This is the accepted version of the article: Lucena, Teresa; Sanz-Sáez, Isabel; Arahal, David R.; [et al.]. «Mesonia oceanica sp. Nov., isolated from oceans during the tara oceans expedition, with a preference for mesopelagic waters». International Journal of Systematic and Evolutionary Microbiology, Vol. 70, issue 7 (2020), p. 4296-4338. 10 pàg. DOI 10.1099/ijsem.0.004296 This version is available at https://ddd.uab.cat/record/251095 under the terms of the license Mesonia oceanica sp. nov. 1 Mesonia oceanica sp. nov., isolated from oceans 2 during the Tara Oceans Expedition, with a preference 3 for mesopelagic waters 4 5 1.1 Author names 6 Teresa Lucena1, Isabel Sanz-Sáez2, David R. Arahal1, Silvia G. Acinas2, Olga Sánchez3, Carlos 7 Pedrós-Alió4, Rosa Aznar1 and María J. Pujalte1* 8 9 1.2 Affiliation 1 10 Departamento de Microbiología y Ecología and Colección Española de Cultivos Tipo (CECT), 11 Universitat de València, Valencia, Spain 2 12 Departament de Biologia Marina i Oceanografia, Institut de Ciències del Mar, ICM-CSIC, 13 Barcelona, Spain 3 14 Departament de Genètica i Microbiologia, Facultat de Biociències, Universitat Autònoma de 15 Barcelona, 08193 Bellaterra, Spain 4 16 Department of Systems Biology, Centro Nacional de Biotecnología (CNB), CSIC, Madrid, 17 Spain 18 19 1.3 Corresponding author 20 maria.j.pujalte@uv.es 21 1.4 Keyword 22 Mesonia, Mesonia oceanica, Flavobacteriaceae, marine bacteria, taxogenomics, 23 mesopelagic zone 24 25 1.5 Repositories: 26 The accession number of Mesonia oceanica ISS653T 16S rRNA gene sequence and draft 27 genome are MH732189 and CABVMM01, respectively. The accession number of Mesonia 28 oceanica ISS1889 16S rRNA gene is MN836382. 29 30 ABSTRACT 31 32 Strain ISS653T, isolated from Atlantic seawater, is a yellow pigmented, non-motile, Gram- 33 negative rod-shaped bacterium, strictly aerobic and chemoorganotrophic, slightly halophilic 34 (1-15% NaCl) and mesophilic (4-37 ºC), oxidase and catalase positive and proteolytic. Its 35 major cellular fatty acids are iso-C15:0, iso-C15:0 2-OH, and iso-C17:0 3-OH; the major identified 36 phospholipid is phosphatidylethanolamine and the major respiratory quinone is MK6. 37 Genome size is 4.28 Mbp and DNA G+C content is 34.9 mol%. 16S rRNA gene sequence 38 similarity places the strain among members of Flavobacteriaceae, with the types of Mesonia 39 phycicola (93.2 %), Salegentibacter mishustinae (93.1 %) and Mesonia mobilis (92.9 %) as 40 closest relatives. Average Amino acid Identity (AAI) and Average Nucleotide Identity (ANI) 41 indexes show highest figures with M. mobilis (81% AAI; 78.9% ANI), M. phycicola (76% AAI; 42 76.3% ANI), M. maritima (72 % AAI, 74.9 % ANI), M. hippocampi (64 % AAI, 70.8 % ANI) and 43 M. algae (68% AAI; 72.2 % ANI). Phylogenomic analysis using the Up-to-date-Bacterial Core 44 Gene set (UBCG) merges strain ISS653T in a clade with species of the genus Mesonia. We 45 conclude that strain ISS653T represents a novel species in the genus Mesonia for which we 46 propose the name Mesonia oceanica sp. nov., and strain ISS653T (= CECT 9532T = LMG 47 31236T) as the type strain. A second strain of the species, ISS1889 (= CECT 30008) was 48 isolated from Pacific Ocean seawater. Data obtained throughout the Tara Oceans expedition 49 indicate that the species is more abundant in the mesopelagic dark ocean than in the photic 50 layer and it is more frequent in the South Pacific, Indian and Atlantic oceans. 51 52 53 The genus Mesonia belongs to the Flavobacteriaceae [1], the bacterial family which includes 54 the largest number of genera to date, 153 according to LPSN [2]. Marine members of the 55 family Flavobacteriaceae play fundamental roles as complex organic matter degraders and 56 in the nutrient turnover in oceans, where many of them are found in association with 57 marine phytoplankton and algal live or detritic material [1, 3] and some display 58 proteorodopsin-based photoheterotrophy [3]. The genus Mesonia was established in 2003 59 to accommodate a group of algal-associated marine bacteria that were different from 60 Salegentibacter and other halophilic flavobacteria [4]. It currently contains eight species 61 with validly published names, all of them isolated from marine environments and 62 organisms: Mesonia algae, the type species [4], M. mobilis [5], M. phycicola [6], M. ostreae 63 [7], M. aquimarina [8], M. hippocampi [9], M. sediminis [10] and M. maritima [11]. The 64 genus is one of the many included in a recent phylogenomic study of the phylum 65 Bacteroidetes [12] and has not been affected by the reclassifications therein proposed. 