Open Access

Genome sequence of the Leisingera aquimarina type strain (DSM 24565T), a member of the marine Roseobacter clade rich in extrachromosomal elements

  • Thomas Riedel
  • , Hazuki Teshima
  • , Jörn Petersen
  • , Anne Fiebig
  • , Karen Davenport
  • , Hajnalka Daligault
  • , Tracy Erkkila
  • , Wei Gu
  • , Christine Munk
  • , Yan Xu
  • , Amy Chen
  • , Amrita Pati
  • , Natalia Ivanova
  • , Lynne A. Goodwin,
  • , Patrick Chain
  • , John C. Detter,
  • , Manfred Rohde
  • , Sabine Gronow
  • , Nikos C. Kyrpides
  • , Tanja Woyke
  • , Markus Göker
  • , Thorsten Brinkhoff
  • and Hans-Peter Klenk
Corresponding author

DOI: 10.4056/sigs.3858183

Received: 30 July 2013

Accepted: 30 July 2013

Published: 30 July 2013


Leisingera aquimarina Vandecandelaere et al. 2008 is a member of the genomically well characterized Roseobacter clade within the family Rhodobacteraceae. Representatives of the marine Roseobacter clade are metabolically versatile and involved in carbon fixation and biogeochemical processes. They form a physiologically heterogeneous group, found predominantly in coastal or polar waters, especially in symbiosis with algae, in microbial mats, in sediments or associated with invertebrates. Here we describe the features of L. aquimarina DSM 24565T together with the permanent-draft genome sequence and annotation. The 5,344,253 bp long genome consists of one chromosome and an unusually high number of seven extrachromosomal elements and contains 5,129 protein-coding and 89 RNA genes. It was sequenced as part of the DOE Joint Genome Institute Community Sequencing Program 2010 and of the activities of the Transregional Collaborative Research Centre 51 funded by the German Research Foundation (DFG).


marinebiofilmovoid-shapedhalotolerantheterotrophicquorum sensingplasmidthiosulfate oxidationcarbon monoxide utilizationRhodobacteraceaeAlphaproteobacteria


Strain R-26159T (= DSM 24565T = LMG 24366T = CCUG 55860T) is the type strain of the species Leisingera aquimarina [1], one of the three species currently with a validly published name in the genus Leisingera; the other ones are the type species L. methylohalidivorans [1,2] and L. nanhaiensis [3]. The genus Leisingera is a member of the widespread Roseobacter clade, present in various marine habitats [4]. Strain R-26159T was isolated from a marine electroactive biofilm grown on a stainless-steel cathode, which was exposed to natural seawater at the ISMAR-CNR Marine Station within the harbor of Genova (Italy) [1]. The genus Leisingera was named after Thomas Leisinger for his work on the bacterial methyl halide metabolism [2]; the species epithet aquimarina refers to the Neolatin adjective marinus, from the sea, from seawater. PubMed records do not currently indicate any follow-up research with strain R-26159T after the initial description of L. aquimarina [1].

Here we present a summary classification and a set of features for L. aquimarina DSM 24565T, together with the description of the genomic sequencing and annotation.

Classification and features of the organism

16S rRNA analysis

A representative genomic 16S rRNA gene sequence of L. aquimarina DSM 24565T was compared using NCBI BLAST [5,6] under default settings (e.g., considering only the high-scoring segment pairs (HSPs) from the best 250 hits) with the most recent release of the Greengenes database [7] and the relative frequencies of taxa and keywords (reduced to their stem [8]) were determined, weighted by BLAST scores. The most frequently occurring genera were Phaeobacter (31.4%), Ruegeria (25.9%), Silicibacter (16.1%), Roseobacter (14.4%) and Nautella (3.9%) (127 hits in total). Regarding the four hits to sequences from other members of the genus, the average identity within HSPs was 99.4%, whereas the average coverage by HSPs was 99.3%. Among all other species, the one yielding the highest score was Leisingera methylohalidivorans (NR_025637), which corresponded to an identity of 99.2% and an HSP coverage of 100.0%. (Note that the Greengenes database uses the INSDC (= EMBL/NCBI/DDBJ) annotation, which is not an authoritative source for nomenclature or classification.) The highest-scoring environmental sequence was FJ202534 (Greengenes short name 'and White Plague Disease-Induced Changes Caribbean Coral Montastraea faveolata kept aquarium 23 days clone SGUS1024'), which showed an identity of 97.8% and an HSP coverage of 100.0%. The most frequently occurring keywords within the labels of all environmental samples which yielded hits were 'coral' (4.7%), 'caribbean' (3.8%), 'faveolata' (3.5%), 'chang' (3.4%) and 'white' (3.3%) (117 hits in total). Environmental samples which yielded hits of a higher score than the highest scoring species were not found, which might indicate that the species is rarely found in the environment.

Figure 1 shows the phylogenetic neighborhood of L. aquimarina in a 16S rRNA gene based tree. The sequences of the four identical 16S rRNA gene copies in the genome do not differ from the previously published 16S rRNA gene sequence AM900415.

