Ribosomal DNA Clone Research Articles

The Relationship Between Dust Sources and Airborne Bacteria in the Southwest of Iran.

The biological agents are carried from deserts and dried lands to long distances by high dust volumes. Their adverse effects can be reduced by specifying and controlling dust sources and their related biological agents. Thus, the current work examined the relationship between the bacteria in air and soil samples by taking samples from the soil surface of two dust sources, as well as from air samples during spring from Khorramshahr and Abadan cities. The dust event is the most influential factor on airborne bacteria. There is an insignificant negative (-0.06), insignificant positive (0.14), and weak positive (0.24) correlation between airborne bacteria and UV radiation, relative humidity, and temperature, respectively. After preparing a 16S ribosomal DNA (rDNA) clone library from the soil and air samples, operational taxonomic unit picking and taxonomic assignment were conducted using QIIME Virtual Box. In the present work, Bacillus was the dominant species. The relationship between dust sources and air samples was determined by principal component analysis. Bacteria in the Hoor-Al-Azim dust source and airborne bacteria on dusty and non-dusty days showed a more significant correlation compared to bacteria in the Shadegan dust source. Source Tracker software was used to estimate the contribution of dust sources. The primary source of dust was associated with the dried areas of Hoor-Al-Azim on the non-dusty and dusty days. Finally, the long transport of airborne bacteria was assessed by moderate resolution imaging spectroradiometer (MODIS) and the back trajectory model of Hybrid Single-Particle Lagrangian Integrated Trajectory (HYSPLIT) data. The research findings can help decision-makers prioritize dust sources to control the adverse effects of dust.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11356-022-21563-6.

Comparison of the effects of biocontrol agent Bacillus subtilis and fungicide metalaxyl–mancozeb on bacterial communities in tobacco rhizospheric soil

Bacillus subtilis Tpb55 is a biocontrol agent isolated from the tobacco phyllosphere that has significant inhibitory effect on tobacco black shank pathogen Phytophthora parasitica var. nicotianae. However, its potential ecological risk remains unknown. In this study, we investigated the impact of B. subtilis Tpb55 and the fungicide metalaxyl–mancozeb on bacterial communities in tobacco rhizospheric soil using amplified ribosomal DNA restriction analysis and clone library analyses of 16S rRNA genes. Our results demonstrate that the treatments affected the bacterial communities in different ways. The dominant phyla Acidobacteria and Proteobacteria comprised 27.11% and 26.51%, respectively, of control soil; 29.61% and 37.99%, respectively, of Tpb55-treated soil; and 20.00% and 39.44%, respectively, of fungicide-treated soil. The most abundant class of Proteobacteria in control soil was Betaproteobacteria (11.45%); in contrast, Alphaproteobacteria (15.08%) and Gammaproteobacteria (16.67%) were the most abundant classes in Tpb55- and fungicide-treated soils, respectively. Bacteria that typically show plant-growth-promoting activity belonging to the genera Bradyrhizobium, Rhizobium and Dyella were enriched in Tpb55 treatment whereas genera known as pesticide-degrading bacteria such as Rhodopseudomonas and Phenylobacterium appeared in fungicide treatment. Tpb55 treatment increased soil bacterial diversity relative to control and fungicide treatment.

Impacts of produced water origin on bacterial community structures of activated sludge

The purpose of this study was to reveal how activated sludge communities respond to influent quality and indigenous communities by treating two produced waters from different origins in a batch reactor in succession. The community shift and compositions were investigated using Polymerase Chain Reaction–denaturing gradient gel electrophoresis (PCR–DGGE) and further 16S ribosomal DNA (rDNA) clone library analysis. The abundance of targeted genes for polycyclic aromatic hydrocarbon (PAH) degradation, nahAc/phnAc and C12O/C23O, was tracked to define the metabolic ability of the in situ microbial community by Most Probable Number (MPN) PCR. The biosystem performed almost the same for treatment of both produced waters in terms of removals of chemical oxygen demand (COD) and PAHs. Sludge communities were closely associated with the respective influent bacterial communities (similarity>60%), while one sludge clone library was dominated by the Betaproteobacteria (38%) and Bacteriodetes (30%) and the other was dominated by Gammaproteobacteria (52%). This suggested that different influent and water quality have an effect on sludge community compositions. In addition, the existence of catabolic genes in sludge was consistent with the potential for degradation of PAHs in the treatment of both produced waters.

