Hypolithic communities: important nitrogen sources in Antarctic desert soils.

Hypolithic microbial communities (i.e. cryptic microbial assemblages found on the undersides of translucent rocks) are major contributors of carbon input into the oligotrophic hyper-arid desert mineral soils of the Eastern Antarctic Dry Valleys. Here we demonstrate, for the first time, that hypolithic microbial communities possess both the genetic capacity for nitrogen fixation (i.e. the presence of nifH genes) and the ability to catalyse acetylene reduction, an accepted proxy for dinitrogen fixation. An estimate of the total contribution of these communities suggests that hypolithic communities are important contributors to fixed nitrogen budgets in Antarctic desert soils.

Growth and phylogenetic properties of novel bacteria belonging to the epsilon subdivision of the Proteobacteria enriched from Alvinella pompejana and deep-sea hydrothermal vents.

Recent molecular characterizations of microbial communities from deep-sea hydrothermal sites indicate the predominance of bacteria belonging to the epsilon subdivision of Proteobacteria (epsilon Proteobacteria). Here, we report the first enrichments and characterizations of four epsilon Proteobacteria that are directly associated with Alvinella pompejana, a deep sea hydrothermal vent polychete, or with hydrothermal vent chimney samples. These novel bacteria were moderately thermophilic sulfur-reducing heterotrophs growing on formate as the energy and carbon source. In addition, two of them (Am-H and Ex-18.2) could grow on sulfur lithoautrotrophically using hydrogen as the electron donor. Optimal growth temperatures of the bacteria ranged from 41 to 45 degrees C. Phylogenetic analysis of the small-subunit ribosomal gene of the two heterotrophic bacteria demonstrated 95% similarity to Sulfurospirillum arcachonense, an epsilon Proteobacteria isolated from an oxidized marine surface sediment. The autotrophic bacteria grouped within a deeply branching clade of the epsilon Proteobacteria, to date composed only of uncultured bacteria detected in a sample from a hydrothermal vent along the mid-Atlantic ridge. A molecular survey of various hydrothermal vent environments demonstrated the presence of two of these bacteria (Am-N and Am-H) in more than one geographic location and habitat. These results suggest that certain epsilon Proteobacteria likely fill important niches in the environmental habitats of deep-sea hydrothermal vents, where they contribute to overall carbon and sulfur cycling at moderate thermophilic temperatures.

Evidence for the presence of thermophilic Fe(III)-reducing microorganisms in deep-sea hydrothermal vents at 13 degrees N (East Pacific Rise).

Microorganisms capable of dissimilatory Fe(III) reduction in the temperature range of 52-90 degrees C were enriched from outer and inner parts of chimney-like structures, tubes of the polychaetous annelid Alvinella sp., and hydrothermal fluids collected at 13 degrees N hydrothermal vent sites on the East Pacific Rise at a depth of 2650 m. Numbers of culturable Fe(III)-reducing thermophiles estimated by the serial dilution technique varied from 10 to 10(7) cells per cm(3) of sample. Phylogenetic analysis of bacterial and archaeal PCR-amplified 16S rDNA genes obtained from Fe(III)-reducing enrichments and separated by denaturing gradient gel electrophoresis revealed sequences related to Deferibacter, Thermotogales (Bacteria) and Thermococcus (Archaea) for which the capacity for Fe(III) reduction had been reported. This was confirmed by isolating a hyperthermophilic iron reducer that belongs to the genus Thermococcus. Other bacterial thermophiles found in the enrichments were related to so far uncultured members of the Clostridiaceae, and epsilon-subdivision of the Proteobacteria.

Chemical speciation drives hydrothermal vent ecology.

The physiology and biochemistry of many taxa inhabiting deep-sea hydrothermal vents have been elucidated; however, the physicochemical factors controlling the distribution of these organisms at a given vent site remain an enigma after 20 years of research. The chemical speciation of particular elements has been suggested as key to controlling biological community structure in these extreme aquatic environments. Implementation of electrochemical technology has allowed us to make in situ measurements of chemical speciation at vents located at the East Pacific Rise (9 degrees 50' N) and on a scale relevant to the biology. Here we report that significant differences in oxygen, iron and sulphur speciation strongly correlate with the distribution of specific taxa in different microhabitats. In higher temperature (> 30 degrees C) microhabitats, the appreciable formation of soluble iron-sulphide molecular clusters markedly reduces the availability of free H2S/HS- to vent (micro)organisms, thus controlling the available habitat.

