Deep sequencing analysis of bacterial community structure of Soldhar hot spring,India

We investigated the bacterial community structure of Soldhar hot spring with extreme high temperature 95°C located in Uttarakhand, India using high throughput sequencing. Bacterial phyla Proteobacteria (88.8%), Deinococcus-Thermus (7.5%), Actinobacteria (2.3%), and Firmicutes (1.07%) were predominated in the sequencing survey, whereas Bacteroidetes, Verrucomicrobia, Aquificae and Acidobacteria were detected in relatively lower abundance in Soldhar hot spring. At the family level, Comamonadaceae (52.5%), Alteromonadaceae (15.9%), and Thermaceae (7.5%) were mostly dominated in the ecosystem followed by Chromatiaceae, Microbacteriaceae, and Cyclobacteriaceae. Besides, members of Rhodobacteraceae, Moraxellaceae, Xanthomonadaceae, Aquificaceae, Enterobacteriaceae, Bacillaceae, Methylophilaceae, etc. were detected as a relatively lower abundance. In the present study we discuss the overall microbial community structure and their relevance to the ecology of the Soldhar hot spring environment.     

Investigation of the microbial ecology of intertidal hot springs by using diversity analysis of 16S rRNA and chitinase genes

The microbial diversity of intertidal hot springs on the seashore of northwest Iceland was examined by combining directed in situ enrichments, artificial support colonization, and mat sampling. Analysis of 16S rRNA genes revealed the presence of clones related to both marine and terrestrial, thermophilic, mesophilic, and psychrophilic microorganisms scattered among 11 bacterial divisions. No archaea were found. The species composition of the enrichments was affected by the length of the hot periods experienced at low tide and was very different from those found in the biomass. A total of 36 chitinase genes were detected by molecular screening of the samples with degenerate primers for glycoside hydrolase family 18. The chitinase gene diversity was at least twofold higher in the enrichment samples than in the controls, indicating that a much higher diversity of hydrolytic genes can be accessed with this approach.     

Comparison of 16S rRNA and protein-coding genes as molecular markers for assessing microbial diversity (Bacteria and Archaea) in ecosystems

PCR amplification of the rRNA gene is the most popular method for assessing microbial diversity. However, this molecular marker is often present in multiple copies in cells presenting, in addition, an intragenomic heterogeneity. In this context, housekeeping genes may be used as taxonomic markers for ecological studies. However, the efficiency of these protein-coding genes compared to 16S rRNA genes has not been tested on environmental data. For this purpose, five protein marker genes for which primer sets are available, were selected (rplB, pyrG, fusA, leuS and rpoB) and compared with 16S rRNA gene results from PCR amplification or metagenomic data from aquatic ecosystems. Analysis of the major groups found in these ecosystems, such as Actinobacteria, Bacteroides, Proteobacteria and Cyanobacteria, showed good agreement between the protein markers and the results given by 16S rRNA genes from metagenomic reads. However, with the markers it was possible to detect minor groups among the microbial assemblages, providing more details compared to 16S rRNA results from PCR amplification. In addition, the use of a set of protein markers made it possible to deduce a mean copy number of rRNA operons. This average estimate is essentially lower than the one estimated in sequenced genomes.     

三七连作根际土壤微生物区系的16S rRNA系统遗传多样性

【目的】针对三七连作土传病害的生物防治技术研究,探讨其根际土壤微生物多样性与根腐病害的关联性。【方法】采集三七连作6年的健康植株与根腐病植株根际土壤,采用多种培养基进行分离、纯化,并对分离菌株提取DNA,用其16S rRNA序列的通用引物进行PCR扩增,进行Blast同源及其系统进化树分析。【结果】从采集样品中分离出的菌株分布于细菌域(Bacteria)中的4个门(Phyla)共40个属(Genera),其中从健康植株组土壤中培养出179株菌,分布于30个属,以伯克氏菌属(Burkholderia)、节杆菌属(Arthrobacter)、链霉菌属(Streptomyces)及芽孢杆菌属(Bacillus)为优势菌群;根腐病株组土样共培养出117株菌,分布于29个属,以罗尔斯顿菌属(Ralstonia——病原菌)、单胞菌属(Sphingomonas)、寡养单胞菌属(Stenotrophomonas)等为优势菌群;其中黄杆菌属(Flavobacterium)、肠杆菌属(Enterobacter)的分离菌株仅从病株组土壤中分离到;发现其可能新种5株;健康组与根腐病组土壤EC值及各元素离子浓度差异明显(P<0.05),健康组土壤中的优势微生物种群分布与土样中的EC值、NO-3、SO2-4、CO2-3、K+及全盐量呈负相关关系(显著度P<0.01)。【结论】研究表明,诱导三七连作土传病害的发生,除病原菌作用影响外,还与土壤理化性质和土壤微生物的种群结构及其优势微生物的种群比例具有密切的关联性,特别是土壤中有益微生物(Burkholderia,Bacillus,Streptomyces等)的丰度,对评价三七土壤健康状况及病害防治预报具有重要的指导意义。     

