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Nuclease activities and cell death processes associated with the development of surface cultures of Streptomyces antibioticus ETH 7451

Area de Microbiología, Departamento de Biologia Funcional, Facultad de Medicina, 33006 Oviedo, Spain¹

Author for correspondence: Jesús Sánchez. Tel: +34 985103555. Fax: +34 985103148. e-mail: JSM{at}sauron.quimica.uniovi.es

The presence and significance of developmentally regulated nucleases in Streptomyces antibioticus ETH 7451 has been studied in relation to the lytic processes occurring during differentiation. The cell-death processes have been followed in surface cultures by a propidium iodide viability assay. This has allowed the visualization of dead (membrane-damaged, red fluorescent) and live (membrane-intact, green fluorescent) mycelium during development, and has facilitated the analysis of the role of nucleases in these processes. A parallel activity-gel analysis showed the appearance of 20–22 kDa, 34 kDa and 44 kDa nucleases, the latter appearing only when aerial mycelium is formed. The appearance of these nucleases shows a remarkable correlation with the death process of the mycelium during differentiation and with chromosomal DNA degradation. The 20–22 kDa enzymes are possibly related to the lytic phenomena taking place in the vegetative substrate mycelium before the emergence of the reproductive aerial mycelium, whereas the function of the 44 kDa nuclease seems to be related to the sporulation step. The 20–22 kDa nucleases require Ca²⁺ for activity and are inhibited by Zn²⁺. The nucleases are loosely bound to the cell wall from where they can be liberated by simple washing. Conceivably, these enzymes work together and co-ordinate to achieve an efficient hydrolysis of DNA from dying cells. The results show that the biochemical reactions related with the lytic DNA degradation during the programmed cell death are notably conserved in Streptomyces. Some of the features of the process and the biochemical characteristics of the enzymes involved are analogous to those taking place during the DNA fragmentation processes in eukaryotic apoptotic cells.

Keywords: programmed cell death, substrate mycelium, chromosomal DNA degradation, viability staining, differentiation

Abbreviations: PI, propidium iodide

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