Downloaded from www.microbiologyresearch.org by IP: 54.242.161.225 On: Sat, 14 May 2016 15:19:22 Streptomyces coelicolor A3(2) plasmid SCP2*: deductions from the complete sequence Iris Haug, 1 Anke Weissenborn, 2 Dirk Brolle, 3 Stephen Bentley, 4 Tobias Kieser 5 and Josef Altenbuchner 1 Correspondence Josef Altenbuchner Josef.Altenbuchner@ po.uni-stuttgart.de 1 Institut fu ¨ r Industrielle Genetik, Universita ¨ t Stuttgart, Allmandring 31, 70569 Stuttgart, Germany 2 Mikrobiologie/Biotechnologie, Eberhard-Karls-Unversita ¨t Tu ¨ bingen, 72076 Tu ¨ bingen, Germany 3 Team Leader Marketing Urology, Pfizer GmbH, PO Box 4949, 76032 Karlsruhe, Germany 4 The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK 5 John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, UK Received 21 May 2002 Revised 13 October 2002 Accepted 17 October 2002 Plasmid SCP2* is a 31 kb, circular, low-copy-number plasmid originally identified in Streptomyces coelicolor A3(2) as a fertility factor. The plasmid was completely sequenced. The analysis of the 31 317 bp sequence revealed 34 ORFs encoding putative proteins from 31 to 710 aa long, most of them lacking similarity to known proteins. Three functional regions had been identified previously: the replication region, the transfer and spreading region, and the stability region. Three genes were identified in the stability region which contribute to the stability of SCP2 as shown by plasmid stability testing. The first gene, mrpA, encodes a new member of the l integrase family of site- specific recombinases. The two genes downstream of mrpA were called parA and parB. The gene product, ParA, shows similarity to a family of ATPases involved in plasmid partition. An increase of plasmid stability could be seen only when both genes were present. By deletion analysis, the replication region could be narrowed down to a 1?6 kb region, consisting of a 650 bp non-coding region and two genes, repI and repII, encoding proteins of 161 and 131 aa. Only RepI exhibits similarities to DNA binding elements and contains a putative helix–turn–helix motif. The traA gene that is essential for DNA transfer and pock formation was identified previously. Upstream of traA, 10 ORFs were found in the same orientation as traA which might be involved in conjugation and DNA spreading, together with one gene in the opposite orientation with similarities to transcrip- tional regulators of DNA transfer. Two transposable elements were found on SCP2*. IS1648 belongs to the IS3 family of insertion sequences. The second element, Tn5417, shows the highest similarity to the Tn4811 element located in the terminal inverted repeats of the Streptomyces lividans chromosome. INTRODUCTION Streptomycetes are Gram-positive soil bacteria that are well known for the production of a huge variety of secondary bioactive metabolites, such as antibiotics, and for their complex life-cycle. Many plasmids have been found in Streptomyces spp. Most of them are self-transmissible fertility factors. Both linear and circular, low- and high- copy-number plasmids occur and some plasmids are integrated into the chromosomes. The first circular plas- mid identified in Streptomyces coelicolor A3(2) was SCP2 (Schrempf et al., 1975), a conjugative low-copy-number plasmid of 31 kb. It is present in one to four copies per chromosome (Bibb et al., 1977) and is stably inherited, being retained by 99?5 % of spores after a single spore-to-spore cycle (Bibb et al., 1980). SCP2*, a spontaneous derivative of SCP2, was first analysed by Bibb & Hopwood (1981). It has a 1000-fold increased chromosome-mobilizing ability and forms more clearly visible pocks compared to SCP2. Pocks are an indicator for the presence of plasmids with DNA transfer ability and can be seen as a region of delayed differentiation when a plasmid-bearing spore germinates in a lawn of a strain lacking the same plasmid. Spontaneous conversion from SCP2 to SCP2* and vice versa has been observed (Bibb et al., 1977). The two forms of the plasmid cannot be distinguished by restriction digestion and the molecular basis of the different phenotypes is unknown. The GenBank accession numbers for the DNA sequences reported in this paper are AL645771 and NC_003904. 0002-5751 G 2003 SGM Printed in Great Britain 505 Microbiology (2003), 149, 505–513 DOI 10.1099/mic.0.25751-0