The improvement of sequencing technologies in the last decade resulted in an exponential increase of completely deciphered bacterial genomes with more than 400 Rhodobacteraceae currently deposited in public data bases. We used the crucial replicases of the two sister plasmids from D. shibae for BLASTP searches and could identify a 185-kb plasmid from Confluentimicrobium naphthalenivorans NS6^T (pNS6001; Jeong et al., 2015) that exhibits a long range synteny with the 126-kb plasmid pDSHI03 (Figure 1). Both replicons share the type IV secretion system (T4SS), toxin/antitoxin modules and a RepABC-2 type replication operon for plasmid maintenance (yellow), which has been replaced in the 191-kb sister plasmid pDSHI01 by a compatible RepABC-9 type equivalent (blue). Cytochrome c biosynthesis and heavy metal detoxification genes are the most conspicuous shared life style determinants of these extrachromosomal replicons (ECRs; Supplementary Table S1). Comprehensive TBLASTN comparisons revealed a protein sequence identity between 92 and 100% for most conserved genes, and the differences reflect their individual evolutionary history in the respective host cell. For example, the selective pressure on the functional genes is indirectly documented by an about 10% lower conservation of several hypothetical proteins, thus providing evidence that the plasmids pNS6001 and pDSHI03 were not recently transferred. However, 74% of the genes from pDSHI03 are shared with the homologous RepABC-2 plasmid from Confluentimicrobium (Supplementary Table S1). The structural backbone of both plasmids is absolutely conserved apart from some unique regions, such as the large 66-kb insertion in pNS6001 (Figure 1). The degree of sequence conservation even exceeds that of the stably coexisting 126-kb and 191-kb sister plasmids of D. shibae that harbor an inverted InDel (see below), but exhibit different compatible replication modules of the RepABC-2 and -9 type representing the ‘heart of a plasmid’ (Wagner-Döbler et al., 2010).

Our BLASTP searches also revealed the presence of a 135-kb contig with RepABC-2 replication module in the draft genome of Roseovarius indicus EhC03 (Rosana et al., 2016) that likely represents a second syntenic plasmid. The contig is less conserved than pNS6001, but still contains 45% of the genes from pDSHI03 including the T4SS (Supplementary Table S2). Systematic TBLASTN based comparisons of the respective replicons showed a slightly lower degree of protein conservation. The benefit of reciprocal BLAST analyses of conserved DNA modules is exemplified by the detection of four pseudogenes in D. shibae. The SAM-dependent methyltransferase from R. indicus (OAO03639.1) and C. naphthalenivorans (WP_054540530.1) matches with two annotated genes of the 126-kb plasmid of D. shibae (Dshi_4003 [WP_050757929.1], Dshi_4008 [WP_012187351.1]) that are separated by a 6-kb fragment containing a type III restriction endonuclease and integrases (Dshi_4004 to Dshi_4007; highlighted in turquoise, Figure 1). This arrangement suggests that the functional gene in Dinoroseobacter was inactivated by a transposition event (Supplementary Table S2). A homologous but inverted insertion is present on the 191-kb plasmid (Dshi_3695 to Dshi_3698) resulting in two annotated SAM-dependent methyltransferase pseudogenes (Dshi_3694 [WP_012187109.1], Dshi_3699 [WP_012187104.1]), which is illustrated by the hourglass shaped syntenic area between the sister plasmids in Figure 1. This case example documents that a thorough comparison of closely related sequences helps to improve the manual genome annotation of model organisms such as D. shibae DFL12^T (Wagner-Döbler et al., 2010).

