Feedback regulation and the intracellular protein profile of Streptomyces griseus in a cycloheximide fermentation.

Two-dimensional gel electrophoresis (2-D PAGE) was used to study the intracellular protein profile of Streptomyces griseus in relation to cycloheximide (CH) biosynthesis. Four proteins (CR1-CR4) were found to be significantly and specifically repressed by addition of the antibiotic (1 g/l at 72 h) to a producing fermentation. Synthesis of these proteins was specific to the idiophase, concurrent with CH production. Initial addition of CH to the production medium resulted in slightly lower synthesis rates of two of the proteins (CR1 and CR2), while significantly delaying the onset of synthesis of the other two (CR3 and CR4). Finally, neutral polymeric resin was added to the fermentation to alleviate feedback regulation of CH synthesis, giving roughly a twofold increase in the antibiotic production rate. Production of proteins CR3 and CR4 was increased approximately tenfold immediately following resin addition, but returned to the control rate of synthesis after 24 h.

High transformation frequency of nonsporulating mutants of Streptomyces griseus.

Three different bld mutants from S. griseus ATCC 10137 were isolated by nitrosoguanidine mutagenesis. They simultaneously lost the capability of antibiotic production and the formation of pigments. The three bld mutants were differently affected by different carbon sources. Two of these mutants showed a high efficiency of transformation with several plasmid vectors, in contrast to the low efficiency of transformation showed by the wild type. We showed that S. griseus ATCC 10137 and the three bld mutants possess an enzymatic activity that protects their DNAs against the digestion by SacI. Antibiotic and pigment production, and low transformability with plasmid DNA were together restored in spontaneous spo+ revertants.

The locomotor capacity of human lymphocytes and its enhancement by cell growth.

The locomotor capacity of human blood lymphocytes taken directly from blood or cultured in various ways was measured by the change from a spherical to a polarized shape which occurs within minutes of adding locomotor stimulants. A minority of lymphocytes, either direct from blood or after culture in human serum albumin or fetal calf serum for up to 72 hr, responded rapidly to such stimulants, but most lymphocytes failed to show any shape change. Colchicine induced the highest proportion of polarized cells, though still below 50%, and deuterium oxide, which stabilizes microtubule assembly, inhibited shape-change, suggesting that microtubules have a regulatory function in the expression of lymphocyte locomotion. However culture in the presence of mitogens, namely, phytohaemagglutinin (PHA), PPD, mixed lymphocyte culture, or anti-T3 (OKT3 greater than or equal to 25 pg/ml), caused a majority of lymphocytes to change shape slowly over a period of hours. In the presence of mitogens, a high proportion of cells was already polarized after 24 hr in culture without addition of further locomotor stimulants. It was concluded that locomotor capacity in lymphocytes is dependent on growth and synthesis for the following reasons. (i) There was a direct relationship between size and locomotor morphology in PHA-cultured lymphocytes. Those lymphocytes that increased in size also became polarized. (ii) Autoradiography showed that the polarized cells were more active in [3H]uridine and [3H]leucine uptake than the spherical cells. This relationship was obvious in PHA-cultured cells but was also evident even in cells direct from blood. The increase in locomotor morphology preceded detectable DNA synthesis ([3H]thymidine uptake). (iii) Increase in locomotor capacity in culture was inhibited by cycloheximide but not by mitomycin c. These findings suggest that those cells most active in RNA and protein synthesis are also the most actively motile, and that, during culture with mitogens, locomotor capacity increases as G1 phase progresses and prior to the commencement of DNA synthesis.

Cycloheximide production by Streptomyces griseus: control mechanisms of cycloheximide biosynthesis.

Cycloheximide accumulation in a fermentation medium has been shown to be the product of the balance between synthesis and degradation of this antibiotic. Glucose has been shown to prevent cycloheximide degradation. Cycloheximide has been shown to interfere with its own synthesis probably due to feedback inhibition. Approaches for increasing cycloheximide titers in the light of these findings are discussed.

Cycloheximide production by Streptomyces griseus: alleviation of end-product inhibition by dialysis-extraction fermentation.

The use of dialysis fermentation with the continuous extraction of the dialysate has resulted in a twofold increase in the cycloheximide titer due to relief from product inhibition. Continuous extraction of the dialysate has eliminated the necessity for large reservoir volumes of fermentation medium normally used in dialysis fermentation. The apparatus used in this procedure is described.