16S rDNA Clone Libraries Research Articles

Paratrechina longicornis ants in a tropical dry forest harbor specific Actinobacteria diversity.

The diversity of Actinobacteria associated with Paratrechina longicornis, an ant species that prefers a high protein diet, in a subtropical dry forest (Guánica, Puerto Rico) was determined by culture methods and by 16S rDNA clone libraries. The results of both methodologies were integrated to obtain a broader view of the diversity. Streptomyces, Actinomadura, Nocardia, Ornithinimicrobium, Tsukamurella, Brevibacterium, Saccharopolyspora, Nocardioides, Microbacterium, Leifsonia, Pseudonocardia, Corynebacterium, Geodermatophilus, Amycolatopsis, and Nonomuraea were found associated with the ants. The genera Streptomyces and Actinomadura were the most abundant. Also, the diversity of Actinobacteria associated with the soil surrounding the nest was determined using 16S rDNA clone libraries. In total, 27 genera of Actinobacteria were associated with the nest soils. A dominant genus was not observed in any of the soil samples. We compared statistically the Actinobacteria communities among P. longicornis nests and each nest with its surrounding soil using the clone libraries data. We established that the communities associated with the ants were consistent and significantly different from those found in the soil in which the ants live.

Evaluating the potential of 18S rDNA clone libraries to complement pyrosequencing data of marine protists with near full-length sequence information

Sequencing of 18S rDNA clone libraries and 454-pyrosequencing are valuable methods used to describe microbial diversity. The massively parallel 454-pyrosequencing generates vast amounts of ribosomal sequence data and has the potential to uncover more organisms, even rare species. However, the relatively short sequence lengths of ∼500 bp are suboptimal for taxonomic annotation and phylogenetic analyses. In this study, we assessed the potential of 18S ribosomal clone libraries to complement corresponding 454-pyrosequencing data with near full-length sequence information. This involved a comparison of protist community compositions in five polar samples suggested by 18S rDNA clone libraries, with the corresponding community compositions suggested by 454-pyrosequencing. The study was conducted with four Arctic water samples, focusing on the eukaryotic picoplankton (0.4–3 µm), and with one sample collected in the Southern Ocean, examining the entire size spectrum (> 0.4 µm). For all individual samples, the protist community compositions suggested by the two different approaches showed significant similarities. Around 70% of the sequences detected by sequencing of clone libraries were also present in the 454-pyrosequencing data set. However, the clone library sequences reflected only ∼20% of the abundant biosphere identified by 454-pyrosequencing and identified ribosomal sequences that were not detected in the 454-pyrosequencing data sets.

Phytoplankton Diversity and Community Composition along the Estuarine Gradient of a Temperate Macrotidal Ecosystem: Combined Morphological and Molecular Approaches

Microscopical and molecular analyses were used to investigate the diversity and spatial community structure of spring phytoplankton all along the estuarine gradient in a macrotidal ecosystem, the Baie des Veys (eastern English Channel). Taxa distribution at high tide in the water column appeared to be mainly driven by the tidal force which superimposed on the natural salinity gradient, resulting in a two-layer flow within the channel. Lowest taxa richness and abundance were found in the bay where Teleaulax-like cryptophytes dominated. A shift in species composition occurred towards the mouth of the river, with the diatom Asterionellopsis glacialis dramatically accumulating in the bottom waters of the upstream brackish reach. Small thalassiosiroid diatoms dominated the upper layer river community, where taxa richness was higher. Through the construction of partial 18S rDNA clone libraries, the microeukaryotic diversity was further explored for three samples selected along the surface salinity gradient (freshwater - brackish - marine). Clone libraries revealed a high diversity among heterotrophic and/or small-sized protists which were undetected by microscopy. Among them, a rich variety of Chrysophyceae and other lineages (e.g. novel marine stramenopiles) are reported here for the first time in this transition area. However, conventional microscopy remains more efficient in revealing the high diversity of phototrophic taxa, low in abundances but morphologically distinct, that is overlooked by the molecular approach. The differences between microscopical and molecular analyses and their limitations are discussed here, pointing out the complementarities of both approaches, for a thorough phytoplankton community description.

