Comparison of rRNA and Polar-Lipid-Derived Fatty Acid Biomarkers for Assessment of 13C-Substrate Incorporation by Microorganisms in Marine Sediments

We determined whether a recently developed method to isolate specific small-subunit (SSU) rRNAs can be used in ¹³C-labeling studies to directly link community structure and function in natural ecosystems. Replicate North Sea sediment cores were incubated at the in situ temperature following addition of ¹³C-labeled acetate, propionate, amino acids, or glucose. Eukaryotic and bacterial SSU rRNAs were separated from total RNA by means of biotin-labeled oligonucleotide probes and streptavidin-coated paramagnetic beads, and the ¹³C content of the isolated rRNA was determined by elemental analysis-isotope ratio mass spectrometry. The SSU rRNA yield with the bead-capture protocol was improved by using helper probes. Incorporation of label into bacterial SSU rRNA was detectable after 2 h of incubation. The labeling was always much greater in bacterial SSU rRNA than in eukaryotic SSU rRNA, suggesting that bacteria were the main consumers of the ¹³C-labeled compounds. Similar results were obtained with the ¹³C-labeled polar-lipid-derived fatty acid (PLFA) approach, except that more label was detected in bacterial PLFA than in bacterial SSU rRNA. This may be attributable to the generally slow growth of sediment microbial populations, which results in low ribosome synthesis rates and relatively few ribosomes per cell. We discuss possible ways to improve the probe-capture protocol and the sensitivity of the ¹³C analysis of the captured SSU rRNA.     

Seasonal and Spatial Variability in Lake Michigan Sediment Small-Subunit rRNA Concentrations

We have used molecular biological methods to study the distribution of microbial small-subunit rRNAs (SSU rRNAs), in relation to chemical profiles, in offshore Lake Michigan sediments. The sampling site is at a depth of 100 m, with temperatures of 2 to 4°C year-round. RNA extracted from sediment was probed with radiolabeled oligonucleotides targeting bacterial, archaeal, and eukaryotic SSU rRNAs, as well as with a universal probe. The coverage of these probes in relation to the present sequence database is discussed. Because ribosome production is growth rate regulated, rRNA concentrations are an indicator of the microbial populations active in situ. Over a 1-year period, changes in sedimentary SSU rRNA concentrations followed seasonal changes in surface water temperature and SSU rRNA concentration. Sedimentary depth profiles of oxygen, reduced manganese and iron, and sulfate changed relatively little from season to season, but the nitrate concentration was approximately fivefold higher in April and June 1997 than at the other times sampling was done. We propose that sediment microbial SSU rRNA concentrations at our sampling site are influenced by seasonal inputs from the water column, particularly the settling of the spring diatom bloom, and that the timing of this input may be modulated by grazers, such that ammonia becomes available to sediment microbes sooner than fresh organic carbon. Nitrate production from ammonia by autotrophic nitrifying bacteria, combined with low activity of heterotrophic denitrifying bacteria in the absence of readily degradable organic carbon, could account for the cooccurrence of high nitrate and low SSU rRNA concentrations.     

Seasonal and spatial variability in Lake Michigan sediment small-subunit rRNA concentrations

We have used molecular biological methods to study the distribution of microbial small-subunit rRNAs (SSU rRNAs), in relation to chemical profiles, in offshore Lake Michigan sediments. The sampling site is at a depth of 100 m, with temperatures of 2 to 4 degrees C year-round. RNA extracted from sediment was probed with radiolabeled oligonucleotides targeting bacterial, archaeal, and eukaryotic SSU rRNAs, as well as with a universal probe. The coverage of these probes in relation to the present sequence database is discussed. Because ribosome production is growth rate regulated, rRNA concentrations are an indicator of the microbial populations active in situ. Over a 1-year period, changes in sedimentary SSU rRNA concentrations followed seasonal changes in surface water temperature and SSU rRNA concentration. Sedimentary depth profiles of oxygen, reduced manganese and iron, and sulfate changed relatively little from season to season, but the nitrate concentration was approximately fivefold higher in April and June 1997 than at the other times sampling was done. We propose that sediment microbial SSU rRNA concentrations at our sampling site are influenced by seasonal inputs from the water column, particularly the settling of the spring diatom bloom, and that the timing of this input may be modulated by grazers, such that ammonia becomes available to sediment microbes sooner than fresh organic carbon. Nitrate production from ammonia by autotrophic nitrifying bacteria, combined with low activity of heterotrophic denitrifying bacteria in the absence of readily degradable organic carbon, could account for the cooccurrence of high nitrate and low SSU rRNA concentrations.     