Variations in the structure of airborne bacterial communities in a downwind area during an Asian dust (Kosa) event

Asian dust (Kosa) events transport airborne microorganisms that significantly impact biological ecosystems, human health, and ice-cloud formation in downwind areas. However, the composition and population dynamics of airborne bacteria have rarely been investigated in downwind areas during Kosa events. In this study, air samplings were sequentially performed at the top of a 10-m high building within the Kosa event arrival area (Kanazawa City, Japan) from May 1 to May 7, 2011, during a Kosa event. The particle concentrations of bacterial cells and mineral particles were ten-fold higher during the Kosa event than on non-Kosa event days. A 16S ribosomal DNA clone library prepared from the air samples primarily contained sequences from three phyla: Cyanobacteria, Firmicutes, and Alphaproteobacteria. The clones from Cyanobacteria were mainly from a marine type of Synechococcus species that was dominant during the first phase of the Kosa event and was continuously detected throughout the Kosa event. The clones from Alphaproteobacteria were mainly detected at the initial and final periods of the Kosa event, and phylogenetic analysis showed that their sequences clustered with those from a marine bacterial clade (the SAR clade) and Sphingomonas spp. During the middle of the Kosa event, the Firmicutes species Bacillus subtilis and Bacillus pumilus were predominant; these species are known to be predominant in the atmosphere above the Chinese desert, which is the source of the dust during Kosa events. The clones obtained after the Kosa event had finished were mainly from Bacillus megaterium, which is thought to originate from local terrestrial areas. Our results suggest that airborne bacterial communities at the ground level in areas affected by Kosa events change their species compositions during a Kosa event toward those containing terrestrial and pelagic bacteria transported from the Sea of Japan and the continental area of China by the Kosa event.

Analysis of chromosomal polymorphism in barley (Hordeum vulgare L. ssp. vulgare) and between H. vulgare and H. chilense using three-color fluorescence in situ hybridization (FISH)

The aim of the present work was to study chromosomal polymorphism within cultivated barley (Hordeum vulgare ssp. vulgare) using three-color fluorescence in situ hybridization (FISH). The physical distribution of the most frequently used, highly repetitive DNA sequences (GAA)7 specific for pericentromeric heterochromatic regions, the ribosomal DNA clone pTa71, specific for the 45S rDNA, and the barley-specific telomere-associated sequence HvT01, was investigated to reveal genetic diversity in metaphase spreads of ten barley genotypes with diverse geographical origin, growth habit and row number. A wild relative of barley, Hordeum chilense was also studied in order to compare the polymorphism between and within Hordeum species. Significant differences in the hybridization patterns of all three DNA probes could be detected between the two related species, but only probes pTa71 and HvT01 showed variation in the intensity and/or position of hybridization sites among genotypes of H. vulgare ssp. vulgare. The extent of polymorphism was less than that earlier reported for molecular markers and was restricted to the long chromosome arms, with differences between the chromosomes. 1H and 3H proved to be the most variable chromosomes and 4H and 6H the most conserved.

Analysis of the Attached Microbial Community on Mucilaginous Cyanobacterial Aggregates in the Eutrophic Lake Taihu Reveals the Importance of Planctomycetes

The phylogenetic diversity of the microbial community assemblage of the carpet-like mucilaginous cyanobacterial blooms in the eutrophic Lake Taihu was investigated. 16S ribosomal DNA clone libraries produced from the DNA of cyanobacterial assemblages that had been washed to remove unattached bacteria contained only cyanobacteria. However, a further treatment which included grinding the freeze-dried material to physically detach cells followed by the removal of larger cells by filtration allowed us to detect a large variety of bacteria within the cyanobacterial bloom community. Interestingly, the dominant members of the microbial community were Planctomycetes followed by Cytophaga-Flavobacterium-Bacteroides (CFB), Betaproteobacteria, and Gammaproteobacteria. The analysis of the 16S ribosomal DNA clone libraries made from enrichment culture revealed much higher phylogenetic diversity of bacteria. Dominant bacterial groups in the enrichment system were identified as members of the Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, and Deltaproteobacteria subdivisions, CFB group, and Planctomycetes. In addition, the clone libraries constructed from Planctomycetes-specific 16S ribosomal RNA primers also verified that the enrichment allowed a diversity of Planctomycetes to proliferate, although the community composition was altered after enrichment.

