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

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 - 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 . 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 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’ 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 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.

Origins of Replication Genome Organization of R. eutropha H16 A principal distinction of the R. eutropha H16 repli- cons is possible based on differences in the individual General Features replication units of Chr1, Chr2 and pHG1 found at the Among the three replicons constituting the R. eutro- origins of replication. Identification of the origins is fa- pha H16 genome, Chr1 and Chr2 correspond in size to cilitated by a pronounced global minimum of the cumu- the two largest replicons present in the closely related lative GC skew [Grigoriev, 1998] for Chr1 and Chr2 and strains R . eutropha JMP134 and R . metallidurans CH34. to some extent also for pHG1. The minimum coincides This also applies to the respective entities of R. sola- with a dnaA gene and a DnaA-binding site located on nacearum GMI1000 and the Burkholderia species (ta- Chr1, but a repA gene and – at least in case of Chr2 – sev- ble 1 ). A replicon roughly equivalent in size to the mega- eral flanking iteron-like putative RepA-binding sites on plasmid pHG1 of strain H16 exists only in the non- Chr2 and pHG1. Additionally, on the smaller replicons it autotrophic strain JMP134, whereas the autotrophic is flanked by parAB gene clusters. DnaA, which is the strain CH34 harbors two smaller megaplasmids. pHG1 is central initiator protein for chromosomal replication in known to be self-transmissible and can be used to trans- bacteria, is often found in direct proximity to the origin fer the capacity for H2 oxidation to other R. eutropha [Fuller et al., 1984; Lee and Bell, 2000]. In contrast, RepA strains [Friedrich et al., 1981]. Consequently, as the genes proteins are typically encoded on plasmids, where they essential for H2 oxidation reside on the megaplasmid, bind to iteron-like sequences around the origin and are curing of R. eutropha H16 for pHG1 results in loss of the required for plasmid DNA replication [del Solar et al., ability to grow lithoautotrophically with H 2 [Hogrefe et 1998]. ParAB proteins are involved in plasmid partition- al., 1984]. In contrast, attempts to remove Chr2 from the ing [Dubarry et al., 2006]. Collectively, these findings genome remained unsuccessful [A. Pohlmann and B. clearly distinguish Chr1 from Chr2 and pHG1 and sug- Friedrich, pers. commun.]. A basic differentiation be- gest a plasmid ancestry of both Chr2 and pHG1. In fact, tween the two chromosomes on the one hand and the the R . solanacearum GMI1000 replicon corresponding to megaplasmid on the other can be made by comparing Chr2 has been designated as a megaplasmid [Salanoubat general coding features of the replicons. All ribosomal et al., 2002]. Chr2 of R . eutropha H16 might thus also be RNA operons of R. eutropha H16 are located on either considered a megaplasmid (Mpl). DnaA Chr1 (CAJ91153) Chr1 (three operons) or Chr2 (two operons). Of the 59 and RepA Chr2 (CAJ94807) of strain H16 are similar to the tRNAs, a complete set of 51 tRNAs for all possible codons corresponding proteins from Chr1 (Q8XTV4) and Chr2 is encoded on Chr1. Seven duplicates are carried on Chr2, ( ϳMpl) (CAD17152) of R. solanacearum (75 and 92% one on pHG1. The two chromosomes share similar values identity, respectively), whereas RepA pHG1 (AAP86121) for G+C content (66.5 and 66.3 mol%, respectively), cod- shows weak resemblance (31% identity) only to a putative ing sequence (88 and 89%, respectively) and fractions of RepA protein (S60672) encoded on the megaplasmid presumably laterally transferred alien DNA (15 and 13%, pMOL28 of R. metallidurans CH34, a closer relative of respectively, as predicted by the SIGI-HMM codon usage R. eutropha H16. analysis tool of Waack et al. [2006]). These values differ significantly from those of pHG1 (G+C: 61.3 mol%; cod- Encoded Functions ing sequence: 81%; alien DNA: 34.0%). Chr1 (four copies) The classification of the R. eutropha H16 replicons and Chr2 (three copies) together carry the same number into two groups with one represented by Chr1 and the of IS elements as the megaplasmid alone, indicating a second comprising Chr2 and pHG1 is supported by stronger genome plasticity of the latter, a conclusion fur- the general distribution of genes on these replicons. As ther supported by the finding that pHG1 contains a large a rule, basic housekeeping functions including DNA rep- ‘junkyard’ region comprising about 70 kbp with 17 rem- lication, transcription, translation and the biosyntheses nants of mobile elements and 22 partial or intact phage- of building blocks such as amino acids, nucleotides and

126 J Mol Microbiol Biotechnol 2009;16:124–135 Fricke /Kusian /Bowien

Downloaded by: Niedersächsische Staats- und Universitätsbibliothek 134.76.162.165 - 10/2/2013 2:40:55 PM Table 1. Genome organization in sequenced species of Ralstonia, Burkholderia, Bordetella, and Pseudomonas

Strain Chromosome 1 Chromosome 2 Replicon 3 Replicon 4 sizea CDSb, n size CDS, n size CDS, n size CDS, n

1 R. eutropha H16c 4.05 3,651 2.91 2,555 0.45 (pHG1)d 420 –e 2 R. eutropha JMP134 3.81 3,439 2.74 2,407 0.63 (megaplasmid) 512 0.09 (pJP4) 87 3 R. metallidurans CH34 3.93 3,601 2.58f 2,313 0.23 (pMOL30) 177 0.17 (pMOL28) 112 4 R. solanacearum GMI1000 3.72 3,440 2.10 1,676 – – Shared CDS (1 to 2)g 2,746 1,419 512 Shared CDS (1 to 3) 2,410 882 4 Shared CDS (1 to 4) 1,902 208 5 B. pseudomallei grouph 3.51–4.13 2,996–3,736 2.33–3.18 2,029–2,611 – – 6 B. thailandensis 3.81 3,276 2.92 2,358 – – 7 B. cepacia complexi 3.29–3.69 2,965–3,334 2.65–3.59 2,346–3,174 1.06–1.40j 918–1,209 0.04–0.16k 45–156 8 B. xenovorans LB400 4.90 4,430 3.36 2,960 1.47 1,312 – Shared CDS (5 to 8) 1,586 476 16 (7–9) Shared CDS (1 to 8) 985 33 0 9 B. bronchiseptica RB50 5.34 4,994 – – – 10 B. pertussis Tohama I 4.09 3,436 – – – Shared CDS (9 to 10) 3,404 Shared CDS (1 to 10) 718 11 P. aeruginosa PAO1 6.26 5,568 – – – 12 P. fluorescens Pf0-1 6.44 5,736 – – – Shared CDS (11 to 12) 3,509 Shared CDS (1 to 12) 471

