Amycolapeptins A and B, Cyclic Nonadepsipeptides Produced by Combined-culture of Amycolatopsis sp. and Tsukamurella pulmonis.

Two nonapeptide natural products, amycolapeptins A (1) and B (2) with a 22-membered cyclic depsipeptide skeleton, ß-hydroxytyrosine, and a highly modified side chain, which were not produced in a monoculture of the rare actinomycete Amycolatopsis sp. 26-4, were discovered in broth of its combined-culture with Tsukamurella pulmonis TP-B0596. The planar structures were elucidated by spectroscopic analyses (extensive 2D-NMR and MALDI-TOF MS/MS). The absolute configurations of component amino acids were unambiguously determined by the highly sensitive advanced Marfey's method we recently developed. Additionally, the structures of unstable/unusual moieties were corroborated by chemical synthesis and CD analysis.

Tripartite Symbiosis of an Anaerobic Scuticociliate with Two Hydrogenosome-Associated Endosymbionts, a Holospora-Related Alphaproteobacterium and a Methanogenic Archaeon.

A number of anaerobic ciliates, unicellular eukaryotes, intracellularly possess methanogenic archaea and bacteria as symbiotic partners. Although this tripartite relationship is of interest in terms of the fact that each participant is from a different domain, the difficulty in culture and maintenance of those host species with symbiotic partners has disturbed both ecological and functional studies so far. In this study, we obtained a stable culture of a small anaerobic scuticociliate, strain GW7. By transmission electron microscopic observation and fluorescent in situ hybridization with domain-specific probes, we demonstrate that GW7 possesses both archaeal and bacterial endosymbionts in its cytoplasm. These endosymbionts are in dependently associated with hydrogenosomes, which are organelle producing hydrogen and ATP under anaerobic conditions. Clone library analyses targeting prokaryotic 16S rRNA genes, fluorescent in situ hybridization with endosymbiont-specific probes, and molecular phylogenetic analyses revealed the phylogenetic affiliations and intracellular localizations of these endosymbionts. The endosymbiotic archaeon is a methanogen belonging to the genus Methanoregula (order Methanomicrobiales); a member of this genus has previously been described as the endosymbiont of an anaerobic ciliate from the genus Metopus (class Armophorea), which is only distantly related to strain GW7 (class Oligohymenophorea). The endosymbiotic bacterium belongs to the family Holosporaceae of the class Alphaproteobacteria, which also comprises several endosymbionts of various aerobic ciliates. For this endosymbiotic bacterium, we propose a novel candidate genus and species, "Candidatus Hydrogenosomobacter endosymbioticus."IMPORTANCE Tripartite symbioses between anaerobic ciliated protists and their intracellular archaeal and bacterial symbionts are not uncommon, but most reports have been based mainly on microscopic observations. Deeper insights into the function, ecology, and evolution of these fascinating symbioses involving partners from all three domains of life have been hampered by the difficulties of culturing anaerobic ciliates in the laboratory and the frequent loss of their prokaryotic partners during long-term cultivation. In the present study, we report the isolation of an anaerobic scuticociliate, strain GW7, which has been stably maintained in our laboratory for more than 3 years without losing either of its endosymbionts. Unexpectedly, molecular characterization of the endosymbionts revealed that the bacterial partner of GW7 is phylogenetically related to intranuclear endosymbionts of aerobic ciliates. This strain will enable future genomic, transcriptomic, and proteomic analyses of the interactions in this tripartite symbiosis and a comparison with endosymbioses in aerobic ciliates.

Population Connectivity in the Common Reef Zoantharian Zoanthus sansibaricus (Anthozoa: Hexacorallia) in Southern Japan.

Tropical and subtropical shallow benthic marine communities are highly diverse and balanced systems that constitute an important natural resource. Knowledge of the genetic diversity, connectivity and reproduction mode of each population is critical to understanding the fate of whole assemblages in times of disturbances. Importantly, the capability of populations to adapt to environmental challenges will be crucial to determining their survival. Here, we report on the population structure of the common reef zoantharian Zoanthus sansibaricus in the northwestern Pacific, by examining populations at three different locations in southern Japan using five highly variable microsatellite markers. Analyses of a population at the species' northern distribution limit combined with analyses of two subtropical populations suggest that habitat characteristics and ocean currents influence the connectivity and genetic diversity of this species. Our findings emphasize the adaptive ability of Z. sansibaricus to different environmental conditions and may help explain the wide distribution and generalist nature of this species.

A quantitative protocol for DNA metabarcoding of springtails (Collembola).

