Microbial diversity with dominance of 16S rRNA gene sequences with high GC contents at 74 and 98 °C subsurface crude oil deposits in Japan

We prepared DNA from the production waters of oil deposits and wellheads of the high- and hypertemperature Japanese oil wells #AR39 (depth, 1230 m; temperature, 74 °C; pressure, 2.92 MPa) and #SR123 (depth, 1687 m; temperature, 98 °C; pressure, 11.3 MPa) to detect indigenous bacterial and archaeal microorganisms. We used PCR to amplify the 16S rRNA genes of microbial communities and characterized them based on their sequences. A few species of microorganisms with high GC contents were detected in samples from oil deposits, whereas the microbial constituents and their GC contents were diverse in wellhead samples. A comparison of the composition of the microbial communities found that the predominant indigenous populations in the #SR123 oil deposit were Thermotoga hypogea-, Thermotoga petrophila- and Thermodesulfobacterium commune-like bacteria with a 61-63% GC content in their 16S rRNA gene sequences, and Archaeoglobus fulgidus-like archaea with a 65% GC content, whereas the major population in #AR39 comprised Thermacetogenium phaeum- and Fervidobacterium pennavorans-like bacteria and Methanothermobacter thermautotrophicus-like archaea with a 60%, 60% and 61% GC content, respectively.     

Evidence that Ammonia-Oxidizing Archaea are More Abundant than Ammonia-Oxidizing Bacteria in Semiarid Soils of Northern Arizona,USA

Autotrophic ammonia-oxidizing communities, which are responsible for the rate-limiting step of nitrification in most soils, have not been studied extensively in semiarid ecosystems. Abundances of soil archaeal and bacterial amoA were measured with real-time polymerase chain reaction along an elevation gradient in northern Arizona. Archaeal amoA was the predominant form of amoA at all sites; however, ratios of archaeal to bacterial amoA ranged from 17 to more than 1,600. Although size of ammonia-oxidizing bacteria populations was correlated with precipitation, temperature, percent sand, and soil C/N, there were no significant relationships between ammonia-oxidizing archaea populations and any of the environmental parameters evaluated in this study. Our results suggest that in these soils, archaea may be the primary ammonia oxidizers, and that ammonia-oxidizing archaea and ammonia-oxidizing bacteria occupy different niches.     

Spatiotemporal variation of bacterial and archaeal communities in sediments of a drinking reservoir,Beijing, China

Bacterial and archaeal assemblages are one of the most important contributors to the recycling of nutrients and the decomposition of organic matter in aquatic sediments. However, their spatiotemporal variation and its driving factors remain unclear, especially for drinking reservoirs, which are strongly affected by human consumption. Using quantitative PCR and Illumina MiSeq sequencing, we investigated the bacterial and archaeal communities in the sediments of a drinking reservoir, the Miyun Reservoir, one of the most important drinking sources for Beijing City. The abundance of bacteria and archaea presented no spatiotemporal variation. With respect to community diversity, visible spatial and temporal differences were observed in archaea, whereas the bacterial community showed minor variation. The bacterial communities in the reservoir sediment mainly included Proteobacteria, Bacteroidetes, Nitrospirae, Acidobacteria, and Verrucomicrobia. The bacterial community structure showed obvious spatial variation. The composition of the bacterial operational taxonomic units (OTUs) and main phyla were dam-specific; the composition of samples in front of the dam were significantly different from the composition of the other samples. The archaeal communities were mainly represented by Woesearchaeota and Euryarchaeota. Distinctly spatial and seasonal variation was observed in the archaeal community structure. The sediment NH₄ ⁺–N, pH, and water depth were identified as the key driving factors of changes in the composition of the bacterial and archaeal communities. Water depth might have the greatest influence on the microbial community structure. The dam-specific community structure may be related to the greater water depth in front of the dam. This finding indicates that water depth might be the greatest contributor to the microbial community structure in the Miyun Reservoir.

