Terpene biosynthesis in marine sponge animals.

Sea sponges are the largest marine source of small-molecule natural products described to date. Sponge-derived molecules, such as the chemotherapeutic eribulin, the calcium-channel blocker manoalide, and antimalarial compound kalihinol A, are renowned for their impressive medicinal, chemical, and biological properties. Sponges contain microbiomes that control the production of many natural products isolated from these marine invertebrates. In fact, all genomic studies to date investigating the metabolic origins of sponge-derived small molecules concluded that microbes-not the sponge animal host-are the biosynthetic producers. However, early cell-sorting studies suggested the sponge animal host may play a role particularly in the production of terpenoid molecules. To investigate the genetic underpinnings of sponge terpenoid biosynthesis, we sequenced the metagenome and transcriptome of an isonitrile sesquiterpenoid-containing sponge of the order Bubarida. Using bioinformatic searches and biochemical validation, we identified a group of type I terpene synthases (TSs) from this sponge and multiple other species, the first of this enzyme class characterized from the sponge holobiome. The Bubarida TS-associated contigs consist of intron-containing genes homologous to sponge genes and feature GC percentage and coverage consistent with other eukaryotic sequences. We identified and characterized TS homologs from five different sponge species isolated from geographically distant locations, thereby suggesting a broad distribution amongst sponges. This work sheds light on the role of sponges in secondary metabolite production and speaks to the possibility that other sponge-specific molecules originate from the animal host.

Metagenomic mining of two Egyptian Red Sea sponges associated microbial community.

The Red Sea is a promising habitat for the discovery of new bioactive marine natural products. Sponges associated microorganisms represent a wealthy source of compounds with unique chemical structures and diverse biological activities. Metagenomics is an important omics-based culture-independent technique that is used as an effective tool to get genomic and functional information on sponge symbionts. In this study, we used metagenomic analysis of two Egyptian Red Sea sponges Hyrtios erectus and Phorbas topsenti microbiomes to study the biodiversity and the biosynthetic potential of the Red Sea sponges to produce bioactive compounds. Our data revealed high biodiversity of the two sponges' microbiota with phylum Proteobacteria as the most dominant phylum in the associated microbial community with an average of 31% and 70% respectively. The analysis also revealed high biosynthetic potential of sponge Hyrtios erectus microbiome through detecting diverse types of biosynthetic gene clusters (BGCs) with predicted cytotoxic, antibacterial and inhibitory action. Most of these BGCs were predicted to be novel as they did not show any similarity with any MIBiG database known cluster. This study highlights the importance of the microbiome of the collected Red Sea sponge Hyrtios erectus as a valuable source of new bioactive natural products.

Microbulbifer spongiae sp. nov., isolated from marine sponge Diacarnus erythraeanus.

A novel bacterium, designated as MI-GT, was isolated from marine sponge Diacarnus erythraeanus. Cells of strain MI-GT are Gram-stain-negative, aerobic, and rod or coccoid-ovoid in shape. MI-GT is able to grow at 10-40 °C (optimum, 28 °C), with 1.0-8.0% (w/v) NaCl (optimum, 4.0%), and at pH 5.5-9.0 (optimum, pH 8.0). The 16S rRNA gene sequence of strain MI-GT shows 98.35, 97.32 and 97.25% similarity to those of Microbulbifer variabilis Ni-2088T, Microbulbifer maritimus TF-17T and Microbulbifer echini AM134T, respectively. Phylogenetic analysis also exhibits that strain MI-GT falls within a clade comprising members of the genus Microbulbifer (class Gammaproteobacteria). The genome size of strain MI-GT is 4478124 bp with a G+C content of 54.51 mol%. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between strain MI-GT and other type strains are 71.61-76.44% (ANIb), 83.27-84.36% (ANIm) and 13.4-18.7% (dDDH), respectively. These values are significantly lower than the recommended threshold values for bacterial species delineation. Percentage of conserved proteins and average amino acid identity values among the genomes of strain MI-GT and other closely related species are 52.04-59.13% and 67.47-77.21%, respectively. The major cellular fatty acids of MI-GT are composed of summed feature 8 (C18 : 1 ω7c or C18 : 1 ω6c), iso-C11 : 0 3-OH, iso-C15 : 0, C16 : 0, and summed feature 9 (C17 : 1 iso ω9c or C16 : 0 10-methyl). The polar lipids of MI-GT mainly consist of phosphatidylethanolamine, phosphatidylglycerol, aminolipid, and two glycolipids. The major respiratory quinone is Q-8. Based on differential phenotypic and phylogenetic data, strain MI-GT is considered to represent a novel species of genus Microbulbifer, for which the name Microbulbifer spongiae sp. nov. is proposed. The type strain is MI-GT (=MCCC 1K07826T=KCTC 8081T).