66 From Tara Oceans expedition (2009–2013) [13] we have isolated in culture a large collection 67 of marine bacteria from different oceanographic regions and depths [14]. Based on such 68 isolation effort, here we present the description of a novel species in this family, based on 69 the phenotypic, genomic and phylogenetic study of the strains ISS653T and ISS1889. 70 71 Isolation 72 73 Strain ISS653T was isolated in September 2012 from surface seawater at the North Atlantic 74 Ocean (36°10'10.2"N 29°01'13.8"W). Sampling strategy and methodology have been 75 described previously [13]. Briefly, the isolate was obtained by plating 100 µl of undiluted 76 and 10x diluted seawater (pre-filtered by 200 µm and 20 µm meshes to remove large 77 plankton) in Marine Agar 2216 plates (BD Diagnostics). Plates were incubated at room 78 temperature (approximately 20ºC) in the dark until no more colonies appeared (10-30 days). 79 Colonies that grew were streaked on agar plates in duplicate to ensure their purity and 80 avoid contamination. The isolates were stored in the broth medium used with glycerol (25% 81 v/v) in cryovials at -80ºC. The culture was regrown on Marine Agar 2216 (BD Diagnostics) at 82 room temperature for 3 days. The strain has been maintained by lyophilisation at the 83 Spanish Type Culture Collection (CECT) as CECT 9532T (= LMG 31236T) where part of the 84 characterization was also conducted. A second strain of the species, ISS1889 (= CECT 30008), 85 was isolated during the same expedition from surface seawater of the Eastern Pacific Ocean 86 (5°15'36.0"S 85°10'04.1"W). A study, including phenotypic, genomic and phylogenetic 87 characterization of the strains was undertaken in order to define their taxonomic position. 88 89 16S RNA phylogeny 90 91 A partial (1328 bp) 16S rRNA gene sequence of strain ISS653T was obtained after DNA 92 extraction as reported previously [15] and deposited under the accession number 93 MH732189. A BLAST search for the closely related taxa, based on this sequence, related the 94 strain to the family Flavobacteriaceae and revealed spec ies of the genera Salengentibacter, 95 Mesonia, Zunongwangia and Gramella species as its closest neighbours, with sequence 96 similarities always lower than 93%. These values suggested that the strain represented a 97 novel taxon. At a different stage, the nearly complete 16S rRNA gene sequence of strain 98 ISS1889 (1425 bp) was obtained through already reported methods [15] and deposited 99 under accession number MN836382. It was found to be identical to that of ISS653T, 100 suggesting that both strains could represent the same species. 101 The sequence (1530 bp) obtained from the genome draft of strain ISS653T, described under 102 the next heading, was subsequently used for determining similarity values with the type 103 strains of closely related species [16] and also to reconstruct phylogenetic trees [17, 18] 104 using ARB. Closest neighbours based on 16S rRNA sequence similarity, as determined by the 105 Identifier tool of EzBiocloud [16] were: Mesonia phycicola (93.2 %), Salegentibacter 106 mishustinae (93.1 %), Mesonia mobilis (92.9 %), Mesonia maritima (92.9 %), 107 Salegentibacter. salarius (92.9 %) and Mesonia aquimarina (92.6 %). A phylogenetic tree 108 based on 16S rRNA gene sequences is shown in Fig. 1. It places strains ISS653T and ISS1889 109 in the clade formed by species of the genus Mesonia, as a sister clade. The low 16S rRNA 110 gene similarities found and the position of the branch in the tree support the taxonomic 111 novelty of the strains at the species level and even suggest that they might represent a 112 novel genus in the family. However, genus boundaries are not defined as clearly as species 113 on genomic and phylogenetic grounds and a careful evaluation of all genomic and 114 phenotypic evidence is required for definition of a genus. We thus, explored some other 115 indexes and phylogenetic approaches to decide whether strains ISS653T and ISS1889 should 116 be classified as representing a novel species of the genus Mesonia or as a novel genus in the 117 family Flavobacteriaceae. 