     

Metagenomic and small-subunit rRNA analyses reveal the genetic diversity of bacteria, archaea, fungi, and viruses in soil

Recent studies have highlighted the surprising richness of soil bacterial communities; however, bacteria are not the only microorganisms found in soil. To our knowledge, no study has compared the diversities of the four major microbial taxa, i.e., bacteria, archaea, fungi, and viruses, from an individual soil sample. We used metagenomic and small-subunit RNA-based sequence analysis techniques to compare the estimated richness and evenness of these groups in prairie, desert, and rainforest soils. By grouping sequences at the 97% sequence similarity level (an operational taxonomic unit [OTU]), we found that the archaeal and fungal communities were consistently less even than the bacterial communities. Although total richness levels are difficult to estimate with a high degree of certainty, the estimated number of unique archaeal or fungal OTUs appears to rival or exceed the number of unique bacterial OTUs in each of the collected soils. In this first study to comprehensively survey viral communities using a metagenomic approach, we found that soil viruses are taxonomically diverse and distinct from the communities of viruses found in other environments that have been surveyed using a similar approach. Within each of the four microbial groups, we observed minimal taxonomic overlap between sites, suggesting that soil archaea, bacteria, fungi, and viruses are globally as well as locally diverse.     

Metagenomic and Small-Subunit rRNA Analyses Reveal the Genetic Diversity of Bacteria, Archaea, Fungi, and Viruses in Soil

Recent studies have highlighted the surprising richness of soil bacterial communities; however, bacteria are not the only microorganisms found in soil. To our knowledge, no study has compared the diversities of the four major microbial taxa, i.e., bacteria, archaea, fungi, and viruses, from an individual soil sample. We used metagenomic and small-subunit RNA-based sequence analysis techniques to compare the estimated richness and evenness of these groups in prairie, desert, and rainforest soils. By grouping sequences at the 97% sequence similarity level (an operational taxonomic unit [OTU]), we found that the archaeal and fungal communities were consistently less even than the bacterial communities. Although total richness levels are difficult to estimate with a high degree of certainty, the estimated number of unique archaeal or fungal OTUs appears to rival or exceed the number of unique bacterial OTUs in each of the collected soils. In this first study to comprehensively survey viral communities using a metagenomic approach, we found that soil viruses are taxonomically diverse and distinct from the communities of viruses found in other environments that have been surveyed using a similar approach. Within each of the four microbial groups, we observed minimal taxonomic overlap between sites, suggesting that soil archaea, bacteria, fungi, and viruses are globally as well as locally diverse.     

Bacterial, archaeal, and eukaryal diversity in the intestines of Korean people

The bacterial, archaeal, and eukaryal diversity in fecal samples from ten Koreans were analyzed and compared by using the PCR-fingerprinting method, denaturing gradient gel electrophoresis (DGGE). The bacteria all belonged to the Firmicutes and Bacteroidetes phyla, which were known to be the dominant bacterial species in the human intestine. Most of the archaeal sequences belonged to the methane-producing archaea but several halophilic archarea-related sequences were also detected unexpectedly. While a small number of eukaryal sequences were also detected upon DGGE analysis, these sequences were related to fungi and stramenopiles (Blastocystis hominis). With regard to the bacterial and archaeal DGGE analysis, all ten samples had one and two prominent bands, respectively, but many individual-specific bands were also observed. However, only five of the ten samples had small eukaryal DGGE bands and none of these bands was observed in all five samples. Unweighted pair group method and arithmetic averages clustering algorithm (UPGMA) clustering analysis revealed that the archaeal and bacterial communities in the ten samples had relatively higher relatedness (the average Dice coefficient values were 68.9 and 59.2% for archaea and bacteria, respectively) but the eukaryal community showed low relatedness (39.6%).     