Paracoccus spongiarum sp. nov., isolated from the marine sponge, Phakellia elegans.

A facultative anaerobic Gram-negative bacterium, designated as strain 2205BS29-5T, was isolated from a marine sponge, Phakellia elegans, in Beomseom on Jeju Island, Republic of Korea, and taxonomically characterized. The cells were catalase and oxidase positive, non-motile, coccoid-rod shaped and capable of poly-ß-hydroxybutyrate production. Growth was observed at 10-37 °C (optimum, 25 °C) and pH 5.0-8.0 (optimum, pH 7.0), and in the presence of 0-9% NaCl (w/v) (optimum, 3.0-4.0%). The major cellular fatty acids and respiratory quinone were identified as summed feature 8 (C18 : 1 ω7c/C18 : 1 ω6c) and Q-10, respectively. The major polar lipids comprised diphosphatidylglycerol, phosphatidylglycerol, four phosphoglycolipids, two unidentified amino lipids and eight unidentified lipids. The DNA G+C content was 67.8%. Strain 2205BS29-5T was most closely represented by Paracoccus amoyensis 11-3T and P. caeni MJ17T with 97.8 and 97.5% 16S rRNA gene sequence similarities, respectively. Phylogenetic analyses based on 16S rRNA genes and whole-genome sequences showed that strain 2205BS29-5T was affiliated with the genus Paracoccus. Genomic analysis showed that strain 2205BS29-5T could synthesize vitamin B family (folate and cobalamin) and ectoine. The average nucleotide identity and digital DNA-DNA hybridization values between strain 2205BS29-5T and P. amoyensis 11-3T were 77.1% and 18.8%, respectively, and with P. caeni MJ17T were 78.4 and 21.2%, respectively. Based on phylogenetic, chemotaxonomic and genome relatedness analyses, strain 2205BS29-5T represents a novel species of the genus Paracoccus, for which the name Paracoccus spongiarum sp. nov. is proposed. The type strain is 2205BS29-5T (=LMG 33062T =KACC 23240T).

Chengkuizengella axinellae sp. nov., a symbiotic bacterium isolated from a marine sponge of the genus Axinella.

A Gram-stain-positive, strictly aerobic, creamy-white colored, endospore-forming and non-motile rods strain, designated as strain 2205SS18-9T, was isolated from a marine sponge, Axinella sp. collected from Seopseom Island, Republic of Korea. Optimal growth of strain 2205SS18-9T was observed at 25-30 °C, pH 6.5-7.0, and in the presence of 3.0% (w/v) NaCl. Cells were oxidase-positive and catalase-negative. Negative for nitrate reduction and indole production. Phylogenetic analyses based on the 16S rRNA gene and whole-genome sequences revealed that strain 2205SS18-9T formed a distinct phyletic lineage in the genus Chengkuizengella, and it was most closely related to Chengkuizengella marina YPA3-1-1T and Chengkuizengella sediminis J15A17T with 97.1 and 96.6% 16S rRNA gene sequence similarities, respectively. The average nucleotide identity and digital DNA-DNA hybridization values between strain 2205SS18-9T and Chengkuizengella marina YPA3-1-1T were 79.0 and 21.6%, respectively. The genomic DNA G + C content was 34.1%. The genome harbors a number of host-adhesion and transporter genes, suggested that strain 2205SS18-9T may interact with its sponge host as a symbiont. Menaquinone-7 was the sole isoprenoid quinone and antieiso-C15:0 (28.5%), iso-C16:0 (25.8%), C16:1 ω7c alcohol (15.0%), and iso-C15:0 (11.2%) were detected as the major fatty acids. Polar lipids included diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, two unidentified aminophospholipids, and an unidentified lipid. The cell-wall peptidoglycan contained lysine, alanine, glutamate, and aspartate. Based on these analyses, strain 2205SS18-9T represents a novel species of the genus Chengkuizengella, for which the name Chengkuizengella axinellae sp. nov. is proposed. The type strain is 2205SS18-9T (= KACC 23238T = LMG 33063T).