118 119 Genome Features 120 121 A draft genome of strain ISS653T was obtained through whole genome sequencing at Centre 122 Nacional d’Anàlisi Genòmica (CNAG) (https://www.cnag.crg.eu/) following procedures 123 described previosuly [15]. The libraries were sequenced on HiSeq 2500 (HiSeq Rapid SBS Kit 124 V2, Illumina) in paired-end mode 2×251+8+8 bp. Primary data analysis, image analysis, base 125 calling and quality scoring of the run were processed using the manufacturer’s software Real 126 Time Analysis (RTA 1.18.66.3), followed by generation FASTQ sequence files. The reads were 127 analysed for quality control using FASTQC, a common quality control tool developed by 128 Babraham Bioinformatics to check raw sequencing data. After filtering, the remaining reads 129 were assembled using SPAdes 3.9.0 software [19]. A plot, coverage versus length of the 130 contigs, was performed to help in the choice of the parameters for contigs filtering. After 131 the filtration of contigs (minimal length 500 bp and coverage 10-50× kmer), evaluation of 132 the final assembly against a reference genome was done with the software QUAST v4.3 [20]. 133 The bioinformatic tool CheckM v1.0.7 [21] was used to assess the genome quality prior to 134 annotation using Prokka v1.12 [22] and RAST v2.0 (Rapid Annotation using Subsystem 135 Technology) [23]. The process of quality assessment of reads, read-processing, assembly 136 and annotation with Prokka was carried out in Linux OS, other tools were accessed online. 137 The minimal standards for the quality of genome sequences and how they can be applied 138 for taxonomic purposes have been observed in this study [24]. 139 The available genomes of type strains of genera of the family Flavobacteriaceae closely 140 related to the novel taxon were retrieved from public databases. Table 1 shows their 141 accession numbers and main characteristics. 142 The draft genome of strain ISS653T has an estimated size of 4.28 Mbp. It is composed of 72 143 contigs with a N50 value of 251,300 nucleotides and final assembly coverage of 452×. 144 CheckM results of contamination and completeness were 0.53 and 99.6 %, respectively. The 145 assembly contains 4030 protein coding sequences and 45 RNA genes. Only one ribosomal 146 RNA operon is detected and its 16S rRNA gene sequence is complete and 100% coincident 147 with the partial sequence previously amplified (Sanger). The G+C molar content is 34.9 %. 148 This genome is the largest one of all available genomes of members of the genus Mesonia, 149 in fact, is about 1.0 Mbp larger than the one of M. phycicola (which had the maximum 150 genome size recorded until now). 151 The presence of four copies of a CTnDOT-like transposon [25] is predicted from the four Tra 152 regions (containing TraJ, K, M, N, I and G genes) identified as part of this Bacteroidetes- 153 specific transposon. It is also present in the genomes of M. maritima (two copies) and M. 154 phycicola (one copy). 155 Further exploration of annotated genome of strain ISS653T allowed the prediction of some 156 potential abilities of the strain, such as multiple degradative polysaccharide capabilities, a 157 trait characteristic of several taxa in the family. Among them, carbohydrate active enzymes 158 (CAZymes) predicted from the genome of strain ISS653T account for pectate lyase (EC 159 4.2.2.2., two copies), polygalacturonase (EC 3.2.1.15), xylan β-1-4 xylosidase (EC 3.2.1.37), 160 endo 1-4 β-xylanase, pectin estearase (EC 3.1.1.11), a pectin degradation protein, KdgF, 161 rhamnogalacturonate acetyl estearase, endoglucanase, β-glucosidase (five copies), mucin 162 desulfating sulfatase (two copies), phytase, glucan 1-4 α-glucosidase (two copies), a 163 polysaccharide deacetylase, glycogen synthase, laminarinase, β-1-4 glucanase, hyaluronan 164 synthase, oligogalacturonate lyase and neopullullanase (Table S2, available in the online 165 version of the article). 166 Apart from these, it is also interesting to note the presence of genes coding for 167 phosphatidylserine decarboxylase (in agreement with the presence of PE as major identified 168 polar lipid) but also of cardiolipin synthase (although no DPG was detected among the 169 identified polar lipids). A large CRISPR region and several Cas proteins are also encoded, as 170 well as a type III restriction-modification system, and various gliding motility-associated ABC 171 transporter permeases. Enzymes involved in carotene metabolism, as phytoene synthase, 172 phytoene dehydrogenase, lycopene β-cyclase and β-carotene hydroxylase are also found. 173 No rhodopsin-related enzymes are predicted. 