rRNA Libraries Research Articles

Reverse Transcription of 16S rRNA To Monitor Ribosome-Synthesizing Bacterial Populations in the Environment

Identification and quantification of phylogenetically defined bacterial populations in the environment are often performed using molecular tools targeting 16S rRNA. Fluorescence in situ hybridization has been used to monitor the expression and processing of rRNA by targeting the 3' tail of precursor 16S rRNA. To expand this approach, we employed reverse transcription of total RNA using primer S-D-Bact-0338-a-A-18. Length heterogeneity detected by slab gel analysis, denaturing high-performance liquid chromatography (DHPLC) was used to differentiate the 5' tail of the precursor from mature 16S rRNA, and the relative abundance of the precursor compared to the abundance of mature 16S rRNA was shown to be a sensitive indicator of the physiologic state of Acinetobacter calcoaceticus ATCC 23055(T). Our results demonstrate that this is a sensitive and reliable method with a detection limit of 10 ng of single-stranded DNA. The assay was also used to differentiate among precursor 16S rRNA levels with mixed pure cultures, as well as to examine the response of a mixed activated sludge culture exposed to fresh growth medium and the antibiotic chloramphenicol. The results of this study demonstrate that this assay is a novel reverse transcription assay that simultaneously measures the mature and precursor 16S rRNA pools for mixed bacterial populations in an engineered environment. Furthermore, collection of the reverse transcription products derived from activated sludge samples by the DHPLC approach enabled identification of the active bacterial genera. Comparison of 16S and precursor 16S rRNA clone library results indicated that the precursor 16S rRNA library is a more sensitive indicator for active bacteria in engineered environmental samples.

Assessment of alkaliphilic haloarchaeal diversity in Sua pan evaporator ponds in Botswana

Cultivation-dependent and molecular-based culture-independent methods were used to assess alkaliphilic haloarchaeal diversity at Sua pan evaporator ponds in Botswana. Isolates belonging to the genera Natrialba, Natronococcus and Natronorubrum were recovered from brine samples by enrichment and identified through a series of biochemical tests as well as sequencing of 16S rRNA fragments. In addition, an environmental 16S rRNA library was constructed from brine samples of two evaporator ponds. The library comprised members of the genera Halorubrum (65%), Natrialba (14%), Natronorubrum (7%) and new phylotypes (14%). The new phylotypes consisted of two clones that exhibited low 16S rRNA similarity (95 – 97%) with known species and could potentially represent new species in the genus Halorubrum, one clone with 91% similarity to Natronolimnobius which could represent a new genus, as well as an unidentifiable phylotype which exhibited 79% similarity to Methanotorris formicicus. Two major differences were observed between cultivation- and molecularbased methods; firstly, Halorubrum species were largely represented in the environmental clone library but no isolates were obtained, and secondly, Natronococcus species were isolated but not detected in the clone library. An overlap between the archaeal isolates and the ribosomal library clones was apparent although the novel phylotypes detected in this study were not recovered through cultivation

Effects of transgenic hybrid aspen overexpressing polyphenol oxidase on rhizosphere diversity.

This study assessed the potential effects of transgenic aspen overexpressing a polyphenol oxidase gene on diversity in rhizosphere communities. Cultivation-independent methods were used to better delineate bacterial and fungal populations associated with transgenic and nontransgenic trees. Gene libraries for the bacterial component of the rhizosphere were established using 16S rRNA and chaperonin-60 (CPN-60) gene sequences, while the fungal community was characterized using 18S rRNA gene sequences. The 16S rRNA gene libraries were dominated by alphaproteobacterial sequences, while the CPN-60 gene libraries were dominated by members of the Bacteroidetes/Chlorobi group. In both the CPN-60 and 16S rRNA libraries, there were differences in only minor components of the bacterial community between transgenic and unmodified trees, and no significant differences in species diversity were observed. Compared to the bacterial gene libraries, greater coverage of the underlying population was achieved with the fungal 18S rRNA libraries. Members of the Zygomycota, Chytridiomycota, Ascomycota, and Basidiomycota were recovered from both libraries. The dominant groups of fungi associated with each tree type were very similar, although there were some qualitative differences in the recovery of less-abundant fungi, likely as a result of the underlying heterogeneity of the fungal population. The methods employed revealed only minor differences between the bacterial and fungal communities associated with transgenic and unmodified trees.

