Ribosomal RNA Gene Clone Research Articles

Structural Analysis of the Bacterial Community of Konoshiro-narezushi by the 16S Ribosomal RNA Gene Clone Library Method

<b>Structural Analysis of the Bacterial Community of Konoshiro-narezushi by the 16S Ribosomal RNA Gene Clone Library Method </b>

Soil bacterial communities in native and regenerated perhumid montane forests

Forest management often results in changes in soil properties and microbial communities. In the present study, we characterized differences in soil bacterial communities caused by forest management practices using 16S ribosomal RNA (rRNA) gene clone libraries. The communities were from a disturbed Chamaecyparis (DCP) forest subjected to harvesting of snags and downed logs, a secondary Chamaecyparis (SCP) plantation subjected to harvesting of old-growth trees, and a secondary cedar plantation (SCD). These forests were compared to a nearby native Chamaecyparis (NCP) forest in a perhumid montane ecosystem. At this locality, the elevation is from 1500 to 2100 m a.s.l., the mean annual precipitation >4000 mm, the mean annual temperature about 12 °C, and the soil pH <4. The phyla Acidobacteria and Proteobacteria predominated among the three disturbed forest soil communities. Several diversity indices and rarefaction curves revealed that the diversity of the SCD community was higher than that of the DCP soils. The diversity of the SCP community was intermediate. The bacterial diversity of the NCP community was lower than communities in the three disturbed forest soils. Analysis of molecular variance revealed that the bacterial community in SCD soils significantly differed from those in the three Chamaecyparis forest soils. Some of the abundant operational taxonomic units (OTUs) significantly differed among the four forest soils. Compared to the three disturbed forest soil communities, the NCP community was dominated by Proteobacteria, which accounted for more than half of the community. These results suggest that the disturbance of forest harvesting and tree species conversion influence the composition of bacterial communities in natural and disturbed forests and increase the diversity of the disturbed forest soil community.

Effects of the antimicrobial tylosin on the microbial community structure of an anaerobic sequencing batch reactor

The effects of the antimicrobial tylosin on a methanogenic microbial community were studied in a glucose-fed laboratory-scale anaerobic sequencing batch reactor (ASBR) exposed to stepwise increases of tylosin (0, 1.67, and 167 mg/L). The microbial community structure was determined using quantitative fluorescence in situ hybridization (FISH) and phylogenetic analyses of bacterial 16S ribosomal RNA (rRNA) gene clone libraries of biomass samples. During the periods without tylosin addition and with an influent tylosin concentration of 1.67 mg/L, 16S rRNA gene sequences related to Syntrophobacter were detected and the relative abundance of Methanosaeta species was high. During the highest tylosin dose of 167 mg/L, 16S rRNA gene sequences related to Syntrophobacter species were not detected and the relative abundance of Methanosaeta decreased considerably. Throughout the experimental period, Propionibacteriaceae and high GC Gram-positive bacteria were present, based on 16S rRNA gene sequences and FISH analyses, respectively. The accumulation of propionate and subsequent reactor failure after long-term exposure to tylosin are attributed to the direct inhibition of propionate-oxidizing syntrophic bacteria closely related to Syntrophobacter and the indirect inhibition of Methanosaeta by high propionate concentrations and low pH.

Change in Bacterial Community Structure in Response to Disturbance of Natural Hardwood and Secondary Coniferous Forest Soils in Central Taiwan

