Fidelity varies in the symbiosis between a gutless marine worm and its microbial consortium.

BACKGROUND: Many animals live in intimate associations with a species-rich microbiome. A key factor in maintaining these beneficial associations is fidelity, defined as the stability of associations between hosts and their microbiota over multiple host generations. Fidelity has been well studied in terrestrial hosts, particularly insects, over longer macroevolutionary time. In contrast, little is known about fidelity in marine animals with species-rich microbiomes at short microevolutionary time scales, that is at the level of a single host population. Given that natural selection acts most directly on local populations, studies of microevolutionary partner fidelity are important for revealing the ecological and evolutionary processes that drive intimate beneficial associations within animal species. RESULTS: In this study on the obligate symbiosis between the gutless marine annelid Olavius algarvensis and its consortium of seven co-occurring bacterial symbionts, we show that partner fidelity varies across symbiont species from strict to absent over short microevolutionary time. Using a low-coverage sequencing approach that has not yet been applied to microbial community analyses, we analysed the metagenomes of 80 O. algarvensis individuals from the Mediterranean and compared host mitochondrial and symbiont phylogenies based on single-nucleotide polymorphisms across genomes. Fidelity was highest for the two chemoautotrophic, sulphur-oxidizing symbionts that dominated the microbial consortium of all O. algarvensis individuals. In contrast, fidelity was only intermediate to absent in the sulphate-reducing and spirochaetal symbionts with lower abundance. These differences in fidelity are likely driven by both selective and stochastic forces acting on the consistency with which symbionts are vertically transmitted. CONCLUSIONS: We hypothesize that variable degrees of fidelity are advantageous for O. algarvensis by allowing the faithful transmission of their nutritionally most important symbionts and flexibility in the acquisition of other symbionts that promote ecological plasticity in the acquisition of environmental resources. Video Abstract.

Evaluation of RNAlater as a Field-Compatible Preservation Method for Metaproteomic Analyses of Bacterium-Animal Symbioses.

Field studies are central to environmental microbiology and microbial ecology, because they enable studies of natural microbial communities. Metaproteomics, the study of protein abundances in microbial communities, allows investigators to study these communities "in situ," which requires protein preservation directly in the field because protein abundance patterns can change rapidly after sampling. Ideally, a protein preservative for field deployment works rapidly and preserves the whole proteome, is stable in long-term storage, is nonhazardous and easy to transport, and is available at low cost. Although these requirements might be met by several protein preservatives, an assessment of their suitability under field conditions when targeted for metaproteomic analyses is currently lacking. Here, we compared the protein preservation performance of flash freezing and the preservation solution RNAlater using the marine gutless oligochaete Olavius algarvensis and its symbiotic microbes as a test case. In addition, we evaluated long-term RNAlater storage after 1 day, 1 week, and 4 weeks at room temperature (22°C to 23°C). We evaluated protein preservation using one-dimensional liquid chromatography-tandem mass spectrometry. We found that RNAlater and flash freezing preserved proteins equally well in terms of total numbers of identified proteins and relative abundances of individual proteins, and none of the test time points was altered, compared to time zero. Moreover, we did not find biases against specific taxonomic groups or proteins with particular biochemical properties. Based on our metaproteomic data and the logistical requirements for field deployment, we recommend RNAlater for protein preservation of field-collected samples targeted for metaproteomic analyses. IMPORTANCE Metaproteomics, the large-scale identification and quantification of proteins from microbial communities, provide direct insights into the phenotypes of microorganisms on the molecular level. To ensure the integrity of the metaproteomic data, samples need to be preserved immediately after sampling to avoid changes in protein abundance patterns. In laboratory setups, samples for proteomic analyses are most commonly preserved by flash freezing; however, liquid nitrogen or dry ice is often unavailable at remote field locations, due to their hazardous nature and transport restrictions. Our study shows that RNAlater can serve as a low-hazard, easy-to-transport alternative to flash freezing for field preservation of samples for metaproteomic analyses. We show that RNAlater preserves the metaproteome equally well, compared to flash freezing, and protein abundance patterns remain stable during long-term storage for at least 4 weeks at room temperature.

Evolution and Maintenance of Mutualism between Tubeworms and Sulfur-Oxidizing Bacteria.