云南热带户用沼气池的原核生物群落结构研究

【目的】揭示云南热带农村户用沼气池中的原核生物(细菌和古菌)的群落结构特征。【方法】采用16S r RNA基因克隆文库技术对云南(北)热带代表性气候区的户用沼气池中的原核生物(细菌和古菌)多样性进行研究。【结果】得到细菌330条有效序列,划分为108个OTUs,文库覆盖度为81.5%;古菌有效序列185条,划分为17个OTUs,文库覆盖度为97.8%。通过Gen Bank数据库进行相似性比对与系统发育分析,结果表明:大部分细菌为未知细菌(Unclassified bacteria,占24.19%),优势细菌类群归属拟杆菌门(Bacteroidetes,占23.58%)、绿弯菌门(Chloroflexi,占21.46%)、厚壁菌门(Firmicutes,占13.91%)和变形菌门(Proteobacteria,占8.74%);古菌主要的优势类群为乙酸盐营养型的甲烷八叠球菌目(Methanosarcinales)的鬃毛甲烷菌属(Methanosaeta,占76.75%);此外还检测到少量未培养的泉古菌门细菌(Crenarchaeota,占9.19%)。【结论】云南(北)热带代表性气候区的农村户用沼气池中的微生物种类十分丰富,不同微生物种类的丰度存在明显差异,并存在明显优势种群,且细菌比古菌具有更丰富的多样性。     

Seasonal changes in nitrogen-cycle gene abundances and in bacterial communities in acidic forest soils

The abundance of genes related to the nitrogen biogeochemical cycle and the microbial community in forest soils (bacteria, archaea, fungi) were quantitatively analyzed via real-time PCR using 11 sets of specific primers amplifying nifH, bacterial amoA, archaeal amoA, narG, nirS, nirK, norB, nosZ, bacterial 16S rRNA gene, archaeal 16S rRNA gene, and the ITS sequence of fungi. Soils were sampled from Bukhan Mountain from September of 2010 to July of 2011 (7 times). Bacteria were the predominant microbial community in all samples. However, the abundance of archaeal amoA was greater than bacterial amoA throughout the year. The abundances of nifH, nirS, nirK, and norB genes changed in a similar pattern, while narG and nosZ appeared in sensitive to the environmental changes. Clone libraries of bacterial 16S rRNA genes were constructed from summer and winter soil samples and these revealed that Acidobacteria was the most predominant phylum in acidic forest soil environments in both samples. Although a specific correlation of environmental factor and gene abundance was not verified by principle component analysis, our data suggested that the combination of biological, physical, and chemical characteristics of forest soils created distinct conditions favoring the nitrogen biogeochemical cycle and that bacterial communities in undisturbed acidic forest soils were quite stable during seasonal change.     

Archaeal and bacterial diversity in hot springs on the Tibetan Plateau, China

The diversity of archaea and bacteria was investigated in ten hot springs (elevation >4600 m above sea level) in Central and Central-Eastern Tibet using 16S rRNA gene phylogenetic analysis. The temperature and pH of these hot springs were 26-81°C and close to neutral, respectively. A total of 959 (415 and 544 for bacteria and archaea, respectively) clone sequences were obtained. Phylogenetic analysis showed that bacteria were more diverse than archaea and that these clone sequences were classified into 82 bacterial and 41 archaeal operational taxonomic units (OTUs), respectively. The retrieved bacterial clones were mainly affiliated with four known groups (i.e., Firmicutes, Proteobacteria, Cyanobacteria, Chloroflexi), which were similar to those in other neutral-pH hot springs at low elevations. In contrast, most of the archaeal clones from the Tibetan hot springs were affiliated with Thaumarchaeota, a newly proposed archaeal phylum. The dominance of Thaumarchaeota in the archaeal community of the Tibetan hot springs appears to be unique, although the exact reasons are not yet known. Statistical analysis showed that diversity indices of both archaea and bacteria were not statistically correlated with temperature, which is consistent with previous studies.     

Application of quantitative real-time PCR for enumeration of total bacterial,archaeal, and yeast populations in kimchi

Kimchi is a Korean traditional fermented food made of brined vegetables, with a variety of spices. Various microorganisms are associated with the kimchi fermentation process. This study was undertaken in order to apply quantitative real-time PCR targeting the 16S and 26S rRNA genes for the investigation of dynamics of bacterial, archaeal, and yeast communities during fermentation of various types of kimchi. Although the total bacterial and archaeal rRNA gene copy numbers increased during kimchi fermentation, the number of yeasts was not significantly altered. In 1 ng of bulk DNA, the mean number of rRNA gene copies for all strains of bacteria was 5.45×10⁶ which was 360 and 50 times greater than those for archaea and yeast, respectively. The total gene copy number for each group of microorganisms differed among the different types of kimchi, although the relative ratios among them were similar. The common dominance of bacteria in the whole microbial communities of various types of kimchi suggests that bacteria play a principal role in the kimchi fermentation process.     