a Replicon sizes in mega-basepairs (Mbp). BX470250; B. pertussis To h a m a I : BX470 2 4 8 ; P. aeruginosa PAO1: AE004091; b CDS, coding sequences. P. fluorescens PfO-1: CP000094. c GenBank accessions: R. eutropha H16: AM260479 (Chr1), AM260479 d Plasmid designations in parentheses. (Chr2), AY305378 (pHG1); R. eutropha JMP134: CP000090 (Chr1), e Replicon not present. CP000091 (Chr2), CP000092 (Mpl); R. metallidurans CH34: CP000352 f Chr2 of R. solanacearum GMI1000 has been classified as megaplasmid (Chr1), CP000353 (Chr2), X71400 (pMOL30), X90708 (pMOL28); R. sola- [Salanoubat et al., 2002]. nacearum GMI1000: AL646052 (Chr), AL646052 (Mpl, corresponding to g Shared CDS, orthologous CDS shared by different replicons from all Chr2); B. mallei ATCC 23344: CP000010 (Chr1), CP000011 (Chr2); B. pseu- genomes indicated in parentheses were determined by calculating BLAST domallei 1710b: CP000124 (Chr1), CP000125 (Chr2); B. pseudomallei score ratios [Rasko et al., 2005]. h K96243: BX571965 (Chr1), BX571966 (Chr2); B. thailandensis E264: Including B. mallei ATCC23344, B. pseudomallei 1710b, B. pseudo- CP000086 (Chr1), CP000085 (Chr2); B. cenocepacia AU 1054: CP000378 mallei K96243. (Chr1), CP000379 (Chr2), CP000380 (Replicon 3 [Chr3]); B. cenocepacia i Including B. cenocepacia AU 1054, B. cenocepacia HI2424, B. cepacia HI2424: CP000458 (Chr1), CP000459 (Chr2), CP000460 (Replicon 3 AMMD, B. sp. 383. [Chr3]); B. cepacia AMMD: CP000440 (Chr1), CP000441 (Chr2), CP000442 j Replicons 3 of the Burkholderia species are megaplasmids but have (Replicon 3 [Chr3]); B. sp. 383: CP000151 (Chr1), CP000152 (Chr2), been designated as chromosomes. CP000150 (Replicon 3 [Chr3]); B. xenovorans LB400: CP000270 (Chr1), k Plasmid 1 of B. cepacia AMMD and B. cenocepacia HI2424. CP000271 (Chr2), CP000273 (Replicon 3 [Chr3]); B. bronchiseptica RB50:

are encoded on Chr1, whereas additional properties tiple copies on Chr1 and Chr2 or pHG1 (see below). While that provide the organism with selective advantages un- the fundamental role of the megaplasmid for two of the der special growth conditions are encoded on Chr2 and most characteristic traits of R. eutropha H16 – facultative pHG1. Single-copy genes in the genome located on Chr2 litho/organoautotrophy and anaerobic growth by deni- and encoding essential functions include peptide chain trification – has been unveiled by conventional genetic release factor 3 (CAJ97353), 4-aminobutyrate amino- studies and confirmed by sequence analysis [Schwartz et transferase (CAJ95772) and argininosuccinate synthase al., 2003], it remained difficult to define the significance (CAJ97313). As a result of redundant genetic information, of Chr2 for the phenotype of R. eutropha H16 until the a number of housekeeping functions are encoded in mul- entire genome sequence had been elucidated. Now it is

