Fibrivirga algicola gen. nov., sp. nov., an algicidal bacterium isolated from a freshwater river

An aerobic, gram-stain-negative, pink-colored, non-motile and rod-shaped algicidal bacterium, designated as JA-25T was isolated from freshwater in Geumgang River, Republic of Korea. Strain JA-25T grew at 15–30 °C and pH 6–9, and did not require NaCl. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain JA-25T belongs to the family ‘Spirosomaceae’ and is most closely related to Fibrella aestuarina BUZ 2T (93.6%). Strain JA-25T showed < 90% sequence similarity to other members of the family ‘Spirosomaceae’. The average nucleotide identity(ANI), in silico DNA-DNA hybridization and average amino acid identity(AAI) values based on the genomic sequences of JA-25T and F. aestuarina BUZ 2T were 74.4, 20.5, and 73.6%, respectively. Strain JA-25T showed an algicidal effect on the marine flagellate alga Heterocapsa triquetra, but no effect on fresh water cyanobacterium (Nostoc). In genome analysis, RIPP-like peptides were detected and predicted to resemble the indolmycin biosynthetic gene cluster, which possibly influence its algicidal effect. Furthermore, a bacteriorhodopsin gene with photoheterotrophic characteristics was detected. The genomic DNA G + C content was 52.5 mol%. The major cellular fatty acids were summed feature 3 (C16:1 ω6c/C16:1 ω7c), C16:1 ω5c, C16:0 (> 10%). The major respiratory quinone was menaquinone 7 and major polar lipids were phosphatidylethanolamine, two unidentified aminolipids, two phospholipids, and five unidentified lipids. Considering the phylogenetic inference, phenotypic, and chemotaxonomic data, strain JA-25T should be classified as a novel species in the novel genus Fibrivirga, with the proposed name Fibrivirga algicola sp. nov. The type strain is JA-25T (= KCCM 43334T = NBRC 114259T).


Introduction
Marine harmful algal blooms occur when toxinproducing algae grow rapidly. These blooms occur worldwide and greatly impact aquatic ecosystems and human health (Patel et al. 2020). Over the last 30 years, harmful algal bloom caused financial losses of over 0.87 billion US dollars because of the massive fish and shellfish mortalities and negative impacts on tourism in China (Yan et al. 2022). The dinoflagellate genus Heterocapsa can cause harmful algal blooms (HABs) and produce toxins that affect the marine ecosystem (Patin et al. 2020). Additionally, several Heterocapsa strains have been found in Korea (Choi et al. 2021;Sakamoto et al. 2021). Thus, to control harmful algal blooms while maintaining the marine ecosystem, specific algicides for Heterocapsa sp. may be useful. The family 'Spirosomaceae' is a member of the order Cytophagales within the phylum Bacteroidetes (García-López et al. 2019). Currently, this family comprises 25 genera including Fibrella on the List of Prokaryotic names with Standing in Nomenclature (https:// lpsn. dsmz. de/ family/ spiro somac eae). Cells in this family are gram-negative, aerobic, or facultative anaerobic, and non-spore forming, with variable motility. The rods have various degrees of curvature, sometimes resulting in the formation of rings, coils, and undulating filaments. Colonies contain a pink or yellow, non-water-soluble pigment. The major quinone is menaquinone 7 and major polar lipid is phosphatidylethanolamine. The G + C content calculated from genome sequences is 35.1-56.4%. The genus Fibrella was proposed by Filippini et al. (2011) and was reported to participate in antagonistic interactions against the cyanobacterium Nostoc muscorum . The freshwater cyanobacterium Nostoc is different from the marine eukaryotic flagellum Heterocapsa in terms of cell envelope and physiology. Unlike strain Fibrella aestuarina BUZ 2 T , strain JA-25 T did not negatively affect Nostoc sp.
In the present study, the novel algicidal strain JA-25 T , which was isolated from freshwater, was evaluated using genotypic, chemotaxonomic, and phenotypic approaches and found to be a representative novel species in the new genus Fibrivirga.

