Crp of Streptomyces coelicolor is the third transcription factor of the large CRP-FNR superfamily able to bind cAMP Adeline Derouaux a , Dominique Dehareng a , Elke Lecocq b , Serkan Halici c , Harald Nothaft c , Fabrizio Giannotta a , Georgios Moutzourelis a , Jean Dusart a , Bart Devreese b , Fritz Titgemeyer c , Jozef Van Beeumen b , Se ´bastien Rigali a, * a Centre d ÕInge ´nierie des Prote ´ines, Universite ´ de Lie `ge, Institut de Chimie B6a, B-4000, Lie `ge, Belgium b Laboratory for Protein Biochemistry and Protein Engineering, Ghent University, K.L. Ledeganckstraat 35, 9000 Ghent, Belgium c Lehrstuhl fu ¨ r Mikrobiologie, Friedrich-Alexander-Universita ¨ t Erlangen-Nu ¨ rnberg, Staudtstrasse 5, 91058 Erlangen, Germany Received 4 October 2004 Available online 6 November 2004 Abstract The chromosomal inactivation of the unique transcription factor of Streptomyces coelicolor that displays a cyclic-nucleotide- binding domain, Crp Sco , led to a germination-defective phenotype similar to the mutant of the adenylate cyclase gene (cya) unable to produce cAMP. By means of cAMP affinity chromatography we demonstrate the specific cAMP-binding ability of Crp Sco , which definitely demonstrate that a Cya/cAMP/Crp system is used to trigger germination in S. coelicolor. However, electromobility shift assays with the purified Crp Sco –cAMP complex and the CRP-like cis-acting element of its own promoter failed. Moreover, we were unable to complement an Escherichia coli crp mutant in trans with Crp Sco . The fact that Vfr from Pseudomonas aeruginosa and GlxR from Corynebacterium glutamicum could complement such an E. coli mutant suggests that the way Crp Sco interacts with DNA should mechanistically differ from its most similar members. This hypothesis was further supported by homology modelling of Crp Sco that confirmed an unusual organisation of the DNA-binding domain compared to the situation observed in Crp Eco . Ó 2004 Elsevier Inc. All rights reserved. Keywords: cAMP; Germination; CRP-FNR family; Cyclic-nucleotide-binding domain; Streptomyces Streptomycetes are Gram-positive soil bacteria with a complex life cycle that includes germination, vegetative mycelial growth, aerial mycelium formation, and spore morphogenesis [1]. In order to restore vegetative growth once conditions become favourable, bacterial spores must be able to sense environmental changes that trigger the germination process. In the initial stages of germina- tion, undefined signals trigger influx of water into spores, resulting in an increase in size and decreased phase brightness followed by germ-tube emergence [2]. Recently, we focused on the study of the unique CRP-FNR member of Streptomyces coelicolor, encoded by orf SCO3571 or the crp gene (noted Crp Sco protein) [3]. Its chromosomal inactivation led to a germination- defective mutant of which the phenotype is similar to the mutant of the adenylate cyclase gene (cya) unable to produce cAMP [3,4]. These similar phenotypes for both mutants and the observation of a peak of cAMP accumulation during germination [4] suggest that cAMP should be regarded as a molecular signal that initiates germination events. However, before concluding that a Cya/cAMP/Crp Sco system is used to trigger germination in S. coelicolor, clear evidence for an interaction of Crp Sco with cAMP still needs to be demonstrated. Furthermore, the cAMP-binding ability of Crp Sco be- came somehow more doubtful as a recent phylogenetic 0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.10.143 * Corresponding author. Fax: +32 4 366 33 64. E-mail address: srigali@ulg.ac.be (S. Rigali). www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 325 (2004) 983–990 BBRC