AbstractTubeworms and sulfur-oxidizing bacteria mutualism, an essential part of the chemosynthetic ecosystem in the deep sea, has several puzzling features. After acquiring sulfur-oxidizing bacteria from the environment, tubeworms become fully dependent on their symbiont bacteria for nutrient intake. Once ingested by the tubeworm larva, no additional symbionts join from the environment, and no symbionts are released until the host tubeworm dies. Despite this very narrow window to acquire symbionts, some tubeworm species can live for >200 years. Such a restricted release of symbionts could lead to a shortage of symbiont bacteria in the environment without which tubeworms could not survive. In our study, we examine the conditions under which this mutualism can persist and whether the host mortality rate evolves toward a low value using a mathematical model for the tubeworm-symbiont bacteria system. Our model reveals that mutualism can persist only when the host mortality rate is within an intermediate range. With cohabitation of multiple symbionts strains in the same host, host mortality rate evolves toward a low value without driving either host or symbiont to extinction when competition among symbionts is weak and their growth within a host is slow. We also find the parameter conditions that lead to unlimited evolutionary escalation of host mortality rate toward coextinction of both tubeworms and symbionts populations (evolutionary double suicide). The generality of this evolutionary fragility in obligate mutualistic systems as well as the contrasting evolutionary robustness in host-parasite systems are discussed.

Methanotrophic bacterial symbionts fuel dense populations of deep-sea feather duster worms (Sabellida, Annelida) and extend the spatial influence of methane seepage.

Deep-sea cold seeps are dynamic sources of methane release and unique habitats supporting ocean biodiversity and productivity. Here, we describe newly discovered animal-bacterial symbioses fueled by methane, between two species of annelid (a serpulid Laminatubus and sabellid Bispira) and distinct aerobic methane-oxidizing bacteria belonging to the Methylococcales, localized to the host respiratory crown. Worm tissue δ13C of -44 to -58‰ are consistent with methane-fueled nutrition for both species, and shipboard stable isotope labeling experiments revealed active assimilation of 13C-labeled methane into animal biomass, which occurs via the engulfment of methanotrophic bacteria across the crown epidermal surface. These worms represent a new addition to the few animals known to intimately associate with methane-oxidizing bacteria and may further explain their enigmatic mass occurrence at 150-million year-old fossil seeps. High-resolution seafloor surveys document significant coverage by these symbioses, beyond typical obligate seep fauna. These findings uncover novel consumers of methane in the deep sea and, by expanding the known spatial extent of methane seeps, may have important implications for deep-sea conservation.

Southernmost records of Escarpia spicata and Lamellibrachia barhami (Annelida: Siboglinidae) confirmed with DNA obtained from dried tubes collected from undiscovered reducing environments in northern Chile.

Deep-sea fishing bycatch enables collection of samples of rare species that are not easily accessible, for research purposes. However, these specimens are often degraded, losing diagnostic morphological characteristics. Several tubes of vestimentiferans, conspicuous annelids endemic to chemosynthetic environments, were obtained from a single batch of deep-sea fishing bycatch at depths of around 1,500 m off Huasco, northern Chile, as part of an ongoing study examining bycatch species. DNA sequences of the mitochondrial cytochrome c oxidase subunit I (COI) gene and an intron region within the hemoglobin subunit B2 (hbB2i) were successfully determined using vestimentiferans' dried-up tubes and their degraded inner tissue. Molecular phylogenetic analyses based on DNA sequence identified the samples as Escarpia spicata Jones, 1985, and Lamellibrachia barhami Webb, 1969. These are the southernmost records, vastly extending the geographical ranges of both species from Santa Catalina Island, California to northern Chile for E. spicata (over 8,000 km), and from Vancouver Island Margin to northern Chile for L. barhami (over 10,000 km). We also determined a 16S rRNA sequence of symbiotic bacteria of L. barhami. The sequence of the bacteria is the same as that of E. laminata, Lamellibrachia sp. 1, and Lamellibrachia sp.2 known from the Gulf of Mexico. The present study provides sound evidence forthe presence of reducing environments along the continental margin of northern Chile.

Metagenomic sequencing suggests a diversity of RNA interference-like responses to viruses across multicellular eukaryotes.