174 The similarity between genomes was assessed using several indexes useful for species and 175 genus delineation. Average Amino acid Identity (AAI) was calculated with the online server 176 ANI/AAI-Matrix [26]. DNA-DNA hybridization (DDH) was estimated in silico with the 177 Genome-to-Genome Distance Calculator (GGDC 2.1), using the BLAST method and 178 recommended formula 2 [27]; Average Nucleotide Identities, according to BLAST (ANIb) 179 were determined in JSpeciesWS [28]. AAI and ANIb values among referenced genomes are 180 presented in Table 2. ANI values confirm, as expected from 16S rRNA gene data, that strain 181 ISS653T does not belong to any of the compared species, since the figures obtained are 182 always lower than 95 % (in fact, lower than 91 %). In silico DDH values for strain ISS653T 183 genome against M. algae, M. hippocampi, M. maritima, M. mobilis, and M. phycicola type 184 strain genomes are 18.0 to 22.4 %, confirming that the strain does not belong to any of the 185 mentioned species. AAI values to other Mesonia spp. are mostly over 70 %, but they range 186 from 64 to 81 %, with 68% to the type species of the genus, M. algae. Finally, phylogenomic 187 relationships among strain ISS653T, members of Mesonia and other representatives of the 188 family Flavobacteriaceae were explored with UBCG, based on the analysis of 92 universal 189 bacterial core gene sequences [29]. The resulting trees, based on amino acid and nucleotide 190 sequences are shown in Fig. 2 and Fig. S1, respectively. In both trees, ISS653T forms a branch 191 deeply embedded within the Mesonia clade; this branch shows the highest possible gene 192 support index (GSI) value on the immediate nodes relating strain ISS653T to M. mobilis and 193 M. phycicola. 194 195 196 Ecology 197 198 Additionally, we explored the biogeography distribution of strain ISS653T across oceans and 199 depths by comparing the amplicon sequencing of the V4-V5 region of the 16S rRNA gene 200 (16S iTAGs, primers 515F-Y and 926R [30]; sequenced by Illumina MiSeq platform (iTAGs) 201 datasets from Tara Oceans [31]. We have been able to compare at 100% similarity the 16S 202 rRNA gene sequences of ISS653T and ISS1889 with zOTUs (zero-radius OYUs, i.e. Operational 203 Taxonomic Units defined at 100% sequence similarity) denoted from high-throughput 204 sequencing of the 16S rRNA of surface and mesopelagic samples (Fig. 3). The two strains 205 were identical to one zOTU that based on rank abundance analysis (Fig. 4) belongs to the 206 rare biosphere in the surface layer (0.0045% of the total reads) but to the mid-abundant 207 biosphere (0.31%) in the mesopelagic. In addition, if we look the average of reads that were 208 100% identical to the mentioned strains per oceanographic region, the higher abundances 209 were found in the mesopelagic samples of the South Pacific Ocean (Fig. 4) where ISS1889 210 was isolated. Interestingly, higher abundances were found in the mesopelagic layer, 211 indicating probably a higher preference of these strains for aphotic layers. 212 213 214 215 Physiology and Chemotaxonomy 216 Phenotypic characterization included morphological, cultural, biochemical, physiological and 217 nutritional screening and was performed by methods described previously [32]. Mesonia 218 algae CECT 9441T, Salegentibacter salegens CECT 9443T, Gramella echinicola CECT 9439T and 219 Zunongwangia profunda CECT 9445T were characterized in parallel for comparative 220 purposes. 221 Flexirubin type pigmentation was tested according to the methods of Bernardet et al. (2002) 222 [33]. In addition, analyses of cellulose degradation, nitrate reduction acid production from 223 carbohydrates in API 50CH/E, APIZYM and API 20NE profile were performed as described 224 previously [15]. Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy 225 (SEM) observations of the morphology and ultrastructure also followed previously described 226 procedures [15]. 227 ISS653T and ISS1889 were Gram-reaction negative, rod-shaped and non-motile, strictly 228 aerobic and chemoorganoheterotrophic, oxidase and catalase positive. They were unable to 229 grow anaerobically, either by carbohydrate fermentation or through nitrate reduction. The 230 strains grew well on Marine Agar and Marine Broth 2216 (BD Diagnostics). Colonies on 231 Marine Agar were yellow, round, with regular borders, with no indication of swarming or 232 gliding. Temperature range for growth was 4 to 37 ºC (30 ºC for ISS1889), with optimum at 233 26-30 ºC. No growth was obtained at 40ºC. pH range was 5 to 8, with optimum at 6-8, very 234 weak growth at 9 and 10 and no growth at 4 and below. The strains grew at total salinities 235 of 1 to 15 %, optimally at 2-4 %, they did not grow at 0.5 % or less nor 18 % or higher 236 salinities. The growth was strictly Na+ -dependent. They are, thus, mesophilic, neutrophilic 237 and slightly halophilic. They