Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Mon, 05 Nov 2018 05:52:27 Journal of General Microbiology (1990), 136, 713-716. Printed in Great Britain 713 A breakdown in macromolecular synthesis preceding differentiation in Streptomyces coelicolor A3(2) C. GRANOZZI,~ R. BILLETTA,lt R. PASS ANTI NO,^ M. SOLLAZZO~ and A. M. PUGLIA~" Dipartimento di Biologia Cellulare e dello Sviluppo, Sezione di Genetica, Universita di Palermo, Via Archiraj 22, Istituto di Biologia dello Sviluppo, CNR, Via Archira$ 20, 90123 Palermo, Italy 90123 Palermo, Italy (Received 15 August 1989; revised 4 December 1989; accepted 20 December 1989) ~ ~~ A transitory cessation of growth was recorded in Sfrepfomyces coelicolor A3(2) at the end of vegetative mycelium formation on solid medium. In the same phase a striking reduction in protein and nucleic acid synthesis was detected. Growth and macromolecularsynthesis resumed, nearly reaching the original values, when morphological differentiation occurred. It is concluded that a physiological stress occurs within the bacterial population just before the onset of the morphological differentiation. Introduction The streptomycetes are morphologically among the most highly differentiated prokaryotes. Their colonies on agar consist of a substrate mycelium and an aerial mycelium which produces spores, the latter representing the propagative phase. The growth cycle at 30°C, from spore to sporulating colony takes about 3-4 d (Hopwood et al., 1973; Chater & Hopwood, 1983). The formation of aerial mycelium occurs about 24 h after seeding spores onto a synthetic medium and coincides with the phase in which secondary metabolites such as pigments and antibiotics are produced. The possibility of common regulatory elements for both morphological and physiological differentiation has been widely investi- gated in the last few years. The isolation and characteri- zation of genes involved in differentiation and the study of pleiotropic mutants defective in both sporulation and antibiotic production have strongly supported inter- dependence of the regulatory pathways of these two aspects of differentiation in Streptomyces coelicolor (Chater, 1989). It is often asserted that secondary metabolism is initiated by nutrient limitation and a reduction in growth rate (Martin & Demain, 1980). The aim of the present work was to measure the rate of nucleic acid and protein synthesis in the critical period in which aerial mycetium first appears. This was achieved by following the incorporation of radiolabelled precur- f Present address : UCSD Medical Center, University of California, 225 Dickinson Street, San Diego, CA 92103-1990. USA. sors into macromolecules by S. coelicolor A3(2) during its growth cycle on solid media. Methods Strains. The mutant strains used throughout the work were derived from the wild-type Streptomyces coelicolor A3(2) (Hopwood et al., 1973; Chater & Hopwood, 1983). They were : strain 44 (hisAZ SCP 1 + SCP2+), strain 1 I (uraAI metAZ strAZ SCPl-SCP2+) and strain 316 (hisD3pheAZ strAI SCPl-SCP2+). Growth conditions. Minimal medium (MM) had the following composition (I-'): NaN03, 1 g; MgS0,.7H20, 0.5 g; KCI, 0.5 g; KH2P04,1 g; trace element solution (FeSO,. 7H20, 1 %; ZnCI2, 17: biotin, 0.1 %), 1 ml; agar, 15 g; and glucose (autoclaved separately as 50%, w/v, solution), 10 g. When required histidine (50 pg mi-'), methionine (37 pg ml-l), phenylalanine (37 pg ml-l) and uracil (7.5 pg ml-l) were added. Complete medium (CM) was as described by Hopwood et al. (1985). General procedures were as described by Hopwood & Sermonti (1962) and Hopwood e? al. (1985). To facilitate harvesting the mycelium and its transfer from one medium to another, the strains were grown on autoclaved cellophane disks (8 cm diam.) lying on solid medium (Sermonti et al., 1971). Growth curves. Spores of the strains were harvested from slant cultures, filtered through glass funnels containing cotton wool, washed by centrifugation and resuspended at a concentration up to lo* ml-l ; lo7 spores were placed on each cellophane disk lying on MM supplied with the required amino acids and incubated at 30 "C. After various times the mycelium was scraped from the cellophane and weighed. Measurement of the rate of DNA, RNA and protein synthesis. Spore suspensions of the strains were seeded on cellophane disks lying on MM and incubated at 30 "C. After various times the cellophane disks with the growing mycelium were transferred onto MM containing: [6- 3H]thymidine (30 pci ml-l; 20-30 Ci mmol-l) for measurement of DNA synthesis; [6-3H]uracil (30 pCi m1-I; 15-30 Ci mmol-l) for 0001-5769 O 1990 SGM