RNA interference (RNAi)-related pathways target viruses and transposable element (TE) transcripts in plants, fungi, and ecdysozoans (nematodes and arthropods), giving protection against infection and transmission. In each case, this produces abundant TE and virus-derived 20-30nt small RNAs, which provide a characteristic signature of RNAi-mediated defence. The broad phylogenetic distribution of the Argonaute and Dicer-family genes that mediate these pathways suggests that defensive RNAi is ancient, and probably shared by most animal (metazoan) phyla. Indeed, while vertebrates had been thought an exception, it has recently been argued that mammals also possess an antiviral RNAi pathway, although its immunological relevance is currently uncertain and the viral small RNAs (viRNAs) are not easily detectable. Here we use a metagenomic approach to test for the presence of viRNAs in five species from divergent animal phyla (Porifera, Cnidaria, Echinodermata, Mollusca, and Annelida), and in a brown alga-which represents an independent origin of multicellularity from plants, fungi, and animals. We use metagenomic RNA sequencing to identify around 80 virus-like contigs in these lineages, and small RNA sequencing to identify viRNAs derived from those viruses. We identified 21U small RNAs derived from an RNA virus in the brown alga, reminiscent of plant and fungal viRNAs, despite the deep divergence between these lineages. However, contrary to our expectations, we were unable to identify canonical (i.e. Drosophila- or nematode-like) viRNAs in any of the animals, despite the widespread presence of abundant micro-RNAs, and somatic transposon-derived piwi-interacting RNAs. We did identify a distinctive group of small RNAs derived from RNA viruses in the mollusc. However, unlike ecdysozoan viRNAs, these had a piRNA-like length distribution but lacked key signatures of piRNA biogenesis. We also identified primary piRNAs derived from putatively endogenous copies of DNA viruses in the cnidarian and the echinoderm, and an endogenous RNA virus in the mollusc. The absence of canonical virus-derived small RNAs from our samples may suggest that the majority of animal phyla lack an antiviral RNAi response. Alternatively, these phyla could possess an antiviral RNAi response resembling that reported for vertebrates, with cryptic viRNAs not detectable through simple metagenomic sequencing of wild-type individuals. In either case, our findings show that the antiviral RNAi responses of arthropods and nematodes, which are highly divergent from each other and from that of plants and fungi, are also highly diverged from the most likely ancestral metazoan state.

Closely coupled evolutionary history of ecto- and endosymbionts from two distantly related animal phyla.

The level of integration between associated partners can range from ectosymbioses to extracellular and intracellular endosymbioses, and this range has been assumed to reflect a continuum from less intimate to evolutionarily highly stable associations. In this study, we examined the specificity and evolutionary history of marine symbioses in a group of closely related sulphur-oxidizing bacteria, called Candidatus Thiosymbion, that have established ecto- and endosymbioses with two distantly related animal phyla, Nematoda and Annelida. Intriguingly, in the ectosymbiotic associations of stilbonematine nematodes, we observed a high degree of congruence between symbiont and host phylogenies, based on their ribosomal RNA (rRNA) genes. In contrast, for the endosymbioses of gutless phallodriline annelids (oligochaetes), we found only a weak congruence between symbiont and host phylogenies, based on analyses of symbiont 16S rRNA genes and six host genetic markers. The much higher degree of congruence between nematodes and their ectosymbionts compared to those of annelids and their endosymbionts was confirmed by cophylogenetic analyses. These revealed 15 significant codivergence events between stilbonematine nematodes and their ectosymbionts, but only one event between gutless phallodrilines and their endosymbionts. Phylogenetic analyses of 16S rRNA gene sequences from 50 Cand. Thiosymbion species revealed seven well-supported clades that contained both stilbonematine ectosymbionts and phallodriline endosymbionts. This closely coupled evolutionary history of marine ecto- and endosymbionts suggests that switches between symbiotic lifestyles and between the two host phyla occurred multiple times during the evolution of the Cand. Thiosymbion clade, and highlights the remarkable flexibility of these symbiotic bacteria.

Bone-Eating Worms Spread: Insights into Shallow-Water Osedax (Annelida, Siboglinidae) from Antarctic, Subantarctic, and Mediterranean Waters.

