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Insights into Stress Ecology and Evolution of Microbial Communities from Uranium-Contaminated
Groundwater Revealed by Metagenomics Analyses
Christopher L. Hemme1,2, Terry J. Gentry1,3, Liyou Wu1,2, Matthew W. Fields4, Chris Detter5,6, Kerrie Barry5, David Watson1, Nikos Kyrpides5, Paul Richardson5, Terry Hazen7, James Tiedje8, Eddy Rubin5 and Jizhong Zhou1,2
Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN1, Institute for Environmental Genomics, University of Oklahoma, Norman, OK2,
Department of Soil & Crop Sciences , Texas A&M University, College Station, TX3, Department of Microbiology, Montana State University, Bozeman, MT4, DOE Joint Genome Institute, Walnut Creek, CA5, Los Alamos National Laboratory, Los Alamos, NM6, Earth Science Division, Lawrence Berkeley National Laboratory, Berkeley, CA7,
Center for Microbial Ecology, Michigan State University, East Lansing, MI 8
http://vimss.lbl.gov/
INTRODUCTION Metabolic Reconstruction of FW106 Community Evolution of Communities in Contaminated Systems
Due to the uncultivated status of the majority of microorganisms in nature, little is known about their genetic properties, biochemical
functions, and metabolic characteristics. Although sequence determination of the microbial community ‘genome’ is now possible with
high throughput sequencing technology, the complexity and magnitude of most microbial communities make meaningful data
acquisition and interpretation difficult. Therefore, we are sequencing groundwater microbial communities with manageable diversity
and complexity (~10-400 phylotypes) at the U.S Department of Energy’s Environmental Remediation Sciences Program (ERSP) Field
Research Center (FRC), Oak Ridge, TN. The microbial community has been sequenced from a groundwater sample contaminated with
very high levels of nitrate, uranium and other heavy metals and pH ~3.7. Sequence analysis of this groundwater sample based on a 16S
rDNA library revealed 10 operational taxonomic units (OTUs) at the 99.6% cutoff with >90% of the OTUs represented by an
unidentified γ-proteobacterial species similar to Frateuria. Additional OTUs were related to a β-proteobacterial species of the genus
Azoarcus. Three clone libraries with different DNA fragment sizes (3, 8 and 40 kb) were constructed, and 50-60 Mb raw sequences were
obtained using a shotgun sequencing approach. The raw sequences were assembled into 2770 contigs totaling ~6 Mb which were
further assembled into 224 scaffolds (1.8 kb-2.4 Mb). Preliminary binning of the scaffolds suggest 4 primary groupings (2 Frateuria-
like γ-proteobacteria, 1 Burkholderia-like β-proteobacteria and 1 Herbaspirillum-like β-proteobacteria). Genes identified from the
sequences were consistent with the geochemistry of the site, including multiple nitrate reductase and metal resistance genes. A low
level of strain diversity was observed in the sample, with little significant polymorphism detected in the ORFs studied. We hypothesize
that the major adaptive response within the community following site contamination resulted from lateral gene transfer events within the
community followed by adaptive evolution of individual genetic elements. These adaptive events likely triggered multiple selective
sweeps within the populations that have reduced the strain heterogeneity of the community. A model is presented elucidating the
mechanisms of community adaptation to contaminated environments.
FW106
Fig. 5. Adaptation of a hypothetical FW106 bacteria to stressors via accumulation of Fig. 6. Proposed model for the evolution of microbial communities
toxin transporters. A) The ancestral state whereby toxin transporters are present in under stressed conditions. Introduction of stressors to a diverse
in single or low copy and which exhibit a given activity and specificity for one or community results in a rapid loss of species diversity. In the case of
more toxic molecules. Upon introduction of high levels of the toxin (e.g. heavy metal severe contamination, this loss could result in the transient isolation of
ions), additional transporter genes are introduced into the cell via lateral gene microcommunities and population bottlenecks which would in turn
transfer and/or gene duplication. B) The selection models posits that selective result in the loss of strain diversity within surviving species. As the
pressures will drive the diversification of duplicated transporters, resulting in surviving community begins to recover, nearly neutral mutations (via
descendent proteins showing changes in activity and/or specificity to the given toxins. single-point mutation, LGT, gene duplication, etc., indicated by red
Such modifications are expected to represent adaptations to specific geochemical dots/lines) will begin to accumulate. The cumulative effects of selective
conditions at a given site. C) The gene dosage model suggests that the accumulation sweeps and background selection will serve to further purge local
of transporters alone provides a general selective advantage to the cell by increasing genomic diversity at linked loci. In cases where the populations remain
FW106 Groundwater Geochemistry the baseline rate of toxin efflux. This model does not require modifications to
duplicate genes via mutation but may represent a transition to the selection model
over long evolutionary time frames. A positive selection screen of the FW106
isolated (e.g. geographical separation, strong geochemical gradients
limiting gene flow, etc.), the cumulative effect of multiple selective
sweeps may result in the emergence of new species specifically adapted
metagenome suggests that the gene duplication model best explains the accumulation to the prevalent conditions.
of metal efflux and nitrate/nitrite antiporters in γI over the short evolutionary time
• Uranium – 51 mg/L (soil ~500 mg/kg) frame (~50 yrs) considered.
