Bacterial diversity of the Inner Mongolian Baer Soda Lake as revealed by 16S rRNA gene sequence analyses

Bacterial diversity associated with Baer Soda Lake in Inner Mongolia of China was investigated using a culture-independent method. Bacterial 16S rRNA gene libraries were generated using bacterial oligonucleotide primers, and 16S rRNA gene sequences of 58 clones were analyzed phylogenetically. The library was dominated by 16S rDNAs of Gram-negative bacteria (24% -Proteobacteria, 31% -Proteobacteria, 33% -Proteobacteria, and 2% -Proteobacteria), with a lower percentage of clones corresponding to Gram-positive bacteria. Forty cloned sequences were similar to that of known bacterial isolates (>97% sequence similarity), represented by the species of the genera Brevundimonas, Comamonas, Alcaligenes, Stenotrophomonas, and Klebsiella. Eighteen cloned sequences showed less affiliation with known taxa (<97% sequence similarity) and may represent novel taxa.Communicated by K. Horikoshi     

Microbial diversity in hot synthetic compost as revealed by PCR-amplified rRNA sequences from cultivated isolates and extracted DNA

High-temperature (>/=60 degrees C) synthetic food waste compost was examined by cultivation-dependent and -independent methods to determine predominant microbial populations. Fluorescent direct counts totaled 6.4 (+/-2.5)x10(10) cells gdw(-1) in a freeze-dried 74 degrees C compost sample, while plate counts for thermophilic heterotrophic aerobes averaged 2.6 (+/-1.0)x10(8) CFU gdw(-1). A pre-lysis cell fractionation method was developed to obtain community DNA and a suite of 16S and 18S rDNA-targeted PCR primers was used to examine the presence of Bacteria, Archaea and fungi. Bacterial 16S rDNA, including a domain-specific 1500-bp fragment and a 300-bp fragment specific for Actinobacteria, was amplified by PCR from all compost samples tested. Archaeal rDNA was not amplified in any sample. Fungal 18S rDNA was only amplified from a separate dairy manure compost that reached a peak temperature of 50 degrees C. Amplified rDNA restriction analysis (ARDRA) was used to screen isolated thermophilic bacteria and a clone library of full-length rDNA fragments. ARDRA screening revealed 14 unique patterns among 63 isolates, with one pattern accounting for 31 of the isolates. In the clone library, 52 unique patterns were detected among 70 clones, indicating high diversity of uncultivated bacteria in hot compost. Phylogenetic analysis revealed that the two most abundant isolates belonged in the genera Aneurinibacillus and Brevibacillus, which are not commonly associated with hot compost. With the exception of one Lactobacillus-type sequence, the clone library contained only sequences that clustered within the genus Bacillus. None of the isolates or cloned sequences could be assigned to the group of obligate thermophilic Bacillus spp. represented by B. stearothermophilus, commonly believed to dominate high-temperature compost. Amplified partial fragments from Actinobacteria, spanning the V3 variable region (Neefs et al. (1990) Nucleic Acids Res. 18, 2237-2242), included sequences related to the genera Saccharomonospora, Gordonia, Rhodococcus and Corynebacterium, although none of these organisms were detected among the isolates or full-length cloned rDNA sequences. All of the thermophilic isolates and sequenced rDNA fragments examined in this study were from Gram-positive organisms.     

Bacterial,archaeal and eukaryotic diversity in Arctic sediment as revealed by 16S rRNA and 18S rRNA gene clone libraries analysis

We studied the microbial diversity in the sediment from the Kongsfjorden, Svalbard, Arctic, in the summer of 2005 based on the analysis of 16S rRNA and 18S rRNA gene clone libraries. The sequences of the cloned 16S rRNA and 18S rRNA gene inserts were used to determine the species identity or closest relatives by comparison with sequences of known species. Compared to the other samples acquired in Arctic and Antarctic, which are different from that of ours, the microbial diversity in our sediment is much higher. The bacterial sequences were grouped into 11 major lineages of the domain Bacteria: Proteobacteria (include α-, β-, γ-, δ-, and ε-Proteobacteria); Bacteroidetes; Fusobacteria; Firmicutes; Chloroflexi; Chlamydiae; Acidobacteria; Actinobacteria; Planctomycetes; Verrucomicrobiae and Lentisphaerae. Crenarchaeota were dominant in the archaeal clones containing inserts. In addition, six groups from eukaryotes including Cercozoa, Fungi, Telonema, Stramenopiles, Alveolata, and Metazoa were identified. Remarkably, the novel group Lentisphaerae was reported in Arctic sediment at the first time. Our study suggested that Arctic sediment as a unique habitat may contain substantial microbial diversity and novel species will be discovered.     

