rRNA Gene Clone Library Analysis Research Articles

Substrate-Specific Clades of Active Marine Methylotrophs Associated with a Phytoplankton Bloom in a Temperate Coastal Environment

Marine microorganisms that consume one-carbon (C(1)) compounds are poorly described, despite their impact on global climate via an influence on aquatic and atmospheric chemistry. This study investigated marine bacterial communities involved in the metabolism of C(1) compounds. These communities were of relevance to surface seawater and atmospheric chemistry in the context of a bloom that was dominated by phytoplankton known to produce dimethylsulfoniopropionate. In addition to using 16S rRNA gene fingerprinting and clone libraries to characterize samples taken from a bloom transect in July 2006, seawater samples from the phytoplankton bloom were incubated with (13)C-labeled methanol, monomethylamine, dimethylamine, methyl bromide, and dimethyl sulfide to identify microbial populations involved in the turnover of C(1) compounds, using DNA stable isotope probing. The [(13)C]DNA samples from a single time point were characterized and compared using denaturing gradient gel electrophoresis (DGGE), fingerprint cluster analysis, and 16S rRNA gene clone library analysis. Bacterial community DGGE fingerprints from (13)C-labeled DNA were distinct from those obtained with the DNA of the nonlabeled community DNA and suggested some overlap in substrate utilization between active methylotroph populations growing on different C(1) substrates. Active methylotrophs were affiliated with Methylophaga spp. and several clades of undescribed Gammaproteobacteria that utilized methanol, methylamines (both monomethylamine and dimethylamine), and dimethyl sulfide. rRNA gene sequences corresponding to populations assimilating (13)C-labeled methyl bromide and other substrates were associated with members of the Alphaproteobacteria (e.g., the family Rhodobacteraceae), the Cytophaga-Flexibacter-Bacteroides group, and unknown taxa. This study expands the known diversity of marine methylotrophs in surface seawater and provides a comprehensive data set for focused cultivation and metagenomic analyses in the future.

Characterization of the Community Structure of a Dechlorinating Mixed Culture and Comparisons of Gene Expression in Planktonic and Biofloc-Associated “Dehalococcoides” andMethanospirillumSpecies

This study sought to characterize bacterial and archaeal populations in a perchloroethene- and butyrate-fed enrichment culture containing hydrogen-consuming "Dehalococcoides ethenogenes" strain 195 and a Methanospirillum hungatei strain. Phylogenetic characterization of this microbial community was done via 16S rRNA gene clone library and gradient gel electrophoresis analyses. Fluorescence in situ hybridization was used to quantify populations of "Dehalococcoides" and Archaea and to examine the colocalization of these two groups within culture bioflocs. A technique for enrichment of planktonic and biofloc-associated biomass was developed and used to assess differences in population distribution and gene expression patterns following provision of substrate. On a per-milliliter-of-culture basis, most D. ethenogenes genes (the hydrogenase gene hupL; the highly expressed gene for an oxidoreductase of unknown function, fdhA; the RNA polymerase subunit gene rpoB; and the 16S rRNA gene) showed no statistical difference in expression between planktonic and biofloc enrichments at either time point studied (1 to 2 and 6 h postfeeding). Normalization of transcripts to ribosome (16S rRNA) levels supported that planktonic and biofloc-associated D. ethenogenes had similar gene expression profiles, with one notable exception; planktonic D. ethenogenes showed higher expression of tceA relative to biofloc-associated cells at 6 h postfeeding. These trends were compared to those for the hydrogen-consuming methanogen in the culture, M. hungatei. The vast majority of M. hungatei cells, ribosomes (16S rRNA), and transcripts of the hydrogenase gene mvrD and the housekeeping gene rpoE were observed in the biofloc enrichments. This suggests that, unlike the comparable activity of D. ethenogenes from both enrichments, planktonic M. hungatei is responsible for only a small fraction of the hydrogenotrophic methanogenesis in this culture.

