INTRODUCTION

Cyanobacteria are widely distributed worldwide and a major causative species of algae bloom in freshwater ecosystems (Robarts and Zohary 1987;Yunes et al. 2003;Havens 2008;You et al. 2013). This algae blooming caused by cyanobacteria induces visual disturbance (Barnett 1984). Some species produce off-flavor compounds, such as geosmin and 2-MIB (Kim et al. 2015), and liver or neurotoxin substances, such as microcystin and anatoxin-a (Suffet et al. 1995;Zander and Pingert 1997). Taxonomic studies on cyanobacteria have emerged due to the harmful effects of these cyanobacteria on the environment (Komárek 2006;Ryu et al. 2018). Taxonomic studies on cyanobacteria have been mainly based on morphological characteristics (Pfannkuche and Lochte 1993;Choi et al. 1998). However, these methods still suffer from light microscopy given their morphological changes in response to various environmental conditions and insufficient taxonomic characteristics (Lehtimäki et al. 2000;Gugger et al. 2002;Willame et al. 2006). Recently, molecular phylogenetic data and ultrastructural and morphological characteristics have been used together to describe cyanobacterial species (Komárek and Anagnostidis 2005;Komárek 2016).

In Korea, studies on cyanobacteria focus on taxa with high environmental impacts, such as the genus Microcystis, which produces toxins, and the genus Anabaena, which generates odorants and neurotoxins. Additionally, only 377 cyanobacterial species have been reported (NIBR 2019), which is less than 10% of the number of cyanobacteria reported worldwide (4,707 species, Guiry and Guiry 2020). Thus, studies on the diversity of cyanobacteria species in Korea are quite insufficient (Ryu et al. 2018).

The genus Cyanobium is an oval-shaped or short rodshaped unicellular cyanobacteria (Rippka and Cohen- Bazire 1983;Komárek et al. 1999). This genus is often considered morphologically similar to the genus Synechococcus (Nägeli 1849;Padisák et al. 1997). However, a difference is noted between the two genera in terms of DNA base composition. The average GC content of Cyanobium is 66-71 moles%, whereas that of Synechococcus is 48-56 moles% (Rippka and Cohen-Bazire 1983).

The genus Cyanobium is an important primary producer in oligotrophic and mesotrophic environments (Jezberová and Komárková 2007). However, only 14 species have been reported worldwide (Guiry and Guiry 2020) due to the absence of distinct morphological characteristics and the low population density (Komárek et al. 1999). Additionally, this genus and species have not yet been reported in Korea.

In this study, morphological and ultrastructural studies and phylogenetic analysis were performed to report the unrecorded genus Cyanobium and the unrecorded species Cyanobium gracile in Korea.

MATERIALS AND METHODS

1. Sampling and clonal culture of Cyanobium gracile

Cultures of C. gracile were established by single-cell isolation from freshwater samples collected at Adongji pond, Korea (35°58ʹ47.4ʺN, 126°46ʹ14.6ʺE) in October 2019. The cultures were grown in BG11 medium at 25°C under a 14 : 10 light : dark cycle and a light intensity of 4,000 lux provided by cool-white fluorescent lamps.

2. Light microscopy

Living C. gracile cells were studied using a Nikon ECLIPSE Ni-U (Nikon, Japan) equipped with differential interference contrast optics. Images were captured using a digital camera (DS-Ri2, Nikon).

3. Transmission electron microscopy

For transmission electron microscopy, the cells were prefixed in a 1 : 1 mixture of 5% (V/V) glutaraldehyde and BG11 culture media for 1 h at 4°C. The glutaraldehydefixed cells were washed 3 times in BG11 culture media and postfixed in 1% (W/V) OsO₄ for 1 h at 4°C. The fixed cells were rinsed three times with distilled water. Dehydration was carried out at 4°C using a graded ethanol series of 50, 60, 70, 80, and 90% for 10 min each and three 10 min changes of pure ethanol. Pellets were then brought to room temperature and transferred through propylene oxide two times for 20 min each with 50% and 75% Spurr’s embedding resin (Spurr 1969) in propylene oxide for 1 h each and 100% overnight. On the following day, pellets were moved to new pure resin and polymerized at 70°C. Blocks were thin-sectioned on a PT-X ultramicrotome (RMC Products, Boeckeler Instruments, Tucson, AZ). Sections of 70 nm thickness were collected on slot copper grids, stained with 3% (w/v) uranyl acetate and Reynold’s lead citrate (Reynolds 1963), and observed using a JEM-1400 Plus at Korea Basic Science Institute (KBSI) operated at 120 kV (JEOL, Tokyo, Japan).

