Community Genomics-Enabled Study of a Low Complexity, Geochemically-Simple Acid Mine Drainage Ecosystem
Department of Environmental Science, Policy and Management, and Earth and Planetary Sciences, University of California, Berkeley, CA; and the DOE Joint Genome Institute, Walnut Creek, CA
Subsurface acid mine drainage (AMD) ecosystems are ideal models for the genome-enabled study of microbial ecology and evolution because they are physically isolated from other ecosystems and are relatively geochemically and biologically simple. We are using culture-independent genome sequencing of an AMD community from the Richmond mine, Iron Mountain, CA, to evaluate the extent and character of lateral gene transfer (LGT) within the community and to resolve microbial community function at the molecular level.
Microbial communities exist in several distinct habitats within the Richmond mine, including biofilms (subaqueous slime streamers and subaerial slimes) and cells attached directly to pyrite granules. All communities investigated to date by 16S rDNA clone libraries comprise only a handful of phylogenetically distinct organisms, typically dominated by the iron-oxidizing genera Leptospirillum and Ferroplasma. A Leptospirillum-dominated biofilm community was chosen for detailed analysis. 16S rDNA clone libraries and fluorescence in situ hybridization (FISH) using group-specific oligonucleotide probes indicated that the community is comprised of only 6 prokaryotic populations (see Figure; the size of colored circles indicates 16S rDNA divergence within populations).
We analyzed 7 Mbp of community genome sequence data from a 3 Kb shotgun library of the biofilm to estimate the community genome size. This analysis used an implementation of the Lander-Waterman equation that took into consideration species abundances determined from the data and by FISH. Results indicate a community genome size of approximately 19 Mbp, suggesting that each population is dominated by a single genome type. The conclusion was robust, even when assembly criteria were varied to improbable extremes and large uncertainties in population structure were included. However, more sequence data are needed to statistically validate the finding. Furthermore, the analysis is insensitive to genome types that occur in low abundance. One isolated member of the AMD biofilm community, Ferroplasma acidarmanus, has been previously sequenced by the JGI and could be used as an internal calibration for the community genome size estimate. We found that approximately 350,000 bp of the 7 Mbp community genome sequence data had ³98% sequence identity to the F. acidarmanus genome, i.e. an ca. 0.2X coverage of this 1.5 Mbp genome. Therefore, a 1X coverage of this genome would require 35 Mbp of community genome data, which can be viewed as a crude estimate of the community genome size. These results suggest that reassembly of the dominant genomes of AMD community members will be tractable with a modest sequencing effort. The apparent population homogeneity may arise due to the specific characteristics of the AMD habitat or may be a widespread phenomenon in microbial ecosystems.
Similarity searches of the initial community genome sequence data revealed many genes consistent with the chemoautotrophic lifestyle of the community, including CO2 fixation genes, nitrogen fixation genes and heme biosynthesis genes (heme is responsible for the pink color of the biofilm). None of these genes had close matches to genes from the F. acidarmanus genome and probably belong to Leptospirillum genomes. The most represented functional categories in the community genome data are DNA replication, recombination and repair (19%; due in part to a large number of transposases that belong to this group), amino acid transport and metabolism (12%) and energy production and conversion (11%). Organism-resolved metabolic pathway information will be used to develop methods to monitor microbial activity in the environment.
LGT is thought to play a crucial role in the ecology and evolution of prokaryotes. The extreme conditions (pH < 1.0, molar concentrations of iron sulfate and mM concentrations of arsenic, copper and zinc, and elevated temperatures of up to 50° C) largely isolate the AMD community from most potential gene donors. Naked DNA, phage and prokaryotes native to neutral pH habitats do not persist at pH <1.0, precluding influx of genes by transformation, transduction and conjugation, respectively. However, prophage have been recognized in the Ferroplasma genome sequence and acidophilic phage have been detected in the biofilm community. Phage may be important vectors for gene exchange. We have initiated a collaboration to sequence the phage community to assess their diversity and enhance our ability to detect prophage in the prokaryote community genome data.
Comparative genome analyses indicate that F. acidarmanus and the ancestor of two acidophilic Thermoplasma species belonging to the Euryarchaeota have traded many genes with phylogenetically remote acidophilic Sulfolobus species (Crenarchaeota). The putatively transferred sets of Sulfolobus genes in Ferroplasma and the Thermoplasma ancestor are distinct, suggesting independent LGT events between organisms living in the same, and adjacent habitats. In both cases, however, the majority of transferred genes are involved in metabolism, particularly energy production/conversion and amino acid transport/metabolism. The lack of genes transferred from the (sequenced) genomes of other prokaryotes is consistent with the hypothesis that extreme acidophiles have limited access to genes from organisms outside their ecotype. Interestingly, Sulfolobus, Ferroplasma and Thermoplasma are all bounded by a single tetraether-dominated membrane, which may facilitate conjugation. To date, no Sulfolobus species have been detected at Iron Mountain, suggesting two possibilities to explain the observed pattern of putatively transferred genes to Ferroplasma from Sulfolobus: 1) Sulfolobus is present at Iron Mountain but in regions currently inaccessible to sampling and/or 2) the transfers occurred prior to introduction of Ferroplasma into the current geological setting. Comparative analyses of the community genome data should improve the resolution of LGT in the community.
Ultimately, our goal is to develop an understanding of how acidophilic organisms evolved and function as communities to control acid mine drainage generation. The community genomics data are essential for this effort.