Molecular diversity reveals previously undetected air-dispersed protist colonists in a Mediterranean area

The molecular diversity of air-dispersed protists was examined through the 18S rRNA gene clone library construction in air samples and samples from experimental water containers passively collecting air-dispersed microorganisms, from July 2007 till October 2008 in three different sites of Northern Greece. The majority of the samplings took place in an urban industrialized coastal city (Thessaloniki). In all the samples, a total of 29 unique phylotypes were detected belonging to 10 known major taxonomic groups. The most abundant phylotypes were affiliated to known taxa of Ciliophora and Chlorophyceae, commonly found in various habitats. Additionally, various previously unnoticed and under-studied taxa, such as Bicosoecida, Oomycetes and Labyrinthulomycetes, were detected. These taxa are potentially important in ecological processes, through dispersal and colonization of various habitats. Multivariate statistical analysis associated the most abundant phylotypes with rainfall, suggesting that rain is a favorable means for reposition of air-dispersed protists. This is the first study investigating the molecular diversity of air-dispersed protists, including algae and heterotrophic protists.

Host-associated bacterial taxa from Chlorobi, Chloroflexi, GN02, Synergistetes, SR1, TM7, and WPS-2 Phyla/candidate divisions

Background and objectiveIn addition to the well-known phyla Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, Spirochaetes, Fusobacteria, Tenericutes, and Chylamydiae, the oral microbiomes of mammals contain species from the lesser-known phyla or candidate divisions, including Synergistetes, TM7, Chlorobi, Chloroflexi, GN02, SR1, and WPS-2. The objectives of this study were to create phyla-selective 16S rDNA PCR primer pairs, create selective 16S rDNA clone libraries, identify novel oral taxa, and update canine and human oral microbiome databases.Design16S rRNA gene sequences for members of the lesser-known phyla were downloaded from GenBank and Greengenes databases and aligned with sequences in our RNA databases. Primers with potential phylum level selectivity were designed heuristically with the goal of producing nearly full-length 16S rDNA amplicons. The specificity of primer pairs was examined by making clone libraries from PCR amplicons and determining phyla identity by BLASTN analysis.ResultsPhylum-selective primer pairs were identified that allowed construction of clone libraries with 96–100% specificity for each of the lesser-known phyla. From these clone libraries, seven human and two canine novel oral taxa were identified and added to their respective taxonomic databases. For each phylum, genome sequences closest to human oral taxa were identified and added to the Human Oral Microbiome Database to facilitate metagenomic, transcriptomic, and proteomic studies that involve tiling sequences to the most closely related taxon. While examining ribosomal operons in lesser-known phyla from single-cell genomes and metagenomes, we identified a novel rRNA operon order (23S-5S-16S) in three SR1 genomes and the splitting of the 23S rRNA gene by an I-CeuI-like homing endonuclease in a WPS-2 genome.ConclusionsThis study developed useful primer pairs for making phylum-selective 16S rRNA clone libraries. Phylum-specific libraries were shown to be useful for identifying previously unrecognized taxa in lesser-known phyla and would be useful for future environmental and host-associated studies.

Spatial distribution of bacterial community in EGSB reactor treating synthetic sulfate-containing wastewater at low organic loading rate

Given that the consumption of organic substances entails costly biodesulfurization, the characteristics of the bacterial community in a reactor should be determined to increase the desulfurizing rate under low organic loading condition. In this study, the bacterial community distribution in the expanded granular sludge bed reactor used to treat sulfate-containing wastewater with low organic loading rate was determined by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and 16S rDNA clone library analyses. DGGE results showed that the predominant bacteria were stable and accounted for similar to 90 % sulfate removal efficiency. Differences in band positions and intensities indicated that the distribution and abundance of bacteria were affected by their positions in the reactor. Typical bands were identified in the bacterial community comprising Desulfovibrio, Desulfomicrobium, Thiomonas, Acinetobacter, Bacteroidetes, and Chloroflexi. Their functions in the reactor were also discussed. The possible links between the functional and microbial responses were also investigated based on the characteristic and spatial distribution of each bacterium in consortium.