Diverse Bacterial PKS Sequences Derived From Okadaic Acid-Producing Dinoflagellates

Okadaic acid (OA) and the related dinophysistoxins are isolated from dinoflagellates of the genus Prorocentrum and Dinophysis. Bacteria of the Roseobacter group have been associated with okadaic acid producing dinoflagellates and have been previously implicated in OA production. Analysis of 16S rRNA libraries reveals that Roseobacter are the most abundant bacteria associated with OA producing dinoflagellates of the genus Prorocentrum and are not found in association with non-toxic dinoflagellates. While some polyketide synthase (PKS) genes form a highly supported Prorocentrum clade, most appear to be bacterial, but unrelated to Roseobacter or Alpha-Proteobacterial PKSs or those derived from other Alveolates Karenia brevis or Crytosporidium parvum.

Diverse Bacterial PKS Sequences Derived From Okadaic Acid-Producing Dinoflagellates

Okadaic acid (OA) and the related dinophysistoxins are isolated from dinoflagellates of the genus Prorocentrum and Dinophysis. Bacteria of the Roseobacter group have been associated with okadaic acid producing dinoflagellates and have been previously implicated in OA production. Analysis of 16S rRNA libraries reveals that Roseobacter are the most abundant bacteria associated with OA producing dinoflagellates of the genus Prorocentrum and are not found in association with non-toxic dinoflagellates. While some polyketide synthase (PKS) genes form a highly supported Prorocentrum clade, most appear to be bacterial, but unrelated to Roseobacter or Alpha-Proteobacterial PKSs or those derived from other Alveolates Karenia brevis or Crytosporidium parvum.

Phylogenetic characterization of bacterial consortia obtained of corroding gas pipelines in Mexico

Characterization of the microbial populations formed in gas pipelines is essential to understand the metallic surface-microbe interaction, their role in metal corrosion, and to implement efficient monitoring and control strategies. Microbial community analysis in a corroded gas pipeline in a petroleum-producing facility in the Southeast region in Mexico was performed by traditional cultivation techniques and identification based on 16S rRNA gene sequence. In all samples, thin bacterial biofilms were observed and pitting corrosion was reveled after removing the biofilms. Six pure or mixed cultures of anaerobic bacteria were obtained and their 16S rRNA libraries were constructed, respectively. At least two members of each RFLP profile were sequenced and the phylogenetic affiliations of cloned bacterial 16S rRNA genes indicated that native biofilms were mainly colonized by Desulfovibrio vulgaris and Desulfovibrio desulfuricans, sulfate-reducing bacteria members; Citrobacter freundii, an Enterobacteriaceae member; Clostridium celerecrescens and Clostridium sporogenes, spore-forming anaerobic species and Cetobacterium somerae, a microaerotolerant, non-spore-forming fusobacteria. Some of these species have been observed consistently in other steel pipelines previously, but Cetobacterium members and C. celerecrescens are described for the fist time in this corroded gas pipeline. The potential role of each species in biofilm formation and steel corrosion is discussed.

