Vallitalea longa sp. nov., an anaerobic bacterium isolated from marine sediment.

A novel bacterium, strain SH18-1T, was isolated from marine sediment collected near Sado Island in the Sea of Japan. This strain was strictly anaerobic, Gram-stain-negative, non-spore-forming, rod-shaped, motile, and mesophilic. It grew at 15-40 °C (optimum, 30-35 °C), at a NaCl concentration of 0.2-5.0 % (w/v; optimum, 1.5-2.5 %), and at pH 5.5-8.5 (optimum, pH 7.0). Results of 16S rRNA gene phylogenetic analysis showed a similarity value of 97.49 % between strain SH18-1T and Vallitalea guaymasensis Ra1766G1T, which was the most closely related species. The genome size of strain SH18-1T was 5.71 Mb and its G+C content was 30.2 mol%. Genome sequence analyses for comparison between strain SH18-1T and V. guaymasensis Ra1766G1T showed values lower than the threshold for species demarcation determined using the Genome-to-Genome Distance Calculator and the Average Nucleotide Identity Calculator. Elemental sulphur, sulphate, thiosulphate, sulphite, fumarate, nitrate, and nitrite were not used as terminal electron acceptors. The major fatty acids in strain SH18-1T were iso-C15 : 0, anteiso-C15 : 0, and C16 : 0, and the detected polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphoglycolipid, glycolipid, three unidentified phospholipids, and one unidentified polar lipid. From these results, strain SH18-1T (=NBRC 115488T=DSM 114058T) is suggested to represent a novel species of the genus Vallitalea and the name Vallitalea longa sp. nov. is proposed.

Sinisalibacter aestuarii sp. nov., isolated from estuarine sediment of the Arakawa River.

A Gram-stain-negative, rod-shaped, non-motile and strictly aerobic bacterium, which showed biofilm-forming ability on polystyrene, designated as strain B-399T, was isolated from the estuarine sediment of the Arakawa River near Tokyo Bay. It grew at pH 6.0-8.5, at 15-35 °C and in the presence of 0-7.5 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain B-399T was clustered in the genus Sinisalibacter and has 96.94 % sequence similarity to Sinisalibacter lacisalsi X12M-4T, which was the only validly described species in this genus. On the basis of our genome sequencing analyses, the average nucleotide identity and digital DNA-DNA hybridization values between strains B-399T and S. lacisalsi X12M-4T were 79.54 and 22.30 %, respectively, which confirms that strain B-399T represents a novel species of the genus Sinisalibacter. The draft genome size and the DNA G+C content of strain B-399T were 4.12 Mb and 65.2 mol%, respectively. The major fatty acids (>10 %) of strain B-399T were C16 : 0, summed feature 8 (C18 : 1 ω6c and/or C18 : 1 ω7c) and C19 : 0 cyclo ω8c. The polar lipids were phosphatidylcholine, phosphatidylglycerol, an unidentified phospholipid, an unidentified aminolipid and unidentified lipids. The respiratory quinone was Q-10. These chemotaxonomic features were almost coincident with those of the genus Sinisalibacter. Therefore, strain B-399T should be classified as representing a new species of the genus Sinisalibacter, for which the name Sinisalibacter aestuarii sp. nov. is proposed. The type strain is B-399T (=NBRC 115629T=DSM 114148T).

Distribution of class IId bacteriocin-producing Virgibacillus salexigens in various environments.