Dominant microbial composition and its vertical distribution in saline meromictic Lake Kaiike (Japan) as revealed by quantitative oligonucleotide probe membrane hybridization

Vertical distributions of dominant bacterial populations in saline meromictic Lake Kaiike were investigated throughout the water column and sediment by quantitative oligonucleotide probe membrane hybridization. Three oligonucleotide probes specific for the small-subunit (SSU) rRNA of three groups of Chlorobiaceae were newly designed. In addition, three general domain (Bacteria, Archaea, and Eukarya)-specific probes, two delta-Proteobacteria-specific probes, a Chlorobiaceae-specific probe, and a Chloroflexi-specific probe were used after optimization of their washing conditions. The abundance of the sum of SSU rRNAs hybridizing with probes specific for three groups of Chlorobiaceae relative to total SSU rRNA peaked in the chemocline, accounting for up to 68%. The abundance of the delta-proteobacterial SSU rRNA relative to total SSU rRNA rapidly increased just below the chemocline up to 29% in anoxic water and peaked at the 2- to 3-cm sediment depth at ca. 34%. The abundance of SSU rRNAs hybridizing with the probe specific for the phylum Chloroflexi relative to total SSU rRNA was highest (31 to 54%) in the top of the sediment but then steeply declined with depth and became stable at 11 to 19%, indicating the robust coexistence of sulfate-reducing bacteria and Chloroflexi in the top of the sediment. Any SSU rRNA of Chloroflexi in the water column was under the detection limit. The summation of the signals of group-specific probes used in this study accounted for up to 89% of total SSU rRNA, suggesting that the DGGE-oligonucleotide probe hybridization approach, in contrast to conventional culture-dependent approaches, was very effective in covering dominant populations.     

Dominant Microbial Composition and Its Vertical Distribution in Saline Meromictic Lake Kaiike (Japan) as Revealed by Quantitative Oligonucleotide Probe Membrane Hybridization

Vertical distributions of dominant bacterial populations in saline meromictic Lake Kaiike were investigated throughout the water column and sediment by quantitative oligonucleotide probe membrane hybridization. Three oligonucleotide probes specific for the small-subunit (SSU) rRNA of three groups of Chlorobiaceae were newly designed. In addition, three general domain (Bacteria, Archaea, and Eukarya)-specific probes, two δ-Proteobacteria-specific probes, a Chlorobiaceae-specific probe, and a Chloroflexi-specific probe were used after optimization of their washing conditions. The abundance of the sum of SSU rRNAs hybridizing with probes specific for three groups of Chlorobiaceae relative to total SSU rRNA peaked in the chemocline, accounting for up to 68%. The abundance of the δ-proteobacterial SSU rRNA relative to total SSU rRNA rapidly increased just below the chemocline up to 29% in anoxic water and peaked at the 2- to 3-cm sediment depth at ca. 34%. The abundance of SSU rRNAs hybridizing with the probe specific for the phylum Chloroflexi relative to total SSU rRNA was highest (31 to 54%) in the top of the sediment but then steeply declined with depth and became stable at 11 to 19%, indicating the robust coexistence of sulfate-reducing bacteria and Chloroflexi in the top of the sediment. Any SSU rRNA of Chloroflexi in the water column was under the detection limit. The summation of the signals of group-specific probes used in this study accounted for up to 89% of total SSU rRNA, suggesting that the DGGE-oligonucleotide probe hybridization approach, in contrast to conventional culture-dependent approaches, was very effective in covering dominant populations.     

