Purification, Characterization, and Role of Nucleases and Serine Proteases in Streptomyces Differentiation

Two exocellular nucleases with molecular masses of 18 and 34 kDa, which are nutritionally regulated and reach their maximum activity during aerial mycelium formation and sporulation, have been detected in Streptomyces antibioticus. Their function appears to be DNA degradation in the substrate mycelium, and in agreement with this proposed role the two nucleases cooperate efficiently with a periplasmic nuclease previously described inStreptomyces antibioticus to completely hydrolyze DNA. The nucleases cut DNA nonspecifically, leaving 5′-phosphate mononucleotides as the predominant products. Both proteins require Mg2+, and the additional presence of Ca2+ notably stimulates their activities. The two nucleases are inhibited by Zn2+and aurin tricarboxylic acid. The 18-kDa nuclease fromStreptomyces is reminiscent of NUC-18, a thymocyte nuclease proposed to have a key role in glucocorticoid-stimulated apoptosis. The 18-kDa nuclease was shown, by amino-terminal protein sequencing, to be a member of the cyclophilin family and also to possess peptidylprolylcis-trans-isomerase activity. NUC-18 has also been shown to be a cyclophilin, and “native” cyclophilins are capable of DNA degradation. The S. antibioticus 18-kDa nuclease is produced by a proteolytic processing from a less active protein precursor. The protease responsible has been identified as a serine protease that is inhibited byN α -p-tosyl-l-lysine chloromethyl ketone and leupeptin. Inhibition of both of the nucleases or the protease impairs aerial mycelium development in S. antibioticus. The biochemical features of cellular DNA degradation during Streptomyces development show significant analogies with the late steps of apoptosis of eukaryotic cells. Two exocellular nucleases with molecular masses of 18 and 34 kDa, which are nutritionally regulated and reach their maximum activity during aerial mycelium formation and sporulation, have been detected in Streptomyces antibioticus. Their function appears to be DNA degradation in the substrate mycelium, and in agreement with this proposed role the two nucleases cooperate efficiently with a periplasmic nuclease previously described inStreptomyces antibioticus to completely hydrolyze DNA. The nucleases cut DNA nonspecifically, leaving 5′-phosphate mononucleotides as the predominant products. Both proteins require Mg2+, and the additional presence of Ca2+ notably stimulates their activities. The two nucleases are inhibited by Zn2+and aurin tricarboxylic acid. The 18-kDa nuclease fromStreptomyces is reminiscent of NUC-18, a thymocyte nuclease proposed to have a key role in glucocorticoid-stimulated apoptosis. The 18-kDa nuclease was shown, by amino-terminal protein sequencing, to be a member of the cyclophilin family and also to possess peptidylprolylcis-trans-isomerase activity. NUC-18 has also been shown to be a cyclophilin, and “native” cyclophilins are capable of DNA degradation. The S. antibioticus 18-kDa nuclease is produced by a proteolytic processing from a less active protein precursor. The protease responsible has been identified as a serine protease that is inhibited byN α -p-tosyl-l-lysine chloromethyl ketone and leupeptin. Inhibition of both of the nucleases or the protease impairs aerial mycelium development in S. antibioticus. The biochemical features of cellular DNA degradation during Streptomyces development show significant analogies with the late steps of apoptosis of eukaryotic cells. The actinomycetes are a large group of filamentous bacteria that are adapted for growth in soil by forming a ramifying network, called a mycelium. Within this group the predominant isolates belong to the genus Streptomyces, which produce a well developed branched mycelium on agar plates, resulting in a compact colony. In the vegetative phase, the filaments often lack cross-walls (substrate mycelium) and thus have several copies of the chromosome. When the colony ages, a characteristic aerial mycelium is formed, in response to unknown signals involving nutrient limitation, which subsequently fragment and/or sporulate by the synchronous formation of cross-walls in the multinucleate sporophores followed by separation of the individual cells directly into spores. This is similar to the growth and differentiation of fungi, and from the morphological and metabolic points of view, Streptomyces can be considered as boundary organisms (1Omura S. Queener S.W. Day L.E. The Bacteria. IX. Academic Press, Inc., New York1986: xvii-xxxiGoogle Scholar). Coincident with the morphological differentiation, the streptomycetes produce numerous compounds (secondary metabolites) within which antibiotics are of commercial relevance. As corresponds to their habitat, these bacteria are nutritionally quite versatile, and most produce extracellular hydrolytic enzymes that permit the utilization of polysaccharides, proteins, fats, and other substrates. In this way, the substrate mycelium promote the solubilization of high molecular weight biopolymers. In addition, the onset of aerial mycelium formation coincides with a noticeable lysis of the substrate hyphae (2Wildermuth H. J. Gen. Microbiol. 1970; 60: 43-50Crossref PubMed Scopus (70) Google Scholar,3Méndez C. Braña A.F. Manzanal M.B. Hardisson C. Can. J. Microbiol. 