Genomic and chromosomal distribution patterns of various repeated DNA sequences in wheat revealed by a fluorescence in situ hybridization procedure

Wheat (Triticum aestivum L.) is an allohexaploid, in which each of the three genomes has a high 1C content. This indicates the presence of multiple tandemly repeated sequences, which should be detectable using in situ hybridization. Some repeats have already been described, but others remain to be recognized. To discover others, 2000 plasmid wheat clones were examined for signal presence after fluorescence in situ hybridization and microscopic signal observation. Among them, 47 clones produced strong discrete signals on wheat chromosomes. Two of the newly identified clones (pTa-535 and pTa-713) were determined to have especially valuable sequences for chromosome identification. In combination with pTa-86 (the pSc119 homologous sequence), these probes enable unambiguous discrimination of all wheat chromosomes including orientation. Four newly identified sequences (pTa-465, pTa-k566, pTa-s120, and pTa-s126) were useful in that they produced discrete signals on various wheat chromosome arms. Two other clones (pTa-k288 and pTa-k229) produced GISH-like (genomic in situ hybridization) signals because they allowed the A, B, and D genomes to be distinguished simultaneously. In addition, centromere, centromere-related, and ribosomal DNA clones were identified. Also described are improvements on slide preparation and reprobing procedures. To enhance discrete signal detection, a new direct fluorescent-labeling procedure, namely the VentR (exo-) terminal extension method, was employed.

16S ribosomal DNA clone libraries to reveal bacterial diversity in anaerobic reactor-degraded tetrabromobisphenol A

Microorganisms able to rapidly degrade tetrabromobisphenol A (TBBPA) were domesticated in an anaerobic reactor and added to gradually increased concentrations of TBBPA. After 240days of domestication, the degradation rate reached 96.0% in cultivated batch experiments lasting 20days. The optimum cultivating temperature and pH were 30°C and 7.0. The bacterial community’s composition and diversity in the reactor was studied by comparative analysis with 16S ribosomal DNA clone libraries. Amplified rDNA restriction analysis of 200 clones from the library indicate that the rDNA richness was high (Coverage C 99.5%) and that evenness was not high (Shannon–Weaver index 2.42). Phylogenetic analysis of 63 bacterial sequences from the reactor libraries demonstrated the presence of Betaproteobacteria (33.1%), Gammaproteobacteria (18.7%), Bacteroidetes (13.9%), Firmicutes (11.4%), Chloroflexi (3.6%), Actinobacteria (0.6%), the candidate division TM7 (4.2%) and other unknown, uncultured bacterial groups (14.5%). Comamonas, Achromobacter, Pseudomonas and Flavobacterium were the dominant types.

Individual chromosome assignment and chromosomal collinearity in Gossypium thurberi, G. trilobum and D subgenome of G. barbadense revealed by BAC-FISH

The experiment on individual chromosome assignments and chromosomal diversity was conducted using a multi-probe fluorescence in situ hybridization (FISH) system in D subgenome of tetraploid Gossypium barbadense (D(b)), G. thurberi (D(1)) and G. trilobum (D(8)), which the later two were the possible subgenome donors of tetraploid cottons. The FISH probes contained a set of bacterial artificial chromosome (BAC) clones specific to 13 individual chromosomes from D subgenome of G. hirsutum (D(h)), a D genome centromere-specific BAC clone 150D24, 45S and 5S ribosomal DNA (rDNA) clones, respectively. All tested chromosome orientations were confirmed by the centromere-specific BAC probe. In D(1) and D(8), four 45S rDNA loci were found assigning at the end of the short arm of chromosomes 03, 07, 09 and 11, while one 5S rDNA locus was successfully marked at pericentromeric region of the short arm of chromosome 09. In D(b), three 45S rDNA loci and two 5S rDNA loci were found out. Among them, two 45S rDNA loci were located at the terminal of the short arm of chromosomes D(b)07 and D(b)09, whilst one 5S rDNA locus was found situating near centromeric region of the short arm of chromosome D(b)09. The positions of the BAC clones specific to the 13 individual chromosomes from D(h) were compared between D(1), D(8) and D(b). The result showed the existence of chromosomal collinearity within D(1) and D(8), and as well between them and D(b). The results will serve as a base for understanding chromosome structure of cotton and polyploidy evolution of cotton genome and will provide bio-information for assembling the sequences of finished and the on-going cotton whole genome sequencing projects.

