Complete genome sequence of Marinobacter adhaerens type strain (HP15), a diatom-interacting marine microorganism

Marinobacter adhaerens HP15 is the type strain of a newly identified marine species, which is phylogenetically related to M. flavimaris, M. algicola, and M. aquaeolei. It is of special interest for research on marine aggregate formation because it showed specific attachment to diatom cells. In vitro it led to exopolymer formation and aggregation of these algal cells to form marine snow particles. M. adhaerens HP15 is a free-living, motile, rod-shaped, Gram-negative gammaproteobacterium, which was originally isolated from marine particles sampled in the German Wadden Sea. M. adhaerens HP15 grows heterotrophically on various media, is easy to access genetically, and serves as a model organism to investigate the cellular and molecular interactions with the diatom Thalassiosira weissflogii. Here we describe the complete and annotated genome sequence of M. adhaerens HP15 as well as some details on flagella-associated genes. M. adhaerens HP15 possesses three replicons; the chromosome comprises 4,422,725 bp and codes for 4,180 protein-coding genes, 51 tRNAs and three rRNA operons, while the two circular plasmids are ~187 kb and ~42 kb in size and contain 178 and 52 protein-coding genes, respectively.


Introduction
Strain HP15 (DSM 23420) is the type strain of the newly established species Marinobacter adhaerens sp. nov. and represents one of 27 species currently assigned to the genus Marinobacter [1]. Strain HP15 was first described by Grossart et al. in 2004 [2] as a marine particle-associated, Gramnegative, gammaproteobacterium isolated from the German Wadden Sea. The organism is of interest because of its capability to specifically attach in vitro to the surface of the diatom Thalassiosira weissflogii-inducing exopolymer and aggregate formation and thus generating marine snow particles [3]. Marine snow formation is an important process of the biological pump, by which atmospheric carbon dioxide is taken up, recycled, and partly exported to the sediments. This sink of organic carbon plays a major role for marine biogeo-chemical cycles [4]. Several studies reported on the formation and properties of marine aggregates [5][6][7][8]. Although it was shown that heterotrophic bacteria control the development and aggregation of marine phytoplankton [3], specific functions of individual bacterial species on diatom aggregation have not been explored thus far. A better understanding of the molecular basis of bacteria-diatom interactions that lead to marine snow formation is currently gained by establishing a bilateral model system, for which M. adhaerens sp. nov. HP15 serves as the bacterial partner of the easy-to-culture diatom, T. weissflogii [3]. Herein, we present a set of features for M. adhaerens sp. nov. HP15 (Table 1) together with its annotated complete genomic sequence, and a detailed analysis of its flagella-associated genes. Standards in Genomic Sciences

Classification and features
M. adhaerens sp. nov. strain HP15 is a motile, Gram-negative, non-spore-forming rod ( Figure 1). Based on its 16S rRNA sequence, strain HP15 was assigned to the Marinobacter genus of Gammaproteobacteria. Two other Marinobacter species were identified based on their interactions with eukaryotes -M. algicola isolated from dinoflagellate cultures [20] and M. bryozoorum derived from Bryozoa [21]. The 16S rRNA gene of strain HP15 is most closely related to those of the type strains of M. flavimaris (99%), M. salsuginis (98%) and M. algicola (96%). These four type strains form a discrete cluster in the phylogenetic tree ( Figure 2). In contrast, DNA-DNA hybridization experiments revealed that the genome of M. adhaerens sp. nov. HP15 showed about 64% binding to that of M. flavimaris [1], which is below the generally accepted species differentiation limit of 70% [25].

Chemotaxonomy
Strain HP15 can grow in artificial sea water with a nitrogen-to-phosphorus ratio of 15:1 supplemented with glucose as the sole carbon source. In presence of diatom cells but without glucose, HP15 utilized diatom-produced carbohydrates as sole source of carbon. Furthermore, M. adhaerens sp. nov. HP15 differed from M. flavimaris and other Marinobacter species in a number of chemotaxonomic properties, such as utilization of glycerol, fructose, lactic acid, gluconate, alanine, and glutamate [1]. Additionally, strain HP15 showed a unique fatty acid composition pattern.