Two-dimensional mapping of photopigment distribution and activity of Chloroflexus-like bacteria in a hypersaline microbial mat

Pigment analysis in an intact hypersaline microbial mat by hyperspectral imaging revealed very patchy and spatially uncorrelated distributions of photopigments Chl a and BChl a/c, which are characteristic photopigments for oxygenic (diatoms and cyanobacteria) and anoxygenic phototrophs (Chloroflexaceae). This finding is in contrast to the expectation that these biomarker pigments should be spatially correlated, as oxygenic phototrophs are thought to supply the Chloroflexaceae members with organic substrates for growth. We suggest that the heterogeneous occurrence is possibly due to sulfide, whose production by sulfate-reducing bacteria may be spatially heterogeneous in the partially oxic photic zone of the mat. We furthermore mapped the near-infra-red-light controlled respiration of Chloroflexaceae under light and dark conditions and found that Chloroflexaceae are responsible for a major part of oxygen consumption at the lower part of the oxic zone in the mat. The presence of Chloroflexaceae was further confirmed by FISH probe and 16S rRNA gene clone library analysis. We assume that species related to the genera Oscillochloris and 'Candidatus Chlorothrix', in contrast to those related to Chloroflexus and Roseiflexus, depend less on excreted photosynthates but more on the presence of free sulfide, which may explain their presence in deeper parts of the mat.

Prokaryotic diversity in Tuz Lake, a hypersaline environment in Inland Turkey

Tuz Lake is an inland thalassohaline water body located in central Anatolia that contributes to 60% of the total salt production in Turkey per year. The microbiota inhabiting this lake has been studied by FISH, denaturing gradient gel electrophoresis of PCR-amplified fragments of 16S rRNA genes, and 16S rRNA gene clone library analysis. Total cell counts per milliliter (1.38 x 10(7)) were in the range of the values normally found for hypersaline environments. The proportion of Bacteria to Archaea in the community detectable by FISH was one to three. 16S rRNA gene clone libraries indicated that the archaeal assemblage was dominated by members of the Square Haloarchaea of the Walsby group, although some other groups were also found. Bacteria were dominated by members of the Bacteroidetes, including Salinibacter ruber-related phylotypes. Because members of Bacteroidetes are widely present in different hypersaline environments, a phylogenetic analysis of 16S rRNA gene sequences from Bacteroidetes retrieved from these environments was carried out in order to ascertain whether they formed a unique cluster. Sequences retrieved from Tuz Lake and a group of sequences from other hypersaline environments clustered together in a branch that could be considered as the 'halophilic branch' within the Bacteroidetes phylum.

Cathodic oxygen reduction catalyzed by bacteria in microbial fuel cells

Microbial fuel cells (MFCs) have the potential to combine wastewater treatment efficiency with energetic efficiency. One of the major impediments to MFC implementation is the operation of the cathode compartment, as it employs environmentally unfriendly catalysts such as platinum. As recently shown, bacteria can facilitate sustainable and cost-effective cathode catalysis for nitrate and also oxygen. Here we describe a carbon cathode open to the air, on which attached bacteria catalyzed oxygen reduction. The bacteria present were able to reduce oxygen as the ultimate electron acceptor using electrons provided by the solid-phase cathode. Current densities of up to 2.2 A m(-2) cathode projected surface were obtained (0.303+/-0.017 W m(-2), 15 W m(-3) total reactor volume). The cathodic microbial community was dominated by Sphingobacterium, Acinetobacter and Acidovorax sp., according to 16S rRNA gene clone library analysis. Isolates of Sphingobacterium sp. and Acinetobacter sp. were obtained using H(2)/O(2) mixtures. Some of the pure culture isolates obtained from the cathode showed an increase in the power output of up to three-fold compared to a non-inoculated control, that is, from 0.015+/-0.001 to 0.049+/-0.025 W m(-2) cathode projected surface. The strong decrease in activation losses indicates that bacteria function as true catalysts for oxygen reduction. Owing to the high overpotential for non-catalyzed reduction, oxygen is only to a limited extent competitive toward the electron donor, that is, the cathode. Further research to refine the operational parameters and increase the current density by modifying the electrode surface and elucidating the bacterial metabolism is warranted.

