Direct Detection of Extracellular Deoxyribonucleases in Different Strains of Streptomyces rimosus

Dušica Vujaklija*, Jelena Žafran, Andreja Mikoč and Vera Gamulin

Department of Molecular Genetics, »Ruđer Bošković« Institute, Bijenička c. 54, 10000 Zagreb, Croatia

Article history:
Received July 25, 1996 
Accepted August 26, 1996

Key words:
Streptomyces rimosus, endonuncleases, extracellular deoxyribonucleases

Summary:
Streptomyces rimosus, an oxytetracycline producer, excretes from the cell small deoxyribonuclease (DNase) with M_r=19 000. The presence of the extracellular enzyme was confirmed by three rapid methods specially developed for this purpose. These tests can be employed for in vivo and in vitro detection of DNases. When bacteria were cultivated on solid medium with addition of DNA and toluidine blue, red zone appears around the colonies with extracellular DNase activity. Using this approach we found among S. rimosus strains, R6-779 with the highest activity and no activity in the strain R6-554; therefore this strain is suitable for complementational cloning of the gene. Activity of the DNase in the liquid media was detected in the microtest which contained only few µL of the filtrate mixed with 5 µg of DNA. Degradation of the DNA was monitored by agarose gel electrophoresis. Electrophoresis of it neon centra ted filtrates on denaturing polyacrylamide gels containing high molecular weight DNA, allowed direct detection of the 19 000 Da enzyme. Activity of the enzyme changes in different fermentation media, but it is not influenced by addition of DNA. Highest activity was observed in the fermentation medium GR2d.