Potential New Antibiotic Sites in the Ribosome Revealed by Deleterious Mutations in RNA of the Large Ribosomal Subunit

The ribosome is the main target for antibiotics that inhibit protein biosynthesis. Despite the chemical diversity of the known antibiotics that affect functions of the large ribosomal subunit, these drugs act on only a few sites corresponding to some of the known functional centers. We have used a genetic approach for identifying structurally and functionally critical sites in the ribosome that can be used as new antibiotic targets. By using randomly mutagenized rRNA genes, we mapped rRNA sites where nucleotide alterations impair the ribosome function or assembly and lead to a deleterious phenotype. A total of 77 single-point deleterious mutations were mapped in 23 S rRNA and ranked according to the severity of their deleterious phenotypes. Many of the mutations mapped to familiar functional sites that are targeted by known antibiotics. However, a number of mutations were located in previously unexplored regions. The distribution of the mutations in the spatial structure of the ribosome showed a strong bias, with the strongly deleterious mutations being mainly localized at the interface of the large subunit and the mild ones on the solvent side. Five sites where deleterious mutations tend to cluster within discrete rRNA elements were identified as potential new antibiotic targets. One of the sites, the conserved segment of helix 38, was studied in more detail. Although the ability of the mutant 50 S subunits to associate with 30 S subunits was impaired, the lethal effect of mutations in this rRNA element was unrelated to its function as an intersubunit bridge. Instead, mutations in this region had a profound deleterious effect on the ribosome assembly.

Novel Rumen Bacterial Diversity in Two Geographically Separated Sub-Species of Reindeer

Svalbard reindeer (Rangifer tarandus platyrhynchus) live under austere nutritional conditions on the high-arctic archipelago of Svalbard, while semi-domesticated Norwegian reindeer (R. tarandus tarandus) migrate between lush coastal summer pastures and inland winter pastures with lichens on mainland Norway. Svalbard reindeer are known to have high rumen concentrations of cellulolytic bacteria, ranging from 15% of the viable population in summer to 35% in winter, compared to only 2.5% in Norwegian reindeer. Their rumen bacterial diversity was investigated through comparative analyses of 16S rRNA gene sequences ( approximately 1.5 kb in length) generated from clone libraries (n = 121) and bacterial isolates (n = 51). LIBSHUFF comparisons of the composition of the two 16S rRNA libraries from Norwegian reindeer showed a significant effect of artificial feeding compared to natural pasture, but failed to yield significant differences between libraries from Norwegian reindeer and Svalbard reindeer. The combined sequences from reindeer were not significantly different from those reported in wild Thompson's gazelle in Kenya but did differ from those reported in domestic cattle in Japan. A total of 90 distinct operational taxonomic units (OTUs) were identified by employing a criterion of 97% similarity, while the Chao1 index estimated the reindeer bacterial rumen population richness at 698 OTUs. The majority of the clone library sequences (92.5%) represented novel strains with <97% identity to any known sequence in the public database, most of them affiliated with the bacterial phylum Firmicutes (low G+C Gram-positives) related to the order Clostridiales (76.7%), while Gram-negative bacteria in the Bacteriodales (Prevotella-Bacteroides group) contributed to 22.5%. Also, six of the isolates were putatively novel strains, possibly representing new species in the Clostridium subphylum (cluster XIVa), Actinomyces and Butyrivibrio.

Temporal change of composition and potential activity of the thermophilic archaeal community during the composting of organic material

To date, composting has been regarded as an aerobic process but it has been shown that composting piles are often sources of atmospheric methane. In order to gain a more comprehensive view on the diversity of methanogenic Archaea in compost, gas chromatographical methods and molecular cloning were used to study relationships of thermophilic archaeal communities and changes in methane production potential during compost maturation. According to the thermophilic methane production potential, wide differences could be detected between differently aged compost materials. In material derived from 3- and 4-week-old piles, low and no thermophilic methane production potential, respectively, was observed at 50 °C. Material from a 6-week-old pile showed the maximum methane production. With compost maturation, the production slowly decreased again with 6 weeks, 8 weeks, and mature compost showing an optimum methane production potential at 60 °C. At 70 °C, only 6-week-old material showed a comparable high production of methane. The 16S rRNA-based phylogenetic surveys revealed an increase of archaeal diversity with compost maturation. In the 6-week-old material, 86% of the sequences in the archaeal 16S rRNA library had the highest sequence similarities to Methanothermobacter spp. and the remaining 14% of the clones were related to Methanosarcina thermophila. Quantification of methanogens in 6-week-old material, on the basis of the methane production rate, resulted in values of about 2×10 7 cells per gram fresh weight. In 8-week-old and mature compost material, the proportion of sequences similar to Methanothermobacter spp. decreased to 34% and 0%, respectively. The mature compost material showed the highest variation in identified sequences, although 33% could be assigned to as yet uncultured Archaea (e.g. Rice cluster I, III, and IV). Our results indicate that compost harbours a diverse community of thermophilic methanogens, with changing composition during the maturation process, presumably due to altered pile conditions. Likewise, compost may act as a potential carrier for thermophilic methanogens in temperate soils because it is widely used as a soil amendment.