Genome Organization of Ralstonia J Mol Microbiol Biotechnol 2009;16:124–135 127 eutropha Downloaded by: Niedersächsische Staats- und Universitätsbibliothek 134.76.162.165 - 10/2/2013 2:40:55 PM evident that Chr2 not only contributes in a fundamental Degradation of Aromatics. R. eutropha H16 has long way to chemoautotrophic and anaerobic capabilities but been known to grow on a remarkable variety of aromatic also carries genes required for motility and the ability to compounds [Johnson and Stanier, 1971]. This trait is utilize a very broad substrate spectrum as well as for comparable to that of the closely related R. eutropha functions in the secondary metabolism of the organism. JMP134, which has been described as a model organism In the following section several of these features will be for the degradation of substituted aromatic pollutants discussed in more detail. [Pemberton et al., 1979]. The ability to degrade e.g. 3- or Facultative Litho-Organoautotrophy. R. eutropha H16 4-hydroxylated benzoates, 4-cresol or biphenyl largely was the first litho/organoautotrophic member of the depends on genetic information contained in Chr2. Genes Burkholderiaceae whose genome sequence had been pub- for the protocatechuate branch of the ␤-ketoadipate path- lished [Pohlmann et al., 2006; Schwartz et al., 2003]. way, the meta-cleavage and the gentisate pathway are car- Genes for key components of the lithoautotrophic me- ried on this replicon. The ␤-ketoadipate pathway is com- tabolism were identified on both Chr2 and pHG1, either plemented by the catechol branch encoded on Chr1. in single copy on only one of the replicons or in two cop- Motility. Differences in the flagellation type of various ies on both. While the genes for the three Ralstonia species are a phenotypic characteristic of this plus auxiliary proteins ( hox and hyp genes) are exclusive- genus. R. eutropha is peritrichously flagellated, while R. ly located on pHG1, the cbb operon containing genes for solanacearum carries polar flagella [Vaneechoutte et al., enzymes catalyzing autotrophic CO2 fixation via the Cal- 2004]. Nevertheless, in the sequenced Ralstonia strains vin-Benson-Bassham cycle is duplicated with one copy biosynthesis of flagella and functions for chemotaxis are on pHG1 and the other on Chr2. However, the main cbb all encoded on Chr2. In contrast, in the Burkholderia spe- gene regulator CbbR (CAJ96185) is only encoded on Chr2 cies, where flagellar gene clusters are located on either [Bowien and Kusian, 2002; Kusian and Bowien, 1997]. Chr1 ( B. mallei ATCC 23344, B. pseudomallei K96243) or Either one of the two cbb operons is sufficient to support Chr2 ( B. thailandensis E264), the distribution of this ge- autotrophic growth of the organism, which is also ob- netic information is less uniform. served with formate as energy source (organoautotro- Secondary Metabolism. Most of the sequenced Burk- phy). At least four different formate dehydrogenases are holderiaceae species are pathogens. R. eutropha H16 is encoded in the genome of strain H16, two on both Chr1 a nonpathogenic organism but still harbors a number and Chr2. of potential toxin gene clusters located on Chr2, most Facultative Anaerobiosis. The reduction of nitrate to of which are fragmentary. These include two genes dinitrogen by R . eutropha H16 under anaerobic growth (CAJ96141-CAJ96142) for a putative insecticidal Tcc tox- conditions is catalyzed by the concerted action of denitri- in similar to that found in Photorhabdus luminescens fying enzymes encoded on Chr2 (nitrate, nitrite and ni- [Bowen et al., 1998] and several cryptic gene clusters for tric oxide reductases) and pHG1 (nitrate, nitric oxide and putative RTX toxins. A nonribosomal polypeptide syn- nitrous oxide reductases) [Schwartz and Friedrich, 2001]. thetase (CAJ96468, CAJ96470-CAJ96472) of unknown A gene for an anaerobic class III ribonucleotide reductase function is also encoded on Chr2. (AAP85992) is carried on pHG1. Degradation of Carbohydrates. Chr2 is indispensable for growth of R. eutropha H16 on sugars and sugar de- G e n o m i c C o m p a r i s o n s rivatives. The degradation of these compounds exclusive- ly involves the Entner-Doudoroff (2-keto-3-deoxy-6- The genome of R. eutropha H16 is structurally charac- phosphogluconate) pathway. Fructose and N -acetylglu- terized by the differentiation between the actual chromo- cosamine are the only sugars utilized by the wild-type some, Chr1, and the two megaplasmid-like replicons strain. Although glucose could formally be metabolized, Chr2 and pHG1. Analogous genome compositions are the organism lacks an uptake system for this hexose. Glu- found in all sequenced Burkholderiaceae strains (Beta- conate, 2-ketogluconate and glucosaminate are the sugar ), where the origins of replication of the acids that serve as substrates. The enzymes for the entire major replicons (Chr1) are associated with dnaA genes Entner-Doudoroff pathway, including a glucokinase and of the smaller replicons (Chr2, Mpl) with repA genes. (CAJ97346), are encoded on Chr2, except for a gluconate Moreover, specific replicative loci (repABC) have been kinase (CAJ92320) the gene of which is located on Chr1 found on many plasmids and second chromosomes in [Pohlmann et al., 2006]. multi-replicon genomes of the Alphaproteobacteria

128 J Mol Microbiol Biotechnol 2009;16:124–135 Fricke /Kusian /Bowien

Downloaded by: Niedersächsische Staats- und Universitätsbibliothek 134.76.162.165 - 10/2/2013 2:40:55 PM Bu. cenocepacia, Chr1 1 Bu. cepacia AMMD, Chr1 2 Bu. sp. 383, Chr1 3 518 Bu. pseudomallei / Bu. mallei, Chr1 4 999 Bu. thailandensis E264, Chr1 5 994 Bu. xenovorans LB400, Chr1 6 R. solanacearum GMI1000, Chr1 7

1000 8 998 R. eutropha JMP134, Chr1 1000R. eutropha H16, Chr1 9 542 R. metallidurans CH34, Chr1 10 1000 R. metallidurans CH34, pMOL28 11 R. eutropha H16, pHG1 12 1000 1000 R. eutropha JMP134, Mpl 13 1000 Bu. xenovorans LB400, Mpl (~Chr3) 14 718 R. metallidurans CH34, pMOL30 15 Bu. cepacia AMMD, Pl 16/ 17 433 R. eutropha JMP134, pJP4 R. solancearum GMI1000, Chr2 (~Mpl) 18 1000 R. metallidurans CH34, Chr2 19 652 1000 R. eutropha H16, Chr2 20 900 R. eutropha JMP134, Chr2 21 1000 Bu. cepacia AMMD, Mpl (~Chr3) 22 958 Bu. sp. 383, Mpl (~Chr3) 23 1000 Bu. cenocepacia, Mpl (~Chr3) 24 822 Bu. xenovorans LB400, Chr2 25 26 1000 Bu. pseudomallei / Bu. mallei, Chr2 1000 Bu. thailandensis E264, Chr2 27 833 Bu. cepacia AMMD, Chr2 28 1000 Fig. 1. Phylogenetic analysis of different repli- Smaller Bu. sp. 383, Chr2 29 0.1 918 cons from Burkholderiaceae genomes by mul- Replicons Bu. cenocepacia, Chr2 30 tiple sequence alignments of the translated a protein sequences of replicon-specific parB ( a ) and repA (b ) genes, respectively. Sequence alignments were performed using the Clust- Bu. xenovorans LB400, Chr2 1 alW program [Chenna et al., 2003]. The scale 570 bar corresponds to 0.1 substitutions per nu- Bu. pseudomallei / Bu. mallei, Chr2 2 1000 cleotide position. Bootstrap values of 1,000 Bu. thailandensis E264, Chr2 3 replications are indicated at interior branch 4 nodes. Chr = Chromosome; Mpl = megaplas- Bu. cepacia AMMD, Chr2 1000 mid; Pl = plasmid. Replicons were designated Bu. cenocepacia, Chr2 5 as chromosomes or megaplasmids according 743 Bu. sp. 383, Chr2 6 to the names of corresponding replicons in R. eutropha H16. Original designations from Bu. cepacia AMMD, Mpl (~Chr3) 7 the GenBank entries are shown in parenthe- 1000 Bu. cenocepacia, Mpl (~Chr3) 8 ses. GenBank accessions: (a ) Putative parB 895 genes: 1 ABK06850; 2 ABI85648; 3 ABB06880; Bu. sp. 383, Mpl (~Chr3) 9 4 5 6 CAH37417; ABC39439; ABE32896; R. metallidurans CH34, pMOL30 10 7 8 9 CAD17113; AAZ62712; CAJ94702; 1000 10 ABF10375; 11 CAI30203; 12 AAP86124; 1000 R. eutropha H16, pHG1, #1 11

