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Link to original content: http://www.ncbi.nlm.nih.gov/pubmed/16000831
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. 2005 Jul;71(7):4121-6.
doi: 10.1128/AEM.71.7.4121-4126.2005.

Analysis of microbial gene transcripts in environmental samples

Rachel S Poretsky  1 , Nasreen Bano, Alison Buchan, Gary LeCleir, Jutta Kleikemper, Maria Pickering, Whitney M Pate, Mary Ann Moran, James T Hollibaugh
Affiliations

Analysis of microbial gene transcripts in environmental samples

Rachel S Poretsky et al. Appl Environ Microbiol. 2005 Jul.

Abstract

We analyzed gene expression in marine and freshwater bacterioplankton communities by the direct retrieval and analysis of microbial transcripts. Environmental mRNA, obtained from total RNA by subtractive hybridization of rRNA, was reverse transcribed, amplified with random primers, and cloned. Approximately 400 clones were analyzed, of which approximately 80% were unambiguously mRNA derived. mRNAs appeared to be from diverse taxonomic groups, including both Bacteria (mainly alpha- and gamma-Proteobacteria) and Archaea (mainly Euryarchaeota). Many transcripts could be linked to environmentally important processes such as sulfur oxidation (soxA), assimilation of C1 compounds (fdh1B), and acquisition of nitrogen via polyamine degradation (aphA). Environmental transcriptomics is a means of exploring functional gene expression within natural microbial communities without bias toward known sequences, and provides a new approach for obtaining community-specific variants of key functional genes.

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Figures

FIG. 1.
FIG. 1.
Taxonomic assignment of SIMO mRNA (A) and bacterial 16S rRNA (B) sequences from the 0.2- to 3.0-μm-size fraction of SIMO bacterioplankton. Archaea, viruses, and fungi were not captured by the 16S rRNA primers used in the rRNA library. No putative taxonomic assignment could be made for 37% of the mRNA clones because they had highest similarity to genes from unclassified organisms. Twenty-three percent of the mRNA clones did not match any database sequence well using a BLASTX E-value cutoff of e−10 and were not included in this figure. Approximately 17% of the rRNA clones could not be readily assigned to a phylum using a cutoff value of 80% similarity to described organisms, while 20% could be classified as Proteobacteria but not assigned a class within this phylum.
FIG. 2.
FIG. 2.
Phylogenetic tree of SIMO-specific soxA sequences and those from representative cultured organisms constructed using the neighbor-joining method of the PHYLIP package (4). The tree is based on the deduced amino acids encoded by the soxA transcripts or genes (positions 8 to 247; Chlorobium tepidum numbering system) and is unrooted, with soxA from Aquifex aeolicus (AE000757) as the outgroup. Bootstrap values of ≥50% are indicated at branch nodes. The scale bar indicates Dayhoff percent accepted mutation (PAM) distance.
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