Figure 1

Phylogenetic tree highlighting the position of L. aquimarina relative to the type strains of the other species within the genus Leisingera and the neighboring genera Phaeobacter and Ruegeria. The tree was inferred from 1,383 aligned characters [9,10] of the 16S rRNA gene sequence under the maximum likelihood (ML) criterion [11]. Rooting was done initially using the midpoint method [12] and then checked for its agreement with the current classification (Table 1). The branches are scaled in terms of the expected number of substitutions per site. Numbers adjacent to the branches are support values from 1,000 ML bootstrap replicates [13] (left) and from 1,000 maximum-parsimony bootstrap replicates [14] (right) if larger than 60%. Lineages with type strain genome sequencing projects registered in GOLD [15] are labeled with one asterisk, those also listed as 'Complete and Published' with two asterisks [16,17]. The genomes of P. caeruleus [58] and P. arcticus [59] are reported in this issue.

Table 1

Classification and general features of L. aquimarina DSM 24565T according to the MIGS recommendations [18].




     Evidence code

     Domain Bacteria

     TAS [19]

     Phylum Proteobacteria

     TAS [20]

     Class Alphaproteobacteria

     TAS [21,22]


     Order Rhodobacterales

     TAS [22,23]

     Family Rhodobacteraceae

     TAS [22,24]

     Genus Leisingera

     TAS [25,26]

     Species Leisingera aquimarina

     TAS [1]


      Subspecific genetic lineage (strain)


     TAS [1]


      Reference for biomaterial

     Vandecandelaere et al. 2008

     TAS [1]

      Current classification

      Gram stain


     TAS [1]

      Cell shape


     TAS [1]



     TAS [1]


     Not reported

      Temperature range

     Mesophile (4 – 37°C)

     TAS [1]

      Optimum temperature




     Halophile, 1-7% NaCl (w/v)

     TAS [1]


      Relationship to oxygen


     TAS [1]

      Carbon source

     Yeast extract, peptone, betaine

     TAS [1]



     Seawater, biofilm

     TAS [1]



     6.5 – 8.0

     TAS [1]


      Biotic relationship

     Free living

     TAS [1]

      Biosafety level


     TAS [27]



     Marine biofilm on stainless steel cathode

     TAS [1]


      Geographic location

     ISMAR-CNR Marine Station, Genoa harbor, Italy

     TAS [1]




     TAS [1]




     TAS [1]



     Not reported

Evidence codes - TAS: Traceable Author Statement (i.e., a direct report exists in the literature); NAS: Non-traceable Author Statement (i.e., not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). Evidence codes are from the Gene Ontology project [28].

Morphology and physiology

Cells of strain R-26159T are Gram-negative, ovoid (1 × 1.4 µm) and contain a single polar flagellum (not visible in Figure 2), which is used for motility. Poly-β-hydroxybutyrate is present in inclusion bodies. Colonies are dark beige-pink in color, round and 1–2 mm in diameter after 3 days incubation on marine agar (MA). Growth occurs after 2 days incubation at 20 °C on MA, but not on Reasoner’ 2A agar (R2A), Nutrient agar (NA), Trypticase-Soy agar (TSA) or Peptone-Yeast Extract-Glucose agar (PYG). The temperature range for growth is 4–37°C whereas no growth occurs at 40°C or higher. The salinity range for growth is 1–7% NaCl. The pH range for growth is 5.5–9.0 with an optimum between 6.5–8. Growth occurs on betaine (1 mM) as a sole carbon source, but not on L-methionine (10 mM). Cells are catalase- and oxidase-positive. Degradation of gelatin is weakly positive but cells do not degrade tyrosine, DNA, starch, casein, chitin, aesculin or Tween 80. The strain shows leucine arylamidase activity; weak alkaline phosphatase, esterase lipase (C8) and naphthol-AS-BI phosphohydrolase activities. No activity is detected for esterase (C4), valine arylamidase, acid phosphatase, α-galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase, lipase (C14), cystine arylamidase, trypsin, α-chymotrypsin, arginine dihydrolase, urease or α-fucosidase. Nitrate is not reduced to nitrite or nitrogen. Indole is not produced and glucose is not fermented. Cells do not assimilate D-glucose, L-arabinose, D-mannose, D-mannitol, N-acetylglucosamine, maltose, potassium gluconate, capric acid, adipic acid, malate, citrate or phenylacetic acid. Cells are susceptible to cefoxitin (30 mg), erythromycin (15 mg), tetracycline (30 mg) and streptomycin (25 mg), but resistant to vancomycin (30 mg), trimethoprim (1.25 mg), clindamycin (2 mg) and gentamicin (30 mg) (all data from [1]).

Figure 2

Scanning electron micrograph of L. aquimarina DSM 24565T.

The utilization of carbon compounds by L. aquimarina DSM 24565T grown at 20°C was also determined for this study using Generation-III microplates in an OmniLog phenotyping device (BIOLOG Inc., Hayward, CA, USA). The microplates were inoculated at 28°C with a cell suspension at a cell density of 95-96% turbidity and dye IF-A. Further additives were vitamin, micronutrient and sea-salt solutions. The exported measurement data were further analyzed with the opm package for R [29,60], using its functionality for statistically estimating parameters from the respiration curves and translating them into negative, ambiguous, and positive reactions. The strain was studied in two independent biological replicates, and reactions with a different behavior between the two repetitions were regarded as ambiguous. At 28°C the strain reacted poorly, with positive reactions only for 1% NaCl, 4% NaCl and lithium chloride. This is in accordance with the comparatively low median of the temperature range of the strain [1].


