Quantifying Microbial Diversity: Morphotypes, 16S rRNA Genes, and Carotenoids of Oxygenic Phototrophs in Microbial Mats

We quantified the diversity of oxygenic phototrophic microorganisms present in eight hypersaline microbial mats on the basis of three cultivation-independent approaches. Morphological diversity was studied by microscopy. The diversity of carotenoids was examined by extraction from mat samples and high-pressure liquid chromatography analysis. The diversity of 16S rRNA genes from oxygenic phototrophic microorganisms was investigated by extraction of total DNA from mat samples, amplification of 16S rRNA gene segments from cyanobacteria and plastids of eukaryotic algae by phylum-specific PCR, and sequence-dependent separation of amplification products by denaturing-gradient gel electrophoresis. A numerical approach was introduced to correct for crowding the results of chromatographic and electrophoretic analyses. Diversity estimates typically varied up to twofold among mats. The congruence of richness estimates and Shannon-Weaver indices based on numbers and proportional abundances of unique morphotypes, 16S rRNA genes, and carotenoids unveiled the underlying diversity of oxygenic phototrophic microorganisms in the eight mat communities studied.     

Diversity of 23S rRNA Genes within Individual Prokaryotic Genomes

Background

The concept of ribosomal constraints on rRNA genes is deduced primarily based on the comparison of consensus rRNA sequences between closely related species, but recent advances in whole-genome sequencing allow evaluation of this concept within organisms with multiple rRNA operons.

Methodology/Principal Findings

Using the 23S rRNA gene as an example, we analyzed the diversity among individual rRNA genes within a genome. Of 184 prokaryotic species containing multiple 23S rRNA genes, diversity was observed in 113 (61.4%) genomes (mean 0.40%, range 0.01%–4.04%). Significant (1.17%–4.04%) intragenomic variation was found in 8 species. In 5 of the 8 species, the diversity in the primary structure had only minimal effect on the secondary structure (stem versus loop transition). In the remaining 3 species, the diversity significantly altered local secondary structure, but the alteration appears minimized through complex rearrangement. Intervening sequences (IVS), ranging between 9 and 1471 nt in size, were found in 7 species. IVS in Deinococcus radiodurans and Nostoc sp. encode transposases. T. tengcongensis was the only species in which intragenomic diversity >3% was observed among 4 paralogous 23S rRNA genes.

Conclusions/Significance

These findings indicate tight ribosomal constraints on individual 23S rRNA genes within a genome. Although classification using primary 23S rRNA sequences could be erroneous, significant diversity among paralogous 23S rRNA genes was observed only once in the 184 species analyzed, indicating little overall impact on the mainstream of 23S rRNA gene-based prokaryotic taxonomy.     

大黄鱼16SrRNA和18SrRNA基因的测定与序列分析

利用多对引物,扩增并测定出大黄鱼16SrRNA基因和18SrRNA基因的部分序列,其长度分别为1202bp和1275bp,16SrRNA基因序列的GC含量为46．12％,18SrRNA基因的Gc含量为53．oo％。将大黄鱼16SrRNA基因序列与GenBank中15种硬骨鱼类的同源序列结合,同时将其18SrRNA基因序列与GenBank中9种脊索动物的同源序列相结合,运用软件获得各自序列间差异百分比,转换和颠换数值等信息。基于这两种基因序列,利用NJ法和BI法,分别构建16种硬骨鱼类和10种脊索动物的分子系统树。18SrRNA构建的系统树包括三大支,一支为哺乳类、鸟类和爬行类共6个物种,一支为两栖类的1个物种,另一支为2种硬骨鱼类。16SrRNA构建的系统树显示大黄鱼所在的石首鱼科与鲈科和盖刺鱼科亲缘关系较近。此外还讨论了这两个基因的序列特征。     

Phylogeny of Molting Protostomes (Ecdysozoa) as Inferred from 18S and 28S rRNA Gene Sequences