Bacterial diversity at different depths in lead-zinc mine tailings as revealed by 16S rRNA gene libraries

Bacterial communities at 10 cm, 100 cm, and 200 cm depths in a 100-year-old lead-zinc tailing heap were evaluated by constructing 16S rRNA gene libraries. In total, 98 operational taxonomic units (OTUs) were identified from 193 clones at a 3% sequence difference level. The OTU number and species richness decreased with the depth. Species composition was significantly different between the three libraries. Fifty-seven percent of the examined clones were Acidobacteria and 27% belonged to Proteobacteria. Other sequences included Chloroflexi, Firmicutes, Chlamydiae, Actinobacteria, Gemmatimonadetes, Nitrospira, and three unclassified OTUs. Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Firmicutes, and Actinobacteria were mainly distributed in the rhizosphere of naturally colonizing plants; however, Deltaproteobacteria, Acidobacteria, and Chloroflexi tended to inhabit the deeper tailings (below the 100 cm-depth).     

Archaeal diversity in a terrestrial acidic spring field revealed by a novel PCR primer targeting archaeal 16S rRNA genes

The phylogenetic diversity of archaeal 16S rRNA genes in a thermoacidic spring field of Ohwakudani, Hakone, Japan, was investigated by PCR-based analysis using a novel Archaea-specific primer designed in the present study. Clone libraries of archaeal 16S rRNA genes were constructed from hot water (78 °C) and mud (28 °C) samples by PCR using a newly designed forward primer and a previously reported forward primer with reverse primers. Most phylotypes found in the libraries from the hot water sample were related to cultured (hyper)thermophiles. The phylotypes and their detection frequencies from the hot water sample were similar for the libraries amplified with the two different primer sets. In contrast, phylotypes having a low similarity (<95%) to cultured Archaea were found in the libraries from the mud sample. Some of the phylotypes were relatively close to members of Thermoplasmata (80-93% similarity) and the others were not clearly affiliated with Crenarchaeota and Euryarchaeota, but related to Thaumarchaeota and Korarchaeota. The phylotypes and their detection frequencies were significantly different between the two libraries of the mud sample. Our results from the PCR-based analysis using the redesigned primer suggest that more diverse, uncultured Archaea are present in acidic environments at a low temperature than previously recognized.     

Bacterial diversity based on 16S rRNA and gyrB genes at Yinshan mine, China

The diversity of bacterial communities at three sites impacted by acid mine drainage (AMD) from the Yinshan Mine in China was studied using comparative sequence analysis of two molecular markers, the 16S rRNA and gyrB genes. The phylogenetic analyses retrieved sequences from six classes of bacteria, Nitrospira, Alphaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, Acidobacteria, and Actinobacteria, as well as sequences related to the plastid of the cyanobacterium Cyanidium acidocaldarium and also some unknown bacteria. The results of phylogenetic analyses based on gyrB and 16S rRNA were compared. This confirmed that gyrB gene analysis may be a useful tool, in addition to the comparative sequence analysis of the 16S rRNA gene, for the analysis of microbial community compositions. Moreover, the Mantel test showed that the geochemical characteristics, especially the pH value and the concentration of iron, strongly influenced the composition of the microbial communities.     

Differentiation of Bartonella species using restriction endonuclease analysis of PCR-amplified 16S rRNA genes

Abstract Differentiation of the four Bartonella species which were formerly classified as Rochalimaea using restriction endonuclease analysis of PCR-amplified citrate synthase gene fragments has previously been described. However, attempts to extend this method to include all members of Bartonella were confounded when amplification of the gene fragment from strains of B. bacilliformis each yielded two products of differing sizes. An alternative differentiation scheme for Bartonella species was developed based on restriction endonuclease analysis of their 16S rRNA genes. As the complete 16S rRNA gene sequences of all extant Bartonella species are available, the usefulness of specific endonucleases could be theoretically predetermined rather than discovered empirically. The potential usefulness of the restriction enzymes Ddel and Mnll was established using this approach, and this potential was confirmed in practice as all eight species could be distinguished from each other.     