Forest management often results in changes in the soil and its microbial communities. In the present study, differences in the soil bacterial community caused by forest management practices were characterized using small subunit (SSU) ribosomal RNA (rRNA) gene clone libraries. The communities were from a native hardwood forest (HWD) and two adjacent conifer plantations in a low-elevation montane, subtropical experimental forest at the Lienhuachi Experimental Forest (LHCEF) in central Taiwan. At this locality, the elevation ranges from 600 to 950 m, the mean annual precipitation is 2,200 mm, the mean annual temperature is 20.8 °C, and the soil pH is 4. The conifer forests included a Cunninghamia konishii Hay (CNH) plantation of 40 years and an old growth Calocedrus formosana (Florin) Florin (CLC) forest of 80 years. A total of 476 clones were sequenced and assigned into 12 phylogenetic groups. Proteobacteria-affiliated clones (53%) predominated in the library from HWD soils. In contrast, Acidobacteria was the most abundant phylum and comprised 39% and 57% in the CLC and CNH libraries, respectively. Similarly, the most abundant OTUs in HWD soils were greatly reduced or absent in the CLC and CNH soils. Based on several diversity indices, the numbers of abundant OTUs and singletons, and rarefaction curves, the diversity of the HWD community (0.95 in evenness and Shannon diversity indices) was somewhat less than that in the CNH soils (0.97 in evenness and Shannon diversity indices). The diversity of the community in CLC soils was intermediate. The differences in diversity among the three communities may also reflect changes in abundances of a few OTUs. The CNH forest soil community may be still in a successional phase that is only partially stabilized after 40 years. Analysis of molecular variance also revealed that the bacterial community composition of HWD soils was significantly different from CLC and CNH soils (p = 0.001). These results suggest that the disturbance of forest conversion and tree species composition are important factors influencing the soil bacterial community among three forest ecosystems in the same climate.

Highly Diverse and Seasonally Dynamic Protist Community in a Pristine Peat Bog

Culture-independent molecular methods based on the amplification, cloning and sequencing of small-subunit ribosomal RNA genes (SSU rDNAs) are powerful tools to study the diversity of microorganisms. Despite so, the eukaryotic microbial diversity of many ecosystems, including peatlands has not yet received much attention. We analysed the eukaryotic diversity by molecular surveys in water from the centre of a pristineSphagnum-dominated peatland in the Jura Mountains of Switzerland during a complete seasonal cycle. The clone libraries constructed from five different temporal samplings revealed a high diversity of protists with representatives of all major eukaryotic phyla. In addition, four sequence types could not be assigned to any known high-level eukaryotic taxon but branched together with a rather good statistic support, raising the possibility of a novel, deep branching eukaryotic clade. The analysis of seasonal patterns of phylotypes showed a clear change in the eukaryotic communities between the warm period (late spring and summer) and the cold period (autumn and winter). Chrysophytes dominated the samples in the cold period while testate amoebae (Arcellinida and Euglyphida) and a few other groups peaked in summer. A few phylotypes (such as a cryptomonad and a perkinsid) were abundant at given sampling times and then almost disappeared, suggesting bloom-like dynamics.

Spatial Bacterial Diversity in a Recent Freshwater Tufa Deposit

16S ribosomal RNA gene clone libraries were generated to assess the prokaryotic diversity within a laminated tufa stromatolite core, obtained from the hardwater rivulet Westerhöfer Bach, Lower Saxony, Germany. Libraries were obtained from the biofilm and six tufa layers, each about 4 to 8 mm thick and 2,351 partial sequences were analyzed. Cyanobacteria and other aerobic organisms dominated the 500 μ m thick biofilm and the first layer, represented by 112 and 154 phylotypes, respectively. DGGE and UniFrac analyses revealed that these two layers were clearly separated from the deeper layers which showed a higher diversity than the tufa surface with less than 350 phylotypes. The species richness of deeper layers was dominated by a broad range of phyla, predominantly of acidobacterial, bacteroidetes and alpha-, beta- and gammaproteobacterial phylotypes. In total, sequences belonging to 26 phyla were detected. Sequences related to anaerobes, including photosynthetic Proteobacteria, are present in low numbers in the deeper layers but their metabolic activities in the dark and water-aerated core remain unclear.

Comparison of the Bacterial Community Structures of Ayu-narezushi Produced by Two Different Manufacturers

We analyzed and compared the bacterial communities in ayu-narezushi (narezushi made using the sweetfish) produced by two different manufacturers in Mie Prefecture, Japan, with the 16S ribosomal RNA gene clone library method. The bacterial diversity of ayu-narezushi 1 (Library 1) was higher than that of ayu-narezushi 2 (Library 2). The predominant lactic acid bacteria were Lactobacillus fuchuensis, Lactococcus piscium, and Leuconostoc gelidum in Library 1 and Lactobacillus sakei in Library 2, suggesting that these species play important roles in the fermentation of the 2 samples of ayu-narezushi. The results of LIBSHUFF indicated a significant difference between 2 libraries (P = 0.001).