13 14 15 629 AAZ65478; ABE37218; CAI11184; R. eutropha JMP134, Mpl 12 16 17 18 ABI92146; AAR31022; CAD17155; 1000 19 20 21 Bu. xenovorans LB400, Mpl (~Chr3) 13 ABF12679; CAJ94810; AAZ64689; 1000 22 23 24 ABI91545; ABB06782; ABF80692; R. metallidurans CH34, pMOL28 14 25 26 27 ABE32970; CAH39837; ABC35021; 1000 15 28 ABI90515; 29 ABB10121; 30 ABF80692. ( b ) R. eutropha H16, pHG1, #2 1 2 982 Putative repA genes: ABE32971; CAH39838; R. solancearum GMI1000, Chr2 (~Mpl)16 3 ABC34521; 4 ABI90516; 5 ABF80038; 1000 17 6 ABB10120; 7 ABI91544; 8 ABF80693; R. metallidurans CH34, Chr2 9 10 11 1000 ABB06783; CAI11185; AAP86122; R. eutropha H16, Chr2 18 12 AAZ65476; 13 ABE37216; 14 CAI30147; Smaller 0.1 849 19 15 AAP86121; 16 CAD17152; 17 ABF12676; Replicons R. eutropha JMP134. Chr2 18 AJ94807; 18 AAZ64686; 19 CP000091. b

Genome Organization of Ralstonia J Mol Microbiol Biotechnol 2009;16:124–135 129 eutropha Downloaded by: Niedersächsische Staats- und Universitätsbibliothek 134.76.162.165 - 10/2/2013 2:40:55 PM R. eutropha R. metallidurans R. solanacearum Bu. pseudomallei Bo. bronchiseptica JMP134 CH34 GMI1000 K96243 RB50 R. eutropha H16, Chr1

Chr1 Chr1 Chr1 Chr1 Chr R. eutropha H16, Chr2

Chr2 Chr2 Chr2 Chr2 R. eutropha H16, pHG1

Mpl pMOL30 pMOL28

Fig. 2. Sequence comparisons of proteins encoded on Chr1, Chr2, generated with MUMmerplot [Delcher et al., 1999]. Direct se- and pHG1 of R. eutropha H16 with those from corresponding rep- quence similarities are depicted in red, inverted similarities in licons of other Ralstonia species, of Burkholderia pseudomallei , blue. Gray lines in the comparisons with Chr1 and Chr2 of R. eu- and of Bordetella bronchiseptica . Comparisons were carried out tropha H16 indicate nucleotide segments of 500 kbp, in the com- using the PROmer tool of the MUMmer package and dot plots parisons with pHG1 segments of 100 kbp.

[MacLellan et al., 2004]. To date, a consistent classifica- the smaller replicons. The resulting phylogenetic trees for tion of the smaller replicons does not exist, since some of ParB ( fig. 1 a) and RepA ( fig. 1 b) conform with the 16S them are designated as megaplasmids (e.g. in R. sola- rRNA-based phylogenetic classification of the organisms. nacearum GMI1000) and others as chromosomes (e.g. in Both trees group the Chr1 replicons of the Ralstonia and the B. cenocepacia complex and B. xenovorans LB400). Burkholderia species on separate branches and the same The interest in the evolutionary origins of the individual applies to the corresponding Chr2 replicons. Further- replicons from different multi-replicon genomes has more, the sequence alignments indicate a relationship be- prompted us to investigate their relationships through tween all Chr1 replicons of these genomes, which is con- comparative sequence analysis. Previously deduced phy- siderably closer than that between all Chr2 replicons. The logenetic trees that were based on multiple sequence relations among the smallest replicons of the Ralstonia alignments of ParAB-like proteins from replicons of dif- genomes are weak and not consistent with the taxonom- ferent B. cenocepacia strains revealed evolutionary rela- ic classifications of the corresponding organisms. For ex- tions between the largest replicons, which could be dis- ample, both ParB and RepA sequences suggest a relation- tinguished from those of the smaller replicons [Dubarry ship between the megaplasmids (ϳ Chr3) of B. xeno- et al., 2006]. This picture was now extended for all se- vorans LB400 and of R. eutropha JMP134 (fig. 1). Only quenced Burkholderiaceae genomes by aligning the se- the Chr3 replicons of the B. cepacia complex show a close quences of putative ParB proteins, encoded on all repli- relationship that is, however, restricted to this taxonomic cons, and of RepA proteins specifically encoded only on group.

130 J Mol Microbiol Biotechnol 2009;16:124–135 Fricke /Kusian /Bowien

Downloaded by: Niedersächsische Staats- und Universitätsbibliothek 134.76.162.165 - 10/2/2013 2:40:55 PM a b

c d

Fig. 3. Sequence comparisons of proteins encoded on the largest P. fluorescens Pf0-1 (horizontal) and P. aeruginosa PAO1 (verti- replicons of different Ralstonia , Burkholderia , Bordetella , and cal). Comparisons were carried out using the PROmer tool of the Pseudomonas species. (a ) R. metallidurans CH34 (horizontal) and MUMmer package and dot plots generated with MUMmerplot R. solanacearum GMI1000 (vertical); ( b ) B. xenovorans LB400 [Delcher et al., 1999]. Direct sequence similarities are depicted in (horizontal) and B. pseudomallei K96243 (vertical); ( c ) B. bronchi- red, inverted similarities in blue. Gray lines indicate nucleotide septica RB50 (horizontal) and B. pertussis Tohama I (vertical); ( d ) segments of 500 kbp.