1985; 31: 446-450Crossref PubMed Scopus (66) Google Scholar). This fact, together with the absence of an increase in dry weight during the development of the aerial mycelium and the displacement of labeled protein precursors from the substrate to the aerial mycelium (3Méndez C. Braña A.F. Manzanal M.B. Hardisson C. Can. J. Microbiol. 1985; 31: 446-450Crossref PubMed Scopus (66) Google Scholar), supports the hypothesis that the aerial mycelium reuses material into the substrate mycelium. which in are to and that have a for The in the substrate mycelium and the of aerial mycelium into can be considered as a Microbiol. PubMed Scopus Google that within a to that to the of the bacteria to the Streptomyces is a of proteolytic enzymes M.B. Microbiol. PubMed Google Scholar). is that extracellular produced in actinomycetes in the of the extracellular S. of in Press, Inc., Scholar). the in the of the substrate mycelium proteins have been The protease activity produced during the late in Streptomyces has been to have a role in cellular on Can. J. Microbiol. PubMed Scopus Google Scholar). other hydrolytic the of in Streptomyces has also been with a role of Academic Press, Scholar). are of their presence H. J. PubMed Scopus Google or biochemical Microbiol. PubMed Scopus Google Scholar). group has a biochemical of several nucleases that for the of DNA and are with the in Streptomyces and Streptomyces antibioticus C. Hardisson C. J. J. Gen. Microbiol. Google C. Hardisson C. J. J. PubMed Google C. Hardisson C. J. Microbiol. Scopus Google S. C. Hardisson C. J. J. Gen. Microbiol. Scopus Google Hardisson C. J. J. PubMed Scopus Google C. S. J. J. PubMed Scopus Google S. J. J. PubMed Scopus Google S. J. PubMed Scopus Google Scholar). these enzymes can the growth of C. Hardisson C. J. J. Gen. Microbiol. Google C. Hardisson C. J. J. PubMed Google C. Hardisson C. J. Microbiol. Scopus Google Scholar), are and their is by which promote high growth and also aerial mycelium formation C. Hardisson C. J. J. Gen. Microbiol. Google C. Hardisson C. J. J. PubMed Google S. C. Hardisson C. J. J. Gen. Microbiol. Scopus Google Scholar). The nucleases are active as and DNA which the high Streptomyces DNA an substrate C. S. J. J. PubMed Scopus Google S. J. J. PubMed Scopus Google S. J. PubMed Scopus Google Scholar). The of the of these enzymes are from S. or S. The have or with and 5′-phosphate C. S. J. J. PubMed Scopus Google S. J. J. PubMed Scopus Google Scholar). The and of the S. antibioticus nuclease with in S. J. PubMed Scopus Google Scholar). of the of this in of S. antibioticus that the of the nuclease differentiation S. C. Hardisson C. J. J. Gen. Microbiol. Scopus Google Scholar). the role for in the of the aerial mycelium with to the substrate mycelium, proposed an function of the S. antibioticus and S. the of DNA from the substrate to the aerial mycelium S. C. Hardisson C. J. J. Gen. Microbiol. Scopus Google C. S. J. J. PubMed Scopus Google S. J. J. PubMed Scopus Google S. J. PubMed Scopus Google Scholar). The periplasmic of these enzymes permit to to the DNA the lysis of the mycelium. other nuclease be to the and produced by the to the S. J. PubMed Scopus Google Scholar). in this two nucleases in the exocellular of S. antibioticus both and The biochemical of the enzymes have been their to the of the periplasmic enzymes described of the nucleases has been identified as a cyclophilin, a of proteins previously proposed to have a role in PubMed Scopus Google J. PubMed Scopus Google C. J. PubMed Google Scholar). a serine protease that is to be in the of active nucleases by proteolytic processing of less active The enzymes in Streptomyces and the of aerial mycelium formation both show significant analogies with the enzymes and steps described during apoptosis in eukaryotic cells. S. was in or and (3Méndez C. Braña A.F. Manzanal M.B. Hardisson C. Can. J. Microbiol. 1985; 31: 446-450Crossref PubMed Scopus (66) Google and in the of activity was by the formation of DNA products. The of DNA in When the of was and the of other and aurin tricarboxylic tricarboxylic protein chloromethyl of for The of of and the the was by PubMed Scopus Google Scholar). The activity was by the increase in the in the of the activity is to an increase of the activity was also by of the of the on DNA aurin tricarboxylic protease protein chloromethyl ketone of of (3Méndez C. Braña A.F. Manzanal M.B. Hardisson C. Can. J. Microbiol. 1985; 31: 446-450Crossref PubMed Scopus (66) Google to the of the exocellular nuclease of S. antibioticus The nucleases directly from the by the agar the of a The resulting was for and the was a to the by for and in of The nucleases a of DNA. for these was from the proteins by the with the followed by PubMed Scopus Google Scholar). activity was by the for in by with and nuclease and as DNA degradation in the substrate mycelium of S. antibioticus was a from the cells and and The by the agar a as described the agar and was and the agar was for The was and the was with an of and with a DNA was from the phase, with with and The was in and with for a and the DNA was in and on a with The nuclease was from by and the proteins in the with The and was in and this The resulting was to a of in and with a of in The was to a of and with a of to The nuclease This was to a of and with a of to The nuclease to which was a The was in to a and with The protein and in with the the nuclease was detected by activity and also by of the of DNA When protein was S. 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