Novel bacterial sulfur oxygenase reductases from bioreactors treating gold-bearing concentrates

The microbial community and sulfur oxygenase reductases of metagenomic DNA from bioreactors treating gold-bearing concentrates were studied by 16S rRNA library, real-time polymerase chain reaction (RT-PCR), conventional cultivation, and molecular cloning. Results indicated that major bacterial species were belonging to the genera Acidithiobacillus, Leptospirillum, Sulfobacillus, and Sphingomonas, accounting for 6.3, 66.7, 18.8, and 8.3%, respectively; the sole archaeal species was Ferroplasma sp. (100%). Quantitative RT-PCR revealed that the 16S rRNA gene copy numbers (per gram of concentrates) of bacteria and archaea were 4.59 x 10(9) and 6.68 x 10(5), respectively. Bacterial strains representing Acidithiobacillus, Leptospirillum, and Sulfobacillus were isolated from the bioreactors. To study sulfur oxidation in the reactors, pairs of new PCR primers were designed for the detection of sulfur oxygenase reductase (SOR) genes. Three sor-like genes, namely, sor (Fx), sor (SA), and sor (SB) were identified from metagenomic DNAs of the bioreactors. The sor (Fx) is an inactivated SOR gene and is identical to the pseudo-SOR gene of Ferroplasma acidarmanus. The sor (SA) and sor (SB) showed no significant identity to any genes in GenBank databases. The sor (SB) was cloned and expressed in Escherichia coli, and SOR activity was determined. Quantitative RT-PCR determination of the gene densities of sor (SA) and sor (SB) were 1,000 times higher than archaeal 16S rRNA gene copy numbers, indicating that these genes were mostly impossible from archaea. Furthermore, with primers specific to the sor (SB) gene, this gene was PCR-amplified from the newly isolated Acidithiobacillus sp. strain SM-1. So far as we know, this is the first time to determine SOR activity originating from bacteria and to document SOR gene in bioleaching reactors and Acidithiobacillus species.

A Molecular Approach to Identify Active Microbes in Environmental Eukaryote Clone Libraries

A rapid method for the simultaneous extraction of RNA and DNA from eukaryote plankton samples was developed in order to discriminate between indigenous active cells and signals from inactive or even dead organisms. The method was tested using samples from below the chemocline of an anoxic Danish fjord. The simple protocol yielded RNA and DNA of a purity suitable for amplification by reverse transcription-polymerase chain reaction (RT-PCR) and PCR, respectively. We constructed an rRNA-derived and an rDNA-derived clone library to assess the composition of the microeukaryote assemblage under study and to identify physiologically active constituents of the community. We retrieved nearly 600 protistan target clones, which grouped into 84 different phylotypes (98% sequence similarity). Of these phylotypes, 27% occurred in both libraries, 25% exclusively in the rRNA library, and 48% exclusively in the rDNA library. Both libraries revealed good correspondence of the general community composition in terms of higher taxonomic ranks. They were dominated by anaerobic ciliates and heterotrophic stramenopile flagellates thriving below the fjord's chemocline. The high abundance of these bacterivore organisms points out their role as a major trophic link in anoxic marine systems. A comparison of the two libraries identified phototrophic dinoflagellates, "uncultured marine alveolates group I," and different parasites, which were exclusively detected with the rDNA-derived library, as nonindigenous members of the anoxic microeukaryote community under study.