Microbial population changes during bioremediation of nitroaromatic- and nitramine-contaminated lagoon

Nitration reactions of aromatic compounds are commonly involved in military industrial processes. Military industries treated their process effluents using lagoon systems for many years. In this study, the sediment of a lagoon was investigated from a bioremediation objective. The physico-chemical characterization of the sediments showed the organic nature of the sediment (25.4% carbon with a C:N=3) highly concentrated in RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine), HMX (octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine) as well as two herbicides Dinoterb (2- tert-butyl-4,6-dinitrophenol) and Dinoseb (2- sec-butyl-4,6-dinitrophenol). Analysis of the 16S rRNA gene clone library revealed the presence of three dominant families, Geobacteriacea, Clostridiaceae and Pseudomonaceae. A bioremediation assay was carried out in anaerobic conditions in order to degrade organic compounds. In these conditions, 100% of Dinoterb and Dinoseb were degraded after 75 days of culture, while RDX and HMX were not consumed. The 16S rRNA gene clone library analysis of this incubation showed a drastic reduction of the final biodiversity composed by clones related to Enterobacteriaceae (especially Leclercia adecarboxylata) and Pseudomonaceae family. It was then suggested that Enterobacteriaceae and Pseudomonaceae were potentially involved in biodegradation of these two herbicides. To confirm this hypothesis, cultures were carried out with isolated species of Pseudomonas putida, Pseudomonas citronellolis and L. adecarboxylata in the presence of Dinoterb. The data confirmed that in the presence of glucose, these microorganisms are able to consume Dinoterb.

Characterization of a filamentous biofilm community established in a cellulose-fed microbial fuel cell

BackgroundMicrobial fuel cells (MFCs) are devices that exploit microorganisms to generate electric power from organic matter. Despite the development of efficient MFC reactors, the microbiology of electricity generation remains to be sufficiently understood.ResultsA laboratory-scale two-chamber microbial fuel cell (MFC) was inoculated with rice paddy field soil and fed cellulose as the carbon and energy source. Electricity-generating microorganisms were enriched by subculturing biofilms that attached onto anode electrodes. An electric current of 0.2 mA was generated from the first enrichment culture, and ratios of the major metabolites (e.g., electric current, methane and acetate) became stable after the forth enrichment. In order to investigate the electrogenic microbial community in the anode biofilm, it was morphologically analyzed by electron microscopy, and community members were phylogenetically identified by 16S rRNA gene clone-library analyses. Electron microscopy revealed that filamentous cells and rod-shaped cells with prosthecae-like filamentous appendages were abundantly present in the biofilm. Filamentous cells and appendages were interconnected via thin filaments. The clone library analyses frequently detected phylotypes affiliated with Clostridiales, Chloroflexi, Rhizobiales and Methanobacterium. Fluorescence in-situ hybridization revealed that the Rhizobiales population represented rod-shaped cells with filamentous appendages and constituted over 30% of the total population.ConclusionBacteria affiliated with the Rhizobiales constituted the major population in the cellulose-fed MFC and exhibited unique morphology with filamentous appendages. They are considered to play important roles in the cellulose-degrading electrogenic community.

Changes in Bacterial Communities of the Marine Sponge Mycale laxissima on Transfer into Aquaculture

The changes in bacterial communities associated with the marine sponge Mycale laxissima on transfer to aquaculture were studied using culture-based and molecular techniques. M. laxissima was maintained alive in flowthrough and closed recirculating aquaculture systems for 2 years and 1 year, respectively. The bacterial communities associated with wild and aquacultured sponges, as well as the surrounding water, were assessed using 16S rRNA gene clone library analysis and denaturing gradient gel electrophoresis (DGGE). Bacterial richness and diversity were measured using DOTUR computer software, and clone libraries were compared using S-LIBSHUFF. DGGE analysis revealed that the diversity of the bacterial community of M. laxissima increased when sponges were maintained in aquaculture and that bacterial communities associated with wild and aquacultured M. laxissima were markedly different than those of the corresponding surrounding water. Clone libraries of bacterial 16S rRNA from sponges confirmed that the bacterial communities changed during aquaculture. These communities were significantly different than those of seawater and aquarium water. The diversity of bacterial communities associated with M. laxissima increased significantly in aquaculture. Our work shows that it is important to monitor changes in bacterial communities when examining the feasibility of growing sponges in aquaculture systems because these communities may change. This could have implications for the health of sponges or for the production of bioactive compounds by sponges in cases where these compounds are produced by symbiotic bacteria rather than by the sponges themselves.

