INTRODUCTION

Suncheon Bay is a representative tidal flat in Korea, composed of a 3.5 km (2.2 mi) long stream, a 2,221 ha (5,490 acres) wide tideland, and a 230 ha (570 acres) wide field of reeds (https://scbay.suncheon.go.kr). Marshes located between terrestrial and water ecosystems have numerous functions of purification, prevention of water pollution, supply of surface water, flow control, and storage and recharge of underground water (You et al. 2012). These characteristics result in reasonable salt content, rich organisms, and healthy water quality. Suncheon Bay is therefore a biodiversity hotspot. Chitinibacter suncheonensis (Kim et al. 2012b), Lutibacter agarilyticus (Park et al. 2013), and Proteiniclasticum aestuarii (Namirimu et al. 2022) have been reported as new bacterial taxa. Brackish water and Phragmites communis Trin (Reed) species were collected and novel or previously unrecorded bacterial species were isolated in Suncheon Bay in 2019-2020. A total of 12 bacterial species belonging to the classes Alphaproteobacteria, Actinobacteria, and Flavobacteria were identified that had not been previously recorded in Korea using phylogenetic analyses based on 16S rRNA gene sequencing. Here, we report the phylogenetic information and phenotypic characteristics of these bacterial species.

MATERIALS AND METHODS

Brackish water and reeds from Suncheon Bay were collected in 2019-2020 to obtain bacterial species that were previously unrecorded (Fig. 1). The collected reed roots were washed thrice with tap water to remove the attached soil. The roots were subsequently immersed in 70% ethanol (Merk, USA) for 3 min, washed with fresh sodium hypochlorite solution (Junsei, Japan) for 5 min, rinsed three times with 70% ethanol for 30 s, and finally washed five times with sterile distilled water, as described by Sun et al. (2008). The surface-sterilized plant samples were then placed in a biological safety cabinet until the surface moisture was evaporated. The samples were aseptically crushed into smaller fragments using a mortar. The brackish water and reed samples were processed separately, spread onto diverse culture media, including Reasoner’s 2A (BD Difco, USA) agar, and incubated at 25°C for three days. All the strains were purified as single colonies and stored in a 20% glycerol suspension at -80°C. The designated strain IDs, sources, culture media, and incubation conditions are summarized in Table 1. The colony morphology of the strains was observed on agar plates with a magnifying glass after the cells had grown to the stationary phase. The cellular morphology and size were examined using transmission electron microscopy (H-7650; Hitachi, Japan) and Gram staining was performed using a Gram staining kit (BD Biosciences, USA). The biochemical characteristics were tested using API 20NE (BioMérieux, marcy I’Etoile, France), as per the instructions provided by the manufacturer. The enzyme characteristics were confirmed using API ZYM (BioMérieux).

The phylogenetic positions of the strains assigned to the phyla Actinobacteria, Bacteroidetes, and Proteobacteria were investigated using 16S rRNA gene analysis, and the 16S rRNA gene was amplified using PCR and sequenced (Baek et al. 2020). The 16S rRNA gene sequences of the strains were analyzed using the EzTaxon-e server (Kim et al. 2012a) and confirmed using the GenBank database (https://ncbi. nlm.nih.gov). The 16S rRNA gene sequences were aligned using EzEditor for phylogenetic analyses (Jeon et al. 2014). A phylogenetic tree was constructed using neighbor-joining (NJ) (Saitou and Nei 1987) in MEGA version 7 (Tamura et al. 2013). The topology of the phylogenetic tree was evaluated using bootstrap analysis (Felsenstein 1985) with 1,000 replications.

