rRNA Gene Phylotypes Research Articles

Mapping Archaeal Diversity in Soda Lakes by Coupling 16S rRNA PCR-DGGE Analysis with Remote Sensing and GIS Technology

The haloarchaeal diversity of four hypersaline alkaline lakes from the Wadi El-Natrun depression (Northern Egypt) was investigated using culture-independent polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) of 16S rRNA gene phylotypes, which was combined with remote sensing and geographic information system (GIS) data to highlight the distribution pattern of the microbial diversity in water and sediment samples. The majority of archaeal sequences identified in all four lakes belonged to the phyla Euryarchaeota and Crenarchaeota. Sediment samples from Beida Lake and water samples from El-Hamra Lake showed the highest levels of archaeal diversity. Sequence similarities ≥ 95% were found between six of the acquired clones and uncultured Halorhabdus, Euryarchaeota, and archaeon clones. In addition, two clones shared a high level of sequence similarity (97%) with unclassified archaea, while other nine clones exhibited 96% to 99% sequence similarity with uncultured archaeon clones, and only one clone showed 97% identity with an uncultured Crenarchaeota. Likewise, 7 DGGE bands presented a sequence similarity of 90 to 98% to Halogranum sp., Halalkalicoccus tibetensis, Halalkalicoccus jeotgali, uncultured Halorubrum, Halobacteriaceae sp., or uncultured haloarchaeon. In conclusion, while the variety of alkaliphilic haloarchaea in the examined soda lakes was restricted, the possibility of uncovering novel species for biotechnological applications from these extreme habitats remains promising.

Characterization of the First Cultured Psychrotolerant Representative of Legionella from Antarctica Reveals Its Unique Genome Structure.

ABSTRACTCulture-independent analysis shows that Legionella spp. inhabit a wide range of low-temperature environments, but to date, no psychrotolerant or psychrophilic strains have been reported. Here, we characterized the first cultivated psychrotolerant representative, designated strain TUM19329T, isolated from an Antarctic lake using a polyphasic approach and comparative genomic analysis. A genome-wide phylogenetic tree indicated that this strain was phylogenetically separate at the species level. Strain TUM19329T shared common physiological traits (e.g., Gram-negative, limited growth on buffered charcoal-yeast extract α-ketoglutarate [BCYEα] agar with l-cysteine requirements) with its relatives, but it also showed psychrotolerant growth properties (e.g., growth at 4°C to 25°C). Moreover, this strain altered its own cellular fatty acid composition to accumulate unsaturated fatty acid at a lower temperature, which may help maintain the cell membrane fluidity. Through comparative genomic analysis, we found that this strain possessed massive mobile genetic elements compared with other species, amounting to up to 17% of the total genes. The majority of the elements were the result of the spread of only a few insertion sequences (ISs), which were spread throughout the genome by a “copy-and-paste” mechanism. Furthermore, we found metabolic genes, such as fatty acid synthesis-related genes, acquired by horizontal gene transfer (HGT). The expansion of ISs and HGT events may play a major role in shaping the phenotype and physiology of this strain. On the basis of the features presented here, we propose a new species—Legionella antarctica sp. nov.—represented by strain TUM19329T (= GTC 22699T = NCTC 14581T).IMPORTANCE This study characterized a unique cultivated representative of the genus Legionella isolated from an Antarctic lake. This psychrotolerant strain had some common properties of known Legionella species but also displayed other characteristics, such as plasticity in fatty acid composition and an enrichment of mobile genes in the genome. These remarkable properties, as well as other factors, may contribute to cold hardiness, and this first cultivated cold-tolerant strain of the genus Legionella may serve as a model bacterium for further studies. It is worth noting that environmentally derived 16S rRNA gene phylotypes closely related to the strain characterized here have been detected from diverse environments outside Antarctica, suggesting a wide distribution of psychrotolerant Legionella bacteria. Our culture- and genome-based findings may accelerate the ongoing studies of the behavior and pathogenicity of Legionella spp., which have been monitored for many years in the context of public health.