The principal cellular fatty acids of strain R-26159T are mono-unsaturated straight chain acids: C18:1 ω7c (71.6%), C14:1 iso E (11.6%), C14:1 2-OH (4.2%), C16:0 2-OH (4.2%), C16:0 (3.5%), an unknown fatty acid of equivalent chain-length 11.799 (2.7%), C12:0 3-OH (2.1%) as well as C10:0 3-OH (2.0%) [26]. Remaining fatty acids were detected in very small fractions only (<1.0%) [1]. The same predominant fatty acids were also found in other members of the Phaeobacter-Leisingera cluster [2,26,30].

Genome sequencing and annotation

Genome project history

This organism was selected for sequencing on the basis of the DOE Joint Genome Institute Community Sequencing Program 2010, CSP 441: “Whole genome type strain sequences of the genera Phaeobacter and Leisingera – a monophyletic group of highly physiologically diverse organisms”. The genome project is deposited in the GenomesOnLine Database [15] and the complete genome sequence was submitted to GenBank. Sequencing, finishing and annotation were performed by the DOE Joint Genome Institute (JGI). A summary of the project information is shown in Table 2.

Table 2

Genome sequencing project information





    Finishing quality

     Improved high quality draft


    Libraries used

     Two genomic libraries: Illumina standard (short PE), Illumina CLIP (long PE)


    Sequencing platforms

     Illumina HiSeq 2000, PacBio


    Sequencing coverage

     699 × Illumina; unknown × PacBio



     Allpath version 39750, Velvet 1.1.05, phrap version SPS - 4.24


    Gene calling method

     Prodigal 1.4, GenePRIMP



    GenBank Date of Release




    NCBI project ID


    Database: IMG



    Source material identifier

     DSM 24565T

    Project relevance

     Tree of Life, carbon cycle, sulfur cycle, environmental

Growth conditions and DNA isolation

A culture of L. aquimarina DSM 24565T was grown in the DSMZ medium 514 (BACTO Marine Broth) [31] at 20°C. Genomic DNA was isolated from 0.5-1 g of cell paste using Jetflex Genomic DNA Purification Kit (GENOMED 600100) following the standard protocol provided by the manufacturer but modified by the use of additional 20 µl proteinase K and 40 minute incubation. DNA is available through the DNA Bank Network [32].

Genome sequencing and assembly

The draft genome was generated using Illumina data [33]. For this genome, we constructed and sequenced an Illumina short-insert paired-end library with an average insert size of 270 bp which generated 13,668,574 reads and an Illumina long-insert paired-end library with an average insert size of 8047.58 +/- 2682.23 bp which generated 11,512,166 reads totaling 3,777 Mbp of Illumina data (Feng Chen, unpublished). All general aspects of library construction and sequencing can be found at the JGI web site [34]. The initial draft assembly contained 64 contigs in 18 scaffold(s). The initial draft data was assembled with Allpaths [35] and the consensus was computationally shredded into 10 kbp overlapping fake reads (shreds). The Illumina draft data was also assembled with Velvet [36], and the consensus sequences were computationally shredded into 1.5 kbp overlapping fake reads (shreds). The Illumina draft data was assembled again with Velvet using the shreds from the first Velvet assembly to guide the next assembly. The consensus from the second Velvet assembly was shredded into 1.5 kbp overlapping fake reads. The fake reads from the Allpaths assembly and both Velvet assemblies and a subset of the Illumina CLIP paired-end reads were assembled using parallel phrap (High Performance Software, LLC) [37]. Possible mis-assemblies were corrected with manual editing in Consed [34,36,37]. Gap closure was accomplished using repeat resolution software (Wei Gu, unpublished), and sequencing of bridging PCR fragments with PacBio (Cliff Han, unpublished) technologies. A total of 57 PCR PacBio consensus sequences were completed to close gaps and to raise the quality of the final sequence. The final assembly is based on 3,777 Mbp of Illumina draft data, which provides an average 699 × coverage of the genome.

Genome annotation

Genes were identified using Prodigal [38] as part of the JGI genome annotation pipeline [39], followed by a round of manual curation using the JGI GenePRIMP pipeline [40]. The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) nonredundant database, UniProt, TIGR-Fam, Pfam, PRIAM, KEGG, COG, and InterPro databases. Additional gene prediction analysis and functional annotation was performed within the Integrated Microbial Genomes - Expert Review (IMG-ER) platform [41].

Genome properties

The genome statistics are provided in Table 3 and Figure 3. The genome consists of a 4.25 Mbp chromosome and seven extrachromosomal elements of 6.2 to 248.9 kbp length with a G+C content of 61.4%. Of the 5,218 genes predicted, 5,129 were protein-coding genes, and 89 RNAs. The majority of the protein-coding genes (80.4%) were assigned a putative function while the remaining ones were annotated as hypothetical proteins. The distribution of genes into COGs functional categories is presented in Table 4.

