Standards in Genomic Sciences (2014) 9:632 -645 DOI:10.4056/sig s.4998989 Draft genome sequence of marine alphaproteobacterial strain HIMB11, the first cultivated representative of a unique lineage within the Roseobacter clade possessing an unusually small genome Bryndan P. Durham1,2, Jana Grote1,3, Kerry A. Whittaker1,4, Sara J. Bender1,5, Haiwei Luo6, Sharon L. Grim 1,7, Julia M. Brown1,8, John R. Casey1,3, Antony Dron1,9, Lennin Florez-Leiva1,10, Andreas Krupke1,11, Catherine M. Luria1,12, Aric H. Mine1,13, Olivia D. Nig ro 1,3, Santhiska Pather1,14, Ag athe Tal armi n 1,15, Emma K. Wear1,16, Thomas S. Weber1,17, Jesse M. Wi lson 1,18, Matthew J. Church 1,3, Edward F. D eLong 1,19, David M. Karl 1,3, Gri eg F. Steward1,3, John M. Eppl ey1,19, Ni kos C. Kyrpides1,20, Stephan Schuster1, and Michael S. Rappé1* 1 Center for Microbial Oceanography: Research and Education, University of Hawaii, Honolulu, Hawaii, USA 2 Department of Microbiology, University of Georgia, Athens, Georgia, USA 3 Department of Oceanography, University of Hawaii, Honolulu, Hawaii, USA 4 Graduate School of Oceanography, University of Rhode Island, Narragansett, Rhode Island, USA 5 School of Oceanography, University of Washington, Seattle, Washington, USA 6 Department of Marine Sciences, University of Georgia, Athens, Georgia, USA 7 Marine Biological Laboratory, Woods Hole, Massachusetts, USA 8 Department of Microbiology, Cornell University, Ithaca, New York, USA 9 Observatoire Océanologique de Villefranche, Villefranche-su r-mer, France 1 Universidad Del Magdalena, Santa Marta, Colombia 1 Max Plank Institute for Marine Microbiology, Bremen, Germany 12 Department of Ecology and Evolutionary Biology, Brown University, Providence, Rhode Island, USA 13 Department of Geophysical Sciences, University of Chicago, Chicago, Illinois, USA 14 School for Marine Science and Technology, University of Massachusetts Dartmouth, Dartmou th, Massachusetts, USA 15 Department of Earth System Science, University of California Irvine, Irvine, CA, USA 16 Marine Science Institute, University of California Santa Barbara, Santa Barbara, California, USA 17 Department of Atmospheric and Ocean Sciences, University of California Los Angeles, Los Ange- les, California, USA 18 School of Natural Sciences, University of California Merced, Merced, California, USA 19 Department of Civil and Environmental Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA 20 Department of Energy Joint Genome Institute, Walnut Creek, California, USA *Correspondence: Michael S. Rappé ([email protected]) Keywords: marine bacterioplankton, Roseobacter, aerobic anoxygenic phototroph, dimethylsulfoniopropionate Strain HIMB11 is a planktonic marine bacterium isolated from coastal seawater in Kaneohe Bay, Oahu, Hawaii belonging to the ubiquitous and versatile Roseobacter clade of the alphaproteobacterial family Rhodobacteraceae. Here we describe the preliminary characteristics of strain HIMB11, including annota- tion of the draft genome sequence and comparative genomic analysis with other members of the Roseobacter lineage. The 3,098,747 bp draft genome is arranged in 34 contigs and contains 3,183 pro- tein-coding genes and 54 RNA genes. Phylogenomic and 16S rRNA gene analyses indicate that HIMB11 represents a unique sublineage within the Roseobacter clade. Comparison with other publicly available genome sequences from members of the Roseobacter lineage reveals that strain HIMB11 has the genomic potential to utilize a wide variety of energy sources (e.g. organic matter, reduced inorganic sulfur, light, carbon monoxide), while possessing a reduced number of substrate transporters. Abbreviations: DMSP - Dimethylsulfoniopropionate, DMS - dimethylsulfide, AAnP - aerobic anoxygenic phototroph, COG - Clusters of Orthologous Groups, PGC -photosynthesis gene cluster, BLUF - blue-light using flavin adenine nucleotide, ABC - ATP-binding cassette, TRAP - tripartite ATP-independent periplasmic, DMTs - drug/metabolite transporters The Genomic Standards Consortium Durham et al. Introduction Bacteria belonging to the Roseobacter lineage of Genome annotation revealed strain HIMB11 to marine Alphaproteobacteria account for a sub- have the genetic potential for bacteriochlorophyll- stantial fraction (ranging ~10 – 25%) of based aerobic anoxygenic phototrophic (AAnP) bacterioplankton cells in surface ocean seawater metabolism and degradation of the algal-derived [1-4], making them one of a relatively small compound DMSP along with production of the number of suitable targets for scientists investi- climate-relevant gas dimethylsulfide (DMS), and gating the ecology of abundant marine bacterial oxidation of the greenhouse gas carbon monoxide groups. Focused genome sequencing efforts have (CO). Collectively, these