Microbiome Aggregated Traits and Assembly Are More Sensitive to Soil Management than Diversity.

ABSTRACTHow soil is managed, particularly for agriculture, exerts stresses upon soil microbiomes, resulting in altered community structures and functional states. Understanding how soil microbiomes respond to combined stresses is important for predicting system performance under different land use scenarios, aids in identification of the most environmentally benign managements, and provides insight into how system function can be recovered in degraded soils. We use a long-established field experiment to study the effects of combined chronic (press) disturbance of the magnitude of organic carbon inputs with acute (pulse) effects of physical disturbance by tillage and chemical disturbance due to inorganic fertilization and pesticide application. We show that because of the variety of ways it can be assessed, biodiversity—here based on microbial small subunit rRNA gene phylotypes—does not provide a consistent view of community change. In contrast, aggregated traits associated with soil microbiomes indicate general loss of function, measured as a reduction of average genome lengths, associated with chronic reduction of organic inputs in arable or bare fallow soils and altered growth strategies associated with rRNA operon copy number in prokaryotes, as well as a switch to pathogenicity in fungal communities. In addition, pulse disturbance by soil tillage is associated with an increased influence of stochastic processes upon prokaryote community assembly, but fungicide used in arable soils results in niche assembly of fungal communities compared to untilled grassland. Overall, bacteria, archaea, and fungi do not share a common response to land management change, and estimates of biodiversity do not capture important facets of community adaptation to stresses adequately.IMPORTANCE Changes in soil microbiome diversity and function brought about by land management are predicted to influence a range of environmental services provided by soil, including provision of food and clean water. However, opportunities to compare the long-term effects of combinations of stresses imposed by different management approaches are limited. We exploit a globally unique 50-year field experiment, demonstrating that soil management practices alter microbiome diversity, community traits, and assembly. Grassland soil microbiomes are dominated by fewer—but phylogenetically more diverse—prokaryote phylotypes which sustain larger genomes than microbiomes in arable or bare fallow soil maintained free of plants. Dominant fungi in grassland soils are less phylogenetically diverse than those in arable or fallow soils. Soil tillage increases stochastic processes in microbiome assembly: this, combined with reduced plant biomass, presents opportunities for organisms with a capacity for pathogenesis to become established in stressed soils.

Enrichment of bacteria and alginate lyase genes potentially involved in brown alga degradation in the gut of marine gastropods

Gut bacteria of phytophagous and omnivorous marine invertebrates often possess alginate lyases (ALGs), which are key enzymes for utilizing macroalgae as carbon neutral biomass. We hypothesized that the exclusive feeding of a target alga to marine invertebrates would shift the gut bacterial diversity suitable for degrading the algal components. To test this hypothesis, we reared sea hare (Dolabella auricularia) and sea snail (Batillus cornutus) for two to four weeks with exclusive feeding of a brown alga (Ecklonia cava). Pyrosequencing analysis of the gut bacterial 16S rRNA genes revealed shifts in the gut microbiota after rearing, mainly due to a decrease in the variety of bacterial members. Significant increases in six and four 16S rRNA gene phylotypes were observed in the reared sea hares and sea snails, respectively, and some of them were phylogenetically close to known alginate-degrading bacteria. Clone library analysis of PL7 family ALG genes using newly designed degenerate primer sets detected a total of 50 ALG gene phylotypes based on 90% amino acid identity. The number of ALG gene phylotypes increased in the reared sea hare but decreased in reared sea snail samples, and no phylotype was shared between them. Out of the 50 phylotypes, 15 were detected only after the feeding procedure. Thus, controlled feeding strategy may be valid and useful for the efficient screening of genes suitable for target alga fermentation.