In order to estimate the degree of conservation of gene those of R. eutropha H16 and R. metallidurans CH34 content and organization between the replicons of dif- (fig. 2) or of B. mallei ATCC 23344 and B. pseudomallei ferent Burkholderiaceae genomes whole-replicon protein K96243 [Holden et al., 2004]. A striking bias in the dis- sequence comparisons were carried out (table 1; fig. 2). tribution of CDS for specific cell functions between Chr1 These comparisons show that the Chr1 replicons not and the smaller replicons as described above for R. eutro- only share the highest fractions of protein-encoding se- pha H16 has also been observed in other Burkholderia- quences (CDS) among the Burkholderiaceae genomes ceae genomes [Holden et al., 2004; Salanoubat et al., (22–33%, compared to 0–2% in all other replicons), but 2002]. Invariably, the majority of essential housekeeping also strong gene synteny. The estimated number of or- functions is encoded on Chr1, additional properties, thologous CDS conserved on the Chr1 replicons decreas- however, preferentially on the smaller replicons. While es from 2,746 found for the two R. eutropha strains, to the conserved localization of CDS for basic metabolic 985 for the 13 completely sequenced Burkholderiaceae features on the largest replicon of the Burkholderiaceae strains and still amounts to 718 for all Burkholderiaceae genomes explains the high degree of gene synteny, it also strains and the single chromosomes of B. bronchiseptica strongly suggests the evolution of these replicons from a RB50 and B. pertussis Tohama I. With the single chro- common ancestor. Chr2 replicons, in contrast, seem to mosomes of Pseudomonas aeruginosa PAO1 and P. fluo- originate from independent Ralstonia- or Burkholderia - rescens Pf0-1 (Gammaproteobacteria) included into the specific, plasmid-like ancestral replicons. The smaller comparison, the number of shared CDS even drops to megaplasmids appear to be mostly species- or even 471 ( table 1 ). For all smaller replicons of the Burkholde- strain-specific. A higher genetic plasticity of the small riaceae a similar degree of conservation is seen only replicons could also have contributed to the lack of con- among the Chr2 replicons of the same genus such as servation among these replicons. Generally, it can be

Genome Organization of Ralstonia J Mol Microbiol Biotechnol 2009;16:124–135 131 eutropha Downloaded by: Niedersächsische Staats- und Universitätsbibliothek 134.76.162.165 - 10/2/2013 2:40:55 PM stated that the specificity of a megaplasmid-like replicon benefit or transfer of essential functions from Chr1 to the for a genus, species or strain increases with decreasing megaplasmid would have imposed a selective pressure on size of this replicon. the presence of the megaplasmid. This genetic stabiliza- tion probably resulted in present large replicons such as Chr2 from R. eutropha H16, hardly distinguishable from Possible Evolution of Burkholderiaceae Genomes the core chromosomes.

If present Burkholderiaceae strains originate from a common ancestral bacterium, its genome consisted most Alternative Evolutionary Strategies probably of a single circular replicon roughly correspond- ing in size, gene content and gene order to the Chr1 rep- The Bordetella species of the family Alcaligenaceae licons of recent Burkholderiaceae genomes. A likely an- within the same taxonomic order () as cestor could have been a simple prototrophic soil/aquatic the Burkholderiaceae and the Pseudomonas species from bacterium. The presence of CDS for elementary compo- the family Pseudomonadaceae of the Gammaproteobac- nents of the TCA cycle and the respiratory chain and of teria display a simpler genome structure with a single numerous transport systems suggests a heterotrophic chromosome, reflecting an alternative strategy of ge- metabolism based on the aerobic utilization of organic nome evolution. Based on phenotypic characterizations substrates as its primary lifestyle. Modest substrate de- present Burkholderia species have been assigned to the mands in conjunction with a lack of metabolic specializa- Pseudomonadaceae prior to their reclassification tion could have facilitated a wide distribution of the through 16S rRNA sequence homology [Yabuuchi et al., ancestral bacterium that allowed its progeny to colonize 1992]. diverse ecological niches. The large size of most Burk- Among the Alcaligenaceae the genomes of three close- holderiaceae genomes indicates later acquisitions of new ly related human pathogens, B. bronchiseptica RB50, B. phenotypic traits with the result of the observed versatile pertussis Tohama I and B. parapertussis 12822 have been instead of restricted metabolic capacities. This might sequenced [Parkhill et al., 2003]. They contain single rep- have been favored by the requirements of a rapidly chang- licons (4.1–5.3 Mbp) comparable in size to Chr1 replicons ing soil or aquatic environment. An adequate genetic in- of the Burkholderiaceae genomes ( table 1 ). Whole-ge- frastructure in this respect consists of a multi-replicon nome protein comparisons between the chromosomes of genome architecture that differentiates between a stable B. bronchiseptica RB50 and B. pertussis Tohama I show a chromosome for basic housekeeping functions and more large fraction of 3,404 orthologous CDS, confirming variable, heterologous megaplasmids for integration of their very close relationship. However, a weak but signif- newly evolving traits. The evolution of species and/or icant gene synteny still exists between Chr1 of R. eutro- strains could have been triggered by the uptake and stable pha H16 and the chromosome of B. bronchiseptica RB50 maintenance of plasmids yielding different phenotypes. (fig. 2), which suggests a common evolutionary origin Such an event could have marked the separation between even of these replicons from different taxonomic fami- for example the Ralstonia and Burkholderia genera – as lies. A putative common ancestor of the Alcaligenaceae suggested by genus-specific relationships of the Chr2 rep- and Burkholderiaceae would most likely have been a sap- licons – and subsequently between species of the same rophytic soil bacterium as can be concluded from the pri- genus – as documented by species-specific relationships mary habitats of most Burkholderiaceae and from the of the Chr3 replicons of for example B. cepacia and B. phenotypic properties of B. bronchiseptica RB50. Of the fungorum. Integration of additional genetic material three Bordetella species, this strain is considered to be might then have gradually enlarged the plasmids to form most similar to a potential precursor organism [Parkhill megaplasmids. Multiple acquisitions of plasmids might et al., 2003], since it has the broadest host spectrum pos- have resulted in phenotypic differences even between sibly associated with the ability to survive in the environ- closely related strains of the same species as seen in the ment [Porter and Wardlaw, 1993]. The smaller genome case of the two R. eutropha strains. The continuous pro- sizes of the obligate pathogens Bordetella pertussis To- cess of genome evolution is reflected by the numbers of hama I and B. parapertussis 12822 compared to B. bron- distinct megaplasmid-like replicons found in single or- chiseptica RB50 and the Burkholderiaceae species sug- ganisms such as R. metallidurans CH34 harboring three gests that differentiation proceeded through genome re- such replicons. Gain of new features with a high selective duction. The speciation probably involved both the

