CORE Metadata, citation and similar papers at core.ac.uk Provided by Springer - Publisher Connector Standards in Genomic Sciences (2013) 9:334-350 DOI:10.4056/sig s.4448212 Genome sequence of Phaeobacter inhibens type strain (T5T), a secondary metabolite producing representative of the marine Roseobacter clade, and emendation of the species description of Phaeobacter inhibens Marco Dog s1, Sonja Vog e t2, Hazuki Teshima 3, Jörn Petersen4, Karen Davenport3, Hajnalka Dalingault3, Amy Chen 5, Amrita Pati6, Natalia Ivanova6, Lynne A. Goodwin3,6, Patrick Chain 3, John C. Detter3,6, Sonja Standfest1, Manfred Rohde7, Sabine Gronow4, Nikos C. Kyrpides6, Tanja Woyke6, Meinh ard Simon 1, Hans-Peter Klenk4, Markus Göker4, Thorsten Brinkhoff1* 1 Institute for Chemistry and Biology of the Marine Environment (ICBM), Oldenburg, Germany 2 Department of Genomic and Applied Microbiology and Göttingen Genomics Laboratory, Institute of Microbiology and Genetics, University of Göttingen, Göttingen, Germany 3 Los Alamos National Laboratory, Bioscience Division, Los Alamos, New Mexico, USA 4 Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany 5 Biological Data Management and Technology Center, Lawrence Berkeley National Laboratory, Berkeley, California, USA 6 DOE Joint Genome Institute, Walnut Creek, California, USA 7 HZI – Helmholtz Centre for Infection Research, Braunschweig, Germany * Correspondence: Thorsten Brinkhoff ([email protected]) Keywords: Anaerobic, motile, rod-shaped, tropodithietic acid, secondary metabolites, Rhodobacterales, Rhodobacteraceae Strain T5T is the type strain of the species Phaeobacter inhibens Marte ns et al. 2006, a sec- ondary metabolite producing bacterium affiliated to the Roseobacter clade. Strain T5T was isolated from a water sample taken at the German Wadden Sea, southern North Sea. Here we describe the complete genome sequence and annotation of this bacterium with a spe- cial focus on the sec ondary metabolism and c ompare it with the g enomes of the Phaeobacter inhibens strains DSM 17395 and DSM 24588 (2.10), selected because of the close phylogenetic relationship based on the 16S rRNA gene sequences of these three strains. The g enome of strain T5T comp rises 4,130,897 bp with 3.92 3 protein-coding genes and shows hig h similarities in genetic and genomic characteristics compared to P. inhibe ns DSM 17395 and DSM 24588 (2.10). Besides the chromosome, strain T5T possesses four plasmids, three of which show a high similarity to the plasmids of the strains DSM 17395 and DSM 24588 (2.10). Analysis of the fourth plasmid suggested horizontal gene transfer. Most of the g enes on this plasmid a re not prese nt in the strains DSM 17395 and DSM 24588 (2.10) including a nitrous oxide reductase, which allows strain T5T a facultative an- aerobic lifestyle. The G+C content was calculated from the genome sequence and differs significantly from the previously published value, thus warranting an emendation of the species description. The Genomic Standards Consortium Dog s et al. Introduction deposited as P. gallaeciensis strains, were recent- Strain T5T was isolated from a water sample tak- ly reclassified as P. inhibens [15]. en on 25th of October 1999 above an intertidal mud flat of the German Wadden Sea (53°42’20’’N, 07°43’11’’E) and found to be closely related to Classification and features the type strain of Roseobacter gallaeciensis [1]. 16S rRNA gene analysis Two years later Martens et al. (2006) reclassified Figure 1 shows the phylogenetic neighborhood of Roseobacter gallaeciensis as Phaeobacter P. inhibens DSM 16374T in a tree based on 16S gallaeciensis and described strain T5T as type rRNA genes. The sequences of the three identical strain of the species Phaeobacter inhibens. As 16S rRNA gene copies differ by one nucleotide found for various Phaeobacter strains [2-7], P. from the previously published 16S rRNA sequence inhibens strain T5T (= DSM 16374T = LMG 22475T (NCBI Accession No. AY177712). = CIP 109289T) is able to produce the antibiotic tropodithietic acid (TDA) [8]. Furthermore, A representative genomic 16S rRNA gene se- strains of P. gallaeciensis and P. inhibens, includ- quence of P. inhibens DSM 16374T was compared ing strain T5T, are able to produce a brownish using NCBI BLAST [16,17] under default settings pigment, which is the basis of the genus name (e.g., considering only the high-scoring segment (phaeos = dark, brown) [1]. The epithet of the pairs (HSPs) from the best 250 hits) with the species name points to the strong inhibitory ac- most recent release of the Greengenes database tivity of P. inhibens against different taxa of ma- [18] and the relative frequencies of taxa and rine bacteria and algae [1]. The genus keywords (reduced to their stem [19]) were de- Phaeobacter is known to have a high potential for termined, weighted by BLAST scores. The most secondary metabolite production, as