Effect of seasonality and physicochemical parameters on bacterial communities in two hot spring microbial mats from Araró, Mexico

In this study, we explored the diversity of culturable bacterial communities residing in hot springs from Araró, México, and analysed the effect of seasonality and related changes in physicochemical parameters of spring water. Two hot springs with unique features, Tina and Bonita, were analysed. Seventy-nine unique 16S rRNA gene phylotypes were detected, belonging to the bacterial phyla Firmicutes, Proteobacteria, and Actinobacteria. A group of dominant phylotypes of the genus Bacillus was recovered in 3 out of 4 of the sampling seasons. Another group of phylotypes was recovered in 2 samplings, while the remaining groups were detected in only 1 season. Ecological indexes for species richness and evenness showed moderate to low diversity in both hot springs, and a Sørensen analysis revealed that the 2 communities shared 64% of their bacterial phylotypes. Physicochemical parameters measured every season showed slight variations, except for temperature and arsenic content. Fluctuations in bacterial composition in the Tina hot spring were correlated mainly with salt content, while diversity in the Bonita hot spring was significantly correlated with temperature, pH, and arsenic content.

Defining boundaries for the distribution of microbial communities beneath the sediment-buried, hydrothermally active seafloor.

Subseafloor microbes beneath active hydrothermal vents are thought to live near the upper temperature limit for life on Earth. We drilled and cored the Iheya North hydrothermal field in the Mid-Okinawa Trough, and examined the phylogenetic compositions and the products of metabolic functions of sub-vent microbial communities. We detected microbial cells, metabolic activities and molecular signatures only in the shallow sediments down to 15.8 m below the seafloor at a moderately distant drilling site from the active hydrothermal vents (450 m). At the drilling site, the profiles of methane and sulfate concentrations and the δ13C and δD isotopic compositions of methane suggested the laterally flowing hydrothermal fluids and the in situ microbial anaerobic methane oxidation. In situ measurements during the drilling constrain the current bottom temperature of the microbially habitable zone to ~45 °C. However, in the past, higher temperatures of 106-198 °C were possible at the depth, as estimated from geochemical thermometry on hydrothermally altered clay minerals. The 16S rRNA gene phylotypes found in the deepest habitable zone are related to those of thermophiles, although sequences typical of known hyperthermophilic microbes were absent from the entire core. Overall our results shed new light on the distribution and composition of the boundary microbial community close to the high-temperature limit for habitability in the subseafloor environment of a hydrothermal field.

Comparative Analysis of Microbial Communities in Iron-Dominated Flocculent Mats in Deep-Sea Hydrothermal Environments.

It has been suggested that iron is one of the most important energy sources for photosynthesis-independent microbial ecosystems in the ocean crust. Iron-metabolizing chemolithoautotrophs play a key role as primary producers, but little is known about their distribution and diversity and their ecological role as submarine iron-metabolizing chemolithotrophs, particularly the iron oxidizers. In this study, we investigated the microbial communities in several iron-dominated flocculent mats found in deep-sea hydrothermal fields in the Mariana Volcanic Arc and Trough and the Okinawa Trough by culture-independent molecular techniques and X-ray mineralogical analyses. The abundance and composition of the 16S rRNA gene phylotypes demonstrated the ubiquity of zetaproteobacterial phylotypes in iron-dominated mat communities affected by hydrothermal fluid input. Electron microscopy with energy-dispersive X-ray microanalysis and X-ray absorption fine structure (XAFS) analysis revealed the chemical and mineralogical signatures of biogenic Fe-(oxy)hydroxide species and the potential contribution of Zetaproteobacteria to the in situ generation. These results suggest that putative iron-oxidizing chemolithoautotrophs play a significant ecological role in producing iron-dominated flocculent mats and that they are important for iron and carbon cycles in deep-sea low-temperature hydrothermal environments. We report novel aspects of microbiology from iron-dominated flocculent mats in various deep-sea environments. In this study, we examined the relationship between Zetaproteobacteria and iron oxides across several hydrothermally influenced sites in the deep sea. We analyzed iron-dominated mats using culture-independent molecular techniques and X-ray mineralogical analyses. The scanning electron microscopy-energy-dispersive X-ray spectroscopy SEM-EDS analysis and X-ray absorption fine structure (XAFS) analysis revealed chemical and mineralogical signatures of biogenic Fe-(oxy)hydroxide species as well as the potential contribution of the zetaproteobacterial population to the in situ production. These key findings provide important information for understanding the mechanisms of both geomicrobiological iron cycling and the formation of iron-dominated mats in deep-sea hydrothermal fields.

