ARTICLES Complete genome sequence of the myxobacterium Sorangium cellulosum Susanne Schneiker1,17, Olena Perlova2,17, Olaf Kaiser1, Klaus Gerth3, Aysel Alici1, Matthias O Altmeyer2, Daniela Bartels4, Thomas Bekel4, Stefan Beyer3, Edna Bode2, Helge B Bode2, Christoph J Bolten5, Jomuna V Choudhuri4,SabrinaDoss6,YasserAElnakady2,BettinaFrank2, Lars Gaigalat1, Alexander Goesmann3, Carolin Groeger6,FrankGross2, Lars Jelsbak7, Lotte Jelsbak8,Jo¨rn Kalinowski4,CarstenKegler2, Tina Knauber6, Sebastian Konietzny4,MarenKopp2,LutzKrause4,DanielKrug2, Bukhard Linke4,TaifoMahmud9, Rosa Martinez-Arias3,AliceCMcHardy4,MichelleMerai2, Folker Meyer4, Sascha Mormann1, Jose Mun˜oz-Dorado10,JuanaPerez10, Silke Pradella3, Shwan Rachid2,Gu¨nter Raddatz11, Frank Rosenau12, Christian Ru¨ckert1, Florenz Sasse3,MarenScharfe3, Stephan C Schuster13,GarretSuen14,AnkeTreuner-Lange6, Gregory J Velicer15,Frank-Jo¨rg Vorho¨lter1,KiraJWeissman2,RoyDWelch14, Silke C Wenzel2, http://www.nature.com/naturebiotechnology David E Whitworth16, Susanne Wilhelm12,ChristophWittmann5,HelmutBlo¨cker3,AlfredPu¨hler1 &RolfMu¨ller2 The genus Sorangium synthesizes approximately half of the secondary metabolites isolated from myxobacteria, including the anti-cancer metabolite epothilone. We report the complete genome sequence of the model Sorangium strain S. cellulosum So ce56, which produces several natural products and has morphological and physiological properties typical of the genus. The circular genome, comprising 13,033,779 base pairs, is the largest bacterial genome sequenced to date. No global synteny with the genome of Myxococcus xanthus is apparent, revealing an unanticipated level of divergence between these myxobacteria. A large percentage of the genome is devoted to regulation, particularly post-translational phosphorylation, which probably supports the strain’s complex, social lifestyle. This regulatory network includes the highest number of eukaryotic protein kinase–like kinases discovered in any organism. Seventeen secondary metabolite loci are encoded in the genome, as well as many enzymes Nature Publishing Group Group Nature Publishing with potential utility in industry. 7 200 © Natural products and their derivatives provide the basis for medicines formation and morphological differentiation culminating in complex targeting a wide range of human diseases. The Gram-negative myxo- multicellular structures called fruiting bodies4. Three myxobacterial bacteria, members of the d-subgroup of proteobacteria, are an suborders are known5 and the availability of the genome sequence of important source of novel classes of secondary metabolites1.Of Myxococcus xanthus (Cystobacterineae)6 enables comparative analysis these, the genus Sorangium is particularly valuable, as 46% of with the Sorangium cellulosum (Sorangiineae) genome to illuminate metabolites isolated from myxobacteria1, including the potent anti- the basis for several important behavioral and metabolic differences. tumor compound epothilone2, derive from this group. The majority These include the ability of Sorangium strains to degrade complex of myxobacterial metabolites are polyketides, nonribosomal polypep- plant materials (Fig. 1). S. cellulosum So ce56, an obligate aerobe, was tides or hybrids of the two structures, many of which are synthesized established previously as a model Sorangium strain7 by virtue of its on gigantic molecular assembly lines composed of polyketide favorable growth characteristics and ability to differentiate reprodu- synthase (PKS) and nonribosomal polypeptide synthetase (NRPS) cibly under laboratory conditions. It synthesizes the cytotoxic multienzymes3. Sorangium strains exhibit additional characteristic chivosazoles7 and the catecholate-type siderophores myxochelins8. features, including ‘social behavior’, cell movement by gliding, biofilm Comparison of the complete genome sequence of strain S. cellulosum 1Department of Genetics, Bielefeld University, PO Box 100131, D-33501 Bielefeld, Germany. 2Department of Pharmaceutical Biotechnology, Saarland University, PO Box 151150, D-66041 Saarbru¨cken, Germany. 3Helmholtz Centre for Infection Research (formerly GBF – German Research Centre for Biotechnology), Inhoffenstrae 7, 38124 Braunschweig, Germany. 4Center for Biotechnology (CeBiTec), Bielefeld University, PO Box 100131, D-33501 Bielefeld, Germany. 5Biochemical Engineering, Saarland University, PO Box 151150, D-66041 Saarbru¨cken, Germany. 6Department of Microbiology and Molecular Biology, University of Giessen, Heinrich-Buff-Ring 26- 32, D-35392 Giessen, Germany. 7Infection Microbiology Group, BioCentrum-DTU, Technical University of Denmark, Lyngby, Denmark. 8Department of Veterinary Pathobiology, University of Copenhagen, DK-1870 Frederiksberg C, Denmark. 