Unveiling antibiofilm potential: proteins from Priestia sp. targeting Staphylococcus aureus biofilm formation.

Staphylococcus aureus is the etiologic agent of many nosocomial infections, and its biofilm is frequently isolated from medical devices. Moreover, the dissemination of multidrug-resistant (MDR) strains from this pathogen, such as methicillin-resistant S. aureus (MRSA) strains, is a worldwide public health issue. The inhibition of biofilm formation can be used as a strategy to weaken bacterial resistance. Taking that into account, we analysed the ability of marine sponge-associated bacteria to produce antibiofilm molecules, and we found that marine Priestia sp., isolated from marine sponge Scopalina sp. collected on the Brazilian coast, secretes proteins that impair biofilm development from S. aureus. Partially purified proteins (PPP) secreted after 24 hours of bacterial growth promoted a 92% biofilm mass reduction and 4.0 µg/dL was the minimum concentration to significantly inhibit biofilm formation. This reduction was visually confirmed by light microscopy and Scanning Electron Microscopy (SEM). Furthermore, biochemical assays showed that the antibiofilm activity of PPP was reduced by ethylenediaminetetraacetic acid (EDTA) and 1,10 phenanthroline (PHEN), while it was stimulated by zinc ions, suggesting an active metallopeptidase in PPP. This result agrees with mass spectrometry (MS) identification, which indicated the presence of a metallopeptidase from the M28 family. Additionally, whole-genome sequencing analysis of Priestia sp. shows that gene ywad, a metallopeptidase-encoding gene, was present. Therefore, the results presented herein indicate that PPP secreted by the marine Priestia sp. can be explored as a potential antibiofilm agent and help to treat chronic infections.

Natural Products Derived from Marine Sponges with Antitumor Potential against Lung Cancer: A Systematic Review.

Non-small-cell lung cancer (NSCLC), the most commonly diagnosed cancer and the leading cause of cancer-related death worldwide, has been extensively investigated in the last decade in terms of developing new therapeutic options that increase patient survival. In this context, marine animals are a source of new, interesting bioactive molecules that have been applied to the treatment of different types of cancer. Many efforts have been made to search for new therapeutic strategies to improve the prognosis of lung cancer patients, including new bioactive compounds and cytotoxic drugs from marine sponges. Their antitumoral effect can be explained by several cellular and molecular mechanisms, such as modulation of the cell cycle or induction of apoptosis. Thus, this systematic review aims to summarize the bioactive compounds derived from marine sponges and the mechanisms by which they show antitumor effects against lung cancer, exploring their limitations and the challenges associated with their discovery. The search process was performed in three databases (PubMed, SCOPUS, and Web of Science), yielding a total of 105 articles identified in the last 10 years, and after a screening process, 33 articles were included in this systematic review. The results showed that these natural sponge-derived compounds are a valuable source of inspiration for the development of new drugs. However, more research in this field is needed for the translation of these novel compounds to the clinic.

Meroterpenoids from Marine Sponge Hyrtios sp. and Their Anticancer Activity against Human Colorectal Cancer Cells.

Two new meroterpenoids, hyrtamide A (1) and hyrfarnediol A (2), along with two known ones, 3-farnesyl-4-hydroxybenzoic acid methyl ester (3) and dictyoceratin C (4), were isolated from a South China Sea sponge Hyrtios sp. Their structures were elucidated by NMR and MS data. Compounds 2-4 exhibited weak cytotoxicity against human colorectal cancer cells (HCT-116), showing IC50 values of 41.6, 45.0, and 37.3 µM, respectively. Furthermore, compounds 3 and 4 significantly suppressed the invasion of HCT-116 cells while also downregulating the expression of vascular endothelial growth factor receptor 1 (VEGFR-1) and vimentin proteins, which are key markers associated with angiogenesis and epithelial-mesenchymal transition (EMT). Our findings suggest that compounds 3 and 4 may exert their anti-invasive effects on tumor cells by inhibiting the expression of VEGFR-1 and impeding the process of EMT.