Estimating high-affinity methanotrophic bacterial biomass, growth, and turnover in soil by phospholipid fatty acid 13C labeling

A time series phospholipid fatty acid (PLFA) 13C-labeling study was undertaken to determine methanotrophic taxon, calculate methanotrophic biomass, and assess carbon recycling in an upland brown earth soil from Bronydd Mawr (Wales, United Kingdom). Laboratory incubations of soils were performed at ambient CH4 concentrations using synthetic air containing 2 parts per million of volume of 13CH4. Flowthrough chambers maintained a stable CH4 concentration throughout the 11-week incubation. Soils were analyzed at weekly intervals by gas chromatography (GC), GC-mass spectrometry, and GC-combustion-isotope ratio mass spectrometry to identify and quantify individual PLFAs and trace the incorporation of 13C label into the microbial biomass. Incorporation of the 13C label was seen throughout the experiment, with the rate of incorporation decreasing after 9 weeks. The delta13C values of individual PLFAs showed that 13C label was incorporated into different components to various extents and at various rates, reflecting the diversity of PLFA sources. Quantitative assessments of 13C-labeled PLFAs showed that the methanotrophic population was of constant structure throughout the experiment. The dominant 13C-labeled PLFA was 18:1omega7c, with 16:1omega5 present at lower abundance, suggesting the presence of novel type II methanotrophs. The biomass of methane-oxidizing bacteria at optimum labeling was estimated to be about 7.2 x 10(6) cells g(-1) of soil (dry weight). While recycling of 13C label from the methanotrophic biomass must occur, it is a slower process than initial 13CH4 incorporation, with only about 5 to 10% of 13C-labeled PLFAs reflecting this process. Thus, 13C-labeled PLFA distributions determined at any time point during 13CH4 incubation can be used for chemotaxonomic assessments, although extended incubations are required to achieve optimum 13C labeling for methanotrophic biomass determinations.     

Distribution of cultivated and uncultivated cyanobacteria and Chloroflexus-like bacteria in hot spring microbial mats.

Oligodeoxynucleotide hybridization probes were developed to complement specific regions of the small subunit (SSU) rRNA sequences of cultivated and uncultivated cyanobacteria and Chloroflexus-like bacteria, which inhabit hot spring microbial mats. The probes were used to investigate the natural distribution of SSU rRNAs from these species in mats of Yellowstone hot springs of different temperatures and pHs as well as changes in SSU rRNA distribution resulting from 1-week in situ shifts in temperature, pH, and light intensity. Synechococcus lividus Y-7c-s SSU rRNA was detected only in the mat of a slightly acid spring, from which it may have been initially isolated, or when samples from a more alkaline spring were incubated in the more acid spring. Chloroflexus aurantiacus Y-400-fl SSU rRNA was detected only in a high-temperature mat sample from the alkaline Octopus Spring or when lower-temperature samples from this mat were incubated at the high-temperature site. SSU rRNAs of uncultivated species were more widely distributed. Temperature distributions and responses to in situ temperature shifts suggested that some of the uncultivated cyanobacteria might be adapted to high-, moderate-, and low-temperature ranges whereas an uncultivated Chloroflexus-like bacterium appears to have broad temperature tolerance. SSU rRNAs of all uncultivated species inhabiting a 48 to 51 degrees C Octopus Spring mat site were most abundant in the upper 1 mm and were not detected below a 2.5-to 3.5-mm depth, a finding consistent with their possible phototrophic nature. However, the effects of light intensity reduction on these SSU rRNAs were variable, indicating the difficulty of demonstrating a phototrophic phenotype in light reduction experiments.     

Distribution of cultivated and uncultivated cyanobacteria and Chloroflexus-like bacteria in hot spring microbial mats

Oligodeoxynucleotide hybridization probes were developed to complement specific regions of the small subunit (SSU) rRNA sequences of cultivated and uncultivated cyanobacteria and Chloroflexus-like bacteria, which inhabit hot spring microbial mats. The probes were used to investigate the natural distribution of SSU rRNAs from these species in mats of Yellowstone hot springs of different temperatures and pHs as well as changes in SSU rRNA distribution resulting from 1-week in situ shifts in temperature, pH, and light intensity. Synechococcus lividus Y-7c-s SSU rRNA was detected only in the mat of a slightly acid spring, from which it may have been initially isolated, or when samples from a more alkaline spring were incubated in the more acid spring. Chloroflexus aurantiacus Y-400-fl SSU rRNA was detected only in a high-temperature mat sample from the alkaline Octopus Spring or when lower-temperature samples from this mat were incubated at the high-temperature site. SSU rRNAs of uncultivated species were more widely distributed. Temperature distributions and responses to in situ temperature shifts suggested that some of the uncultivated cyanobacteria might be adapted to high-, moderate-, and low-temperature ranges whereas an uncultivated Chloroflexus-like bacterium appears to have broad temperature tolerance. SSU rRNAs of all uncultivated species inhabiting a 48 to 51 degrees C Octopus Spring mat site were most abundant in the upper 1 mm and were not detected below a 2.5-to 3.5-mm depth, a finding consistent with their possible phototrophic nature. However, the effects of light intensity reduction on these SSU rRNAs were variable, indicating the difficulty of demonstrating a phototrophic phenotype in light reduction experiments.     

