Genomes to Life Contractor-Grantee Workshop III
February 6-9, 2005, Washington, D.C.
Environmental Genomics
65
Whole Community Proteomics Study of an Acid Mine Drainage Biofilm Reveals Key Roles for “Hypothetical” Proteins in a Natural Microbial Biofilm
Jill Banfield*1 (jill@eps.berkeley.edu),Rachna J. Ram1, Gene W. Tyson1, Eric Allen1, Nathan VerBerkmoes2,3, Michael P. Thelen1, Brett J. Baker1, Manesh Shah3, Robert Hettich3, and Robert C. Blake II4
1University of California, Berkeley CA; 2University of Tennessee-Oak Ridge National Laboratory, Oak Ridge, TN; 3Oak Ridge National Laboratory, Oak Ridge, TN; and 4Xavier University of Louisiana, New Orleans, LA
We are studying relatively simple, low diversity microbial communities associated with extremely acidic, metal-rich mine drainage system to develop an understanding of adaptation, evolution, and the linkages between microbial activity and environmental geochemistry. In order to assess the genetic potential of an entire natural biofilm sample we partially reconstructed the genomes of five dominant organisms (Tyson et al. 2004). Comparative genomic analyses have revealed the structure of each population and provided insights into the processes that create and remove genome heterogeneity. The genomes contain large blocks dominated by genes that encode hypothetical proteins. These are regions inserted in one species or strain relative to others and are inferred to be of phage origin. Phage insertion appears to be the most rapid process leading to strain heterogeneity, and possibly diversification. Within strain populations, gene order is largely retained, and insertions or loss of single genes are rare events. Bacterial populations are dominated by a single clonal type. Homologous recombination is a key force shaping the genomes of archaeal populations in the system, but is rare between different species.
We have characterized the protein complement of a natural microbial community similar to that studied genomically in order to determine which genes are expressed and functionally important. By combining mass spectrometry-based “shotgun” proteomics with community genomics we confidently identified at least 1700 proteins from five dominant species. Proteins involved in protein refolding and response to oxidative stress were abundant, indicating that damage to biomolecules is a key challenge for survival. We validated more than 400 hypothetical proteins, a small subset of which are encoded within blocks of genes apparently acquired by lateral transfer. Entire operons encoding expressed, novel, lineage-specific proteins may be important for acid, metal and radical tolerance. 26% of the detected Leptospirillum group II proteins were hypothetical. An extracellular fraction was dominated by a novel protein shown to be a cytochrome central to iron oxidation and AMD formation. Sequencing of DNA encoding cytochrome regions for which peptides were not recovered revealed two amino acid substitutions. Using the strain variant sequence, 100% peptide coverage of the mature protein was achieved. Thus, an iterative genomic and proteomic approach analyses enable detailed in situ analyses of activity within natural microbial consortia.
* Presenting author