Metagenomic analysis of the uterine bacterial microbiota in healthy and metritic postpartum dairy cows

At present, many bacterial species are validly known as etiological agents of dairy cattle metritis, yet the vast uncultured fraction has received no attention so far. The purpose of this study was to use culture-independent methods to describe and compare the uterine bacterial composition in healthy and metritic postpartum Holstein dairy cows. Both group-specific 16S ribosomal DNA PCR-denaturing gradient gel electrophoresis (DGGE) and clone library sequencing of broad-range 16S ribosomal DNA PCR revealed differences in the bacterial communities comparing healthy and metritic cows. Bacterial diversity in healthy and metritic uteri was greater and more complex than described previously by traditional culture methods. Sequences were assigned to 5 major groups (Gammaproteobacteria, Firmicutes, Fusobacteria, Bacteroidetes, and Tenericutes) and to uncultured bacteria. Additionally, DGGE suggested the presence of Actinobacteria. Most clone sequences in the metritic status libraries were affiliated with the phylum Fusobacteria. Many components, especially from other phyla, have not previously been isolated from cases of metritis. In the clone libraries from the healthy status dairy cows, Gammaproteobacteria was the most prominent group and most sequences showed high identity with Mannheimia varigena, Pasteurella hemolytica, and members of the phylum Tenericutes. Our data showed that the uterine bacterial community in postpartum dairy cows differed considerably between healthy and metritic cows and described the occurrence of a previously unrecognized extent of this diversity in the bovine intrauterine microbiota.

Revealing archaeal diversity patterns and methane fluxes in Admiralty Bay, King George Island, and their association to Brazilian Antarctic Station activities

The study of Antarctic archaeal communities adds information on the biogeography of this group and helps understanding the dynamics of biogenic methane production in such extreme habitats. Molecular methods were combined to methane flux determinations in Martel Inlet, Admiralty Bay, to assess archaeal diversity, to obtain information about contribution of the area to atmospheric methane budget and to detect possible interferences of the Antarctic Brazilian Station Comandante Ferraz (EACF) wastewater discharge on local archaeal communities and methane emissions. Methane fluxes in Martel Inlet ranged from 3.2 to 117.9 μmol CH 4 m −2 d −1, with an average of 51.3±8.5 μmol CH 4 m −2 d −1 and a median of 57.6 μmol CH 4 m −2d −1. However, three negative fluxes averaging −11.3 μmol CH 4 m −2 d −1 were detected in MacKellar Inlet, indicating that Admiralty Bay can be either a source or sink of atmospheric methane. Denaturing gradient gel electrophoresis (DGGE) showed that archaeal communities at EACF varied with depth and formed a group separated from the reference sites. Granulometric analysis indicated that differences observed may be mostly related to sediment type. However, an influence of wastewater input could not be discarded, since higher methane fluxes were found at CF site, suggesting stimulation of local methanogenesis. DGGE profile of the wastewater sample grouped separated from all other samples, suggesting that methanogenesis stimulation may be due to changes in environmental conditions rather than to the input of allochtonous species from the wastewater. 16S ribosomal DNA clone libraries analysis showed that all wastewater sequences were related to known methanogenic groups belonging to the hydrogenotrophic genera Methanobacterium and Methanobrevibacter and the aceticlastic genus Methanosaeta. EACF and Botany Point sediment clone libraries retrieved only groups of uncultivated Archaea, with predominance of Crenarchaeota representatives (MCG, MG1, MBG-B, MBG-C and MHVG groups). Euryarchaeota sequences found were mostly related to the LDS and RC-V groups, but MBG-D and DHVE-5 were also present. No representatives of cultivated methanogenic groups were found, but coverage estimates suggest that a higher number of clones would have to be analyzed in order to cover the greater archaeal diversity of Martel Inlet sediment. Nevertheless, the analysis of the libraries revealed groups not commonly found by other authors in Antarctic habitats and also indicated the presence of groups of uncultivated archaea previously associated to methane rich environments or to the methane cycle.