Table 3

Genome Statistics



      % of Total

Genome size (bp)



DNA coding region (bp)



DNA G+C content (bp)



Number of scaffolds


Extrachromosomal elements


Total genes



RNA genes



rRNA operons


tRNA genes



Protein-coding genes



Genes with function prediction (proteins)



Genes in paralog clusters



Genes assigned to COGs



Genes assigned Pfam domains



Genes with signal peptides



Genes with transmembrane helices



CRISPR repeats


Figure 3

Graphical map of the chromosome. From bottom to the top: Genes on forward strand (colored by COG categories), Genes on reverse strand (colored by COG categories), RNA genes (tRNAs green, rRNAs red, other RNAs black), GC content (black), GC skew (purple/olive).

Table 4

Number of genes associated with the general COG functional categories








       Translation, ribosomal structure and biogenesis




       RNA processing and modification








       Replication, recombination and repair




       Chromatin structure and dynamics




       Cell cycle control, cell division, chromosome partitioning




       Nuclear structure




       Defense mechanisms




       Signal transduction mechanisms




       Cell wall/membrane/envelope biogenesis




       Cell motility








       Extracellular structures




       Intracellular trafficking and secretion, and vesicular transport




       Posttranslational modification, protein turnover, chaperones




       Energy production and conversion




       Carbohydrate transport and metabolism




       Amino acid transport and metabolism




       Nucleotide transport and metabolism




       Coenzyme transport and metabolism




       Lipid transport and metabolism




       Inorganic ion transport and metabolism




       Secondary metabolites biosynthesis, transport and catabolism




       General function prediction only




       Function unknown




       Not in COGs

Insights into the genome

Genome sequencing of Leisingera aquimarina DSM 24565T reveals the presence of seven plasmids with sizes between 6 kb and 249 kb (Table 5). The circular conformation of the chromosome and the two smallest extrachromosomal elements has been experimentally validated. The six larger plasmids contain characteristic replication modules [42] of the RepABC-, DnaA-like, RepA- and RepB-type comprising a replicase as well as the parAB partitioning operon [43]. The respective replicases that mediate the initiation of replication are designated according to the established plasmid classification scheme [44]. The different numbering of e.g. the replicases RepC-8, RepC-13 and RepC-14 from RepABC-type plasmids corresponds to specific plasmid compatibility groups that are required for a stable coexistence of the replicons within the same cell [45]. The cryptic 6 kb plasmid pAqui_G6 contains a solitary RepA-II type replicase without a partitioning module, but replicon maintenance in the daughter cells is probably ensured by its postsegregational killing system (PSK) consisting of a typical operon with two small genes encoding a stable toxin and an unstable antitoxin [46]. PSK systems are also located on pAqui_C182 and pAqui_F126 (Tab. 6).

Table 5

General genomic features of the chromosome and extrachromosomal replicons from Leisingera aquimarina strain DSM 24565T.




    Length (bp)

    GC (%)


   No. Genes#













































    DnaA-like I












*Circularity not experimentally validated; #deduced from automatic annotation.

Table 6

Integrated Microbial Genome (IMG) locus tags of L. aquimarina DSM 24565T genes for the initiation of replication, toxin/antitoxin modules and two representatives of type IV secretion systems (T4SS) that are required for conjugation. The locus tags are accentuated in blue1,2,3.

     Replication Initiation

     Plasmid Stability

    Type IV Secretion



     Locus Tag










     Aqui _3705

    Aqui _3720³






     Aqui _4685²

    Aqui _4598³






























    DnaA-like I











¹solitary replicase without partitioning module; ²traC gene of F factor conjugation system; 3presence of adjacent DNA relaxase VirD2.

The locus tags of all replicases, plasmid stability modules and the large virB4 and virD4 genes of type IV secretion systems are presented in Table 6. A characteristic T4SS comprising the relaxase VirD2 and the coupling protein VirD4 as well as the complete virB gene cluster for the transmembrane channel is located on the chromosome [47]. Its functional role is unclear, but very closely related T4SS are detected on plasmids of e.g. Dinoroseobacter shibae DSM 16493T [61], Leisingera nanhaiensis DSM 24252T and Phaeobacter caeruleus DSM 24564T [48]. Furthermore, the largest plasmid pAqui_A249 contains the complete F factor conjugation transfer (tra) region with 20 genes (Aqui_4678 to Aqui_4697). It exhibits only weak homology with the typical type IV secretion system of the Roseobacter clade, which is represented by the chromosomal counterpart, but it resembles the F sex factor of Escherichia coli that is the paradigm for bacterial conjugation [49].