Estimating High-Affinity Methanotrophic Bacterial Biomass, Growth, and Turnover in Soil by Phospholipid Fatty Acid 13C Labeling

A time series phospholipid fatty acid (PLFA) ¹³C-labeling study was undertaken to determine methanotrophic taxon, calculate methanotrophic biomass, and assess carbon recycling in an upland brown earth soil from Bronydd Mawr (Wales, United Kingdom). Laboratory incubations of soils were performed at ambient CH₄ concentrations using synthetic air containing 2 parts per million of volume of ¹³CH₄. Flowthrough chambers maintained a stable CH₄ concentration throughout the 11-week incubation. Soils were analyzed at weekly intervals by gas chromatography (GC), GC-mass spectrometry, and GC-combustion-isotope ratio mass spectrometry to identify and quantify individual PLFAs and trace the incorporation of ¹³C label into the microbial biomass. Incorporation of the ¹³C label was seen throughout the experiment, with the rate of incorporation decreasing after 9 weeks. The δ¹³C values of individual PLFAs showed that ¹³C label was incorporated into different components to various extents and at various rates, reflecting the diversity of PLFA sources. Quantitative assessments of ¹³C-labeled PLFAs showed that the methanotrophic population was of constant structure throughout the experiment. The dominant ¹³C-labeled PLFA was 18:1ω7c, with 16:1ω5 present at lower abundance, suggesting the presence of novel type II methanotrophs. The biomass of methane-oxidizing bacteria at optimum labeling was estimated to be about 7.2 × 10⁶ cells g⁻¹ of soil (dry weight). While recycling of ¹³C label from the methanotrophic biomass must occur, it is a slower process than initial ¹³CH₄ incorporation, with only about 5 to 10% of ¹³C-labeled PLFAs reflecting this process. Thus, ¹³C-labeled PLFA distributions determined at any time point during ¹³CH₄ incubation can be used for chemotaxonomic assessments, although extended incubations are required to achieve optimum ¹³C labeling for methanotrophic biomass determinations.     

Detection and Capturing of <Superscript>14</Superscript>C Radioactively-Labeled Small Subunit rRNA from Mixed Microbial Communities of a Microbial Mat Using Magnetic Beads

Carbon cycling in the hypersaline microbial mats from Chiprana Lake, Spain is primarily dependent on phototrophic microorganisms with the ability to fix CO₂ into organics that can be further utilized by aerobic as well as anaerobic heterotrophic bacteria. Here, mat pieces were incubated in seawater amended with ¹⁴C sodium bicarbonate and the incorporation of the radiocarbon in the small subunit ribosomal RNA (SSU rRNA) of mat organisms was followed using scintillation counter and autoradiography. Different domains of SSU rRNA were separated from the total RNA by means of streptavidin-coated magnetic beads and biotin-labeled oligonucleotide probes. The ¹⁴C label was detected in isolated RNA by both scintillation counter and autoradiography, however the latter technique was less sensitive. Using scintillation counter, the radiolabel incorporation increased with time with a maximum rate of 0.18 Bq ng⁻¹ detected after 25 days. The bacterial SSU rRNA could be captured using the magnetic beads, however the hybridization efficiency was around 20%. The captured RNA was radioactively labeled, which could be mainly due to the fixation of radiocarbon by phototrophic organisms. In conclusion, the incubation of microbial mats in the presence of radiolabeled bicarbonate leads to the incorporation of the ¹⁴C label into RNA molecules through photosynthesis and this label can be detected using scintillation counter. The used approach could be useful in studying the fate of fixed carbon and its uptake by other microorganisms in complex microbial mats, particularly when species-specific probes are used and the hybridization efficiency and RNA yield are further optimized.     