Archaeal abundance across a pH gradient in an arable soil and its relationship to bacterial and fungal growth rates

Soil pH is one of the most influential factors for the composition of bacterial and fungal communities, but the influence of soil pH on the distribution and composition of soil archaeal communities has yet to be systematically addressed. The primary aim of this study was to determine how total archaeal abundance (quantitative PCR [qPCR]-based estimates of 16S rRNA gene copy numbers) is related to soil pH across a pH gradient (pH 4.0 to 8.3). Secondarily, we wanted to assess how archaeal abundance related to bacterial and fungal growth rates across the same pH gradient. We identified two distinct and opposite effects of pH on the archaeal abundance. In the lowest pH range (pH 4.0 to 4.7), the abundance of archaea did not seem to correspond to pH. Above this pH range, there was a sharp, almost 4-fold decrease in archaeal abundance, reaching a minimum at pH 5.1 to 5.2. The low abundance of archaeal 16S rRNA gene copy numbers at this pH range then sharply increased almost 150-fold with pH, resulting in an increase in the ratio between archaeal and bacterial copy numbers from a minimum of 0.002 to more than 0.07 at pH 8. The nonuniform archaeal response to pH could reflect variation in the archaeal community composition along the gradient, with some archaea adapted to acidic conditions and others to neutral to slightly alkaline conditions. This suggestion is reinforced by observations of contrasting outcomes of the (competitive) interactions between archaea, bacteria, and fungi toward the lower and higher ends of the examined pH gradient.     

Different response of bacteria,archaea and fungi to process parameters in nine full-scale anaerobic digesters

Biogas production is a biotechnological process realized by complex bacterial, archaeal and likely fungal communities. Their composition was assessed in nine full-scale biogas plants with distinctly differing feedstock input and process parameters. This study investigated the actually active microbial community members by using a comprehensive sequencing approach based on ribosomal 16S and 28S rRNA fragments. The prevailing taxonomical units of each respective community were subsequently linked to process parameters. Ribosomal rRNA of bacteria, archaea and fungi, respectively, showed different compositions with respect to process parameters and supplied feedstocks: (i) bacterial communities were affected by the key factors temperature and ammonium concentration; (ii) composition of archaea was mainly related to process temperature; and (iii) relative abundance of fungi was linked to feedstocks supplied to the digesters. Anaerobic digesters with a high methane yield showed remarkably similar bacterial communities regarding identified taxonomic families. Although archaeal communities differed strongly on genus level from each other, the respective digesters still showed high methane yields. Functional redundancy of the archaeal communities may explain this effect. 28S rRNA sequences of fungi in all nine full-scale anaerobic digesters were primarily classified as facultative anaerobic Ascomycota and Basidiomycota. Since the presence of ribosomal 28S rRNA indicates that fungi may be active in the biogas digesters, further research should be carried out to examine to which extent they are important players in anaerobic digestion processes.     

Molecular assessment of complex microbial communities degrading long chain fatty acids in methanogenic bioreactors

Microbial diversity of anaerobic sludge after extended contact with long chain fatty acids (LCFA) was studied using molecular approaches. Samples containing high amounts of accumulated LCFA were obtained after continuous loading of two bioreactors with oleate or with palmitate. These sludge samples were then incubated in batch assays to allow degradation of the biomass-associated LCFA. In addition, sludge used as inoculum for the reactors was also characterized. Predominant phylotypes of the different samples were monitored using denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene fragments. Fingerprinting analysis showed changes in bacterial and archaeal communities during LCFA accumulation and degradation. Full-length 16S rRNA gene sequences of 22 clones, representing the predominant bacteria and archaea, were determined. Most bacterial clones (80%) clustered within the Clostridiaceae. Two major groups of methanogens were identified: hydrogen- and formate-utilizing organisms, closely related to Methanobacterium, and acetoclastic organisms closely related to Methanosaeta and Methanosarcina. Quantification by FISH and real-time PCR showed that the relative abundance of archaea increased during degradation of biomass-accumulated LCFA. These results provide insight into the importance and dynamics of balanced communities of bacteria and methanogens in LCFA-accumulation/degradation cycles.     

