The strains used in this study are listed in Table 1 with indication of their origins and references of previous studies. Marine Agar (MA) and Marine Broth (MB) were used as routine cultivation media and incubations were done at 26°C.

The complete 16S rRNA gene sequence of strains CECT 5113^T, CECT 5114, CECT 5118^T, and CECT 5120 were 1,465, 1,465, 1,469, and 1,469 nucleotides in length, respectively. These sequences extracted from the genome were compared with corresponding sequences of the type strains within the Roseobacter group using alignments retrieved from SILVA and LTP (Yarza et al., 2010) latest updates as references. When necessary, additional sequences were retrieved from the GenBank/EMBL/DDBJ databases. Alignments were corrected manually based on secondary structure information. Sequence similarities were calculated in ARB based on sequence similarities without the use of an evolutionary substitution model. Phylogenetic analysis using alternative treeing methods (maximum-parsimony, maximum-likelihood, and distance matrix) and data subsets were performed using the appropriate ARB tools (Ludwig et al., 2004).

Genomic DNA was isolated using Real Pure Spin kit (Durviz) following the standard protocol recommended by the manufacturer. The integrity of the extracted DNA was checked by visualization in a 2.0% (w/v) agarose gel electrophoresis. Its purity and quantity was checked by measuring the absorbance at 260 and 280 nm with a spectrophotometer Nanodrop2000c (Thermo Scientific) and calculating the ratio A260/A280. Genome sequencing was achieved at Central Service of Support to Experimental Research (SCSIE) of the University of Valencia (Valencia, Spain) using an Illumina Miseq technology with 2 × 250 paired-end reads. The Illumina reads were analyzed for quality control using FASTQC, a common quality control tool developed by Babraham Bioinformatics to check raw sequencing data, which is wrapped in Galaxy Orione Server (Cuccuru et al., 2014). After filtering, the remaining reads were assembled using several software choices for comparative purposes: (i) Spades 3.0.0 (Bankevich et al., 2012) incorporated as a tool in Galaxy Orione Server, (ii) Seqman Ngen 12.0.1 (DNAstar), and (iii) Velvet 1.0.0 de novo assembler (Zerbino and Birney, 2008). After performance evaluation and comparison of metrics, the best assembly for each organism was further processed. The bioinformatic tool CheckM (Parks et al., 2015) was used to assess the genome quality prior to annotation using Prokka v1.4.0 (Seemann, 2014), an open source software tool within Galaxy Orione Server, and RAST v2.0 (Rapid Annotation using Subsystem Technology; Aziz et al., 2008).

The similarity between genomes was assessed using several indices useful for species delineation. Thus, the DNA-DNA hybridization (DDH) was estimated in silico with the Genome-to-Genome Distance Calculator (GGDC 2.0) using the BLAST method and recommended formula 2 (Meier-Kolthoff et al., 2013); the average nucleotide identities according to MUMmer (ANIm) and BLAST (ANIb) were determined in JSpeciesWS (Richter et al., 2015); and OrthoANI values were calculated with the standalone Orthologous Average Nucleotide Identity Tool (OAT) (Lee et al., 2016).

The phylogenetic relationship of the genomes was explored with BCG54 using default settings. This software tool is available for download at EzBioCloud (Yoon et al., 2017) and employs a set of bacterial core genes, namely 54, that are single-copy and commonly present in all bacterial genomes.