We performed several experiments using three strains of Virgibacillus salexigens, namely, P2, NT N53, and C-20MoT (DSM 11483T), which were isolated from completely different sources, in relation to bacteriocin production ability. Results of whole-genome sequencing analysis revealed that all strains have very similar sequences encoding class IId bacteriocin. Although a partial amino acid sequence of the purified bacteriocin produced by strain P2 isolated from fermented food was previously reported, whole-genome sequencing and the N-terminal sequencing results in this study showed that its complete amino acid sequence consisted of 48 residues, which corresponded to that of the hypothetical bacteriocin encoded by the gene in Virgibacillus massiliensis strain Vm-5T (DSM 28587T) isolated from the human gut. From the results of 16S rRNA gene sequencing and whole-genome sequencing analyses, we taxonomically confirmed Vm-5T to be a strain of V. salexigens, and its broth culture showed antibacterial activity. Strain NT N53 isolated from the deep-sea floor produced two bacteriocins, namely, NTN-A and NTN-B. The results of N-terminal sequencing, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and whole-genome sequencing analyses showed that their amino acid sequences differed in only one residue, and NTN-A showed the same sequence as the bacteriocin produced by strain P2. Although strain C-20MoT isolated from a solar saltern had the coding sequence very similar to that of NTN-A, its broth culture showed no antibacterial activity. This finding suggests that class IId bacteriocin-producing or bacteriocin-gene-encoding V. salexigens strains are widely distributed in distinct environment sources with different geographical and material properties.

Streptomyces otsuchiensis sp. nov., a biosurfactant-producing actinobacterium isolated from marine sediment.

A novel actinobacterium producing biosurfactant, designated OTB305T, was isolated from marine sediment sampled at Otsuchi Bay, Iwate Prefecture, Japan and its taxonomic position was examined using a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequences exhibited that strain OTB305T was closely related to Streptomyces bohaiensis JCM 19630T (98.8 %) and Streptomyces lonarensis DSM 42084T (98.8 %). The chemotaxonomic characteristics of strain OTB305T corresponded to those of the genus Streptomyces as follows: the diamino acid of the cell-wall peptidoglycan was ll-diaminopimelic acid; whole-cell hydrolysates contained glucose and lacked characteristic major sugars; the predominant isoprenoid quinones were MK-9(H8) and MK-9(H6); the polar lipids were phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and an unidentified phospholipid; the major cellular fatty acids were iso-C16 : 0, C16 : 0 and C16 : 1 ω7c; and the genomic DNA G+C content was 72.83 mol%. However, genomic relatedness analysis based on the average nucleotide identity and some phenotypic characteristics revealed that strain OTB305T was distinguished from closely related Streptomyces species. Therefore, strain OTB305T represents a novel species of the genus Streptomyces, for which the name Streptomyces otsuchiensis sp. nov. is proposed. The type strain is OTB305T (=NBRC 113255T=TBRC 9682T).

Lysinimicrobium sediminis sp. nov., an actinobacterium isolated from estuary sediment.

A novel Gram-stain-positive actinobacterium, designated HT7-17T, was isolated from a sediment sample collected from the estuary of the Tama River, Japan, and its taxonomic position was investigated using a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequence comparisons revealed that strain HT7-17T was closely related to members of the genus Lysinimicrobium, with a similarity range of 97.1-98.2 %. The peptidoglycan type of strain HT7-17T was A4α, the predominant menaquinone was demethylmenaquinone DMK-9(H4) and the major fatty acids were anteiso-C15 : 0, C16 : 0 and iso-C16 : 0. The DNA G+C content was 69.9 mol%. These chemotaxonomic features corresponded to those of the genus Lysinimicrobium. Meanwhile, the differences in some phenotypic characteristics, along with the result of DNA-DNA hybridization, indicated that strain HT7-17T should be distinguished from the recognized species of the genus Lysinimicrobium. Therefore, strain HT7-17T represents a novel species of the genus Lysinimicrobium, for which the name Lysinimicrobium sediminis sp. nov. is proposed. The type strain is HT7-17T (=NBRC 112286T=TBRC 7037T).

Diversity of the bacterial community in Myanmar traditional salted fish yegyo ngapi.