Archaeal protein translocation crossing membranes in the third domain of life.

Proper cell function relies on correct protein localization. As a first step in the delivery of extracytoplasmic proteins to their ultimate destinations, the hydrophobic barrier presented by lipid-based membranes must be overcome. In contrast to the well-defined bacterial and eukaryotic protein translocation systems, little is known about how proteins cross the membranes of archaea, the third and most recently described domain of life. In bacteria and eukaryotes, protein translocation occurs at proteinaceous sites comprised of evolutionarily conserved core components acting in concert with other, domain-specific elements. Examination of available archaeal genomes as well as cloning of individual genes from other archaeal strains reveals the presence of homologues to selected elements of the bacterial or eukaryotic translocation machines. Archaeal genomic searches, however, also reveal an apparent absence of other, important components of these two systems. Archaeal translocation may therefore represent a hybrid of the bacterial and eukaryotic models yet may also rely on components or themes particular to this domain of life. Indeed, considering the unique chemical composition of the archaeal membrane as well as the extreme conditions in which archaea thrive, the involvement of archaeal-specific translocation elements could be expected. Thus, understanding archaeal protein translocation could reveal the universal nature of certain features of protein translocation which, in some cases, may not be readily obvious from current comparisons of bacterial and eukaryotic systems. Alternatively, elucidation of archaeal translocation could uncover facets of the translocation process either not yet identified in bacteria or eukaryotes, or which are unique to archaea. In the following, the current status of our understanding of protein translocation in archaea is reviewed.     

Springtime dynamics,productivity and activity of prokaryotes in two Arctic fjords

In the Kongsfjorden–Krossfjorden system (Spitsbergen), increasing temperatures enhance glacier melting and concomitant intrusion of freshwater. These altered conditions affect the timing, intensity, and composition of the phytoplankton spring bloom in Kongsfjorden; yet, the effects on prokaryotes (bacteria and archaea) are not well understood. The aim of this study was to examine springtime prokaryote communities in both fjords as a function of hydrographic and phytoplankton variability. Prokaryote community composition was studied in two consecutive years by molecular fingerprinting of the 16S rRNA gene. In addition, we measured bacterial abundance, productivity (³H-Leucine uptake), and single-cell activity using catalyzed reporter deposition fluorescence in situ hybridization combined with microautoradiography. Differences in bacterial and archaeal communities were found between Kongsfjorden and Krossfjorden. Furthermore, an increase in productivity, abundance, and proportion of active bacterial cells was observed during the course of spring. Bacteroidetes were the most abundant bacterial group among the assessed taxa in both Kongsfjorden and Krossfjorden. Multivariate analysis of the microbial community fingerprints revealed a strong temporal shaping of both the bacterial and archaeal communities in addition to a spatial separation between the two fjords. A significant part of the observed bacterial variation could be explained by cyanobacterial biomass, as deduced from pigment analysis, and by phosphate concentration. Archaea were mainly controlled by abiotic factors. We speculate that the bacterial response to hydrographic changes and glacier meltwater is mediated through shifts in phytoplankton abundance and composition, whereas archaea are directly influenced by abiotic environmental variables.     

