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Living on Thin Air

Prof Gregory Cook (left) and Dr Chris Greening (right)

Prof Gregory Cook (left) and Dr Chris Greening (right) have been investigating the role of hydrogen in the adaptation of myco­bacteria to starvation and hypoxia. Credit: Sharron Bennett

By Chris Greening

Soil bacteria can survive lengthy periods without food or water by metabolising hydrogen. How they do this has wider implications for understanding the biology of soils, the chemistry of the atmosphere and the development of artificial catalysts to harness hydrogen as a fuel source.

Mycobacteria are extraordinarily hardy bacteria: they can survive for great lengths of time in the absence of growth. Still the biggest bacterial killer, Mycobacterium tuberculosis resists drug treatment and evades immune detection by entering latent states. Also abundant in soils, mycobacteria such as M. smegmatis can withstand sudden downturns in their environment. Both organisms can cope with starvation and hypoxia for months or even years.

Despite much study, little is understood about how bacteria generate energy during persistence. Four years ago, Prof Gregory Cook’s laboratory at The University of Otago secured funding from the Royal Society of New Zealand to investigate the role of molecular hydrogen in the adaptation of myco­bacteria to starvation and hypoxia. Together with Dr Michael Berney and Honour’s student Kiel Hards, I investigated the enzymes that metabolise hydrogen in these organisms during the course of my PhD.

We initially studied mycobacterial hydrogen metabolism because we thought there would be scope to make medically significant findings in this area. After all, pioneering work from Prof Robert Maier of The University of Georgia showed that hydrogen oxidation was crucial to the pathogenesis of causative agents of stomach ulcers (Helicobacter pylori) and food poisoning (Salmonella enterica). We hypothesised that M. tuberculosis might use similar mechanisms to persist in human lung tissues during tuberculosis infections.

We discovered early in the project that hydrogen metabolism probably has no role in the pathogenesis of M. tuberculosis. But we pressed on, and eventually realised that researching the hydrogenases of soil mycobacteria could have environmental significance.

Four years later, the findings of our study have contributed to the general understanding of how soil bacteria survive starvation and hypoxia. Furthermore these findings, published in five primary papers, revealed surprising new facets about the processes that control the composition of the atmosphere. Our work might also have implications for building fuel cells.

Microbial Hydrogen Metabolism

All organisms require energy to grow and survive. In the case of humans and other higher beings, energy generation is a relatively inflexible process: we can only respire using organic carbon sources (e.g. sugars) as a fuel and oxygen as an oxidant. While fermentation can transiently occur when oxygen becomes limiting, the end-product (lactic acid) must be recycled.

Energy generation is a much more flexible and colourful process in the microbial world. There is great variety within and between bacteria in the methods they use to generate energy: gases, sulfur and metals serve as fuels; compounds such as nitrate enable respiration in the absence of oxygen; and fermentation can be sustained through the excretion of end-products. This flexibility enables bacteria to adapt to their niches and withstand environmental pressures.

Molecular hydrogen metabolism is a particularly interesting aspect of microbial energy generation. As the most fundamental chemical compound, hydrogen has several impressive properties: it yields more energy per unit weight than any other molecule, and it is also a highly diffusible gas. For these reasons, this molecule has been selected for a dual role in bacterial cells. Some bacteria can use hydrogen as a fuel to drive cellular processes (hydrogen consumption), while others excrete hydrogen through fermentation reactions when oxidant is unavailable for respiration (hydrogen production).

Hydrogenases are the catalysts that allow microbes to consume and produce hydrogen. First discovered by bacterial physiologist Marjory Stephenson in the 1930s, the unique properties of these enzymes continue to fascinate biologists, chemists, geologists and engineers alike. While hydrogen is a relatively simple molecule, cells require very complex enzymes to efficiently metabolise it. Hydrogenases are some of the most specialised and efficient enzymes on the planet: they are synthesised through complex pathways, employ crystal-like catalytic sites, and use sophisticated inorganic chemistry.

It has been hypothesised that hydrogen was the first fuel for the genesis of life. In the billions of years since, hydrogenases have been retained in most bacterial classes and have evolved to become more specialised. A single hydrogenase can break down up to 10,000 molecules of hydrogen per second and convert the energy released into biologically useful forms. In contrast, industrial processes continue to rely on expensive, easily-damaged platinum-based catalysts to activate hydrogen. We have many lessons to learn from nature about how to best activate this precious energy source.

Switching to an Environmental Focus

Since M. tuberculosis is notoriously difficult to work with, we initially focused on using the faster-growing, non-infectious bacterium M. smegmatis as a model to study mycobacterial hydrogen metabolism. Although M. smegmatis and M. tuberculosis occupy different niches, they are closely related and share many similarities in their metabolism. However, hydrogen seems to be an exception to this rule.

