Genome sequence of Planktotalea frisia type strain (SH6-1T), a representative of the Roseobacter group isolated from the North Sea during a phytoplankton bloom

Planktotalea frisia SH6-1T Hahnke et al. (Int J Syst Evol Microbiol 62:1619–24, 2012) is a planktonic marine bacterium isolated during a phytoplankton bloom from the southern North Sea. It belongs to the Roseobacter group within the alphaproteobacterial family Rhodobacteraceae. Here we describe the draft genome sequence and annotation of the type strain SH6-1T. The genome comprises 4,106,736 bp and contains 4128 protein-coding and 38 RNA genes. The draft genome sequence provides evidence for at least three extrachromosomal elements, encodes genes for DMSP utilization, quorum sensing, photoheterotrophy and a type IV secretion system. This indicates not only adaptation to a free-living lifestyle of P. frisia but points also to interactions with prokaryotic or eukaryotic organisms.


Introduction
The Roseobacter group features a global distribution in marine ecosystems like the water column and biological surfaces comprising up to 25% of marine microbial communities [1][2][3]. Members of this group exhibit numerous metabolic capabilities; besides aerobic anoxygenic photosynthesis and the production of bacteriochlorophyll a, they are also capable of oxidizing carbon monoxide, degrading aromatic compounds and catabolizing organic sulfur compounds [4]. Some representatives of this group are also able to synthesize secondary metabolites and to produce quorum sensing molecules like acylated homoserine lactones [5][6][7]. Genomic analysis showed that almost half of the marine Roseobacter genomes encode a type IV secretion system [4], thus, assuming to play a role in interactions of bacteria with other prokaryotic and eukaryotic cells including phytoplankton [8].
A recent study on genomic contents of the Roseobacter group identified a cluster of eight purely pelagic roseobacters which are distinct from the other members of this group [9]. One member of this cluster is strain HTCC2083, isolated from the coastal northwest Pacific Ocean [10]. Planktotalea frisia, the type species of the genus Planktotalea [11], is the closest relative of HTCC2083. P. frisia has been isolated from the southern North Sea, with highest abundances in spring and summer and constitutes up to 0.9% of the bacterioplankton [12].
In order to expand the knowledge on roseobacters prominent in marine pelagic systems we sequenced the genome of P. frisia and present the draft version together with its annotations. Even though SH6-1 T was originally allocated to the free-living fraction [13], experimental studies in which SH6-1 T was grown in presence of axenic algae cultures suggested specific interactions with different phytoplankton species. Furthermore, this representative of the Roseobacter group occurred mainly free-living during a phytoplankton bloom in the North Sea but also in the particle-associated fraction after the breakdown of a Phaeocystis bloom [12]. Thus, our special focus was on genomic features related to the lifestyle of this organism and we had a closer look on genes involved in sulfur cycling such as degradation of dimethylsulfoniopropionate and genes indicating biofilm formation, motility, chemotaxis and quorum sensing pointing to a surface-attached lifestyle.

Organism information
Classification and features Figure 1 shows the phylogenetic neighborhood of P. frisia DSM 23709 T in a 16S rRNA gene sequence-based tree analyzed using NCBI-BLAST [14] and ARB [15]. The sequence of the single 16S rRNA gene copy in the genome does not differ from the previously published 16S rRNA gene sequence (FJ882052).
Strain SH6-1 T (= DSM 23709 T = LMG 25294 T ) was isolated from a water sample of the southern North Sea (54°42' N, 06°48′ E) during a phytoplankton bloom from a water depth at 2 m in May 2007 [11].
Cells of P. frisia SH6-1 T are Gram-negative irregular rods with a width of 0.4 to 1 μm and a length of 0.5 to 4 μm (Fig. 2) [11]. On seawater agar colonies are small, circular, convex and whitish with a shiny surface. SH6-1 T is a marine, aerobic bacterium with a temperature range of 4-32°C and an optimum growth rate at 20-25°C. The salinity range for this strain is between 1.25 and 8% NaCl. The optimal pH range for growth is 7.5-9.0 with pH 6.0 being the lowest possible pH at which growth occurs under the tested conditions.

Genome project history
The genome was sequenced within the Collaborative Research Center "Ecology, Physiology and Molecular Biology of the Roseobacter clade: Towards a Systems Biology Understanding of a Globally Important Clade of Marine Bacteria" funded by Deutsche Forschungsgemeinschaft. The genome project was deposited in the Genomes OnLine Database [16] and in the Integrated Microbial Genomes database [17]. The Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession number MLCB00000000. The version described here is version MLCB01000000. A summary of the project information is shown in Table 2.

Growth conditions and genomic DNA preparation
A culture of SH6-1 T was grown in DSMZ medium 1282 (SH Seawater medium) [11] at 20°C. Genomic DNA was isolated using a Power Soil DNA Isolation kit (MoBio) following the standard protocol provided by the manufacturer but modified by the addition of 100 μl Tris for cell lysis. DNA is available from DSMZ through DNA Bank Network [18].

