C-MORE Summer Course 2011: Comparative genomics of Roseobacter strain HIMB11 reveals metabolic specialization in a coastal marine isolate Presenters Origin and Isolation Metabolism of HIMB11 Revealed by Genome Specialized Substrate The planktonic, marine bacterium Roseobacter Annotation Transport Indicated by strain HIMB11 is a member of the ubiquitous and versatile Roseobacter lineage of the Table 2. HIMB11 genome statistics. Comparative Genomics alphaproteobacterial family Rhodobacteraceae. Strain HIMB11 was HIMB11 is a CO-oxidizing HIMB11 expands our understanding of isolated from surface seawater collected off the AAnP, capable of harvesting Roseobacter diversity given its coast of Kaneohe Bay, Oahu, and its genome Attribute Value % of was sequenced using a 454 GS FLX Ti total energy from CO and light, seemingly streamlined metabolic platform to 121x coverage. Analysis of the 3.1 respectively. capabilities. Key Mb genome, comprising 3,182 protein-coding Size 3114138 bp 100 genes and 57 functional RNA genes, reveals G+C content 1548876 bp 49.74 lowest abundance strain HIMB11 to be a DMSP-degrading, CO- Coding region 2825259 bp 90.72 for a given oxidizing, aerobic anoxygenic phototroph COG (AAnP). While members of the Roseobacter Total genes 3239 100 lineage are typically considered ecological RNA genes 57 1.76 generalists, features of the strain HIMB11 Protein-coding 3182 98.24 genome indicate a more specialized lifestyle. genes Compared to 36 other Roseobacter genomes currently sequenced, strain HIMB11 harbors a Genes (with): Roseobacter low number of TRAP transporters and an Function prediction 2566 79.22 unusually high number of antibiotic/drug and Assigned to COGs 2533 78.2 secondary metabolite transporters. We highest abundance hypothesize that HIMB11 may rely on a more Assigned to Pfam 2563 79.13 limited suite of substrates for growth, possibly domains • ABC, ATP- Signal peptides 920 28.4 in association with a reluctant phytoplankter binding cassette host. Transmembrane 654 20.19 • DMT, helices Drug/metabolite HIMB11 was isolated from transporter • TRAP, Tripartite surface seawater in Kaneohe ATP-independent Carbon Metabolism: The glycolysis pathway is ABC sugar ABC amino DMT TRAP periplasmic Bay, Oahu during May of incomplete; however, HIMB11 possesses a acid 2005. complete pathway for gluconeogenesis and can perform anapleurotic CO fixation. Authors 2 Figure 4. Heat map of clusters of orthologous groups (COGs) A B D Figure 2. Genes for acquisition of N, P, and for common Roseobacter carbohydrate and amino acid Durham, B.P. Nitrogen Metabolism: HIMB11 is capable of S in HIMB11, as well as energy acquisition. transporters. The number of COG hits is normalized to genome Whittaker, K.A. transporting reduced and organic nitrogen DMSP, dimethylsulfoniopropionate; C-O-P, size. HIMB11 is outlined with a red box. Bender, S.J. sources including: ammonia, urea, phosphoesters; C-P, phosphonates; Pi, Brown, J.M. polyamines, amino acids, etc. (Figure 2). Casey, J.R. phosphate. Dron, A. Flores-Leiva, L.R. Phosphorus Metabolism: HIMB11 utilizes both Grim, S.L. C inorganic & organic phosphorus sources Krupke, A. based on the presence of a high-affinity Pi Luria, C.M. transporter and alkaline phosphatase, Mine, A.H. respectively. Pather, S. Talarmin, A. Sulfur Metabolism: HIMB11 appears to rely Wear, E.K. solely on organic sources of sulfur, including Weber, T.S. Wilson, J.M. amino acids and DMSP. Whittaker, K.A. Figure 1. Photomicrograph of DAPI-stained Additional modes of energy acquisition HIMB11 (A); location of Kaneohe Bay, Oahu include anoxygenic photosynthesis, CO (B, C); maximum-likelihood phylogenomic tree oxidation, aromatics degradation, and sulfur derived from 719 concatenated shared oxidation. Figure 5. Gene neighborhoods for the three COG1593 TRAP orthologous genes using RAxML (D) showing transporters in HIMB11. Genes indicative of substrate are the relationship between HIMB11 (indicated HIMB11 may play an integral labeled and colored blue (arabinose), green (benzoate), and with a star) and other roseobacters. Bootstrap red (tyrosine/phenylalanine), respectively. values are indicated at nodes. Candidatus role in carbon and sulfur Partnerships and Puniceispirillum marinum IMCC1322, cycling through its role in the Compared to other roseobacters, TRAP Leveraged belonging to the SAR116 clade, is used as an Funding outgroup. breakdown on DMSP as well transporters are low in abundance as production of DMS. whereas DMTs are enriched in HIMB11, The Agouron Institute Table 1. General features of HIMB11. possibly due to enhanced secondary Gordon and Betty Figure 3. DMSP is a major source of both Moore Foundation Feature Description carbon and sulfur for marine bacterioplankton metabolite transport. in ocean surface waters. There are two C-MORE Gram stain Negative competing pathways of DMSP degradation, the demethylation pathway that leads to National Science Foundation Cell shape Irregular short rods assimilation of sulfur, and the cleavage Cell size 0.8 * 0.3-0.5 μm pathway that leads to the release of Conclusions Motility Flagellum dimethylsulfide (DMS). DMS is a climate-active Sporulation Non-sporulating gas which has been implicated in the formation • HIMB11 has the potential to play a role in the cycling of climatically important gases, DMS, CO, and CO . Temp. range Mesophilic of climate-cooling aerosols and clouds. 2 Carbon source Ambient DOC HIMB11 harbors both sides of the pathway, with • Roseobacters are often considered ecological generalists; Energy source Mixotrophic genes indicated in bold. however, HIMB11 maintains a more streamlined, reduced 1 genome indicative of a specialized lifestyle. Habitat Seawater Adapted from Moran, et. al., 2012 Salinity ~35.0 ‰ • Reduced genome size and transporter abundance, in particular TRAP transporters, indicate HIMB11 uses a Oxygen Aerobic conservative suite of substrates. Biotic relationship Free-living Acknowledgements • Overabundance of antibiotic/drug and secondary Location Kaneohe Bay, Oahu metabolite transporters (DMTs) suggests that HIMB11 may Collection Date May 18, 2005 We would like to thank Matthew Church, Ed DeLong, Dave Karl, Mike Rappé, Grieg o Steward, John Eppley, Jana Grote, Nikos Kyrpides, Stephan Shuster, and the entire C- interact directly with other organisms, possibly with Latitude 21.44 N phytoplankton, as do many roseobacters. o MORE staff for support and instruction during the 2011 summer course. Longitude -157.78 W • Future genome analysis will focus on identifying all central Depth ~1 m We also would like to thank Mary Ann Moran and Haiwei Luo at University of Georgia for metabolic pathways, interrogating more transporter genes, helpful instruction on Roseobacter genomics and bioinformatics analyses. targeting mobile elements, and linking genomic information to the ecology and physiology of HIMB11. We received support from the University of Hawaii School of Ocean and Earth Science and Technology (SOEST), the Center for Microbial Oceanography: Research and Education (C-MORE), the Gordon and Betty Moore Foundation, the Agouron Institute, and 1. Moran, Mary Ann, et. al., 2012. Genomic Insights into Bacterial DMSP Transformations. Ann. Rev. Mar. Sci. Vol. 4: 523-542. the National Science Foundation. Cruises and Time Series: Agouron 2011 (Kilo Moana) .