# Presentation: 627036

## Insights into Stress Ecology and Evolution of Microbial Communities from Uranium-Contaminated Groundwater Revealed by Metagenomics Analyses 

** ****INTRODUCTION**

**  ****Uranium – 51 mg/L (soil ~500 mg/kg)**
**  ****Nitrate – 2,331 mg/L**
**  ****Sulfate – 1997 mg/L**
**  ****Total Organic Carbon (TOC) – 244 mg/L**
**  ****Total Inorganic Carbon (TIC) – 284 mg/L**
**  ****pH – 3.7**
**  ****Heavy Metals and Organics – ****μ****g to mg/L**

- 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.

** ****~70 Mb raw sequence**
** ****~8 Mb assembled sequence**
** ****2770 contigs**
** ****224 scaffolds (1.8 kb-2.4 Mb)**
** ****5 preliminary phylotypes based on identification of anchor ****	****genes (16S rRNA, 23S rRNA, *****gyrB*****, *****recA*****, *****rpoB*****, *****ileS*****,  *****fusA*****)**
** ****~70 of metagenomic sequences assigned to these 5 phylotypes**
** ****Estimated Genome Coverage*******
** ****FRC Gamma Group I *****(Frateuria *****I) *****–***** 9.7X**
** ****FRC Beta Group I (*****Burkholderia*****) – 1.1X**
** ****FRC Gamma Group II (*****Frateuria *****II) - <0.1X**
** ****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**

**FW106**

**Insights into Stress Ecology and Evolution of Microbial Communities from Uranium-Contaminated Groundwater Revealed by Metagenomics Analyses **
-  **Christopher L. Hemme****1,2****, Terry J. Gentry****1,3****,  Liyou Wu****1,2****, Matthew W. Fields****4****, Chris Detter****5,6****, Kerrie Barry****5****, David Watson****1****, Nikos Kyrpides****5****, Paul Richardson****5****, Terry Hazen****7****, James Tiedje****8****, Eddy Rubin****5**** and Jizhong Zhou****1,2**** ****Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN****1****, Institute for Environmental Genomics, University of Oklahoma, Norman, OK****2****, **
**Department of Soil & Crop Sciences** **, Texas A&M University, College Station, TX****3****, Department of Microbiology, Montana State University, Bozeman, MT****4****, DOE Joint Genome Institute, Walnut Creek, CA****5****, Los Alamos National Laboratory, Los Alamos, NM****6****, Earth Science Division, Lawrence Berkeley National Laboratory, Berkeley, CA****7****, **
**Center for Microbial Ecology, Michigan State University, East Lansing, MI****8**

**Fig. 1.  Phylogeny of the predicted 16S rRNA sequences obtained from the assembled metagenomic sequence.  The phylogeny defines the four primary phylotypes of the community and was used for preliminary  binning of  the remaining the 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.**

** ****Evolution of Communities in Contaminated Systems**

**Fig. 3.  Genomic region of Contig 2766 (****γ****I) showing ****β****-proteobacterial metal resistance genes associated with mobilee elements.  Genes are colored as follows:  yellow, ****γ****-proteobacteria; green, ****β****-proteobacteria; violet, ****α****-proteobacteria; orange, ****δ****-proteobacteria; grey, actinobacteria; white, unassigned; red, mobile elements.**

**Fig. 4.  Amino acid phylogeny of CzcD Co****2+****/Zn****2+****/Cd****2+**** efflux protein.  FW106 sequences are outlined in red, all other colors are as described in Fig. 3.  The phylogeny suggests **

**Fig. 2.  Metabolic reconstruction of FW106 community.  Important stressors present at FW106 are shown in blue, likely carbon sources in green.  The presence of specific enzymes indicated by color: magenta, ****γ****I; yellow, ****β****I; white, unassigned; grey, not identified.  The community 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 were only found in ****β****I, 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 chemiosmotic gradient and B) multiple organic acid metabolism pathways designed to degrade protonated acids that permeate the cell under acidic conditions.**

** ****Metabolic Reconstruction of FW106 Community**

** ****FW106 Groundwater Geochemistry**

** ****Metagenome Statistics**

** ****FW106 Phylotypes**

** ****Evidence for Lateral Gene Transfer and Gene Duplication**

**  ****Introduction of high levels of multiple stressors has resulted in the irreversible loss of species and strain biodiversity at FW106**
**  ****Selective pressures imposed by stressors have resulted in rapid adaptations to general and specific stresses (metals, acidity, organic solvents, nitrate/nitrite)**
**  ****The community employs a heterotrophic, respiratory lifestyle coupling denitrification to metabolism of simple and complex carbohydrates permeating from soil**
**  ****The accumulation of metal and nitrite transporters via LGT and/or gene duplication provides a selective advantage to the cell by increasing the baseline rate of toxin efflux**
**  ****Sharp geochemical gradients at the FRC serve as *****de facto***** barriers to gene flow, resulting in the isolation of microbial communities and the eventual emergence of species adapted to specific local conditions**

**Future Directions**

**  ****Comparative metagenomics of FW106 to metagenome of the pristine FW301 community**
**  ****Quantitative analysis of geochemical resistance genes between two communities**
**  ****Genomic sequencing of FRC isolates to test hypotheses on LGT, gene duplication, etc.**
**  ****MLST surveys to test speciation hypotheses**