Phylogenetic relationships within the group of molting protostomes were reconstructed by comparing the sets of 18S and 28S rRNA gene sequences considered either separately or in combination. The reliability of reconstructions was estimated from the bootstrap indices for major phylogenetic tree nodes and from the degree of congruence of phylogenetic trees obtained by different methods. By either criterion, the phylogenetic trees reconstructed on the basis of both 18 and 28S rRNA gene sequences were better than those based on the 18S or 28S sequences alone. The results of reconstruction are consistent with the phylogenetic hypothesis classifying protostomes into two major clades: molting Ecdysozoa (Priapulida + Kinorhyncha, Nematoda + Nematomorpha, Onychophora + Tardigrada, Myriapoda + Chelicerata, and Crustacea + Hexapoda) and nonmolting Lophotrochozoa (Plathelminthes, Nemertini, Annelida, Mollusca, Echiura, and Sipuncula). Nematomorphs (Nematomorpha) do not belong to the clade Cephalorhyncha (Priapulida + Kinorhyncha). It is concluded that combined data on the 18S and 28S rRNA gene sequences provide a more reliable basis for phylogenetic inferences.__________Translated from Molekulyarnaya Biologiya, Vol. 39, No. 4, 2005, pp. 590–601.Original Russian Text Copyright © 2005 by Petrov, Vladychenskaya.     

A Comprehensive Census of Microbial Diversity in Hot Springs of Tengchong,Yunnan Province China Using 16S rRNA Gene Pyrosequencing

The Rehai and Ruidian geothermal fields, located in Tengchong County, Yunnan Province, China, host a variety of geochemically distinct hot springs. In this study, we report a comprehensive, cultivation-independent census of microbial communities in 37 samples collected from these geothermal fields, encompassing sites ranging in temperature from 55.1 to 93.6°C, in pH from 2.5 to 9.4, and in mineralogy from silicates in Rehai to carbonates in Ruidian. Richness was low in all samples, with 21–123 species-level OTUs detected. The bacterial phylum Aquificae or archaeal phylum Crenarchaeota were dominant in Rehai samples, yet the dominant taxa within those phyla depended on temperature, pH, and geochemistry. Rehai springs with low pH (2.5–2.6), high temperature (85.1–89.1°C), and high sulfur contents favored the crenarchaeal order Sulfolobales, whereas those with low pH (2.6–4.8) and cooler temperature (55.1–64.5°C) favored the Aquificae genus Hydrogenobaculum. Rehai springs with neutral-alkaline pH (7.2–9.4) and high temperature (>80°C) with high concentrations of silica and salt ions (Na, K, and Cl) favored the Aquificae genus Hydrogenobacter and crenarchaeal orders Desulfurococcales and Thermoproteales. Desulfurococcales and Thermoproteales became predominant in springs with pH much higher than the optimum and even the maximum pH known for these orders. Ruidian water samples harbored a single Aquificae genus Hydrogenobacter, whereas microbial communities in Ruidian sediment samples were more diverse at the phylum level and distinctly different from those in Rehai and Ruidian water samples, with a higher abundance of uncultivated lineages, close relatives of the ammonia-oxidizing archaeon “Candidatus Nitrosocaldus yellowstonii”, and candidate division O1aA90 and OP1. These differences between Ruidian sediments and Rehai samples were likely caused by temperature, pH, and sediment mineralogy. The results of this study significantly expand the current understanding of the microbiology in Tengchong hot springs and provide a basis for comparison with other geothermal systems around the world.     

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.     

Design and Experimental Application of a Novel Non-Degenerate Universal Primer Set that Amplifies Prokaryotic 16S rRNA Genes with a Low Possibility to Amplify Eukaryotic rRNA Genes

The deep sequencing of 16S rRNA genes amplified by universal primers has revolutionized our understanding of microbial communities by allowing the characterization of the diversity of the uncultured majority. However, some universal primers also amplify eukaryotic rRNA genes, leading to a decrease in the efficiency of sequencing of prokaryotic 16S rRNA genes with possible mischaracterization of the diversity in the microbial community. In this study, we compared 16S rRNA gene sequences from genome-sequenced strains and identified candidates for non-degenerate universal primers that could be used for the amplification of prokaryotic 16S rRNA genes. The 50 identified candidates were investigated to calculate their coverage for prokaryotic and eukaryotic rRNA genes, including those from uncultured taxa and eukaryotic organelles, and a novel universal primer set, 342F-806R, covering many prokaryotic, but not eukaryotic, rRNA genes was identified. This primer set was validated by the amplification of 16S rRNA genes from a soil metagenomic sample and subsequent pyrosequencing using the Roche 454 platform. The same sample was also used for pyrosequencing of the amplicons by employing a commonly used primer set, 338F-533R, and for shotgun metagenomic sequencing using the Illumina platform. Our comparison of the taxonomic compositions inferred by the three sequencing experiments indicated that the non-degenerate 342F-806R primer set can characterize the taxonomic composition of the microbial community without substantial bias, and is highly expected to be applicable to the analysis of a wide variety of microbial communities.     