4. DNA extraction, amplification, and sequencing

Approximately 10 mL aliquots of culture media were obtained in the exponential growth phase. Cells were harvested by centrifugation (1,330×g, model 5415; Eppendorf, Hamburg, Germany) for 1 min at room temperature followed by washing three times with sterilized distilled water. According to the manufacturer’s protocol, total genomic DNA was extracted from the pellet using InstaGenetm Matrix (BIO-RAD, CA, USA). Polymerase chain reaction (PCR) was performed using 27F/1492R universal primers to amplify 16S SSU rDNA (Edwards et al. 1989). PCR amplification was performed with a total volume of 30 μL containing EF-Taq (SolGent, Daejeon, Korea), each dNTP, 10·Ex Taq Buffer, each primer, and 20 ng of template DNA. The 16S SSU rDNA gene was amplified using a DNA Engine Tetrad 2 Peltier Thermal Cycler (BIO-RAD, CA, USA) with the following conditions: initial denaturation at 95°C for 2 min; 35 cycles each of 95°C for 2 min, 55°C for 1 min, and 72°C for 1 min; final extension at 72°C for 10 min; and holding at 4°C. According to the manufacturer’s protocol, the PCR products were purified using a Multiscreen filter plate (Millipore Corp., MA, USA). The purified template was sequenced with PRISM BigDyeTM Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, CA, USA). The 16S SSU rDNA gene sequence alignment was edited using the Genetic Data Environment (GDE 2.2) program (Smith et al. 1994), and the aligned sequence was registered in GenBank (Accession Number MT644519).

5. Phylogenetic analyses

Sequence data of 14 strains (Table 1) were used for the analysis of MODELTEST v.3.7 (Posada and Cradall 1998), and maximum likelihood (ML). ML analysis was performed with RAxML v8.2.4 (Stamatakis 2014) using the general time reversible plus gamma (GTR+G) model with random sequence addition 1,000 times followed by a heuristic search using tree-bisection reconnection (TRB) branch swapping. Bayesian analysis was performed using MrBayes v3.2 (Ronquist et al. 2012) to construct random inference trees with the GTR+G+I model 2,000,000 times. The phylogenetic tree was constructed every 1,000 cycles, and the burn-in point was graphically identified based on the likelihood score in the phylogenetic tree (Tracer v1.5; http://tree.bio.ed.ac.uk/software/tracer/).

RESULTS AND DISCUSSION

1. Taxonomic summary

Phylum Cyanobacteria Stanier ex Cavalier-Smith, 2002

Class Cyanophyceae Schaffner, 1909

Order Synechococcales Hoffmann, Komárek & Kastovsky, 2005

Family Synechococcaceae Komárek & Anagnostidis, 1995

Genus CyanobiumRippka & Cohen-Bazire, 1983

Cyanobium gracileRippka & Cohen-Bazire 1983

Holotype. Type strain deposited at Pasteur Culture Collection (PCC), PCC6307.

Material examined. Freshwater was collected from the Adongji pond, Adong-ri, Gaejeong-myeon, Gunsan-si, Jeollabuk- do, Republic of Korea (35°58ʹ47.4ʺN, 126°46ʹ 14.6ʺE) on October 16, 2019.

Diagnosis. Cells are a pale blue-green single cell. The cell shape is ovate, oval, or short rod-shaped without gelatinous envelopes. Cells solitary or in twos after division, not in colonies. 1.08-3.87 μm long and 0.75-1.51 μm wide. The thylakoid membranes are stacked in three and arranged along the cell walls. Obligatory photoautotroph.

Distribution. North America (Smith 2010) and the Republic of Korea.

Voucher slide. Two slides of gelatin-embedded specimens were deposited at Nakdonggang National Institute of Biological Resources, Korea (NNIBRCY894 and NNIBRCY 895).

2. Morphology and ultrastructure

Cyanobium gracile was pale blue-green and ovate-, oval-, or rod-shaped (Figs. 1, 2). Synechococcus species, similar to Cyanobium species in morphology, generally have a long cylindrical shape and are occasionally asymmetrical (Komárek et al. 1999), but C. gracile cells were observed to be symmetrical (Figs. 1, 2). When the cell divided by simple dichotomy, the cell was elongated rod-shaped or eight-shaped (Figs. 1B, 2). C. gracile cells were 1.08-3.87 μm (n=75, mean=1.84±0.43 μm) long and 0.75-1.51 μm (n=75, mean=1.08±0.18 μm) wide. Cells were larger than that of the type strain PCC6307 (0.4-2.4×0.25-0.4 μm, Komárek et al. 1999). The nucleolus was observed in the center of the cell of C. gracile. Peripheral thylakoid membranes were stacked in three, and each of the thylakoid membranes was arranged in parallel (Fig. 3). This thylakoid architecture is a typical characteristic of the genus Cyanobium (Gantt and Conti 1969;Komárek and Cepak 1998). Additionally, an electron opaque material, which was presumed to be polyphosphate granules, was observed in the cytoplasm. These granules were distributed between the cell wall and the outer thylakoid membrane (Fig. 3).

3. Phylogeny

BLAST analysis indicated that the 16S SSU rDNA sequence of C. gracile showed 100% similarity to the reference sequences of CP003495, NR_102447, AF216944, MT488300, DQ275599, and NR_114406. Bayesian and maximum likelihood (ML) analyses were performed with the 16S SSU rDNA sequence of C. gracile and 13 references (Fig. 4). In the phylogenetic tree, C. gracile formed a monophyletic clade with C. gracile PCC9604 and PCC6307 (ML=100, pp=1.00). In addition, C. gracile formed a sister group with four strains of Prochlorococcus marinus and two strains of Synechococcus sp. (ML=100, pp=1.00).