A method for transformation of Geobacillus debilis by conjugation was developed using a recombinant plasmid, pNW33N-pxyl-bs2-mob, derived from pNW33N. The plasmid includes the mob region of RP4 for mobilization, is mobilized from E. coli S17-1 to G. debilis, and can stably propagate in G. debilis trans-conjugants grown at 50 oC and 55 oC, in the presence of thiamphenicol. Successful conjugation was depended on the cell density and viability of G. debilis when harvested for conjugation, as well as the metabolic activity of E. coli S17-1 used for conjugation. Substantial reduction in size of the plasmid DNA was observed when G. debilis transconjugants were cultured at 60 oC in the presence of thiamphenicol, and uniform rearrangement of the plasmid DNA was observed after culturing G. debilis transconjugants in the presence of spectinomycin, even at 50 oC.

A method for transformation of Geobacillus debilis by conjugation was developed using a recombinant plasmid, pNW33N-pxyl-bs2-mob, derived from pNW33N. The plasmid includes the mob region of RP4 for mobilization, is mobilized from E. coli S17-1 to G. debilis, and can stably propagate in G. debilis trans-conjugants grown at 50 oC and 55 oC, in the presence of thiamphenicol. Successful conjugation was depended on the cell density and viability of G. debilis when harvested for conjugation, as well as the metabolic activity of E. coli S17-1 used for conjugation. Substantial reduction in size of the plasmid DNA was observed when G. debilis transconjugants were cultured at 60 oC in the presence of thiamphenicol, and uniform rearrangement of the plasmid DNA was observed after culturing G. debilis transconjugants in the presence of spectinomycin, even at 50 oC.

Growth of Escherichia coli A-l under aerobic conditions in an enriched medium with a total amount of 0.2 per cent glucose was biphasic and asparaginase II activity was detected after depletion of ammonia from the growth medium in the second phase of growth. Glucose was exhausted two hours before ammonia and three hours before asparaginase II activity was detected. The concentration of 3',5'-cyclic adenosine monophosphate was found to fluctuate when the dissolved oxygen in the medium reached a low level, when glucose and ammonia were exhausted, and when the cells entered the second stationary phase of growth. Culture tube studies of the growth of E_j_ coli A-l in three per cent nutrient broth with varied concentrations of ammonium chloride and potassium nitrate gave lower specific activity of asparaginase II when this was compared to that seen in three per cent nutrient broth alone. The addition of glucose to the same medium before asparaginase II activity was detected resulted in the production of acid by E. coli A-l with cessation of growth; however, addition after L-asparaginase synthesis had started did not affect the specific activity of the enzyme. The addition of ammonium chloride suppressed L-asparaginase synthesis, but addition after enzyme synthesis started had no affect. These findings suggest that asparaginase II is produced by E. coli A-l in response to low concentrations of ammonia and that exogenously supplied nitrogen compounds may play a major role in the regulation of this enzyme. It is suggested that E. coli A-l produced L-asparaginase in order to obtain ammonia for the synthesis of glutamine from glutamate. The synthesis of glutamine from glutamate is the first step of a highly branched pathway which ultimately leads to the synthesis of many of the important macromolecules of the cell.