Osedax, commonly known as bone-eating worms, are unusual marine annelids belonging to Siboglinidae and represent a remarkable example of evolutionary adaptation to a specialized habitat, namely sunken vertebrate bones. Usually, females of these animals live anchored inside bone owing to a ramified root system from an ovisac, and obtain nutrition via symbiosis with Oceanospirillales gamma-proteobacteria. Since their discovery, 26 Osedax operational taxonomic units (OTUs) have been reported from a wide bathymetric range in the Pacific, the North Atlantic, and the Southern Ocean. Using experimentally deployed and naturally occurring bones we report here the presence of Osedax deceptionensis at very shallow-waters in Deception Island (type locality; Antarctica) and at moderate depths near South Georgia Island (Subantarctic). We present molecular evidence in a new phylogenetic analysis based on five concatenated genes (28S rDNA, Histone H3, 18S rDNA, 16S rDNA, and cytochrome c oxidase I-COI-), using Maximum Likelihood and Bayesian inference, supporting the placement of O. deceptionensis as a separate lineage (Clade VI) although its position still remains uncertain. This phylogenetic analysis includes a new unnamed species (O. 'mediterranea') recently discovered in the shallow-water Mediterranean Sea belonging to Osedax Clade I. A timeframe of the diversification of Osedax inferred using a Bayesian framework further suggests that Osedax diverged from other siboglinids during the Middle Cretaceous (ca. 108 Ma) and also indicates that the most recent common ancestor of Osedax extant lineages dates to the Late Cretaceous (ca. 74.8 Ma) concomitantly with large marine reptiles and teleost fishes. We also provide a phylogenetic framework that assigns newly-sequenced Osedax endosymbionts of O. deceptionensis and O. 'mediterranea' to ribospecies Rs1. Molecular analysis for O. deceptionensis also includes a COI-based haplotype network indicating that individuals from Deception Island and the South Georgia Island (ca. 1,600 km apart) are clearly the same species, confirming the well-developed dispersal capabilities reported in other congeneric taxa. In addition, we include a complete description of living features and morphological characters (including scanning and transmission electron microscopy) of O. deceptionensis, a species originally described from a single mature female, and compare it to information available for other congeneric OTUs.

Census of bacterial microbiota associated with the glacier ice worm Mesenchytraeus solifugus.

The glacier ice worm, Mesenchytraeus solifugus, is a unique annelid, inhabiting only snow and ice in North American glaciers. Here, we analyzed the taxonomic composition of bacteria associated with M. solifugus based on the 16S rRNA gene. We analyzed four fixed-on-site and 10 starved ice worm individuals, along with glacier surface samples. In total, 1341 clones of 16S rRNA genes were analyzed for the ice worm samples, from which 65 bacterial phylotypes (99.0% cut-off) were identified. Of these, 35 phylotypes were closely related to sequences obtained from their habitat glacier and/or other components of cryosphere; whereas three dominant phylotypes were affiliated with animal-associated lineages of the class Mollicutes. Among the three, phylotype Ms-13 shared less than 89% similarity with database sequences and was closest to a gut symbiont of a terrestrial earthworm. Using fluorescence in situ hybridization, Ms-13 was located on the gut wall surface of the ice worms. We propose a novel genus and species, 'Candidatus Vermiplasma glacialis', for this bacterium. Our results raise the possibility that the ice worm has exploited indigenous glacier bacteria, while several symbiotic bacterial lineages have maintained their association with the ice worm during the course of adaptive evolution to the permanently cold environment.

Effects of benthic macrofauna bioturbation on the bacterial community composition in lake sediments.

Benthic macrofauna are considered to be an important part of the lacustrine ecosystem, and bioturbation may greatly affect the biogeochemical processes and microbial activities in sediments. In the present study, the bacterial community composition in sediments inhabited by 3 different types of benthic macrofauna (Corbicula fluminea, Chironomidae larvae, and tubificid worms) in the shallow and eutrophic Lake Taihu was studied to investigate the different effects of bioturbation on the composition of these communities. Microcosms were constructed, and culture-independent methods, including terminal restriction fragment length polymorphism (T-RFLP) and clone library analysis, were performed to evaluate the bacterial communities. Analysis of similarities (ANOSIM) and multidimensional scaling (MDS) analysis of T-RFLP patterns demonstrated that differences in the bacterial community composition between the control and the macrofauna-inhabited sediments were not as great as expected, although the chemical properties of the sediments changed remarkably. Nevertheless, the dominant bacterial group in each type of macrofauna-inhabited sediment was different. Acidobacteria, Betaproteobacteria, and Deltaproteobacteria were the dominant bacterial groups in sediments inhabited by C. fluminea, tubificid worms, and Chironomidae larvae, respectively. The data obtained in this study are helpful for understanding the effects of bioturbation in a shallow, eutrophic lake.