Multiple marker parallel tag environmental DNA sequencing reveals a highly complex eukaryotic community in marine anoxic water

Sequencing of ribosomal DNA clone libraries amplified from environmental DNA has revolutionized our understanding of microbial eukaryote diversity and ecology. The results of these analyses have shown that protist groups are far more genetically heterogeneous than their morphological diversity suggests. However, the clone library approach is labour-intensive, relatively expensive, and methodologically biased. Therefore, even the most intensive rDNA library analyses have recovered only small samples of much larger assemblages, indicating that global environments harbour a vast array of unexplored biodiversity. High-throughput parallel tag 454 sequencing offers an unprecedented scale of sampling for molecular detection of microbial diversity. Here, we report a 454 protocol for sampling and characterizing assemblages of eukaryote microbes. We use this approach to sequence two SSU rDNA diversity markers-the variable V4 and V9 regions-from 10 L of anoxic Norwegian fjord water. We identified 38 116 V4 and 15 156 V9 unique sequences. Both markers detect a wide range of taxonomic groups but in both cases the diversity detected was dominated by dinoflagellates and close relatives. Long-tailed rank abundance curves suggest that the 454 sequencing approach provides improved access to rare genotypes. Most tags detected represent genotypes not currently in GenBank, although many are similar to database sequences. We suggest that current understanding of the ecological complexity of protist communities, genetic diversity, and global species richness are severely limited by the sequence data hitherto available, and we discuss the biological significance of this high amplicon diversity.

A novel microbial habitat of alkaline black liquor with very high pollution load: Microbial diversity and the key members in application potentials

A microbial community which developed naturally in alkaline black liquor was investigated by culture-based and culture-independent techniques. The community was effective in lowering pH, color, and chemical oxygen demand (COD) of black liquor, and the community activities varied in different seasons. Both 16S ribosomal DNA (rDNA) clone library and polymerase chain reaction–denaturing gradient gel electrophoresis (PCR–DGGE) analyses suggested that the seasonal bacterial communities had obvious differences in diversities and compositions. Clostridium species were suggested to be the key agents in black liquor treatment. Moreover, the isolates of the genera Halomonas and Bacillus were shown to be effective in treating very heavily polluted black liquor. The strains of Halomonas, Clostridium and especially Bacillus, might be the key producers of xylanase and CMCase in the community. The worldwide problem of black liquor treatment and renewable resource utilization would benefit from these microorganisms in application potentials.

New Insights into the Diversity of Marine Picoeukaryotes

Over the last decade, culture-independent surveys of marine picoeukaryotic diversity based on 18S ribosomal DNA clone libraries have unveiled numerous sequences of novel high-rank taxa. This newfound diversity has significantly altered our understanding of marine microbial food webs and the evolution of eukaryotes. However, the current picture of marine eukaryotic biodiversity may be significantly skewed by PCR amplification biases, occurrence of rDNA genes in multiple copies within a single cell, and the capacity of DNA to persist as extracellular material. In this study we performed an analysis of the metagenomic dataset from the Global Ocean Survey (GOS) expedition, seeking eukaryotic ribosomal signatures. This PCR-free approach revealed similar phylogenetic patterns to clone library surveys, suggesting that PCR steps do not impose major biases in the exploration of environmental DNA. The different cell size fractions within the GOS dataset, however, displayed a distinct picture. High protistan diversity in the <0.8 µm size fraction, in particular sequences from radiolarians and ciliates (and their absence in the 0.8–3 µm fraction), suggest that most of the DNA in this fraction comes from extracellular material from larger cells. In addition, we compared the phylogenetic patterns from rDNA and reverse transcribed rRNA 18S clone libraries from the same sample harvested in the Mediterranean Sea. The libraries revealed major differences, with taxa such as pelagophytes or picobiliphytes only detected in the 18S rRNA library. MAST (Marine Stramenopiles) appeared as potentially prominent grazers and we observed a significant decrease in the contribution of alveolate and radiolarian sequences, which overwhelmingly dominated rDNA libraries. The rRNA approach appears to be less affected by taxon-specific rDNA copy number and likely better depicts the biogeochemical significance of marine protists.