were able to hydrolyse gelatine and casein but the hydrolyses of 238 Tween 80 (negative for ISS1889) and DNA were weak. Alginate, starch, cellulose and agar 239 were not hydrolysed. Good growth was obtained on Baumann’s Basal Medium with yeast 240 extract (positive control of the carbon source screening) but it was negative with all the 241 substrates used (D-ribose, L-arabinose, D-xylose, D-glucose, D-fructose, D-galactose, D- 242 trehalose, D-mannose, L-rhamnose, maltose, D-cellobiose, lactose, sucrose, D-melibiose, 243 amygdalin, salicin, N-acetyl-D-glucosamine, D-gluconate, D-glucuronate, D-galacturonate, D- 244 saccharate, D-glycerol, D-mannitol, D-sorbitol, myo-inositol, acetate, pyruvate, propionate, 245 butyrate, citrate, t-aconitate, 2 ketoglutarate, succinate, fumarate, malate, lactate, 3- 246 hydroxybutyrate, glycine, L-leucine, L-alanine, L-glutamate, L-serine, L-arginine, L-tyrosine, 247 L-treonine, L-aspartate, L-citrulline, L-ornithine, L-histidine, L-lysine, L-sarcosine and 248 putrescine) suggesting that ISS653T may have a growth factor requirement. ISS1889 was not 249 tested for carbon source utilization in this medium. On the assimilation tests of API 20NE, 250 both strains grew well with malate and, less conspicuously, on glucose, mannose, maltose, 251 arabinose, mannitol, N-acetyl D-glucosamine, gluconate and adipate, but not on caprate or 252 citrate; phenyacetate grow was positive only for ISS653T. Both strains were positive for 253 PNPG test (β-galactosidase activity) and esculin and gelatin hydrolysis, but negative for 254 indole, arginine dihydrolase and urease. On APIZYM, both strains were positive for alkaline 255 and acid phosphatases, leucine and valine arylamidases, and α- and β-glucosidases and were 256 negative for other tests. Acid production from carbohydrates on API50CH/E strips was very 257 scarce, with positive reaction only for aesculin hydrolysis and a slight acidification in D- 258 glucose, D-mannose, amygdalin, salicin, D-cellobiose and maltose wells after 48 h incubation. 259 Strain ISS1889 was almost identical except for the acid production from arbutin and 2- 260 ketoglutarate (weak). They also showed slight differences in minor fatty acids (Table S1). 261 Fig. 3 displays SEM and TEM images of strain ISS653T cells: they are regular straight rods 262 with rounded ends, 0.5-0.6 × 0.9-1.5 µm in size, appearing singly or in pairs and showing the 263 profile typical for a Gram-negative bacterium in TEM images. No appendages or internal 264 structures were seen. Additional images are included in Fig. S2. 265 Fatty acid methyl esters were extracted from ISS653T and ISS1889 biomass grown in Marine 266 Agar at 26 ºC after 72 h incubation. Extracts were prepared according to standard protocols 267 as described for the MIDI Microbial Identification System [34] at the CECT. Cellular fatty acid 268 content was analysed by gas chromatography with an Agilent 6850 chromatographic unit, 269 with the MIDI Microbial Identification System using the TSBA6 method [35] and identified 270 using the Microbial Identification Sherlock software package. Table S1 shows the cellular 271 fatty acids detected in the two strains, that include iso-C15:0, summed feature 3 and iso-C17:0 272 3-OH as major components, followed by iso-C15:1 G, summed feature 9 (C16:0 10-methyl/iso- 273 C17:1 ω9c) and iso-C15:0 3-OH. An issue has been reported [36] about the peak names for 274 summed feature 3 in flavobacteria that makes us believe it might correspond to iso-C15:0 2- 275 OH, and so it is reported as such in the species description. The fatty acid profile of the 276 strains closely resembles those of other Mesonia species and all the fatty acids present in 277 percentages higher than 5% are found in similar amounts in the six species reported by [11]. 278 Analysis of respiratory quinones and major polar lipids of ISS653T were carried out by the 279 Identification Service and Dr. Brian Tindall, DSMZ, Braunschweig. Germany. Detailed 280 methods for the analyses have been reported previously [15]. MK6 was identified as the 281 major quinone and phosphatidyl ethanolamine (PE) was the only identified polar lipid, 282 among others detected (three unidentified lipids, two glycolipids and three aminolipids, Fig. 283 S3). Both MK6 and PE are typical chemotaxonomic features of the genus Mesonia, present 284 in all species so far described. 