• Nitrate – 2,331 mg/L
• Sulfate – 1997 mg/L
• Total Organic Carbon (TOC) – 244 mg/L Conclusions
• Total Inorganic Carbon (TIC) – 284 mg/L
• pH – 3.7
• Heavy Metals and Organics – μg to mg/L
• Introduction of high levels of multiple stressors has resulted in the irreversible
Fig. 2. Metabolic reconstruction of FW106 community. Important stressors present at FW106 are shown in blue, likely carbon sources in loss of species and strain biodiversity at FW106
green. The presence of specific enzymes indicated by color: magenta, γI; yellow, βI; white, unassigned; grey, not identified. The community • Selective pressures imposed by stressors have resulted in rapid adaptations to
Metagenome Statistics shows specific adaptations for degradation of 1,2-dichloroethene and a diverse number metal resistance mechanisms. Fermentation does not
appear to occur with the community instead based on respiration of nitrate (denitrification) or oxygen. Assimilatory nitrate reductase genes general and specific stresses (metals, acidity, organic solvents, nitrate/nitrite)
were only found in βI, suggesting that this bacteria may serve as a keystone species producing biological nitrogen for the rest of the
community. The most likely mechanism of acid resistance is a combination ofA) proton and cation transporters acting to maintain the • The community employs a heterotrophic, respiratory lifestyle coupling
• ~70 Mb raw sequence chemiosmotic gradient and B) multiple organic acid metabolism pathways designed to degrade protonated acids that permeate the cell under
denitrification to metabolism of simple and complex carbohydrates permeating
• ~8 Mb assembled sequence acidic conditions.
• 2770 contigs from soil
• 224 scaffolds (1.8 kb-2.4 Mb) • The accumulation of metal and nitrite transporters via LGT and/or gene
• 5 preliminary phylotypes based on identification of anchor genes (16S rRNA, 23S rRNA, gyrB,
duplication provides a selective advantage to the cell by increasing the baseline
recA, rpoB, ileS, fusA)
• ~70 of metagenomic sequences assigned to these 5 phylotypes
Evidence for Lateral Gene Transfer and Gene Duplication rate of toxin efflux
• Estimated Genome Coverage*
• FRC Gamma Group I (Frateuria I) – 9.7X
• Sharp geochemical gradients at the FRC serve as de facto barriers to gene flow,
• FRC Beta Group I (Burkholderia) – 1.1X resulting in the isolation of microbial communities and the eventual emergence of
• FRC Gamma Group II (Frateuria II) - <0.1X species adapted to specific local conditions
• FRC Beta Group II (Herbispirillum) - <0.1X
• FRC Alpha Group I (Afipia) - <0.1X
* Genome coverage estimated using reference genome sizes as follows: Burkholderia, 7.0 Mb; Herbispirillum, 6.8 Mb; Xanthomonas, 4.2 Mb; Bradyrhizobium, 9.1
Mb
Future Directions
FW106 Phylotypes • Comparative metagenomics of FW106 to metagenome of the pristine FW301
community
• Quantitative analysis of geochemical resistance genes between two communities
Fig. 1. Phylogeny of the predicted 16S rRNA
sequences obtained from the assembled
• Genomic sequencing of FRC isolates to test hypotheses on LGT, gene
metagenomic sequence. The phylogeny defines the duplication, etc.
four primary phylotypes of the community and was
used for preliminary binning of the remaining the • MLST surveys to test speciation hypotheses
contigs. Taxon labels are colored as follows: Red,
genes predicted from the metagenome (2 gene
fragments mapping to FRC Gamma Group I and
FRC Beta Group I are not shown); Blue and Green,
genes experimentally isolated from locations within
the FRC.
ACKNOWLEDGEMENT
ESPP is part of the Virtual Institute for Microbial Stress and Survival
supported by the U. S. Department of Energy, Office of Science, Office of
Biological and Environmental Research, Genomics:GTL Program through
Fig. 3. Genomic region of Contig 2766 (γI) showing β-proteobacterial metal resistance
genes associated with mobilee elements. Genes are colored as follows: yellow, γ-
contract DE-AC02-05CH11231 between Lawrence Berkeley National
proteobacteria; green, β-proteobacteria; violet, α-proteobacteria; orange, δ-
proteobacteria; grey, actinobacteria; white, unassigned; red, mobile elements. Laboratory and the U. S. Department of Energy.
Fig. 4. Amino acid phylogeny of CzcD Co2+/Zn2+/Cd2+ efflux
protein. FW106 sequences are outlined in red, all other colors
are as described in Fig. 3. The phylogeny suggests