We developed a novel protocol with superior quantitative analysis results for DNA metabarcoding of Collembola, a major soil microarthropod order. Degenerate PCR primers were designed for conserved regions in the mitochondrial cytochrome c oxidase subunit I (mtCOI) and 16S ribosomal RNA (mt16S) genes based on published collembolan mitogenomes. The best primer pair was selected based on its ability to amplify each gene, irrespective of the species. DNA was extracted from 10 natural communities sampled in a temperate forest (with typically 25-30 collembolan species per 10 soil samples) and 10 mock communities (with seven cultured collembolan species). The two gene regions were then amplified using the selected primers, ligated with adapters for 454 technology, and sequenced. Examination of the natural community samples showed that 32 and 36 operational taxonomic units (defined at a 90% sequence similarity threshold) were recovered from the mtCOI and mt16S data, respectively, which were comparable to the results of the microscopic identification of 25 morphospecies. Further, sequence abundances for each collembolan species from the mtCOI and mt16S data of the mock communities, after normalization by using a species as the internal control, showed good correlation with the number of individuals in the samples (R = 0.91-0.99), although relative species abundances within a mock community sample estimated from sequences were skewed from community composition in terms of the number of individuals or biomass of the species. Thus, this protocol enables the comparison of collembolan communities in a quantitative manner by metabarcoding.

Precezomycin, a novel antibiotic biosynthetic precursor of cezomycin, from actinomycete Kitasatospora putterlickiae 10-13.

A novel antibiotic biosynthetic precursor of cezomycin, named precezomycin (1), was isolated from culture broth of actinomycete Kitasatospora putterlickiae 10-13. The planar structure was determined by 1D/2D NMR and HR(ESI)MS data analyses, and the absolute configurations were established by TDDFT calculation of ECD spectra. Precezomycin (1) exhibited moderate antibacterial activity against gram-positive bacteria including Staphylococcus aureus and Bacillus subtilis. The discovery of 1 extends the natural product family of cezomycin and provides a new insight into understanding the biosynthetic process of these polyether ionophore metabolites.

Amoxetamide A, a new anoikis inducer, produced by combined-culture of Amycolatopsis sp. and Tsukamurella pulmonis.

Cancer cells including colorectal cancer cells are resistant to anoikis, an anchorage-independent programmed death, which enables metastasis and subsequent survival in a new tumor microenvironment. In this study, we identified a new anoikis inducer, amoxetamide A (1) with a ß-lactone moiety, that was produced by combined-culture of Amycolatopsis sp. 26-4 and mycolic acid-containing bacteria (MACB) Tsukamurella pulmonis TP-B0596. The structure of 1 including the stereochemistry of C8 was determined by MS and NMR spectroscopy and modified Mosher's method, and the absolute configurations of C11 and C12 were suggested as 11R and 12S, respectively, by GIAO NMR calculations. Amoxetamide A (1) exhibited anoikis-inducing activity in human colorectal cancer HT-29 cells in anchorage-independent culture conditions.

New antimalarial iromycin analogs produced by Streptomyces sp. RBL-0292.

Two new antimalarial compounds, named prenylpyridones A (1) and B (2), were discovered from the actinomycete cultured material of Streptomyces sp. RBL-0292 isolated from the soil on Hamahiga Island in Okinawa prefecture. The structures of 1 and 2 were elucidated as new iromycin analogs having α-pyridone ring by MS and NMR analyses. Compounds 1 and 2 showed moderate in vitro antimalarial activity against chloroquine-sensitive and chloroquine-resistant Plasmodium falciparum strains, with IC50 values ranging from 80.7 to 106.7 µM.

Antibiotics production by an actinomycete isolated from the termite gut.

As well as the search for new antibiotics, a new resource or strains for the known antibiotics is also important. Microbial symbionts in the gut of termites could be regarded as one of the feasible resource for such purpose. In this study, antibiotic-producing actinomycetes were screened from symbionts of the termite gut. 16SrRNA sequence analysis for the 10 isolates revealed that they belong to actinomycetes such as Streptomyces sp., Kitasatospora sp., and Mycobacterium sp. A culture broth from one of the isolate, namely strain CA1, belonging to the genera Streptomyces exhibited antagonistic activity against actinomycetes (Micrococcus spp.), gram-positive bacteria (Bacillus spp.), and yeast (Candida spp.). The structures of 2 compounds isolated from the culture broth of the strain CA1 were identified as those of actinomycin X2 and its analog, D. This study is the first to report that some symbionts of the termite gut are antibiotic-producing actinomycetes, and suggest that the termite gut is a feasible resource for bioprospecting.

Complete Genome Sequence of "Candidatus Hydrogeosomobacter endosymbioticus," an Intracellular Bacterial Symbiont of the Anaerobic Ciliate Scuticociliate GW7.

The bacterium "Candidatus Hydrogenosomobacter endosymbioticus" is an intracellular symbiont of anaerobic scuticociliate GW7, which is associated with hydrogenosome together with methanogenic archaea. Here, we report a complete genome sequence of the symbiont consisting of 827 kbp. Knowing this sequence would contribute to the understanding of the metabolic interactions and evolution of the tripartite symbiosis.

