Mapping Selenium Respiration Pathways in Gram-negative Bacteria

Imagine for a moment, the conditions necessary to sustain life. What comes to mind? Water? Oxygen? Sunlight? Think again. Many of the world’s smallest organisms have evolved and adapted to live under extreme conditions where these basic building blocks are scarce or absent altogether.

Visiting MnDRIVE scholar Clive Butler is determined to crack the code in one such extremophile, the bacterium Thauera selenatis. Like many microorganisms, T. selenatis exhibits remarkable respiratory flexibility. In the absence of oxygen, it’s able to substitute compounds based upon nitrogen or sulfur in a process called anaerobic respiration, which is familiar to most of us as the “rotten egg” smell from the anaerobic respiration of sulfur.

More importantly, T. selenatis is part of a small class of organisms that can breathe selenium oxides; a nonmetal trace element required for some protein synthesis. This trait alone makes the organism worthy of attention, but the bug is also a remarkably efficient bio-machine able to secrete tiny selenium nanoparticles with unexpected physical and optical characteristics. Currently very expensive and highly toxic to produce, these nanoparticles are sought after in scientific research and advanced manufacturing.

To better understand how T. selenatis is able to accomplish this remarkable feat, Butler is spending his research sabbatical working in the lab of UMN microbiologist, Jeff Gralnick (Microbiology/BTI). Gralnick and Butler have known each other for several years and follow each other’s research. Butler, an associate professor at the University of Exeter, is a biochemist looking at protein structure and function who is also interested in exploring molecular genetics and the ability to clone and manipulate an organism’s genetic code. When the opportunity for a study leave arose, Butler approached Gralnick about a possible collaboration. Leveraging Gralnick’s expertise in molecular genetics, the pair set out to create knockout mutants in a related organism called Aeromonas hydorphila, also reported to respire selenate and iron, which could help shed light on the inner working of T. selenatis and some of the iron-reducers studied in the Gralnick lab.

Unlike the mutants in science-fiction movies, these genetic mutants are bacterial strains engineered to express or suppress specific genes and traits. They help scientists isolate biochemical processes to better understand their sequence and function.

“Getting an idea of those mechanisms will give us a leg up on how this organism lives and how it survives,” says Butler. Later this month when he returns to his lab in Exeter, Butler will use the techniques learned in the Gralnick lab to investigate the missing links in the selenium respiration cycle for T. selenatis.

Butler traces his interest in science to a childhood accident that damaged his right arm. The original injury, which occurred when Butler was seven, led to tissue damage from internal swelling and required extensive reconstructive surgery. Over the next ten years as Butler’s arm continued to grow, complications arose, and the process repeated itself.

“From seven to sixteen,” Butler explains, “I spent my life in and out of hospitals around doctors and medical professionals. I became quite interested in science, medicine, and biological processes in general. Why don’t these muscles work anymore? Why doesn’t the arm do what it should do? In school, science was one of the few things I was pretty good at. Sports or the trades were out of the question. In today’s society, we’re more accepting of limitations, but this happened in the seventies, so it tended shut some doors and open others.”

By the time he entered university, his interest had shifted to biochemistry.

“One of things the arm brought home is just how dependent we are on oxygen. At a human level, if we don’t have oxygen we suffocate and die, but oxygen is also necessary at a cellular level. One of the great things about bacteria is they are really flexible with how they use energy. They often find themselves in a situation where they are deprived of oxygen and have to find a way to conserve energy and live and grow. They have to adapt. That adaptability is intriguing from a personal perspective because the injury forced me to adapt and move on with my life.”

Right handed before the accident, Butler learned to use his left hand for most daily tasks. Today he considers himself a lefty. If someone throws a ball his way, he instinctively uses his now dominant left hand. With the encouragement of his father, an amateur painter, Butler began to draw as a way of building eye-hand coordination. He became a rather accomplished draftsman and continues to draw and paint in his spare time, though science remains his primary focus.

His interest in selenate respiration began about 15 years ago after the first strain of T. selenatis was isolated from selenium-contaminated drainage waters in California’s San Joaquin Valley, by the late Prof. Joan Macy. Like many organisms that thrive in extreme environments, T. selenatis evolved to take advantage of available resources. In this case, a soluble and potentially toxic selenium oxide known as selenate.

Because selenium is an essential micronutrient necessary for some enzyme and amino acid production, many micro-organisms have evolved selenium uptake mechanisms. However, few have evolved as T. selenatis, which uses selenium in the respiratory chain that converts organic nutrients into cellular energy.

An important micronutrient, selenium is a basic building block for the 21st amino acid, selenocysteine, which is found in nearly every life form on earth. It’s widely used in feed supplements and has important industrial applications in glass production and the manufacture of circuit boards, batteries, and solar cells. Some studies suggest it may even have anti-microbial effects, but despite its beneficial role, selenium is not entirely trouble-free. Selenium oxides are frequently a byproduct of mining and fossil fuel production. They are highly soluble and toxic in large quantities and represent a potential environmental risk. Soon after its discovery, scientist began looking at T. selenatis as a potential tool for bioremediation of selenate contamination in the water district surrounding the San Joaquin Valley.