The distribution and characterization of bacteria including lactic acid bacteria (LAB) in the traditional and popular salted fish yegyo ngapi in Myanmar were studied to clarify the contribution of these bacteria to the curing and ripening of this product. Samples of yegyo ngapi purchased from a market in Yangon were used. Most of the isolates obtained using de Man, Rogosa and Sharpe medium containing 10 % NaCl were identified as coccoid LAB on the basis of their basic phenotypic characteristics. From the results of 16S rRNA gene sequencing and PCR-restriction fragment length polymorphism analysis of this gene, most of the isolates were identified as the halophilic LAB Tetragenococcus muriaticus. Analyses of the 16S rRNA gene based on the clone library using DNA extracted from salted fish products were also performed. The results of these molecular-analysis-based techniques showed that spore-forming and non-spore-forming anaerobic bacteria including the genera Clostridium and Halanaerobium in addition to T. muriaticus were also frequently found in bacterial communities. These findings suggest that the anaerobic condition during curing and ripening resulted in bacterial communities composed of strictly anaerobic bacteria and halophilic LAB, and that these bacteria might also contribute to the manufacturing processes of this product. In addition, DNA sequences similar to that of Clostridium botulinum were found in the clone library analysis. Therefore, despite no reports of botulism poisoning from the region where the samples were taken, closer surveillance should be carried out from the viewpoint of food safety.

Production of bacteriocin by Virgibacillus salexigens isolated from "terasi": a traditionally fermented shrimp paste in Indonesia.

A natural antibacterial-substance-producing gram-positive bacterium was isolated from terasi shrimp paste, a popular fermented product in Indonesia. This strain, a spore-forming and strictly aerobic bacterium, was identified as Virgibacillus salexigens by 16S rRNA gene sequence analysis. The antibacterial substance purified from the precipitated product in the culture supernatant of the strain using ammonium sulfate showed a broad inhibition spectrum against gram-positive bacteria, including a typical foodborne bacterium, namely, Listeria monocytogenes. The antibacterial activity of the substance was inactivated by treatments with various proteolytic enzymes. It was stable after heating or pH treatment, and approximately 60% of the initial activity remained even after heating at 121 °C for 15 min. In addition, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis indicated that its monoisotopic mass weight was 5318.4 Da (M+H)(+). On the basis of the results obtained by the automated Edman degradation technique and MALDI-TOF MS analysis, the substance can be classified as a member of Class IId bacteriocins, but it could not be identified as any of the previously purified substances except for the putative bacteriocin predicted from the draft genome sequence data of gram-positive bacteria such as Virgibacillus and Bacillus strains.

Hyaluromycin, a new hyaluronidase inhibitor of polyketide origin from marine Streptomyces sp.

Hyaluromycin (1), a new member of the rubromycin family of antibiotics, was isolated from the culture extract of a marine-derived Streptomyces sp. as a HAase inhibitor on the basis of HAase activity screening. The structure of 1 was elucidated through the interpretation of NMR data for the compound and its 3″-O-methyl derivative in combination with an incorporation experiment with [1,2-13C2]acetate. The compound's absolute configuration was determined by the comparison of its circular dichroism (CD) spectrum with those of other rubromycins. Hyaluromycin (1) consists of a γ-rubromycin core structure possessing a 2-amino-3-hydroxycyclopent-2-enone (C5N) unit as an amide substituent of the carboxyl function; both structural units have been reported only from actinomycetes. Hyaluromycin (1) displayed approximately 25-fold more potent hyaluronidase inhibitory activity against hyaluronidase than did glycyrrhizin, a known inhibitor of plant origin.

New polycyclic tetramate macrolactams with antimycobacterial activity produced by marine-derived Streptomyces sp. KKMA-0239.

During our screening for anti-mycobacterial agents against Mycobacterium avium complex (MAC), two new polycyclic tetramate macrolactams (PTMs), named hydroxycapsimycin (1) and brokamycin (2), were isolated along with the known PTM, ikarugamycin (3), from the culture broth of marine-derived Streptomyces sp. KKMA-0239. The relative structures of 1 and 2 were elucidated by spectroscopic data analyses, including 1D and 2D NMR. Furthermore, the absolute configuration of 1 was confirmed by a single-crystal X-ray diffraction analysis. Compounds 2 and 3 exhibited moderate antimycobacterial activities against MAC, including clinically isolated drug-resistant M. avium.