Microbial Ecology of the Hive and Pollination Landscape: Bacterial Associates from Floral Nectar, the Alimentary Tract and Stored Food of Honey Bees (Apis mellifera)

Nearly all eukaryotes are host to beneficial or benign bacteria in their gut lumen, either vertically inherited, or acquired from the environment. While bacteria core to the honey bee gut are becoming evident, the influence of the hive and pollination environment on honey bee microbial health is largely unexplored. Here we compare bacteria from floral nectar in the immediate pollination environment, different segments of the honey bee (Apis mellifera) alimentary tract, and food stored in the hive (honey and packed pollen or “beebread”). We used cultivation and sequencing to explore bacterial communities in all sample types, coupled with culture-independent analysis of beebread. We compare our results from the alimentary tract with both culture-dependent and culture-independent analyses from previous studies. Culturing the foregut (crop), midgut and hindgut with standard media produced many identical or highly similar 16S rDNA sequences found with 16S rDNA clone libraries and next generation sequencing of 16S rDNA amplicons. Despite extensive culturing with identical media, our results do not support the core crop bacterial community hypothesized by recent studies. We cultured a wide variety of bacterial strains from 6 of 7 phylogenetic groups considered core to the honey bee hindgut. Our results reveal that many bacteria prevalent in beebread and the crop are also found in floral nectar, suggesting frequent horizontal transmission. From beebread we uncovered a variety of bacterial phylotypes, including many possible pathogens and food spoilage organisms, and potentially beneficial bacteria including Lactobacillus kunkeei, Acetobacteraceae and many different groups of Actinobacteria. Contributions of these bacteria to colony health may include general hygiene, fungal and pathogen inhibition and beebread preservation. Our results are important for understanding the contribution to pollinator health of both environmentally vectored and core microbiota, and the identification of factors that may affect bacterial detection and transmission, colony food storage and disease susceptibility.

Composition and diversity analysis of the gut bacterial community of the Oriental armyworm, Mythimna separata, determined by culture-independent and culture-dependent techniques.

The intestinal bacteria community structure and diversity of the Oriental armyworm, Mythimna separata (Walker) (Lepidoptera: Noctuidae), was studied by analysis of a 16S rDNA clone library, denaturing gradient gel electrophoresis,and culture-dependent techniques. The 16S rDNA clone library revealed a bacterial community diversity comprising Cyanobacteria, Firmicutes, Actinobacteria, Gracilicutes and Proteobacteria, among which Escherichia coli (Migula) (Enterobacteriales: Enterobacteriaceae) was the dominant bacteria. The intestinal bacteria isolated by PCR-denaturing gradient gel electrophoresis were classified to Firmicutes, Proteobacteria, and Gracilicutes, and E. coli was again the dominant bacteria. The culture-dependent technique showed that the intestinal bacteria belonged to Firmicutes and Actinobacteria, and Staphylococcus was the dominant bacteria. The intestinal bacteria of M. separata were widely distributed among the groups Cyanobacteria, Firmicutes, Actinobacteria, Gracilicutes, Proteobacteria, and Gracilicutes. 16S rDNA clone library, denaturing gradient gel electrophoresis, and culture-dependent techniques should be integrated to obtain precise results in terms of the microbial community and its diversity.

Long-Term Rice and Green Manure Rotation Alters the Endophytic Bacterial Communities of the Rice Root

This study focuses on the effects of long-term rice rotated with milk vetch being as green manure on the composition of bacteria in rice roots. The endophytic bacterial communities in rice roots of the rice-rice-milk vetch (R-R-MV) and the rice-rice-winter fallow (R-R-WF) crop rotations with a 28-year research history were investigated using combined culture-dependent and culture-independent methods. It was found that the endophytic bacterial population in rice roots with the green manure was significantly higher than that of without it. There were 169 and 77 strains of endophytic bacteria that were isolated from rice roots of the R-R-MV and the R-R-WF, respectively. The 16S rRNA gene analysis shows that the 77 R-R-WF bacteria belong to 15 species of 14 genera while the other 169 R-R-MV bacteria belong to 21 species of 19 genera, in which Herbaspirillum and Cedecea were two mutually dominant populations and Burkholderia, Pseudomonas, Sphingomonas, and Pantoea accounted for large proportions of the endophytic bacteria in rice roots through R-R-MV rotation. The analysis of 16S rDNA clone libraries showed that the Shannon-Weaver diversity index of endophytic bacteria in R-R-MV approximates that in R-R-WF rotation, whereas the richness indexes of Chao 1 and ACE in R-R-MV rotation system were significantly higher than those in R-R-WF rotation. The diversity of endophytic bacteria was richer in R-R-MV. Both the culture-dependent and the culture-independent method revealed significant effect of long-term different tillage systems on the microbial community.