Halenaquinol Blocks Staphylococcal Protein A Anchoring on Cell Wall Surface by Inhibiting Sortase A in Staphylococcus aureus.

Sortase A (SrtA) is a cysteine transpeptidase that binds to the periplasmic membrane and plays a crucial role in attaching surface proteins, including staphylococcal protein A (SpA), to the peptidoglycan cell wall. Six pentacyclic polyketides (1-6) were isolated from the marine sponge Xestospongia sp., and their structures were elucidated using spectroscopic techniques and by comparing them to previously reported data. Among them, halenaquinol (2) was found to be the most potent SrtA inhibitor, with an IC50 of 13.94 µM (4.66 µg/mL). Semi-quantitative reverse transcription PCR data suggest that halenaquinol does not inhibit the transcription of srtA and spA, while Western blot analysis and immunofluorescence microscopy images suggest that it blocks the cell wall surface anchoring of SpA by inhibiting the activity of SrtA. The onset and magnitude of the inhibition of SpA anchoring on the cell wall surface in S. aureus that has been treated with halenaquinol at a value 8× that of the IC50 of SrtA are comparable to those for an srtA-deletion mutant. These findings contribute to the understanding of the mechanism by which marine-derived pentacyclic polyketides inhibit SrtA, highlighting their potential as anti-infective agents targeting S. aureus virulence.

Identification of Axinellamines A and B as Anti-Tubercular Agents.

Tuberculosis remains a significant global health pandemic. There is an urgent need for new anti-tubercular agents to combat the rising incidence of drug resistance and to offer effective and additive therapeutic options. High-throughput screening of a subset of the NatureBank marine fraction library (n = 2000) identified a sample derived from an Australian marine sponge belonging to the order Haplosclerida that displayed promising anti-mycobacterial activity. Bioassay-guided fractionation of the organic extract from this Haplosclerida sponge led to the purification of previously identified antimicrobial pyrrole alkaloids, axinellamines A (1) and B (2). The axinellamine compounds were found to have a 90% minimum inhibitory concentration (MIC90) of 18 µM and 15 µM, respectively. The removal of protein and complex carbon sources reduced the MIC90 of 1 and 2 to 0.6 and 0.8 µM, respectively. The axinellamines were not toxic to mammalian cells at 25 µM and significantly reduced the intracellular bacterial load by >5-fold. These data demonstrate that axinellamines A and B are effective anti-tubercular agents and promising targets for future medicinal chemistry efforts.

Aurantoside L, a New Tetramic Acid Glycoside with Anti-Leishmanial Activity Isolated from the Marine Sponge Siliquariaspongia japonica.

A new tetramic acid glycoside, aurantoside L (1), was isolated from the sponge Siliquariaspongia japonica collected at Tsushima Is., Nagasaki Prefecture, Japan. The structure of aurantoside L (1) composed of a tetramic acid bearing a chlorinated polyene system and a trisaccharide part was elucidated using spectral analysis. Aurantoside L (1) showed anti-parasitic activity against L. amazonensis with an IC50 value of 0.74 µM.

Hydroxytakakiamide and Other Constituents from a Marine Sponge-Associated Fungus Aspergillus fischeri MMERU23, and Antinociceptive Activity of Ergosterol Acetate, Acetylaszonalenin and Helvolic Acid.

