We describe a new molecular approach to analyzing the genetic diversity of complex microbial populations. This technique is based on the separation of polymerase chain reaction-amplified fragments of genes coding for 16S rRNA, all the same length, by denaturing gradient gel electrophoresis (DGGE). DGGE analysis of different microbial communities demonstrated the presence of up to 10 distinguishable bands in the separation pattern, which were most likely derived from as many different species constituting these populations, and thereby generated a DGGE profile of the populations. We showed that it is possible to identify constituents which represent only 1% of the total population. With an oligonucleotide probe specific for the V3 region of 16S rRNA of sulfate-reducing bacteria, particular DNA fragments from some of the microbial populations could be identified by hybridization analysis. Analysis of the genomic DNA from a bacterial biofilm grown under aerobic conditions suggests that sulfate-reducing bacteria, despite their anaerobicity, were present in this environment. The results we obtained demonstrate that this technique will contribute to our understanding of the genetic diversity of uncharacterized microbial populations.

Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA

16S ribosomal RNA gene (rDNA) amplicon analysis remains the standard approach for the cultivation-independent investigation of microbial diversity. The accuracy of these analyses depends strongly on the choice of primers. The overall coverage and phylum spectrum of 175 primers and 512 primer pairs were evaluated in silico with respect to the SILVA 16S/18S rDNA non-redundant reference dataset (SSURef 108 NR). Based on this evaluation a selection of 'best available' primer pairs for Bacteria and Archaea for three amplicon size classes (100-400, 400-1000, ≥ 1000 bp) is provided. The most promising bacterial primer pair (S-D-Bact-0341-b-S-17/S-D-Bact-0785-a-A-21), with an amplicon size of 464 bp, was experimentally evaluated by comparing the taxonomic distribution of the 16S rDNA amplicons with 16S rDNA fragments from directly sequenced metagenomes. The results of this study may be used as a guideline for selecting primer pairs with the best overall coverage and phylum spectrum for specific applications, therefore reducing the bias in PCR-based microbial diversity studies.

Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies

The Ribosomal Database Project (RDP) provides researchers with quality-controlled bacterial and archaeal small subunit rRNA alignments and analysis tools. An improved alignment strategy uses the Infernal secondary structure aware aligner to provide a more consistent higher quality alignment and faster processing of user sequences. Substantial new analysis features include a new Pyrosequencing Pipeline that provides tools to support analysis of ultra high-throughput rRNA sequencing data. This pipeline offers a collection of tools that automate the data processing and simplify the computationally intensive analysis of large sequencing libraries. In addition, a new Taxomatic visualization tool allows rapid visualization of taxonomic inconsistencies and suggests corrections, and a new class Assignment Generator provides instructors with a lesson plan and individualized teaching materials. Details about RDP data and analytical functions can be found at http://rdp.cme.msu.edu/.

The Ribosomal Database Project: improved alignments and new tools for rRNA analysis

The distal human intestine represents an anaerobic bioreactor programmed with an enormous population of bacteria, dominated by relatively few divisions that are highly diverse at the strain/subspecies level. This microbiota and its collective genomes (microbiome) provide us with genetic and metabolic attributes we have not been required to evolve on our own, including the ability to harvest otherwise inaccessible nutrients. New studies are revealing how the gut microbiota has coevolved with us and how it manipulates and complements our biology in ways that are mutually beneficial. We are also starting to understand how certain keystone members of the microbiota operate to maintain the stability and functional adaptability of this microbial organ.

