A Substance Effecting Differentiation in Streptomyces griseus

Abstract

A conidium-producing variant of Streptomyces griseus, strain 45-H, produces a substance, factor C, which is capable of inducing conidium formation in the hyphae of a conidium-non-producing mutant, strain 52-1. Factor C can be determined quantitatively on the basis of this biological effect. The biologically active substance can be purified by ion-exchange chromatography on cellulose phosphate combined with affinity chromatography on DNA-agarose. The purified substance is concentrated at least 1700 times. The molecular weight of factor C, estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, is about 34500. On determining the amino acid composition of factor C 60 % of the amino acids were found to be hydrophobic.Cytodifferentiation has been studied in Streptomyces griseus strains [l, 21. From the fermentation liquid of the conidium-producing S. griseus 45-H strain a substance (designated factor C) has been isolated which, if added to the culture medium of the otherwise non-differentiating S. griseus 52-1 strain, has been found to elicit various cytomorphological signs of conidium formation [3,4].In our previous papers [3-61 we have already described the most apparent features of the effect of this endogenous substance which induces differentiation.In the present paper the process of the purification and the chemical properties of factor C are described. MATERIALS AND METHODS Production of Factor CStrain 45-H, a mutant of Streptomyces griseus, was cultivated in 10 1 of filtered soybean medium in a MicroFerm fermentor, model MF-114 (New Brunswick Scientific Co., Inc., U.S.A.), operating at an airflow of 6 l/min at 27 "C. Cultivation was continued for 66-72 h when conidia, or reproductive hyphae, appeared in a large number. Detection of Factor CFactor C can be detected on the basis of its effect inducing cytomorphological changes in the submerged culture of S. griseus 52-1. Its effect was evaluated with a phase-contrast microscope in 72-h submerged cultures of the test strain after glutaraldehyde fixation. The test was regarded positive if formation of reproductive branches could be detected in the test strain which otherwise did not produce conidia. Estimation of the Quantity of Factor CSerial dilution was performed and the amount of factor C found in 1 ml of the highest dilution still definitely positive was taken as unit. Removal of the MyceliaThe pH of the culture filtrate of S. griseus 45-H was adjusted to 6.5 with 2 M HzS04 and after the addition of 0.001 M EDTA the mycelia were centrifuged by a continuous-action rotor at 21 000 x g in an MSE high-speed 18 centrifuge at 4°C. After centrifugation the pH of the culture filtrate was adjusted to 4.5 with 2 M HzS04. Ion-Exchange ChromatographyTwo steps of ion-exchange chromatography were used consecutively.Batchwise Method. 60 g precyclized, equilibrated and fines-free Whatman P-1 cellulose phosphate were added to the 10-1 culture filtrate. Then the mixture was kept in a cold room for 2 h while being stirred continu-

Structure-activity relationships of virginiae butanolide C, an inducer of virginiamycin production in Streptomyces virginiae.

“…At or above the effective concentration, the diameter of clear zone increased, by which we determined the minimumeffective concentration of each analogue (Table 1). By comparing the minimumeffective concentrations for 8, 9 and 10, it was clear that cis VB-Ctype analogues (10) were 10-fold more active than the corresponding trans isomers (9), and 9 were 10-fold more active than A-factor type analogues (8). Therefore, 2£-cis configuration is important for inducing activity.…”

“…In Fig. 3 is shown an example of the bioassay in which several concentrations of A-factor type analogues (8) were tested. Whenthe concentration of analogues tested was insufficient for virginiamycinproduction, no clear zone was observed (8 mmcorresponds to the diameter of the cups).…”

ssgA Is Essential for Sporulation of Streptomyces coelicolor A3(2) and Affects Hyphal Development by Stimulating Septum Formation

“…One of the best-characterized proteins involved is factor C, which was identified as a 34-kDa protein that restores submerged sporulation to an S. griseus mutant. Although antibodies against factor C cross-react with proteins in a wide variety of prokaryotic and eukaryotic organisms, no homologue has yet been identified in any of the databases (5,6). More recently, a mutant of S. griseus (designated SY1) that produced submerged spores in rich as well as in minimal liquid media was identified.…”

Streptomyces griseus 45H, a producer of the extracellular autoregulator protein factor C, is a member of the species Streptomyces albidoflavus

“…S. griseus 45H has been described as a strain that sporulates well both on solid medium and in liquid culture, the latter being a rather rare property of Streptomyces strains. In addition, the strain secreted a substance, named factor C, into the culture medium that also induced cytodifferentiation of S. griseus 52-1 and stimulated sporulation in liquid culture.The activity of factor C was shown to be due to a protein (Biró et al, 1980). The cloning and sequencing of the facC gene (Birkó et al, 1999), which encodes this extracellular signalling protein, revealed a gene with no close relatives in the database at that time.…”

“…The activity of factor C was shown to be due to a protein (Biró et al, 1980). The cloning and sequencing of the facC gene (Birkó et al, 1999), which encodes this extracellular signalling protein, revealed a gene with no close relatives in the database at that time.…”