N-169. PCR-Independent Analysis of Active Microbial Communities

E. L. Brodie1, K. C. Goldfarb1, R. A. Daly2,1, J. Pett-Ridge3, N. H. Nguyen2, T. Z. DeSantis1, Y. M. Piceno1, S. Gross4, M. M. Blackwell4, G. L. Andersen1;
1Lawrence Berkeley Natl. Lab., Berkeley, CA, 2Univ. of California, Berkeley, CA, 3Lawrence Livermore Natl. Lab., Livermore, CA, 4Louisiana State Univ., Baton Rouge, LA.

Background: Molecular analyses of complex microbial communities have revolutionized our understanding of ecosystem processes. While community genomic approaches are providing PCR-independent analysis of genetic diversity within low-complexity populations, no method currently exists to monitor the active fraction of complex microbial populations without PCR amplification in a high-throughput and high-coverage manner. Here we present the integration of a direct donor-mediated rRNA labeling procedure with high-density phylogenetic microarray (PhyloChip) hybridization. Methods: Total RNA from a range of sample types (pure cultures, soils, arthropod intestinal contents) was extracted using a modified CTAB method and purified. Total RNA was fragmented, dephosphorylated and each fragment end-labeled with multiple biotin moieties using a donor molecule. Labeled RNA was hybridized to PhyloChip microarrays overnight and arrays were washed, stained and scanned. Organisms with probe-sets exhibiting 90% or greater positive probe pairs were considered present. Results: Following optimization of fragmentation, dephosphorylation, labeling and hybridization conditions, PhyloChip hybridization intensity demonstrated a linear relationship to RNA concentration, independent of organism hybridized. Tropical forest soils known to produce high quantities of methane were incubated with and without electron donor and a methanogenesis inhibitor. Inhibition of methane production corresponded to a decrease in the relative intensity of probe-sets for methanogenic archaea as hypothesized. Direct hybridization of RNA extracted from various gut regions of passalid beetles demonstrated that methanogenic archaea were most active in the anterior hindgut. Conclusions: Together these data demonstrate the potential to analyze the active fraction of complex microbial populations without the need for primers or PCR amplification. This approach may be applied to any microbial system without a priori knowledge of the community composition.