https://science.sciencemag.org/content/sci/307/5717/1915.full.pdf

Host-Bacterial Mutualism in the Human Intestine

For centuries, biologists have studied patterns of plant and animal diversity at continental scales. Until recently, similar studies were impossible for microorganisms, arguably the most diverse and abundant group of organisms on Earth. Here, we present a continental-scale description of soil bacterial communities and the environmental factors influencing their biodiversity. We collected 98 soil samples from across North and South America and used a ribosomal DNA-fingerprinting method to compare bacterial community composition and diversity quantitatively across sites. Bacterial diversity was unrelated to site temperature, latitude, and other variables that typically predict plant and animal diversity, and community composition was largely independent of geographic distance. The diversity and richness of soil bacterial communities differed by ecosystem type, and these differences could largely be explained by soil pH (r(2) = 0.70 and r(2) = 0.58, respectively; P < 0.0001 in both cases). Bacterial diversity was highest in neutral soils and lower in acidic soils, with soils from the Peruvian Amazon the most acidic and least diverse in our study. Our results suggest that microbial biogeography is controlled primarily by edaphic variables and differs fundamentally from the biogeography of "macro" organisms.

/pdf/the-diversity-and-biogeography-of-soil-bacterial-communities-1lkjoz8774.pdf

The diversity and biogeography of soil bacterial communities

A set of oligonucleotide primers capable of initiating enzymatic amplification (polymerase chain reaction) on a phylogenetically and taxonomically wide range of bacteria is described along with methods for their use and examples. One pair of primers is capable of amplifying nearly full-length 16S ribosomal DNA (rDNA) from many bacterial genera; the additional primers are useful for various exceptional sequences. Methods for purification of amplified material, direct sequencing, cloning, sequencing, and transcription are outlined. An obligate intracellular parasite of bovine erythrocytes, Anaplasma marginale, is used as an example; its 16S rDNA was amplified, cloned, sequenced, and phylogenetically placed. Anaplasmas are related to the genera Rickettsia and Ehrlichia. In addition, 16S rDNAs from several species were readily amplified from material found in lyophilized ampoules from the American Type Culture Collection. By use of this method, the phylogenetic study of extremely fastidious or highly pathogenic bacterial species can be carried out without the need to culture them. In theory, any gene segment for which polymerase chain reaction primer design is possible can be derived from a readily obtainable lyophilized bacterial culture.

16S ribosomal DNA amplification for phylogenetic study.

Of the three primary phylogenetic domains--Archaea (archaebacteria), Bacteria (eubacteria), and Eucarya (eukaryotes)--Archaea is the least understood in terms of its diversity, physiologies, and ecological panorama. Although many species of Crenarchaeota (one of the two recognized archaeal kingdoms sensu Woese [Woese, C. R., Kandler, O. & Wheelis, M. L. (1990) Proc. Natl. Acad. Sci. USA 87, 4576-4579]) have been isolated, they constitute a relatively tight-knit cluster of lineages in phylogenetic analyses of rRNA sequences. It seemed possible that this limited diversity is merely apparent and reflects only a failure to culture organisms, not their absence. We report here phylogenetic characterization of many archaeal small subunit rRNA gene sequences obtained by polymerase chain reaction amplification of mixed population DNA extracted directly from sediment of a hot spring in Yellowstone National Park. This approach obviates the need for cultivation to identify organisms. The analyses document the existence not only of species belonging to well-characterized crenarchaeal genera or families but also of crenarchaeal species for which no close relatives have so far been found. The large number of distinct archaeal sequence types retrieved from this single hot spring was unexpected and demonstrates that Crenarchaeota is a much more diverse group than was previously suspected. The results have impact on our concepts of the phylogenetic organization of Archaea.

https://www.pnas.org/content/pnas/91/5/1609.full.pdf

Remarkable archaeal diversity detected in a Yellowstone National Park hot spring environment

Phylogenetic analysis of ribosomal RNA sequences obtained from uncultivated organisms of a hot spring in Yellowstone National Park reveals several novel groups of Archaea, many of which diverged from the crenarchaeal line of descent prior to previously characterized members of that kingdom. Universal phylogenetic trees constructed with the addition of these sequences indicate monophyly of Archaea, with modest bootstrap support. The data also show a specific relationship between low-temperature marine Archaea and some hot spring Archaea. Two of the environmental sequences are enigmatic: depending upon the data set and analytical method used, these sequences branch deeply within the Crenarchaeota, below the bifurcation between Crenarchaeota and Euryarchaeota, or even as the sister group to Eukaryotes. If additional data confirm either of the latter two placements, then the organisms represented by these ribosomal RNA sequences would merit recognition as a new kingdom, provisionally named "Korarchaeota."

https://www.pnas.org/content/pnas/93/17/9188.full.pdf

Perspectives on archaeal diversity, thermophily and monophyly from environmental rRNA sequences.