Association of Mycoplasma ovipneumoniae Infection with Population-Limiting Respiratory Disease in Free-Ranging Rocky Mountain Bighorn Sheep ( Ovis canadensis canadensis )

Bronchopneumonia is a population-limiting disease in bighorn sheep in much of western North America. Previous investigators have isolated diverse bacteria from the lungs of affected sheep, but no single bacterial species is consistently present, even within single epizootics. We obtained high-quality diagnostic specimens from nine pneumonic bighorn sheep in three populations and analyzed the bacterial populations present in bronchoalveolar lavage specimens of seven by using a culture-independent method (16S rRNA gene amplification and clone library analyses). Mycoplasma ovipneumoniae was detected as a predominant member of the pneumonic lung flora in lambs with early lesions of bronchopneumonia. Specific PCR tests then revealed the consistent presence of M. ovipneumoniae in the lungs of pneumonic bighorn sheep in this study, and M. ovipneumoniae was isolated from lung specimens of five of the animals. Retrospective application of M. ovipneumoniae PCR to DNA extracted from archived formalin-fixed, paraffin-embedded lung tissues of historical adult bighorn sheep necropsy specimens supported the association of this agent with bronchopneumonia (16/34 pneumonic versus 0/17 nonpneumonic sheep were PCR positive [P < 0.001]). Similarly, a very strong association was observed between the presence of one or more M. ovipneumoniae antibody-positive animals and the occurrence of current or recent historical bronchopneumonia problems (seropositive animals detected in 9/9 versus 0/9 pneumonic and nonpneumonic populations, respectively [P < 0.001]). M. ovipneumoniae is strongly associated with bronchopneumonia in free-ranging bighorn sheep and is a candidate primary etiologic agent for this disease.

Microbial life in Champagne Pool, a geothermal spring in Waiotapu, New Zealand

Surveys of Champagne Pool, one of New Zealand's largest terrestrial hot springs and rich in arsenic ions and compounds, have been restricted to geological and geochemical descriptions, and a few microbiological studies applying culture-independent methods. In the current investigation, a combination of culture and culture-independent approaches were chosen to determine microbial density and diversity in Champagne Pool. Recovered total DNA and adenosine 5'-triphosphate (ATP) content of spring water revealed relatively low values compared to other geothermal springs within New Zealand and are in good agreement with low cell numbers of 5.6 +/- 0.5 x 10(6) cells/ml obtained for Champagne Pool water samples by 4',6-diamidino-2-phenylindole (DAPI) staining. Denaturing Gradient Gel Electrophoresis (DGGE) and 16S rRNA (small-subunit ribosomal nucleic acid) gene clone library analyses of environmental DNA indicated the abundance of Sulfurihydrogenibium, Sulfolobus, and Thermofilum-like populations in Champagne Pool. From these results, media were selected to target the enrichment of hydrogen-oxidizing and sulfur-dependent microorganisms. Three isolates were successfully obtained having 16S rRNA gene sequences with similarities of approximately 98% to Thermoanaerobacter tengcongensis, 94% to Sulfurihydrogenibium azorense, and 99% to Thermococcus waiotapuensis, respectively.