9College of Pharmacy, Oregon State University, Corvallis, Oregon 97331-3507 USA. 10Departamento de Microbiologia, Facultad de Ciencias, Universidad de Granada, E-18071 Granada, Spain. 11Max-Planck-Institute for Biological Cybernetics, Spemannstr. 34, D-72076 Tuebingen, Germany. 12Institute for Molecular Enzyme Technology, Heinrich-Heine-Universita¨tDu¨sseldorf, Stetternicher Forst, D-52426 Ju¨lich, Germany. 13Max-Planck-Institute for Developmental Biology, Spemannstr. 35, D-72076, Tu¨bingen, Germany. 14Department of Biology, Syracuse University, Syracuse, New York 13244, USA. 15Department of Biology, Indiana University, Bloomington, Indiana 47405 USA. 16Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK. 17These authors contributed equally to this work. Correspondence should be addressed to R.M. ([email protected]). Received 7 May; accepted 4 October; published online 28 October 2007; doi:10.1038/nbt1354 NATURE BIOTECHNOLOGY VOLUME 25 NUMBER 11 NOVEMBER 2007 1281 ARTICLES So ce56 with the genomes of M. xanthus and other d-proteobacteria online). The first and third largest families encode regulatory proteins: provides new insights into the complex regulatory networks governing serine/threonine/tyrosine protein kinases and histidine kinases (498 myxobacterial lifestyles and secondary metabolism. The sequence also members), and putative s54-dependent regulators (80 members), opens opportunities to engineer high-titer producers of clinically respectively. The second largest family (124 members) encompasses relevant natural products. hypothetical proteins and other proteins annotated as polymorphic GC-rich repetitive sequences that participate in cell surface inter- RESULTS actions in mycobacteria10. The 63 members of the fourth Architecture and features of the S. cellulosum So ce56 genome largest paralogous family belong to the functional category of The genome sequence of S. cellulosum So ce56 was obtained by whole secondary metabolism. genome shotgun sequencing, and the assembly was validated by a complete fosmid map (Supplementary Fig. 1 online). The main Phylogenomic analysis of S. cellulosum So ce56 features of the genome sequence are shown in Figure 2 and We attempted to use phylogenomic analysis to deduce functions for Table 1. At 13,033,799 base pairs, the circular chromosome is the B35% of unassigned genes in the S. cellulosum So ce56 genome. the largest bacterial genome described to date by B4 Mb. The Phylogenomic maps are a three-dimensional landscape in which S. cellulosum genome contains 9,367 predicted protein coding mountains correspond to proteins with a shared evolutionary history. sequences (CDS), numbered from the initiation codon of the dnaA As CDSs that cluster have a high likelihood of being functionally gene. The average GC content of the chromosome is 71.4%, consistent linked, such maps can be used to make functional predictions for with the strong GC bias observed in other d-proteobacteria. In the uncharacterized CDSs on the basis of coinheritance. A phylogenomic absence of the GC-skew inversion typically seen at the replication map of S. cellulosum So ce56 was constructed by aligning the predicted origin of bacterial chromosomes (Fig. 2), it was not possible to discern S. cellulosum proteome against each of the predicted proteomes from the location of oriC. Simultaneous analysis of the genome for GC 446 sequenced bacterial species. To permit meaningful comparisons, content, dinucleotide frequency and codon usage using the PAI-IDA we retained predicted proteins in the map only if their phylogenetic Program 1.1 (ref. 9) revealed a relatively uniform pattern over the first profiles matched those from at least five other bacteria. The present http://www.nature.com/naturebiotechnology two-thirds of the chromosome (to 8.5 Mb), but nine anomalous iteration contains 5,862 CDSs, representing B62% of the predicted sections in the last third of the genome (Supplementary Fig. 2 proteome. Retained proteins were clustered using a combination of online). A majority of insertion sequences (16 of 28) occur in this force-directed placement and multidimensional scaling11 into region, suggesting that foreign elements may have been preferentially 39 mountains, as visualized by VxInsight12 (Supplementary Fig. 3 introduced by horizontal transfer into this section of the chromosome. online). The breadth and height of each mountain signify the tightness A putative function could be assigned to 4,895 (52.2%) of the of clustering and the number of proteins, respectively. Each mountain encoded proteins on the basis of manual annotation. Of the rest, 1,224 was then assigned an overall function by consideration of the Gene (13.1%) showed similarity to conserved hypothetical proteins in other Ontology