Comprehensive genetic selection revealed essential bases in the peptidyl-transferase center.

During protein synthesis, the ribosome catalyzes peptide-bond formation. Biochemical and structural studies revealed that conserved nucleotides in the peptidyl-transferase center (PTC) and its proximity may play a key role in peptide-bond formation; the exact mechanism involved remains unclear. To more precisely define the functional importance of the highly conserved residues, we used a systematic genetic method, which we named SSER (systematic selection of functional sequences by enforced replacement), that allowed us to identify essential nucleotides for ribosomal function from randomized rRNA libraries in Escherichia coli cells. These libraries were constructed by complete randomization of the critical regions in and around the PTC. The selected variants contained natural rRNA sequences from other organisms and organelles as well as unnatural functional sequences; hence providing insights into the functional roles played by these essential bases and suggesting how the universal catalytic mechanism of peptide-bond formation could evolve in all living organisms. Our results highlight essential bases and interactions, which are shaping the PTC architecture and guiding the motions of the tRNA terminus from the A to the P site, found to be crucial not only for the formation of the peptide bond but also for nascent chain elongation.

Phylogenetic Comparisons of Bacterial Communities from Serpentine and Nonserpentine Soils

I present the results of a culture-independent survey of soil bacterial communities from serpentine soils and adjacent nonserpentine comparator soils using a variety of newly developed phylogenetically based statistical tools. The study design included site-based replication of the serpentine-to-nonserpentine community comparison over a regional scale ( approximately 100 km) in Northern California and Southern Oregon by producing 16S rRNA clone libraries from pairs of samples taken on either side of the serepentine-nonserpentine edaphic boundary at three geographical sites. At the division level, the serpentine and nonserpentine communities were similar to each other and to previous data from forest soils. Comparisons of both richness and Shannon diversity produced no significant differences between any of the libraries, but the vast majority of phylogenetically based tests were significant, even with only 50 sequences per library. These results suggest that most samples were distinct, consisting of a collection of lineages generally not found in other samples. The pattern of results showed that serpentine communities tended to be more similar to each other than they were to nonserpentine communities, and these differences were at a lower taxonomic scale. Comparisons of two nonserpentine communities generally showed differences, and some results suggest that the geographical site may control community composition as well. These results show the power of phylogenetic tests to discern differences between 16S rRNA libraries compared to tests that discard DNA data to bin sequences into operational taxonomic units, and they stress the importance of replication at larger scales for inferences regarding microbial biogeography.

Molecular diversity studies of bacterial communities of oil polluted microbial mats from the Etang de Berre (France)

The biodiversity of microbial mats inhabiting the oil-contaminated lagoon Etang de Berre was determined by molecular approaches. The fingerprint of denaturing gradient gel electrophoresis (DGGE) and automatic ribosomal intergenic spacer analysis (ARISA) of mats exposed to different pollution levels showed specific microbial communities for each site but similar diversity richness. Species composition of the mats were compared by constructing 16S rRNA libraries. Amplified rDNA restriction analysis (ARDRA) of clone libraries confirmed their similar level of diversity richness. Phylogenetic analysis of the 16S rRNA sequences showed that the classes gamma and alpha of Proteobacteria were abundantly present in both sites whereas phylotypes related to the delta-Proteobacteria and to the uncultured WS3 group were mainly found in the site with the highest pollution. Identification of the species involved in oil degradation by combining culture-based approaches and DGGE, showed that enrichment cultures were constituted by members of the Rhodobacterales and species related to Rhodococcus, Sphingomonas, Xanthomonas and Microbacterium, all of them known for their ability to degrade hydrocarbons. Our findings suggest that oil pollution has not affected the biodiversity richness of the mats. However, the populations involved in hydrocarbon degradation represent a minor fraction of the mat communities in the Etang de Berre.