BIODIVERSITY OF GUT MICROFLORA OF Oreochromis niloticus BASED ON CULTURE-INDEPENDENT rRNA GENE ANALYSES AT LAKE NASSER, EGYPT

The diversity of Oreochromis niloticus gut microbiome domains, eukaryotes, bacteria and archaea, was studied to understand the contribution of microbiota to the health of the fish. Fishes were collected from four different Khors, Kalabsha, Wadi Abyad, Tushka and Korosko, of Lake Nasser, Egypt. The approach of this study depends on culture-independent PCR/DGGE and sequence of small subunit of rRNA genes, 18S rRNA gene and 16S rRNA gene. The DGGE patterns displayed 5, 12 and 5 band groups, phylotypes, for eukaryotic 18S rRNA gene, bacterial and archaeal 16S rRNA genes, respectively, in gut contents from the studied khors. DGGE showed bands, which were common and specific for each site and could be used as a bar code to certify the origin of the fish. Statistical analyses, using binary matrix, showed numbers of DGGE bands, 1, 2 and 2, for eukaryotes, bacteria and archaea, respectively, were commonly occurred in all studied khors. The DGGE phylotype, 3.Euk.Kr characterized eukaryotes in Khor Korosko. Phylogenetic analyses showed that two of eukaryotic phylotypes, 1.Euk.Kl.Kr and 2.Euk.Common, were belonged to crustacean Ostracoda. Bacterial phylotypes in all studied khors were located in the branch of cyanobacteria, alpha proteobacteria, but most of them constituted unique phylogenetic lineages within the branch of uncultured environmental bacteria. All archaeal phylotypes were located in the branch of methanogenic uncultured euryarchaeota. Some helminthes, of the genera Neoechinorhynchus and Catenula, -like rRNA gene phylotypes were recorded in guts from Kalabsha, Tushka and Korosko, suggesting common gut parasitic worms. The DGGE patterns and sequence analyses showed high similarities of eukaryote, bacteria and archaea rRNA gene phylotype compositions in fish guts from distant khors, implicating core gut microbiome. This is the first survey of all microbiome domains in tilapia guts at Lake Nasser based on molecular approaches.

Composition of Eukaryotic and Prokaryotic rRNA Gene Phylotypes in Guts of Adults and Fingerlings of Mugil cephalus, Inhabiting an Egyptian Mediterranean Estuary

To understand the contribution of gut microflora to the feeding and health of Mugil cephalus in a Mediterranean estuary, we have explored and evaluated the diversity of rRNA genes in gut contents of both of adults and fingerlings. Bulk DNAs were extracted from the fish guts, followed by PCR, cloning and sequencing of eukaryote 18S rRNA gene; bacterial and archaeal 16S rRNA genes. Rarefaction analyses recorded 11, 18 and 13 phylotype groups of rRNA genes for eukaryotes, bacteria and archaea, respectively, in the adult guts. The fingerling guts contained 6 and 11 phylotype groups of rRNA genes for eukartyotes and bacteria, respectively, while lacked archaea. Adult and fingerling guts were dominated by zooplankton-like 18S rRNA gene phylotypes, belonging to those of rotifers, genus Brachionus and copepods, genus Apocyclops. Both of diatoms, genus Cyclotella- and fish-like rRNA gene phylotypes were recorded only in adult guts. Phylotypes, which belonged to uncultured proteobacteria and cyanobacteria, covered most of the bacterial 16S rRNA gene composition of adult guts. Bacteroidetes-like phylotypes characterized the fingerling guts. The study showed the first record of uncultured thaumarchaeote-like 16S rRNA genes in the fish guts, opening a window for exploring thaumarchaeote biological significances in fish metabolism. The fish guts represented a reservoir for geographic distant species and reflected the plankton ecosystem variability at the studied estuary. The pollution in the estuary could shape a fraction of the fish gut microbiota. To our knowledge, this work presented the first complete profile of rRNA gene diversities for all known microbiome domains in fish guts.