Organic loading rate and food-to-microorganism ratio shape prokaryotic diversity in a demo-scale up-flow anaerobic sludge blanket reactor treating domestic wastewater

We investigated the microbial community in an up-flow anaerobic sludge blanket (UASB) reactor treating domestic wastewater (DW) during two different periods of organic loading rate (OLR) and food-to-microorganism (F/M) ratio. 16S rDNA clone libraries were generated, and quantitative real-time PCR (qPCR) analyses were performed. Fluctuations in the OLR and F/M ratio affected the abundance and the composition of the UASB prokaryotic community, mainly at the species level, as well as the performance of the UASB reactor. The qPCR analysis suggested that there was a decrease in the bacterial cell number during the rainy season, when the OLR and F/M ratio were lower. However, the bacterial diversity was higher during this time, suggesting that the community degraded more diversified substrates. The diversity and the abundance of the archaeal community were higher when the F/M ratio was lower. Shifts in the methanogenic community composition might have influenced the route of methane production, with methane produced by acetotrophic methanogens (dry season), and by hydrogenotrophic, methylotrophic and acetotrophic methanogens (rainy season). This study revealed higher levels of bacterial diversity, metabolic specialization and chemical oxygen demand removal efficiency of the DW UASB reactor during the rainy season.

Comparison of the Norway lobster (Nephrops norvegicus) gut bacterial communities using 16S rDNA clone libraries and pyrosequencing

By comparing 16S rDNA cloning and 454 pyrosequencing in the Nephrops norvegicus midgut, several common bacterial OTUs were detected. However, when only one method is to be selected, it needs to be considered whether the revealing of rare OTUs or their accurate phylogenetic relationships is mostly preferred.

Bacterial Diversity in the High Arsenic Groundwater Systems of Jianghan Plain and Datong Basin by 16S rDNA Clone Library

To compare the difference bacterial diversity between Jianghan plain and Datong basin. The high arsenic sediment samples in this study were collected from different depths of borehole. The bacterial diversity in high arsenic aquifer sediments was studied by 16S rDNA clone library. The dominant bacterial community included Burkholderiales, Pseudomonadales, and Enterobacteriales in Jianghan plain, and Burkholderiales,Enterobacteriales and Actinomycetales in Datong basin.

Bacteria Community Analysis by Quantitative Real-Time PCR of Fermenting Wax Gourd and its Changes of Organic Acids

The bacterial communities and predominating species during the fermentation of wax gourd were investigated by using 16S rDNA clone library technology combined with a quantitative real-time polymerase chain reaction (qRT-PCR) method. At the beginning of wax gourd pickling, Acinetobacter spp. were the predominant bacteria (∼107 cells/mL). During the next 5–20 days of fermentation, the predominating bacteria were Weissella spp. (∼107–108 cells/mL). Lactobacillus spp. were a significant portion (∼106 cells/mL) of the bacterial flora only at day 10 of the fermentation. At days 15 and 20 of fermentation, W. paramesenteroides was the main species (∼107 cells/mL), identified by using qRT-PCR. Lactic, citric and acetic acids levels were low at the beginning of fermentation and increased significantly (P < 0.05). The increase in lactic acid to 0.33 g/L at day 20 was likely to have occurred because of the sudden increase in W. paramesenteroides and Weissella spp. 5 days earlier. Practical Applications The microorganisms play an important role in the processing and preservation of pickled vegetable foods. The predominant species and its organic acids concentration in the fermentation process of pickled wax gourd were investigated using the 16S rDNA clone library technology combined with quantitative real-time polymerase chain reaction. This method can provide quicker information about the bacterial communities involved in traditional wax gourd fermentation and data regarding flavor formation of pickled vegetables. The formation of organic acids and antimicrobial activity by specific types of lactic acid bacteria may facilitate linking the microbial ecosystem of pickled wax gourd to quality control. It can also provide the microbial and theory identification for quality control of pickled wax gourd processing.

