Genome sequence of the Litoreibacter arenae type strain (DSM 19593T), a member of the Roseobacter clade isolated from sea sand

Litoreibacter arenae Kim et al. 2012 is a member of the genomically well-characterized Rhodobacteraceae clade within the Roseobacter clade. Representatives of this clade are known to be metabolically versatile and involved in marine carbon-producing and biogeochemical processes. They form a physiologically heterogeneous group of Alphaproteobacteria and were mostly found in coastal or polar waters, especially in symbiosis with algae, in microbial mats, in sediments or together with invertebrates and vertebrates. Here we describe the features of L. arenae DSM 19593T, including novel aspects of its phenotype, together with the draft genome sequence and annotation. The 3,690,113 bp long genome consists of 17 scaffolds with 3,601 protein-coding and 56 RNA genes. This genome was sequenced as part of the activities of the Transregional Collaborative Research Centre 51 funded by the German Research Foundation (DFG).


Introduction
Strain GA2-M15 T (= DSM 19593 = KACC 12675) is the type strain of the species Litoreibacter arenae [1,2]. The genus Litoreibacter is a member of the highly abundant marine Roseobacter lineage, which plays an important role in the global carbon and sulfur cycles, and thus for the climate on Earth. Phylogenetically, this alphaproteobacterial genus is related to the genera Jannaschia, Octadecabacter and Thalassobius [1]. Strain GA2-M15 T was isolated from a sea-sand sample from the coast of Homi Cape, Pohang City, South Korea as Thalassobacter arenae [1], which was later on reclassified into Litoreibacter arenae [2]. The name for the genus Litoreibacter was constructed from litoreus ('belonging to the seashore') and bacter ('a rod') [3]. The species epithet arenae refers to the Neolatin adjective arenae, 'of sand'. Current PubMed records do not indicate any follow-up research with strain GA2-M15 T after the initial description of T. arenae [1] and its reclassification into L. arenae [2]. Here we present a summary classification and a set of features for L. arenae DSM 19593 T , including novel aspects of its phenotype, together with the description of the genomic sequencing and annotation. Standards in Genomic Sciences