RESULTS AND DISCUSSION

A total of 300 bacterial strains were isolated from the brackish water and reed species from Suncheon Bay during bacterial screening. Based on the similarity of the 16S rRNA gene sequences, 12 strains of previously unrecorded bacterial species were recorded in Korea. Based on the 16S rRNA gene sequence comparisons and phylogenetic analyses, the 12 strains were assigned to the classes Actinobacteria, Alphaproteobacteria, and Flavobacteria. The three strains of the class Actinobacteria were distributed in two orders - Corynebacteriales and Micrococcales. The eight strains of the class Alphaproteobacteria were distributed in three orders - Rhizobiales, Rhodobacterales, and Sphingomonadales. A strain of Flavobacteria was distributed in the order Flavobacteriales (Table 1). According to the isolation sources, six strains were obtained from brackish water and six strains were obtained from the reed species (Table 1). At the genus level, six unreported species from brackish water belonged to the genera Altererythrobacter (two species), Paracoccus, Rhodovulum, Rhizobium, Alphaproteobacteria, and Microbacterium of Actinobacteria. The remaining six unreported species from the reeds belonged to the genera Martelella, Rhizobium, and Jiella of Alphaproteobacteria, Mycolicibacterium, and Demequina of Actinobacteria, and Confluentibacter of Flavobacteria. A phylogenetic tree of the previously unrecorded species was constructed among the related taxa and is presented in Figures 2 and 3, respectively.

As expected from the high 16S rRNA gene sequence similarities between the 12 strains and their closest relatives, each strain formed a robust phylogenetic clade with the most closely related species in the trees. The 12 bacterial species were gram-negative or gram-positive, and rod, shortrod, or oval-shaped in Figure 4.

This study proposes that the following are the previously unrecorded bacterial species: Mycolicibacterium helvum, Microbacterium diaminobutyricum, Demequina lutea, Confluentibacter sediminis, Jiella aquimaris, Martelella mediterranea, Rhizobium wuzhouenes, Rhizobium oryziradicis, Paracoccus sediminilitoris, Rhodovulum algae, Altererythrobacter flavus, and Altererythrobacter oceanensis from Suncheon Bay, Korea.

Description of Altererythrobacter oceanensis SBII-40

The cells are gram-negative, non-flagellated, and rodshaped. The colonies are circular, convex with an entire edge, and yellow-colored after three days on R2A agar at 25°C. It is positive for esculin hydrolysis and β-galactosidase but negative for nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, urease, gelatinase, and oxidase. It does not utilize D-glucose, L-arabinose, D-mannose, D-mannitol, N-acetyl-glucosamine, D-malt- ose, potassium gluconate, capric acid, adipic acid, malic acid, trisodium citrate, or phenylacetic acid. Strain SBII-40 displays the highest 16S rRNA gene sequence similarity with Altererythrobacter oceanensis Y2^T (99.65%). Strain SBII- 40 (=NNIBR2019641BA32) was isolated from the brackish water of the Suncheon Bay Wetland Reserve, Korea. The GenBank accession number of 16S rRNA gene sequence is MN559424.

Description of Paracoccus sediminilitoris SBII-64

The cells are gram-negative, non-flagellated, and rodshaped. The colonies are circular, convex with an entire edge, and ivory-colored after three days on R2A agar at 25°C. It is positive for urease, esculin hydrolysis, β-galactosidase, and oxidase, but negative for nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, and gelatinase. L-arabinose, D-mannose, D-mannitol, D-maltose, potassium gluconate, malic acid, and trisodium citrate are utilized. It does not utilize D-glucose, N-acetyl-glucosamine, capric acid, adipic acid, or phenylacetic acid. Strain SBII-64 displays the highest 16S rRNA gene sequence similarity with Paracoccus sediminilitoris DSL-16^T (99.71%). Strain SBII-64 (=NNIBR2019641BA31) was isolated from the brackish water of the Suncheon Bay Wetland Reserve, Korea. The GenBank accession number of 16S rRNA gene sequence is MN559423.

Description of Mycolicibacterium helvum SWHRII-18

The cells are gram-positive, non-flagellated, and rodshaped. The colonies are circular, convex with an entire edge, and yellow-colored after three days on R2A agar at 25°C. It is positive for nitrate reduction, but negative for indole production, glucose fermentation, arginine dihydrolase, urease, esculin hydrolysis, gelatinase, β-galactosidase, and oxidase. It does not utilize D-glucose, L-arabinose, D-mannose, D-mannitol, N-acetyl-glucosamine, D-maltose, potassium gluconate, capric acid, adipic acid, malic acid, and trisodium citrate. Strain SWHRII-18 displays the highest 16S rRNA gene sequence similarity with Mycolicibacterium helvum DL739^T (99.56%). Strain SWHRII-18 (=NNIBR2019641BA23) was isolated from the reeds of Suncheon Bay Wetland Reserve, Korea. The GenBank accession number of 16S rRNA gene sequence is MN559 415.