All determinations were performed in duplicate in non-simultaneous assays. Cell morphology was determined on wet mounts prepared from 24 to 48 h MA cultures of the strains, by using phase contrast microscopy in a Leica DMRB fluorescence microscopy. Colony morphology and pigmentation were recorded from 48 h MA cultures. PHB accumulation was determined according to Spiekermann et al. (1999). Ranges of temperature (4, 15, 26, 37, and 40°C) and salinities (3.5–10%) were determined in MA incubated up to 7 days. Marine Agar was supplemented with NaCl to attain total salinities of 4, 5, 6, 7, 8, 9, and 10%. Optimal values were taken from the fastest grown plates. Specific ionic requirements were tested by assessing the growth abilities of the strain on solid media with defined combinations of four sea salts (NaCl, MgCl₂, CaCl₂, and KCl), according with already reported methods (Macián et al., 2005). Extracellular hydrolytic activities on casein, starch, Tween-80, and DNA were determined after 6 d incubation by using Marine Agar supplemented with 10% (v/v) casein suspension, or 0.2% (w/v) soluble starch, for the first substrates. Tween-80 Agar (Smibert and Krieg, 2007) and DNAse agar (Oxoid) were supplemented with sea salts (Marine Cation Supplement, Farmer and Hickman-Brenner, 2006). Activity on starch was revealed after lugol addition and HCl 1 N was used to show DNAse activity. Oxidase test was performed with Oxoid oxidase discs and catalase was tested with 10 vol. H₂O₂. API 20NE and API ZYM strips were inoculated with cell suspensions prepared in 3.5% Seasalts (Oxoid) and AUX Medium for API 20NE assimilation tubes was supplemented with a concentrated Seasalts solution to give the same salinity used in the cell suspension. Sole carbon and energy sources used for growth were tested on Basal Medium Agar as described by Baumann and Baumann (1981).

Fatty acid methyl esters were extracted from biomass grown for 48 h on MA at 26°C and prepared according to standard protocols as described for the MIDI Microbial Identification System (Sasser, 1990) at the CECT. Cellular fatty acid content was analyzed by gas chromatography with an Agilent 6850 chromatographic unit, with the MIDI Microbial Identification System using the TSBA6 method (MIDI, 2008) and identified using the Microbial Identification Sherlock software package.

Strains CECT 5113^T, CECT 5114, CECT 5118^T, and CECT 5120 were affiliated to the genus Thalassobius based on partial 16S rRNA gene sequence comparison performed among routine identification and authentication procedures at CECT. The strains had been isolated from the same samples and geographic location that rendered the type strain of T. mediterraneus (CECT 5383^T = XSM19^T). In fact, they were provisionally identified as T. mediterraneus or Thalassobius sp. strains in the CECT catalog. The similarities of their respective 16S rRNA gene sequences to T. mediterraneus CECT 5383^T were low (only 96.6%) for strains CECT 5113^T and CECT 5114, which showed a closer position to T. maritimus (98.3%). On the other hand, strains CECT 5118^T and CECT 5120 showed a 99.6% similarity to T. mediterraneus CECT 5383^T.

When a phylogenetic tree based on almost complete (genome derived) 16S rRNA gene sequences was built with the whole set of Roseobacter group species plus these four strains (Figure 1), a clear affiliation to the genus Thalassobius could be seen, as the sequences are included among Thalassobius spp. As expected, strains CECT 5113^T and CECT 5114 join T. maritimus GSW-M6^T with a high bootstrap support while strains CECT 5118^T and CECT 5120 merge with T. mediterraneus CECT 5383^T, also with high bootstrap, according with their similarity levels toward these species. The Thalassobius species, however, do not only include the sequences of these four strains, but also the types of the five Shimia species (all forming a monophyletic cluster within the Thalassobius group), plus the two species of Thalassococcus, which remain as a pair related to T. maritimus.