Understanding the selenate reduction pathways could allow scientists to develop highly efficient microbial filters for use in bioremediation and the recovery of selenium from mining or industrial processes. Equally intriguing, however, is the potential to develop microbial factories or bio-machines capable of producing selenium nanoparticles perhaps combined with other materials like cadmium.

“In theory, using synthetic biology you could insert both transport and production proteins into an organism like E. coli to create a biological machine that would produce a range of nanomaterials,” explains Butler. “The advantage would be getting the organism to select the materials, process them, and spit them out, which means you don’t have to go through the downstream processes of breaking up the cell to extract material.”

Early on, Butler hypothesized that selenium was processed at the very heart of the cell in the cytoplasm, where it would be available both for cell respiration and protein synthesis. Over several years, Butler and his group at the University of Exeter observed and documented the two-step process whereby T. selenatis absorbs selenate (Se0₄^2-) from the surrounding environment and converts it to elemental selenium. The first phase, selenate reduction, takes place in the cell’s outer compartment, the periplasm, and was fairly common among selenate reducing bacteria. The second phase, in which the byproduct selenite (Se0₃^2-) was further reduced to elemental selenium, took place in the cytoplasm at the very heart of the cell.

“Most respiratory processes involving oxygen or nitrogen produce aqueous or gaseous end products, making elimination from the cell possible through diffusion,” Butler explains. “Organisms that produce non-soluble respiratory products must evolve a mechanism to avoid entombment of the waste product within the cell.”

Once inside the cell, Butler points out, it is typically very difficult to excrete elemental solids like selenium. The alternative is the slow accumulation of selenium within the cell wall, which would eventually overwhelm the cell and destroy the bacteria. This clearly wasn’t the case in T. selenatis.

When Butler’s group began tracking the accumulation of selenium within T. selenatis, they found that selenium deposits formed inside the cell in the early-growth phase, but shifted to the outside during later stages of cell growth. Under an electron microscope, they also found that the selenium had formed into distinct nanospheres that were not quite round and strikingly uniform in size of 130 nanometers (± 28nm). The particles were quite different from structures observed when mineral accumulation takes place outside of the cell where there is little or no regulation of size or shape.

The evidence suggested that selenium particles were formed inside the cell membrane. This behavior left Butler with an even more puzzling question. How was the cell able to consistently regulate particle size within a very narrow band observed in the lab? The answer, Butler discovered, lay in a novel protein called SefA.

SefA is part of a family of proteins, which Butler refers to as minerallins, that play a role in mineral formation. SefA has been found in other selenium respiring organisms. It has also been isolated from dental plaque and likely plays a role in both the formation and transport of mineral structures, though as Butler admits, this idea is still theoretical.

In T. selenatis, SefA appeared to play an important role in regulating both nanosphere assembly and particle size. A series of physiological experiments and biochemical assays linked SefA to the selenite reduction stage. SefA production rose when selenite levels where high and SefA even accompanied the export of selenium particles from the cell.

Butler’s confirmed his hunch about SefA through in vitro experiments. His team found that the were able to control selenium particle size by regulating the amount of SefA protein in solution. Evidence for SefA’s role in particle formation was further strengthened by synthetic biology experiments in which the research team inserted SefA producing genes into a strain of E. coli. The new strain behaved as expected, and produced nanoparticles similar to those found in T. selenatis. 

Exactly how SefA regulates particle size is still a mystery. Butler suspects that SefA may be coating the particles, and perhaps forming a protein cage with an optimal size of 130 nm. 

“We have done some experiments placing tags on the SefA protein to interfere with its function. If you put a tag on one end, you get much smaller particles. If you put a tag on both ends, there is no regulation at all. The protein itself is definitely controlling size, which is probably due to the way it assembles the particles. Unless we get down to the atomic structures, we won’t know for sure.”

Using atomic force microscopy, Butler’s team hopes to probe the surface and test its rigidity. If the particles are solid, it presents a very different set of challenges than if the particles are made of a sponge-like materials that could be compressed as they cross the cell wall.

Butler also hopes to use high-resolution optical microscopy to study single cells, and observe as they respire selenate to determine how particles leave the cell. They may be secreted randomly across the membrane, but he has also observed pearl like particles streaming off the cell wall, which suggests a single point of secretion. If this proves true, it raises new questions. “What controls the flow?” Butler asks. “How does it that happen in a microbe with no digestive track or organ-level development?” 

Back in Exeter, Butler will continue to create his knockout mutants, producing bacterial strains that selectively eliminate proteins he suspects are involved in selenium transport. With luck, he will identify the elusive proteins responsible for moving selenium nanoparticles across the cytoplasm and outer membrane without damaging the cell. Someday, researchers may use this knowledge to create the perfect bio-machine. Until then, Butler’s work continues to evolve as he studies the biochemical processes of one very small and highly adaptable creature.