Microbial Communities Can Be Described by Metabolic Structure: A General Framework and Application to a Seasonally Variable, Depth-Stratified Microbial Community from the Coastal West Antarctic Peninsula.

Taxonomic marker gene studies, such as the 16S rRNA gene, have been used to successfully explore microbial diversity in a variety of marine, terrestrial, and host environments. For some of these environments long term sampling programs are beginning to build a historical record of microbial community structure. Although these 16S rRNA gene datasets do not intrinsically provide information on microbial metabolism or ecosystem function, this information can be developed by identifying metabolisms associated with related, phenotyped strains. Here we introduce the concept of metabolic inference; the systematic prediction of metabolism from phylogeny, and describe a complete pipeline for predicting the metabolic pathways likely to be found in a collection of 16S rRNA gene phylotypes. This framework includes a mechanism for assigning confidence to each metabolic inference that is based on a novel method for evaluating genomic plasticity. We applied this framework to 16S rRNA gene libraries from the West Antarctic Peninsula marine environment, including surface and deep summer samples and surface winter samples. Using statistical methods commonly applied to community ecology data we found that metabolic structure differed between summer surface and winter and deep samples, comparable to an analysis of community structure by 16S rRNA gene phylotypes. While taxonomic variance between samples was primarily driven by low abundance taxa, metabolic variance was attributable to both high and low abundance pathways. This suggests that clades with a high degree of functional redundancy can occupy distinct adjacent niches. Overall our findings demonstrate that inferred metabolism can be used in place of taxonomy to describe the structure of microbial communities. Coupling metabolic inference with targeted metagenomics and an improved collection of completed genomes could be a powerful way to analyze microbial communities in a high-throughput manner that provides direct access to metabolic and ecosystem function.

Genotyping of uncultured archaea in a polluted site of Suez Gulf, Egypt, based on 16S rRNA gene analyses

Culture-independent 16S rRNA gene analysis approach was used to explore and evaluate archaea in a polluted site, El-Zeitia, Suez Gulf, Egypt. Metagenomic DNA was extracted from a sediment sample. Archaeal 16S rRNA gene was PCR amplified using universal archaeal primers, followed by cloning and direct analyses by sequencing. Rarefaction analysis showed saturation, recording 21 archaeal 16S rRNA gene phylotypes, which represented the total composition of archaea in the studied sample. Phylogenetic analysis showed that all recorded phylotypes belonged to two archaeal phyla. Sixteen phylotypes were located in the branch of methanogenic Euryarchaeota and more closely related to species of the genera Methanosaeta and Methanomassiliicoccus. Five phylotypes were affiliated to the new archaeal phylum Thaumarchaeota, which represented by species Candidatus nitrosopumilus. The recorded phylotypes had unique sequences, characterizing them as new phylogenetic lineages. This work is the first investigation of uncultured archaea in the Suez Gulf, and implicated that the environmental characteristics shaped the diversity of archaeal 16S rRNA genes in the studied sample.

Microbial communities at the borehole observatory on the Costa Rica Rift flank (Ocean Drilling Program Hole 896A)