Bacterial diversity and biogeography in deep-sea surface sediments of the South Atlantic Ocean

Microbial biogeographic patterns in the deep sea depend on the ability of microorganisms to disperse. One possible limitation to microbial dispersal may be the Walvis Ridge that separates the Antarctic Lower Circumpolar Deep Water from the North Atlantic Deep Water. We examined bacterial communities in three basins of the eastern South Atlantic Ocean to determine diversity and biogeography of bacterial communities in deep-sea surface sediments. The analysis of 16S ribosomal RNA (rRNA) gene clone libraries in each basin revealed a high diversity, representing 521 phylotypes with 98% identity in 1051 sequences. Phylotypes affiliated with Gammaproteobacteria, Deltaproteobacteria and Acidobacteria were present in all three basins. The distribution of these shared phylotypes seemed to be influenced neither by the Walvis Ridge nor by different deep water masses, suggesting a high dispersal capability, as also indicated by low distance-decay relationships. However, the total bacterial diversity showed significant differences between the basins, based on 16S rRNA gene sequences as well as on terminal restriction fragment length polymorphism fingerprints. Noticeably, both geographic distance and environmental heterogeneity influenced bacterial diversity at intermediate (10-3000 km) and large scales (>3000 km), indicating a complex interplay of local contemporary environmental effects and dispersal limitation.

Bacterial diversity in water injection systems of Brazilian offshore oil platforms

Biogenic souring and microbial-influenced corrosion is a common scenario in water-flooded petroleum reservoirs. Water injection systems are continuously treated to control bacterial contamination, but some bacteria that cause souring and corrosion can persist even after different treatments have been applied. Our aim was to increase our knowledge of the bacterial communities that persist in the water injection systems of three offshore oil platforms in Brazil. To achieve this goal, we used a culture-independent molecular approach (16S ribosomal RNA gene clone libraries) to analyze seawater samples that had been subjected to different treatments. Phylogenetic analyses revealed that the bacterial communities from the different platforms were taxonomically different. A predominance of bacterial clones affiliated with Gammaproteobacteria, mostly belonging to the genus Marinobacter (60.7%), were observed in the platform A samples. Clones from platform B were mainly related to the genera Colwellia (37.9%) and Achromobacter (24.6%), whereas clones obtained from platform C were all related to unclassified bacteria. Canonical correspondence analyses showed that different treatments such as chlorination, deoxygenation, and biocide addition did not significantly influence the bacterial diversity in the platforms studied. Our results demonstrated that the injection water used in secondary oil recovery procedures contained potentially hazardous bacteria, which may ultimately cause souring and corrosion.

Microbial diversity in ostrich ceca as revealed by 16S ribosomal RNA gene clone library and detection of novel Fibrobacter species

The ostrich ( Struthio camelus) is a herbivorous bird and although the hindgut is known as the site for fiber digestion, little is known about the microbial diversity in the ostrich hindgut. Our aim was to analyze the microbial diversity in ostrich ceca using a 16S ribosomal RNA gene (rDNA) clone library approach. A total of 310 clones were sequenced and phylogenetically analyzed and were classified into 110 operational taxonomy units (OTUs) based on a 98% similarity criterion. The similarity of the sequences ranged from 86 to 99% and 95 OTUs had less than 98% similarity to the sequences in the public databases. Coverage and the Shannon–Wiener index ( H′) of the library were 83.9% and 4.29, respectively. The sequences were assigned to the following 6 phyla: Firmicutes (50.9% of the total number of sequences), Bacteroidetes (39.4%), Fibrobacteres (6.5%), Euryarchaeota (1.9%), Spirochaetes (1.0%), and Verrucomicrobia (0.3%); approximately 90% of the sequences were affiliated with Firmicutes and Bacteroidetes. The only OTU of Fibrobacteres (OTU 107), had 93 and 90% similarity to Fibrobacter succinogenes and F. intestinalis, respectively, suggesting a new species of Fibrobacter in ostrich ceca. Clostridium coccoides and C. leptum formed major groups within the Firmicutes. There was no OTU with high similarity (≥98%) to the 16S rDNA of cultivated fibrolytic bacteria in our library. Although two OTUs were affiliated with Euryarchaeota, no sequence was affiliated with methanogenic Archaea. This study presents the very complex ostrich cecal microbial community, in which the majority of the bacterial species have not yet been cultivated.