Pseudoalteropeptide A, a novel lipopeptide from the marine bacterium Pseudoalteromonas piscicida SWA4_PA4 isolated from marine seaweed.

A new lipopeptide, pseudoalteropeptide A (1) was isolated from the marine bacterium Pseudoalteromonas piscicida SWA4_PA4. The structure was elucidated by spectroscopic analyses including NMR and MSMS spectra. It showed moderate iron chelating activity as well as cytotoxic activity against Jurkat human T lymphocyte cells. isolation/marine bacterium/natural product/structure elucidation.

Thioamycolamides A-E, Sulfur-Containing Cycliclipopeptides Produced by the Rare Actinomycete Amycolatopsis sp.

A series of novel sulfur-containing cycliclipopeptides named thioamycolamides A-E, with thiazoline, thioether rings, and fatty acid moieties, were identified from the culture broth of the rare actinomycete Amycolatopsis sp. 26-4. The planar structural elucidation was accomplished by HRMS and 1D/2D NMR spectroscopic data analyses. The absolute configurations were unambiguously determined by Marfey's method, CD spectroscopy, and synthesis of partial structures. Moreover, their growth inhibitory activities against human tumor cell lines were investigated.

Application of random amplified polymorphic DNA (RAPD) analysis coupled with microchip electrophoresis for high-resolution identification of Monascus strains.

Monascus fungi are commonly used for a variety of food products in Asia, and are also known to produce some biologically active compounds. Since the use of Monascus is expected to increase in food industries, strain-level identification and management of Monascus will be needed in the near future. In the present study, random amplified polymorphic DNA (RAPD) analysis coupled with microchip electrophoresis was applied for this purpose. Evaluations of the analysis stability revealed that reproducible results could be obtained, although template DNA fragmentation could influence the resulting RAPD pattern. RAPD analysis using 15 Monascus strains consisting of four species, M. ruber, M. pilosus, M. purpureus, and M. kaoliang showed that each strain generated a unique RAPD pattern, which allows strain-level identification of Monascus. In addition, the phylogenetic tree constructed from RAPD patterns reflected M. ruber-M. pilosus and M. purpureus-M. kaoliang clusters inferred from both ITS and beta-tubulin gene sequences, which indicated that the RAPD pattern could reflect their phylogenetic traits to a certain extent. On the other hand, RAPD analysis did not support the monophyletic clustering of the four Monascus species used in this study, which suggests the necessity of reexamination of species boundaries in Monascus.

Specific DNA binding of a potential transcriptional regulator, inosine 5'-monophosphate dehydrogenase-related protein VII, to the promoter region of a methyl coenzyme m reductase I-encoding operon retrieved from Methanothermobacter thermautotrophicus strain DeltaH.

Two methyl coenzyme M reductases (MCRs) encoded by the mcr and mrt operons of the hydrogenotrophic methanogen Methanothermobacter thermautotrophicus DeltaH are expressed in response to H(2) availability. In the present study, cis elements and trans-acting factors responsible for the gene expression of MCRs were investigated by using electrophoretic mobility shift assay (EMSA) and affinity particle purification. A survey of their operator regions by EMSA with protein extracts from mrt-expressing cultures restricted them to 46- and 41-bp-long mcr and mrt upstream regions, respectively. Affinity particle purification of DNA-binding proteins conjugated with putative operator regions resulted in the retrieval of a protein attributed to IMP dehydrogenase-related protein VII (IMPDH VII). IMPDH VII is predicted to have a winged helix-turn-helix DNA-binding motif and two cystathionine beta-synthase domains, and it has been suspected to be an energy-sensing module. EMSA with oligonucleotide probes with unusual sequences showed that the binding site of IMPDH VII mostly overlaps the factor B-responsible element-TATA box of the mcr operon. The results presented here suggest that IMPDH VII encoded by MTH126 is a plausible candidate for the transcriptional regulator of the mcr operon in this methanogen.

High cell density cultivation of recombinant Escherichia coli for hirudin variant 1 production.

A synthetic medium, TK-25, for high cell density cultivation (HCDC) of Escherichia coli K-12 was modified to support HCDC of strain JM109. By optimizing the culture conditions, the cell concentration of 65 g/l in 14 h was obtained in the optimized medium, namely TK-10, with glucose-fed batch cultivation. When these conditions were further applied for HCDC of E. coli JM109 harboring pUC-based recombinant plasmid which expresses a hirudin variant, HV-1-fused protein under the control of trp promoter, it grew to 24 g/l of dried cells expressed as an inclusion body as 15.9% of the total protein, corresponding to 1908 mg/l hirudin-fused protein.

High cell density cultivation of recombinant E. coli for hirudin variant 1 production by temperature shift controlled by pUC18-based replicative origin.