An unreported prenylated indole derivative hydroxytakakiamide (4) was isolated, together with the previously described ergosterol (1), ergosterol acetate (2), and (3R)-3-(1H-indol-3-ylmethyl)-3, 4-dihydro-1H-1,4-benzodiazepine-2,5-dione (3), from the column fractions of the crude ethyl acetate extract of the culture of a marine sponge-associated fungus, Aspergillus fischeri MMERU 23. The structure of 4 was elucidated by the interpretation of 1D and 2D NMR spectral data and high-resolution mass spectrum. The absolute configuration of the stereogenic carbon in 3 was proposed to be the same as those of the co-occurring congeners on the basis of their biogenetic consideration and was supported by the comparison of its sign of optical rotation with those of its steroisomers. The crude ethyl acetate extract and 2 were evaluated, together with acetylaszonalenin (5) and helvolic acid (6), which were previously isolated from the same extract, for the in vivo antinociceptive activity in the mice model. The crude ethyl acetate extract exhibited antinociceptive activity in the acetic acid-induced writhing and formalin tests, while 2, 5, and 6 displayed the effects in the late phase of the formalin test. On the other hand, neither the crude ethyl acetate extract nor 2, 5, and 6 affected the motor performance of mice in both open-field and rotarod tests. Additionally, docking studies of 2, 5, and 6 were performed with 5-lipoxygenase (5-LOX) and phosphodiesterase (PDE) enzymes, PDE4 and PDE7, which are directly related to pain and inflammatory processes. Molecular docking showed that 6 has low affinity energy to PDE4 and PDE7 targets while retaining high affinity to 5-LOX. On the other hand, while 2 did not display any hydrogen bond interactions in any of its complexes, it achieved overall better energy values than 6 on the three antinociceptive targets. On the other hand, 5 has the best energy profile of all the docked compounds and was able to reproduce the crystallographic interactions of the 5-LOX complex.

Irciniaplysins A-D: New Psammaplysin Derivatives from Philippine Marine Sponge Ircinia sp.

Four new psammaplysin derivatives (1-4) with fatty acyl substituents, designated irciniaplysins A-D, and three known psammaplysins (5-7) were isolated from a marine sponge Ircinia sp. Their structures were elucidated using extensive spectroscopic analyses. The positions of the double bonds and the branch points of the fatty acyl side chains were determined by GC-MS analysis of their fatty acid methyl ester (FAME) derivatives. Irciniaplysins A (1) and B (2) contained an unusual long-chain fatty acyl substituent with a 5,9-diene unit. The isolated compounds were evaluated for their cytotoxic activity against the human colorectal carcinoma (HCT 116) cells, however, none of these compounds showed significant activity.

Marcytoglobosins A and B, Cytochalasans From a Marine Sponge Associated Chaetomium globosum 162105 Fungus.

Two new cytochalasans, marcytoglobosins A (1) and B (2) were isolated from the marine sponge associated fungus Chaetomium globosum 162105, along with six known compounds (3-8). The complete structures of two new compounds were determined based on 1D/2D NMR and HR-MS spectroscopic analyses coupled with ECD calculations. All eight isolates were evaluated for their antibacterial activity. Among them, compounds 3-8 displayed antibacterial effects against Staphylococcus epidermidis, Bacillus thuringiensis, Pseudomonas syringae pv. Actinidiae, Vibrio alginolyticus, and Edwardsiella piscicida with minimum inhibitory concentration (MIC) values ranging from 10 to 25 µg/mL.

Two New Isocoumarins Isolated from the Marine-Sponge-Derived Fungus Setosphaeria sp. SCSIO41009.

Two new dihydroisocoumarins, exserolides L and M (1 and 2), along with six known compounds (3-8) were isolated from the extract of the marine-sponge-derived fungus Setosphaeria sp. SCSIO41009. Their structures were established by spectroscopic analyses. The absolute configurations of two new compounds were determined by modified Mosher's method and ECD data. Compounds 1 and 4 showed significant antiviral activities against A/Puerto Rico/8/34 H274Y (H1 N1) with IC50 values of 4.07±0.76 µM and 20.06±4.85 µM, respectively.

One New Quinazoline-Containing Diketopiperazine Isolated from the Marine-Sponge-Derived Fungus Penicillium sp. SCSIO41043.