Genome sequencing and annotation
Genome project history M. adhaerens HP15 was selected for sequencing because of its phylogenetic position, its particular feature as a diatom-interacting marine organism [3], and its feasible genetic accessibility to act as a model organism. The respective genome project is deposited in the Genome OnLine Database [19] and the complete genome sequence in GenBank. The main project information is summarized in Table 2. plus all type strains of the genus Marinobacter and the type species of the neighbor order Pseudomonadales. Sequence selection and alignment improvements were carried out using the Living Tree Project database [22] and the ARB software package [23]. The tree was inferred from 1,531 alignment positions using RAxML [24] with GTRGAMMA model. Support values from 1,000 bootstrap replicates are displayed above branches if larger than 50%. The scale bar indicates substitutions per site.

Growth conditions and DNA isolation
M. adhaerens sp. nov. HP15 was grown in 100 ml Marine Broth medium [26] at 28°C. A total of 23 µg DNA was isolated from the cell paste using a Qiagen DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions.

Genome sequencing and assembly
The Marinobacter adhaerens sp. nov.  Phylum Proteobacteria TAS [11] Class Gammaproteobacteria TAS [12,13] Current classification Order Alteromonadales TAS [12,14] Family Alteromonadaceae TAS [15][16][17] Genus Evidence codes -IDA: inferred from Direct Assay (first time in publication); 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 of the species, or anecdotal evidence). These evidence codes are from the Gene Ontology project [19]. If evidence code is IDA, then the property was directly observed for a live isolate by one of the authors or an expert mentioned in the acknowledgements.

Genome annotation
Potential protein-coding genes were identified using GLIMMER v3.02 [27], transfer RNA genes were identified using tRNAScan-SE [28] and ribosomal RNA genes were identified via BLAST searches [29] against public nucleotide databases. The annotation of the genome sequence was performed with the GenDB v2.2.1 system [30]. For each predicted gene, similarity searches were performed against public sequence databases (nr, SwissProt, KEGG) and protein family databases (Pfam, InterPro, COG). Signal peptides were predicted with SignalP v3.0 [31,32] and transmembrane helices with TMHMM v2.0 [33]. Based on these observations, annotations were derived in an automated fashion using a fuzzy logic-based approach [34]. Finally, the predictions were manually checked with respect to missing genes in intergenic regions and putative sequencing errors, and the annotations were manually curated using the Artemis 11.3.2 program and refined for each putative gene [35].

Genome properties
The genome of strain HP15 comprises three circular replicons: the 4,422,725 bp chromosome and two plasmids of ~187 kb and ~42 kb, respectively (Table 3A and Figure 3). The genome possesses a 56.9% GC content (Table 3B). Of the 4,482 predicted genes, 4,422 were protein coding genes, and 60 RNAs; 391 pseudogenes were also identified. The majority of the protein-coding genes (67.5%) were assigned with a putative function, while those remaining were annotated as hypothetical proteins. The distribution of genes into COGs functional categories is presented in Table 4.  a) The total is based on either the size of the genome in base pairs or the total number of protein coding genes in the annotated genome. b) Also includes 54 pseudogenes and 5 other genes.

Flagella-associated gene clusters of M. adhaerens HP15
Because M. adhaerens HP15 was experimentally shown to adhere to diatom cells, gene clusters coding for secretion, assembly, and mechanistic function of the polar flagellum were analyzed in detail ( Figure 4). Besides several other chemotactic mechanisms and various cell surface interactions, bacterial flagella and other cell appendages had previously been shown to be instrumental for chemotactic movement towards and adhesion to biotic surfaces [36,37]. The amino acid sequences of proteins encoded by the three identified gene clusters showed significant similarities to orthologous and experimentally well-described gene products of P.
aeruginosa PAO1 and various other bacterial species as determined by BLASTP algorithm comparison using the Blosum 62 substitution matrix [29]. Not surprisingly, hook and motor switch complex components were most conserved. However, gene products involved in flagellar filament formation encoded by Cluster II also showed 53 to 78% similarity to the respective PAO1 proteins. Mutagenesis of flagella-associated genes of M. adhaerens HP15 will be carried out in the near future to study the role of flagella in bacteria-diatom interactions and to further our understanding of the cell-to-cell communication between those organisms.  a) The total is based on the total number of protein coding genes in the annotated genome