The copy number of a plasmid, pUC-based vector, was previously shown to be affected by culture temperature. In this study, intracellular hirudin variant 1 (f-HV1) fused to porcine adenylate kinase protein was produced using recombinant Escherichia coli by temperature shift cultivation coupled with a high cell density cultivation technique for E. coli JM109. The optimal temperature for cellular growth suppressing f-HV1 production was 33 degrees C, resulting in a final dried cell concentration of 45.7 g/l, with a specific growth rate of 0.54 1/h. Optimizing the temperature-shift conditions (temperature shifted to an OD660 nm of 15 from 33 degrees C to 37 degrees C) resulted in the production of f-HV1 up to 4763 mg/l as an inclusion body with dried cell concentration of 44 g/l in 18 h.

Geochemical and Microbiological Evidence for Microbial Methane Production in Deep Aquifers of the Cretaceous Accretionary Prism.

Accretionary prisms are thick layers of sedimentary material piled up at convergent plate boundaries. Large amounts of anaerobic groundwater and methane (CH4) are contained in the deep aquifers associated with accretionary prisms. In order to identify microbial activity and CH4 production processes in the deep aquifers associated with the Cretaceous accretionary prism in Okinawa Island, Japan, we performed geochemical and microbiological studies using anaerobic groundwater and natural gas (mainly CH4) samples collected through four deep wells. Chemical and stable hydrogen and oxygen isotope analyses of groundwater samples indicated that the groundwater samples obtained from each site originated from ancient seawater and a mixture of rainwater and seawater, respectively. Additionally, the chemical and stable carbon isotopic signatures of groundwater and natural gas samples suggested that CH4 in the natural gas samples was of a biogenic origin or a mixture of biogenic and thermogenic origins. Microscopic observations and a 16S rRNA gene analysis targeting microbial communities in groundwater samples revealed the predominance of dihydrogen (H2)-producing fermentative bacteria and H2-utilizing methanogenic archaea. Moreover, anaerobic cultures using groundwater samples suggested a high potential for CH4 production by a syntrophic consortium of H2-producing fermentative bacteria and H2-utilizing methanogenic archaea through the biodegradation of organic substrates. Collectively, our geochemical and microbiological data support the conclusion that the ongoing biodegradation of organic matter widely contributes to CH4 production in the deep aquifers associated with the Cretaceous accretionary prism.

Genome Sequence of Lactobacillus paracasei Strain LC-Ikematsu, Isolated from a Pineapple in Okinawa, Japan.

The draft genome sequence of Lactobacillus paracasei strain LC-Ikematsu, isolated from a pineapple in Okinawa, was determined. The total length of the 87 contigs was 3.08 Mb with a G+C content of 46.2% and 2,946 coding sequences. The genome analysis revealed its biosynthetic ability of 11 amino acids.

Complete mitochondrial genome of Cacospongia mycofijiensis (Dictyoceratida: Demospongiae): the first report for the sponge family Thorectidae.

We sequenced a complete mitochondrial genome of the marine sponge, Cacospongia mycofijiensis, which is the first report for the family Thorectidae in the class Demospongiae. The mitogenome was obtained from a de novo assembly of shotgun genome sequencing using Illumina Miseq technology, which reconstructed a circular genome with 97 × of sequence coverage. The assembled mitochondrial genome consisting of 16,227 bp includes 14 protein-coding genes, 2 rRNAs and 2 tRNAs. This complete mitogenome sequence will be useful especially for the phylogenic studies of Demospongiae.

Complete Genome Sequence of the Intracellular Bacterial Symbiont TC1 in the Anaerobic Ciliate Trimyema compressum.

A free-living ciliate, Trimyema compressum, found in anoxic freshwater environments harbors methanogenic archaea and a bacterial symbiont named TC1 in its cytoplasm. Here, we report the complete genome sequence of the TC1 symbiont, consisting of a 1.59-Mb chromosome and a 35.8-kb plasmid, which was determined using the PacBio RSII sequencer.

Termite-regulated fungal monoculture in fungus combs of a macrotermitine termite Odontotermes formosanus.

The mechanism of the exclusive growth of Termitomyces in fungus combs with fungi-growing termites, O. formosanus was examined using laboratory scale fungus combs. In the combs without the termites, vigorous growth of unidentified fungi was observed although no significant change was found in the case of the combs with termites. In addition, these results were reproducible even when incubated in a separated dish, suggesting that the physicochemical conditions were not the reason for the growth. With the molecular based analysis for the microbial communities in the combs, monoculture of the Termitomyces in the combs with termites was confirmed while the bacterial communities were independent either with or without termites. Possible mechanism of the exclusive growth of Termitomyces, such as the selective grazing of pathogenic fungi or contribution of antifungal activity giving actinomycetes were also discussed.