The ENIGMA Science Focus Area (SFA) is a multi-disciplinary research effort working to develop a deeper understanding of subsurface microbiology within the Y-12 Area of the Oak Ridge Reservation (ORR). This subsurface site is contaminated with high concentrations of multiple heavy metals, nitrate, and low pH. ENIGMA researchers are currently examining the influence of these environmental stressors on critical microbially-mediated processes within the ORR ecosystem. Due to the high concentrations of nitrate at the site, they are particularly interested in studying how these anthropogenic perturbations influence microbial transformations of nitrogen compounds such as nitrate, nitrite, and nitrous oxide. To accomplish these goals, ENIGMA is organized into three aims that reflect the different research scales (i.e., field level to molecular level) that ENIGMA projects iteratively cycle through. Within these aims, the development and implementation of emerging tools and techniques by ENIGMA researchers further facilitates the advancement of key scientific goals. The following series of recent studies conducted by ENIGMA researchers highlights this framework in action.
Field Scale Microbial Ecology
Researchers working within ENIGMA Aim 1 are characterizing the microbial ecology of the contaminated ORR subsurface by collecting data on both the microbial community and geochemistry (i.e, pH, nitrate levels, heavy metal levels) of the site. These studies aim to uncover how anthropogenic perturbations influence the native microbial community present at this site. In 2019, a high-resolution survey was performed within the highly contaminated “Area 3” of the ORR. This study, led by the lab of Terry Hazen (University of Tennessee and Oak Ridge National Lab), generated data on the hydrology and geology, geochemistry, and microbial communities present at the site. This work was published, in part, in the journals Environmental Microbiology and Frontiers in Environmental Science.
For these studies, researchers in the lab of Jizhong Zhou (University of Oklahoma) analyzed microbial communities within Area 3 by extracting microbial DNA from environmental samples. A technique called polymerase chain reaction (PCR) was used to target and make many copies of a specific region of this DNA known as the 16S gene. This gene serves as a molecular name tag, as researchers can identify each microbial species by its 16S sequence. This work revealed which organisms were present and how abundant they were in the survey samples. Working with Mike Adams at the University of Georgia, postdoctoral researcher Jennifer Goff further analyzed these microbial community data and noticed that the most abundant species in the Area 3 sediments was the bacterium Bacillus cereus. Additional comparisons of 16S gene sequences revealed that a bacterium isolated by the Adams lab from this site, B. cereus str. CPTF, was a representative of this highly abundant species. Several ENIGMA researchers, led by Goff, set out to further characterize B. cereus isolate in order to understand how it survives in this high-stress environment.
Genomic Characterization Advances Tool Development
Based on Aim 1 field observations, researchers within ENIGMA Aim 2 are working to isolate a collection of microorganisms reflective of the biodiversity found in the contaminated ORR subsurface. The B. cereus str. CPTF isolated by the Adams lab is one member of this larger collection known as the Environmental Atlas. Like a detailed collection of maps that might be found on the shelves of your local bookstore, ENIGMA’s Environmental Atlas is more than a list of the microorganisms found at the site. Once completed, it will include high-resolution microscopy images, growth data, and genomic data for each ORR isolate. For strain CPTF, Goff conducted the laboratory work to parameterize its growth within the context of the unique ORR geochemistry. This strain was found to be tolerant to the high concentrations of nitrate, nitrite, and various heavy metals that are found in the ORR subsurface. Strain CPTF also can respirate, or breathe, using nitrate in the absence of oxygen.
In order to determine the genetic basis for strain CPTF’s ability to survive in high heavy metal conditions, the genome had to first be sequenced and assembled. Lauren Lui, a research scientist in the lab of Adam Arkin (University of California, Berkeley, Lawrence Berkeley National Laboratory), and Torben Nielsen, a scientist at Lawrence Berkeley National Laboratory, used what is known as a third-generation sequencing technology to generate a high-quality genome for strain CPTF. The sequencing technology they used, called Nanopore sequencing, can generate reads hundreds of times longer than short-read sequencing methods. Similar to how having larger puzzle pieces makes it easier to complete a puzzle, having longer reads makes it easier to finish assembling genomes. Genomes assembled with only short-read sequencing are often still fragmented, and not completely circular (most bacterial genomes are circular, as compared to linear chromosomes like the human genome). Lui and Nielsen were able to completely finish the genome of strain CPTF.
The completion of the strain CPTF genome facilitated the discovery of the large number of mobile genetic elements harbored by this strain. Mobile genetic elements are pieces of DNA capable of moving within the genome of an organism or between the genome of two different organisms. This can introduce new genes and rearrange an organism’s genome, resulting in the emergence of new traits. Mobile genetic elements like plasmids, viruses, and transposable elements can be powerful drivers of rapid evolution under high stress conditions, like the ORR subsurface. The ability to take up and maintain a large number of mobile genetic elements may have contributed to the resilience of this dominant species at the site.
Omics-Facilitated Laboratory Simulations
ENIGMA Aim 3 researchers are investigating ecologically important microbial phenomena on a molecular level in the laboratory. Using strain CPTF, Goff set out to explore the impact of mixed metal exposure on cellular physiology and microbially-catalyzed nitrate removal. The ORR subsurface is contaminated both with nitrate and a mixture of heavy metals that includes Uranium, Aluminium, Cadmium, Cobalt, Nickel, Manganese, Iron, and Copper. While numerous sites like the ORR subsurface are contaminated with a mixture of heavy metals, historically researchers have only considered individual metal toxicity in bacteria. In collaboration with ENIGMA researchers Christopher Petzold and Yan Chen (Lawrence Berkeley National Lab), the growth and cellular response of strain CPTF to an ORR-inspired metal mixture (developed using geochemical data from the site) was compared to individual metal exposure.
Petzold and Chen used their high-throughput platform to examine the impacts of these exposures on the proteome (the entire set of proteins expressed by a cell) of strain CPTF. Manual proteomic sample preparation methods limit capacity and often lead to poor data quality at high sample loads. Petzold and Chen have developed step-by-step protocols to prepare samples for high-throughput proteomic analysis of bacterial cells. These protocols have been developed to facilitate rapid, low variance sample preparation of hundreds of samples and can be easily implemented on widely-available automated liquid handlers, enabling flexibility for future protocol development. ENIGMA researchers have also applied this protocol to mock mixtures of ENIGMA synthetic community samples to assess community composition using the metaproteomics. This method estimates the community structure by calculating the percentage of protein contribution from each member. Other Aim 3 researchers are now applying this ability to generate high-quality, high-throughput, reproducible metaproteomic data to their synthetic community analyses.
By combining this proteomics approach with other laboratory experiments, ENIGMA researchers discovered that metal mixture exposure disrupted cellular physiology to an extent that was greater than the summation of the individual metal exposures. Specifically, exposure to multiple metals disrupted iron homeostasis, causing CPTF cells to behave like they were starved for iron. This disturbance of iron metabolism inhibited the activity of two iron-utilizing enzymes key for microbial nitrate removal in the ORR subsurface. These findings, recently published in The ISME Journal and featured in a US Department of Energy Science Highlight. , underscored the need to consider complex mixture effects when studying heavy metal stress in microorganisms.