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Erick Cardenas,James R. Cole,James M. Tiedje,Joon Hong Park 대한환경공학회 2009 Environmental Engineering Research Vol.14 No.1
Microorganisms play an important role in the geochemical cycles, industry, environmental cleanup, and biotechnology among other fields. Given the high microbial diversity, identification of the microorganism is essential in understanding and managing the processes. One of the most popular and powerful method for microbial identification is comparative 16S rRNA gene analysis. Due to the highly conserved nature of this essential gene, sequencing and later comparison of it against known rRNA databases can provide assignment of the bacteria into the taxonomy, and identity of its closest relatives. Isolation and sequencing of 16S rRNA genes directly from natural environments (either from DNA or RNA) can also be used to study the structure of the whole microbial community. Nowadays, novel sequencing technologies with massive outputs are giving researchers worldwide the chance to study the microbial world with a depth that was previously too expensive to achieve. In this article we describe commonly used research approaches for the study of individual microorganisms and microbial communities using the tools provided by Ribosomal Database Project website.
Sul, Woo Jun,Cole, James R,Jesus, Ederson da C,Wang, Qiong,Farris, Ryan J,Fish, Jordan A,Tiedje, James M National Academy of Sciences 2011 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF Vol.108 No.35
<P>High-throughput sequencing of 16S rRNA genes has increased our understanding of microbial community structure, but now even higher-throughput methods to the Illumina scale allow the creation of much larger datasets with more samples and orders-of-magnitude more sequences that swamp current analytic methods. We developed a method capable of handling these larger datasets on the basis of assignment of sequences into an existing taxonomy using a supervised learning approach (taxonomy-supervised analysis). We compared this method with a commonly used clustering approach based on sequence similarity (taxonomy-unsupervised analysis). We sampled 211 different bacterial communities from various habitats and obtained 1.3 million 16S rRNA sequences spanning the V4 hypervariable region by pyrosequencing. Both methodologies gave similar ecological conclusions in that β-diversity measures calculated by using these two types of matrices were significantly correlated to each other, as were the ordination configurations and hierarchical clustering dendrograms. In addition, our taxonomy-supervised analyses were also highly correlated with phylogenetic methods, such as UniFrac. The taxonomy-supervised analysis has the advantages that it is not limited by the exhaustive computation required for the alignment and clustering necessary for the taxonomy-unsupervised analysis, is more tolerant of sequencing errors, and allows comparisons when sequences are from different regions of the 16S rRNA gene. With the tremendous expansion in 16S rRNA data acquisition underway, the taxonomy-supervised approach offers the potential to provide more rapid and extensive community comparisons across habitats and samples.</P>
Park, Joonhong,Congeevaram, Shankar,Ki, Dong-Won,Tiedje, James M. The Korean Society of Toxicogenomics and Toxicopro 2006 Molecular & cellular toxicology Vol.2 No.1
In this study we attempted to develop a novel genomic method to selectively isolate target functional microbial genomes from environmental samples. For this purpose, stable isotope probing (SIP) was applied in selectively isolating organic pollutant-assimilating populations. When soil microbes were fed with $^{13}C-labeled $ biphenyl, biphenyl-utilizing cells were incorporated with the heavy carbon isotope. The heavy DNA portion was successfully separated by CsCl equilibrium density gradient. And the diversity in the heavy DNA was sufficiently reduced, being suitable for the current DNA microarray techniques to detect biphenyl-utilizing populations in the soil. In addition, we proposed a new way to get more genetic information by combining this SIP method with selective metagenomic approach. The increased selective power of these new DNA isolation methods will be expected to provide a good quality of new genetic information, which, in turn, will result in development of a variety of biomarkers that may be used in assessing ecotoxicology issues including the impacts of organic hazards, and antibiotic-resistant pathogens on human and ecological systems.
Sul, Woo Jun,Park, Joonhong,Quensen III, John F.,Rodrigues, Jorge L. M.,Seliger, Laurie,Tsoi, Tamara V.,Zylstra, Gerben J.,Tiedje, James M. American Society for Microbiology 2009 Applied and environmental microbiology Vol.75 No.17
<B>ABSTRACT</B><P>Stable isotope probing with [<SUP>13</SUP>C]biphenyl was used to explore the genetic properties of indigenous bacteria able to grow on biphenyl in PCB-contaminated River Raisin sediment. A bacterial 16S rRNA gene clone library generated from [<SUP>13</SUP>C]DNA after a 14-day incubation with [<SUP>13</SUP>C]biphenyl revealed the dominant organisms to be members of the genera <I>Achromobacter</I> and <I>Pseudomonas</I>. A library built from PCR amplification of genes for aromatic-ring-hydroxylating dioxygenases from the [<SUP>13</SUP>C]DNA fraction revealed two sequence groups similar to <I>bphA</I> (encoding biphenyl dioxygenase) of <I>Comamonas testosteroni</I> strain B-356 and of <I>Rhodococcus</I> sp. RHA1. A library of 1,568 cosmid clones was produced from the [<SUP>13</SUP>C]DNA fraction. A 31.8-kb cosmid clone, detected by aromatic dioxygenase primers, contained genes of biphenyl dioxygenase subunits <I>bphAE</I>, while the rest of the clone's sequence was similar to that of an unknown member of the <I>Gammaproteobacteria</I>. A discrepancy in G+C content near the <I>bphAE</I> genes implies their recent acquisition, possibly by horizontal transfer. The biphenyl dioxygenase from the cosmid clone oxidized biphenyl and unsubstituted and <I>para</I>-only-substituted rings of polychlorinated biphenyl (PCB) congeners. A DNA-stable isotope probing-based cosmid library enabled the retrieval of functional genes from an uncultivated organism capable of PCB metabolism and suggest dispersed dioxygenase gene organization in nature.</P>
Novel biphenyl-oxidizing bacteria and dioxygenase genes from a korean tidal mudflat.
Lee, Tae Kwon,Lee, Jaejin,Sul, Woo Jun,Iwai, Shoko,Chai, Benli,Tiedje, James M,Park, Joonhong American Society for Microbiology 2011 Applied and environmental microbiology Vol.77 No.11
<P>Gene-targeted FLX titanium pyrosequencing integrated with stable isotope probing (SIP) using [(13)C]biphenyl substrate revealed that tidal mudflat sediments harbor novel aromatic ring hydroxylating dioxygenases (ARHD). More than 80% of the detected ARHD genes comprise four clades (0.5 distance) with 49 to 70% amino acid identity to sequences in public databases. The 16S rRNA sequences enriched in the (13)C fraction were from the Betaproteobacteria, bacilli (primarily Paenibacillus-like), and unclassified phyla.</P>