Analysis of bacterial community and bacterial nutritional enzyme activity associated with the digestive tract of wild Chilean octopus (Octopus mimusGould, 1852)

Information on the bacterial community associated with octopus is very scarce, unlike fish and other molluscs. This study revealed the bacterial community associated with digestive tract of wild Chilean octopus Octopus mimus using a culture-dependent method and 16S rDNA clone library. Moreover, we analysed the bacterial nutritional enzyme activity of culturable bacteria. A culture-dependent method showed that the composition of the culturable bacterial community was substantially different between female and male octopus. The predominant species in female octopus were Vibrionaceae and Streptococcaceae, whereas only Vibrionaceae was dominated in male octopus. Bacterial nutritional enzyme activities of culturable bacteria from male octopus were much higher than female octopus. The 16S rDNA clone library analysis showed that the bacterial community of male octopus exhibited a higher diversity than that of female octopus. The genus Mycoplasma was the predominant bacteria in the digestive tract of all octopus samples. The results obtained in this study raise the possibility that each octopus has different food consumption due to different bacterial community and nutritional enzyme activity, although Mycoplasma sp. is one of the predominant bacteria in the digestive tract. Moreover, our results are useful for the future of microbiological investigation associated with the octopus and for probiotics in the octopus aquaculture.

Polyvinyl alcohol biodegradation under denitrifying conditions

Polyvinyl alcohol was biodegraded under denitrifying conditions with a microbial community originated from a municipal wastewater treatment plant. The derived microbial consortium was capable of polyvinyl alcohol degradation under both denitrifying and aerobic conditions. The community dynamics was monitored by temperature gradient gel electrophoresis, and a principal utilizing organism was identified and assigned as Steroidobacter sp. PD. The possible role of Steroidobacter sp. PD was also investigated by sequencing the 16S rDNA clone library prepared from the degrading community. qPCR analysis showed that the fraction of the microorganism in the community was very low initially (0.02%) and had reached to about 16% by the end of the biodegradation experiment. The study revealed that polyvinyl alcohol can be biodegraded in a water environment not only under aerobic but also under denitrifying conditions.

Molecular Genetic Diversity and Quantitation of Methanogen in Ruminal Fluid of Buffalo (Bubalus bubalis) Fed Ration (Wheat Straw and Concentrate Mixture Diet)

High roughage diet causes more methane emissions; however, the total methanogen abundance is not influenced by roughage proportion. Technologies to reduce methane emissions are lacking, and development of inhibitors and vaccines that mitigate rumen-derived methane by targeting methanogens relies on present knowledge of the methanogens. In this work, we have investigated molecular diversity of rumen methanogens of Surti buffalo. DNA from rumen fluid was extracted, and 16S rRNA encoding genes were amplified using methanogen specific primer to generate 16S rDNA clone libraries. Seventy-six clones were randomly selected and analysed by RFLP resulting in 21 operational taxonomic units (OTUs). BLAST analysis with available sequences in database revealed sequences of 13 OTUs (55 clones) showing similarity with Methanomicrobium sp, 3 OTUs (15 clones) with Methanobrevibacter sp. The remaining 5 OTUs (6 clones) belonged to uncultured archaea. The phylogenetic analysis indicated that methanogenic communities found in the library were clustered in the order of Methanomicrobiales (18 OTUs) and Methanobacteriales (3 OTUs). The population of Methanomicrobiales, Methanobacteriales, and Methanococcales were also observed, accounting for 1.94%, 0.72%, and 0.47% of total archaea, respectively.

Bacterial community structures associated with a natural spring phytoplankton bloom in the Yellow Sea, China

Bacterial community structures associated with a spring phytoplankton bloom were investigated using denaturing gradient gel electrophoresis (DGGE) and 16S rDNA clone libraries. Statistical and phylogenetic analyses applied on both molecular methods revealed differences in bacterial community composition between the bloom station and post-bloom station, as well as between two bloom stages (bloom- and decay-) at bloom station. At the class level, the bacterial community at the bloom station was dominated by Alphaproteobacteria, Flavobacteria and Gammaproteobacteria, whereas Alphaproteobacteria and Gammaproteobacteria were dominant at the post-bloom station. At order level, no obvious predominant subgroup was found at the post-bloom station. In contrast, predominant subgroups were observed in bloom samples and they changed over the course of bloom. Rhodobacterales (mainly Roseobacter) and Flavobacteriales (mainly Flavobacterium) were the predominant subgroups in the bloom period, whereas Roseobacter became the unique predominant subgroup in the decay-bloom period. Rhodobacterales and Flavobacteriales, which were dominant in the bloom-associated bacterial communities in the Yellow Sea, were also reported as dominant during bloom conditions in other ocean regions, suggesting that they play an important role in bloom events.