A close revision of 16S rRNA trees presented along the proposals of new species on these three genera reveal large variations in the relative positions of the taxa included in the analysis, depending on the particular set of selected taxa and the treeing method. The instability of the branching is always noticeable: for example, Nogi et al. (2015) used four Thalassobius spp. and no Thalassococcus to build their Neighbor Joining tree with the five species of Shimia: Thalassobius spp. appear as closest neighbors, forming a monophyletic cluster with the Shimia spp. By contrast, Hameed et al. (2013) presented a Neighbor Joining tree in which neighbors of Shimia are not Thalassobius spp., but Nautella italica and Lentibacter algarum. More examples of instability could be seen when comparing other papers describing Roseobacter group members. It is clear that 16S rRNA gene sequences alone are unable to resolve the phylogeny of a group displaying such complexity and overburden of genera descriptions. It seems particularly important to include not only taxa showing the highest 16S rRNA sequence similarity to the strains being considered, but a large representative set of members of the group. A more robust phylogeny is likely to be achieved by the use of whole genome information and selection of a large, optimized set of genes allowing more sound phylogenetic resolution of the clade, an approach currently in development. Genomic sequences are also valuable for the circumscription of the isolates at the species level and for providing insights into their biology, as will be reported in the coming subheadings.

Genome length, G+C molar content, protein and rRNA genes are summarized in Table 2 for all the genomes used. Strains CECT 5113^T and CECT 5114 possess genomes in the lower size and G+C range: <3.5 Mb genome size and <55 mol% G+C molar content. They are similar to T. maritimus, the phylogenetically closest species, which is also under 3.5 Mb genome length and was until now the one with lower G+C content (56.3 mol%). Other type strains except for T. mediterraneus have genomes larger than 3.9 Mb and G+C higher than 58 mol%. Strains CECT 5118^T and CECT 5120 had larger genomes, 4.3–4.4 Mb, and G+C molar content is among the higher in the genus, 59.8 mol%, surpassed only by the value of the T. aestuarii type strain (60.4 mol%).

ANIb, ANIm, OrthoANI, and digital DDH were determined in order to relate the strains CECT 5113^T, CECT 5114, CECT 5118^T, and CECT 5120 to the eight strains of Thalassobius, Shimia, and Thalassococcus sp. with publicly available genomes. Results of these determinations are reported in Table 3 (ANIb, ANIm, digital DDH) and Figure 2 (OrthoANI).

The four strains formed two very tight pairs related by almost 100% with all ANI calculations: strains CECT 5113^T and CECT 5114 were 99.8–100% related to each other and showed a maximum relatedness to the genome of the type strain of T. maritimus (as expected from 16S rRNA gene sequence similarity), with values of 75.0–75.1% (ANIb), 75.6–75.7% (OrthoANI), and 19.5–20.9% (digital DDH). Values with other species were even lower. According with the currently established boundaries for genomic species definition (95–96 for ANI, 70% for digital DDH) these values qualify both strains as members of a single genomic species, different from any of their closest relatives.

Strains CECT 5118^T and CECT 5120 also show high levels of reciprocal relatedness (99.9% ANI and 98.5% digital DDH). T. mediterraneus, the species most related in terms of 16S rRNA gene sequence, is also the closest, with 79.1% ANIb, 79.9% OrthoANI, and 23.2% digital DDH relatedness to them. All other Thalassobius, Shimia, and Thalassococcus species compared show even lower values. Thus, strains CECT 5118^T and CECT 5120 should also be considered as yet another new species from a genomic point of view.

In order to substantiate the recognition of the two novel species, a wide phenotypic characterization of the strains was undertaken, including the type strains of T. mediterraneus, T. gelatinovorus, T. aestuarii, and T. maritimus.

Strains CECT 5113^T, CECT 5114, CECT 5118^T, and CECT 5120 were Gram-reaction (lyse in 3% KOH) and Gram-staining negative coccobacilli or short rods. Motility was negative in all cases and previous data (Ortigosa et al., 1994) on flagella stain indicate that the strains do not synthetize flagella. All four strains grow well on Marine Agar forming circular, slightly convex colonies with entire margin, which develop in 2 days incubation at 26°C. Strain CECT 5120 was slower and grew to a density lower than the three other strains. Strain CECT 5118^T produced a beige to brown diffusible pigment in prolonged incubations. All strains were able to accumulate polyhydroxyalkanoates (PHA) as revealed by the Nile Red plate assay on Marine Agar plus D-glucose (Spiekermann et al., 1999).