The 243 kb RepABC-13 type plasmid pAqui_B243 is predominated by seven ABC-transporters. Even more conspicuous is the presence of a couple of pentose phosphate pathway genes including an operon of genes of the Entner-Doudoroff pathway (Aqui_4914 to Aqui_4917; EC; EC; EC; EC that is generally used in Roseobacters to convert D-fructose-6-phosphate to D-glyceraldehyde-3-phosphate [50]. The exclusive missing gene within this operon is the chromosome encoded 6-phosphogluconolactonase (Aqui_2983; EC The presence of a glycolytic 6-phosphofructokinase (PFK; Aqui_4950; EC is a genetic novelty in this group of marine bacteria, because the current opinion was that “the typical pfk gene is absent from all sequenced Roseobacter clade genomes and glucose is hence probably catabolized via the Entner–Doudoroff pathway and not via classical glycolysis” [51]. However, the putative functionality of the Embden-Meyerhoff-Parnas pathway (glycolysis) has to be validated e.g. via pulse-chase experiments with 13C labeled glucose [52]. Finally, the plasmid pAqui_B243 contains the phosphoenolpyruvate synthase (Aqui_4951; EC that is required together with the chromosomal phosphoenolpyruvate carboxylase (Aqui_0364; EC for prokaryotic CO2 fixation and the formation of oxaloacetate from pyruvate.

The 148 kb RepB-I type plasmid pAqui_D148 contains a complete rhamnose operon [47] and many genes that are required for polysaccharide biosynthesis. This extrachromosomal replicon also harbors two siderophore synthetase genes (Aqui_4320; Aqui_4321), two outer membrane receptors for Fe-transport (Aqui_4319; Aqui_4360) and genes of a putative ABC-type Fe3+ siderophore transport system (Aqui_4361 to Aqui_4364).

The 140 kb RepA-I type plasmid pAqui_E140 is largely predominated by glycosyltransferases, polysaccharide biosynthesis as well as cell-wall biogenesis genes, and it contains an operon for GDP-mannose metabolism (Aqui_5058 to Aqui_5055).

The 126 kb DnaA-like I replicon pAqui_F126 contains a large type VI secretion system (T6SS) with a size of about 30 kb. The role of this export system that has been first described in the context of bacterial pathogenesis, but recent findings indicate a more general physiological role in defense against eukaryotic cells and other bacteria in the environment [53]. Homologous T6S systems are present on the DnaA-like I plasmids of Leisingera methylohalidivorans DSM 14336T (pMeth_A285) and Phaeobacter caeruleus DSM 24564T (pCaer_C109) as well as the RepC-8 type plasmid of Phaeobacter daeponensis DSM23529T (pDaep_A276).

Genome analysis of strain L. aquimarina DSM 24565T revealed further the presence of genes encoding LuxI as well as LuxR homologues, which are involved in quorum sensing (QS), an already known feature of several members of the Roseobacter clade [54]. QS is a bacterial communication system used by many bacterial species to coordinate special behaviors based on bacterial population density [54]. Whereas two genes encode a N-acyl-L-homoserine lactone synthase (LuxI, Aqui_0074, Aqui_4264), some genes were identified to encode LuxR homologues (response and transcriptional regulators, e.g., Aqui_0075 and Aqui_3114).

Furthermore, several genes forming a putative operon are involved in the oxidation of (e.g., Aqui_3422 to Aqui_3426) indicating the oxidation of thiosulfate into sulfate to produce energy. Additionally genes for carbon monoxide utilization (Aqui_2391 and Aqui_2392, Aqui_2518, Aqui_2520, Aqui_3522, Aqui_5216 and Aqui_5217) were observed.

Interestingly, also a gene encoding a sensor of blue light using FAD (BLUF, Aqui_2375) was detected, indicating possible blue-light depending signal transduction.

As indicated by the 16S rRNA gene sequence analysis (Figure 1), the classification of some Leisingera and Phaeobacter species might need to be reconsidered. We conducted a preliminary phylogenomic analysis with GGDC [55-57] applied to the genome of L. aquimarina DSM 24565T and the draft genomes of the type strains of the other Leisingera and Phaeobacter species. The results shown in Table 7 indicate that the DNA-DNA hybridization (DDH) similarities calculated in silico of L. aquimarina to Phaeobacter caeruleus and P. daeponensis species are higher than those to L. nanhaiensis, confirming the 16S rRNA gene sequence analysis. Thus a taxonomic revision of L. aquimarina might be warranted.

Table 7

DDH similarities between L. aquimarina DSM 24565T and the other Leisingera and Phaeobacter species (with genome-sequenced type strains)

Reference species

     formula 1

     formula 2

      formula 3

L. nanhaiensis (2512047090)




L. methylohalidivorans (2512564009)




P. arcticus (2516653081)




P. caeruleus (2512047087)




P. daeponensis (2516493020)




P. gallaeciensis (AOQA01000000)




P. inhibens (2516653078)




DDH similarities were calculated in silico with the GGDC server version 2.0 [57]. The standard deviations indicate the inherent uncertainty in estimating DDH values from intergenomic distances based on models derived from empirical test data sets (which are always limited in size); see [56] for details. The distance formulas are explained in [56]. The numbers in parentheses are IMG object IDs (GenBank accession number in the case of P. gallaeciensis) identifying the underlying genome sequences.