The microbiology of subsurface, hydrothermally influenced basaltic crust flanking mid-ocean ridges has remained understudied, due to the difficulty in accessing the subsurface environment. The instrumented boreholes resulting from scientific ocean drilling offer access to samples of the formation fluids circulating through oceanic crust. We analyzed the phylogenetic diversity of bacterial communities of fluid and microbial mat samples collected in situ from the observatory at Ocean Drilling Program Hole 896A, drilled into ~6.5 million-year-old basaltic crust on the flank of the Costa Rica Rift in the equatorial Pacific Ocean. Bacterial 16S rRNA gene sequences recovered from borehole fluid and from a microbial mat coating the outer surface of the fluid port revealed both unique and shared phylotypes. The dominant bacterial clones from both samples were related to the autotrophic, sulfur-oxidizing genus Thiomicrospira. Both samples yielded diverse gamma- and alphaproteobacterial phylotypes, as well as members of the Bacteroidetes, Planctomycetes, and Verrucomicrobia. Analysis of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) genes (cbbL and cbbM) from the sampling port mat and from the borehole fluid demonstrated autotrophic carbon assimilation potential for in situ microbial communities; most cbbL genes were related to those of the sulfur-oxidizing genera Thioalkalivibrio and Thiomicrospira, and cbbM genes were affiliated with uncultured phylotypes from hydrothermal vent plumes and marine sediments. Several 16S rRNA gene phylotypes from the 896A observatory grouped with phylotypes recovered from seawater-exposed basalts and sulfide deposits at inactive hydrothermal vents, but there is little overlap with hydrothermally influenced basaltic boreholes 1026B and U1301A on the Juan de Fuca Ridge flank, suggesting that site-specific characteristics of Hole 896A (i.e., seawater mixing into borehole fluids) affect the microbial community composition.

Impacts of poultry house environment on poultry litter bacterial community composition.

Viral and bacterial pathogens are a significant economic concern to the US broiler industry and the ecological epicenter for poultry pathogens is the mixture of bedding material, chicken excrement and feathers that comprises the litter of a poultry house. This study used high-throughput sequencing to assess the richness and diversity of poultry litter bacterial communities, and to look for connections between these communities and the environmental characteristics of a poultry house including its history of gangrenous dermatitis (GD). Cluster analysis of 16S rRNA gene sequences revealed differences in the distribution of bacterial phylotypes between Wet and Dry litter samples and between houses. Wet litter contained greater diversity with 90% of total bacterial abundance occurring within the top 214 OTU clusters. In contrast, only 50 clusters accounted for 90% of Dry litter bacterial abundance. The sixth largest OTU cluster across all samples classified as an Arcobacter sp., an emerging human pathogen, occurring in only the Wet litter samples of a house with a modern evaporative cooling system. Ironically, the primary pathogenic clostridial and staphylococcal species associated with GD were not found in any house; however, there were thirteen 16S rRNA gene phylotypes of mostly Gram-positive phyla that were unique to GD-affected houses and primarily occurred in Wet litter samples. Overall, the poultry house environment appeared to substantially impact the composition of litter bacterial communities and may play a key role in the emergence of food-borne pathogens.

Microbial Communities and Chemosynthesis in Yellowstone Lake Sublacustrine Hydrothermal Vent Waters

Five sublacustrine thermal spring locations from 1 to 109 m water depth in Yellowstone Lake were surveyed by 16S ribosomal RNA gene sequencing in relation to their chemical composition and dark CO2 fixation rates. They harbor distinct chemosynthetic bacterial communities, depending on temperature (16–110°C) and electron donor supply (H2S <1 to >100 μM; NH3 <0.5 to >10 μM). Members of the Aquificales, most closely affiliated with the genus Sulfurihydrogenibium, are the most frequently recovered bacterial 16S rRNA gene phylotypes in the hottest samples; the detection of these thermophilic sulfur-oxidizing autotrophs coincided with maximal dark CO2 fixation rates reaching near 9 μM C h−1 at temperatures of 50–60°C. Vents at lower temperatures yielded mostly phylotypes related to the mesophilic gammaproteobacterial sulfur oxidizer Thiovirga. In contrast, cool vent water with low chemosynthetic activity yielded predominantly phylotypes related to freshwater Actinobacterial clusters with a cosmopolitan distribution.