Microbial diversity of cold-seep sediments in Sagami Bay, Japan, as determined by 16S rRNA gene and lipid analyses

Microbial communities in Calyptogena sediment and microbial mats of Sagami Bay, Japan, were characterized using 16S rRNA gene sequencing and lipid biomarker analysis. Characterization of 16S rRNA gene isolated from these samples suggested a predominance of bacterial phylotypes related to Gammaproteobacteria (57-64%) and Deltaproteobacteria (27-29%). The Epsilonproteobacteria commonly found in cold seeps and hydrothermal vents were only detected in the microbial mat sample. Significantly different archaeal phylotypes were found in Calyptogena sediment and microbial mats; the former contained only Crenarchaeota clones (100% of the total archaeal clones) and the latter exclusively Euryarchaeota clones, including the anaerobic oxidation of methane archaeal groups ANME-2a and ANME-2c. Many of these lineages are as yet uncultured and undescribed groups of bacteria and archaea. Phospholipid fatty acid analysis suggested the presence of sulphate-reducing and sulphur-oxidizing bacteria. Results of intact glyceryl dialkyl glyceryl tetraether lipid analysis indicated the presence of nonthermophilic marine planktonic archaea. These results suggest that the microbial community in the Sagami Bay seep site is distinct from previously characterized cold-seep environments.     

Characterization of the bacterial and archaeal communities in rice field soils subjected to long-term fertilization practices

The bacterial and archaeal communities in rice field soils subjected to different fertilization regimes for 57 years were investigated in two different seasons, a non-planted, drained season (April) and a rice-growing, flooded season (August), by performing soil dehydrogenase assay, real-time PCR assay and pyrosequencing analysis. All fertilization regimes increased the soil dehydrogenase activity while the abundances of bacteria and archaea increased in the plots receiving inorganic fertilizers plus compost and not in those receiving inorganic fertilizers only. Rice-growing and flooding decreased the soil dehydrogenase activity while they increased the bacterial diversity in rice field soils. The bacterial communities were dominated by Chloroflexi, Proteobacteria, and Actinobacteria and the archaeal communities by Crenarchaeota at the phylum level. In principal coordinates analysis based on the weighted Fast UniFrac metric, the bacterial and archaeal communities were separated primarily by season, and generally distributed along with soil pH, the variation of which had been caused by long-term fertilization. Variations in the relative abundance according to the season or soil pH were observed for many bacterial and archaeal groups. In conclusion, the microbial activity, prokaryotic abundance and diversity, and prokaryotic community structure in the rice field soils were changed by season and long-term fertilization.     

Co-evolution of the archaeal tRNA-dependent amidotransferase GatCAB with tRNA(Asn)

The important identity elements in tRNA(Gln) and tRNA(Asn) for bacterial GatCAB and in tRNA(Gln) for archaeal GatDE are the D-loop and the first base pair of the acceptor stem. Here we show that Methanothermobacter thermautotrophicus GatCAB, the archaeal enzyme, is different as it discriminates Asp-tRNA(Asp) and Asp-tRNA(Asn) by use of U49, the D-loop and to a lesser extent the variable loop. Since archaea possess the tRNA(Gln)-specific amidotransferase GatDE, the archaeal GatCAB enzyme evolved to recognize different elements in tRNA(Asn) than those recognized by GatDE or by the bacterial GatCAB enzyme in their tRNA substrates.     

Different Effects of Transgenic Maize and Nontransgenic Maize on Nitrogen-Transforming Archaea and Bacteria in Tropical Soils

The composition of the rhizosphere microbiome is a result of interactions between plant roots, soil, and environmental conditions. The impact of genetic variation in plant species on the composition of the root-associated microbiota remains poorly understood. This study assessed the abundances and structures of nitrogen-transforming (ammonia-oxidizing) archaea and bacteria as well as nitrogen-fixing bacteria driven by genetic modification of their maize host plants. The data show that significant changes in the abundances (revealed by quantitative PCR) of ammonia-oxidizing bacterial and archaeal communities occurred as a result of the maize host being genetically modified. In contrast, the structures of the total communities (determined by PCR-denaturing gradient gel electrophoresis) were mainly driven by factors such as soil type and season and not by plant genotype. Thus, the abundances of ammonia-oxidizing bacterial and archaeal communities but not structures of those communities were revealed to be responsive to changes in maize genotype, allowing the suggestion that community abundances should be explored as candidate bioindicators for monitoring the possible impacts of cultivation of genetically modified plants.