Parasphingopyxis lamellibrachiae gen. nov., sp. nov., isolated from a marine annelid worm.

A Gram-stain-negative, aerobic, motile, orange-pigmented, slightly halophilic, rod-shaped bacterium, designated strain JAMH 0132(T), was isolated from the trophosome of a tubeworm in Kagoshima Bay, Japan, and its taxonomic position was investigated using a polyphasic approach. The novel strain grew optimally at 28-30 °C and with about 2.0 % (w/v) NaCl. Chemotaxonomic analysis showed that Q-10 was the predominant respiratory quinone and that C(18 : 1)ω7c, C(16 : 0) 2-OH and C(16 : 0) were the major fatty acids. Sphingoglycolipid, phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol and phosphatidylcholine were the major polar lipids. The genomic DNA G+C content was 60.1 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain JAMH 0132(T) belonged to the family Sphingomonadaceae, within the class Alphaproteobacteria. The novel strain appeared most closely related to Sphingopyxis baekryungensis SW-150(T) (95.1 % 16S rRNA gene sequence similarity) and showed less sequence similarity with representatives of the genera Blastomonas, Sphingomonas, Sphingosinicella and Novosphingobium (<94.8 %). In having no detectable polyamine, strain JAMH 0132(T) differed from members of all genera currently in the family Sphingomonadaceae. On the basis of its phenotypic properties and phylogenetic distinctiveness, strain JAMH 0132(T) represents a novel species of a new genus in the family Sphingomonadaceae for which the name Parasphingopyxis lamellibrachiae gen. nov., sp. nov. is proposed. The type strain of Parasphingopyxis lamellibrachiae gen. nov., sp. nov. is JAMH 0132(T) (= JCM 15549(T) = NCIMB 14486(T)).

Chemical and microbiological changes during vermicomposting of coffee pulp using exotic (Eudrilus eugeniae) and native earthworm (Perionyx ceylanesis) species.

Coffee pulp is the main solid residue from the wet processing of coffee berries. Due to presence of anti-physiological and anti-nutritional factors, coffee pulp is not considered as adequate substrate for bioconversion process by coffee farmers. Recent stringent measures by Pollution Control authorities, made it mandatory to treat all the solid and liquid waste emanating from the coffee farms. A study was conducted to evaluate the efficiency of an exotic (Eudrilus eugeniae) and a native earthworm (Perionyx ceylanesis) from coffee farm for decomposition of coffee pulp into valuable vermicompost. Exotic earthworms were found to degrade the coffee pulp faster (112 days) as compared to the native worms (165 days) and the vermicomposting efficiency (77.9%) and vermicompost yield (389 kg) were found to significantly higher with native worms. The multiplication rate of earthworms (280%) and worm yield (3.78 kg) recorded significantly higher with the exotic earthworms. The percentage of nitrogen, phosphorous, potassium, calcium and magnesium in vermicompost was found to increase while C:N ratio, pH and total organic carbon declined as a function of the vermicomposting. The plant nutrients, nitrogen (80.6%), phosphorus (292%) and potassium (550%) content found to increase significantly in the vermicompost produced using native earthworms as compared to the initial values, while the calcium (85.7%) and magnesium (210%) content found to increase significantly in compost produced utilizing exotic worms. Vermicompost and vermicasts from native earthworms recorded significantly higher functional microbial group's population as compared to the exotic worms. The study reveals that coffee pulp can be very well used as substrate for vermicomposting using exotic (Eudrilus eugeniae) and native earthworm (Perionyx ceylanesis).

Bacterial exopolysaccharides from extreme marine habitats: production, characterization and biological activities.