Isolation and culture conditions
Freshwater was collected from the Geumgang River in South Korea (35° 50′ 38.1″ N,127° 24′ 51.8″ E). The freshwater was diluted in 50 mM phosphate buffer and spread on Reasoner's 2A agar (R2A; MBcell, Seoul, Korea) plates. The plates were incubated at 25 °C for 2 weeks, after which the colonies were streaked onto R2A agar at least three times to obtain pure colonies. The identity of the colonies was determined using 16S rRNA sequencing. Based on the 16S rRNA sequencing results, one colony designated as JA-25 T was selected for polyphasic characterization. Strain JA-25 T was routinely cultured on R2A agar at 25 °C for 3 days for phenotypic, physiological, and chemotaxonomic analyses and maintained in glycerol suspensions (20%, v/v)  16S rRNA gene phylogenetic analyses Chromosomal DNA was extracted from strain JA-25 T using a DNA extraction kit (iNtRON Biotechnology, Gyeonggi-do, South Korea). The 16S rRNA gene was amplified using AccuPower PCR PreMix (Bioneer, Daejeon, Korea) and the bacteria-specific primers 27F (5′-AGA GTT TGATCMTGG CTC AG-3′) and 1492R (5′-AAG GAG GTG ATC CAG CCG C-3′). 16S rRNA gene sequencing was performed as previously described (Roh et al. 2008). The almost full-length 16S rRNA gene sequence (1,451 bp) was assembled and curated using SeqMan Pro 14 (DNASTAR, Madison, WI, USA). Phylogenetic neighbors were identified, and pairwise sequence similarities were calculated using EzBioCloud (Yoon et al. 2017). Phylogenetic relationships between closely related species were determined using the MEGA6 program (Tamura et al. 2013). Phylogenetic trees were constructed using the neighbor-joining (Saitou and Nei 1987), maximum-parsimony (Fitch 1971), and maximum-likelihood methods (Felsenstein 1981), and evolutionary distances were calculated using Kimura's two-parameter model. To evaluate the stability of the phylogenetic trees, bootstrap analysis was performed by obtaining a consensus tree based on 1,000 randomly generated trees.

Whole-genome sequencing analyses
The genomic DNA of strain JA-25 T was extracted and purified using a QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). The purified genomic DNA was quantified using a Qubit 3.0 fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). A genome library was prepared using the Nextra DNA Flex Library Prep Kit (Illumina, San Diego, CA, USA). The average library size was 550 bp, and draft genome sequencing was performed on a Novaseq 6000 platform (Illumina) with 101 bp paired-end reads according to the manufacturer's protocols. Lowquality reads were trimmed using a quality threshold of Q20. The raw data were assembled using Unicycler 0.4.7 (Wick et al. 2017). The average nucleotide identity was calculated using JSpecies (Richter and Rosselló-Móra 2009), and the distance (1-similarity) was visualized in the R environment ver. 4.0.4 (https:// www.r-proje ct. org/). The average amino acid identity (AAI) values were calculated with a 20% identity cut-off using the AAI Calculator (http:// enve-omics. ce. gatech. edu/ aai/ index). The digital DNA-DNA hybridization (dDDH) was calculated using Genome-to-Genome Distance Calculator 2.1 (https:// ggdc. dsmz. de/ ggdc. php) using formula 2.