Molecular Detection of Anaerobic Ammonium-Oxidizing (Anammox) Bacteria in High-Temperature Petroleum Reservoirs

Anaerobic ammonium-oxidizing (anammox) process plays an important role in the nitrogen cycle of the worldwide anoxic and mesophilic habitats. Recently, the existence and activity of anammox bacteria have been detected in some thermophilic environments, but their existence in the geothermal subterranean oil reservoirs is still not reported. This study investigated the abundance, distribution and functional diversity of anammox bacteria in nine out of 17 high-temperature oil reservoirs by molecular ecology analysis. High concentration (5.31–39.2 mg l−1) of ammonium was detected in the production water from these oilfields with temperatures between 55°C and 75°C. Both 16S rRNA and hzo molecular biomarkers indicated the occurrence of anammox bacteria in nine out of 17 samples. Most of 16S rRNA gene phylotypes are closely related to the known anammox bacterial genera Candidatus Brocadia, Candidatus Kuenenia, Candidatus Scalindua, and Candidatus Jettenia, while hzo gene phylotypes are closely related to the genera Candidatus Anammoxoglobus, Candidatus Kuenenia, Candidatus Scalindua, and Candidatus Jettenia. The total bacterial and anammox bacterial densities were 6.4 ± 0.5 × 103 to 2.0 ± 0.18 × 106 cells ml−1 and 6.6 ± 0.51 × 102 to 4.9 ± 0.36 × 104 cell ml−1, respectively. The cluster I of 16S rRNA gene sequences showed distant identity (<92%) to the known Candidatus Scalindua species, inferring this cluster of anammox bacteria to be a new species, and a tentative name Candidatus “Scalindua sinooilfield” was proposed. The results extended the existence of anammox bacteria to the high-temperature oil reservoirs.

Bacteria from drinking water supply and their fate in gastrointestinal tracts of germ-free mice: A phylogenetic comparison study

Microorganisms in drinking water sources may colonize in gastrointestinal (GI) tracts and this phenomenon may pose a potential health risk especially to immunocompromised population. The microbial community diversity of the drinking water was compared with the GI tracts of the mice using phylogenetic and statistical analyses of 16S rRNA gene sequences. A group of germ-free mice were fed with drinking water from public water supply that passed through an automated watering system with documented biofilm accumulation. From drinking water and GI tracts of the germ-free mice, 179 bacteria were isolated and 75 unique 16S rRNA gene phylotypes were sequenced as operational taxonomic unit (OTU, >97% similarity). Three major groups of the genus Acidovorax (21%), Variovorax (42%) and Sphingopyxis (15%) were found in drinking water. Three major groups of the genus Ralstonia (24%), Staphylococcus (20%) and Bosea (22%) were found in GI tracts. Ralstonia (6%, 24%), Sphingopyxis (15%, 2%), Bacillus (3%, 5%), Escherichia coli (3%, 2%) and Mesorhizobium (3%, 5%) were found in both sources – drinking water and GI tract. A lineage-per-time plot shows that the both bacterial communities have convex shape lines, suggesting an excess of closely related ecotypes. A significant F ST test (0.00000–0.00901) coupled with an insignificant P test (0.07–0.46) implies that the tree contained several clades of closely related bacteria. Both phylogenetic and statistical results suggest a correlation between the bacterial communities originating in the drinking water and those associated with the GI tracts. The GI tract showed a higher genetic diversity than the drinking water, but a similar lineage-per-time plot was obtained overall. It means a sudden evolutionary transformation and colonization occurred with high selective forces.

Microbial community of acetate utilizing denitrifiers in aerobic granules

The aerobic sludge granules cultivated at high organic loading rates could effectively convert 100-700 mg l(-1) nitrite to nitrogen gas with 400 or 1,200 mg l(-1) dosed acetate. The denitrifying microbial community structure of the so-cultivated granules was investigated by 16S rRNA gene sequences and localized using fluorescence in situ hybridization (FISH). The 16S rRNA gene phylotypes in the clone library and FISH probes used exhibited high diversity among the bacteria and denitrifying communities, with the members of Betaproteobacteria predominant that were closely related to families Comamonadaceae, Nitrosomonadaceae, Alcaligenaceae, and Rhodocyclaceae. The confocal laser scanning microscope and staining test revealed that active microbial community principally distributed at 200-250 microm beneath the outer surface, embedded in extracellular polymeric substances.