Many marine bacteria produce exopolysaccharides (EPS) as a strategy for growth, adhering to solid surfaces, and to survive adverse conditions. There is growing interest in isolating new EPS producing bacteria from marine environments, particularly from extreme marine environments such as deep-sea hydrothermal vents characterized by high pressure and temperature and heavy metal presence. Marine EPS-producing microorganisms have been also isolated from several extreme niches such as the cold marine environments typically of Arctic and Antarctic sea ice, characterized by low temperature and low nutrient concentration, and the hypersaline marine environment found in a wide variety of aquatic and terrestrial ecosystems such as salt lakes and salterns. Most of their EPSs are heteropolysaccharides containing three or four different monosaccharides arranged in groups of 10 or less to form the repeating units. These polymers are often linear with an average molecular weight ranging from 1 x 10(5) to 3 x 10(5) Da. Some EPS are neutral macromolecules, but the majority of them are polyanionic for the presence of uronic acids or ketal-linked pyruvate or inorganic residues such as phosphate or sulfate. EPSs, forming a layer surrounding the cell, provide an effective protection against high or low temperature and salinity, or against possible predators. By examining their structure and chemical-physical characteristics it is possible to gain insight into their commercial application, and they are employed in several industries. Indeed EPSs produced by microorganisms from extreme habitats show biotechnological promise ranging from pharmaceutical industries, for their immunomodulatory and antiviral effects, bone regeneration and cicatrizing capacity, to food-processing industries for their peculiar gelling and thickening properties. Moreover, some EPSs are employed as biosurfactants and in detoxification mechanisms of petrochemical oil-polluted areas. The aim of this paper is to give an overview of current knowledge on EPSs produced by marine bacteria including symbiotic marine EPS-producing bacteria isolated from some marine annelid worms that live in extreme niches.

Gut bacterium of Dendrobaena veneta (Annelida: Oligochaeta) possesses antimycobacterial activity.

The new bacterial strain with antimycobacterial activity has been isolated from the midgut of Dendrobaena veneta (Annelida). Biochemical and molecular characterization of isolates from 18 individuals identified all as Raoultella ornithinolytica genus with 99% similarity. The bacterium is a possible symbiont of the earthworm D. veneta. The isolated microorganism has shown the activity against four strains of fast-growing mycobacteria: Mycobacterium butiricum, Mycobacterium jucho, Mycobacterium smegmatis and Mycobacterium phlei. The multiplication of the gut bacterium on plates with Sauton medium containing mycobacteria has caused a lytic effect. After the incubation of the cell free extract prepared from the gut bacterium with four strains of mycobacteria in liquid Sauton medium, the cells of all tested strains were deformed and divided to small oval forms and sometimes created long filaments. The effect was observed by the use of light, transmission and scanning microscopy. Viability of all examined species of mycobacteria was significantly decreased. The antimycobacterial effect was probably the result of the antibiotic action produced by the gut bacterium of the earthworm. The application of ultrafiltration procedure allowed to demonstrate that antimicrobial substance with strong antimycobacterial activity from bacterial culture supernatant, is a protein with the molecular mass above 100 kDa.

Molecular characterization of bacteria associated with the trophosome and the tube of Lamellibrachia sp., a siboglinid annelid from cold seeps in the eastern Mediterranean.

Specimens of Lamellibrachia (Annelida: Siboglinidae) were recently discovered at cold seeps in the eastern Mediterranean. In this study, we have investigated the phylogeny and function of intracellular bacterial symbionts inhabiting the trophosome of specimens of Lamellibrachia sp. from the Amon mud volcano, as well as the bacterial assemblages associated with their tube. The dominant intracellular symbiont of Lamellibrachia sp. is a gammaproteobacterium closely related to other sulfide-oxidizing tubeworm symbionts. In vivo uptake experiments show that the tubeworm relies on sulfide for its metabolism, and does not utilize methane. Bacterial communities associated with the tube form biofilms and occur from the anterior to the posterior end of the tube. The diversity of 16S rRNA gene phylotypes includes representatives from the same divisions previously identified from the tube of the vent species Riftia pachyptila, and others commonly found at seeps and vents.

Molecular characteristics of the tubeworm, Ridgeia piscesae, from the deep-sea hydrothermal vent.