Phenotypic and chemotaxonomic characteristics
To investigate its morphological and physiological properties, strain JA-25 T was routinely cultivated on R2A agar at 25 °C. Gram staining was performed using the non-staining method described by Buck with a Gram staining kit (bioMérieux, Marcy-l'Etoile, France). Growth under anaerobic conditions was determined after incubation for 1 week in a GasPak EZ Anaerobe Pouch System (BD, Franklin Lakes, NJ, USA) on R2A. Cell morphology was observed using a transmission electron microscope (H-7650;, Hitachi, Tokyo, Japan). Gliding motility was investigated as described by Bernardet et al. (2002) using a modified R2A medium. Growth was assessed under various conditions, including different temperature (10 °C, 15 °C, 20 °C, 37 °C, 40 °C, and 45 °C) and pH (pH 5, 6, 7, 8, 9, 10, and 11) values, on R2A agar. The pH values were adjusted by adding the following buffers as needed: 10 mM MES for pH 5.0 and 6.0; 10 mM bis-Tris propane for pH 7.0, 8.0, and 9.0; and 10 mM CAPS for pH 10.0 and 11.0. NaCl tolerance (0, 1, 2, 3, and 5%, w/v) was tested on R2A agar. Production of flexirubin-type pigments was determined according to the reversible color shift to red, purple, or brown when yellow or orange colonies were covered with aqueous 20% KOH solution (Siddiqi et al. 2016). Hydrolysis of CM-cellulose and starch was tested by adding 0.5% of the respective polysaccharides to R2A agar medium. API ZYM, API 20NE, and 50CH strips (bioMérieux) were used to determine the physiological and biochemical characteristics of the strain. The API 20NE and 50CH results were recorded after 3 days, and those of API ZYM were recorded after 1 day at 25 °C. Catalase and oxidase activities were measured using a 3% (v/v) hydrogen peroxide solution and 1% (w/v) tetramethyl-p-phenylenediamine (bioMérieux), respectively. Quinones in strain JA-25 T grown on R2A medium were analyzed using high-performance liquid chromatography (Waters, Milford, MA, USA) as described by Hirashi et al. (1996). The cellular fatty acid composition was analyzed according to the instructions provided in the Sherlock Microbial Identification System (Miller 1982), using strain JA-25 T and F. aestuarina DSM 22563 T cultivated under the same conditions on R2A agar at 25 °C for 3 days. Fatty acids were analyzed using gas chromatography (Hewlett Packard 6890; Agilent Technologies, Santa Clara, CA, USA) and identified using the Microbial Identification software package (Sasser 1990) based on the TSBA6 database. Polar lipids of strain JA-25 T were extracted and separated using two-dimensional thin-layer chromatography on a silica gel glass plate (Merck, Darmstadt, Germany) as described by Xin et al. (2000). The separated polar lipid spots were detected by spraying the plate with 5% ethanolic molybdophosphoric acid (for total polar lipids) and ninhydrin (aminolipids) as described by Minnikin et al. (1984). Phospholipids were detected by spraying the plate with Zinzadze reagent (Minnikin et al. 1984).

Algicidal activity assay
The algicidal activity assay was performed as described previously by Cho and Kim (2018) with slight modifications. Strain JA-25 T was cultured in R2A broth medium for 3 days, after which the bacterial cells were collected at ~ 3000 × g. The bacterial cell pellets were washed three times with algal medium and then diluted with algal medium. Bacteria at concentrations J1 (6 mg/mL), J2 (3 mg/mL), J3 (1.5 mg/mL), and J4 (0.75 mg/mL) were added to 24-well plates containing harmful algae in exponential growth phase. Equal amounts of algal medium were used as controls. The plates were cultivated under the culture conditions of harmful algae. Algicidal activity was calculated every day using the following equation, and the assay was performed in triplicate.

Algicidal rate
where N c and N t are the numbers of algal cells in the control and treatment groups, respectively.

Results and discussion
Phylogenetic and phylogenomic analyses Strain JA-25 T showed the highest 16S rRNA gene sequence similarity to the type strain of F. aestuarina BUZ 2 T (93.6%), followed by Spirosoma migulaei 15J9-8 T (89.4%). The pairwise 16S rRNA gene sequence similarity values between strain JA-25 T and the other members of the family 'Spirosomaceae' were below 90.0%. Strain JA-25 T clustered with F. aestuarina BUZ 2 T in the neighbor-joining tree ( Fig. 1), and this cluster was independent from other related genera in the family 'Spirosomaceae'. The maximum likelihood tree, neighbor-joining, and maximum-parsimony trees all showed the same topology. For 16S rRNA signature nucleotide analysis, approximately 25 species were selected based on their 16 s rRNA sequence similarity and phylogenetic tree topology (Fig. 1). Analysis of the 16S rRNA signature nucleotide patterns revealed shared patterns between strain JA-25 T    on protein sequences showed that relatedness values ranged from 61.8 to 73.6% (Table S1), satisfying the threshold range of 60-80% for separating different genera as proposed by Luo et al. (2014) and Rodriguez and Konstantinidis (2014); these results supported that the strain is a novel genus in the family 'Spirosomaceae'. Genome annotation was performed using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP). A total of 5,440 genes, including 5,393 coding sequences, 66 pseudogenes, and 47 RNA genes were annotated in Fibrella sp. JA-25 T . The assembly contained 1, 3, and 1 of full-length 5S, 16S, and 23S rRNA genes, respectively; 40 tRNA genes; and two non-coding RNAs. OrthoVenn2 (https:// ortho venn2. bioin fotoo lkits. net/) was used to analyze shared or independent gene clusters between JA-25 T and the closest relative F. aestuarina BUZ 2 T . Both strains shared 3,840 gene clusters. Strain JA-25 T and BUZ 2 T had 66 and 77 independent gene clusters, respectively. Interestingly, the bacteriorhodopsin (NZ_WAEL01000002.1; 312,795-313,550) and Brp/Blh family beta-carotene dioxygenase (NZ_ WAEL01000002.1; 314,744-313,797) genes were only found in JA-25 T and not in F. aestuarina BUZ 2 T is absent (Table 1).