132 J Mol Microbiol Biotechnol 2009;16:124–135 Fricke /Kusian /Bowien

Downloaded by: Niedersächsische Staats- und Universitätsbibliothek 134.76.162.165 - 10/2/2013 2:40:55 PM acquisition of virulence-associated genes and the simul- Pf0-1 indicate a bias towards the integration of addition- taneous loss of CDS for basic metabolic functions al CDS resulting in an overall increase of genome size. A required for survival outside of the human host. The high large fraction of the orthologous CDS found in the two number of IS elements found in the genomes of B. pertus- Pseudomonas genomes might therefore result from inde- sis Tohama I (261 copies) and B. parapertussis 12822 (112 pendent gene acquisition events. copies) compared to B. bronchiseptica RB50 (none) led to For the symbiotic nitrogen fixer Sinorhizobium meli- the assumption that acquisition of IS elements was a crit- loti of the genetically diverse class of Alphaproteobacte- ical prerequisite in the evolutive process of genome decay ria [Sällström and Andersson, 2005], mutant strains [Preston et al., 2004]. This notion is supported by the re- with reduced numbers of replicons were obtained from sults of whole-genome protein comparisons performed standard cultures of the wild-type strain [Guo et al., between the genomes of B. bronchiseptica RB50 and B. 2003]. Mutants with only one or two replicons instead of pertussis Tohama I. While general gene synteny is appar- three could be stably cultivated and reverted to the orig- ent between the two genomes, it displays frequent discon- inal genome structure. Interestingly, the mutants dis- tinuity between short sequence stretches and thus docu- played the same phenotype as the wild type, except for a ments a high incidence of rearrangement events (fig. 3). slight reduction of growth efficiency in minimal medi- The IS elements might be responsible for recombinatory um. The genome rearrangements were shown to take activity showing, however, a local restriction. Rearrange- place at cointegration sites consisting of repeated DNA ments between very distant chromosomal loci or integra- sequences with more than 95% identity over at least 1 tion and deletion of single genes, which would be repre- kbp in length. Reiterated DNA elements meeting these sented by single dots in the graphical output of the com- criteria are present in all three replicons of R. eutropha parison only occurred at low frequencies. H16, in the genomes of B. bronchiseptica RB50 and P. Like the Burkholderiaceae, the Pseudomonadaceae aeruginosa PAO1 and also in any bacterial genome con- are a phenotypically heterogeneous family of ubiquitous taining multi-copy CDS or more than one ribosomal soil and aquatic bacteria. Although genome sizes within RNA gene cluster or IS element. Therefore, large ge- the Pseudomonadaceae vary between species and even nomes might generally be subject to more dynamic strains (Pseudomonas stutzeri : 3.7–4.7 Mbp [Ginard et structural changes than smaller ones, suggesting that the al., 1997]; P. aeruginosa: 5.2–7.1 Mbp [Schmidt et al., documented architectures of Burkholderiaceae genomes 1996]), large megaplasmid-like secondary replicons have represent relatively stable, currently dominant organiza- not been found. A considerable degree of restriction site tional states of the genomes. In any case, it appears as if polymorphism has been documented even between dif- single- and multi-replicon bacteria are equally competi- ferent strains of the same species [Ginard et al., 1997], tive in colonizing similar habitats. Future research will which at the same time seems not to affect the overall hopefully reveal why these organisms developed differ- content of housekeeping genes [Kiewitz and Tümmler, ent strategies for the evolution of their genomes and why 2000]. Whole-genome protein comparisons between P. a specific genome organization is beneficial for one group aeruginosa PAO1 and P. fluorescens Pf0-1, which docu- of bacteria but not for the other. ment the high number of orthologous CDS, also demon- strate the almost complete lack of gene synteny (fig. 3). Chr1 of R. eutropha H16 shares a large fraction of CDS Acknowledgements with the genomes of P. aeruginosa PAO1 (1,136 CDS) and P. fluorescens Pf0-1 (1,128 CDS), but shows no gene syn- The work in the authors’ laboratories was performed within the Ralstonia eutropha genome project as part of the GenoMik teny with any Pseudomonas species (data not shown). The Competence Network Göttingen financed by the Bundesministe- missing conservation of gene order even among Pseudo- rium für Bildung und Forschung (BMBF). Further support came monas species indicates a genome organization, which is from the Niedersächsisches Ministerium für Wissenschaft und surprisingly different from that of the Ralstonia and Bor- Kultur. detella species. Continuous gene turnover with simulta- neous acquisitions and losses of single CDS, preserving the total numbers of conserved CDS, could be one expla- nation, a less restricted recombinatory activity compared to the Bordetella strains another. In general, the large genome sizes of P. aeruginosa PAO1 and P. fluorescens

Genome Organization of Ralstonia J Mol Microbiol Biotechnol 2009;16:124–135 133 eutropha Downloaded by: Niedersächsische Staats- und Universitätsbibliothek 134.76.162.165 - 10/2/2013 2:40:55 PM References

Aragno M, Schlegel HG: The mesophilic hydro- Godoy D, Randle G, Simpson AJ, Aanensen DM, Madison LL, Huisman GW: Metabolic engineer- gen-oxidizing (Knallgas) bacteria; in Balows Pitt TL, Kinoshita R, Spratt BG: Multilocus ing of poly(3-hydroxyalkanoates): from

A, Trüper HG, Dworkin M, et al (eds): The sequence typing and evolutionary relation- DNA to plastic. Microbiol Mol Biol Rev 1999;

Prokaryotes. New York, Springer, 1992, pp ships among the causative agents of melioi- 63: 21–53. 344–384. dosis and glanders, Burkholderia pseudo- Nierman WC, DeShazer D, Kim HS, Tettelin H, Bowen D, Rocheleau TA, Blackburn M, Andreev mallei and Burkholderia mallei . J Clin Nelson KE, Feldblyum T, Ulrich RL, Ron-

O, Golubeva E, Bhartia R, Ffrench-Constant Microbiol 2003; 41: 2068–2079. ning CM, Brinkac LM, Daugherty SC, Da- RH: Insecticidal toxins from the bacterium Goris J, De Vos P, Caballero-Mellado J, Park J, vidsen TD, Deboy RT, Dimitrov G, Dodson

Photorhabdus luminescens . Science 1998; Falsen E, Quensen JF 3rd, Tiedje JM, Van- RJ, Durkin AS, Gwinn ML, Haft DH, Khou-