Towards bioremediation of toxic unresolved complex mixtures of hydrocarbons: identification of bacteria capable of rapid degradation of alkyltetralins

Unresolved complex mixtures (UCMs) of aromatic hydrocarbons are widespread, but often overlooked, environmental contaminants. Since UCMs are generally rather resistant to bacterial degradation, bioremediation of UCM-contaminated sites by bacteria is a challenging goal. Branched chain alkyltetralins are amongst the individual classes of components of aromatic UCMs which have been identified in hydrocarbon-contaminated sediments and a number of synthetic alkyltetralins have proved toxic in laboratory studies. Thus, alkyltetralins should perhaps be amongst the targets for UCM bioremediation strategies. The slow degradation of several alkyltetralins by a microbial consortium has been reported previously; however, the bacteria involved remain unidentified and no single strain capable of alkyltetralin biodegradation has been isolated. The present project therefore aimed to enrich and identify bacterial consortia and single strains of bacteria from a naturally hydrocarbon-contaminated site (Whitley Bay, UK), which were capable of the degradation of two synthetic alkyltetralins (6-cyclohexyltetralin (CHT) and 1-(3’-methylbutyl)-7-cyclohexyltetralin (MBCHT)). Bacteria were enriched from sediment collected from Whitley Bay, UK by culturing with CHT and MBCHT for a period of 4 months. Biodegradation experiments were then established and degradation of model compounds monitored by gas chromatography–mass spectrometry. Internal standards allowed the generation of quantitative data. 16S rRNA gene clone libraries were constructed from individual enrichments to allow assessment of microbial community structure. Selective media containing MBCHT were used to isolate single bacterial strains. These strains were then tested in liquid culture for their ability to degrade MBCHT. The consortia obtained through enrichment culture were able to degrade 87% of CHT and 76% of MBCHT after only 46 days compared with abiotic controls. The 16S ribosomal RNA gene clone libraries of these bacteria were dominated by sequences of Rhodococcus spp. Using selective media, a strain of Rhodococcus was then isolated that was also able to biodegrade 63% of MBCHT in only 21 days. The present report describes the isolation of a single bacterial strain able to degrade the resistant MBCHT. Although significant losses of MBCHT were observed, putative metabolites were not detectable. Rhodococcus sp. have been reported previously to be able to biodegrade a range of hydrocarbon compounds. Due to their environmental persistence and toxicity, aromatic UCMs require bioremediation. The culturing and identification of such bacteria capable of rapid degradation of alkyltetralins may be an important step toward the development of bioremediation strategies for sites contaminated with toxic UCMs.

Molecular analysis of the root canal microbiota associated with endodontic treatment failures

The failure of endodontic treatment is usually caused by persistent/secondary intraradicular infections and Enterococcus faecalis has been considered to be the main pathogen involved. Nevertheless, the breadth of bacterial diversity involved with endodontic treatment failures remains to be consistently explored by culture-independent approaches. This study determined the intraradicular microbiota of root-canal-treated teeth with post-treatment apical periodontitis using 16S ribosomal RNA gene clone library analysis. Bacteria were present in all cases, confirming the infectious etiology of post-treatment disease. Seventy-four bacterial taxa belonging to six phyla were found in the nine cases investigated. Of these, 55% were identified as as-yet-uncultivated phylotypes, which also made up a significant proportion of the microbiota in many cases. Twenty-five new phylotypes were identified. Most teeth harbored a mixed consortium, with a mean number of 10 taxa per case. Only 11 taxa were found in more than one case, revealing a high interindividual variability in the composition of the microbiota. The current findings revealed new candidate endodontic pathogens, including as-yet-uncultivated bacteria and taxa other than E. faecalis, which may participate in the mixed infections associated with post-treatment apical periodontitis.