All strains grew optimally at 26°C and with salinities ranging from that in Marine Agar (3.4% aprox.) to 5% (3.5% of mixed salts in MA plus additional 1.6% NaCl). When cultured in media of the same nutritional content as MA (1% Tryptone and 0.3% yeast extract) but deprived of salts or supplemented with 2% NaCl or 2% KCl none of the strains was able to grow, indicating a strictly halophilic nature. Strains CECT 5118^T and CECT 5120 were also unable to grow when the medium was supplemented with mixtures of 2% NaCl plus 0.9% MgCl₂, 0.2% CaCl₂, or 0.1% KCl or combinations of the four salts. Strains CECT 5113^T and CECT 5114 did not grow with 2% NaCl or 2% KCl or with 2% NaCl plus MgCl₂ or CaCl₂ separately, but were able to grow when both divalent cations, calcium, and magnesium chlorides, were added to NaCl.

The strains were poorly reactive in API ZYM and API 20NE supplemented with Marine Cations Supplement, which contains the four principal marine cations as sulfate and chloride salts. In order to better fulfill the ionic requirements of the strains, API strips were replicated using Ocean Salts (Oxoid) solution, so the final salinity was 3.5%, but reactivity on the miniaturized tests systems did not improve.

Anaerobic growth with nitrate as alternative electron-acceptor and a mixture of succinate, acetate and lactate as substrates was assayed in Baumann's denitrification medium (Baumann and Baumann, 1981) with negative results for all four strains. T. gelatinovorus CECT 4357^T was the only positive strain. Results for nitrate to nitrite reduction in API 20NE strips were in accordance with the results obtained in Baumann's medium. None of the strains was able to ferment D-glucose, a result that, in combination to the inability to live anaerobically with nitrate, usually qualifies a strain as strict aerobe.

A large number of sole carbon and energy sources for growth were tested on Baumann's Basal medium Agar supplemented with 0.2% carbohydrates, or 0.1% organic acids, amino acids, and amines. The list of used substrates is included in the species descriptions. As a general rule, organic acids were the most widely used carbon sources, although most amino acids and some carbohydrates were also used by the strains. This behavior was already described as characteristic for the genus. The spectrum of carbon sources is useful to differentiate species within the group, as indicated in Table 4.

Hydrolytic extracellular activities were very scarce among Thalassobius strains, with two exceptions: T. gelatinovorus CECT 4357^T and T. aestuarii CECT 8650^T were able to degrade gelatin, but other strains did not show hydrolysis on any of the substrates tested: gelatin, casein, Tween-80, starch, or DNA.

Cellular fatty acid composition was determined for the eight strains alongside with the result reflected in Table 5. The main fatty acid for all strains is included in the Summed Feature 8, corresponding to C_{18 : 1} ω7c/C_{18 : 1} ω6c, which accounts for 72–88% of the total. It is also the dominant fatty acid in the whole Rhodobacteraceae family. The second most abundant, common fatty acid, is C_{16 : 0} (3–9%) while C_{18 : 0} is also common to all species but accounts for 1–2.6%. The rest of fatty acids have a differential distribution among species, with C_{18 : 1} ω7c 11methyl, C_{10 : 0} 3OH, and C_{12 : 1} 2OH amounting up to 9% in some species.

Differentiation among species of the genus based on experimental phenotypic tests could be achieved as indicated in Table 4.

After annotation with RAST and Prokka, using the SEED Viewer (Overbeek et al., 2014) and BIOiPLUG to explore annotated genomes and Metacyc as a resource for metabolic pathway information (Caspi et al., 2016), the following features could be predicted from genome sequences:

Selected genome-predicted differences for these two pairs and other Thalassobius, Shimia, and one Thalassococcus spp. are summarized in Table 6.

