Mesonia aestuariivivens sp. nov., isolated from a tidal flat

A Gram-negative, aerobic, non-flagellated and ovoid or rod-shaped bacterial strain (JHPTF-M18T), which was isolated from a tidal flat sediment in Republic of Korea, was taxonomically characterized. Strain JHPTF-M18T grew optimally at 25 °C, at pH 7.0–7.5 and in the presence of 2.0–3.0% (w/v) NaCl. 16S rRNA gene sequence analysis showed that strain JHPTF-M18T forms a phylogenetic lineage within the radiation comprising type strains of Mesonia species. The 16S rRNA gene of strain JHPTF-M18T shared sequence similarities of 97.7% with that of type strain of M. mobilis and 92.5–96.8% with those of type strains of the other nine Mesonia species. The DNA G+C content was 33.1% based on its genomic sequence. AAI, ANI and dDDH values between strain JHPTF-M18T and the type strains of M. mobilis, M. hitae, M. oceanica, M. phycicola and M. algae were 72.1–83.7%, 73.1–79.7% and 18.5–22.8%, respectively. Strain JHPTF-M18T contained MK-6 as the predominant menaquinone and iso-C15:0, iso-C17:0 3-OH and summed feature 3 (C16:1ω7c and/or C16:1ω6c) as its major fatty acids. Major polar lipids of strain JHPTF-M18T were phosphatidylethanolamine and two unidentified lipids. Strain JHPTF-M18T was separated from recognized Mesonia species by its phenotypic properties together with the phylogenetic and genetic distinctiveness. Based on data presented in this study, strain JHPTF-M18T is considered to represent a novel species of the genus Mesonia. The name Mesonia aestuariivivens sp. nov. is proposed for JHPTF-M18T (=KACC 22185T = NBRC 115119T).


Introduction
The genus Mesonia was created by Nedashkovskaya et al. (2003) with the assignment of Mesonia algae as the type species and belonged to the family Flavobacteriaceae of the phylum Bacteroidetes (Bernardet 2011). The genus Mesonia currently consists of ten species with validly published names at July 2022 (https:// lpsn. dsmz. de/ genus/ meson ia; Parte 2018). Members of the genus Mesonia are known to be Gram-stain-negative, aerobic, catalase-and oxidase-positive and rod-shaped and to contain menaquinone-6 as predominant isoprenoid quinone, phosphatidylethanolamine as only major phospholipid identified and DNA G + C contents of 31.4-42.1 mol% (Kang and Lee 2010;Kolberg et al. 2015;Lee et al. 2012;Lucena et al. 2020;Nedashkovskaya et al. 2003Nedashkovskaya et al. , 2006Zhou et al. 2021). Isolation sources of Mesonia species described so far include green alga, seaweed, seawater, diseased Barbour's Seahorse and sea cucumber culture pond (Kang and Lee 2010;Kolberg et al. 2015;Lee et al. 2012;Lucena et al. 2020;Nedashkovskaya et al. 2003Nedashkovskaya et al. , 2006Wang et al. 2015). Recently, in the course of screening novel bacteria from a tidal flat at Seocheon on the Yellow Sea of Korean peninsula, many bacterial isolates have Communicated by Erko Stackebrandt. been obtained followed by identified by 16S rRNA sequence analysis. Of these bacterial isolates, one strain (designated as JHPTF-M18 T ) which showed the closest affiliation to members of the genus Mesonia, was selected for further taxonomic study. In this study, strain JHPTF-M18 T is characterized further using a polyphasic characterization.

Bacterial strains and culture conditions
A tidal flat sediment was collected in July 2020 from Seocheon (

Sequencing and phylogenetic analysis of 16S rRNA gene
Chromosomal DNA extraction was performed using a Wizard Genomic DNA isolation kit (Promega) according to the manufacturer's instruction. The 16S rRNA gene amplification was performed as described previously (Yoon et al. 1997) using PCR in which 9F (5'-GAG TTT GAT CCT GGC TCA G-3') and 1512R (5'-ACG GTT ACC TTG TTA CGA CTT-3') were used. Sequencing of the 16S rRNA gene followed by phylogenetic analysis was carried out as described by Yoon et al. (2003). Similarity between 16S rRNA gene sequences was calculated using alignment obtained using Clustal W program.

