J Mol Microbiol Biotechnol 2009;16:124–135 Published online: October 29, 2008 DOI: 10.1159/000142899 The Genome Organization of Ralstonia eutropha Strain H16 and Related Species of the Burkholderiaceae a, b a a Wolfgang Florian Fricke Bernhard Kusian Botho Bowien a b Institut für Mikrobiologie und Genetik, and Göttingen Genomics Laboratory, Georg-August-Universität Göttingen, Göttingen , Germany Key Words between the smaller replicons is considerably lower, sug- ,Ralstonia eutropha strain H16 ؒ Genome organization ؒ gesting a species-specific origin of Chr2. The megaplasmids -Cupriavidus necator H16 ؒ Burkholderiaceae ؒ however, in most cases do not show any taxonomically re Burkholderiaceae genomes lated similarities. Based on the results of the comparative studies, a hypothesis for the evolution of the multi-replicon genomes of the Burkholderiaceae is proposed. Abstract Copyright © 2008 S. Karger AG, Basel Ralstonia eutropha strain H16 is a facultatively chemolithoau- totrophic, hydrogen-oxidizing bacterium belonging to the family Burkholderiaceae of the Betaproteobacteria . The ge- Introduction nome of R . eutropha H16 consists of two chromosomes (Chr1, Chr2) and one megaplasmid (pHG1), and thus shows a multi- Ralstonia eutropha strain H16 (reclassified as Cupria- replicon architecture, which is characteristic for all members vidus necator H16 [Vandamme and Coenye, 2004]) is a of the Burkholderiaceae sequenced so far. The genes for ubiquitous nonpathogenic soil and freshwater bacterium housekeeping cell functions are located on Chr1. In contrast, belonging to the Burkholderiaceae family within the class many characteristic traits of R. eutropha H16 such as the abil- of the Betaproteobacteria . The organism is the most thor- ity to switch between alternative lifestyles and to utilize a oughly studied representative of the ‘Knallgas’ bacteria broad variety of growth substrates are primarily encoded on that oxidize hydrogen in the presence of molecular oxy- the smaller replicons Chr2 and pHG1. The latter replicons gen [Bowien and Schlegel, 1981]. Adapted to rapidly also differ from Chr1 by carrying a repA -associated origin of changing environmental conditions, R. eutropha H16 replication typically found on plasmids. Relationships be- evolved a high metabolic versatility with the capability to tween the individual replicons from various Burkholderiace- switch between different nutritional modes, taking ad- ae genomes were studied by multiple sequence alignments vantage of various energy and carbon sources for growth. and whole-replicon protein comparisons. While strong con- Strain H16 is an aerobic facultative chemolitho-organo- servation of gene content and order among the largest autotroph able to utilize hydrogen or formate, respective- replicons indicate a common ancestor, the resemblance ly, as energy sources for carbon dioxide assimilation. Di- © 2008 S. Karger AG, Basel Botho Bowien 1464–1801/09/0162–0124$26.00/0 Institut für Mikrobiologie und Genetik Fax +41 61 306 12 34 Georg-August-Universität Göttingen E-Mail [email protected] Accessible online at: Grisebachstrasse 8, DE–37077 Göttingen (Germany) www.karger.com www.karger.com/mmb Tel. +49 5541 3938 15, Fax +49 551 3998 42, E-Mail [email protected] Downloaded by: Niedersächsische Staats- und Universitätsbibliothek 134.76.162.165 - 10/2/2013 2:40:55 PM verse organic compounds such as tricarboxylic acid tropha JMP134 (reclassified as Cupriavidus pinatubonen- (TCA) cycle intermediates, fatty acids, sugar acids, and sis JMP134 [Sato et al., 2006]) does not grow autotrophi- aromatics support heterotrophic growth of the strain cally and has originally been isolated based on its [Aragno and Schlegel, 1992; Schwartz and Friedrich, unusual ability to degrade aromatic pesticides [Pember- 2006]. The metabolism of R. eutropha H16 is strictly re- ton et al., 1979]; R. solanacearum GMI100 is a plant patho- spiratory under both oxic and anoxic conditions. During gen with a very broad host range of over 200 species [Hay- anoxia respiration is maintained by a complete denitrifi- ward, 2000]; B. pseudomallei K96243 is the causative agent cation pathway using oxidized nitrogen compounds as of melioidosis [Dance, 1991; Holden et al., 2004] and coex- terminal electron acceptors [Cramm, 2007]. Another ists with the avirulent B. thailandensis E264 as a soil- characteristic physiological property of R. eutropha H16 borne pathogen endemic in Southeast Asia and Northern is its ability to accumulate large amounts of polyhydroxy- Australia [Brett et al., 1998]; B. mallei ATCC 23344 is an alkanoates (PHA) as internal storage material, whenever obligate parasite of horses, mules and donkeys that causes the carbon source is in surplus but other nutrients or ox- glanders [Nierman et