285 286 287 288 In summary, phylogenetic, genomic and phenotypic distinctiveness of the strains are 289 indication of them representing, at least, a novel species, a novelty confirmed by 16S rRNA 290 gene sequence similarity (less than 94 %) and ANI values (less than 95 %) exhibited between 291 strain ISS653T and the rest of the species of the genus Mesonia spp. and the neighboring 292 taxa. In fact, the strains initially appeared to represent a novel genus in the family due to the 293 low 16S rRNA gene similarity and to the topology of the 16S rRNA gene-based tree (Fig. 1). 294 However, when considering genomic indexes, such as AAI, and the UBCG phylogenomic 295 analysis, the relationship to their neighbors seems closer than anticipated by the 16S rRNA 296 analysis: instead of representing a marginal and distant branch, ISS653T merges in the core 297 of the genus Mesonia. Its AAI values to other species of the genus Mesonia fall in the lower 298 range of AAIs among congeneric species of Flavobacteriaceae, as, for example, species of 299 the genus Psychroflexus spp. (62-91 % intragenus AAIs, data not shown). AAI has been used 300 to define genus boundaries in the family, and particularly with the genus Chryseobacterium 301 and related genera, in a recent paper [40]. These authors propose a cutoff value of 76% AAI 302 for assignment of a novel species to an existing genus and consider that all type strain of 303 congeneric species should present at least a 74% AAI to each other. By following this 304 proposal, the novel species characterized in our study would constitute a novel genus, 305 different from Mesonia, and this genus would encompass M. mobilis and M. phycicola. The 306 species M. hippocampi and M. maritima should, in turn, be classified in two novel, different 307 genera, while M. algae would be the only representative of the genus Mesonia sensu stricto. 308 However, such major arrangements should wait until genome sequences for the type strains 309 of all species of the genus Mesonia are available. For the time being, we consider the best 310 option is to describe the novel species as a member of the genus Mesonia, provided that the 311 genus does not become polyphyletic with this inclusion and that the novel species fits well 312 with the genus description: the presence of iso-C15:0 as major fatty acid, a genomic DNA G+C 313 content of 34.9%, PE as the major identified polar lipid, MK-6 as the major respiratory 314 quinone, the pigmentation type, the aerobic chemoorganotrophic metabolism, a positive 315 responese for catalase, oxidase and alkaline phosphatase and the strict requirement of 316 sodium for growth are features that qualify the strains as members of the genus Mesonia. 317 On the other hand, several traits shown in Table 3 allow to differentiate them 318 phenotypically from any other Mesonia species. 319 In consequence, we propose a novel species of the genus Mesonia, with the name Mesonia 320 oceanica and strain ISS653T = CECT 9532T = LMG 31236T as the type strain. 321 322 Protologue 323 DESCRIPTION OF MESONIA OCEANICA SP. NOV. 324 Mesonia oceanica (o.ce.a’ni.ca, N.L. fem. adj. oceanica, of or pertaining to the ocean). 325 Cells are Gram-reaction negative, rod-shaped, 0.5-0.6 µm × 0.9-1.5 µm and non-motile. 326 Strictly aerobic and chemoorganotrophic; positive for catalase and oxidase. Colonies in 327 Marine Agar medium are regular and yellow pigmented. Flexirubin-type pigments are not 328 produced. Mesophilic, neutrophilic and slightly halophilic, with optima at: 26 ºC (range: 4-30 329 ºC, 40 ºC negative), 6-8 pH (range: 5-8, pH 4, negative, pH 9 and 10, weak) and 2-4 % total 330 salinity (range: 1 to 15 %, 0.5 and 18 % negative). Requires sodium ions for growth. Nitrate is 331 not reduced to nitrite or N2. Hydrolyses esculin, casein, gelatin, Tween 80 (weakly) and DNA 332 (weakly), but not alginate, cellulose (as filter paper) or agar. Indole production from 333 tryptophan, arginine dihydrolase and uresase are negative. PNPG test (β-galactosidase) is 334 positive in API 20NE. Assimilates malate, as well as glucose, mannose, maltose, arabinose, 335 mannitol, N-acetyl D-glucosamine, gluconate and adipate, but not caprate or citrate on API 336 20NE. The type strain was unable to grow in minimal medium (Basal Medium) with any of 337 52 sole carbon and energy sources, but grew with yeast extract. The following 338 carbohydrates are metabolised with weak acid production in aerobic API 50CH/E tubes: D- 339 glucose, D-mannose, amygdalin, salicin, D-cellobiose and maltose. Enzymatic activities 340 displayed on API ZYM are alkaline and acid phosphatases, leucine and valine arylamidases, 341 and α- and β-glucosidases. Naphthol-AS-BI-phosphohydrolase, estearase