Phylogenetic analysis of intestinal bacteria in the Chinese mitten crab (Eriocheir sinensis)

To identify the dominant intestinal bacteria in the Chinese mitten crab, and to investigate the differences in the intestinal bacteria between pond-raised and wild crabs. The diversity of intestinal bacteria in the Chinese mitten crabs was investigated by denaturing gradient gel electrophoresis (DGGE) fingerprinting, 16S rRNA gene clone library analysis and real-time quantitative PCR. The principal component analysis of DGGE profiles indicated that substantial intersubject variations existed in intestinal bacteria in pond-raised crab. The sequencing of 16S rRNA genes revealed that 90-95% of the phylotypes in the clone libraries were affiliated with Proteobacteria and Bacteroidetes. Some genera were identified as unique in wild crabs and in pond-raised crabs, whereas Bacteroidetes was found to be common in all sampled crab groups. Real-time quantitative PCR indicated that the abundance of Bacteroides and the total bacterial load were approximately four-to-10 times higher in pond-raised crabs than in wild crabs. A significant portion of the phylotypes shared low similarity with previously sequenced organisms, indicating that the bacteria in the gut of Chinese mitten crabs are yet to be described. The intestinal bacteria of pond-raised crabs showed higher intersubject variation, total diversity and abundance than that observed in wild crabs. The high proportion of the clones of Proteobacteria and Bacteroidetes in the clone library is an indication that these bacteria may be the dominant population in the gut of the Chinese mitten crab. This study demonstrated obvious differences in the intestinal bacterial composition of pond-raised crabs and wild crabs. This knowledge will increase our understanding of the effects of aquaculture operations on bacterial community composition in the crab gut and provide necessary data for the development of probiotic products for crab cultivation.

Selective Phylogenetic Analysis Targeting 16S rRNA Genes of Hyperthermophilic Archaea in the Deep-Subsurface Hot Biosphere

International drilling projects for the study of microbial communities in the deep-subsurface hot biosphere have been expanded. Core samples obtained by deep drilling are commonly contaminated with mesophilic microorganisms in the drilling fluid, making it difficult to examine the microbial community by 16S rRNA gene clone library analysis. To eliminate mesophilic organism contamination, we previously developed a new method (selective phylogenetic analysis [SePA]) based on the strong correlation between the guanine-plus-cytosine (G+C) contents of the 16S rRNA genes and the optimal growth temperatures of prokaryotes, and we verified the method's effectiveness (H. Kimura, M. Sugihara, K. Kato, and S. Hanada, Appl. Environ. Microbiol. 72:21-27, 2006). In the present study we ascertained SePA's ability to eliminate contamination by archaeal rRNA genes, using deep-sea hydrothermal fluid (117 degrees C) and surface seawater (29.9 degrees C) as substitutes for deep-subsurface geothermal samples and drilling fluid, respectively. Archaeal 16S rRNA gene fragments, PCR amplified from the surface seawater, were denatured at 82 degrees C and completely digested with exonuclease I (Exo I), while gene fragments from the deep-sea hydrothermal fluid remained intact after denaturation at 84 degrees C because of their high G+C contents. An examination using mixtures of DNAs from the two environmental samples showed that denaturation at 84 degrees C and digestion with Exo I completely eliminated archaeal 16S rRNA genes from the surface seawater. Our method was quite useful for culture-independent community analysis of hyperthermophilic archaea in core samples recovered from deep-subsurface geothermal environments.

Characterization of the microbial diversity in a permafrost sample from the Canadian high Arctic using culture-dependent and culture-independent methods

A combination of culture-dependent and culture-independent methodologies (Bacteria and Archaea 16S rRNA gene clone library analyses) was used to determine the microbial diversity present within a geographically distinct high Arctic permafrost sample. Culturable Bacteria isolates, identified by 16S rRNA gene sequencing, belonged to the phyla Firmicutes, Actinobacteria and Proteobacteria with spore-forming Firmicutes being the most abundant; the majority of the isolates (19/23) were psychrotolerant, some (11/23) were halotolerant, and three isolates grew at -5 degrees C. A Bacteria 16S rRNA gene library containing 101 clones was composed of 42 phylotypes related to diverse phylogenetic groups including the Actinobacteria, Proteobacteria, Firmicutes, Cytophaga - Flavobacteria - Bacteroides, Planctomyces and Gemmatimonadetes; the bacterial 16S rRNA gene phylotypes were dominated by Actinobacteria- and Proteobacteria-related sequences. An Archaea 16S rRNA gene clone library containing 56 clones was made up of 11 phylotypes and contained sequences related to both of the major Archaea domains (Euryarchaeota and Crenarchaeota); the majority of sequences in the Archaea library were related to halophilic Archaea. Characterization of the microbial diversity existing within permafrost environments is important as it will lead to a better understanding of how microorganisms function and survive in such extreme cryoenvironments.