Genomic analysis
A TruSeq DNA LT Sample Prep kit (Illumina) was used to prepare a library for genomic sequencing. The library was sequenced using Illumina MiSeq platform. Sequencing data were assembled with SPAdes (Bankevich et al. 2012). Contamination of genome sequence was assessed using Con-tEst16S (Lee et al. 2017). Library construction and sequencing were performed by Chunlab Inc. (Republic of Korea). Phylogenomic tree based on genomic sequences and tree based on AAI were constructed with UBCG (Na et al. 2018) and EzAAI (Kim et al. 2021) in the EzBioCloud, respectively. The ANI value based on BLAST + was calculated using JSpecies WS (http:// jspec ies. riboh ost. com/ jspec iesws/; Richter et al. 2015) or OrthoANI

Chemotaxonomic characterization
Extraction and HPLC analysis of isoprenoid quinones were performed as described by Komagata and Suzuki (1987) and Park et al. (2014), respectively. Fatty acid analysis was performed as described by Park et al. (2014) using the standard MIDI protocol (Sherlock Microbial Identification System, version 6.2B), GC (Hewlett Packard 6890) and TSBA6 database of the Microbial Identification System (Sasser 1990). Extraction of polar lipids was carried out according to procedures described by Minnikin et al. (1984). They were separated by two-dimensional TLC using the solvent systems as described by Embley and Wait (1994). The TLC plates were sprayed with various reagents as described by Park et al. (2014), and individual polar lipids were visualized followed by identified with heating at 150 °C for 3 min.

Morphological, cultural, physiological and biochemical characterization
Cell shape, Gram reaction, pH range for growth, anaerobic growth, growth at various concentrations of NaCl, requirement for Mg 2+ ions, hydrolysis of gelatin and urea, susceptibility to antibiotics were investigated as described by Park et al. (2014). Growth at 4, 10, 20, 25, 28, 30, 35, 37 and 40 °C was measured on MA to estimate the optimal temperature and temperature range for growth. Nitrate reduction and hydrolysis of aesculin and Tween 80 were investigated as described previously (Lányí 1987) using artificial seawater (Bruns et al. 2001) for the preparation of the media.
Hydrolysis of other substrates was tested as described by Barrow and Feltham (1993) with the modification that MA Page 3 of 6 550 was used. Activity of catalase and oxidase was determined as described by Lányí (1987). The presence of flexirubintype pigments was investigated as described previously (Bernardet et al. 2002;Reichenbach 1992). Spectral analysis of in vivo pigment absorption was performed as described previously (Jung et al. 2016). Other enzyme activities were determined using the API ZYM system (bioMe´rieux); the results were checked after incubation for 8 h at 25 °C. Acid production from carbohydrates was tested as described by Leifson (1963).

Results and discussion
Phylogenetic analysis based on 16S rRNA gene sequence  (2006) and Kim et al. (2014).

Genomic features
The genome size of strain JHPTF-M18 T obtained from the assembly of sequencing reads was 3,328,752 bp with a sequencing depth of coverage of 482.93X. The genomic sequence of strain JHPTF-M18 T contained 73 contigs with N50 length of 192,421 bp. The complete 16S rRNA gene sequence extracted from the genomic data using ContEst16S (Lee et al. 2017) was found to be identical to respective 16S rRNA gene information previously obtained by Sanger sequencing. This indicated that strain JHPTF-M18 T and its genomic data were not mislabelled and did not originate from any source of contamination . Based on its genomic sequence data, the DNA G + C content of strain JHPTF-M18 T was 33.1%, a value in the range reported for Mesonia species (Lucena et al. 2020