Considering both the experimentally determined features and the presence of genes predicting given phenotypic traits in the genomes, there are enough differences between strains and their closely related species for unequivocally differentiate the genomic groups previously delineated (Tables 4, 6). Genomic, phylogenetic and phenotypic information thus confirm that the four strains represent two novel species. They fulfill the basic characteristics defining the genus Thalassobius (Arahal et al., 2005) and have Thalassobius species as their closest phylogenetic neighbors in both cases, thus, they should be recognized as new Thalassobius species: we propose the name of Thalassobius activus, for strains CECT 5113^T and CECT 5114, with CECT 5113^T as type strain. Strains CECT 5118^T and CECT 5120 constitute another new species, for which we propose the name of Thalassobius autumnalis, with CECT 5118^T as type strain. Protologues for both species are included at the end of the section.

As previously pointed, another goal of this study was to study the relationships between the species of the genera Thalassobius, Shimia, and Thalassococcus by using genome-derived data. All publicly available type strain genomes of species in these three genera have been considered together with the genome from an unclassified Shimia species (Kanukollu et al., 2016) that has been proven to constitute a separate genomic species (Tables 2, 3). The tree generated with BCG54 using Thalassobacter stenotrophicus CECT 5294^T (CYRX01) and Nereida ignava CECT 5292^T (CVQV01) as outgroup is shown in Figure 3. The tree confirms the phylogenetic relationships previously suggested by 16S rRNA gene sequence comparison and genomic relatedness, confirming the close position of T. activus to T. maritimus and of T. autumnalis to T. mediterraneus. The grouping of T. mediterraneus plus T. autumnalis with T. gelatinovorus, a regular finding with other approaches, is also confirmed. All three Shimia species appear inside the Thalassobius genus, as closest relatives of T. aestuarii, while T. halodurans separates from Thalassobius and Shimia, and merges with the pair used as an outgroup, discarding the topology that Thalassococcus spp. occupied in the 16S rRNA tree. The results obtained from the BCG54 tree do not sustain monophyly for Thalassobius as currently defined. Furthermore, monophyly for Thalassobius—Shimia grouping is not resolved, as the group formed by T. mediterraneus-T. autumnalis-T. gelatinovorus does not merge with the rest of species before the last node. A better resolution of this node might be attained if the genomes of all Shimia, Thalassobius, and Thalassococcus were available, but, for the moment, we only can conclude that there are three possible monophyletic groups to be considered: the core Thalassobius spp. or Thalassobius spp. sensu stricto (containing the type species T. mediterraneus plus T. autumnalis and T. gelatinovorus), the one of T. maritimus plus T. activus sp. nov. and the one of Shimia spp. plus T. aestuarii. A strict taxonomic procedure would split Thalassobius spp. in three genera, with T. aestuarii (and perhaps others, as T. abyssi, T. aquaeponti, and T. litoreus) being reclassified as new combinations of Shimia, and T. maritimus plus T. activus being proposed as a new genus. But as already commented, this would be premature, since the addition of more type strain genomes would eventually reveal if the whole group constitutes a monophyletic lineage at higher levels, thus allowing a reclassification of all Shimia spp as Thalassobius new combinations, a solution that, in our opinion, would be also possible and more clarifying than the splitting option, which would imply to describe more and more indistinguishable genera within the Roseobacter group.

A survey of the gene content of the Thalassobius spp., Shimia spp., and T. halodurans draft genomes was conducted in order to enlighten the taxonomic status of the three genera. We looked for traits especially used for taxonomic work at the genus level. Genomic traits that are differential among species and/or genera are presented in Table 6. A list of features shared by all three genera representatives is included as Table 7.