MiCA: A Web-Based Tool for the Analysis of Microbial Communities Based on Terminal-Restriction Fragment Length Polymorphisms of 16S and 18S rRNA Genes

A web-based resource, Microbial Community Analysis (MiCA), has been developed to facilitate studies on microbial community ecology that use analyses of terminal-restriction fragment length polymorphisms (T-RFLP) of 16S and 18S rRNA genes. MiCA provides an intuitive web interface to access two specialized programs and a specially formatted database of 16S ribosomal RNA sequences. The first program performs virtual polymerase chain reaction (PCR) amplification of rRNA genes and restriction of the amplicons using primer sequences and restriction enzymes chosen by the user. This program, in silico PCR and Restriction (ISPaR), uses a binary encoding of DNA sequences to rapidly scan large numbers of sequences in databases searching for primer annealing and restriction sites while permitting the user to specify the number of mismatches in primer sequences. ISPaR supports multiple digests with up to three enzymes. The number of base pairs between the 5′ and 3′ primers and the proximal restriction sites can be reported, printed, or exported in various formats. The second program, APLAUS, infers a plausible community structure(s) based on T-RFLP data supplied by a user. APLAUS estimates the relative abundances of populations and reports a listing of phylotypes that are consistent with the empirical data. MiCA is accessible at .     

Molecular Characterization of the Diversity and Distribution of a Thermal Spring Microbial Community by Using rRNA and Metabolic Genes

The diversity and distribution of a bacterial community from Coffee Pots Hot Spring, a thermal spring in Yellowstone National Park with a temperature range of 39.3 to 74.1°C and pH range of 5.75 to 6.91, were investigated by sequencing cloned PCR products and quantitative PCR (qPCR) of 16S rRNA and metabolic genes. The spring was inhabited by three Aquificae genera—Thermocrinis, Hydrogenobaculum, and Sulfurihydrogenibium—and members of the Alpha-, Beta-, and Gammaproteobacteria, Firmicutes, Acidobacteria, Deinococcus-Thermus, and candidate division OP5. The in situ chemical affinities were calculated for 41 potential metabolic reactions using measured environmental parameters and a range of hydrogen and oxygen concentrations. Reactions that use oxygen, ferric iron, sulfur, and nitrate as electron acceptors were predicted to be the most energetically favorable, while reactions using sulfate were expected to be less favorable. Samples were screened for genes used in ammonia oxidation (amoA, bacterial gene only), the reductive tricarboxylic acid (rTCA) cycle (aclB), the Calvin cycle (cbbM), sulfate reduction (dsrAB), nitrogen fixation (nifH), nitrite reduction (nirK), and sulfide oxidation (soxEF1) by PCR. Genes for carbon fixation by the rTCA cycle and nitrogen fixation were detected. All aclB sequences were phylogenetically related and spatially correlated to Sulfurihydrogenibium 16S rRNA gene sequences using qPCR (R² = 0.99). This result supports the recent finding of citrate cleavage by enzymes other than ATP citrate lyase in the rTCA cycle of the Aquificaceae family. We briefly consider potential biochemical mechanisms that may allow Sulfurihydrogenibium and Thermocrinis to codominate some hydrothermal environments.     