Chemotaxonomic characteristics
The predominant isoprenoid quinone detected in strain JHPTF-M18 T was menaquinone-6 (MK-6), at a peak area ratio of approximately 95%, consistent with the results shown in the genus Mesonia (Lucena et al. 2020;Nedashkovskaya et al. 2003). The major fatty acids (> 10% of the total fatty acids in all growth phases) found in strain JHPTF-M18 T were iso-C 15:0 , iso-C 17:0 3-OH and summed feature 3 (C 16:1 ω7c and/or C 16:1 ω6c) ( Table S1). The fatty acid profiles of strain JHPTF-M18 T were similar to those of the type strains of M. mobilis and M. algae in that iso-C 15:0 , iso-C 17:0 3-OH are major fatty acids, even though there were differences in the proportions of some fatty acids, e.g. iso-C 16:0 , anteiso-C 17:1 ω9c and summed feature 3 (C 16:1 ω7c and/or C 16:1 ω6c) ( Table S1). The major polar lipids detected in stain JHPTF-M18 T were phosphatidylethanolamine and two unidentified lipids; minor amounts of eight other unidentified lipids, two unidentified aminolipids and one unidentified aminophospholipid were also present (Fig. S2). The polar lipid profile of strain JHPTF-M18 T was similar to that of the type strain of M. algae in that phosphatidylethanolamine is the only major phospholipid identified and one unidentified lipid is major component, but distinguished from that of the type strain of M. algae by the absence of one unidentified glycolipid as a major component (Fig. S2).

Conclusion
Combined results obtained from the phylogenetic, genomic and chemotaxonomic analyses made it reasonable to assign strain JHPTF-M18 T as a member of the genus Mesonia ( Fig. 1; Figs. S1 & S2; Table S1). Strain JHPTF-M18 T was distinguished from the type strains of M. mobilis and M. algae by differences in several phenotypic characteristics, including hydrolysis and acid production from some substrates, activity of some enzymes and susceptibility to some antibiotics (Table 1). Based on the polyphasic taxonomic data presented, strain JHPTF-M18 T is considered to represent a novel species of the genus Mesonia, for which we propose the name Mesonia aestuariivivens sp. nov.
Cells are ovoid or rod-shaped measuring approximately 0.2-0.4 μm in diameter and 0.3-3.0 μm in length. Gram-staining reaction is negative. Spores are not formed. No flagellum is found. Non-motile by gliding. Colonies on MA are circular, slightly convex, smooth, glistening, vivid yellow in colour and 0.5-1.0 mm after incubation for 3 days at 25 °C. Grows optimally at 25 °C and pH 7.0-7.5. Growth occurs at 4 and 37 °C but not at 40 °C, and occurs at pH 5.5 but not at pH 5.0. Growth occurs in the presence of 0.5-14.0% (w/v) NaCl with an optimum of approximately 2.0-3.0% (w/v) NaCl. Mg 2+ ions are not required for growth. Anaerobic growth does not occur on MA and on MA supplemented with nitrate. Catalase-and oxidase-positive. Nitrate is not reduced to nitrite. Aesculin, gelatin, Tween 80 and L-tyrosine are hydrolysed, but casein, hypoxanthine, starch, urea and xanthine are not. Flexirubin-type pigments are not produced. Carotenoid pigments are produced. Acid is not produced from L-arabinose, D-cellobiose, D-fructose, D-galactose, D-glucose, lactose, maltose, D-mannose, D-melezitose, melibiose, L-rhamnose, D-raffinose, D-ribose, sucrose, D-trehalose, D-xylose, myoinositol, D-mannitol and D-sorbitol. In assays with the API ZYM system, activities of alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, cystine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase and -glucosidase are present, but activities of other enzymes are absent. The predominant menaquinone is MK-6. The major fatty acids (> 10% of total fatty acids) are iso-C 15:0 , iso-C 17:0 3-OH and summed feature 3 (C 16:1 ω7c and/ or C 16:1 ω6c). The major polar lipids are phosphatidylethanolamine and two unidentified lipids. The DNA G + C content of the type strain is 33.1% (from genome sequence data).
The type strain, JHPTF-M18 T (= KACC 22185 T = NBRC 115119 T ), was isolated from a tidal flat sediment collected from Seocheon on the Yellow Sea, South Korea.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence and GenBank accession number for the whole genome shotgun sequence of strain JHPTF-M18 T are MW364546 and JAHWDF000000000, respectively.

Conflicts of interest
The authors declare that there are no conflicts of interest.