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Denaturing gradient gel electrophoresis profiles of 16S rRNA-defined populations inhabiting a hot spring microbial mat community.

ABSTRACT: Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene segments was used to profile microbial populations inhabiting different temperature regions in the microbial mat community of Octopus Spring, Yellowstone National Park. DGGE allowed a rapid evaluation of the distributions of amplifiable sequence types. Profiles were essentially identical within regions of the mat defined by one temperature range but varied between sites with different temperature ranges. Individual DGGE bands were sequenced, and the sequences were compared with those previously obtained from the mat by cloning and from cultivated Octopus Spring isolates. Two known cyanobacterial populations and one known green nonsulfur bacterium-like population were detected by DGGE, as were many new cyanobacterial and green nonsulfur and green sulfur bacterium-like populations and a novel bacterial population of uncertain phylogenetic affiliation. The distributions of several cyanobacterial populations compared favorably with results obtained previously by oligonucleotide probe analyses and suggest that adaptation to temperature has occurred among cyanobacteria which are phylogenetically very similar.

SUBMITTER: Ferris MJ

PROVIDER: S-EPMC167804 | biostudies-other | 1996 Feb

REPOSITORIES: biostudies-other

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Denaturing gradient gel electrophoresis profiles of 16S rRNA-defined populations inhabiting a hot spring microbial mat community.

Ferris M J MJ Muyzer G G Ward D M DM

Applied and environmental microbiology 19960201 2

Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene segments was used to profile microbial populations inhabiting different temperature regions in the microbial mat community of Octopus Spring, Yellowstone National Park. DGGE allowed a rapid evaluation of the distributions of amplifiable sequence types. Profiles were essentially identical within regions of the mat defined by one temperature range but varied between sites with different temperature ranges. Individual DGG ...[more]

PMID: 8593039

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Project description:The compositions of Octopus Spring and Mushroom Spring (Yellowstone National Park, Wyoming, USA) microbial mats have been thoroughly studied, but the compositions of the effluent waters that flow above the mats have not. In this study, cells in the mats and overflowing waters of both springs were investigated at multiple sites where Synechococcus spp. are the dominant cyanobacteria (ca. 72°C to ca. 50°C), and on several dates. In addition to microscopic analyses of stained and autofluorescent cells, 16S rRNA gene sequencing was used to characterize the major taxa present and a protein-encoding gene (psaA) was sequenced and analyzed by ecotype simulation to predict species of Synechococcus The mats of both springs were similar in terms of the downstream distribution of predominant taxa detected previously. However, waters above these mats were predominated by taxa that reside in upstream mats or communities above the upper-temperature limit of the mat. A disturbance/recolonization study was performed at a site normally predominated by Synechococcus species adapted to low temperatures. After removing indigenous Synechococcus cells, Synechococcus species adapted to higher temperatures, which were predominant in the water overflowing this site, colonized the newly forming mat. Differences in recolonization under reduced and UV-screened irradiance suggested that, in addition to physical transport, environmental conditions likely select for species that are better adapted to these different conditions and can influence mat recovery. A transport model was developed and used to predict that, in Mushroom Spring, erosion predominates in the narrower and deeper upstream effluents and deposition predominates over erosion in wider and shallower downstream effluents.IMPORTANCE In flowing aquatic systems, cell erosion and deposition are important to the dispersal of cells from one location to another. Very little is known about microbial dispersal and the physical processes that underlie it. This study demonstrates its importance to colonization of downstream surfaces and especially to the recolonization and functioning of disturbed sites. Ecological systems in flowing environments are often, roughly speaking, pseudosteady, in that nutrients enter the system and by-products leave at relatively steady rates. Over time, material inputs and outputs must balance. Measurements of input fluxes (e.g., growth rates and proxies, such as photosynthesis rates) are frequent. However, erosion and deposition of cells are seldom measured and ecological significance is sometimes neglected. The importance of these parameters is immediately evident in any attempt to construct a model of long-time community behavior, as spatial ecological structure is significantly impacted and can be dominated by migration of organisms, even in small numbers.

Project description:The effect of developing chrysanthemum roots on the presence and activity of bacterial populations in the rhizosphere was examined by using culture-independent methods. Nucleic acids were extracted from rhizosphere soil samples associated with the bases of roots or root tips of plants harvested at different stages of development. PCR and reverse transcriptase (RT) PCR were used to amplify 16S ribosomal DNA (rDNA) and 16S rRNA, respectively, and the products were subjected to denaturing gradient gel electrophoresis (DGGE). Prominent DGGE bands were excised and sequenced to gain insight into the identities of predominantly present (PCR) and predominantly active (RT-PCR) bacterial populations. The majority of DGGE band sequences were related to bacterial genera previously associated with the rhizosphere, such as Pseudomonas, Comamonas, Variovorax, and Acetobacter, or typical of root-free soil environments, such as Bacillus and Arthrobacter. The PCR-DGGE patterns observed for bulk soil were somewhat more complex than those obtained from rhizosphere samples, and the latter contained a subset of the bands present in bulk soil. DGGE analysis of RT-PCR products detected a subset of bands visible in the rDNA-based analysis, indicating that some dominantly detected bacterial populations did not have high levels of metabolic activity. The sequences detected by the RT-PCR approach were, however, derived from a wide taxonomic range, suggesting that activity in the rhizosphere was not determined at broad taxonomic levels but rather was a strain- or species-specific phenomenon. Comparative analysis of DGGE profiles grouped all DNA-derived root tip samples together in a cluster, and within this cluster the root tip samples from young plants formed a separate subcluster. Comparison of rRNA-derived bacterial profiles showed no grouping of root tip samples versus root base samples. Rather, all profiles derived from 2-week-old plant rhizosphere soils grouped together regardless of location along the root.

Project description:Recent studies of bacterial speciation have claimed to support the biological species concept-that reduced recombination is required for bacterial populations to diverge into species. This conclusion has been reached from the discovery that ecologically distinct clades show lower rates of recombination than that which occurs among closest relatives. However, these previous studies did not attempt to determine whether the more-rapidly recombining close relatives within the clades studied may also have diversified ecologically, without benefit of sexual isolation. Here we have measured the impact of recombination on ecological diversification within and between two ecologically distinct clades (A and B') of Synechococcus in a hot spring microbial mat in Yellowstone National Park, using a cultivation-free, multi-locus approach. Bacterial artificial chromosome (BAC) libraries were constructed from mat samples collected at 60°C and 65°C. Analysis of multiple linked loci near Synechococcus 16S rRNA genes showed little evidence of recombination between the A and B' lineages, but a record of recombination was apparent within each lineage. Recombination and mutation rates within each lineage were of similar magnitude, but recombination had a somewhat greater impact on sequence diversity than mutation, as also seen in many other bacteria and archaea. Despite recombination within the A and B' lineages, there was evidence of ecological diversification within each lineage. The algorithm Ecotype Simulation identified sequence clusters consistent with ecologically distinct populations (ecotypes), and several hypothesized ecotypes were distinct in their habitat associations and in their adaptations to different microenvironments. We conclude that sexual isolation is more likely to follow ecological divergence than to precede it. Thus, an ecology-based model of speciation appears more appropriate than the biological species concept for bacterial and archaeal diversification.

Dataset Information

Denaturing gradient gel electrophoresis profiles of 16S rRNA-defined populations inhabiting a hot spring microbial mat community.

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Denaturing gradient gel electrophoresis profiles of 16S rRNA-defined populations inhabiting a hot spring microbial mat community.

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