Isolation of small-subunit rRNA for stable isotopic characterization

Small-subunit ribosomal RNA (SSU rRNA) has several characteristics making it a good candidate biomarker compound: it is found in bacteria, archaea and eukaryotes; it is quickly degraded extracellularly, hence SSU rRNA extracted from a sample probably derives from the currently active population; it includes both conserved and variable regions, allowing the design of capture probes at various levels of phylogenetic discrimination; and rRNA sequences from uncultured species can be classified by comparison with the large and growing public database. Here we present a method for isolation of specific classes of rRNAs from mixtures of total RNA, employing biotin-labelled oligonucleotide probes and streptavidin-coated paramagnetic beads. We also show that the stable carbon isotope composition of Escherichia coli total RNA and SSU rRNA reflects that of the growth substrate for cells grown on LB, M9 glucose and M9 acetate media. SSU rRNA is therefore a promising biomarker for following the flow of carbon, and potentially nitrogen, in natural microbial populations. Some possible applications are discussed.     

A comparison of the crystal structures of eukaryotic and bacterial SSU ribosomal RNAs reveals common structural features in the hypervariable regions

While the majority of the ribosomal RNA structure is conserved in the three major domains of life--archaea, bacteria, and eukaryotes, specific regions of the rRNA structure are unique to at least one of these three primary forms of life. In particular, the comparative secondary structure for the eukaryotic SSU rRNA contains several regions that are different from the analogous regions in the bacteria. Our detailed analysis of two recently determined eukaryotic 40S ribosomal crystal structures, Tetrahymena thermophila and Saccharomyces cerevisiae, and the comparison of these results with the bacterial Thermus thermophilus 30S ribosomal crystal structure: (1) revealed that the vast majority of the comparative structure model for the eukaryotic SSU rRNA is substantiated, including the secondary structure that is similar to both bacteria and archaea as well as specific for the eukaryotes, (2) resolved the secondary structure for regions of the eukaryotic SSU rRNA that were not determined with comparative methods, (3) identified eukaryotic helices that are equivalent to the bacterial helices in several of the hypervariable regions, (4) revealed that, while the coaxially stacked compound helix in the 540 region in the central domain maintains the constant length of 10 base pairs, its two constituent helices contain 5+5 bp rather than the 6+4 bp predicted with comparative analysis of archaeal and eukaryotic SSU rRNAs.     

Use of isotopic and molecular techniques to link toluene degradation in denitrifying aquifer microcosms to specific microbial populations

Microcosms were inoculated with sediments from both a petroleum-hydrocarbon (PHC)-contaminated aquifer and from a nearby pristine aquifer and incubated under anoxic denitrifying conditions with [methyl-13C]toluene. These microcosms served as a laboratory model system to evaluate the combination of isotope (13C-labeling of polar-lipid-derived fatty acids) and molecular techniques (16S rRNA-targeting gene probes) to identify the toluene-metabolizing population. After total depletion of toluene, the following bacterial phospholipid fatty acids (PLFA) were 13C-enriched: 16:1omega7c, 16:1omega7t, 16:0, cy17:0, and 18:1omega7c. Pure culture experiments demonstrated that these compounds were also found in PLFA profiles of PHC-degrading Azoarcus spp. (beta-Proteobacteria) and related species. The origin of the CO2 evolved in the microcosms was determined by measurements of stable carbon isotope ratios. Toluene represented 11% of the total pool of mineralized substrates in the contaminated sediment and 54% in the pristine sediment. The microbial community in the microcosm incubations was characterized by using DAPI staining and whole-cell hybridization with specific fluorescently labeled 16S rRNA-targeted oligonucleotide probes. Results revealed that 6% of the DAPI-stained cells in the contaminated sediment and 32% in the pristine sediment were PHC-degrading Azoarcus spp. In biotic control microcosms (incubated under denitrifying conditions, no toluene added), Azoarcus spp. cells remained at less than 1% of the DAPI-stained cells. The results show that isotope analysis in combination with whole-cell hybridization is a promising approach to identify and to quantify denitrifying toluene degraders within microbial communities.     

Sequence-specific cleavage of small-subunit (SSU) rRNA with oligonucleotides and RNase H: a rapid and simple approach to SSU rRNA-based quantitative detection of microorganisms