280: 2129–2132. damme P: Classification of the biphenyl- and ri H, Kolonay JF, Madupu R, Mohammoud Y, Bowien B, Kusian B: Genetics and control of CO 2 polychlorinated biphenyl-degrading strain Nelson WC, Radune D, Romero CM, Sarria assimilation in the chemoautotroph Ralsto- LB400T and relatives as Burkholderia xeno- S, Selengut J, Shamblin C, Sullivan SA, White

nia eutropha . Arch Microbiol 2002; 178: 85– vorans sp. nov. Int J Syst Evol Microbiol O, Yu Y, Zafar N, Zhou L, Fraser CM: Struc-

93. 2004; 54: 1677–1681. tural flexibility in the Burkholderia mallei

Bowien B, Schlegel HG: Physiology and bio- Grigoriev A: Analyzing genomes with cumula- genome. Proc Natl Acad Sci USA 2004; 101:

chemistry of aerobic hydrogen-oxidizing tive skew diagrams. Nucleic Acids Res 1998; 14246–14251.

bacteria. Annu Rev Microbiol 1981; 35: 405– 26: 2286–2290. Parkhill J, Sebaihia M, Preston A, Murphy LD, 452. Guo X, Flores M, Mavingui P, Fuentes SI, Her- Thomson N, Harris DE, Holden MT, Church- Brett PJ, DeShazer D, Woods DE: Burkholderia nandez G, Davila G, Palacios R: Natural ge- er CM, Bentley SD, Mungall KL, Cerdeno- thailandensis sp. nov., a Burkholderia pseu- nomic design in Sinorhizobium meliloti : Tarraga AM, Temple L, James K, Harris B, domallei -like species. Int J Syst Bacteriol novel genomic architectures. Genome Res Quail MA, Achtman M, Atkin R, Baker S,

1998; 48: 317–320. 2003; 13: 1810–1817. Basham D, Bason N, Cherevach I, Chilling- Chenna R, Sugawara H, Koike T, Lopez R, Gib- Hayward AC: Ralstonia solanacearum; in Ency- worth T, Collins M, Cronin A, Davis P, son TJ, Higgins DG, Thompson JD: Multiple clopedia of Microbiology. London, Academ- Doggett J, Feltwell T, Goble A, Hamlin N, sequence alignment with the Clustal series of ic Press, 2000, vol 4, pp 32–42. Hauser H, Holroyd S, Jagels K, Leather S,

programs. Nucleic Acids Res 2003; 31: 3497– Hogrefe C, Römermann D, Friedrich B: Alcali- Moule S, Norberczak H, O’Neil S, Ormond 3500. genes eutrophus genes (Hox). D, Price C, Rabbinowitsch E, Rutter S, Sand-

Cramm R: Genomic view of energy metabolism J Bacteriol 1984; 158: 43–48. ers M, Saunders D, Seeger K, Sharp S, Sim- in Ralstonia eutropha H16. J Mol Microbiol Holden MT, Titball RW, Peacock SJ, Cerdeno- monds M, Skelton J, Squares R, Squares S, Biotechnol 2009;16:38–52 . Tarraga AM, Atkins T, Crossman LC, Pitt T, Stevens K, Unwin L, Whitehead S, Barrell Dance DA: Melioidosis: the tip of the iceberg? Churcher C, Mungall K, Bentley SD, Sebai- BG, Maskell DJ: Comparative analysis of the

Clin Microbiol Rev 1991; 4: 52–60. hia M, Thomson NR, Bason N, Beacham IR, genome sequences of Bordetella pertussis , Del Solar G, Giraldo R, Ruiz-Echevarria MJ, Es- Brooks K, Brown KA, Brown NF, Challis GL, Bordetella parapertussis and Bordetella bron-

pinosa M, Diaz-Orejas R: Replication and Cherevach I, Chillingworth T, Cronin A, chiseptica . Nat Genet 2003; 35: 32–40. control of circular bacterial plasmids. Mi- Crossett B, Davis P, DeShazer D, Feltwell T, Pemberton JM, Corney B, Don RH: Evolution

crobiol Mol Biol Rev 1998; 62: 434–464. Fraser A, Hance Z, Hauser H, Holroyd S, Ja- and spread of pesticide degrading ability Delcher AL, Kasif S, Fleischmann RD, Peterson gels K, Keith KE, Maddison M, Moule S, among soil microorganisms; in Timmis KN, J, White O, Salzberg SL: Alignment of whole Price C, Quail MA, Rabbinowitsch E, Ruth- Pühler A (eds): Plasmids of Medical, Envi-

genomes. Nucleic Acids Res 1999; 27: 2369– erford K, Sanders M, Simmonds M, Songsi- ronmental and Commercial Importance. 2376. vilai S, Stevens K, Tumapa S, Vesaratchavest Amsterdam, Elsevier, 1979, pp 287–299. Dubarry N, Pasta F, Lane D: ParABS systems of M, Whitehead S, Yeats C, Barrell BG, Oyston Pohlmann A, Fricke WF, Reinecke F, Kusian B, the four replicons of Burkholderia cenocepa- PC, Parkhill J: Genomic plasticity of the Liesegang H, Cramm R, Eitinger T, Ewering cia : new chromosome centromeres confer causative agent of melioidosis, Burkholderia C, Pötter M, Schwartz E, Strittmatter A, Voss

partition specificity. J Bacteriol 2006;188: pseudomallei . Proc Natl Acad Sci USA 2004; I, Gottschalk G, Steinbüchel A, Friedrich B,

1489–1496. 101: 14240–14245. Bowien B: Genome sequence of the bioplas- Friedrich B, Hogrefe C, Schlegel HG: Naturally Johnson BF, Stanier RY: Dissimilation of aro- tic-producing ‘Knallgas’ bacterium Ralsto-

occurring genetic transfer of hydrogen-oxi- matic compounds by Alcaligenes eutrophus . nia eutropha H16. Nat Biotechnol 2006; 24:

dizing ability between strains of Alcaligenes J Bacteriol 1971; 107: 468–475. 1257–1262.

eutrophus . J Bacteriol 1981; 147: 198–205. Kiewitz C, Tümmler B: Sequence diversity of Porter JF, Wardlaw AC: Long-term survival of Fuller RS, Funnell BE, Kornberg A: The dnaA Pseudomonas aeruginosa: impact on popula- Bordetella bronchiseptica in lake water and protein complex with the E. coli chromo- tion structure and genome evolution. J Bac- in buffered saline without added nutrients.

somal replication origin (oriC ) and other teriol 2000; 182: 3125–3135. FEMS Microbiol Lett 1993; 110: 33–36.