Study of bacterial communities in Antarctic coastal waters by a combination of 16S rRNA and 16S rDNA sequencing

An ecological study on distribution of Antarctic bacterial communities was determined by 16S-based phylogenetic analyses of clone libraries derived from RNA and DNA extracted from two different marine areas and compared between each other. Superficial seawater samples were collected from four stations in Ross Sea, three of them located in Rod Bay and one in Evans Cove; for each station two clone libraries (16S rDNA and 16S rRNA) were prepared and evident divergences between DNA and RNA libraries of each site were obtained. Of all phylotypes 93.6% were found in RNA libraries; in contrast, only 31 phylotypes (70.5%) were retrieved from total microbial community (DNA libraries). DNA and RNA sequences related to gamma-Proteobacteria and Bacteroidetes groups, typical for Antarctic sea-ice bacterial communities, were detected in analysed sites. 16S rDNA and rRNA libraries derived from the two different areas were enriched by picophytoplanktonic 16S sequences of plastid and mitochondrion origins, reflecting that the algal blooms occurred during sampling (Antarctic summer 2003). The finding in Rod Bay libraries of high percentage of DNA clones apparently affiliated with beta-Proteobacteria typical for activated sludges and well water could be explained by the presence of a sewage depuration system at this site. Obtained results clearly demonstrate that combination of 16S rDNA and 16S rRNA gene sequencing is preferred approach to have a more reliable vision on the composition of microbial communities.

Relationships Between Microbial Community Structure and Soil Processes Under Elevated Atmospheric Carbon Dioxide

There is little current understanding of the relationship between soil microbial community composition and soil processes rates, nor of the effect climate change and elevated CO(2) will have on microbial communities and their functioning. Using the eastern cottonwood (Populus deltoides) plantation at the Biosphere 2 Laboratory, we studied the relationships between microbial community structure and process rates, and the effects of elevated atmospheric CO(2) on microbial biomass, activity, and community structure. Soils were sampled from three treatments (400, 800, and 1200 ppm CO(2)), a variety of microbial biomass and activity parameters were measured, and the bacterial community was described by 16S rRNA libraries. Glucose substrate-induced respiration (SIR) was significantly higher in the 1200 ppm CO(2) treatment. There were also a variety of complex, nonlinear responses to elevated CO(2). There was no consistent effect of elevated CO(2) on bacterial diversity; however, there was extensive variation in microbial community structure within the plantation. The southern ends of the 800 and 1200 ppm CO(2) bays were dominated by beta-Proteobacteria, and had higher fungal biomass, whereas the other areas contained more alpha-Proteobacteria and Acidobacteria. A number of soil process rates, including salicylate, glutamate, and glycine substrate-induced respiration and proteolysis, were significantly related to the relative abundance of the three most frequent bacterial taxa, and to fungal biomass. Overall, variation in microbial activity was better explained by microbial community composition than by CO(2) treatment. However, the altered diversity and activity in the southern bays of the two high CO(2) treatments could indicate an interaction between CO(2) and light.

Improved template representation in cpn60 polymerase chain reaction (PCR) product libraries generated from complex templates by application of a specific mixture of PCR primers

Some classes of high G+C content organisms such as the Actinobacteria, which are known through culture-based studies to be present in large numbers in particular microbial communities, are under-represented or even absent from 16S rRNA or cpn60 polymerase chain reaction (PCR) product libraries derived from these templates. Using reference cpn60 sequence data from organisms with high G+C content genomes, a pair of PCR primers were designed which, when used in combination with the previously developed degenerate, universal cpn60 primers, improve the representation of templates with high G+C content. The primers were validated using a combination of traditional and quantitative real-time PCR on both manufactured template mixtures and biological samples. The development and optimization of this specific primer mixture represents an improvement of established methods and a significant advance in the ability to generate cpn60 PCR product libraries that more closely represent the sequence diversity in complex templates.