Bacterial reduction and persistence after endodontic treatment procedures

Bacteria that persist after endodontic disinfection procedures may lead to treatment failure. Over 50% of the bacteria found in endodontic infections are as-yet-uncultivated so investigations of bacteria that endure treatment procedures should include techniques that side-step cultivation. This culture-independent study evaluated the bacterial reduction promoted by intracanal disinfection procedures and identified the taxa persisting after treatment. Samples taken from the infected canals of teeth with apical periodontitis before treatment (S1), after instrumentation using NaOCl as irrigant (S2) and after interappointment medication with a calcium hydroxide paste (S3) were subjected to 16S rRNA gene clone library and real-time polymerase chain reaction analyses. The S2 and S3 samples from five of the 15 canals showed negative results. In the other cases, instrumentation and instrumentation/medication promoted a significant reduction (99.67% and 99.85%, respectively) in the number of bacteria when compared to S1 samples. Forty-three distinct bacterial taxa were identified, of which 24 (56%) were as-yet-uncultivated phylotypes. Nineteen of these 43 taxa (including eight as-yet-uncultivated phylotypes) were disclosed in post-treatment samples, with streptococci being the most prevalent taxa. Findings demonstrated that culture-independent methods provide a detailed insight into the effects of intracanal disinfection protocols, helping to define more effective strategies to deal with endodontic bacteria, including as-yet-uncultivated phylotypes.

Microbial community associated with the colonial ascidian Cystodytes dellechiajei

The ascidian Cystodytes dellechiajei (Della Valle, 1877) (phylum Chordata, class Ascidiacea, family Polycitoridae) is a colonial tunicate that inhabits benthic rock environments in the Atlantic, Pacific and Indian Oceans, as well as the Mediterranean Sea. Its life cycle has two phases, the adult sessile colony and the free-living larva. Both adult zooids and larvae are surrounded by a protective tunic that contains several eukaryotic cell lines, is composed mainly of acidic mucopolysacharides associated with collagen and elastin-like proteins, and is covered by a thin cuticle. The microbiota associated with the tunic tissues of adult colonies and larva of C. dellechiajei has been examined by optical, confocal and electron microscopy and by fluorescence in situ hybridization (FISH), denaturing gradient gel electrophoresis (DGGE), and 16S rRNA gene clone library analysis. Microscopy analyses indicated the presence inside the tunic, both for the adult and the larva, of a dense community of Bacteria while only the external surface of colony cuticle was colonized by diatoms, rodophyte algae and prokaryotic-like epiphytes. Transmission electron microscopy showed tunic eukaryotic cells that were engulfing and lysing bacteria. 16S rRNA gene analyses (DGGE and clone libraries) and FISH indicated that the community inside the tunic tissues of the adults and larvae was dominated by Alphaproteobacteria. Bacteria belonging to the phyla Gammaproteobacteria and Bacteroidetes were also detected in the adults. Many of the 16S rRNA gene sequences in the tunic tissues were related to known aerobic anoxygenic phototrophs (AAP), like Roseobacter sp. and Erythrobacter sp. In order to check whether the gene pufM, coding for the M subunit of the reaction centre complex of aerobic anoxygenic photosynthesis, was being expressed inside the ascidian tissues, two libraries, one for an adult colony and one for larva, of cDNA from the expressed pufM gene were also constructed. The sequences most frequently (64% for colony and 67% for larva) retrieved from these libraries presented > 90% aa identity with the pufM gene product of the Roseobacter-like group, a cluster of AAP widely detected in marine planktonic environments.