16S rRNA基因高通量测序法分析黄河太岁细菌的多样性

【背景】太岁在我国的记载由来已久,《神农本草经》记载其具有扶正固本、轻身不老的功效。但是,太岁作为一种生物体,它的组成分类尚不明确,因而其药用价值得不到有效的科学验证。因此,利用各种生物技术和手段来客观地分析太岁的成分,为利用和开发太岁提供科学依据。【目的】检测太岁(编号D15112285)中所含细菌的种类,探究太岁中可能存在的原核微生物的种类及其之间的关系。【方法】采用Illumina MiSeq 2×250系统对黄河太岁的细菌16S rRNA基因(V4区)进行研究,利用FLASH等软件对数据进行分析。【结果】共获得OTU(Operational taxonomical unit)626条,涉及19门49纲80目107科112属。在属的水平上前十的优势菌群有Bacteroides、Coprococcus、Escherichia、Ruminococcus、Lactobacillus、[Ruminococcus]、Oscillospira、Faecalibacterium、Shewanella和Halomonas。【结论】黄河太岁中存在多种不同种类的细菌。     

Bacterial diversity in saline-alkali ponds rearing common carp (Cyprinus carpio) as revealed by 16S rRNA gene sequences

The bacterial diversity in saline-alkali ponds rearing common carp was investigated using the 16S rRNA gene clone library technique. Phylogenetic analysis of the most common and dominant sequences recovered indicated that these sequences fell into the following major lineages, including Proteobacteria (α-, β-, γ-), Actinobacteria, Cyanobacteria, Planctomycetes, Fibrobacteres, Bacteroidetes, Chloroflexi, and unclassified bacteria. Sequence analysis showed that the bacterial diversity was abundant, and the sequences belonging to β-Proteobacteria, α-Proteobacteria and Actinobacteria were predominant. The most sequences in the saline-alkali rearing ponds exhibited low similarity with known bacterial 16S rRNA genes, suggesting that these sequences may represent novel bacteria. In addition, the majority of our sequences were most closely affiliated with sequences retrieved from inland waters of China. These results suggest that the saline-alkali ponds rearing common carp are specific ecologic niches and the distribution of the bacteria may be influenced by geographical factors. This study reports the bacterial diversity in saline-alkali ponds rearing common carp by the culture-independent technique for the first time; therefore, it provides important information for understanding the microbial ecology in saline-alkali rearing ponds and managing the microbial community composition to promote and maintain the health of aquaculture environments.     

Molecular diversity of 16S rRNA and gyrB genes in copper mines

The molecular diversities of the microbial communities from four sites impacted by acid mine drainage (AMD) at Dexing Copper Mine in Jiangxi province of China were studied using 16S rRNA sequences and gyrB sequences. Of the four sampled sites, each habitat exhibited distinct geochemical characteristics and the sites were linked geographically allowing us to correlate microbial community structure to geochemical characteristics. In the present study, we examined the molecular diversity of 16S rRNA and gyrB genes from water at these sites using a PCR-based cloning approach. We found that the microbial community appears to be composed primarily of Proteobacteria, Acidobacteria, Actinobacteria, Nitrospira, Firmicutes, Chlorella and unknown phylotypes. Of clones affiliated with Nitrospira, Leptospirillum ferrooxidans, Leptospirillum ferriphilum and Leptospirillum group III were all detected. Principal-component analysis (PCA) revealed that the distribution of the microbial communities was influenced greatly by geochemical characteristics. The overall PCA profiles showed that the sites with similar geochemical characteristics had more similar microbial community structures. Moreover, our results also indicated that gyrB sequence analysis may be very useful for differentiating very closely related species in the study of microbial communities. H. Yin and L. Cao contributed equally to this work.     

