Electric vehicles (EVs) use high-powered rechargeable batteries to store electrical energy that powers their motors. Most EV batteries use a specific mixture of metals to hold the battery’s charge, called nanoscale lithiated nickel manganese cobalt oxides, aka NMC materials. NMC materials can store a large amount of energy, making them ideal for batteries, but the metals themselves can be toxic in soil and water if the batteries are not properly recycled.
NMC batteries leave behind a specific combination of toxic metals, so researchers can study how bacteria respond to this waste when it enters the environment. By understanding how bacteria adapt to toxic metals, scientists hope to predict how new technologies like EV batteries will affect the environment.
In 2019, a team of researchers from the University of Minnesota reported that a common soil bacterium called Shewanella oneidensis can rapidly evolve resistance to NMC materials, but they didn’t know how. True beneficial gene evolution usually develops over thousands of generations, but Shewanella oneidensis adapted much more quickly. The team recently conducted a follow-up study to determine the genetic mechanisms behind this bacterium’s unique metal resistance.
They started by analyzing the complete DNA sequences of 33 Shewanella colonies that were resistant to either pure NMC waste or individual metals like nickel and cobalt, using a process called whole-genome sequencing. They expected to find DNA mutations in genes that control how bacteria take up and expel toxic metals that would give the resistant Shewanella an advantage in surviving NMC metal toxicity. However, they didn’t find any DNA mutations in metal-related genes. Instead, they found that a specific DNA region was duplicated 10 to 15 times in all 33 NMC-resistant or metal-resistant colonies. This result suggested to the researchers that a temporary adaptation, not true gene evolution, was responsible for the bacteria’s metal resistance.
The researchers wanted to know more about the duplicated DNA region in metal-resistant Shewanella, so they focused on 2 gene duplication mechanisms that might serve as temporary adaptations in Shewanella. The first mechanism directly duplicates DNA regions to create extra copies of a gene, via a process called gene duplication amplification. The second mechanism moves DNA segments, called mobile genetic elements, into different DNA sections to create extra copies of a gene.
To test whether either of these mechanisms was causing the gene duplication in Shewanella, the team grew the bacteria in different liquid media containing only cobalt, only nickel, or cobalt and nickel together. The researchers used a technique that measures the exact amount of DNA in a given sequence, called quantitative polymerase chain reaction (qPCR), to calculate how many duplicated DNA regions were in each genome sequence. They found that neither cobalt nor nickel alone triggered DNA duplication. But the bacteria grown in the media with cobalt and nickel together had 8 additional copies of the DNA region. This suggested that something about the combination of nickel and cobalt triggered gene duplication.
To determine whether this gene duplication only occurred with nickel and cobalt, the team substituted copper and zinc for nickel in the growth media. They exposed Shewanella cells to different combinations of these metals using a grid pattern in a 96-well plate, a technique called a checkerboard assay. Then they repeated the qPCR process to measure the duplicated DNA region. They found that no other combinations of metals triggered gene duplication, suggesting that nickel and cobalt are especially toxic together, and that the gene duplication responds to this combined toxicity.
Finally, the researchers identified the function of the duplicated DNA region. They found that one of the genes within the region turned on a special protein that pushes metal ions and other toxins out of the cell, called an efflux pump protein. The team hypothesized that this efflux pump protein was responsible for removing cobalt and nickel from the bacteria’s cells. To test this theory, they grew modified Shewanella cells without the efflux pump gene in media containing cobalt, nickel, or cobalt and nickel together. They found that the modified bacteria grew well in the nickel-only treatment, but didn’t grow in the cobalt-only or combined treatments, which indicated that the efflux pump protein is necessary for Shewanella to resist cobalt toxicity.
Based on their experiments, the team concluded that nickel and cobalt together trigger a gene duplication event in Shewanella oneidensis. This gene duplication event increases the number of efflux proteins in Shewanella that can expel cobalt and underlies its unique NMC resistance. They suggested that future researchers study temporary adaptations similar to gene duplication in bacteria to track how they respond to electronic waste and other new technologies.