Genome sequencing and assembly
The draft genome sequence was generated using Illumina sequencing technology. For this genome, we constructed and sequenced an Illumina paired-end library with the Illumina Nextera XT library preparation kit and sequencing of the library using Genome Analyzer IIx were performed as described by the manufacturer (Illumina, San Diego, CA, USA). A total of 4.6 million paired-end reads were derived from sequencing and trimmed using Trimmomatic version 0.32 [19]. De novo assembly of all trimmed reads with SPAdes version 3.5.0 [20] resulted in 227 contigs and 150-fold coverage.

Genome annotation
Genes were identified as part of the genome annotation pipeline of the Integrated Microbial Genomes (IMG-ER) platform using Prodigal v2.50 [21]. The predicted CDS were translated used to search the CDD, KEGG, UniProt, TIGRFam, Pfam and InterPro databases. These data sources were combined to assert a product description for each predicted protein.

Genome properties
The genome consists of 227 contigs with a total length of 4,106,736 bp and a G + C content of 53.77% (Table 3). Of the 4166 genes predicted, 4128 were protein-coding genes, and 38 RNA genes. No pseudogenes or CRISPR elements were found. For the majority of the proteincoding genes (78.06%) a putative function could be assigned and the others were annotated as hypothetical proteins. The genome statistics are provided in Table 3 and Fig. 3. The distribution of genes into COGs functional categories is presented in Table 4.

Insights from the genome sequence
Genome sequencing of Planktotalea frisia SH6-1 T resulted in 227 contigs with sizes between 0.51 kb and 181 kb. A detailed view on plasmid organization was not possible due to the number and length of contigs of the draft genome, but scanning the genome for typical plasmid repABC-type replication modules from Rhodobacterales [28] resulted in three modules, suggesting that this strain carries at least three extrachromosomal elements. Phage-mediated horizontal gene transfer is known to drive genomic diversity of bacteria and prophagelike structures are common in marine bacteria [29]. The genome of strain SH6-1 T carries a complete gene transfer agent cluster (PFRI_24010-24170) organized similar to the first genetically characterized GTA agent of Rhodobacter capsulatus RcGTA [30] containing 14 of the 15 genes but lacking the orfg1 gene. RcGTA-like genes are present in all taxonomic orders of Alphaproteobacteria and within the Roseobacter group, except in most strains of the Pelagic Roseobacter Cluster, i.e. Planktomarina temperata, Planktomicrobium forsetii, Rhodobacterales bacterium HTCC2255 and HTCC2083 [3,4,9]. Only strain HTCC2150 of the PRC members encodes the GTAlike gene cluster [4].
Genes encoding type IV secretion systems (T4SSs), facilitating the transfer of proteins and nucleoprotein a 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). These evidence codes are from the Gene Ontology project [61]  complexes by the formation of a pilus, were found in half of the analyzed genomes of marine representatives of the Roseobacter group [4,8,31]. Vir proteins are essential components for conjugation and hypothesized to play a role in the cell-cell contact between roseobacters and phytoplankton cells [8]. The T4SS seems to be a unique pattern of marine organisms within the Roseobacter group, some Erythrobacteraceae and Caulobacteraceae [32]. The genome of strain SH6-1 T also encodes the complete T4SS for translocating DNA or proteins into other cells. It includes the virB operon (virB1 to − 11, excluding virB7; PFRI_11620-11730) mediating the transmembrane channel formation and the virD2 and virD4 relaxase and coupling proteins (PFRI_35220, PFRI_ 35230) analogous to the archetypal Agrobacterium tumefaciens VirB/D4 system [33]. The presence of the Vir gene cluster in the genome of P. frisia indicates that this strain is able to transfer DNA and proteins into prokaryotic and/or eukaryotic cells. Flagellar synthesis as well as motility seem to be of importance for surface attachment and biofilm formation in many Proteobacteria [34][35][36]. The genome of P. frisia SH6-1 T exhibits some genes for flagellar synthesis but covering only 8 of 30 analyzed COG flagellar families. Analysis of the corresponding genes revealed that the flagellar loci are located at the terminus of the single contigs as it is also the case for Roseobacter sp. strain MED193 with only 11 of 30 genes grouping into COG flagellar families [31].
Hence, a precise statement about the existence of a complete set and therefore a flagellum for strain SH6-1 T is not possible but should not be excluded due to the detection of slight wobbling under laboratory conditions [11]. The genome of strain P. frisia reveals, however, no genes encoding proteins associated to chemotaxis and the ability to move towards certain chemicals in the environment.
Roseobacters are well known to be involved in the transformation of dimethylsulfoniopropionate, a metabolite produced primarily by marine phytoplankton, either by demethylation or cleavage [4,8,37]. Strain SH6-1 T harbors genes for both, the cleavage and the demethylation pathway, indicating its ability to utilize DMSP. Two genes encoding for the dimethylsulfoniopropionate demethylase converting DMSP into methylmercaptopropionate [38,39] are present but genes encoding the subsequent degradation of MMPA to acetaldehyde are absent from the draft genome sequence. Genes encoding for the alternative DMSP cleavage pathway are present in P. frisia, DddP (PFRI_00730), DddQ (PFRI_14360) and DddW (PFRI_38540) producing dimethylsulfide and acrylate, which is in contrast to previous studies where no DMS formation for P. frisia was detected [13].
Carbon monoxide can be an additional potential electron donor, which is formed by photolysis of dissolved organic matter. Only Roseobacter strains containing both the definitive form I and putative form II of the CO dehydrogenases large subunit (coxL) are capable of oxidizing CO under laboratory conditions [40]. Planktotalea frisia exhibits both gene structures the form I (coxMSL; PFRI_33480-33500) as well as form II (coxSLM; PFRI_ 01330-01350), but form I is lacking the downstream genes coxDEF detected in other genomes of the marine Roseobacter group [40]. Hence, it needs to be proved if this strain is able to use CO as an additional electron donor.
Inorganic sulfur compounds play an important role for mixotrophic growth in the marine environment with thiosulfate as common compound in seawater. The Roseobacter group makes use of the oxidation of thiosulfate to sulfate using the periplasmic Sox multienzyme complex like Ruegeria pomeroyi [41]. The genome of P. frisia SH6-1 T encodes proteins associated to a set of sox genes (soxRSVWXYZABCDEF; PFRI_19680, PFRI_14240, PFRI_ 37660-37740) suggesting that reduced sulfur compounds can be a complementary energy source.
Quite a few marine bacteria are capable of using light as an additional energy source. Proteorhodopsins are widely distributed in major bacterial groups like Flavobacteriia, Alphaproteobacteria and Gammaproteobacteria [43] and aerobic anoxygenic phototrophs are widely distributed within the Roseobacter group [2,44] and also for P. frisia genes encoding subunits of the photosynthetic reactions center complex (pufML) were detected via specific PCR [13]. Genes for a functional photosynthetic gene cluster (PFRI_28770-28970, PFRI_19280-19350, PFRI_19150-19250) were found in the genome of SH6-1 T . They include bch and crt genes coding for the bacteriochlorophyll and carotenoid biosynthetic pathways, puf genes coding for the subunits of the light harvesting complex and the reaction center complex, hem genes and also genes for sensor proteins. Due to the structure of the puf-operon and presence of the additional pufX gene, only reported for the anaerobic Rhodobacter lineage so far, P. frisia can be assigned to the phylogroup E according to Yutin et al. [45] occurring only in coastal oceans. In addition, two genes encoding blue light sensors using FAD (BLUF; PFRI_28190, PFRI_41660) are also present in the genome of strain SH6-1 T indicating possible blue light-dependent signal transduction.
To analyze the lifestyle of P. frisia the genome was also screened for genes associated with quorum sensing (QS). QS systems mediated by N-acyl-L-homoserine lactones (AHLs) provide significant benefits to the group and influence bacterial social traits such as virulence, motility and biofilm formation in many Proteobacteria including the Roseobacter group [46][47][48][49]. Genome analysis revealed the presence of genes encoding an Nacyl-L-homoserine lactone synthetase (luxI homolog; PFRI_23420) and a response regulator (luxR homolog; PFRI_23430) indicating that P. frisia can perform QS.