Bacterial community structure associated with the Antarctic soft coral, Alcyonium antarcticum

The structure and composition of microbial communities inhabiting the soft coral Alcyonium antarcticum were investigated across three differentially contaminated sites within McMurdo Sound, Antarctica. Diverse microbial communities were revealed at all sites using culture-based analysis, denaturing gradient gel electrophoresis (DGGE), 16S rRNA gene clone-library analysis, and FISH. Phylogenetic analysis of isolates and retrieved sequences demonstrated close affiliation with known psychrophiles from the Antarctic environment and high similarity to Gammaproteobacteria clades of sponge-associated microorganisms. The majority of bacteria detected with all techniques reside within the Gammaproteobacteria, although other phylogenetic groups including Alpha- and Betaproteobacteria, Bacteroidetes, Firmicutes, Actinomycetales, Planctomycetes, and Chlorobi and bacteria from the functional group of sulfate-reducing bacteria were also present. Multivariate (nMDS) analysis of DGGE banding patterns and principal component analysis of quantitative FISH data revealed no distinct differences in community composition between differentially contaminated sites. Rather, conserved coral-associated bacterial groups were observed within and between sites, providing evidence to support specific coral-microbial interactions. This is the first investigation of microbial communities associated with Antarctic soft corals, and the results suggest that spatially stable microbial associations exist across an environmental impact gradient.

Microbial Diversity in Maras Salterns, a Hypersaline Environment in the Peruvian Andes

Maras salterns are located 3,380 m above sea level in the Peruvian Andes. These salterns consist of more than 3,000 little ponds which are not interconnected and act as crystallizers where salt precipitates. These ponds are fed by hypersaline spring water rich in sodium and chloride. The microbiota inhabiting these salterns was examined by fluorescence in situ hybridization (FISH), 16S rRNA gene clone library analysis, and cultivation techniques. The total counts per milliliter in the ponds were around 2 x 10(6) to 3 x 10(6) cells/ml, while the spring water contained less than 100 cells/ml and did not yield any detectable FISH signal. The microbiota inhabiting the ponds was dominated (80 to 86% of the total counts) by Archaea, while Bacteria accounted for 10 to 13% of the 4',6'-diamidino-2-phenylindole (DAPI) counts. A total of 239 16S rRNA gene clones were analyzed (132 Archaea clones and 107 Bacteria clones). According to the clone libraries, the archaeal assemblage was dominated by microorganisms related to the cosmopolitan square archaeon "Haloquadra walsbyi," although a substantial number of the sequences in the libraries (31% of the 16S rRNA gene archaeal clones) were related to Halobacterium sp., which is not normally found in clone libraries from solar salterns. All the bacterial clones were closely related to each other and to the gamma-proteobacterium "Pseudomonas halophila" DSM 3050. FISH analysis with a probe specific for this bacterial assemblage revealed that it accounted for 69 to 76% of the total bacterial counts detected with a Bacteria-specific probe. When pond water was used to inoculate solid media containing 25% total salts, both extremely halophilic Archaea and Bacteria were isolated. Archaeal isolates were not related to the isolates in clone libraries, although several bacterial isolates were very closely related to the "P. halophila" cluster found in the libraries. As observed for other hypersaline environments, extremely halophilic bacteria that had ecological relevance seemed to be easier to culture than their archaeal counterparts.

