Dataset Information

A census of rRNA genes and linked genomic sequences within a soil metagenomic library.

ABSTRACT: We have analyzed the diversity of microbial genomes represented in a library of metagenomic DNA from soil. A total of 24,400 bacterial artificial chromosome (BAC) clones were screened for 16S rRNA genes. The sequences obtained from BAC clones were compared with a collection generated by direct PCR amplification and cloning of 16S rRNA genes from the same soil. The results indicated that the BAC library had substantially lower representation of bacteria among the Bacillus, alpha-Proteobacteria, and CFB groups; greater representation among the beta- and gamma-Proteobacteria, and OP10 divisions; and no rRNA genes from the domains Eukaryota and Archaea. In addition to rRNA genes recovered from the bacterial divisions Proteobacteria, Verrucomicrobia, Firmicutes, Cytophagales, and OP11, we identified many rRNA genes from the BAC library affiliated with the bacterial division Acidobacterium; all of these sequences were affiliated with subdivisions that lack cultured representatives. The complete sequence of one BAC clone derived from a member of the Acidobacterium division revealed a complete rRNA operon and 20 other open reading frames, including predicted gene products involved in cell division, cell cycling, folic acid biosynthesis, substrate metabolism, amino acid uptake, DNA repair, and transcriptional regulation. This study is the first step in using genomics to reveal the physiology of as-yet-uncultured members of the Acidobacterium division.

SUBMITTER: Liles MR

PROVIDER: S-EPMC154537 | biostudies-literature | 2003 May

REPOSITORIES: biostudies-literature

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A census of rRNA genes and linked genomic sequences within a soil metagenomic library.

Liles Mark R MR Manske Brian F BF Bintrim Scott B SB Handelsman Jo J Goodman Robert M RM

Applied and environmental microbiology 20030501 5

We have analyzed the diversity of microbial genomes represented in a library of metagenomic DNA from soil. A total of 24,400 bacterial artificial chromosome (BAC) clones were screened for 16S rRNA genes. The sequences obtained from BAC clones were compared with a collection generated by direct PCR amplification and cloning of 16S rRNA genes from the same soil. The results indicated that the BAC library had substantially lower representation of bacteria among the Bacillus, alpha-Proteobacteria, a ...[more]

PMID: 12732537

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Project description:Core histone hyperacetylation, in particular of H4, is concentrated in the promoter-upstream regions of active genes and in certain cases is locuswide. Antibodies to hyperacetylated H4 were used to immunoprecipitate dinucleosomal chromatin derived from K562 human erythroleukemic cells by micrococcal nuclease digestion. The extracted DNA was made into a genomic library and was expected to contain sequences from genes active in K562 cells (an active, 'aDNA' library). Clones (180) were randomly selected from the library; 24 of 103 tested (23%) contained highly repeated sequences, as determined by their hybridization to total genomic DNA, and were not analyzed further. An additional 10 clones (6%) were shown to contain no insert DNA. The remaining 146 were sequenced and compared with the nucleic acid databases and in all six frames to the protein databases: Sixeen clones could be assigned to known genes, the majority of which (12) were tissue specific. All but 2 of these 16 corresponded to segments 5' of the coding sequences, as expected if H4 acetylation is concentrated at promoter regions. Thirty-three clones (23%) displayed high sequence identity to cDNAs in the expressed sequence tag database (dbEST). Northern blots and reverse transcription (RT)-PCR were used to determine the proportion of clones representing sequences expressed in K562 cells: Although only 1 of 34 tested clones showed a band in Northern hybridization, RT-PCR demonstrated that at least 12 of 40 tested clones (30%) were present in the mRNA population. Because a further 8 of these 40 clones were identified as gene fragments by database sequence comparisons, it follows that about half of this subset of 40 clones is derived from genes. The aDNA library is thus very gene rich and not skewed toward the most highly expressed sequences, as in mRNA libraries. The aDNA library is also rich in promoters and could be a valuable source of such sequences, particularly those that lack CpG islands or other features that allow their specific selection.

Project description:To access the genetic and biochemical potential of soil microorganisms by culture-independent methods, a 24,546-member library in Escherichia coli with DNA extracted directly from soil had previously been constructed (M. R. Rondon, P. R. August, A. D. Bettermann, S. F. Brady, T. H. Grossman, M. R. Liles, K. A. Loiacono, B. A. Lynch, I. A. MacNeil, M. S. Osburne, J. Clardy, J. Handelsman, and R. M. Goodman, Appl. Environ. Microbiol. 66:2541-2547, 2000). Three clones, P57G4, P89C8, and P214D2, produced colonies with a dark brown melanin-like color. We fractionated the culture supernatant of P57G4 to identify the pigmented compound or compounds. Methanol extracts of the acid precipitate from the culture supernatant contained a red and an orange pigment. Structural analysis revealed that these were triaryl cations, designated turbomycin A and turbomycin B, respectively; both exhibited broad-spectrum antibiotic activity against gram-negative and gram-positive organisms. Mutagenesis, subcloning, and sequence analysis of the 25-kb insert in P57G4 demonstrated that a single open reading frame was necessary and sufficient to confer production of the brown, orange, and red pigments on E. coli; the predicted product of this sequence shares extensive sequence similarity with members of the 4-hydroxyphenylpyruvate dioxygenase (4HPPD) family of enzymes. Another member of the same family of genes, lly, which is required for production of the hemolytic pigment in Legionella pneumophila, also conferred production of turbomycin A and B on E. coli. We further demonstrated that turbomycin A and turbomycin B are produced from the interaction of indole, normally secreted by E. coli, with homogentisic acid synthesized by the 4HPPD gene products. The results demonstrate successful heterologous expression of DNA extracted directly from soil as a means to access previously uncharacterized small organic compounds, serving as an example of a chimeric pathway for the generation of novel chemical structures.

Dataset Information

A census of rRNA genes and linked genomic sequences within a soil metagenomic library.

Publications

A census of rRNA genes and linked genomic sequences within a soil metagenomic library.

Similar Datasets

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