A strict anaerobic extreme thermophilic hydrogen-producing culture enriched from digested household waste

The aim of this study was to enrich, characterize and identify strict anaerobic extreme thermophilic hydrogen (H(2)) producers from digested household solid wastes. A strict anaerobic extreme thermophilic H(2) producing bacterial culture was enriched from a lab-scale digester treating household wastes at 70 degrees C. The enriched mixed culture consisted of two rod-shaped bacterial members growing at an optimal temperature of 80 degrees C and an optimal pH 8.1. The culture was able to utilize glucose, galactose, mannose, xylose, arabinose, maltose, sucrose, pyruvate and glycerol as carbon sources. Growth on glucose produced acetate, H(2) and carbon dioxide. Maximal H(2) production rate on glucose was 1.1 mmol l(-1) h(-1) with a maximum H(2) yield of 1.9 mole H(2) per mole glucose. 16S ribosomal DNA clone library analyses showed that the culture members were phylogenetically affiliated to the genera Bacillus and Clostridium. Relative abundance of the culture members, assessed by fluorescence in situ hybridization, were 87 +/- 5% and 13 +/- 5% for Bacillus and Clostridium, respectively. An extreme thermophilic, strict anaerobic, mixed microbial culture with H(2)-producing potential was enriched from digested household wastes. This study provided a culture with a potential to be applied in reactor systems for extreme thermophilic H(2) production from complex organic wastes.

Bacterial diversity in surface sediments from the Pacific Arctic Ocean

In order to assess bacterial diversity within four surface sediment samples (0-5 cm) collected from the Pacific Arctic Ocean, 16S ribosomal DNA clone library analysis was performed. Near full length 16S rDNA sequences were obtained for 463 clones from four libraries and 13 distinct major lineages of Bacteria were identified (alpha, beta, gamma, delta and epsilon-Proteobacteria, Acidobacteria, Bacteroidetes, Chloroflexi, Actinobacteria, Firmicutes, Planctomycetes, Spirochetes, and Verrucomicrobia). alpha, gamma, and delta-Proteobacteria, Acidobacteria, Bacteroidetes, Actinobacteria were common phylogenetic groups from all the sediments. The gamma-Proteobacteria were the dominant bacterial lineage, representing near or over 50% of the clones. Over 35% of gamma-Proteobacteria clones of four clone library were closely related to cultured bacterial isolates with similarity values ranging from 94 to 100%. The community composition was different among sampling sites, which potentially was related to geochemical differences.

Characterization of Bacterial Community Structure and Diversity in Rhizosphere Soils of Three Plants in Rapidly Changing Salt Marshes Using 16S rDNA

The structure and diversity of the bacterial communities in rhizosphere soils of native Phragmites australis and Scirpus mariqueter and alien Spartina alterniflora in the Yangtze River Estuary were investigated by constructing 16S ribosomal DNA (rDNA) clone libraries. The bacterial diversity was quantified by placing the clones into operational taxonomic unit (OTU) groups at the level of sequence similarity of > 97%. Phylogenetic analysis of the resulting 398 clone sequences indicated a high diversity of bacteria in the rhizosphere soils of these plants. The members of Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, and Deltaproteobacteria of the phylum Proteobacteria were the most abundant in rhizobacteria. Chao 1 nonparametric diversity estimator coupled with the reciprocal of Simpson's index (1/D) was applied to sequence data obtained from each library to evaluate total sequence diversity and quantitatively compare the level of dominance. The results showed that Phragmites, Scirpus, and Spartina rhizosphere soils contained 200, 668, and 382 OTUs, respectively. The bacterial communities in the Spartina and Phragmites rhizosphere soils displayed species dominance revealed by 1/D, whereas the bacterial community in Scirpus rhizosphere soil had uniform distributions of species abundance. Overall, analysis of 16S rDNA clone libraries from the rhizosphere soils indicates that the changes in bacterial composition may occur concomitantly with the shift of species composition in plant communities.

The archaebacterial communities in Antarctic bathypelagic sediments

Abstract 16S ribosomal DNA clone library analysis was performed to assess archaeal diversity within three surficial sediment samples obtained from the bathypelagic zone (depth: 2165–3406 m) of the Weddell Sea, Antarctica. The nearly complete 16S rDNA gene (1440 bp) was obtained for 146 clones and 46 phylotypes were defined. The majority of the sequences (>99%) formed three clusters within the Marine Group I Crenarchaeota. The most important cluster, with 78.8% of the clones, included Candidatus Nitrosopumilus maritimus, a mesophilic archaeon able to oxidize ammonia. The most important subgroup in that cluster was the APA4-0cm subgroup (with 62.3% of the clones). This subgroup might represent important Crenarchaeota in the functioning of the bathypelagic sedimentary ecosystems of the Weddell Sea because it dominated the clone libraries in all sampling stations, and was found in sediments separated by very large geographic distances. Only one clone grouped within the Euryarchaeota. This euryarchaeal clone could not be affiliated with any of the previously defined clusters and might represent a novel euryarchaeal lineage.