A phylogenetic RaxML tree of the RNA polymerase beta subunit (RpoB) from 69 Roseobacter strains was calculated in order to reveal the relationships of the host cells of the syntenic RepABC-2 type plasmids (Supplementary Figure S1). The taxon sampling included the natural hosts D. shibae, C. naphtahalenivorans as well as R. indicus and largely corresponded to that of a recent phylogenomic analysis of 65 Roseobacter genomes (Michael et al., 2016). Our single gene phylogeny recovered all seven clades as monophyletic groups and many subtrees were even supported with a solid bootstrap proportion (BP). Furthermore, it also mirrors the branching pattern of a phylogenomic analysis based on 44 roseobacters with a different taxon sampling including three single-cell genomes from an uncultured streamlined lineage from the ocean surface (Luo et al., 2014). This outcome documents that the phylogenetic position of novel Rhodobacteraceae genomes can rapidly be estimated by a single gene RpoB analysis, which is based on 1374 amino acid (aa) alignment positions only, thus serving as a first proxy for complex phylogenomic studies with more than 200,000 aa positions (Case et al., 2007; Michael et al., 2016). The broader taxon sampling of eight genome sequenced strains from clade 5 (former analyses were restricted to two or three strains in this particular subgroup; Luo et al., 2014; Michael et al., 2016; Simon et al., 2017) recovered a very close branching of Confluentimicrobium with Rhodovulum sp. NI22 and a sistergroup relationship with the genus Actibacterium (77% BP; Figure 2A). The basal position of Dinoroseobacter shows a considerable evolutionary distance to Confluentimicrobium. The absence of syntenic RepABC-2 replicons and even plasmids of the same compatibility group from the remaining six Roseobacter strains in clade 5 is in agreement with a horizontal recruitment by D. shibae and C. naphthalenivorans from taxa outside of clade 5, a hypothesis that is based on the distinct codon usage (CU; see below) and thus more plausible than the alternative explanation of a common ancestry followed by differential plasmid losses. The phylogenetic position of Roseovarius indicus in clade 3 indicates that the syntenic RepABC-2 plasmids (Figure 1; Supplementary Tables S1, S2) are migrating within the Roseobacter group. This prediction is supported by the experimental conjugation of pDSHI03 into P. inhibens DSM 17395 (Patzelt et al., 2016) thereby connecting clades 1 and 5 (Figure 2A). Syntenic RepABC-2 plasmid have thus been experimentally transferred between the most distant clades, and can naturally be detected in members of clade 5 and 3, thus documenting independent events of lateral gene transfer in the ocean.

The relative synonymous codon usage (RSCU) of the chromosome and ECRs from D. shibae and C. naphthalenivorans was analyzed using methods described previously (Petersen et al., 2013) and a plot of the averaged RSCU of protein-coding genes of all ten replicons is shown in Figure 2B. The principal component (PC) analysis confirmed the classification of D. shibae’s 152-kb and 72-kb ECRs as chromids, which exhibit a RSCU comparable to that of the chromosome (Harrison et al., 2010), whereas the three more distinct replicons of 86-kb, 126-kb and 191-kb represent authentic plasmids. The RSCU analysis of all Confluentimicrobium replicons showed a distinct localization of the three ECRs compared with the chromosome providing evidence that they also represent typical plasmids. This conclusion is independently supported by the presence of structurally conserved T4SS on the chromosome and all three ECRs (Supplementary Table S3) that contain RepABC operons of different compatibility groups (pNS6001 [185-kb, RepABC-2], pNS6002 [157-kb, RepABC-9], pNS6003 [156-kb, RepABC-5]). RepABC modules represent the most frequently mobilized plasmid type among the four characteristic replication systems of Roseobacter ECRs (RepA, RepB, DnaA-like, RepABC; Petersen et al., 2013). Based on the detection of a RepABC-9 operon on another replicon of Confluentimicrobium, the 157-kb plasmid pNS6002, which is homologous and functionally equivalent to that of the 191-kb sister plasmid pDSHI01 from D. shibae (blue module, Figure 1), we compared both plasmids via BLASTN, but observed only a limited structural conservation for the replication module and the generally conserved T4SS. However, the presence of a co-existing RepABC-2 and -9 plasmid pair in two Roseobacter species reflects a stable co-evolution of these ECRs. The close grouping of the syntenic 126-kb pDSHI03 and 185-kb pNS6001 plasmids in the RSCU, which is highlighted with red arrows in Figure 2B, suggests a common origin of both replicons. The large distance of both replicons to their respective chromosomes unequivocally documents that neither D. shibae nor Confluentimicrobium is the genuine host cell of this conjugative RepABC-2 type plasmid.

Dinoroseobacter shibae DFL12^T was isolated from a culture of the dinoflagellate Prorocentrum lima maintained at the Biological Research Station of the Alfred-Wegener-Institute for Polar Research (AWI) at Helgoland in the North Sea (Biebl et al., 2005). The algal culture was a gift from the Toralla Marine Science Station (ECIMAT) of the University of Vigo, Spain, where the dinoflagellate had been isolated from the North Atlantic Ocean. C. naphtalenivorans NS6^T was isolated from heavily polluted tidal flat sediments in the South Sea, a part of the Pacific Ocean adjacent to South Korea (Jeong et al., 2015), at a distance of roughly 8000 km to Vigo, Spain. Thus, today those two species do not share a similar habitat or location. Roseovarius indicus EhC03 was isolated from a culture of the coccolithophore alga Emiliania huxleyi M217 maintained at the Plymouth Algal Collection, United Kingdom. The algae had originally been isolated from surface water of the South Pacific (NCBI BioSample SAMN04965936; Rosana et al., 2016). The synteny of the R. indicus RepABC-2 plasmid with pDSHI03 of D. shibae is interesting and it could be speculated if those plasmids share conserved traits that are beneficial for the association of roseobacters with eukaryotic microalgae from phylogenetically distant phyla, e.g., haptophytes (Emiliania huxleyi) and dinoflagellates (Prorocentrum lima).