Description of Demequina lutea SWMRII-32

The cells are gram-positive, non-flagellated, and rodshaped. The colonies are circular, convex with an entire edge, and yellow-colored after three days on R2A agar at 25°C. It is positive for glucose fermentation, esculin hydrolysis, β-galactosidase, and oxidase but negative for nitrate reduction, indole production, arginine dihydrolase, urease, and gelatinase. D-Glucose, L-arabinose, D-mannose, D-mannitol, and D-maltose are utilized. It does not utilize N-acetyl-glucosamine, potassium gluconate, capric acid, adipic acid, malic acid, trisodium citrate, or phenylacetic acid. Strain SWMRII-32 displays the highest 16S rRNA gene sequence similarity with Demequina lutea NBRC 106155^T (99.31%). Strain SWMRII-32 (=NNIBR2019641BA24) was isolated from the reeds of the Suncheon Bay Wetland Reserve, Korea. The GenBank accession number of 16S rRNA gene sequence is MN559416.

Description of Jiella aquimaria SWMRII-46

The cells are gram-negative, non-flagellated, and rodshaped. The colonies are circular, convex with an entire edge, and yellow-colored after three days on R2A agar at 25°C. It is positive for urease, β-galactosidase, and oxidase but negative for nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, esculin hydrolysis, and gelatinase. D-Glucose, D-mannitol, potassium gluconate, malic acid, and trisodium citrate are utilized. It does not utilize L-arabinose, D-mannose, N-acetyl-glucosamine, D-maltose, capric acid, adipic acid, and phenylacetic acid. Strain SWMRII-46 displays the highest 16S rRNA gene sequence similarity with Jiella aquimaria LZB041^T (99.15%). Strain SWMRII-46 (=NNIBR2019641BA27) was isolated from the reeds of the Suncheon Bay Wetland Reserve, Korea. The GenBank accession number of 16S rRNA gene sequence is MN559419.

Description of Rhizobium oryziradicis SWMRII-48

The cells are gram-negative, non-flagellated, and rodshaped. The colonies are circular, convex with an entire edge, and ivory colored after 3 days on R2A agar at 25°C. It is positive for urease, esculin hydrolysis, β-galactosidase, and oxidase, but negative for nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, and gelatinase. D-Glucose, L-arabinose, D-mannose, D-mannitol, D-maltose, malic acid, and trisodium citrate are utilized. It does not utilize N-acetyl-glucosamine, potassium gluconate, capric acid, adipic acid, or phenylacetic acid. Strain SWMRII- 48 displays the highest 16S rRNA gene sequence similarity with Rhizobium oryziradicis N19^T (99.22%). Strain SWMRII-48 (=NNIBR2019641BA30) was isolated from the reeds of the Suncheon Bay Wetland Reserve, Korea. The GenBank accession number of 16S rRNA gene sequence is MN559422.

Description of Martelella mediterranea SWSII-22

The cells are gram-negative, non-flagellated, and rodshaped. Colonies circular, convex with an entire edge, and ivory colored after three days on R2A agar at 25°C. It is positive for nitrate reduction, urease, esculin hydrolysis, β-galactosidase, and oxidase, but negative for indole production, glucose fermentation, arginine dihydrolase, and gelatinase. L-Arabinose, D-mannitol, and D-maltose are utilized. It does not utilize D-glucose, D-mannose, N-acetyl-glucosamine, potassium gluconate, capric acid, adipic acid, malic acid, trisodium citrate, and phenylacetic acid. Strain SWSII- 22 displays the highest 16S rRNA gene sequence similarity with Martelella mediterranea DSM 17316^T (99.21%). Strain SWSII-22 (=NNIBR2019641BA28) was isolated from the reeds of the Suncheon Bay Wetland Reserve, Korea. The GenBank accession number of 16S rRNA gene sequence is MN559420.