The authors would like to gratefully acknowledge the assistance of Iliana Schröder for growing L. aquamarina cultures and Evelyne-Marie Brambilla for DNA extraction and quality control (both at the DSMZ). The work conducted by the U.S. Department of Energy Joint Genome Institute was supported by the Office of Science of the U.S. Department of Energy under contract No. DE-AC02-05CH11231; the work conducted by the members of the Roseobacter consortium was supported by the German Research Foundation (DFG) Transregio-SFB 51. We also thank the European Commission which supported phenotyping via the Microme project 222886 within the Framework 7 program.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


  1. Vandecandelaere I, Segaert E, Mollica A, Faimali M and Vandamme P.. Leisingera aquimarina sp. nov., isolated form a marine electroactive biofilm, and emended descriptions of Leisingera methylohalidivorans Schaefer et al. 2002, Phaeobacter daeponensis Yoon et al. 2007 and Phaeobacter inhibens Martens et al. 2006. Int J Syst Evol Microbiol. 2008; 58:2788-2793 View ArticlePubMed
  2. Schaefer JK, Goodwin KD, McDonald IR, Murrell JC and Oremland RS. Leisingera methylohalidivorans gen. nov., sp. nov., a marine methylotroph that grows on methyl bromide. Int J Syst Evol Microbiol. 2002; 52:851-859 View ArticlePubMed
  3. Sun F, Wang B, Liu X, Lai Q, Du Y, Li G, Luo J and Shao Z. Leisingera nanhaiensis sp. nov., isolated from marine sediment. Int J Syst Evol Microbiol. 2010; 60:275-280 View ArticlePubMed
  4. Buchan A, González JM and Moran MA. Overview of the marine Roseobacter lineage. Appl Environ Microbiol. 2005; 71:5665-5677 View ArticlePubMed
  5. Altschul SF, Gish W, Miller W, Myers EW and Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990; 215:403-410PubMed
  6. Korf I, Yandell M, Bedell J. BLAST, O'Reilly, Sebastopol, 2003.
  7. DeSantis TZ, Hugenholtz P, Larsen N, Rojas M, Brodie EL, Keller K, Huber T, Dalevi D, Hu P and Andersen GL. Greengenes, a Chimera-Checked 16S rRNA Gene Database and Workbench Compatible with ARB. Appl Environ Microbiol. 2006; 72:5069-5072 View ArticlePubMed
  8. Porter MF. An algorithm for suffix stripping. Program: electronic library and information systems 1980; 14:130-137.
  9. Lee C, Grasso C and Sharlow MF. Multiple sequence alignment using partial order graphs. Bioinformatics. 2002; 18:452-464 View ArticlePubMed
  10. Castresana J. Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Mol Biol Evol. 2000; 17:540-552 View ArticlePubMed
  11. Stamatakis A, Hoover P and Rougemont J. A rapid bootstrap algorithm for the RAxML web-servers. Syst Biol. 2008; 57:758-771 View ArticlePubMed
  12. Hess PN and De Moraes Russo CA. An empirical test of the midpoint rooting method. Biol J Linn Soc Lond. 2007; 92:669-674 View Article
  13. Pattengale ND, Alipour M, Bininda-Emonds ORP, Moret BME and Stamatakis A. How Many Bootstrap Replicates Are Necessary? Lect Notes Comput Sci. 2009; 5541:184-200 View Article
  14. Swofford DL. PAUP*: Phylogenetic Analysis Using Parsimony (*and Other Methods), Version 4.0 b10. Sinauer Associates, Sunderland, 2002.
  15. Pagani I, Liolios K, Jansson J, Chen IM, Smirnova T, Nosrat B, Markowitz VM and Kyrpides NC. The GenomesOnLine Database (GOLD) v.4: status of genomic and metagenomic projects and their associated metadata. Nucleic Acids Res. 2012; 40:D571-D579 View ArticlePubMed
  16. Ruiz-Ponte C, Cilia V, Lambert C and Nicolas JL. Roseobacter gallaciensis sp. nov., a marine bacterium isolated from rearings and collectors of the scallop Pecten maximus. Int J Syst Bacteriol. 1998; 48:537-542 View ArticlePubMed
  17. Moran MA, Buchan A, González JM, Heidelbarg JF, Witman WB, Kiene JR, Henriksen JR, King GM, Belas R and Fuqua C. Genome sequence of Silicibacter pomeroyi reveals adaptation to the marine environment. Nature. 2004; 432:910-913 View ArticlePubMed
  18. Field D, Garrity G, Gray T, Morrison N, Selengut J, Sterk P, Tatusova T, Thomson N, Allen MJ and Angiuoli SV. The minimum information about a genome sequence (MIGS) specification. Nat Biotechnol. 2008; 26:541-547 View ArticlePubMed
  19. Woese CR, Kandler O and Weelis ML. Towards a natural system of organisms. Proposal for the domains Archaea and Bacteria. Proc Natl Acad Sci USA. 1990; 87:4576-4579 View ArticlePubMed
