Biomedical subjects
Publications and source records attributed to D Riesenberg.
High-cell-density cultivations of Escherichia coli in glucose-mineral-salt media produce more than 100 g dry cells litre-1 in special fed-batch modes with feeding of glucose and ammonia only. The specific growth rate can be adjusted to allow optimum recombinant protein generation.
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A defined medium was developed which, by means of a specific fed-batch mode, allows growth of the recombinant Escherichia coli strain TG1 (pBB210) up to a cell density of 60 g dry weight/l. Apart from glucose and aqueous ammonia fed as carbon and nitrogen sources, it was necessary to supply other nutrients or O2-enriched air. Aqueous ammonia also served for pH control. The pO2 level was kept at 20% saturation via closed-loop controls operating the two output variables of stirrer speed and glucose feeding rate. This fed-batch method prevented significant accumulation of acetate and other metabolic by-products. The recombinant E. coli expressed interferon alpha 1 more efficiently at a lower specific growth rate (muPr approximately 0.15 h-1) than at the maximum specific growth rate (mu max = 0.45 h-1). Therefore, fermentation in the batch phase at mu max was only allowed to continue up to a medium cell density. In the succeeding fed-batch phase, the specific growth rate was reduced to muPr by increasing the stirrer speed according to an empirically developed time scale.
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Explore the source record for details and available documents.
Explore the source record for details and available documents.
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Explore the source record for details and available documents.
Explore the source record for details and available documents.
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Aradioimmunoassay for (p)ppGpp was set up and applied to Streptomyces hygroscopicus. The (p)ppGpp specific antibodies obtained from rabbits immunized with ppGpp-human serum albumin could not distinguish between guanosine-5'-triphosphate-3'-diphosphate (pppGpp) and guanosine-5'-diphosphate-3'-diphosphate (ppGpp) whereas the cross reacitivity against structurally related nucleotides was negligible. Therefore, the antibodies were used for determination of (p)ppGpp, i.e. the sum of pppGpp and ppGpp. The pretreatment of biological samples for quantification of (p)ppGpp included formic acid extraction of cell pool followed by step gradient elution on DEAE-Sephadex A25 (Cl-) column for separation of (p)ppGpp from nucleotides with three or less phosphate groups. The very sensitive radioimmunoassay allowed determination of very low amounts: 1--50 pmoles (p)ppGpp per assay tube. The application of the RIA to S. hygroscopicus revealed a basal level in exponentially growing mycelium of about 2 pmoles (p)ppGpp per mg dry weight. The equilibrated pool size after induction of stringent response with serine hydroxamate was found to be about 30 pmoles (p)ppGpp per mg dry weight.
Cobalt ions synergistically supported the inducing effect of A-factor 1a on the formation of aerial mycelium and anthracyclines by the blocked S. griseus mutant 86. This suggested an involvement of effector 1a in the transport or metabolism of this trace element. In accordance with the proposed role of 1a as an autoregulatory secondary metabolite, the parent strain S. griseus JA 3933 secreted 1a into the medium after a decrease of the specific growth rate and before the onset of antibiotic production.
The mixed culture kinetics of stringent and relaxed Escherichia coli cells were investigated in a glucose-limited chemostat at different dilution rates. Independent of the dilution rate the stringent cells competed out the relaxed cells. But the number of generations necessary for displaying the relaxed cells by the stringent ones increased with increasing dilution rate. The results are discussed as a consequence of the regulatory role of guanosine-5'-diphosphate-3'-diphosphate (ppGpp) which is known to be present at different concentrations in stringent and relaxed cells under conditions of nutrient limitation. In addition, it is postulated that the coefficient of the maintenance metabolism according to PIRT (1965) is slower in stringent cells than in relaxed cells of E. coli.
We describe optimized conditions for isolation of relaxed mutants of bacteria with 4-thiouridine-containing tRNAs. The results presented here imply that besides the knowledge of the action spectra for near UV-induced growth inhibition of stringent and relaxed cells the fluence--fluence rate dependence of growth inhibition is an essential factor for optimizing the enrichment of relaxed mutants. We investigated systematically the dependence of growth inhibition of both stringent and relaxed strains of E. coli on the wavelength lambda, on the fluence F and on the fluence rate I within the ranges lambda = 301-365 nm, F = 0-48 kJ X m-2 and I = 0-60 W X m-2. The optimized conditions for selection of relaxed mutants of E. coli are lambda = 334 nm, F = 48 kJ X m-2 and I = 60 W X m-2. These optimized parameters determined for E. coli may be used in general to select relaxed mutants of other bacterial species by combining the turbidostat technique with near UV irradiation (Riesenberg et al. 1983).
The kinetics of growth, extracellular alpha-amylase formation and pool sizes of guanosine polyphosphates (p)ppGpp and adenosine phosphates (ATP and AMP) were determined during discontinuous cultivation of Bacillus subtilis 44. The results indicate a positive involvement of (p)ppGpp in the regulation of the expression of the alpha-amylase gene.