DNA sites. Cell 1984; 38: 889–900. Kusian B, Bowien B: Organization and regula- Preston A, Parkhill J, Maskell DJ: The Bordetel- Ginard M, Lalucat J, Tümmler B, Römling tion of cbb CO2 assimilation genes in auto- lae: lessons from genomics. Nat Rev Micro-

U: Genome organization of Pseudomonas trophic bacteria. FEMS Microbiol Rev 1997; biol 2004; 2: 379–390.

stutzeri and resulting taxonomic and evolu- 21: 135–155. Rasko DA, Myers GS, Ravel J: Visualization of tionary considerations. Int J Syst Bacteriol Lee DG, Bell SP: ATPase switches controlling comparative genomic analyses by BLAST

1997; 47: 132–143. DNA replication initiation. Curr Opin Cell score ratio. BMC Bioinformatics 2005; 6: 2.

Biol 2000; 12: 280–285. Reinecke F, Steinbüchel A: Ralstonia eutropha MacLellan SR, Sibley CD, Finan TM: Second H16 as model organism for PHA metabolism chromosomes and megaplasmids in bacte- and for biotechnological production of tech- ria; in Funnell BE, Phillips GJ (eds): Plasmid nically interesting biopolymers. J Mol Mi- Biology. Washington, American Society for crobiol Biotechnol 2009;16:91–108 . Microbiology Press, 2004, pp 529–542.

134 J Mol Microbiol Biotechnol 2009;16:124–135 Fricke /Kusian /Bowien

Downloaded by: Niedersächsische Staats- und Universitätsbibliothek 134.76.162.165 - 10/2/2013 2:40:55 PM Salanoubat M, Genin S, Artiguenave F, Gouzy J, Schwartz E, Friedrich B: A physical map of the Vandamme P, Holmes B, Vancanneyt M, Coenye Mangenot S, Arlat M, Billault A, Brottier P, megaplasmid pHG1, one of three genomic T, Hoste B, Coopman R, Revets H, Lauwers Camus JC, Cattolico L, Chandler M, Choisne replicons in Ralstonia eutropha H16. FEMS S, Gillis M, Kersters K, Govan JR: Occur-

N, Claudel-Renard C, Cunnac S, Demange Microbiol Lett 2001; 201: 213–219. rence of multiple genomovars of Burkholde- N, Gaspin C, Lavie M, Moisan A, Robert C, Schwartz E, Friedrich B: The H2 -metabolizing ria cepacia in cystic fibrosis patients and pro- Saurin W, Schiex T, Siguier P, Thebault P, prokaryotes; in Dworkin M, Falkow S, posal of Burkholderia multivorans sp. nov.

Whalen M, Wincker P, Lev y M, Weissenbach Rosenberg E, et al (eds): The Prokaryotes. Int J Syst Bacteriol 1997; 47: 1188–1200. J, Boucher CA: Genome sequence of the New York, Springer, 2006, pp 496–563. Vaneechoutte M, Kämpfer P, De Baere T, Falsen plant pathogen Ralstonia solanacearum . Na- Schwartz E, Henne A, Cramm R, Eitinger T, E, Verschraegen G: Wautersia gen. nov., a

ture 2002; 415: 497–502. Friedrich B, Gottschalk G: Complete nucleo- novel genus accommodating the phylogenet- Sällström B, Andersson SGE: Genome reduction tide sequence of pHG1: a Ralstonia eutropha ic lineage including Ralstonia eutropha and in the alpha-Proteobacteria . Curr Opin Mi- H16 megaplasmid encoding key enzymes of related species, and proposal of Ralstonia

crobiol 2005; 8: 579–585. H 2 -based lithoautotrophy and anaerobiosis. [ Pseudomonas ] syzygii [Roberts et al., 1990]

Sato Y, Nishihara H, Yoshida M, Watanabe M, J Mol Biol 2003; 332: 369–383. comb. nov. Int J Syst Evol Microbiol 2004; 54: Rondal JD, Concepcion RN, Ohta H: Cupria- Stanier RY, Palleroni NJ, Doudoroff M: The aer- 317–327. vidus pinatubonensis sp. nov. and Cupriavi- obic pseudomonads: a taxonomic study. J Waack S, Keller O, Asper R, Brodag T, Damm C,

dus laharis sp. nov., novel hydrogen-oxidiz- Gen Microbiol 1966; 43: 159–271. Fricke WF, Surovcik K, Meinicke P, Merkl R: ing, facultatively chemolithotrophic bacteria Steinbüchel A, Schlegel HG: Physiology and mo- Score-based prediction of genomic islands in isolated from volcanic mudflow deposits lecular genetics of poly(␤ -hydroxyalkanoic prokaryotic genomes using hidden Markov

from Mt. Pinatubo in the Philippines. Int J acid) synthesis in Alcaligenes eutrophus . Mol models. BMC Bioinformatics 2006; 7: 142.

Syst Evol Microbiol 2006; 56: 973–978. Microbiol 1991; 5: 535–542. Yabuuchi E, Kosako Y, Oyaizu H, Yano I, Hotta Schmidt KD, Tümmler B, Römling U: Compara- Vandamme P, Coenye T: of the genus H, Hashimoto Y, Ezaki T, Arakawa M: Pro- tive genome mapping of Pseudomonas aeru- Cupriavidus : a tale of lost and found. Int J posal of Burkholderia gen. nov. and transfer

ginosa PAO with P. aeruginosa C, which be- Syst Evol Microbiol 2004; 54: 2285–2289. of seven species of the genus Pseudomonas longs to a major clone in cystic fibrosis homology group II to the new genus, with patients and aquatic habitats. J Bacteriol the type species Burkholderia cepacia [Pal-

1996; 178: 85–93. leroni and Holmes, 1981] comb. nov. Micro-

biol Immunol 1992; 36: 1251–1275.

Genome Organization of Ralstonia J Mol Microbiol Biotechnol 2009;16:124–135 135 eutropha Downloaded by: Niedersächsische Staats- und Universitätsbibliothek 134.76.162.165 - 10/2/2013 2:40:55 PM