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.     

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.     

Comparative genetic diversity of the narG,nosZ, and 16S rRNA genes in fluorescent pseudomonads

The diversity of the membrane-bound nitrate reductase (narG) and nitrous oxide reductase (nosZ) genes in fluorescent pseudomonads isolated from soil and rhizosphere environments was characterized together with that of the 16S rRNA gene by a PCR-restriction fragment length polymorphism assay. Fragments of 1,008 bp and 1,433 bp were amplified via PCR with primers specific for the narG and nosZ genes, respectively. The presence of the narG and nosZ genes in the bacterial strains was confirmed by hybridization of the genomic DNA and the PCR products with the corresponding probes. The ability of the strains to either reduce nitrate or totally dissimilate nitrogen was assessed. Overall, there was a good correspondence between the reductase activities and the presence of the corresponding genes. Distribution in the different ribotypes of strains harboring both the narG and nosZ genes and of strains missing both genes suggests that these two groups of strains had different evolutionary histories. Both dissimilatory genes showed high polymorphism, with similarity indexes (Jaccard) of between 0.04 and 0.8, whereas those of the 16S rRNA gene only varied from 0.77 to 0.99. No correlation between the similarity indexes of 16S rRNA and dissimilatory genes was seen, suggesting that the evolution rates of ribosomal and functional genes differ. Pairwise comparison of similarity indexes of the narG and nosZ genes led to the delineation of two types of strains. Within the first type, the similarity indexes of both genes varied in the same range, suggesting that these two genes have followed a similar evolution. Within the second type of strain, the range of variations was higher for the nosZ than for the narG gene, suggesting that these genes have had a different evolutionary rate.     

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 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.     

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.     

Mitochondrial 16S rRNA sequence diversity of hominoids

We determined nucleotide sequences of the 16S rRNA gene of mitochondrial DNA (mtDNA) (about 1.6 kb) for 35 chimpanzee, 13 bonobo, 10 gorilla, 16 orangutan, and 23 gibbon individuals. We compared those data with published sequences and estimated nucleotide diversity for each species. All the ape species showed higher diversity than human. We also constructed phylogenetic trees and networks. The two orangutan subspecies were clearly separated from each other, and Sumatran orangutans showed much higher nucleotide diversity than Bornean orangutans. Some gibbon species did not form monophyletic clusters, and variation within species was not much different from that among species in the subgenus Hylobates.     

Characterization of a Yellowstone hot spring microbial community by 5S rRNA sequences

The microorganisms inhabiting a 91 degrees C hot spring in Yellowstone National Park were characterized by sequencing 5S rRNAs isolated from the mixed, natural microflora without cultivation. By comparisons of these sequences with reference sequences, the phylogenetic relationships of the hot spring organisms to better characterized ones were established. Quantitation of the total 5S-sized rRNAs revealed a complex microbial community of three dominant members, a predominant archaebacterium affiliated with the sulfur-metabolizing (dependent) branch of the archaebacteria, and two eubacteria distantly related to Thermus spp. The archaebacterial and the eubacterial 5S rRNAs each constituted about half the examined population.     