All three genera representatives share characteristics owned by the whole Rhodobacteraceae family, as the peptidoglycan composition (with m-DAP at 3rd position), predominant ubiquinone (Q10), major cellular fatty acid (C_{18 : 1} ω7c/C_{18 : 1} ω6c) and a G+C molar content over 50%. Maybe, the lack of minCDE genes should be added to this list of traits common to all Rhodobacteraceae: the absence of these genes in various Rhodobacteraceae was already noted by Lutkenhaus (2007) and we have been unable, so far, to find any member of this group that contained this part of the cytoskeletal machinery. Obviously, these shared common traits (symplesiomorphies) should not be considered as relevant to back a genera fusion proposal. But there are other that are shared by these particular genera (synapomorphies): we would highlight, for example, the GTA gene set composition, characterized by the common absence of capsid gene, which seems to be exclusive of these genera in contrast to the other >40 genomes of Roseobacter group members that could be compared in the Seed Viewer. The ability to accumulate polyhydroxyalkanoates, although not exclusive, would be another common character, not shared with all other Rhodobacteraceae. Other candidate traits are indicated in Table 7.

On the other hand, several genomic differences were found that allow species discrimination, as well as differentiation of the three phylogenetic clusters revealed by gene and multigenic trees: polar lipid synthesis, for example, pull apart the T. aestuarii-Shimia spp. group by the exclusive presence of DPG synthetic ability, while Thalassococcus could be differentiated by the unique presence of gene psd, encoding a phosphatidyl ethanolamine-synthetizing enzyme, phosphatidyl serine decarboxylase (EC 4.1.1.65), which is absent from all other species. The absence of genes encoding for PE biosynthesis in all Thalassobius species was surprising, as Park et al. (2012) reported the detection of PE in the type strains of four species (T. maritimus, T. aestuarii, T. gelatinovorus, and T. mediterraneus) by using TLC chromatography. But genomes of these type strains did not contain phosphatidylserine decarboxylase gene and PE synthesis is not predicted by RAST for any of the eight Thalassobius strains whose genomes have been explored. Genes for key enzymes of the alternative routes for PE synthesis, ethanolamine phosphotransferase (EC 2.7.8.1) or L-serine phosphatidylethanolamine phosphatidyl transferase (EC 2.7.8.29) were also undetected. Undoubtedly, this is a point that needs more attention, because of the importance that is given to polar lipid composition in bacterial taxonomy.

Absence of carbon monoxide dehydrogenase and hvrA gene are characteristic of T. maritimus-T. activus, while, the presence of aryl sulfatase is exclusive of the Shimia spp.-T. aestuarii group.

It is interesting to highlight that T. aestuarii shares with T. gelatinovorus the ability to degrade aromatic compounds, as confirmed through Aromadeg (Duarte et al., 2014) showing a complete that both type strains contain genes coding for extradiol dioxygenases of Viccinal Chelate superfamily and Lig B superfamily (for degrading monocyclic substrates in the first case and homoprorocatechuate and protocatechuate, in the second) and Rieske non-heme iron oxygenases of the phthalate dioxygenases family. However, only T. aestuarii DSM 15283^T genome harbors an extradiol dioxygenase of the Cupin superfamily, which anables to degrade gentisate and, on the other side, T. gelatinovorus CECT 4357^T possesses an additional phthalate oxygenase and a benzoate oxygenase. T. halodurans DSM 26915^T genome also encodes two extradiol dioxygenases of Viccinal Chelate superfamily and one Rieske non-heme iron oxygenases of the phthalate dioxygenases. The three Shimia genomes were also checked with Aromadeg (default parameters) but no protein had matches with the database.