Description of Rhizobium wuzhouense SBIII-10

The cells are gram-negative, non-flagellated, and rodshaped. The colonies are circular, convex with an entire edge, and cream white after 3 days on R2A agar at 25°C. It is positive for urease, β-glucosidase, β-galactosidase, and oxidase, but negative for nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, and gelatinase. D-Glucose, L-arabinose, D-mannose, D-mannitol, N-acetyl-glucosamine, D-maltose, malic acid, and trisodium citrate are utilized. It does not utilize potassium gluconate, capric acid, adipic acid, or phenylacetic acid. Strain SBIII-10 displays the highest 16S rRNA gene sequence similarity with Rhizobium wuzhouense W44^T (99.48%). Strain SBIII-10 (=NNIBR2020641BA486) was isolated from the brackish water of the Suncheon Bay Wetland Reserve, Korea. The GenBank accession number of 16S rRNA gene sequence is MT893380.

Description of Rhodovulum algae 20SBII-15

The cells are gram-negative, non-flagellated, and rodshaped. The colonies are circular, convex with an entire edge, and purple-colored after three days on R2A agar at 25°C. It is positive for indole production, urease, β-glucosidase, and oxidase, but negative for nitrate reduction, glucose fermentation, arginine dihydrolase, gelatinase, and β-galactosidase. It does not utilize D-glucose, L-arabinose, D-mannose, D-mannitol, N-acetyl-glucosamine, D-maltose, potassium gluconate, capric acid, adipic acid, malic acid, trisodium citrate, or phenylacetic acid. Strain 20SBII-15 displays the highest 16S rRNA gene sequence similarity with Rhodovulum algae JA877^T (99.93%). Strain 20SBII-15 (=NNIBR2020641BA489) was isolated from the brackish water of the Suncheon Bay Wetland Reserve, Korea. The GenBank accession number of 16S rRNA gene sequence is MT893 377.

Description of Microbacterium diaminobutyricum 20SBII-23

The cells are gram-positive, non-flagellated, and rodshaped. The colonies are circular, convex with an entire edge, and yellow-colored after three days on R2A agar at 25°C. It is positive for β-glucosidase and β-galactosidase, but negative for nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, urease, and gelatinase. L-Arabinose, D-mannose, D-maltose, and potassium gluconate are utilized. It does not utilize D-glucose, D-mannitol, N-acetyl-glucosamine, capric acid, adipic acid, malic acid, trisodium citrate, phenylacetic acid, or oxidase. Strain 20SBII-23 displays the highest 16S rRNA gene sequence similarity with Microbacterium diaminobutyricum R263^T (100%). Strain 20SBII-23 (=NNIBR2020641BA488) was isolated from the brackish water of the Suncheon Bay Wetland Reserve, Korea. The GenBank accession number of 16S rRNA gene sequence is MT893378.

Description of Altererythrobacter flavus 20SBII-24

The cells are gram-positive, non-flagellated, and rodshaped. The colonies are circular, convex with an entire edge, and yellow-colored after three days on R2A agar at 25°C. It is positive for β-glucosidase and oxidase, but negative for nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, urease, gelatinase, and β-galactosidase. D-Glucose, D-maltose, and adipic acidare utilized. It does not utilize L-arabinose, D-mannose, D-mannitol, N-acetylglucosamine, potassium gluconate, capric acid, malic acid, trisodium citrate, and phenylacetic acid. Strain 20SBII-24 displays the highest 16S rRNA gene sequence similarity with Altererythrobacter flavus MS1-4^T (99.93%). Strain 20SBII-24 (=NNIBR2020641BA487) was isolated from the brackish water of the Suncheon Bay Wetland Reserve, Korea. The GenBank accession number of 16S rRNA gene sequence is MT893379.

Description of Confluentibacter sediminis 20SMRII-2

The cells are gram-positive, non-flagellated, and rodshaped. The colonies are circular, convex with an entire edge, and yellow after three days on R2A agar at 25°C. It is positive for β-glucosidase and β-galactosidase, but negative for nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, urease, and gelatinase. D-Glucose, L-arabinose, and D-mannose are utilized. It does not utilize D-mannitol, N-acetyl-glucosamine, D-maltose, potassium gluconate, capric acid, adipic acid, malic acid, trisodium citrate, phenylacetic acid, and oxidase. Strain 20SMRII-2 displays the highest 16S rRNA gene sequence similarity with Confluentibacter sediminis DSL-48^T (99.86%). Strain 20SMRII-2 (=NNIBR2020641BA490) was isolated from the reeds of the Suncheon Bay Wetland Reserve, Suncheon, Korea. The GenBank accession number of 16S rRNA gene sequence is MT893376.