Conclusions
In addition to biogeochemically important features reported previously from other sequenced strains of the Roseobacter group e.g. [3,41,50,51], genome analysis of P. frisia SH6-1 T , which is closely related to a member of Fig. 3 Planktotalea frisia SH6-1 T artificial circular chromosome map. Genome comparison of P.frisia SH6-1 T with 6 genome sequenced members of the Pelagic Roseobacter Cluster [9]. Circles (from outside to inside): 1 and 2: Genes encoded by the leading and lagging strand of P.frisia SH6-1 T marked in COG colors in the artificial chromosome map; 3-7: The presence of orthologous genes is indicated for the genomes of Rhodobacterales bacterium HTCC2083, Planktomarina temperata RCA23 T , Rhodobacteraceae bacterium HIMB11, CHAB-I-5 SB2 and Rhodobacteraceae bacterium HTCC2255. The similarity of orthologous genes is illustrated in red to light yellow and singletons in grey (grey: >e − 10 -1; light yellow: <e − 50 -> e − 10 ; gold: <e − 50 -> e − 90 ; light orange: <e − 90 -> e − 100 ; orange: <e − 100 -> e − 120 ; red: <e − 120 -0). The two innermost circles represent the GC-content and the GC-skew the Pelagic Roseobacter Cluster [9], HTCC2083, revealed the presence of at least three extrachromosomal elements and genes associated with quorum sensing and type IV secretion systems.
Correspondingly, we assume that this strain can switch between free-living and an algal host associated lifestyle.

Abbreviations
AHLs: Acyl homoserine lactones; DMSP: Dimethylsulfoniopropionate; IMG: Integrated microbial genomes; QS: Quorum sensing; T4SS: Type IV secretion system The total is based on the total number of protein coding genes in the genome