In a recent phylogenomic study, Simon et al. (2017) analyse the evolutionary adaptation of Rhodobacteraceae to marine and non-marine habitats. Among other things, they conclude that the so-called Roseobacter group is not monophyletic, but their members derived from a common marine ancestor shared with other non-marine, non-halophilic Rhodobacteraceae (Rhodobacter and Paracoccus, mainly), the later representing an adaptation to non-marine habitats. A selection of genome-predicted, habitat-correlated enzymes is shown in their Table 1, including several lost in non-marine habitats plus others that were gained in marine habitats. The analysis of Simon et al. (2017) included no Thalassobius, Shimia, or Thalassococcus (the genome labeled as “Thalassiobium sp. R2A62” is completely unrelated to any true Thalassobius), so we have explored the distribution of the marine-related enzymes they reported in the genomes employed in this paper, as all of them pertain to marine inhabitants. Results are indicated in Table 8. As it can readily be observed, the five enzymes classified under the category of “Lost in non-marine habitats” are all present in the genomes of Thalassobius spp. (6 species), Shimia spp (3 species), and in the single Thalassococcus species included. The only significant exception was the genes for large, medium, and small carbon monoxide dehydrogenase chains, absent from the genomes of T. maritimus and T. activus sp. nov. (both strains), a trait already highlighted as characteristic of this small clade (Figure 3). On the other hand, enzymes apparently “gained in marine habitats” are not widely represented in Thalassobius or Shimia genomes. A few interesting exceptions are the betaine homocysteine S-methyl transferase (EC 2.1.1.5), involved in glycine-betaine catabolism, present in all these genomes, and the nitrile hydratase (EC 4.2.1.84), absent only from the genomes T. activus sp. nov. and T. halodurans, but present in the rest. Two additional occurrences are worth to note: γ-butyrobetaine dioxygenase (EC 1.14.11.1), participating in carnitine biosynthesis, is found exclusively in the genomes of Thalassobius spp. sensu stricto (T. mediterraneus, T. autumnalis sp. nov., and T. gelatinovorus) while aryl sulfatase (EC 3.1.6.1) is present only in T. aestuarii and Shimia spp. (as already mentioned); thus, these two enzymes could be used as discriminant traits to differentiate the respective groups.

So, this analysis proves there is room for either a proposal of reunification of (at least) Shimia spp. with Thalassobius spp. under the name of Thalassobius, but also for the splitting of Thalassobius-Shimia in three genera (one of them a new one for T. maritimus-T. activus). However, any decision should be postponed until the gap in genomes of type strains pertaining to the group is closed.

Thalassobius activus (ac.ti'vus. L. masc. adj. activus, active, referring to the metabolic activity of the type strain).

Cells are Gram-reaction-negative, non- motile coccobacilli. Aerobic and chemoorganotroph, they grow on Marine Agar as regular unpigmented circular colonies. Cells accumulate PHB. Mesophilic, able to grow from 15 to 26°C (optimum, 26°C), but not a 4 or 37°C. Slightly halophilic, requires sodium, calcium and magnesium salts for growth. Maximum salinity for growth is 6% (optimum 3.5–5%), no growth is obtained without salt, at 7% or above. Oxidase and catalase are positive. Fermentation of carbohydrates, nitrate reduction and denitrification are negative. Negative for urea, casein, gelatin, starch, Tween-80 and DNA hydrolysis. Indole production from tryptophan and arginine dihydrolase are negative, PNPG and alkaline phosphatase are positive.

Carbon sources used for growth included D-cellobiose, sucrose, D-melibiose, D-mannitol, m-inositol, acetate, pyruvate, propionate, butyrate, t-aconitate, citrate, 2-oxoglutarate, succinate, lactate, 3-hydroxybutyrate, glycine, L-leucine, L-serine, L-threonine, L-arginine, L-glutamate, L-alanine, L-tyrosine, L-ornithine, 4-aminobutyrate, and L-aspartate. No growth is obtained with D-ribose, L-arabinose, D-xylose, D-glucose, D-fructose, D-galactose, D-trehalose, D-mannose, L-rhamnose, maltose, lactose, salicin, amygdalin, D-glycerol, D-sorbitol, D-gluconate, D-glucuronate, D-galacturonate, D-glycerate, D-saccharate, L-citrulline, L-histidine, L-lysine, L-sarcosine, or putrescine.