An effective method based on wet-heat treatment for the selective isolation of Micromonospora from estuarine sediments.

Several methods for the isolation of Micromonospora from soil samples have been developed; however, it is unclear whether these methods are optimal for estuarine samples. In this study, we optimized the conditions of a wet-heat method for the selective isolation of Micromonospora from estuarine sediments. Sediments were collected from the Arakawa River (estuarine sediments) and Tokyo Bay (marine sediments). Sediment samples were wet-heated at 45, 55, or 65 °C for 30 min and then incubated at 27 °C for 3 weeks. After incubation, most of the actinomycete colonies were macroscopically determined to be of the genus Micromonospora or Streptomyces. In contrast to the treatment at 55 °C, treatment at 65 °C drastically reduced the number of Streptomyces colonies but increased the number of Micromonospora colonies from the estuarine sediments. This procedure allowed us to grow cultures that were composed of more than 90 % Micromonospora. In addition, treatment at 65 °C did not affect the diversity of Micromonospora species compared with treatment at 55 °C. These results indicate that the wet-heat method, which involves pre-treating the sediment at 65 °C for 30 min, is a very simple and effective method for the selective enrichment of a large number of diverse Micromonospora from estuarine sediments. Our results may lead to the isolation of new Micromonospora species, which produce novel bioactive compounds, from different estuarine sediments.

Microplastic pollution in the gastrointestinal tract of giant river catfish Sperata seenghala (Sykes, 1839) from the Meghna River, Bangladesh.

Freshwater rivers are considered the major route for microplastics (MPs), yet limited studies have been reported on MPs in freshwater river fish, especially in Bangladesh. This research reveals the intake of MPs by the giant river catfish Sperata seenghala, collected from the Meghna River, which is the only outlet of the Ganges-Brahmaputra River. Three locations, namely, Chandpur Sadar, Bhola Sadar, and Char Fasson, along the Meghna River, were selected in order to investigate the gastrointestinal tracts (GIT) of the fish. Ninety percent (n=27) of fish (n=30) were contaminated, with fragment-shaped MPs (65%) as the most abundant among the four types. A total of 179 MP particles were detected using micro-Fourier transformed infrared spectroscopy (µ-FTIR), with an average of 5.96 ± 1.32 MP particles per fish. Among the four size groups, the highest proportion of MPs (54%) occurred in the 45-100 µm group; the dominant color among the seven color groups was white (30%). The highest quantity of MPs was found in the relatively densely populated Chandpur Sadar region. Polypropylene-polyethylene copolymer (PP-PE, 23%) was proportionally dominant among the 15 types. No significant relationship was found between the total number of observed MPs and the GIT weight. This study will help us to understand MP pollution in S. seenghala that may transmit to the human body through the food chain.

Haneummycin, a new 22-membered macrolide lactam antibiotic, produced by marine-derived Streptomyces sp. KM77-8.

A new antibiotic named haneummycin (1) was isolated from a culture broth of marine-derived Streptomyces sp. KM77-8 by solvent extraction and HPLC using a C4 column. The structure of 1 was elucidated including relative stereochemistry as a new 22-membered macrolide lactam associated with a cyclopentanone and three sugars by various spectroscopic analyses, such as MS and NMR. Compound 1 displayed significant antibacterial activities against Gram-positive bacteria including vancomycin-resistant Enterococcus faecium (VRE) and methicillin-resistant Staphylococcus aureus (MRSA) with both MIC values of 8.0 µg ml-1.

New dihydronaphthothiophene derivatives by the biological transformation of seriniquinone using marine-derived actinomycete Streptomyces albogriseolus OM27-12.