Microbial communities in iron‐silica‐rich microbial mats at deep‐sea hydrothermal fields of the Southern Mariana Trough

The abundance, diversity and composition of bacterial and archaeal communities in the microbial mats at deep-sea hydrothermal fields were investigated, using culture-independent 16S rRNA and functional gene analyses combined with mineralogical analysis. Microbial mats were collected at two hydrothermal areas on the ridge of the back-arc spreading centre in the Southern Mariana Trough. Scanning electron microscope and energy dispersive X-ray spectroscopic (SEM-EDS) analyses revealed that the mats were mainly composed of amorphous silica and contained numerous filamentous structures of iron hydroxides. Direct cell counting with SYBR Green I staining showed that the prokaryotic cell densities were more than 10(8) cells g(-1). Quantitative polymerase chain reaction (Q-PCR) analysis revealed that Bacteria are more abundant than Archaea in the microbial communities. Furthermore, zetaproteobacterial cells accounted for 6% and 22% of the prokaryotic cells in each mat estimated by Q-PCR with newly designed primers and TaqMan probe. Phylotypes related to iron-oxidizers, methanotrophs/methylotrophs, ammonia-oxidizers and sulfate-reducers were found in the 16S rRNA gene clone libraries constructed from each mat sample. A variety of unique archaeal 16S rRNA gene phylotypes, several pmoA, dsrAB and archaeal amoA gene phylotypes were also recovered from the microbial mats. Our results provide insights into the diversity and abundance of microbial communities within microbial mats in deep-sea hydrothermal fields.

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

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

Prokaryotic Populations and Activities in an Interbedded Coal Deposit, Including a Previously Deeply Buried Section (1.6–2.3 km) Above ∼ 150 Ma Basement Rock

A largely terrestrial, lignite/coal-bearing, 148 m core from the Waikato Basin, New Zealand, was studied, with a multidisciplinary approach, for subsurface microbiology. The top ≈76 m was Latest Miocene-Late Pleistocene (≈0.4–5.5 Ma) sediments, which overlay an unconformity and a previously deeply buried (1,600–2,300 m, × 55–75°C) ≈69 m section of moderately indurated, Late Eocene-Early Oligocene (≈32–35 Ma) deposits. Below this is weathered, Late Jurassic metasedimentary basement rock (145.5–157.0 Ma). Similar cell numbers (mean 1.2 × 106 cm −3 ), high viability (4–32%), intact phospholipids (biomarkers for living Bacteria) and activity (sulphate reduction, DNA replication) occurred heterogeneously throughout the core, including the weathered basement rock. Substantial numbers of viable anaerobic heterotrophic and lignite-utilizing bacteria (means 3.4 × 10 4 , 3.0 × 10 3 cm −3 ) were present throughout the core. This is similar to some deep terrestrial formations but contrasts with the generally exponentially decreasing prokaryotic populations in sub-seafloor sediments. For Bacteria, ≈76% of the 16S rRNA gene phylotypes were similar above (31.98 m) and below (133.55 m) the 76 m unconformity, which together with similar cell numbers indicates limited deep burial impact/palaeosterilization, or effective re-colonisation. Archaeal populations were not dominant being only detected with general primers at 31.98 m and those detected with methanogen functional primers were different above and below the ≈76 m unconformity. Both dominant bacterial (Proteobacteria, Actinobacteria, Firmicutes, Chloroflexi) and archaeal (Miscellaneous Crenarchaeotic Group, Methanosarcinales and Methanobacteriales) sequences were similar to those previously detected in both marine and terrestrial subsurface environments, reflecting the changing depositional conditions of the formation. However, the presence of ANME sequences had not been previously found in the terrestrial subsurface. A large proportion of the bacterial 16S rDNA diversity was cultured (43% of commonest genera). Prokaryotic populations and activity changed with lithology and depth and substrates (formate, acetate, oxalate) may diffuse from high-carbon, lignite/coaly layers to support bacterial populations in adjacent sandy or clay-silt layers.