Bacterial diversity and community structure in an aerated lagoon revealed by ribosomal intergenic spacer analyses and 16S ribosomal DNA sequencing

We investigated the bacterial community structure in an aerated plug-flow lagoon treating pulp and paper mill effluent. For this investigation, we developed a composite method based on analyses of PCR amplicons containing the ribosomal intergenic spacer (RIS) and its flanking partial 16S rRNA gene. Community percent similarity was determined on the basis of RIS length polymorphism. A community succession was evident in the lagoon, indicated by a progressive community transition through seven sample locations. The most abrupt changes in community structure were associated with a temperature change from 39 to 35 degrees C and with increases in dissolved oxygen. The temporal differences in community structure, based on summer and winter samplings, were greater than the spatial differences during either season. Clone libraries of rDNA-RIS amplicons were constructed from each of three summer samples. Among 90 clones analyzed (30 clones from each sample), 56 phylotypes were distinguished by restriction fragment length polymorphism. Indices of phylotype richness, evenness, and diversity all increased in clone libraries from the beginning to the end of the lagoon. A representative clone of each phylotype was phylogenetically analyzed on the basis of its partial 16S rRNA gene sequence (ca. 450 bp). Phylogenetic analysis confirmed the increase in diversity and further indicated increasing richness of bacterial divisions. Pioneers in the community spatial succession appeared to include thermotolerant, microaerophilic methanol-oxidizing bacteria related to the genus Methylobacillus, as well as thermotolerant, microaerophilic nitrogen-fixing bacteria related to the genus Azospirillum.     

Uncultured bacterial diversity in a seawater recirculating aquaculture system revealed by 16S rRNA gene amplicon sequencing

Bacterial diversity in a seawater recirculating aquaculture system (RAS) was investigated using 16S rRNA amplicon sequencing to understand the roles of bacterial communities in the system. The RAS was operated at nine different combinations of temperature (15°C, 20°C, and 25°C) and salinity (20‰, 25‰, and 32.5‰). Samples were collected from five or six RAS tanks (biofilters) for each condition. Fifty samples were analyzed. Proteobacteria and Bacteroidetes were most common (sum of both phyla: 67.2% to 99.4%) and were inversely proportional to each other. Bacteria that were present at an average of ≥ 1% included Actinobacteria (2.9%) Planctomycetes (2.0%), Nitrospirae (1.5%), and Acidobacteria (1.0%); they were preferentially present in packed bed biofilters, mesh biofilters, and maturation biofilters. The three biofilters showed higher diversity than other RAS tanks (aerated biofilters, floating bed biofilters, and fish tanks) from phylum to operational taxonomic unit (OTU) level. Samples were clustered into several groups based on the bacterial communities. Major taxonomic groups related to family Rhodobacteraceae and Flavobacteriaceae were distributed widely in the samples. Several taxonomic groups like [Saprospiraceae], Cytophagaceae, Octadecabacter, and Marivita showed a cluster-oriented distribution. Phaeobacter and Sediminicola-related reads were detected frequently and abundantly at low temperature. Nitrifying bacteria were detected frequently and abundantly in the three biofilters. Phylogenetic analysis of the nitrifying bacteria showed several similar OTUs were observed widely through the biofilters. The diverse bacterial communities and the minor taxonomic groups, except for Proteobacteria and Bacteroidetes, seemed to play important roles and seemed necessary for nitrifying activity in the RAS, especially in packed bed biofilters, mesh biofilters, and maturation biofilters.     

Bacterial community composition of a shallow hypertrophic freshwater lake in China, revealed by 16S rRNA gene sequences

The phylogenetic composition of a bacterial community from a hypertrophic freshwater lake in China was investigated by sequencing cloned 16S rRNA genes. Three hundred and thirty-six bacterial clones from four clone libraries in different months (March, May, July and September in 2004) were classified into 142 operational taxonomic units, most of which were affiliated with bacterial divisions commonly found in freshwater ecosystem, e.g. Alpha-, Beta-, Gamma- and Deltaproteobacteria, Bacteriodetes and Actinobacteria. The results showed that the composition of bacterial community in the July library was the most diverse one. Actinobacteria was the most significant lineage in Lake Taihu, with dominant numbers of operational taxonomic units in the May, July and September libraries. Phylogenetic analysis suggested that 53 sequences were grouped into six novel clusters which may represent specific populations indigenous to the environment. Coverage analyses indicated that the clone libraries could provide a fine inventory of bacterial diversity in the lake.     