Classification and features 16S rRNA gene analysis
A representative genomic 16S rRNA gene sequence of L. arenae DSM 19593 T was compared using NCBI BLAST [4,5] under default settings (e.g., considering only the high-scoring segment pairs (HSPs) from the best 250 hits) with the most recent release of the Greengenes database [6] and the relative frequencies of taxa and keywords (reduced to their stem [7]) were determined, weighted by BLAST scores. The most frequently occurring genera were Jannaschia (38.1%), Thalassobacter (15.4%), Octadecabacter (11.7%), Roseovarius (10.7%) and Roseobacter (10.2%) (28 hits in total). Regarding the three hits to sequences from other members of the genus, the average identity within HSPs was 96.0%, whereas the average coverage by HSPs was 98.7%. Among all other species, the one yielding the highest score was 'Octadecabacter orientus' (DQ167247), which corresponded to an identity of 99.2% and an HSP coverage of 99.6%. (Note that the Greengenes database uses the INSDC (= EMBL/NCBI/DDBJ) annotation, which is not an authoritative source for nomenclature or classification). The highestscoring environmental sequence was FJ664800 (Greengenes short name 'Quantitative dynamics cells plankton-fed microbial fuel cell clone plankton D11'), which showed an identity of 97.0% and an HSP coverage of 99.6%. The most frequently occurring keywords within the labels of all environmental samples which yielded hits were 'lake' (9.9%), 'tin' (9.8%), 'xiaochaidan' (9.4%), 'microbi' (2.6%) and 'sea' (2.5%) (222 hits in total). Environmental samples which yielded hits of a higher score than the highest scoring species were not found. Figure 1 shows the phylogenetic neighborhood of L. arenae in a 16S rRNA sequence based tree. The sequence of the single 16S rRNA gene in the genome does not differ from the previously published 16S rDNA sequence (EU342372).
The substrate utilization and resistance patterns of L. arenae DSM 19593 T were also determined for this study using Generation-III microplates in an OmniLog phenotyping device (BIOLOG Inc., Hayward, CA, USA). The microplates were inoculated at 28°C with a cell suspension at a cell density of 95-96% turbidity and dye IF-A. Further additives were vitamin, micronutrient and sea salt solutions. The exported measurement data were further analyzed with the opm package for R [26,27], using its functionality for statistically estimating parameters from the respiration curves such as the maximum height, and automatically translating these values into negative, ambiguous, and positive reactions. The strain was studied in two independent biological replicates, and reactions with a different behavior between the two repetitions, if any, were regarded as ambiguous.  [8,9] of the 16S rRNA gene sequence under the maximum likelihood (ML) criterion [10]. Rooting was done initially using the midpoint method [11] and then checked for its agreement with the current classification ( Table 1). The branches are scaled in terms of the expected number of substitutions per site. Numbers adjacent to the branches are support values from 650 ML bootstrap replicates [12] (left) and from 1,000 maximum-parsimony bootstrap replicates [13] (right) if larger than 60%. Lineages with type strain genome sequencing projects registered in GOLD [14] are labeled with one asterisk, those also listed as 'Complete and Published' with two asterisks [15]. L. arenae DSM 19593 T showed positive reactions for pH 6, 1% NaCl, 4% NaCl, D-galactose, 3-O-methyl-Dglucose, D-fucose, L-fucose, L-rhamnose, 1% sodium lactate, myo-inositol, rifamycin SV, L-aspartic acid, Lglutamic acid, L-histidine, L-serine, D-glucuronic acid, glucuronamide, quinic acid, L-lactic acid, citric acid, αketo-glutaric acid, D-malic acid, L-malic acid, nalidixic acid, lithium chloride, acetic acid and sodium formate. No reaction was found for dextrin, D-maltose, Dtrehalose, D-cellobiose, β-gentiobiose, sucrose, Dturanose, stachyose, pH 5, D-glucose-6-phosphate, D-fructose-6phosphate, D-aspartic acid, D-serine, troleandomycin, minocycline, gelatin, glycyl-L-proline, L-alanine, Larginine, L-pyroglutamic acid, lincomycin, guanidine hydrochloride, niaproof, pectin, D-galacturonic acid, L-galactonic acid-γ-lactone, D-gluconic acid, mucic acid, D-saccharic acid, vancomycin, tetrazolium violet, tetrazolium blue, p-hydroxy-phenylacetic acid, methyl pyruvate, D-lactic acid methyl ester, bromo-succinic acid, potassium tellurite, tween 40, γ-amino-n-butyric acid, α-hydroxy-butyric acid, β-hydroxy-butyric acid, α-keto-butyric acid, acetoacetic acid, propionic acid, aztreonam, butyric acid and sodium bromate. The measured utilization of carbon sources differs in some aspects from the one recorded in [1]. Lhistidine and L-rhamnose were reported in [1] not to support bacterial growth, whereas in the Omnilog measurements both substrates yielded a positive reaction. This may be due to the higher sensitivity of respiratory measurements [28]. The utilization of propionate, D-fructose, D-glucose, D-mannose, Dmannitol, melibiose and glycerol reported by [1] could not be confirmed by the Omnilog measurements. Changes in the substrate-utilization pattern may arise from distinct cultivation conditions such as growth medium and temperature.

Genome sequencing and annotation
Genome project history The strain was first chosen for genome sequencing in the Genomic Encyclopedia of Bacteria and Archaea (GEBA) phase I project according the GEBA criteria [31,32], but then sequenced as part of the "Ecology, Physiology and Molecular Biology of the Roseobacter clade project: Towards a Systems Biology Understanding of a Globally Important Clade of Marine Bacteria" funded by the German Research Council (DFG). Project information is stored in the Genomes OnLine Database [33]. The Whole Genome Shotgun (WGS) sequence is deposited in Genbank and the Integrated Microbial Genomes database (IMG) [34]. A summary of the project information is shown in Table 2.

Growth conditions and DNA isolation
A culture of DSM 19593 T was grown aerobically in DSMZ medium 514 [35] at 28°C. Genomic DNA was isolated using Jetflex Genomic DNA Purification Kit (GENOMED 600100) following the standard protocol provided by the manufacturer but modified by an incubation time of 60 min, incubation on ice over night on a shaker, the use of additional 50 µl proteinase K, and the addition of 100 µl protein precipitation buffer. DNA is available from DSMZ through the DNA Bank Network [36].