and estearase 342 lipase are weakly positive; lipase, cystine arylamidase, trypsin, α-chymotrypsin, α- 343 galactosidase, β-galactosidase, β-glucuronidase, N-acetyl-β-glucosaminidase, α- 344 mannosidase and α-fucosidase are negative. Major polar lipids are phosphatidyl 345 ethanolamine (PE), two unidentified glycolipids, three unidentified aminolipids and three 346 unidentified lipids. Major respiratory quinone is MK6. Major cellular fatty acids include iso- 347 C15:0, iso-C15:0 2-OH (although reported as summed feature 3 (C16:1 ω7c/ω6c) and iso-C17:0 3- 348 OH. 349 The type strain is ISS653T (=CECT 9532T =LMG 31236T), which was isolated from surface 350 seawater of the Atlantic Ocean during the Tara Oceans Expedition. Strain ISS1889 (=CECT 351 30008) is an additional strain of the species. The G+C DNA content of the type strain is 34.9 352 mol% and its genome size is 4.28 Mbp. The GenBank/EMBL/DDBJ accession numbers for the 353 whole genome sequence and 16S rRNA gene of strain ISS653T are CABVMM01 and 354 MH732189, respectively. 355 356 357 AUTHOR STATEMENTS 358 359 1.6 Authors and contributors 360 Conceptualization: I.S.S., S.G.A. and M.J.P. Data curation, formal analysis and investigation: 361 T.L., I.S.S., S.G.A., O.S. and M.J.P. Funding acquisition: S.G.A. and C.P.A. Project 362 administration and resources: D.R.A., S.G.A., R.A. and M.J.P. Supervision and writing original 363 draft: M.J.P. All authors contributed to review and editing. 364 365 1.7 Conflicts of interest 366 The authors declare that there are no conflicts of interest. 367 368 1.8 Funding information 369 Sequencing was funded by grant CTM2016-80095-C2-1-R to CPA from the Spanish 370 Ministerio de Economía y Competitividad. Additional funding was obtained by grant 371 CTM2017-87736-R to SGA from the Spanish Ministerio de Economía y Competitividad. Other 372 parts of this work received no specific grant from any funding agency. 373 374 1.9 Ethical approval 375 Not applicable 376 377 1.10 Consent for publication 378 Not applicable. 379 380 1.11 Acknowledgements 381 We thank our fellow scientists, the crew and chief scientists of the different cruise legs 382 involved in Tara Oceans for collecting the culture samples used in this study. This is a Tara 383 Oceans contributed paper number 105. 384 385 386 ABBREVIATIONS 387 388 CECT, Colección Española de Cultivos Tipo; LMG, Laboratorium voor Microbiologie, 389 Universiteit Gent; ANI, Average Nucleotide Identity; DDH, DNA-DNA hybridization; AAI, 390 Average Amino acid Identity; SCSIE, Servicio Central de Soporte a la Investigación 391 Experimental; RAST, Rapid Annotation using Subsystem Technology; GGDC, Genome-to- 392 Genome-Distance Calculator; BLAST, Basic Local Alignment Search Tool; UBCG, Up-to-date- 393 Bacterial Core Gene; GSI, Gene Support Index; LTP, The All-Species Living Tree Project; NCBI, 394 National Center for Biotechnology Information; PE, Phosphatidylethanolamine. 395 396 397 REFERENCES 398 399 1. Bernardet JF. Flavobacteriaceae. In Bergey’s Manual of Systematics of Archaea and 400 Bacteria. 2015. John Wiley & Sons, Inc. doi: 10.1002/9781118960608.fbm.00069 401 2. Parte AC. 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Wangia profunda gen. nov., sp. nov., a 504 novel marine bacterium of the family Flavobacteriaceae isolated from southern Okinawa 505 Trough deep-sea sediment. FEMS Microbiol Lett 2007;271:53–58. 506 40. Nicholson AC, Gulvik CA, Whitney AM, Humrighouse BW, Bell ME, et al. Division of the 507 genus Chryseobacterium: Observation of discontinuities in amino acid identity values, a 508 possible consequence of major extinction events, guides transfer of nine species to the 509 genus Epilithonimonas, eleven species to the genus Kaistella, and three species to the 510 genus Halpernia gen. nov., with description of Kaistella daneshvariae sp. nov. and 511 Epilithonimonas vandammei sp. nov. derived from clinical specimens. Int J Syst Evol 512 Microbiol 2020, doi: 10.1099/ijsem.0.003935 . 513 514 515 516 FIGURES AND TABLES 517 518 Fig. 1. Phylogenetic reconstruction based on the 16S rRNA gene using the Neighbor joining 519 method. Sequence accession numbers are given in parentheses. Bar, number of 520 substitutions per position. 521 Fig. 2. Phylogenetic tree generated with UBCG [29] by using amino acid sequences. The 522 numbers at the nodes indicate the gene support index (GSI, maximal value is 92). Genome 523 accession numbers are indicated in parentheses. Bar, 0.05 substitutions per position. 