Comparison of bacterial diversity along the human intestinal tract by direct cloning and sequencing of 16S rRNA genes

Bacterial diversity of the mucosal biopsies from human jejunum, distal ileum, ascending colon and rectum were compared by analysis of PCR-amplified 16S rDNA clone libraries. A total of 347 clones from the mucosal biopsies were partially sequenced and assigned to six phylogenetic phyla of the domain Bacteria: Firmicutes, Bacteroidetes, Proteobacteria, Fusobacteria, Verrucomicrobia, and Actinobacteria. The jejunum sample had least microbial diversity compared to the other samples and a trend towards highest diversity in ascending colon was observed. The clone libraries of distal ileum, ascending colon and rectum were not significantly different from each other (P>0.0043), but they differed significantly from the jejunum library (P=0.001). The population of sequences retrieved from jejunal biopsies was dominated by sequences closely related to Streptococcus (67%), while the population of sequences derived from distal ileum, ascending colon and rectum were dominated by sequences affiliated with Bacteroidetes (27-49%), and Clostridium clusters XIVa (20-34%) and IV (7-13%). The results indicate that the microbial community in jejunum is different from those in distal ileum, ascending colon and rectum, and that the major phylogenetic groups are similar from distal ileum to rectum.     

Selective phylogenetic analysis targeting 16S rRNA genes of hyperthermophilic archaea in the deep-subsurface hot biosphere

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

Genetic diversity of Acacia tortilis ssp. raddiana rhizobia in Tunisia assessed by 16S and 16S-23S rDNA genes analysis

AIMS: In order to understand the genetic diversity of Acacia tortilis ssp. raddiana-rhizobia in Tunisia, isolates from nine geographical locations were obtained and analysed. METHODS AND RESULTS: Characterization using restriction fragment length polymorphism analysis (RFLP) of PCR-amplified 16S rRNA gene and the intergenic spacer (IGS) between the 16S and 23S rRNA genes was undertaken. Symbiotic efficiency of the strains was also estimated. Analysis of the 16S rRNA by PCR-RFLP showed that the isolates were phylogenetically related to Ensifer ssp., Rhizobium tropicii-IIA, and Rhizobium tumefaciens species. Analysis of 16S-23S spacer by PCR-RFLP showed a high diversity of these rhizobia and revealed eleven additional groups, which indicates that these strains are genetically very diverse. Full 16S rRNA gene-sequencing showed that the majority of strains form a new subdivion inside the genera Ensifer, with Ensifer meliloti being its nearest neighbour. Nodulation test performed on the plant host demonstrated differences in the infectivity among the strains. CONCLUSION: Rhizobial populations that nodulate specifically and efficiently Acacia tortilis ssp. raddiana in representative soils of Tunisia is dominated by E. meliloti-like genomospecies. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper provides the first clear characterization and symbiotic efficiency data of rhizobia strains nodulating A. tortilis in Tunisia.     

16S rRNA sequences of uncultivated hot spring cyanobacterial mat inhabitants retrieved as randomly primed cDNA.

Cloning and analysis of cDNAs synthesized from rRNAs is one approach to assess the species composition of natural microbial communities. In some earlier attempts to synthesize cDNA from 16S rRNA (16S rcDNA) from the Octopus Spring cyanobacterial mat, a dominance of short 16S rcDNAs was observed, which appear to have originated only from certain organisms. Priming of cDNA synthesis from small ribosomal subunit RNA with random deoxyhexanucleotides can retrieve longer sequences, more suitable for phylogenetic analysis. Here we report the retrieval of 16S rRNA sequences from three formerly uncultured community members. One sequence type, which was retrieved three times from a total of five sequences analyzed, can be placed in the cyanobacterial phylum. A second sequence type is related to 16S rRNAs from green nonsulfur bacteria. The third sequence type may represent a novel phylogenetic type.     

16S rRNA sequences of uncultivated hot spring cyanobacterial mat inhabitants retrieved as randomly primed cDNA.

Cloning and analysis of cDNAs synthesized from rRNAs is one approach to assess the species composition of natural microbial communities. In some earlier attempts to synthesize cDNA from 16S rRNA (16S rcDNA) from the Octopus Spring cyanobacterial mat, a dominance of short 16S rcDNAs was observed, which appear to have originated only from certain organisms. Priming of cDNA synthesis from small ribosomal subunit RNA with random deoxyhexanucleotides can retrieve longer sequences, more suitable for phylogenetic analysis. Here we report the retrieval of 16S rRNA sequences from three formerly uncultured community members. One sequence type, which was retrieved three times from a total of five sequences analyzed, can be placed in the cyanobacterial phylum. A second sequence type is related to 16S rRNAs from green nonsulfur bacteria. The third sequence type may represent a novel phylogenetic type.