Genome sequencing and assembly
The genome was sequenced using one Illumina PE library ( Table 2). Illumina sequencing [37] was performed on a GA IIx platform with 150 cycles. The paired-end library contained 520 bp insert size. To correct sequencing errors and improve quality of the reads, clipping was performed using fastq-mcf [38] and quake [39]. After this step, 4,717,610 reads with a median length of 124 bp were assembled using velvet [40]. The resulting draft genome consisted of 71 contigs organized in 45 scaffolds. The initial draft sequences were separated into artificial Sanger reads of 1,000 nt size plus 75 nt overlap. The number of gaps was reduced by manual editing in phred/phrap/consed version 20.0 [41]. The final assembly was composed of 17 contigs organized in 15 scaffolds. (The version deposited at Genbank contains two scaffolds less, which did not meet the requirements for the minimal contig length. The additional fragments 'thalar_Contig12.1' and 'thalar_Contig18_1.4' can be found in the IMG database). The combined sequences provided a 195× coverage of the genome. Evidence codes -TAS: Traceable Author Statement (i.e., a direct report exists in the literature); NAS: Nontraceable 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). Evidence codes are from the Gene Ontology project [25]. Standards in Genomic Sciences

Genome annotation
Genes were identified using Prodigal [42] as part of the JGI genome annotation pipeline [43]. The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) nonredundant database, UniProt, TIGR-Fam, Pfam, PRIAM, KEGG, COG, and InterPro databases. Identification of RNA genes were carried out by using HMMER 3.0rc1 [44] (rRNAs) and tRNAscan-SE 1.23 [45] (tRNAs). Other non-coding genes were predicted using INFER-NAL 1.0.2 [46]. Additional gene prediction analysis and functional annotation was performed within the Integrated Microbial Genomes -Expert Review (IMG-ER) platform [34]. CRISPR elements were detected using CRT [47] and PILER-CR [48].

Genome properties
The genome statistics are provided in Table 3 and Figure 3. The genome consists of a 3.56 Mbp chromosome with a G+C content of 60%, and a 140 kbp plasmid with a G+C content 59%. Of the 3,657 genes predicted, 3,601 were protein-coding genes, and 56 RNAs. The majority of the protein-coding genes (81.8%) were assigned a putative function while the remaining ones were annotated as hypothetical proteins. The distribution of genes into COGs functional categories is presented in Table 4.

Insights into the genome
The genome comprises a single extrachromosomal element (with not yet validated circularity), 'thalar_Contig204.17', which is 139.9 kbp in size containing 130 protein-coding genes including a large RTX-toxin gene and a F0F1-type ATPase operon. It contains the typical replication modules.
Its replication system is of the ABC-9 type with the compatibility group RepC-9 [49]. This type of repABC operon was found in two representatives of the genera Octadecabacter and Roseobacter, respectively, as well as in Dinoroseobacter shibae [49]. The presence of the replication-initiation gene DnaA (Thalar_03034) reveals the chromosomal origin of the largest scaffold, namely, the 1.091 Mbp long scaffold 'thalar_Contig148.14'. However, the scaffold harbors neither a plasmid stability module nor a type IV secretion system. Genome analysis of strain DSM 19593 T revealed the presence of genes encoding proteins associated to carbon monoxide utilization (thalar_00241, thalar_00242, thalar_02265, thalar_03324, thalar_03325, thalar_03395, thalar_03397) as well as genes forming a putative operon, which are involved in the oxidation of sulfur (thalar_01786 to_01792) indicating the oxidation of sulfur to produce energy. Additional gene sequences of interest encode a homogentisate 1,2-dioxygenase (thalar_03573), several haloacid dehalogenase superfamily proteins (thalar_00489, thalar_00580, thalar_01120, thalar_01943, thalar_02401) and a 2-haloalkanoic acid dehalogenase type II (thalar_00287). The presence of such genes could indicate the respiratory degradation of recalcitrant compounds by strain DSM 19593 T in its ecological niche. Further genes encoding a N-acyl-L-homoserine lactone synthetase (thalar_00160) and a response regulator (thalar_00161) associated to quorum sensing were observed [50][51][52][53]. Genome analysis of strain DSM 19593 T also revealed the presence of genes encoding a bacteriophage associated genes (e.g., thalar_00003 to 00007). A gene encoding a sensor of blue light using FAD (BLUF, thalar_02670) was also detected, indicating possible blue-light dependent signal transduction.