competence is maximally expressed. These were a change in steady state biomass concentrations precisely at the growth rate of maximal competence, and a change in the amount of acetate excreted as a metabolite of glucose underoxidation.

Data from the above studies were applied to studies in which a genetic transformation system was sought for C. beijerinckii NRRL B-592. No natural transformation was observed; consequently, a protoplast formation and regeneration system was successfully developed for this organism. This system, however, would not allow gene transfer by protoplast transformation or fusion.

Plasmid pHR106 DNA was introduced to intact cells of C. beijerinckii by electroporation and an analysis by Southern hybridization indicated that the plasmid had integrated into the chromosome. Recombinant derivatives of pHR106 harboring cloned inserts were tested for their ability to transfer and recombine into the chromosome after transfer to C. beijerinckii. These plasmids were transferred and expressed in C. beijerinckii cells, but may no longer integrate. The use of pHR106 and insert-containing derivatives to transform wild-type C. beijerinckii is discussed. The plasmid-based transformation system outlined in these studies offers the best way yet described to introduce cloned genes to this organism.

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