Seriniquinone was originally isolated as a melanoma-selective anti-cancer agent from a culture broth of marine bacteria. Pharmacological studies on its selectivity and unique target are ongoing. A new dihydronaphthothiophene (1) was synthesized by the biological transformation of seriniquinone using marine-derived actinomycete Streptomyces albogriseolus OM27-12, and its derivatives (2-4) were chemically synthesized. Compounds 1-4 exhibited selective cytotoxic activity against melanoma and improved solubility.

Development of an in vivo-mimic silkworm infection model with Mycobacterium avium complex.

In vivo-mimic silkworm infection models with Mycobacterium avium and Mycobacterium intracellulare were newly established to evaluate the therapeutic effects of anti-M. avium complex (MAC) antibiotics. Silkworms raised at 37°C died within 72 hours of an injection of M. avium or M.intracellulare (2.5 × 107 colony-forming unit (CFU)/larva·g) into the hemolymph. Clinical anti-mycobacterial (tuberculosis) antibiotics were evaluated under these conditions. Clarithromycin, kanamycin, streptomycin, amikacin, and ciprofloxacin exerted therapeutic effects in a dose-dependent manner, which was consistent with those in the mouse model. Furthermore, three effective actinomycete culture broths were selected in the screening program of our microbial broth library using the silkworm model, and four active metabolites, ohmyungsamycins A and B (1 and 2), chartreusin (3), and griseoviridin (4), were identified. Among these compounds, 1 showed the lowest 50% effective dose (ED50) value (8.5 µg/larva·g), while 3 had the best ED50/minimum inhibitory concentration (MIC) ratio (7.4). These results indicate that silkworm models are a useful tool for identifying anti-MAC antibiotics candidates with veritable therapeutic effects.

Diversity of salt-tolerant tellurate-reducing bacteria in a marine environment.

Tellurium (Te) has been increasingly used as a semiconductor material in copious amounts, with a concomitant increase in its discharge from industrial effluents and mining wastewater into the environment. However, soluble Te, such as tellurate (VI) and tellurite (IV), is toxic to organisms. Thus, highly efficient technologies need to be developed for a double-benefit detoxification and recovery of soluble Te from industrial and mining wastewater. Since industrial wastewater contains high concentrations of salt, salt-tolerant microorganisms that metabolize rare metals such as Te have been the subject of focus for the effective detoxification and recovery of Te. In the present study, a total of 52 salt-tolerant tellurate-reducing microorganisms were isolated from marine environmental samples. Of these, 18 strains achieved greater than, or equal to, 50% removal of water-soluble Te from a medium containing 0.4 mM tellurate after 72 h incubation. The 18 isolated strains belonged to 13 species of the following 9 genera: Sulfitobacter, Ruegeria, Hoeflea, Alteromonas, Marinobacter, Pseudoalteromonas, Shewanella, Idiomarina, and Vibrio. No microorganism has been reported to reduce tellurate and tellurite from six of the aforementioned genera, namely, Sulfitobacter, Ruegeria, Alteromonas, Marinobacter, Idiomarina, and Vibrio. Especially, one of the isolates Sulfitobacter sp. strain TK39B, removed 82% (w/w) of soluble Te with a 4% NaCl tolerance. These results showed that salt-tolerant tellurate-reducing bacteria that can be used in the detoxification and recovery of Te are widely present in the marine environment.

Akazamicin, a cytotoxic aromatic polyketide from marine-derived Nonomuraea sp.

In our screening program on marine-derived actinomycetes, Nonomuraea sp. AKA32 isolated from deep-sea water collected from a depth of 800 m in Sagami Bay, Japan was found to produce compounds cytotoxic to cancer cells. Activity-guided purification led to the isolation of a new aromatic polyketide, akazamicin (1), along with two known compounds, actinofuranone C (2) and N-formylanthranilic acid (3). Structures of these compounds were elucidated through the interpretation of NMR and MS spectroscopic data. Compounds 1, 2, and 3 displayed cytotoxicity against murine B16 melanoma cell line with the IC50 value of 1.7, 1.2, and 25 µM, respectively.