indicated by frequently occurring genera were Ruegeria biosynthesis of TDA and N-acyl homoserine lac- (32.5%), Phaeobacter (28.8%), Silicibacter tones (AHLs), as well as presence of genes coding (13.6%), Roseobacter (13.3%) and Nautella for polyketide synthases (PKS) and nonribosomal (3.5%) (141 hits in total). Regarding the single peptide synthetases (NRPS) [2,7-10]. Biosynthe- hit to sequences from the species, the average sis of many different bioactive natural products identity within HSPs was 99.8%, whereas the av- is mediated by PKSs or NRPSs, including antibiot- erage coverage by HSPs was 99.3%. Regarding ics, toxins and siderophores. Moreover, produc- the nine hits to sequences from other species of tion of volatile compounds is widespread over the genus, the average identity within HSPs was the Roseobacter clade. It displays a particularly 99.0%, whereas the average coverage by HSPs high proportion of volatile sulfur-containing was 99.2%. Among all other species, the one compounds and thus seems to play an important yielding the highest score was P. gallaeciensis role in the sulfur cycle of the ocean [11]. The sul- (NZ_ABIF01000004), which corresponded to an fur-containing TDA, for which the sulfur precur- identity of 100.0% and an HSP coverage of sor has not yet been determined, plays an im- 100.0%. (Note that the Greengenes database uses portant role in the mutualistic symbioses of P. the INSDC (= EMBL/NCBI/DDBJ) annotation, inhibens and marine algae [12]. p-Coumaric acid which is not an authoritative source for nomen- causes the organism to switch from a state of mu- clature or classification). The highest-scoring en- tualistic symbiosis to a pathogenic lifestyle in vironmental sequence was AJ296158 which toxicity is mediated via the production of (Greengenes short name 'Spain:Galicia isolate str. the algicidal roseobacticides, which, like p- PP-154'), which showed an identity of 99.8% and coumaric, is also a sulfur-containing metabolite an HSP coverage of 100.0%. The most frequently [13,14]. occurring keywords within the labels of all envi- ronmental samples which yielded hits were Here we present the genome of P. inhibens strain 'microbi' (3.1%), 'marine' (2.6%), 'coral' (2.3%), T5T with particular emphasis on the genes in- 'biofilm' (2.1%) and 'membrane, structure, swro' volved in secondary metabolism and comparison (1.8%) (100 hits in total). Environmental sam- with the recently published genomes of the P. ples which yielded hits of a higher score than the inhibens strains DSM 17395 and DSM 24588 highest scoring species were not found. (2.10) [3]. DSM 17395 and DSM 24588, originally http://standardsing enomics.org 335 Phaeobacter inhibens type strain (T5T) Morphology and physiology Chemotaxonomy Cells of T5T are ovoid rods, 1.4-1.9 x 0.6-0.8 µm There are no significant differences between the (Figure 2). Furthermore, T5T cells show the typical fatty-acid profile of strain T5T and other repre- multicellular star-shaped structure described pre- sentatives of the Roseobacter clade [1]. Strain T5T viously for P. gallaeciensis and other Roseobacter- has the highest profile similarity to P. gallaeciensis clade organisms [2,4,47] (Figure 2). Cells of T5T CIP 105210T [1]. The principal cellular fatty acids are motile by means of a polar flagellum. T5T is a of strain T5T are the following saturated Gram-negative, marine, facultatively anaerobic, branched-chain fatty acids: C (73.77%), 11- mesophilic bacterium with an optimal growth methyl C (7.45%), C18:1ω7c16:0 (3.83%), C18:0 temperature between 27 and 29 °C and an optimal (3.14%), 218:1ω7c-OH C16:0 (3.10%), C14:1 (2.19%), 3-OH salinity between 0.51 and 0.68 M. The pH range C10:0 (1.71%), 3-OH C12:0 (1.59%), 3-OH C14 :1/ 3 for growth is 6.0 – 9.5, with an optimum at 7.5. On oxo-C14:0 (0.87%), C (0.76%) and an unam- T marine agar T5 forms smooth and convex colo- biguously identified18.1ω9c fatty acid (1.59%) [1]. The nies with regular edges and brownish pigmenta- major polar lipids of strain T5T comprise tion on ferric citrate containing media. T5T utilizes phosphatidylglycerol, phosphatidylethanolamine, pentoses, hexoses, disaccharides and most amino phosphatidylcholine, an aminolipid and two uni- acids as carbon and energy sources. No vitamin dentified lipids [1]. requirements were observed [1]. Figure 1. Phylogenetic tree highlighting the position of P. inh ibens relative to the type strains of the other species with- in the genus Phaeobacter and the neighboring genera Leisingera and Ruegeria [1,20-33]. The tree was inferred from 1,385 aligned characters [34, 35] of the 16S rRNA gene sequence under the maximum likelihood (ML) criterion [36]. Rooting was done initially using the midpoint method [37] and then checked for its agreement with the current classi- fication (Table 1). The branches are scaled in terms of the expected number of substitutions per site.