Postinoculation Protozoan Establishment and Association Patterns of Methanogenic Archaea in the Ovine Rumen

Association patterns between archaea and rumen protozoa were evaluated by analyzing archaeal 16S rRNA gene clone libraries from ovine rumen inoculated with different protozoa. Five protozoan inoculation treatments, fauna free (negative control), holotrich and cellulolytic protozoa, Isotricha and Dasytricha spp., Entodinium spp., and total fauna (type A) were tested. We used denaturing gradient gel electrophoresis, quantitative PCR, and phylogenetic analysis to evaluate the impact of the protozoan inoculants on the respective archaeal communities. Protozoan 18S ribosomal DNA clone libraries were also evaluated to monitor the protozoal population that was established by the inoculation. Phylogenetic analysis suggested that archaeal clones associated with the fauna-free, the Entodinium, and the type A inoculations clustered primarily with uncultured phylotypes. Polyplastron multivesiculatum was the predominant protozoan strain established by the holotrich and cellulolytic protozoan treatment, and this resulted predominantly in archaeal clones affiliated with uncultured and cultured methanogenic phylotypes (Methanosphaera stadtmanae, Methanobrevibacter ruminantium, and Methanobacterium bryantii). Furthermore, the Isotricha and Dasytricha inoculation treatment resulted primarily in archaeal clones affiliated with Methanobrevibacter smithii. This report provides the first assessment of the influence of protozoa on archaea within the rumen microbial community and provides evidence to suggest that different archaeal phylotypes associate with specific groups of protozoa. The observed patterns may be linked to the evolution of commensal and symbiotic relationships between archaea and protozoa in the ovine rumen environment. This report further underscores the prevalence and potential importance of a rather large group of uncultivated archaea in the ovine rumen, probably unrelated to known methanogens and undocumented in the bovine rumen.

Bioaccumulation and secondary gold nugget formation

Experimental evidence suggests that microorganisms are important in the cycling of Au (1). Recent microcosm studies with auriferous soils from Australian mine sites have shown that the resident microbiota are capable of solubilising up to 80 wt.% of the Au contained in these soils (1). A number of common soil bacteria, actinomycetes, archaea, and fungi have been shown to precipitate Au(I/III)-complexes under a wide range of experimental conditions (2). Morphological evidence for the occurrence of micrometre sized bacterioform structures on secondary Au grains has been reported in earlier studies (3). However, neither the biological origin of these structures nor associated organisms or processes have been identified. Gold grains for this study were obtained from the auriferous soils overlying mineralisation at the Tomakin Parkand the Hit or Miss Gold Mine in moderate New South Wales and tropical Queensland, respectively. The grains were 200 to 2200 μm in diameter and generally showed little sign of physical transport. Analyses of gold grain digests that amounted to an average fineness of 99.2% established their secondary origin. Scanning electron microscopy (SEM) revealed bacterioform on untreated grains from both sites. The presence of active bacterial biofilms on the surface of Au grains was confirmed using confocal stereo laser microscopy (CSLM) combined with nucleic acid staining. Isolation, amplification and separation of 16S ribosomal DNA from these biofilms and identification of the organisms present using DNA sequence analysis showed that unique, sitespecific bacterial communities are associated with these Au grains that differ from those dominating the surrounding soil. 16S ribosomal DNA clones bearing 99 % similarity to a known metallophilic bacterium are present on all 16S rDNA positive grains from both locations. The ability of this metallophilic bacterium to accumulate AuCl4 complexes was tested under in-vitro conditions, and biologically active cells typically accumulated twice as much Au as lysed cell preparations. These results indicate that bacteria that are associated with Au grains may be able to actively reduce AuCl4 and accumulate metallic Au, and thus contribute to the authigenic formation of secondary Au nuggets in the regolith.