Retrieval of entire genes from environmental DNA by inverse PCR with pre-amplification of target genes using primers containing locked nucleic acids.

We had been unsuccessful to amplify desired nucleotide sequences from various environmental DNA samples by using the inverse polymerase chain reaction (IPCR) technique, most probably because the copy numbers of target DNA sequences had been quite low. To enrich the target DNA sequences prior to IPCR, a rolling-circle amplification was used with a site-specific primer containing locked nucleic acids (LNAs). This pre-amplified IPCR (PAI-PCR) method increased the sensitivity of PCR almost 10,000 times compared with the standard IPCR in model experiments using Escherichia coli. We then applied the PAI-PCR method to isolate glycosyl hydrolase genes from DNAs extracted from vermiform appendixes of horses and termite guts. The flanking sequences of the target genes were amplified and cloned successfully using PAI-PCR, whereas standard IPCR resulted in no amplification.

Long-term monitoring of the succession of a microbial community in activated sludge from a circulation flush toilet as a closed system.

The microbial diversity and community succession of a circulation flush toilet were investigated by terminal restriction fragment length polymorphism and cloning analyses. Clonal libraries of 16S rRNA gene on day 3 and day 127 were constructed. On day 3, 102 clones were sequenced; Proteobacteria and Bacteroidetes accounted for 27% and 45%, respectively. On day 127, Proteobacteria had increased to 43% and Bacteroidetes had decreased to 26% of a total of 100 clones. Terminal restriction fragment length polymorphism peaks were identified by in silico analysis of clone libraries. The relative abundances of Nitrosomonas increased from 1% to 6% with commencement of nitrification and denitrification. Similarly, the relative abundance of terminal restriction fragments generated from Xanthomonas increased from 3% to 10%. Therefore, these bacteria could play a prominent role in this process. To reveal the relationship between stability of the microbial community and performance of the system, microbial community succession was visualized by multidimensional scaling analysis. The microbial community structure changed markedly, particularly during the start-up period of the system. The plots then became stable after the start of nitrification and denitrification. This result suggests that the succession of microbial community structure had a correlation with the performance of the system.

Bacterial community structures of deep-sea water investigated by molecular biological techniques.

The aim of the present study was to investigate the bacterial community structures of deep-sea water (DSW) and surface seawater (SSW) samples in Japan by molecular biological techniques. DGGE analyses and pyrosequencing analysis revealed that bacterial community structures of DSW were diverse and differed from those of SSW. This is the first report on the horizontal variation of bacterial community structures of DSW throughout Japan. In addition, pyrosequencing analysis revealed that the number of phyla in DSW was larger than that in SSW, and specific phyla, such as Firmicutes and Planctomycetes, were characterized by a higher proportion of the bacterial community structure in DSW than in SSW. Taken together, these results indicate that a variety of bacteria that are specifically adapted to the DSW environments can be expected to be found in DSW, and DSW would thus be a potential resource for novel or unique microorganisms and compounds.

7-Chlorofolipastatin, an inhibitor of sterol O-acyltransferase, produced by marine-derived Aspergillus ungui NKH-007.

A new depsidone, named 7-chlorofolipastatin, and five known structurally related depsidones were isolated from the culture broth of the marine-derived fungus Aspergillus ungui NKM-007 by solvent extraction and HPLC using an octadecylsilyl column. The structure of 7-chlorofolipastatin was elucidated by various spectroscopic data including 1D and 2D NMR spectroscopy. 7-Chlorofolipastatin inhibited sterol O-acyltransferase (SOAT) 1 and 2 isozymes in cell-based and enzyme assays using SOAT1- and SOAT2-expressing Chinese hamster ovary (CHO) cells.