Phenotypic and chemotaxonomic characteristics
Strain JA-25 T was a gram-negative, catalase-positive, catalase-positive, and oxidase-negative. Colonies of strain JA-25 T grown on R2A were circular, smooth, and pink. The cells were rod-shaped (0.8-1.2 μm wide and 1.5-3.5 μm long), as shown in Figure S3. Gliding motility was absent, and the production of flexirubin pigments was negative. Optimal growth of strain JA-25 T was observed at 25 °C and pH 7.0 in medium containing 0% (w/v) NaCl. Strain JA-25 T produced a pink pigment with absorbance peaks at 480 and 510 nm, with a major peak at 482 nm, which is similar to F. aestuarina BUZ 2 T . The isolation source, colony color, and growth temperature and pH ranges were used to distinguish between closely related genera and strain JA-25 T . A detailed comparison of the characteristics of JA-25 T with those of its close relatives in the family 'Spirosomaceae' is provided in Table 1. The cellular fatty acid profiles of strain JA-25 T and Fibrella aestuarina BUZ 2 T are shown in Table 3. The dominant fatty acids of strain JA-25 T were summed feature 3 (comprising C16:1 ω6c and/ or C16:1 ω6c; 40.8%), C16:1 ω5c (21.4%) and C16:0 (10%). Although the overall fatty acid composition of the strain JA-25 T was similar to that of strain BUZ 2 T , it can be distinguished from its closest phylogenetic neighbors by its smaller proportion of summed feature 3 and larger proportion of C16:0. Menaquinone 7 was the predominant respiratory quinone in strain JA-25 T . The major polar lipids of strain JA-25 T were phosphatidylethanolamine, two unidentified aminolipids, two phospholipids, and five unidentified lipids ( Figure S4). Catalase was positive but oxidase was negative. Nitrate reduction was negative. In API ZYM tests, alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, cysteine arylamidase, trypsin, α-chymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, α-galactosidase, β-galactosidase, α-glucosidase, β-glucosidase and N-acetyl-β-glucosaminidase were positive. In API 50CH assays, acid was produced from d-xylose, d-galactose, d-glucose, d-fructose, mannose, methyl-d-mannoside, methyl-d-glucopyranoside, N-acetyl-glucosamine, amygdalin, arbutin, salicin, cellobiose, maltose, sucrose, trehalose, inulin, melizitose, d-raffinose, turanose, and gentibiose.

Algicidal activity
Strain JA-25 T showed algicidal activity after 4 days of co-culture. The concentrations of J1 (6 mg/mL) and J2 (3 mg/mL) showed the highest algicidal activity toward the harmful algae H. triquetra (Fig. 2). Meaningful algicidal activity was not observed in the bacterial culture supernatant or bacterial cell extracts against harmful algae (data not shown). Algicidal activity was more effective in bacterial co-culture. Further studies are needed to understand these results, such as by changing the extraction methods and bacterial culture conditions. The amount of bacterial inoculum is an important factor affecting algicidal activity, as demonstrated by the differences between treatment groups (Hu et al. 2019). Under a scanning electron microscope, a large number of algal cells surrounded by microorganisms was observed, and burst algal cells to which microorganisms were attached were observed. Although additional studies are necessary, the mechanisms of the algicidal effects may be based on direct interaction (Hu et al. 2019).

Conclusion
A strain phylogenetically related to algicidal strain JA-25 T was characterized using a polyphasic approach. The DNA GC contents, mena-quinone, and major polar lipids of strain JA-25 T were showed that this strain belongs to the family 'Spirosomaceae' description. Genome analysis based on the ANI and AAI values suggested that strain JA-25 T is a novel species in a new genus. Thus, based on the phylogenetic inference, phenotypic, and chemotaxonomic Description of Fibrivirga gen. nov.