524 Fig. 3. Distribution of strains ISS653T and ISS1889. (A). Rank abundance based on the 525 number of reads per zOTU denoted from the 16S rRNA sequences. Color indicates the layer 526 were the zOTUs come from: light-blue, surface; turquoise, mesopelagic. Orange circle and 527 square indicate the position in the rank of the zOTU 100% identical to the two strains. (B). 528 Heatmap indicating the average of reads 100% identical to the isolates per oceanographic 529 region in surface and mesopelagic (Meso) samples. Lighter colors indicate lower number of 530 reads, while strong indicate higher number of reads. 531 Fig. 4. SEM and TEM images of strain ISS653T. Samples were gold sputter coated in order to 532 visualize them with SEM Zeiss MERLIN Fe. Visualizations were done at the Microscopy 533 service of the Universitat Autònoma de Barcelona 534 (http://sct.uab.cat/microscopia/en/content/inici). 535 536 Table 1. Genomes used in the study and their main characteristics. Strain Accession number Size G+C Protein RNA (Mbp) (mol%) encoding encoding genes genes Mesonia oceanica GCF_902499555 4.28 34.9 3854 45 T ISS653 (CABVMM01) Mesonia algae DSM GCF_003253545 3.09 33.1 2859 70 15361T (QKYV01) Mesonia mobilis GCF_000423405 3.21 35.2 2875 46 T DSM 19841 (AUHX01) Mesonia phycicola GCF_900141885 3.23 31.4 2911 47 T DSM 21425 (FQYY01) Mesonia maritima jgi.1227497* 3.14 33.7 2946 67 DSM 102814T Mesonia hippocampi jgi.1220074* 2.59 34.5 2378 45 T DSM 29568 Salegentibacter GCF_900142975 4.01 37.2 3415 58 T salegens ACAM 48 (LT670848.1) Gramella echinicola GCF_000423065 3.51 36.9 3112 50 DSM 19838T (AUHG01) Zunongwangia GCF_000023465 5.13 36.2 4270 60 T profunda SM-A87 (CP001650.1) 537 538 *These sequence data were produced by the US Department of Energy Joint Genome 539 Institute (http://www.jgi.doe.gov/) in collaboration with the user community and are part of 540 The One Thousand Microbial Genomes Phase 4 Project (KMG-4) by M. Göker. 541 542 Table 2. Average Amino acid Identity (AAI, yellow cells) and Average Nucleotide Identity 543 (ANIb, blue cells) indexes among genomes of type strains of Flavobacteriaceae species 544 related to ISS653T. Values relating Mesonia oceanica with all other species are shown in bold 545 type. 546 1 2 3 4 5 6 7 8 9 T 1 Mesonia oceanica ISS653 78.9 76.3 74.9 70.8 72.2 70.0 68.8 72.3 2 Mesonia mobilis DSM 19841T 81 78.4 74.4 70-9 72.4 69.7 68.6 70.7 3 Mesonia phycicola DSM 21425T 76 80 73.4 71.5 73.5 69.6 68.9 70.0 4 Mesonia maritima DSM 102814T 72 72 70 71.3 72.9 70.4 69.0 70.9 5 Mesonia hippocampi DSM 29568T 64 65 65 65 71.0 69.1 68.2 68.9 6 Mesonia algae DSM 15361T 68 70 70 70 65 69.5 68.8 69.6 7 Salegentibacter salegens ACAM 48T 63 64 63 64 61 63 71.1 71.3 8 Gramella echinicola DSM 19838T 62 63 63 63 61 63 70 70.2 9 Zunongwangia profunda SM-A87T 65 64 63 64 60 62 68 67 547 548 549 Table 3. Differential characteristics between strains ISS653T and ISS1889 and its closest 550 phylogenomic relatives. 1, Mesonia oceanica ISS653T and ISS1889; 2, M. algae CECT 9441T; 551 3, M. mobilis [5]; 4, M. phycicola [6]; 5, M. ostreae [7]; 6, M. aquimarina [8]; 7, M. 552 hippocampi [9]; 8, M. sediminis [10]; 9, M. maritima [11]; 10, Salegentibacter salegens CECT 553 9443T; 11, Gramella echinicola CECT 9439T; 12, Zunongwangia profunda CECT 9445T. All data 554 from this study unless indicated. +: positive; - negative; nd: not determined; Y: yellow; W: 555 white; Or: orange; G: gliding; Fl: flagellated. All strains were Gram-reaction-negative, 556 aerobic, chemoorganotrophic bacteria, positive for oxidase, catalase, alkaline and acid 557 phosphatases and leucine arylamidase; and negative for cellulose hydrolysis, α- 558 galactosidase, β-glucuronidase, α-mannosidase and α-fucosidase. 1 2 3 4 5 6 7 8 9 10 11 12 Pigment colour Y Y Y Y W Y Or Y Y Y Y Y Flexirubin - - - - - - + nd - - - - Motility - - +, G - - - - - +, Fl - +, G - Temp. range (ºC) 4-30 4-34 10-30 10-30 4-31 10-30 4-36 4-42 10-35 4-28 4-37 15-37 NaCl range (% w/v) 1-15 1-15 0.5-15 0.5-12 0-12 0.5-12 1-10 0.5-7 0.5-11 1-12 1-12 0.5-15 Hydrolysis of: Esculin + - + + - - - + - + + + Casein + + + - - + nd nd + w + - DNA w - + + - - nd nd + w + + Tween 80 w + + - - + nd + + - - + Enzymatic activity (API ZYM) a b c Valine arylamidase + + + + + + - + + + + + a b c Trypsin - - - - - - + w - - w + a b c β-Galactosidase - - - - - - - - - + - + a b c α-Glucosidase + - + + - - - - - - + + b c β-Glucosidase + - - - - + - - - nd w + d d d G+C mol% 34.9 33.1 35.2 31.4 42.1 41.4 34.5 40.7 33.7 37.2 36.9 36.2 a 559 data from [38]; bdata from [39] ; cdata from [40]; ddetermined by HPLC (all other values, 560 from WGS)