Diversity of the Bacterial Community Found in Sammanarezushi (Saury Narezushi Revealed by the 16S rRNA Gene Clone Library

Narezushi is one of Japanese traditional foods and is made by fermenting salted fish meat and cooked rice together. In this study, the microbial diversity of samma-narezushi (narezushi using saury, Cobolabis saira) was analyzed by the 16S ribosomal RNA gene (rDNA) clone library. Randomly selected 89 clones were sequenced and phylogenetically analyzed. The sequences were classified into 12 operational taxonomy units (OTUs) at the 97% identity threshold. Most of the clones (89%) were lactic acid bacteria (LAB) and included Lactobacillus sakei, Leuconostoc gelidum, Lactococcus lactis subsp. lactis and L. pisium. The most predominant clone was L. sakei at 72% of the clones. The second most abundant clone was L. gelidum. These results suggest that L. sakei mainly contributes to the lactic acid fermentation process, taste and flavor of samma-narezushi. The present study showed that the culture independent method is useful for the microbial diversity analysis of narezushi.

Predominance of β‐proteobacteria in summer melt pools on Arctic pack ice

The diversity and community structure of bacteria in melt pools on Arctic pack ice floes were dominated by bproteobacteria. Thirty‐five percent of the pure cultures isolated in 1997 from pack ice floes north of Svalbard and in the Fram Strait were from the β‐proteobacteria group. Within this group, there were only two phylotypes clustering within the widespread Beta I cluster, also known as the Comamonadaceae clade. One phylotype, most closely related to Aquaspirillum arcticum (96.0‐97.3% identical), was frequent among cultures isolated from 10 melt pools. A 16S ribosomal RNA (rRNA) gene clone library, constructed from a melt pool that was sampled 2 yr later in the Fram Strait, was also dominated by β‐proteobacteria, in particular the same recurrent isolate phylotype designated “MP‐BetaI”. Fluorescence in situ hybridization of 20 melt pools corroborated the cultivation and cloning data. b‐Proteobacteria were the most abundant bacterial group, constituting ~49% of the bacteria that were stained by 4'6‐diamidino‐2‐phenylindole (DAPI). α‐ and γ‐proteobacteria accounted for only 2% each, the Cytophaga‐Flavobacterium group accounted for 9%, and the Actinobacteria spp. accounted for 9%. Approximately 63% of the β‐proteobacterial fraction that was found in the melt pools was determined with a newly developed probe to be the recurrent β‐proteobacterial MP‐BetaI phylotypes, indicating that it is particularly adapted for success in this extreme environment.

Cloning of ribosomal RNA genes from an Indian isolate ofGiardia lamblia and the use of intergenic nontranscribing spacer regions in the differentiation ofGiardia from other enteric pathogens

The ribosomal RNA genes from an Indian isolate ofGiardia lamblia have been cloned and characterized with respect to size, composition and copy number. Southern blotting and rDNA cloning ofGiardia lamblia revealed that genes coding for ribosomal RNA (rRNA) are exceptionally small and are encoded within a 5.6 kb genome fragment repeat. The rDNA repeat unit of this isolate was found to be highly G-C rich like other human isolates and the physical map showed severalSmaI sites. There are 132 copies of the rDNA repeat unit per cell in a head to tail arrangement. Two fragments corresponding to intergenic (0.2 kb and 0.3 kb) region and one (0.8 kb) containing both an intergenic region and a small part of the small subunit ribosomal RNA (SS rRNA) have been identified within the rDNA. These were used in heterogeneity studies ofGiardia isolated from two geographic locations as well as in the analysis of cross reactivity with other enteric organisms. In Southern blots, all the three fragments were found to be highly specific for the differential diagnosis ofGiardia spp. from the other enteric pathogens. These findings should help in developing a sensitive and more specific method for the diagnosis of giardiasis over currently available techniques.