Characterization of a Culturable Alphaproteobacterial Symbiont Common to Many Marine Sponges and Evidence for Vertical Transmission via Sponge Larvae

A closely related group of alphaproteobacteria were found to be present in seven genera of marine sponges from several locations and were shown to be transferred between sponge generations through the larvae in one of these sponges. Isolates of the alphaproteobacterium were cultured from the sponges Axinella corrugata, Mycale laxissima, Monanchora unguifera, and Niphates digitalis from Key Largo, Florida; Didiscus oxeata and Monanchora unguifera from Discovery Bay, Jamaica; an Acanthostronglyophora sp. from Manado, Indonesia; and Microciona prolifera from the Cheasapeake Bay in Maryland. Isolates were very similar to each other on the basis of 16S rRNA gene sequence (>99% identity) and are closely related to Pseudovibrio denitrificans. The bacterium was never isolated from surrounding water samples and was cultured from larvae of M. laxissima, indicating that it is a vertically transmitted symbiont in this sponge. Denaturing gradient gel electrophoresis, 16S rRNA gene clone library analysis, and fluorescent in situ hybridization with probes specific to the alphaproteobacterium confirmed the presence of this bacterium in the M. laxissima larvae. The alphaproteobacterium was densely associated with the larvae rather than being evenly distributed throughout the mesohyl. This is the first report of the successful culture of a bacterial symbiont of a sponge that is transferred through the gametes.

Phylogenetic analysis and in situ identification of bacteria community composition in an acidic Sphagnum peat bog.

The Bacteria community composition in an acidic Sphagnum peat bog (pH 3.9 to 4.5) was characterized by a combination of 16S rRNA gene clone library analysis, rRNA-targeted fluorescence in situ hybridization (FISH), and cultivation. Among 84 environmental 16S rRNA gene clones, a set of only 16 cloned sequences was closely related (>or=95% similarity) to taxonomically described organisms. Main groups of clones were affiliated with the Acidobacteria (24 clones), Alphaproteobacteria (20), Verrucomicrobia (13), Actinobacteria (8), Deltaproteobacteria (4), Chloroflexi (3), and Planctomycetes (3). The proportion of cells that hybridized with oligonucleotide probes specific for members of the domains Bacteria (EUB338-mix) and Archaea (ARCH915 and ARC344) accounted for only 12 to 22% of the total cell counts. Up to 24% of the EUB338-positive cells could be assigned by FISH to specific bacterial phyla. Alphaproteobacteria and Planctomycetes were the most numerous bacterial groups (up to 1.3x10(7) and 1.1x10(7) cells g-1 peat, respectively). In contrast to conventional plating techniques, a novel biofilm-mediated enrichment approach allowed us to isolate some representatives of predominant Bacteria groups, such as Acidobacteria and Planctomycetes. This novel strategy has great potential to enable the isolation of a significant proportion of the peat bog bacterial diversity.

Microbial community of a mesophilic propionate-degrading methanogenic consortium in chemostat cultivation analyzed based on 16S rRNA and acetate kinase genes

We constructed a mesophilic anaerobic chemostat that was continuously fed with synthetic wastewater containing propionate as the sole source of carbon and energy. Steady-state conditions were achieved below the critical dilution rate of 0.3 d (-1) with almost complete substrate degradation. The propionate-degrading methanogenic communities in the chemostat at dilution rates of 0.01, 0.08, and 0.3 d (-1) were analyzed using molecular biological techniques. Fluorescence in situ hybridization with archaeal and bacterial domain-specific probes showed that archaeal cells predominated throughout the three dilution rates. Archaeal-16S rRNA gene clone library analysis and quantitative real-time polymerase chain reaction studies showed that hydrogenotrophic methanogen rRNA genes closely related to Methanoculleus was detected at a dilution rate of 0.01 d (-1) , whereas rRNA genes closely related to the Methanoculleus and Methanospirillum genera were detected at dilution rates of 0.08 and 0.3 d (-1) . The aceticlastic methanogen, Methanosaeta , was detected throughout the three dilution rates. Bacterial-rRNA gene clone library analysis and denaturing gradient gel electrophoresis demonstrated that rRNA genes affiliated with the genus Syntrophobacter predominated at the low dilution rate, whereas rRNA genes affiliated with the phylum Firmicutes predominated at the higher dilution rates. A significant number of rRNA genes affiliated with the genus Pelotomaculum were detected at dilution rate of 0.3 d (-1) . The diversity of genes encoding acetate kinase agreed closely with the results of the rRNA gene analysis. The dilution rates significantly altered the archaeal and bacterial communities in the propionate-fed chemostat.