features indicate the po- provided significant insights into the functional tential for strain HIMB11 to participate in the bio- and ecological roles for this group [5-7]. In 2004, geochemical cycling of sulfur and carbon, and con- the first member of this group to have its genome comitantly affect global climate processes. sequenced, Ruegeria pomeroyi (basonym Silicibacter pomeroyi) strain DSS-3 [8], revealed Classification and features strategies used by the Roseobacter group for nu- Strain HIMB11 was isolated by a high-throughput, trient acquisition in the marine environment. To dilution-to-extinction approach [15] from surface date, over 40 genomes have been sequenced seawater collected near the coast of Oahu, Hawaii, from members of the Roseobacter lineage. Com- USA, in the tropical North Pacific Ocean. The strain parative analysis among 32 of these genomes in- was isolated in seawater sterilized by tangential dicates that members of this group are ecological flow filtration and amended with low concentra- generalists, having relatively plastic require- tions of inorganic nitrogen and phosphorus (1.0 ments for carbon and energy metabolism, which µM NH4Cl, 1.0 µM NaNO3, and 0.1 µM KH2PO4). may allow them to respond to a diverse range of environmental conditions [9]. For example, Comparative analysis of the HIMB11 16S rRNA members of the Roseobacter lineage have the ge- gene sequence to those from cultured, sequenced nomic potential to obtain energy via oxidation of roseobacters indicates that HIMB11 occupies a organic substrates, oxidation of inorganic com- unique lineage that is divergent from the 16S pounds, and/or sunlight-driven electron transfer rRNA gene sequences of Roseobacter strains al- via bacteriochlorophyll a, proteorhodopsin, or ready in culture (Figure 1). Based on the National xanthorhodopsin phototrophic systems. Genome Center for Biotechnology Information (NCBI) analyses as well as culture experiments have also non-redundant database, the HIMB11 16S rRNA revealed a variety of mechanisms by which gene sequence is most similar (~99% nucleotide roseobacters may associate and interact with identity) to a large number of environmental phytoplankton and other eukaryotes. These in- gene clones obtained from various marine envi- clude genes involved in uptake of compounds ronments that exclusively fall in the Roseobacter produced by algae such as peptides, amino acids, lineage of Alphaproteobacteria. putrescine, spermidine, and DMSP [10,11], as well as genes for chemotaxis, attachment, and Because of the significant sequence variation in secretion [12]. 16S rRNA genes (up to 11%) and the prevalence of horizontal gene transfer within the clade, estab- Strain HIMB11 was isolated from surface sea- lishing a taxonomic framework for roseobacters water collected from Kaneohe Bay off the coast of remains a challenge [9]. When genome sequence Oahu, Hawaii, USA in May, 2005. Subsequent 16S data is available, it is often more informative to rRNA gene sequence comparisons revealed it to perform a phylogenomic analysis based on shared be a member of the Roseobacter clade of marine orthologs versus 16S rRNA phylogenetic analysis bacterioplankton [13] that was highly abundant alone [9,21]. A maximum likelihood tree con- after a storm-induced phytoplankton bloom in the structed using 719 shared orthologous protein bay [14]. Here, we present a preliminary set of sequences supported the 16S rRNA gene-based features for strain HIMB11, a description of the analysis by revealing that HIMB11 formed a draft genome sequence and annotation, and a unique sublineage of the Roseobacter clade (Fig- comparative analysis with 35 other genome se- ure 2). quences from members of the Roseobacter lineage. http://standardsing enomics.org 633 Alphaproteobacterial strain HIMB11 Figure 1. Phylogenetic relationships between HIMB11 and bacterial strains belonging to the Roseobacter clade. SSU rRNA gene sequences were aligned with version 111 of the ‘All-Species Living Tree’ project SSU rRNA gene database [16] using the ARB software package [17]. The phy- logeny was constructed from nearly full-length gene sequences using the RAxML maximum likeli- hood method [18] within ARB, filtered to exclude alignment positions that contained gaps or am- biguous nucleotides in any of the sequences included in the tree. Bootstrap analyses were deter- mined by RAxML [19] via the raxmlGUI graphical front end [20]. The scale bar corresponds to 0.05 substitutions per nucleotide position. Open circles indicate nodes with bootstrap support be- tween 50-80%, while closed circles indicate bootstrap support >80%, from 500 replicates. A varie- ty of Archaea were used as outgroups. 6 34 Standards in Genomic Sciences Durham et al. HIMB11 cells