  20. Garrity GM, Bell JA, Lilburn T. Phylum XIV. Proteobacteria phyl nov. In: Brenner DJ, Krieg NR, Stanley JT, Garrity GM (eds), Bergey’s Manual of Sytematic Bacteriology, second edition. Vol. 2 (The Proteobacteria), part B (The Gammaproteobacteria), Springer, New York, 2005, p. 1.
  21. Garrity GM, Bell JA, Lilburn T. Class I. Alphaproteobacteria class. nov. In: Brenner DJ, Krieg NR, Stanley JT, Garrity GM (eds), Bergey’s Manual of Sytematic Bacteriology, second edition. Vol. 2 (The Proteobacteria), part C (The Alpha-, Beta-, Delta-, and Epsilonproteobacteria), Springer, New York, 2005, p. 1.
  22. Validation list No. 107. List of new names and new combinations previously effectively, but not validly, published. Int J Syst Evol Microbiol. 2006; 56:1-6 View ArticlePubMed
  23. Garrity GM, Bell JA, Lilburn T. Order III. Rhodobacterales ord. nov. In: Brenner DJ, Krieg NR, Staley JT, Garrity GM (eds), Bergey’s Manual of Systematic Bacteriology, second edition. vol. 2 (The Proteobacteria), part C (The Alpha-, Beta-, Delta-, and Epsilonproteobacteria), Springer, New York, 2005, p. 161.
  24. Garrity GM, Bell JA, Lilburn T. Family I. Rhodobacteraceae fam. nov. In: Brenner DJ, Krieg NR, Staley JT, Garrity GM (eds), Bergey’s Manual of Systematic Bacteriology, second edition. vol. 2 (The Proteobacteria), part C (The Alpha-, Beta-, Delta-, and Epsilonproteobacteria), Springer, New York, 2005, p. 161.
  25. Schaefer JK, Goodwin KD, McDonald IR, Murrell JC and Oremland RS. Leisingera methylohalidivorans gen. nov., sp. nov., a marine methylotroph that grows on methyl bromide. Int J Syst Evol Microbiol. 2002; 52:851-859 View ArticlePubMed
  26. Martens T, Heidorn T, Pukall R, Simon M, Tindall BJ and Brinkhoff T. Reclassification of Roseobacter gallaeciensis Ruiz-Ponte et al 1998 as Phaeobacter gallaeciensis gen. nov., comb. nov., description of Phaeobacter inhibens sp. nov., reclassification of Ruegeria algicola (Lafay et al. 1995) Uchino et al. 1999 as Marinovum algicola gen. nov., comb. nov., and emended descriptions of the genera Roseobacter, Ruegeria and Leisingera. Int J Syst Evol Microbiol. 2006; 56:1293-1304 View ArticlePubMed
  27. . Classification of Bacteria and Archaea in risk groups. TRBA. 2010; 466:93
  28. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS and Eppig JT. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet. 2000; 25:25-29 View ArticlePubMed
  29. Vaas LAI, Sikorski J, Michael V, Göker M and Klenk HP. Visualization and curve-parameter estimation strategies for efficient exploration of phenotype microarray kinetics. PLoS ONE. 2012; 7:e34846 View ArticlePubMed
  30. Yoon JH, Kang SJ, Lee SY and Oh TK. Phaeobacter daeponensis sp. nov., isolated from a tidal flat of the Yellow Sea in Korea. Int J Syst Evol Microbiol. 2007; 57:856-861 View ArticlePubMed
  31. List of growth media used at the DSMZ: Web Site
  32. Gemeinholzer B, Dröge G, Zetzsche H, Haszprunar G, Klenk HP, Güntsch A, Berendsohn WG and Wägele JW. The DNA Bank Network: the start from a German initiative. Biopreserv Biobank. 2011; 9:51-55 View Article
  33. Bennett S. Solexa Ltd. Pharmacogenomics. 2004; 5:433-438 View ArticlePubMed
  34. . Web Site
  35. Butler J, MacCallum I, Kleber M, Shlyakhter IA, Belmonte MK, Lander ES, Nusbaum C and Jaffe DB. ALLPATHS: de novo assembly of whole-genome shotgun microreads. Genome Res. 2008; 18:810-820 View ArticlePubMed
  36. Zerbino DR and Birney E. Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res. 2008; 18:821-829 View ArticlePubMed
  37. Phrap and Phred for Windows. MacOS, Linux, and Unix. Web Site
  38. Hyatt D, Chen GL, LoCascio PF, Land ML, Larimer FW and Hauser LJ. Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics. 2010; 11:119 View ArticlePubMed
  39. Mavromatis K, Ivanova NN, Chen IM, Szeto E, Markowitz VM and Kyrpides NC. The DOE-JGI Standard operating procedure for the annotations of microbial genomes. Stand Genomic Sci. 2009; 1:63-67 View ArticlePubMed
  40. Pati A, Ivanova NN, Mikhailova N, Ovchinnikova G, Hooper SD, Lykidis A and Kyrpides NC. GenePRIMP: a gene prediction improvement pipeline for prokaryotic genomes. Nat Methods. 2010; 7:455-457 View ArticlePubMed
  41. Markowitz VM, Ivanova NN, Chen IMA, Chu K and Kyrpides NC. IMG ER: a system for microbial genome annotation expert review and curation. Bioinformatics. 2009; 25:2271-2278 View ArticlePubMed