The 16S rRNAs from 29 cyanobacteria and the cyanelle of the phytoflagellate Cyanophora paradoxa were partially sequenced by a dideoxynucleotide-terminated, primer extension method A least-squares distance matrix analysis was used to infer phylogenetic trees that include green chloroplasts (those of euglenoids, green algae, and higher plants) The results indicate that many diverse forms of cyanobacteria diverged within a short span of evolutionary distance Evolutionary depth within the surveyed cyanobacteria is substantially less than that separating the major eubacterial taxa, as though cyanobacterial diversification occurred significantly after the appearance of the major eubacterial groups Three of the five taxonomic sections defined by Rippka et al (R Rippka, J Deruelles, J B Waterbury, M Herdman, and R Y Stanier, J Gen Microbiol 111:1-61, 1979) (sections II [pleurocapsalean], IV [heterocystous, filamentous, nonbranching], and V [heterocystous, filamentous, branching]) are phylogenetically coherent However, the other two sections (I [unicellular] and III [nonheterocystous, filamentous]) are intermixed and hence are not natural groupings Our results not only support the conclusion of previous workers that the cyanobacteria and green chloroplasts form a coherent phylogenetic group but also suggest that the chloroplast lineage, which includes the cyanelle of C paradoxa, is not just a sister group to the free-living forms but rather is contained within the cyanobacterial radiation

Evolutionary relationships among cyanobacteria and green chloroplasts.

Techniques based on amplification of 16S rRNA genes for comparing bacterial communities are now widely used in microbial ecology, but calibration of these techniques with traditional tools, such as cultivation, has been conspicuously absent. In this study, we compared levels of bacterial community diversity in two pinyon rhizosphere soil samples and two between-tree (interspace) soil samples by analyzing 179 cultivated bacterial isolates and 801 16S rRNA genes amplified from extracted soil DNA. Phylotypes were defined by performing a restriction fragment length polymorphism analysis of 16S rRNA gene sequences with the enzymes RsaI and BstUI. The average level of 16S rRNA gene sequence similarity of members of a phylotype was 86.6% based on an analysis of partial sequences. A total of 498 phylotypes were identified among the 16S ribosomal DNA (rDNA) clones, while 34 phylotypes occurred among the cultivated isolates. Analysis of sequences from a subset of the phylotypes showed that at least seven bacterial divisions were represented in the clone libraries, whereas the isolates represented only three. The phylotype richness, frequency distribution (evenness), and composition of the four culture collections and the four clone libraries were investigated by using a variety of diversity indices. Although cultivation and 16S rRNA cloning analyses gave contradictory descriptions of the relative phylotype richness for one of the four environments, the two methods identified qualitatively consistent relationships when levels of evenness were compared. The levels of phylotype similarity between communities were uniformly low (15 to 31%). Both methods consistently indicated that one environment was distinct from the other three. Our data illustrate that while 16S rDNA cloning and cultivation generally describe similar relationships between soil microbial communities, significant discrepancies can occur.

https://aem.asm.org/content/aem/65/4/1662.full.pdf

Susan M. Barns

Papers

16S ribosomal DNA amplification for phylogenetic study.

Remarkable archaeal diversity detected in a Yellowstone National Park hot spring environment

Perspectives on archaeal diversity, thermophily and monophyly from environmental rRNA sequences.

Evolutionary relationships among cyanobacteria and green chloroplasts.

Levels of bacterial community diversity in four arid soils compared by cultivation and 16S rRNA gene cloning.