Rhizosphere bacterial communities associated with healthy and Heterodera glycines-infected soybean roots

Bacterial communities in rhizosphere soil of soybean plants that were healthy, infected with soybean cyst nematode (SCN, Heterodera glycines), and infected with SCN but treated with Purpureocillium lilacinus YES-2 were investigated with community-level physiological profile (Biolog) and 16S rDNA clone library analyses. Biolog data indicated significant differences in substrate utilization patterns of the rhizosphere bacterial communities associated with healthy, SCN-infected, and SCN-infected plus P. lilacinus-treated plants; among the three treatments, substrate richness and catabolic diversity were lowest in the rhizosphere of healthy soybeans. Analysis of 16S rDNA profiles placed the soybean rhizosphere bacteria into seven groups: Proteobacteria, Actinobacteria, Acidobacteria, Firmicutes, Bacteroidetes, Chloroflexi, and an unclassified bacterial clade. The percentages of Rhizobiales and Actinobacteria clones were greater in the rhizosphere of healthy plants than SCN-infected plants, while the opposite was true for the proportions of Bacteroidetes and Firmicutes clones. Addition of P. lilacinus did significantly affect the rhizosphere bacterial community of SCN-infected plants. These results suggest that rhizosphere bacterial community may play an important role in the changes of soybean rhizosphere biological conditions during the infection process. Further studies will identify more specific changes in the rhizosphere bacterial community during the establishment and progression of SCN disease, and relate these changes to potential effects on disease management, soybean health, and soybean productivity.

Characterization of bacterial communities in hybrid upflow anaerobic sludge blanket (UASB)–membrane bioreactor (MBR) process for berberine antibiotic wastewater treatment

Biodegradation of berberine antibiotic was investigated in upflow anaerobic sludge blanket (UASB)-membrane bioreactor (MBR) process. After 118days of operation, 99.0%, 98.0% and 98.0% overall removals of berberine, COD and NH4(+)-N were achieved, respectively. The detailed composition of the established bacterial communities was studied by using 16S rDNA clone library. Totally, 400 clones were retrieved and grouped into 186 operational taxonomic units (OTUs). UASB was dominated by Firmicutes and Bacteroidetes, while Proteobacteria, especially Alpha- and Beta-proteobacteria were prevalent in the MBRs. Clostridium, Eubacterium and Synergistes in the UASB, as well as Hydrogenophaga, Azoarcus, Sphingomonas, Stenotrophomonas, Shinella and Alcaligenes in the MBRs were identified as potential functional species in biodegradation of berberine and/or its metabolites. The bacterial community compositions in two MBRs were significantly discrepant. However, the identical functions of the functional species ensured the comparable pollutant removal performances in two bioreactors.

A novel lignin degradation bacterial consortium for efficient pulping

A lignin degradation bacterial consortium named LDC was screened from the sludge of a reeds pond by a restricted subculture. It could break down 60.9% lignin in reeds at 30°C under conditions of static culture within 15 days. In order to analyze the diversity of LDC, plate isolation, 16S rDNA clone library and ARDRA (Amplified Ribosomal DNA Restriction Analysis) were performed. Six bacterial strains were isolated from LDC and eighteen DNA phylotypes were identified from 230 bacterial analyzed clones. They were classified into Clostridiales(9.1%), Geovibrio thiophilus (5.1%), Desulfomicrobium (10.9%), Pseudomonas sp. (25.2%), Azoarcus sp. (5.1%), Thauera (5.1%), Paenibacillus sp. (5.1%), Cohnella sp. (2.2%), Acinetobacter sp. (3.1%), Microbacterium (7.8%), and uncultured bacterium (21.3%). In addition, physical characteristics of paper hand-sheets between biological pretreatment and chemical pretreatment were compared. The results showed that LDC had the capability of lignin degradation and was efficient for pulping, which would provide a new choice for biopulping.