One new quinazoline-containing diketopiperazine (1), along with 24 known compounds including nine alkaloids (2-9, and 25), thirteen lactones (10-22), aspterric acid (23), and catechol (24), were isolated from the marine sponge-derived Penicillium sp. SCSIO41043. Their planar structures were unequivocally elucidated by spectroscopic analysis. The absolute configuration of 1 was determined by a comparison of reported and experimental electronic circular dichroism (ECD) spectra. Compound 16 was found to notably inhibit the growth of five pathogenic bacteria and fungi with MIC values ranging from 0.5-16.0 µg/mL. Compounds 7, 17, 20, and 22 demonstrated moderate activity against Micrococcus luteus with MIC values ranging from 35.6 to 71.1 µg/mL. Moreover, 1-3 displayed different degrees of antioxidant activity with EC50 values of 0.98, 0.60, 0.46 mg/mL, respectively.

A new 3,4-dinorsteroid from the marine sponge Cliona sp.

A new steroid, 2a-oxa-2-oxo-5ß-hydroxy-3,4-dinor-24-methylcholesta-22E-ene (1), together with 10 known ones (2-11), was isolated from the marine sponge Cliona sp. The structures of these compounds were determined by the spectroscopic methods (UV, IR, MS, and NMR) and X-ray diffraction analysis. Compound 1 was the third example of 3,4-dinorsteroid with a hemiketal at C-5 that was isolated from the natural source. In addition, the antibacterial activities of these compounds were also evaluated. However, none of them exhibited significant inhibition effects.

Qipengyuania spongiae sp. nov., isolated from marine sponge Cinachyrella kuekenthali.

A novel bacterial strain, designated as PHS-Z21T, was isolated from the marine sponge Cinachyrella kuekenthali collected from PG Dave's Rock, Philippines. Cells of PHS-Z21T are Gram-stain-negative, non-motile, pale-yellow-pigmented, short rods. PHS-Z21T is able to grow at 10-40 ℃ (optimum, 30 ℃), pH 5.5-9.0 (optimum, pH 8.5) and with 3-9 % (w/v) NaCl (optimum, 4 %). Its 16S rRNA gene sequence shows 98.6 % similarity to Qipengyuania nanhaisediminis CGMCC 1.7715T, 98.5 % similarity to Qipengyuania vulgaris 022-2-10T and 98.4 % similarity to Qipengyuania flava SW-46T, respectively. The phylogenetic tree based on 16S rRNA gene sequences reveals that PHS-Z21T is clustered with Q. flava SW-46T. The total genome of PHS-Z21T is approximately 2 932 896 bp in size with a DNA G+C content of 64.7 %. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values among PHS-Z21T and other type strains are 70.0-77.3 % (ANIb), 83.3-86.8 % (ANIm) and 13.0-26.9 % (dDDH), respectively. The dDDH and ANI values are below the standard cutoff criteria for delineating bacterial species. Percentage of conserved proteins (POCP) values between the genome of strain PHS-Z21T and those of members of the genera Qipengyuania, Erythrobacter, Altererythrobacter and Alteriqipengyuania were 62.0-74.5 %, 55.8-63.2 %, 60.7-66.9 % and 63.9-66.8%, respectively, while the AAI values were 68.4-74.3 %, 63.8-65.9 %, 66.3-68.3 % and 64.7-66.9%, respectively. The major fatty acids of PHS-Z21T are composed of summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c), C18 : 1ω7c 11-methyl, C16 : 0 and summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c). The polar lipids of PHS-Z21T mainly consist of diphosphatidylglycerol, glycolipid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol and glycophospholipid. The respiratory lipoquinone was identified as Q-10. On the basis of the phenotypic and phylogenetic data, strain PHS-Z21T represents a novel species of the genus Qipengyuania, for which the name Qipengyuania spongiae sp. nov. is proposed. The type strain is PHS-Z21T (=MCCC 1K07849T=KCTC 92590T).

Brevibacterium spongiae sp. nov., isolated from marine sponge Hymeniacidon sp.