Characterization of a novel spirochete associated with the hydrothermal vent polychaete annelid, Alvinella pompejana.

A highly integrated, morphologically diverse bacterial community is associated with the dorsal surface of Alvinella pompejana, a polychaetous annelid that inhabits active high-temperature deep-sea hydrothermal vent sites along the East Pacific Rise (EPR). Analysis of a previously prepared bacterial 16S ribosomal DNA (rDNA) library identified a spirochete most closely related to an endosymbiont of the oligochete Olavius loisae. This spirochete phylotype (spirochete A) comprised only 2.2% of the 16S rDNA clone library but appeared to be much more dominant when the same sample was analyzed by denaturing gradient gel electrophoresis (DGGE) and the terminal restriction fragment length polymorphism procedure (12 to 18%). PCR amplification of the community with spirochete-specific primers used in conjunction with DGGE analysis identified two spirochete phylotypes. The first spirochete was identical to spirochete A but was present in only one A. pompejana specimen. The second spirochete (spirochete B) was 84.5% similar to spirochete A and, more interestingly, was present in the epibiont communities of all of the A. pompejana specimens sampled throughout the geographic range of the worm (13 degrees N to 32 degrees S along the EPR). The sequence variation of the spirochete B phylotype was less than 3% for the range of A. pompejana specimens tested, suggesting that a single spirochete species was present in the A. pompejana epibiotic community. Additional analysis of the environments surrounding the worm revealed that spirochetes are a ubiquitous component of high-temperature vents and may play an important role in this unique ecosystem.

Bacterial symbiont transmission in the wood-boring shipworm Bankia setacea (Bivalvia: Teredinidae).

The Teredinidae (shipworms) are a morphologically diverse group of marine wood-boring bivalves that are responsible each year for millions of dollars of damage to wooden structures in estuarine and marine habitats worldwide. They exist in a symbiosis with cellulolytic nitrogen-fixing bacteria that provide the host with the necessary enzymes for survival on a diet of wood cellulose. These symbiotic bacteria reside in distinct structures lining the interlamellar junctions of the gill. This study investigated the mode by which these nutritionally essential bacterial symbionts are acquired in the teredinid Bankia setacea. Through 16S ribosomal DNA (rDNA) sequencing, the symbiont residing within the B. setacea gill was phylogenetically characterized and shown to be distinct from previously described shipworm symbionts. In situ hybridization using symbiont-specific 16S rRNA-directed probes bound to bacterial ribosome targets located within the host gill coincident with the known location of the gill symbionts. These specific probes were then used as primers in a PCR-based assay which consistently detected bacterial rDNA in host gill (symbiont containing), gonad tissue, and recently spawned eggs, demonstrating the presence of symbiont cells in host ovary and offspring. These results suggest that B. setacea ensures successful inoculation of offspring through a vertical mode of symbiont transmission and thereby enables a broad distribution of larval settlement.

Genetic variation among endosymbionts of widely distributed vestimentiferan tubeworms.

Vestimentiferan tubeworms thriving in sulfidic deep-sea hydrothermal vents and cold seeps are constrained by their nutritional reliance on chemoautotrophic endosymbionts. In a recent phylogenetic study using 16S ribosomal DNA, we found that endosymbionts from vent and seep habitats form two distinct clades with little variation within each clade. In the present study, we used two different approaches to assess the genetic variation among biogeographically distinct vestimentiferan symbionts. DNA sequences were obtained for the noncoding, internal transcribed spacer (ITS) regions of the rRNA operons of symbionts associated with six different genera of vestimentiferan tubeworms. ITS sequences from endosymbionts of host genera collected from different habitats and widely distributed vent sites were surprisingly conserved. Because the ITS region was not sufficient for distinguishing endosymbionts from different habitats or locations, we used a DNA fingerprinting technique, repetitive-extragenic-palindrome PCR (REP-PCR), to reveal differences in the distribution of repetitive sequences in the genomes of the bacterial endosymbionts. Most of the endosymbionts displayed unique REP-PCR patterns. A cladogram generated from these fingerprints reflected relationships that may be influenced by a variety of factors, including host genera, geographic location, and bottom type.