CHROMOPHYTE PLASTID 16S RIBOSOMAL RNA GENES FOUND IN A CLONE LIBRARY FROM ATLANTIC OCEAN SEAWATER

ABSTRACT In recent years, the cloning of ribosomal RNA genes from natural plankton communities has provided insight into the biodiversity of marine bacterioplankton. Small eukaryotic phytoplankton, like bacterioplankton, can be difficult to cultivate or identify routinely by morphological characteristics. We used bacteria‐specific 16S rRNA primers to amplify genes from picoplankton samples collected on 0.2–μm filters by filtration from a depth of 10 m in the pelagic region over the continental shelf off Cape Hatteras, North Carolina. Nucleic acid sequencing and probe hybridization revealed that chromophyte plastid genes comprised 25% of the genes in a library of 170 clones. The plastid genes belonged to two groups within the Chromophyta: the Prymnesiophyceae and the Bacillariophyceae. Comparisons revealed substantial diversity among the bacillariophyte gene sequences, but the species from which the genes originated could not be identified because few sequences from cultured bacillariophytes are available. The prymnesiophyte genes could not be identified either, although they were most similar (similarity = 0.94) to plastid genes from the coccolithophorid Emiliania huxleyi (Lohmann) Hay and Mohler strain PML92D. These results provide evidence of abundant chromophyte plastid 16S rRNA genes in the water over the continental shelf off Cape Hatteras. The results also suggest that plastid 16S rRNA genes may provide suitable genetic markers for studying phytoplankton biodiversity and biogeography.

Eosinophilic colitis associated with larvae of the pinworm Enterobius vermicularis

Various helmintic parasites, most of which are uncommon in economically developed countries, can cause abdominal pain and eosinophilic inflammation of the bowel. A homosexual man presented with severe abdominal pain and haemorrhagic colitis, eosinophilic inflammation of the ileum and colon, and numerous unidentifiable larval nematodes in diarrhoeal stool. His symptoms resolved with anthelmintic treatment alone. Using comparative morphology and molecular cloning of nematode ribosomal RNA genes, we identified the parasites as larvæ of the pinworm Enterobius vermicularis, which are rarely observed oe associated with disease. Occult enterobiasis is widely prevalent and may be a cause of unexplained eosinophilic enterocolitis. Lancet 1995; 346: 410–12

Cloning and Characterization of Ribosomal RNA Genes from Three Species of Haemonchus (Nematoda: Trichostrongyloidea) and Identification of PCR Primers for Rapid Differentiation

Genomic DNAs prepared from adult worms of Haemonchus contortus, Huemonchus placei, and Haemonchus similis were used to clone and map complete ribosomal RNA gene repeats from each species. The lengths of the repeating units were estimated at 7.2, 7.2, and 7.4 kb, respectively, with a second and distinct repeat 6.9 kb in length identified from H. contortus only. Restriction enzyme recognition sites within all ribosomal RNA genes were fully conserved except for the loss of a SalI site within the large subunit rRNA gene of the 6.9-kb H. contortus repeat where only minor differences were observed in the restriction enzyme recognition sequences within the external spacer DNAs. Sequence analysis of the subcloned small subunit ribosomal DNAs from each species demonstrated 100% conservation within the 1758-bp fragments with only limited sequence variability observed within the adjacent 5′ external-transcribed spacer. Enzymatic amplification of external spacer sequences using primers complementary and proximal to the 3′-end of the large subunit and the 5′-end of the small subunit rDNAs enabled rapid differentiation of individual worms of H. contortus from H. placei by utilizing the size variability within this region of the repeat.

Cloning and restriction analysis of ribosomal RNA genes from Mycobacterium smegmatis

A genomic library of Mycobacterium smegmatis DNA was constructed in phage EMBL3. A clone (λ HB85) containing rRNA genes was isolated using as probes, fragments of E. coli rRNA cistron B. This cloned DNA fragment was mapped by restriction analysis and was shown to contain one complete set of rRNA genes (rRNA A). The physical mapping of the second set of rRNA genes of M. smegmatis (rRNA B) was done by restriction analysis of total chromosomal DNA. The two sets of rRNA genes showed highly conserved restriction sites within the respective sets but not in the flanking regions. The two rRNA sets of genes are organised like in the other eubacteria in the order 16S-23S-5S.

Cloning and restriction analysis of ribosomal RNA genes from Mycobacterium smegmatis

A genomic library of Mycobacterium smegmatis DNA was constructed in phage EMBL3. A clone (gamma HB85) containing rRNA genes was isolated using as probes, fragments of E. coli rRNA cistron B. This cloned DNA fragment was mapped by restriction analysis and was shown to contain one complete set of rRNA genes (rRNA A). The physical mapping of the second set of rRNA genes of M. smegmatis (rRNA B) was done by restriction analysis of total chromosomal DNA. The two sets of rRNA genes showed highly conserved restriction sites within the respective sets but not in the flanking regions. The two rRNA sets of genes are organised like in the other eubacteria in the order 16S-23S-5S.