Bacterioplankton community structure in the Arctic waters as revealed by pyrosequencing of 16S rRNA genes

Fjords and open oceans are two typical marine ecosystems in the Arctic region, where glacial meltwater and sea ice meltwater have great effects on the bacterioplankton community structure during the summer season. This study aimed to determine the differences in bacterioplankton communities between these two ecosystems in the Arctic region. We conducted a detailed census of microbial communities in Kongsfjorden (Spitsbergen) and the Chukchi Borderland using high-throughput pyrosequencing of the 16S rRNA gene. Gammaproteobacteria and Bacteroidetes were the dominant members of the bacterioplankton community in Kongsfjorden. By contrast, the most abundant bacterial groups in the surface seawater samples from the Chukchi Borderland were Alphaproteobacteria and Actinobacteria. Differences in bacterial communities were found between the surface and subsurface waters in the investigation area of the Chukchi Borderland, and significant differences in bacterial community structure were also observed in the subsurface water between the shelf and deep basin areas. These results suggest the effect of hydrogeographic conditions on bacterial communities. Ubiquitous phylotypes found in all the investigated samples belonged to a few bacterial groups that dominate marine bacterioplankton communities. The sequence data suggested that changes in environmental conditions result in abundant rare phylotypes and reduced amounts of other phylotypes.     

Genetic diversity of Datisca cannabina-compatible Frankia strains as determined by sequence analysis of the PCR-amplified 16S rRNA gene.

The presence of Frankia strains in soil samples collected from northern areas of Pakistan was detected by inoculating Coriaria nepalensis and Datisca cannabina plants. The abundance of compatible Frankia strains in some areas was indicated by profuse nodulation of the host plants, whereas soil samples from other localities failed to result in nodulation. An oligonucleotide probe (COR/DAT) directed against the 16S rRNA gene of the endophytes of Coriaria and Datisca spp. that did not cross-react with the RNA gene of Frankia strains isolated from other hosts was developed. Genetic diversity among Frankia strains nodulating D. cannabina was determined by sequence analysis of the partial 16S rRNA gene amplified from nodules induced by soil samples from different localities by PCR. Four types of Frankia sequences and one non-Frankia sequence were detected by hybridization with a Frankia genus probe and the COR/DAT probe as well as by sequence analysis of the cloned PCR products.     

Phylogenetic clustering of soil microbial communities by 16S rRNA but not 16S rRNA genes

We evaluated phylogenetic clustering of bacterial and archaeal communities from redox-dynamic subtropical forest soils that were defined by 16S rRNA and rRNA gene sequences. We observed significant clustering for the RNA-based communities but not the DNA-based communities, as well as increasing clustering over time of the highly active taxa detected by only rRNA.     

Bacterial diversity of a Carolina bay as determined by 16S rRNA gene analysis: confirmation of novel taxa.

Carolina bays are naturally occurring shallow elliptical depressions largely fed by rain and shallow ground water. To identify members of the domain Bacteria which inhibit such an environment, we used PCR to construct a library of 16S rRNA genes (16S rDNAs) cloned from DNA extracted from the sediments of Rainbow bay, located on the Savannah River Site, near Aiken, S.C. Oligonucleotides complementary to conserved regions of 16S rDNA were used as primers for PCR, and gel-purified PCR products were cloned into vector pGEM-T. Partial sequencing of the cloned 16S rDNAs revealed an extensive amount of phylogenetic diversity within this system. Of the 35 clones sequenced, 32 were affiliated with five bacterial groups: 11 clustered with the Proteobacteria division (including members of the alpha, beta, and delta subdivisions), 8 clustered with the Acidobacterium subdivision of the Fibrobacter division (as categorized by the Ribosomal Database Project's taxonomic scheme, version 5.0), 7 clustered with the Verrucomicrobium subdivision of the Planctomyces division, 3 clustered with the gram-positive bacteria (Clostridium and relatives subdivision), and 3 clustered with the green nonsulfur bacteria. One sequence branched very deeply from the Bacteria and was found not to be associated with any of the major divisions when phylogenetic trees were constructed. Two clones did not consistently cluster with specific groups and may be chimeric sequences. None of the clones exhibited an exact match to any of the 16S rDNA sequences deposited in the databases, suggesting that most of the bacteria in Rainbow Bay are novel species. In particular, the clones related to the Acidobacterium subdivision and the Verrucomicrobium subdivision confirm the presence of novel taxa discovered previously in other molecular surveys of this type.