Diversity of dissimilatory bisulfite reductase genes of bacteria associated with the deep-sea hydrothermal vent polychaete annelid Alvinella pompejana.

A unique community of bacteria colonizes the dorsal integument of the polychaete annelid Alvinella pompejana, which inhabits the high-temperature environments of active deep-sea hydrothermal vents along the East Pacific Rise. The composition of this bacterial community was characterized in previous studies by using a 16S rRNA gene clone library and in situ hybridization with oligonucleotide probes. In the present study, a pair of PCR primers (P94-F and P93-R) were used to amplify a segment of the dissimilatory bisulfite reductase gene from DNA isolated from the community of bacteria associated with A. pompejana. The goal was to assess the presence and diversity of bacteria with the capacity to use sulfate as a terminal electron acceptor. A clone library of bisulfite reductase gene PCR products was constructed and characterized by restriction fragment and sequence analysis. Eleven clone families were identified. Two of the 11 clone families, SR1 and SR6, contained 82% of the clones. DNA sequence analysis of a clone from each family indicated that they are dissimilatory bisulfite reductase genes most similar to the dissimilatory bisulfite reductase genes of Desulfovibrio vulgaris, Desulfovibrio gigas, Desulfobacterium autotrophicum, and Desulfobacter latus. Similarities to the dissimilatory bisulfite reductases of Thermodesulfovibrio yellowstonii, the sulfide oxidizer Chromatium vinosum, the sulfur reducer Pyrobaculum islandicum, and the archaeal sulfate reducer Archaeoglobus fulgidus were lower. Phylogenetic analysis separated the clone families into groups that probably represent two genera of previously uncharacterized sulfate-reducing bacteria. The presence of dissimilatory bisulfite reductase genes is consistent with recent temperature and chemical measurements that documented a lack of dissolved oxygen in dwelling tubes of the worm. The diversity of dissimilatory bisulfite reductase genes in the bacterial community on the back of the worm suggests a prominent role for anaerobic sulfate-reducing bacteria in the ecology of A. pompejana.

Use of methacrylate De-embedding protocols for in situ hybridization on semithin plastic sections with multiple detection strategies.

New plastic resins are gradually replacing traditional paraffin-embedding in situ hybridization (ISH) strategies. One unique resin that has not been fully investigated or exploited with respect to light microscopic ISH is a methacrylate mixture. The advantage of this resin is its ability to be removed from tissues postsectioning, dramatically increasing hybridization signal compared to that obtained in other plastics. The goal of this study was to evaluate the general applicability of the methacrylate embedding acetone de-embedding (MEADE) technique for ISH investigations. Several high-resolution, high-sensitivity ISH protocols are described, using both end-labeled oligonucleotides and randomly primed DNA probes (200-400 BPS), signal amplification by catalyzed reporter deposition (CARD), and chromogenic and fluorescent detection methods. With slight modification, the MEADE ISH technique permitted localization of bacterial symbionts in fragile gill tissue and collagen transcripts in foot tissue of two marine bivalves. MEADE ISH has proved extremely versatile and will likely be suitable for many future applications.

Molecular Identification and Localization of Filamentous Symbiotic Bacteria Associated with the Hydrothermal Vent Annelid Alvinella pompejana.

Alvinella pompejana is a polychaetous annelid that inhabits high-temperature environments associated with active deep-sea hydrothermal vents along the East Pacific Rise. A unique and diverse epibiotic microflora with a prominent filamentous morphotype is found associated with the worm's dorsal integument. A previous study established the taxonomic positions of two epsilon proteobacterial phylotypes, 13B and 5A, which dominated a clone library of 16S rRNA genes amplified by PCR from the epibiotic microbial community of an A. pompejana specimen. In the present study deoxyoligonucleotide PCR primers specific for phylotypes 13B and 5A were used to demonstrate that these phylotypes are regular features of the bacterial community associated with A. pompejana. Assaying of other surfaces around colonies of A. pompejana revealed that phylotypes 13B and 5A are not restricted to A. pompejana. Phylotype 13B occurs on the exterior surfaces of other invertebrate genera and rock surfaces, and phylotype 5A occurs on a congener, Alvinella caudata. The 13B and 5A phylotypes were identified and localized on A. pompejana by in situ hybridization, demonstrating that these two phylotypes are, in fact, the prominent filamentous bacteria on the dorsal integument of A. pompejana. These findings indicate that the filamentous bacterial symbionts of A. pompejana are epsilon Proteobacteria which do not have an obligate requirement for A. pompejana.

