Marine organisms often form symbiotic relationships with various microorganisms to adapt and thrive in harsh environments. These symbiotic microbes contribute to host survival by providing nutrition, modulating the hosts’ immune system, and supporting overall physiological stability. Advances in high-throughput sequencing technologies have enabled a deeper understanding of the structure and function of symbiotic microbial communities, as well as host-microbe interactions. Notably, symbiotic bacteria associated with marine invertebrates such as corals and sponges are recognized as a potential source of useful bioactive compounds, including antibiotics and enzymes. However, obtaining high-quality microbial DNA from host tissues still remains a technical challenge due to the presence of unknown substances. This study focuses on optimizing sample preparation and DNA extraction procedures and additional purification to improve the recovery of microbial DNA while minimizing host DNA contamination. Comparison between several methods was conducted using sponge samples to evaluate DNA quality and microbial recovery. A sample designated as 2110BU-001 was collected from the east coast of the Republic of Korea and used for culture-independent microbial cell isolation. Total bacterial DNA was extracted by using a manual Phenol-Chloroform protocol and three commercial kits. DNA extracted using the standard manual method showed both the highest yield and the largest fragment size. However, PCR (Polymerase chain reaction) test showed that quality of manually extracted DNA was not enough for sequencing. Therefore, the quality of DNA was improved through additional purification steps. Briefly, host eukaryotic cells were removed by mechanical process and almost only bacterial DNA was successfully obtained by combination of manual extraction method and further purification processes. The established protocol was successfully introduced to extraction of metagenomic DNA from mussel and jellyfish microbiomes, indicating that it can be widely applied to various marine organisms.
Two Gram-stain-negative, obligately aerobic, non-motile, short rod-shaped bacteria, designated IMCC43871T and IMCC45206T, were isolated from coastal surface seawater collected from the Yellow Sea and the South Sea of Korea, respectively. The two strains shared 99.2% 16S rRNA gene sequence similarity with each other and exhibited ≤ 98.4% similarity to three described Rubrivirga species. Average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH) values between IMCC43871T and IMCC45206T were 88.5% and 36.3%, respectively, confirming that they represent two distinct species. Their ANI (≤ 77.7%) and dDDH (≤ 21.4%) values relative to the type strains of the genus Rubrivirga further supported the recognition of strains IMCC43871T and IMCC45206T as two novel species within the genus. The complete genomes of IMCC43871T (4.17 Mb, 71.8% G + C content) and IMCC45206T (4.17 Mb, 72.8% G + C content) fall within the known genomic range of the genus. Cellular fatty acid, quinone, and polar lipid profiles were consistent with the chemotaxonomic features of the genus Rubrivirga, supporting their affiliation with the genus. Based on phylogenetic, genomic, and phenotypic evidence, strains IMCC43871T and IMCC45206T are proposed as two novel species, Rubrivirga aquatilis sp. nov. and Rubrivirga halophila sp. nov., respectively. The type strains are IMCC43871T (= KCTC 102072T = NBRC 116463T) and IMCC45206T (= KCTC 92925T = NBRC 116172T = CCTCC AB 2023136T).
Two Gram-stain-negative, strictly aerobic, non-motile, rod-shaped bacteria, designated D3-12ᵀ and G2-2ᵀ, were isolated from the phycosphere of marine red algae. Both strains exhibited catalase- and oxidase-positive activities. Strain D3-12ᵀ grew optimally at 30°C, pH 7.0, and 2.0–3.0% (w/v) NaCl, while strain G2-2ᵀ showed optimal growth at 30°C, pH 7.0, and 2.0% NaCl. Ubiquinone-10 was the sole respiratory quinone in both strains. The major fatty acids (> 5%) in strain D3-12ᵀ were feature 8 (C18:1 ω7c and/or C18:1 ω6c), 11-methyl-C18:1 ω7c, and C16:0, while strain G2-2ᵀ contained summed feature 8 and C16:0. The predominant polar lipids in strain D3-12ᵀ were phosphatidylcholine, phosphatidylglycerol, and phosphatidylethanolamine, whereas strain G2-2ᵀ contained phosphatidylglycerol and diphosphatidylglycerol. The genomic DNA G + C content was 59.9% for strain D3-12ᵀ and 60.2% for strain G2-2ᵀ. Phylogenetic analyses based on 16S rRNA and whole-genome sequences placed both strains into distinct lineages within the family Roseobacteraceae, separate from previously described genera. Genome-based relatedness metrics, including average nucleotide identity, digital DNA-DNA hybridization, average amino acid identity, and percentage of conserved proteins, further confirmed that these strains represent novel genera. Based on phenotypic, chemotaxonomic, and molecular characteristics, strains D3-12ᵀ and G2-2ᵀ are proposed as novel genera: Phycobium rhodophyticola gen. nov., sp. nov. (D3-12ᵀ = KACC 22712ᵀ = JCM 35528ᵀ) and Aliiphycobium algicola gen. nov., sp. nov. (G2-2ᵀ = KACC 22602ᵀ = JCM 35752ᵀ). Additionally, metabolic features relevant to interactions with marine algae, including genes associated with carbohydrate-active enzymes, vitamin biosynthesis, phenylacetic acid production, and bacterioferritin synthesis, were bioinformatically investigated.
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