Ridgeia piscesae, living around the extremely harsh hydrothermal vent in deep sea, is an ideal model for studying the adaptative mechanism to extreme environment. For insights of its molecular characteristics, a cDNA library of R. piscesae was constructed. A total of 879 expressed sequence tags (ESTs) were sequenced and 199 genes were identified for the first time. They were found to be involved in basal metabolism, adaptation and defense, or signal transduction. Among them, we found 23 various chitin-binding proteins, which are the major component of the chitinous tube that prevents the tubeworms from predators and surrounding extreme environment. Additionally, high polymorphism also exists in other genes, such as myohemerythrin, lysozyme. The gene-expression profile might help to further understand the molecular basis of tubeworm physiology. It will also lay a good foundation for functional studies on the adaptation to extreme environments.

Neptunomonas japonica sp. nov., an Osedax japonicus symbiont-like bacterium isolated from sediment adjacent to sperm whale carcasses off Kagoshima, Japan.

Novel bacterial species were isolated from sediments adjacent to sperm whale carcasses off Kagoshima, Japan, at a depth of 226-246 m. The isolated strains, JAMM 0745T, JAMM 1380, JAMM 1475 and JAMM 1610, were Gram-negative, rod-shaped, non-spore-forming and motile by means of a single polar or subterminal flagellum. Phylogenetic analysis based on 16S rRNA gene sequences of the novel isolates indicated a relationship to a symbiotic bacterial clone of the polychaete Osedax japonicus (99.6-99.9 % sequence similarity) and these bacteria were closely related to members of the genus Neptunomonas (95.6-96.0 % similarity) within the class Gammaproteobacteria. The novel strains were able to produce isoprenoid quinone Q-8 as the major quinone component. The predominant fatty acids were C16 : 0, C16 : 1 and C18 : 1, with C18 : 2 and C20 : 2 present in smaller amounts. The DNA G+C contents of the four novel strains were about 43.6-43.8 mol%. Based on the taxonomic differences observed, the four isolated strains appear to represent a novel species of the genus Neptunomonas. The name Neptunomonas japonica sp. nov. (type strain JAMM 0745T=JCM 14595T=DSM 18939T) is proposed for the novel strains.

Enzymic approach to eurythermalism of Alvinella pompejana and its episymbionts.

The equilibrium model, which describes the influence of temperature on enzyme activity, has been established as a valid and useful tool for characterizing enzyme eurythermalism and thermophily. By introducing K(eq), a temperature-dependent equilibrium constant for the interconversion between E(act), the active form of enzyme, and E(inact), a reversibly inactive form of enzyme, the equilibrium model currently provides the most complete description of the enzyme-temperature relationship; its derived parameters are intrinsic and apparently universal and, being derived under reaction conditions, potentially have physiological significance. One of these parameters, T(eq), correlates with host growth temperature better than enzyme stability does. The vent-dwelling annelid Alvinella pompejana has been reported as an extremely eurythermal organism, and the symbiotic complex microbial community associated with its dorsal surface is likely to experience similar environmental thermal conditions. The A. pompejana episymbiont community, predominantly composed of epsilonproteobacteria, has been analyzed metagenomically, enabling direct retrieval of genes coding for enzymes suitable for equilibrium model applications. Two such genes, coding for isopropylmalate dehydrogenase and glutamate dehydrogenase, have been isolated from the A. pompejana episymbionts, heterologously expressed, and shown by reverse transcription-quantitative PCR to be actively expressed. The equilibrium model parameters of characterized expression products suggested that enzyme eurythermalism constitutes part of the thermal adaptation strategy employed by the episymbionts. Moreover, the enzymes' thermal characteristics correspond to their predicted physiological roles and the abundance and expression of the corresponding genes. This paper demonstrates the use of the equilibrium model as part of a top-down metagenomic approach to studying temperature adaptation of uncultured organisms.

Ultrasonic depolymerization of an exopolysaccharide produced by a bacterium isolated from a deep-sea hydrothermal vent polychaete annelid.

Low frequency ultrasound was used to depolymerize a high-molecular-weight exopolysaccharide (EPS) produced by a deep-sea hydrothermal bacterium Alteromonas macleodii subsp. fijiensis biovar deepsane. The influence of several parameters was examined including the duration of ultrasonic irradiation, EPS concentration, reaction temperature and volume of the sonicated solution. With the aim of optimizing the depolymerization, the native EPS was simultaneously treated with hydrogen peroxide and ultrasound. This study identified the sonication conditions that produce low-molecular-weight derivatives from the native EPS (>10(6)Da) with good reproducibility.