Phylogenetic characterization of the epibiotic bacteria associated with the hydrothermal vent polychaete Alvinella pompejana.

Alvinella pompejana is a polychaetous annelid that inhabits active deep-sea hydrothermal vent sites along the East Pacific Rise, where it colonizes the walls of actively venting high-temperature chimneys. An abundant, morphologically diverse epibiotic microflora is associated with the worm's dorsal integument, with a highly integrated filamentous morphotype clearly dominating the microbial biomass. It has been suggested that this bacterial population participates in either the nutrition of the worm or in detoxification of the worm's immediate environment. The primary goal of this study was to phylogenetically characterize selected epibionts through the analysis of 16S rRNA gene sequences. Nucleic acids were extracted from bacteria collected from the dorsal surface of A. pompejana. 16S rRNA genes were amplified with universal bacterial primers by the PCR. These genes were subsequently cloned, and the resulting clone library was screened by restriction fragment length polymorphism analysis to identify distinct clone types. The restriction fragment length polymorphism analysis identified 32 different clone families in the library. Four of these families were clearly dominant, representing more than 65% of the library. Representatives from the four most abundant clone families were chosen for complete 16S rRNA gene sequencing and phylogenetic analysis. These gene sequences were analyzed by a variety of phylogenetic inference methods and found to be related to the newly established epsilon subdivision of the division Proteobacteria. Secondary structural model comparisons and comparisons of established signature base positions in the 16S rRNA confirmed the placement of the Alvinella clones in the epsilon subdivision of the Proteobacteria.

Vertical transmission of a chemoautotrophic symbiont in the protobranch bivalve, Solemya reidi.

Invertebrates that contain endosymbiotic chemoautotrophic bacteria are widely distributed in a variety of reducing marine habitats (i.e., hydrothermal vents and cold seeps). The mechanisms of symbiont transmission and incorporation during early host development are poorly understood primarily because these critical early life stages have not been procured. Solemya reidi is a gutless protobranch bivalve found inhabiting pulpmill effluent and sewage outfall sites. Endosymbiotic sulfur oxidizing bacteria are contained within specialized host cells of the gill filaments. These bivalves have the advantage over their deep-sea counterparts in that they can be spawned and cultured axenically, providing the opportunity to examine the ontogenetic process of symbiont incorporation. The 16S rDNA of the symbiont from Solemya reidi was sequenced from a PCR product obtained using two bacterial-specific primers. Phylogenetic analysis of this sequence identified a hypervariable region of the molecule that was unique to this symbiont. An 18-base oligonucleotide probe was synthesized and tested for specificity and sensitivity to detect its symbiont-specific target. Immobilized cellular RNAs from a range of taxa were used to screen the specificity of the Solemya probe. At high stringency, the symbiont-specific probe hybridized only to RNA extracted from gill tissue of S. reidi. When matched with a bacterial-specific forward primer in PCR amplifications, the symbiont-specific probe successfully amplified S. reidi symbiont 16S rDNA genes in genomic DNA isolated from the host ovary, eggs, and 1- and 4-day-old larvae. Amplifications detected the presence of symbiont target in the germ tissue and larvae. Nonradioactive in situ hybridizations were performed on 3-day-old larvae with the symbiont-specific probe. Detection of hybrids localized the symbionts to the epithelial test cells of the larvae. These results suggest that the symbionts are vertically transmitted with the egg and are incorporated as a post-metamorphosis event.

Transovarial inheritance of endosymbiotic bacteria in clams inhabiting deep-sea hydrothermal vents and cold seeps.