  42. del Solar G, Giraldo R, Ruiz-Echevarria MJ, Espinosa M and Diaz-Orejes R. Replication and control of circular bacterial plasmids. Microbiol Mol Biol Rev. 1998; 62:434-464PubMed
  43. Petersen J, Brinkmann H, Berger M, Brinkhoff T, Päuker O and Pradella S. Origin and evolution of a novel DnaA-like plasmid replication type in Rhodobacterales. Mol Biol Evol. 2011; 28:1229-1240 View ArticlePubMed
  44. Petersen J. Phylogeny and compatibility: plasmid classification in the genomics era. Arch Microbiol. 2011; 193:313-321PubMed
  45. Petersen J, Brinkmann H and Pradella S. Diversity and evolution of repABC type plasmids in Rhodobacterales. Environ Microbiol. 2009; 11:2627-2638 View ArticlePubMed
  46. Zielenkiewicz U and Ceglowski P. Mechanisms of plasmid stable maintenance with special focus on plasmid addiction systems. Acta Biochim Pol. 2001; 48:1003-1023PubMed
  47. Giraud MF and Naismith JH. The rhamnose pathway. Curr Opin Struct Biol. 2000; 10:687-696 View ArticlePubMed
  48. Petersen J, Frank O, Göker M and Pradella S. Extrachromosomal, extraordinary and essential-the plasmids of the Roseobacter clade. Appl Microbiol Biotechnol. 2013; 97:2805-2815 View ArticlePubMed
  49. Lawley TD, Klimke WA, Gubbins MJ and Frost LS. F factor conjugation is a true type IV secretion system. FEMS Microbiol Lett. 2003; 224:1-15 View ArticlePubMed
  50. Zech H, Thole S, Schreiber K, Kalhöfer D, Voget S, Brinkhoff T, Simon M, Schomburg D and Rabus R. Growth phase-dependent global protein and metabolite profiles of Phaeobacter gallaeciensis strain DSM 17395, a member of the marine Roseobacter-clade. Proteomics. 2009; 9:3677-3697 View ArticlePubMed
  51. Petersen J, Brinkmann H, Bunk B, Michael V, Päuker O and Pradella S. Think pink: photosynthesis, plasmids and the Roseobacter clade. Environ Microbiol. 2012; 14:2661-2672 View ArticlePubMed
  52. Fürch T, Preusse M, Tomasch J, Zech H, Wagner-Döbler I, Rabus R and Wittmann C. Metabolic fluxes in the central carbon metabolism of Dinoroseobacter shibae and Phaeobacter gallaeciensis, two members of the marine Roseobacter clade. BMC Microbiol. 2009; 9:209 View ArticlePubMed
  53. Schwarz S, Hood RD and Mougous JD. What is type VI secretion doing in all those bugs? Trends Microbiol. 2010; 18:531-537 View ArticlePubMed
  54. Wagner-Döbler I, Thiel V, Eberl L, Allgaier M, Bodor A, Meyer S, Ebner S, Hennig A, Pukall R and Schulz S. Discovery of complex mixtures of novel long-chain quorum sensing signals in free-living and host-associated marine alphaproteobacteria. ChemBioChem. 2005; 6:2195-2206 View ArticlePubMed
  55. Auch AF, Von Jan M, Klenk HP and Göker M. Digital DNA-DNA hybridization for microbial species delineation by means of genome-to-genome sequence comparison. Stand Genomic Sci. 2010; 2:117-134 View ArticlePubMed
  56. Auch AF, Klenk HP and Göker M. Standard operating procedure for calculating genome-to-genome distances based on high-scoring segment pairs. Stand Genomic Sci. 2010; 2:142-148 View ArticlePubMed
  57. Meier-Kolthoff JP, Auch AF, Klenk HP and Göker M. Genome sequence-based species delimitation with confidence intervals and improved distance functions. BMC Bioinformatics. 2013; 14:60 View ArticlePubMed
  58. Beyersmann PG, Chertkov O, Petersen J, Fiebig A, Chen A, Pati A, Ivanova N, Lapidus A, Goodwin LA and Chain P. Genome sequence of Phaeobacter caeruleustype strain (DSM 24564T), a surface-associated member of the marine Roseobacter clade. Stand Genomic Sci. 2013; (this issue).
  59. Freese HM, Dalingault H, Petersen J, Pradella S, Davenport K, Teshima H, Chen A, Pati A, Ivanova N and Goodwin LA. Genome sequence of the plasmid and phage-gene rich marine Phaeobacter arcticus type strain (DSM 23566T). Stand Genomic Sci. 2013; (this issue).
  60. Vaas LAI, Sikorski J, Hofer B, Fiebig A, Buddruhs N, Klenk HP and Göker M. opm: An R package for analyzing OmniLog Phenotype Microarray data. Bioinformatics. 2013; 29:1823-1824 View ArticlePubMed
  61. Wagner-Döbler I, Ballhausen B, Berger M, Brinkhoff T, Buchholz I, Bunk B, Cypionka H, Daniel R, Drepper D and Gerdts G. The complete genome sequence of the algal symbiont Dinoroseobacter shibae: a hitchhiker’s guide to life in the sea. ISME J. 2010; 4:61-77 View ArticlePubMed