A novel bacterial strain, designated as WHS-Z9T, was isolated from marine sponge Hymeniacidon sp. collected from Weihai (37° 25' N, 121° 58' E), Shandong Province, PR China. Cells of strain WHS-Z9T were Gram-stain-positive, non-spore-forming, non-motile, short-rod-shaped and light yellow-pigmented. The strain could grow at 10-40 °C (optimum, 20 °C), pH 4.5-9.5 (optimum, pH 8.5) and 2-14 % (w/v) NaCl (optimum, 4 %). The 16S rRNA gene sequence of strain WHS-Z9T showed 98.7  % similarity to that of Brevibacterium epidermidis NBRC 14811T, 98.5  % to Brevibacterium sediminis FXJ8.269T and 98.4 % to Brevibacterium oceani BBH7T. The phylogenetic tree based on 16S rRNA gene sequences revealed that strain WHS-Z9T was clustered with Brevibacterium limosum o2T. The whole genome of WHS-Z9T was approximately 4 217 721 bp in size with a G+C content of 65.2  %. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values among WHS-Z9T and other Brevibacterium type strains were 83.3-85.5 % (ANI based on blast), 86.4-87.9  % (ANI based on MUMmer) and 41.9-57.5 % (dDDH). Percentage of conserved protein values between the genomes of strain WHS-Z9T and members of genera Brevibacterium were 76.8-82.9 %, while the average amino acid identity (AAI) values were 83.7-87.0  %. The dDDH, ANI, AAI and POCP values were below the standard cut-off criteria for the delineation of bacterial species. The sole respiratory quinone in strain WHS-Z9T was MK-8(H2), and the predominant fatty acids were anteiso-C15 : 0 and anteiso-C17 : 0. The major polar lipids of WHS-Z9T consisted of diphosphatidylglycerol and glycolipid. The diagnostic cell-wall diamino acid of strain WHS-Z9T was meso-diaminopimelic acid. Based on the data obtained in this study, strain WHS-Z9T (=MCCC 1K07845T=KCTC 49848T) should be classified as the type strain of a novel species of the genus Brevibacterium, for which the name Brevibacterium spongiae sp. nov. is proposed.

Muricauda myxillae sp. nov., isolated from a marine sponge (Myxilla rosacea), and reclassification of Flagellimonas hymeniacidonis as Muricauda symbiotica nom. nov.

A Gram-stain-negative, aerobic and non-motile rods strain, designated as strain 2012CJ39-3T, was isolated from a marine sponge, Myxilla rosacea, collected on Chuja Island, Republic of Korea. Optimal growth of strain 2012CJ39-3T was observed at 25 °C, pH 7.0 and in the presence of 2.0-3.0 % (w/v) NaCl. Strain 2012CJ39-3T contained menaquinone-6 as the respiratory quinone, iso-C15 : 1 G, iso-C15 : 0, and iso-C17 : 0 3-OH as the predominant fatty acids, and phosphatidylethanolamine, an unidentified phospholipid, an unidentified glycolipid, three unidentified aminolipids and nine unidentified lipids as major polar lipids. The genomic DNA G+C content was 38.4 mol%. Results of phylogenetic analyses based on the 16S rRNA gene and whole-genome sequences revealed that strain 2012CJ39-3T formed a distinct phyletic lineage in the genus Muricauda. Strain 2012CJ39-3T was most closely related to Flagellimonas hymeniacidonis 176CP5-101T, Muricauda spongiicola 2012CJ35-5T, Muricauda algicola AsT0115T, Muricauda flava DSM 22638T and Muricauda parva SW169T with 96.5, 96.4, 96.3, 95.8 and 95.6 % 16S rRNA gene sequence similarity, respectively. The average nucleotide identity and digital DNA-DNA hybridization values between strain 2012CJ39-3T and M. spongiicola 2012CJ35-5T, F. hymeniacidonis 176CP5-101T, M. algicola AsT0115T, M. flava DSM 22638T and M. parva SW169T were 75.6, 74.2, 78.6, 75.3 and 74.8 % and 27.4, 19.9, 36.3, 24.2 and 18.9 %, respectively. Based on these results, strain 2012CJ39-3T represents a novel species of the genus Muricauda, for which the name Muricauda myxillae sp. nov. is proposed. The type strain is 2012CJ39-3T (=KACC 22644T= LMG 32582T). In addition, Flagellimonas hymeniacidonis is reclassified as Muricauda symbiotica nom. nov.