Influence of the culture medium composition on the excreted/secreted proteases from Streptomyces violaceoruber

Abstract

Streptomyces violaceoruber produces two different classes of mycelium, the substrate and the aerial mycelium. Since proteases have been associated with morphological turnover processes in other Streptomyces species, the presence of excretory/secretory proteolytic activities was investigated here in S. violaceoruber culture supernatants. Various polypeptide bands, with apparent molecular masses ranging from 40 to 180 kDa, were detected in soy trypticase broth (STB) culture media supernatants following 72 h of growth, using Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Zymograms showed the presence of five proteolytic enzymes (Spvio1–5), which migrated as bands of 167.7, 130.7, 110.7, 48.3 and 40.9 kDa, respectively. The characterization of these proteases by specific inhibitors showed that Spvio1–4 belong to the serine protease group and Spvio5 corresponds to a cysteine protease. Additionally, Spvio2 and 5 were inhibited by a mixture of EDTA and EGTA, indicating that both require divalent cations. The protease pattern obtained in STB enriched with glucose was identical to that obtained in STB. However, Spvio3 and 4 were absent when nitrogen was added to the culture medium. Cell death was fluorescently detected following 72 h of S. violaceoruber growth in STB and in STB that was enriched with glucose. On the contrary, no cell death was detected in nitrogen-enriched STB media. Additionally, the formation of the aerial mycelium was impaired in solid cultures of STB media enriched with nitrogen. These results demonstrate that the composition of the media influences the morphological turnover of the colony and the pattern of excreted/secreted proteases from S. violaceoruber, and suggest that Spvio3 and 4 are involved in the aerial mycelium formation.