A rapid and simple approach to the small-subunit (SSU) rRNA-based quantitative detection of a specific group of microorganisms in complex ecosystems has been developed. The method employs sequence-specific cleavage of rRNA molecules with oligonucleotides and RNase H. Defined mixtures of SSU rRNAs were mixed with an oligonucleotide (referred to as a "scissor probe") that was specifically designed to hybridize with a particular site of targeted rRNA and were subsequently digested with RNase H to proceed to sequence-dependent rRNA scission at the hybridization site. Under appropriate reaction conditions, the targeted rRNAs were correctly cut into two fragments, whereas nontargeted rRNAs remained intact under the same conditions. The specificity of the cleavage could be properly adjusted by controlling the hybridization stringency between the rRNA and the oligonucleotides, i.e., by controlling either the temperature of the reaction or the formamide concentration in the hybridization-digestion buffer used for the reaction. This enabled the reliable discrimination of completely matched rRNA sequences from single-base mismatched sequences. For the detection of targeted rRNAs, the resulting RNA fragment patterns were analyzed by gel electrophoresis with nucleotide-staining fluorescent dyes in order to separate cleaved and intact rRNA molecules. The relative abundance of the targeted SSU rRNA fragments in the total SSU rRNA could easily be calculated without the use of an external standard by determining the signal intensity of individual SSU rRNA bands in the electropherogram. This approach provides a fast and easy means of identification, detection, and quantification of a particular group of microbes in clinical and environmental specimens based on rRNA.     

Label-free detection of 16S ribosomal RNA hybridization on reusable DNA arrays using surface plasmon resonance imaging

In this paper, we describe the detection of bacterial cell-extracted 16S ribosomal RNA (rRNA) using an emerging technology, surface plasmon resonance (SPR) imaging of DNA arrays. Surface plasmon resonance enables detection of molecular interactions on surfaces in response to changes in the index of refraction, therefore eliminating the need for a fluorescent or radioactive label. A variation of the more common SPR techniques, SPR imaging enables detection from multiple probes in a reusable array format. The arrays developed here contain DNA probes (15-21 bases) designed to be complementary to 16S rRNA gene sequences of Escherichia coli and Bacillus subtilis as well as to a highly conserved sequence found in rRNAs from most members of the domain Bacteria. We report species-specific hybridization of cell-extracted total RNA and in vitro transcribed 16S rRNA to oligonucleotide probes on SPR arrays. We tested multiple probe sequences for each species, and found that success or failure of hybridization was dependent upon probe position in the 16S rRNA molecule. It was also determined that one of the probes intended to bind 16S rRNA also bound an unknown protein. The amount of binding to these probes was quantified with SPR imaging. A detection limit of 2 micro g ml-1 was determined for fragmented E. coli total cellular RNA under the experimental conditions used. These results indicate the feasibility of using SPR imaging for 16S rRNA identification and encourage further development of this method for direct detection of other RNA molecules.     

Sequence-Specific Cleavage of Small-Subunit (SSU) rRNA with Oligonucleotides and RNase H: a Rapid and Simple Approach to SSU rRNA-Based Quantitative Detection of Microorganisms

A rapid and simple approach to the small-subunit (SSU) rRNA-based quantitative detection of a specific group of microorganisms in complex ecosystems has been developed. The method employs sequence-specific cleavage of rRNA molecules with oligonucleotides and RNase H. Defined mixtures of SSU rRNAs were mixed with an oligonucleotide (referred to as a “scissor probe”) that was specifically designed to hybridize with a particular site of targeted rRNA and were subsequently digested with RNase H to proceed to sequence-dependent rRNA scission at the hybridization site. Under appropriate reaction conditions, the targeted rRNAs were correctly cut into two fragments, whereas nontargeted rRNAs remained intact under the same conditions. The specificity of the cleavage could be properly adjusted by controlling the hybridization stringency between the rRNA and the oligonucleotides, i.e., by controlling either the temperature of the reaction or the formamide concentration in the hybridization-digestion buffer used for the reaction. This enabled the reliable discrimination of completely matched rRNA sequences from single-base mismatched sequences. For the detection of targeted rRNAs, the resulting RNA fragment patterns were analyzed by gel electrophoresis with nucleotide-staining fluorescent dyes in order to separate cleaved and intact rRNA molecules. The relative abundance of the targeted SSU rRNA fragments in the total SSU rRNA could easily be calculated without the use of an external standard by determining the signal intensity of individual SSU rRNA bands in the electropherogram. This approach provides a fast and easy means of identification, detection, and quantification of a particular group of microbes in clinical and environmental specimens based on rRNA.     