Major cellular fatty acids are C_{18 : 1} ω7c/ω6c and C_{10 : 0} 3OH.

The predominant respiratory quinone, Q10 was inferred from annotated gene encoding decaprenyldiphosphate synthase (EC 2.5.1.91). Major polar lipids, PG and PC, were inferred from annotated genes encoding CTP-phosphatidate cytidyl transferase (EC 2.7.7.41), phosphatidyl glycerophosphate synthase (EC 2.7.8.5), phosphatidyl glycerophosphatase (EC 3.1.3.27), and phosphatidyl choline synthase (EC 2.7.8.24), in the genomes of strains CECT 5113^T and CECT 5114.

The G+C content of the DNA is 54.4–54.5 mol% (partial genome sequences).

The type strain, CECT 5113^T (=11SM13^T =LMG 29900^T) was isolated from coastal seawater, Mediterranean Sea, at Vinaroz coast, Spain.

Thalassobius autumnalis (au.tum.na'lis. L. adj. autumnus, fall, autumn, after the season of isolation).

Cells are Gram-reaction negative, non-motile coccobacilli, aerobic, and chemoorganotroph. Grows on Marine Agar forming regular circular colonies. Some strains produce a brown diffusible pigment on prolonged incubation. Polyhydroxybutyrate (PHB) is accumulated in the cells. Mesophilic and slightly halophilic, growth is observed from 15 to 37°C (optimum, 26°C), but not at 4 or 40°C and up to 6% total salinity in media supplemented with sea salts (optimum 3.5–5%). Does not grow at 7% or more salinity. It displays complex ionic requirements, as it is unable to grow either without added salts or with addition of simple salts (NaCl, KCl, MgCl₂, CaCl₂). Oxidase and catalase positive. Nitrate reduction to nitrite or N₂ is negative. Does not ferment carbohydrates. Negative for urea, casein, gelatin, starch, Tween-80, and DNA hydrolysis. Indole production from tryptophan and arginine dihydrolase are negative, PNPG (β-galactosidase) and leucine arylamidase are positive.

The following sole carbon and energy sources are used for growth: D-ribose, D-xylose, D-glucose, D-mannose, D-galactose, D-cellobiose, sucrose, salicin, N-acetyl-D-glucosamine, D-glycerol, m-inositol, acetate, pyruvate, propionate, butyrate, t-aconitate, citrate, 2-oxoglutarate, succinate, malate, lactate, 3-hydroxybutyrate, L-leucine, L-serine, L-glutamate, L-alanine, L-tyrosine, L-ornithine, 4-aminobutyrate, L-sarcosine, and putrescine. Growth is negative with L-arabinose, D-fructose, D-trehalose, L-rhamnose, maltose, lactose, D-melibiose, D-amygdalin, D-mannitol, D-sorbitol, D-gluconate, D-glucuronate, D-galacturonate, D-glycerate, D-saccharate, glycine, L-threonine, L-arginine, L-citrulline, L-histidine, L-lysine, and L-aspartate.

Major cellular fatty acids are C_{18 : 1} ω7c/ω6c and C_{18 : 1} ω7c 11-methyl.

The predominant respiratory quinone, Q10 was inferred from annotated gene encoding decaprenyldiphosphate synthase (EC 2.5.1.91). Major polar lipids, PG and PC, were inferred from annotated genes encoding CTP-phosphatidate cytidyl transferase (EC 2.7.7.41), phosphatidyl glycerophosphate synthase (EC 2.7.8.5), phosphatidyl glycerophosphatase (EC 3.1.3.27), and phosphatidyl choline synthase (EC 2.7.8.24), in the genomes of strains CECT 5118^T and CECT 5120.

The G+C content of the DNA is 59.8 mol% (partial genome sequences).

The type strain, CECT 5118^T (=XSM11^T =LMG 29904^T) was isolated from coastal seawater, Mediterranean Sea, at Vinaroz coast, Spain.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.