Clustering Binary Oligonucleotide Fingerprint Vectors for DNA Clone Classification Analysis

We considered the problem of clustering binarized oligonucleotide fingerprints that attempts to identify clusters. Oligonucleotide fingerprinting is a powerful DNA array based method to characterize cDNA and rRNA libraries and has many applications including gene expression profiling and DNA clone classification. DNA clone classification is the main application for the problem considered in this paper. Most of the existing approaches for clustering use normalized real intensity values and thus do not treat positive and negative hybridization signals equally. This is demonstrated in a series of recent publications where a discrete approach typically useful in the classification of microbial rRNA clones has been proposed. In the discrete approach, hybridization intensities are normalized and thresholds are set such that a value of 1 represents hybridization, a value of 0 represents no hybridization, and an N represents unknown, which is also called a missing value. A combinatorial optimization problem is then formulated attempting to cluster the fingerprints and resolve the missing values simultaneously. It has been examined that missing values cause much difficulty in clustering analysis and most clustering methods are very sensitive to them. In this paper, we turned a little back to the traditional clustering problem, which takes in no missing values but with the revised goal to stabilize the number of clusters and maintain the clustering quality. We adopted the binarizing scheme used in the discrete approach as it is shown to be typically useful for the clone classifications. We formulated such a problem into another combinatorial optimization problem. The computational complexity of this new clustering problem and its relationships to the discrete approach and the traditional clustering problem were studied. We have designed an exact algorithm for the new clustering problem, which is an A* search algorithm for finding a minimum number of clusters. The experimental results on two commonly tested real datasets demonstrated that the A* search algorithm runs fast and performs better than some popular hierarchical clustering methods, in terms of separating clones that have different characteristics with respect to the given oligonucleotide probes.

Diversity and succession of the intestinal bacterial community of the maturing broiler chicken.

The diversity of bacterial floras in the ilea and ceca of chickens that were fed a vegetarian corn-soy broiler diet devoid of feed additives was examined by analysis of 1,230 partial 16S rRNA gene sequences. Nearly 70% of sequences from the ileum were related to those of Lactobacillus, with the majority of the rest being related to Clostridiaceae (11%), Streptococcus (6.5%), and Enterococcus (6.5%). In contrast, Clostridiaceae-related sequences (65%) were the most abundant group detected in the cecum, with the other most abundant sequences being related to Fusobacterium (14%), Lactobacillus (8%), and Bacteroides (5%). Statistical analysis comparing the compositions of the different 16S rRNA libraries revealed that population succession occurred during some sampling periods. The significant differences among cecal libraries at 3 and 7 days of age, at 14 to 28 days of age, and at 49 days of age indicated that successions occurred from a transient community to one of increasing complexity as the birds aged. Similarly, the ileum had a stable bacterial community structure for birds at 7 to 21 days of age and between 21 to 28 days of age, but there was a very unique community structure at 3 and 49 days of age. It was also revealed that the composition of the ileal and cecal libraries did not significantly differ when the birds were 3 days old, and in fact during the first 14 days of age, the cecal microflora was a subset of the ileal microflora. After this time, the ileum and cecum had significantly different library compositions, suggesting that each region developed its own unique bacterial community as the bird matured.

Functional genetic selection of the decoding center in E. coli 16S rRNA.

Ribosomal RNAs (rRNAs) play crucial roles in protein biosynthesis. The decoding center of 16S rRNA in 30S subunit affords a place for interaction between mRNA and tRNA, and contributes to the fidelity of the decoding by monitoring the codon-anticodon base pairing. The helices 18 and 44 in 16S rRNA are known to be major components of the decoding center. To investigate functional role of the conserved sequence in rRNAs, we employed a new genetic method that allows us to identify and select from randomized E. coli rRNA libraries those rRNA sequences absolutely required for the ribosome function. Functional consensus sequences were identified in both helices, providing us with a new insight into the decoding mechanism.