Vesicomyid clams are conspicuous fauna at many deep-sea hydrothermal-vent and cold-seep habitats. All species examined have specialized gill tissue harboring endosymbiotic bacteria, which are thought to provide the hosts' sole nutritional support. In these species mechanisms of symbiont inheritance are likely to be key elements of dispersal strategies. These mechanisms have remained unresolved because the early life stages are not available for developmental studies. A specific 16S rRNA-directed oligodeoxynucleotide probe (CG1255R) for the vesocomyid endosymbionts was used in a combination of sensitive hybridization techniques to detect and localize the endosymbionts in host germ tissues. Symbiont-specific polymerase chain reaction amplifications, comparative gene sequencing, and restriction fragment length polymorphisms were used to detect and confirm the presence of symbiont target in tissue nucleic acid extracts. Nonradioactive in situ hybridizations were used to resolve the position of the bacterial endosymbionts in host cells. Symbiont 16S rRNA genes were consistently amplified from the ovarial tissue of three species of vesicomyid clams: Calyptogena magnifica, C. phaseoliformis, and C. pacifica. The nucleotide sequences of the genes amplified from ovaries were identical to those from the respective host symbionts. In situ hybridizations to CG1255R labeled with digoxigenin-11-dUTP were performed on ovarial tissue from each of the vesicomyid clams. Detection of hybrids localized the symbionts to follicle cells surrounding the primary oocytes. These results suggest that vesicomyid clams assure successful, host-specific inoculation of all progeny by using a transovarial mechanism of symbiont transmission.

Identification and localization of bacterial endosymbionts in hydrothermal vent taxa with symbiont-specific polymerase chain reaction amplification and in situ hybridization techniques.

Invertebrates that contain endosymbiotic chemoautotrophic eubacteria are widely distributed in a variety of reducing marine habitats, including deepsea hydrothermal vents. The mechanisms of symbiont transmission in these invertebrates are not understood. To test the hypothesis that symbionts are transmitted via the eggs of hosts, we used group-specific hybridization probes complementary to 16S ribosomal RNAs (rRNAs) to look for symbionts in eggs and ovaries. 16S rRNA sequences were examined for domains unique to the symbionts of three vent animals: Calyptogena magnifica, Bathymodiolus thermophilus, and Riftia pachyptila. Three 16S rRNA-directed oligodeoxynucleotide hybridization probes (CG-1255R, RP-1243R, BT-1255R) specific for these endosymbionts were synthesized and evaluated by dot-blot hybridization. At higher stringencies, all three probes showed a high degree of specificity for their target endosymbionts rRNAs. The probes were also used as polymerase chain reaction (PCR) primers for detection of the symbiont 16S rRNA genes in genomic DNA isolated from host tissues known to contain symbionts. All three symbiont-specific probes were highly sensitive and specific as PCR primers; they successfully amplified 1 pg target DNA. However, all amplifications of extracted egg DNA from the vestimentiferan R. pachyptila with either universal eubacterial (Eub A/B) or the Riftia symbiont-specific (RP-1243R/Eub B) primer sets were unsuccessful. Nonradioactive in situ hybridizations were performed on ovarian tissue from the vestimentiferan Ridgea piscesae using RP-1243R, 3' end-labeled with digoxigenin-11-dUTP (Boehringer Mannheim). The probe was subsequently detected with an alkaline phosphatase-conjugated immunoglobulin G antibody specific for the digoxigenin moeity. The probe bound only to the tissue of R. pisceasae coincident with the known location of symbiont cells and was not detected in any region of the ovary. These data indicate that transovarial symbiont transmission in the vestimentiferans does not take place and that symbiont acquisition is probably a post-spawning event.

Mussel growth supported by methane as sole carbon and energy source.

Symbioses between chemoautotrophic bacteria and several specialized marine invertebrates are well documented. However, none of these symbioses have been demonstrated to provide sufficient energy and carbon to the host to enable it to grow. Growth rates of seep mussels collected from hydrocarbon seeps off the coast of Louisiana were measured in a controlled environment where methane was the sole carbon and energy source. The growth rates increased to a maximum of 17.2 micrometers per day in response to methane and approached zero in the absence of methane. These mussels contain methanotrophic symbiotic bacteria in their gills, which suggests that these bacteria provide their hosts with a net carbon flux originating from methane.

Peritoneal lavage as a diagnostic procedure for intra-abdominal trauma.

Peritoneal lavage is an established emergency diagnostic tool in cases of suspected damage inside the abdomen due to blunt and occasionally penetrating abdominal trauma. This is a relatively safe procedure which is often useful in recognizing intra-abdominal bleeding requiring surgical exploration. The procedure requires careful patient selection and technique, together with awareness of the limitations of the procedure and the ability to interpret the returns of peritoneal lavage.