al., 2004]; the B. cepacia complex ygen are limiting [Reinecke and Steinbüchel, 2007; Stein- comprises genetically distinct but phenotypically similar büchel and Schlegel, 1991]. Commercial production of species with pathogenic and both beneficial and detri- PHA polyesters as biodegradable thermoplastics is an es- mental effects on plants [Stanier et al., 1966; Vandamme tablished biotechnological application of the organism et al., 1997]; like R. eutropha JMP134, B. xenovorans LB400 [Madison and Huisman, 1999]. Three independent repli- can utilize a number of substituted aromatic compounds cons, chromosome 1 (Chr1; 4.05 Mbp), chromosome 2 as sources of energy and carbon [Goris et al., 2004]. (Chr2; 2.91 Mbp) and megaplasmid pHG1 (0.45 Mbp), Phenotypic diversity within the Burkholderiaceae is constitute the genome (7.41 Mbp) of the strain [Pohl- accompanied by the multi-replicon genome structure as mann et al., 2006]. The megaplasmid carries the genetic a common organizational feature found in all sequenced information required for lithoautotrophy and denitrifi- strains of this family ( table 1 ). The genomes of the organ- cation – two of the most characteristic features of the or- isms consist of at least two ( R. solanacearum GMI1000, ganism [Schwartz et al., 2003]. B. pseudomallei group) and up to four ( R. eutropha At the time this review was written, in addition to JMP134, R . metallidurans CH34, B. cepacia complex) in- R. eutropha H16, the genomes of 18 strains of the Burk- dependent circular replicons. Accordingly, the total ge- holderiaceae had been completely sequenced and were ac- nome sizes vary considerably within the range of 5.8–9.7 cessible through the NCBI webpage [http://www.ncbi. Mbp. Relatively large size differences also exist among nlm.nih.gov]. These include three members of the genus the individual replicons and are most evident for the Ralstonia ( R. eutropha JMP134, R . metallidurans CH34, (mega)plasmids ( table 1 ). The multipartite genome struc- R. solanacearum GMI1000 [Salanoubat et al., 2002]) and ture of the Burkholderiaceae appears to be even more 15 representatives of the genus Burkholderia ( B. thailan- characteristic as three sequenced genomes of Bordetella densis E264, B. vietnamiensis G4, B. xenovorans LB400, species belonging to the related family Alcaligenaceae eight strains from the Burkholderia pseudomallei group within the Betaproteobacteria show a completely differ- including B. pseudomallei strains 1106a, 1710b, 668 and ent organization. The genomes of the respiratory tract K96243 [Holden et al., 2004] and B. mallei ATCC 23344 pathogens B. bronchiseptica RB50, B. parapertussis 12822 [Nierman et al., 2004], NCTC 10229, NCTC 10247 and and B. pertussis Tohama I harbor only a single circular SAVP1 and four strains of the Burkholderia cepacia com- chromosome corresponding in size (4.1–5.3 Mbp) rough- plex including B. cepacia AMMD, B. cenocepacia strains ly to the largest replicons of the Burkholderiaceae ge- HI2424 and AU 1054 and Burkholderia sp. 383). Except nomes [Parkhill et al., 2003]. for B. mallei ATCC 2344 – an animal pathogen that ap- This contribution evaluates the sequence of the R. eu- parently evolved by genomic reduction from a single clone tropha H16 genome to highlight some specific features of of B. pseudomallei [Godoy et al., 2003] – all strains are each replicon. Differences in coding properties and orga- basically free-living soil bacteria characterized by their nization of basic replication units found between Chr1 on ability to occupy alternative environmental niches offer- one side and Chr2 and the megaplasmid pHG1 on the ing them selective benefits: R. eutropha H16 and R . metal- other are pointed out and compared with other Burkhol- lidurans CH34 (reclassified as Cupriavidus metallidurans deriaceae genomes. The comparative analysis is extend- CH34 [Vandamme and Coenye, 2004]) are the only facul- ed to multiple sequence alignments of individual pro- tative chemolitho-organoautotrophs in this group; R . eu- teins associated with specific replicons and finally to Genome Organization of Ralstonia J Mol Microbiol Biotechnol 2009;16:124–135 125 eutropha Downloaded by: Niedersächsische Staats- und Universitätsbibliothek 134.76.162.165 - 10/2/2013 2:40:55 PM whole-replicon protein comparisons with the BLAST type integrase genes [Schwartz et al., 2003]. Bidirectional score ratio approach [Rasko et al., 2005]. These data pro- BLAST comparisons revealed a high degree of redundan- vide the basis for the discussion of a hypothetical model cy between the R. eutropha H16 replicons with 782, 99 for the evolution of multipartite genome structures in the and 115 gene homologues located on Chr1 and Chr2, Burkholderiaceae . Chr1 and pHG1, and Chr2 and pHG1, respectively.