DNA sequence and secondary structure of the mitochondrial small subunit ribosomal RNA coding region including a group-IC2 intron from the cultivated basidiomycete Agrocybe aegerita

Due to their structural complexity and their evolutionary dimension, rRNAs are the most investigated nucleic acids in prokaryotes, eukaryotes and organelles. However, no complete sequence of a mitochondrial small subunit (SSU) rRNA was available in the basidiomycotina subdivision. The mitochondrial gene encoding the SSU rRNA of the cultivated basidiomycete Agrocybe aegerita was cloned and its complete nucleotide sequence achieved; the 5′- and 3′-ends were localized by nuclease S1 mapping, leading to a size of 3277 nt. The secondary structure of the SSU rRNA (1906 nt in size) possessed all the helices and loops of the prokaryotic model; a unique modification was found in a conserved nucleotide predicted by the model: the nt 487 was A instead of C. The same modification, has been found in all the partial basidiomycete mitochondrial sequences available in databases. The Agrocybe aegerita SSU rRNA was characterized by large and unusual extensions leading to additional helices in the variable domains V4, V6 and V9, which were the longest of the known prokaryotic or mitochondrial SSU rRNAs. Nucleotide sequence analysis indicated a 1371-bp intron, belonging to subgroup-IC2, located in a conserved loop in the 3′-part of the SSU rRNA. This intron, which is the second example reported in a fungal mitochondrial SSU rDNA, encoded a putative protein (407 aa) sharing homologies with endonucleases involved in group-I intron mobility. This report constitutes the first complete mitochondrial SSU rRNA sequence and secondary structure of any member of the basidiomycotina subdivision.     

A discontinuous small subunit ribosomal RNA in Tetrahymena pyriformis mitochondria

We show here that in the mitochondria of Tetrahymena pyriformis, the small subunit (SSU) rRNA is discontinuous, being comprised of two separate components which we term "alpha" (a novel low molecular weight RNA, approximately equal to 200 nucleotides long) and "beta" (a previously described 14 S RNA). The SSU alpha rRNA has been sequenced in its entirety; it represents the immediate 5'-terminal domain of conventional SSU rRNA. The sequences at the ends of the SSU beta rRNA have also been determined; they show that this molecule corresponds to the 3'-terminal 7/8 of conventional SSU rRNA. A 2.5-kilobase pair XbaI restriction fragment of T. pyriformis mitochondrial DNA which contains the SSU alpha and SSU beta rRNA genes was cloned and its complete nucleotide sequence was determined. This revealed that the genes encoding the two segments of SSU rRNA are separated by a 54-base pair (A + T)-rich spacer. The alpha and beta sequences can be fitted to a generalized secondary structure model for eubacterial 16 S rRNA, with the two RNA species associating through long range interactions to form base-paired regions characteristic of SSU rRNA. In this model, the spacer is situated in a region of pronounced primary and secondary structural variation among SSU rRNAs. The significance of these findings with respect to rRNA biosynthesis and processing and the possible evolutionary relationship between spacers and variable regions in rRNA genes is discussed.     

Successional changes in an evolving anaerobic chlorophenol-degrading community used to infer relationships between population structure and system-level processes.

The response of a complex methanogenic sediment community to 2-chlorophenol (2-CP) was evaluated by monitoring the concentrations of this model contaminant and important metabolic intermediates and products and by using rRNA-targeted probes to track several microbial populations. Key relationships between the evolving population structure, formation of metabolic intermediates, and contaminant mineralization were identified. The nature of these relationships was intrinsically linked to the metabolism of benzoate, an intermediate that transiently accumulated during the mineralization of 2-CP. Before the onset of benzoate fermentation, reductive dehalogenation of 2-CP competed with methanogenesis for endogenous reducing equivalents. This suppressed H(2) levels, methane production, and archaeal small-subunit (SSU)-rRNA concentrations in the sediment community. The concentrations of bacterial SSU rRNA, including SSU rRNA derived from "Desulfovibrionaceae" populations, tracked with 2-CP levels, presumably reflecting changes in the activity of dehalogenating organisms. After the onset of benzoate fermentation, the abundance of Syntrophus-like SSU rRNA increased, presumably because these syntrophic organisms fermented benzoate to methanogenic substrates. Consequently, although the parent substrate 2-CP served as an electron acceptor, cleavage of its aromatic nucleus also influenced the sediment community by releasing the electron donors H(2) and acetate. Increased methane production and archaeal SSU-rRNA levels, which tracked with the Syntrophus-like SSU-rRNA concentrations, revealed that methanogenic populations in particular benefited from the input of reducing equivalents derived from 2-CP.