A Comparison of Methods for Clustering 16S rRNA Sequences into OTUs

Recent studies of 16S rRNA sequences through next-generation sequencing have revolutionized our understanding of the microbial community composition and structure. One common approach in using these data to explore the genetic diversity in a microbial community is to cluster the 16S rRNA sequences into Operational Taxonomic Units (OTUs) based on sequence similarities. The inferred OTUs can then be used to estimate species, diversity, composition, and richness. Although a number of methods have been developed and commonly used to cluster the sequences into OTUs, relatively little guidance is available on their relative performance and the choice of key parameters for each method. In this study, we conducted a comprehensive evaluation of ten existing OTU inference methods. We found that the appropriate dissimilarity value for defining distinct OTUs is not only related with a specific method but also related with the sample complexity. For data sets with low complexity, all the algorithms need a higher dissimilarity threshold to define OTUs. Some methods, such as, CROP and SLP, are more robust to the specific choice of the threshold than other methods, especially for shorter reads. For high-complexity data sets, hierarchical cluster methods need a more strict dissimilarity threshold to define OTUs because the commonly used dissimilarity threshold of 3% often leads to an under-estimation of the number of OTUs. In general, hierarchical clustering methods perform better at lower dissimilarity thresholds. Our results show that sequence abundance plays an important role in OTU inference. We conclude that care is needed to choose both a threshold for dissimilarity and abundance for OTU inference.     

Phylogenetic Analysis of Sequences of the 12S and 16S rRNA Mitochondrial Genes in the Family Bovidae: New Evidence

The phylogeny of the family Bovidae has been inferred from our data on the 12S and 16S rRNA mtDNA gene sequences and from the results of other authors. A considerable (2460 bp) length of the analyzed fragments of these conserved genes and the use of different methods of cladogram construction allowed us to verify the systematic position of the genera Saiga, Pantholops, Procapra, and Oreamnos. Saigas were shown to be phylogenetically far closer to gazelles than black-tailed gazelles and pygmy antelopes. In general, the genetic analysis data are in agreement with the results of morphological studies.     

Metazoan Relationships on the Basis of 18S rRNA Sequences: A Few Years Later...

SYNOPSIS. The 18S rRNA database is continuously growing andnew tree construction methods are being developed. The presentstudy aims at assessing what effect the addition of recentlydetermined animal 18S rRNA sequences and the use of a recentlydeveloped distance matrix calculation method have on the resultsof some previously published case studies on metazoan phytogeny.When re-assessing three case studies, part of their conclusionswas confirmed on the basis of the present 18S rRNA data set:1) the monophyly of Arthropoda; 2) the monophyly of the Vestimentifera-Pogonophoraand their protostome character; 3) the doubt about the monophyleticorigin of Echiura-Sipuncula and 4) the coelomate characterofNemertea. Yet, it is also demonstrated that some of the previousresults are no longer warranted when updating the analyses:1) the monophyly of both the Annelida and the Eutrochozoa; 2)the sister-group relationship of Echiura to Vestimentifera-Pogonophoraand 3) the polyphyly of the Mesozoa and their close relationshipto Myxozoa and Nematodes. In addition, some new very preliminaryevidence is provided for: 1) a common ancestry of Platyhelminthesand Mesozoa and the monophyly of the latter group and 2) themonophyly of Clitellata, Hirudinida and Oligochaeta. Finally,doubt is casted on the monophyly of the Polychaeta and the polychaeteorders Spionida, Phyllodocida, and Sabellidae. Of course, thesehypotheses also need further testing.     

Use of 16S rRNA and rpoB Genes as Molecular Markers for Microbial Ecology Studies

Several characteristics of the 16S rRNA gene, such as its essential function, ubiquity, and evolutionary properties, have allowed it to become the most commonly used molecular marker in microbial ecology. However, one fact that has been overlooked is that multiple copies of this gene are often present in a given bacterium. These intragenomic copies can differ in sequence, leading to identification of multiple ribotypes for a single organism. To evaluate the impact of such intragenomic heterogeneity on the performance of the 16S rRNA gene as a molecular marker, we compared its phylogenetic and evolutionary characteristics to those of the single-copy gene rpoB. Full-length gene sequences and gene fragments commonly used for denaturing gradient gel electrophoresis were compared at various taxonomic levels. Heterogeneity found between intragenomic 16S rRNA gene copies was concentrated in specific regions of rRNA secondary structure. Such “heterogeneity hot spots” occurred within all gene fragments commonly used in molecular microbial ecology. This intragenomic heterogeneity influenced 16S rRNA gene tree topology, phylogenetic resolution, and operational taxonomic unit estimates at the species level or below. rpoB provided comparable phylogenetic resolution to that of the 16S rRNA gene at all taxonomic levels, except between closely related organisms (species and subspecies levels), for which it provided better resolution. This is particularly relevant in the context of a growing number of studies focusing on subspecies diversity, in which single-copy protein-encoding genes such as rpoB could complement the information provided by the 16S rRNA gene.