Genome comparisons revealed that none of the three enzymes in M. smegmatis were found in M. tuberculosis. This was initially disappointing as much of the work in our laboratory has depended on translating findings about the metabolism of the soil bacterium into its disease-causing relative.

But then I realised that the hydrogenases of M. smegmatis could have environmental implications. Studies led by Prof Ralf Conrad and Dr Philippe Constant of the Max-Planck Institute had revealed that the soil bacterium Streptomyces avermitilis was able to scavenge the extremely low concentrations of hydrogen found in the Earth’s atmosphere. Furthermore, they inferred that a unique type of hydrogenase was responsible for this high-affinity activity. Much to our excitement, this hydrogenase was also encoded in M. smegmatis.

In collaboration with Prof Conrad, I was able to show that M. smegmatis could also scavenge atmospheric hydrogen. We showed that this activity depended on two hydrogen-oxidising hydrogenases. This study, published in Proceedings of the National Academy of Sciences USA, showed for the first time that hydrogenases could have sufficiently high affinities to scavenge atmospheric hydrogen. This had several major biological and geological implications.

Resolving the Hydrogen Cycle’s Sink

Hydrogen is maintained at a constant trace concentration throughout the Earth’s atmosphere of 500 parts per billion. This concentration in turn influences the wider composition of the atmosphere, including the abundance of the greenhouse gases methane and nitrous oxide.

Analogous to the water cycle and carbon cycle, the global hydrogen cycle rapidly turns over hydrogen in the Earth’s atmosphere. This cycle is the result of biological, chemical and anthropogenic components. In general, few biological processes have a net influence on the hydrogen cycle, since most hydrogen produced is immediately recycled by bacteria. Instead, abiotic processes such as fossil fuel burning are responsible for the vast majority of net hydrogen released into the atmosphere.

Nevertheless, it has long been known that soil activity is responsible for net consumption of about 80% of global hydrogen from the Earth’s atmosphere. However, efforts to isolate the organisms responsible for atmospheric hydrogen oxidation have failed for several decades. The soil bacteria known to oxidise hydrogen were only capable of recycling the high concentrations of hydrogen produced by biological processes. Therefore, the joint discoveries of the Cook and Conrad laboratories finally resolved the organisms and enzymes serving as the main net sink in this cycle.

These findings open up some major questions for future work. First of all, how do mycobacterial hydrogenases manage to scavenge hydrogen at atmospheric concentrations as low as 500 parts per billion? Secondly, how do they achieve this without being inhibited by oxygen, which is traditionally a poison for hydrogenases? The properties of these enzymes might depend on unique catalytic chemistry and cellular interactions.

Gaining an understanding of these adaptations may have industrial implications because high-affinity oxygen-insensitive hydrogenases (or chemicals mimicking them) could be useful for hydrogen sensors and fuel cells. And while it remains a distant and uncertain possibility, there remains hope that hydrogen will one day overtake fossil fuels as a power source. Hydrogen has many advantages over traditional fossil fuels: it can be renewably produced, creates no harmful emissions, and has a higher energy content. However, much more primary research is needed to fully appreciated how best to store and harness this precarious gas.

Surviving Starvation and Hypoxia

As well as exploring the geological importance of hydrogen scavenging, we also wanted to understand the biological function of this process. When M. smegmatis is starved of energy, it switches from growth to survival modes. We have shown that expression of the genes encoding the two hydrogen-scavenging hydrogenases increases during this switch. While mutants lacking these enzymes grow normally, their long-term survival is half that of the natural bacterium.

Despite its low concentration, hydrogen is ubiquitous and unlimited in the Earth’s atmosphere – unlike practically all other fuel sources. Hence hydrogen represents a dependable fuel source for long-term survival. While trace levels of hydrogen are insufficient for cellular growth, it provides just enough energy for cells to remain energised when all other energy sources are exhausted.

Equally interesting are our collaborator’s findings that high-affinity hydrogenases are primarily expressed in the resilient spores of S. avermitilis. Could hydrogen scavenging be a widespread mechanism that enables soil bacteria to survive nutrient deprivation?

Hydrogen metabolism has an even more significant role in the adaptation of mycobacteria to hypoxia. Just like humans, mycobacteria require oxygen to grow. However, environmental and infectious species alike can still persist when oxygen becomes deprived. For instance, M. smegmatis will run out of oxygen when soils are waterlogged.

In a second publication in Proceedings of the National Academy of Sciences USA we showed that a combination of hydrogen production and recycling allows M. smegmatis to withstand hypoxia. Its three hydrogenases become active when oxygen is limiting. The hydrogenase expressed under this condition produces hydrogen; it allows M. smegmatis to be metabolically active when oxygen is unavailable.

However, the two high-affinity hydrogen-oxidising hydrogenases can still recycle this hydrogen when trace amounts of oxygen are reintroduced. Reflecting the importance of these processes, mutants unable to produce and recycle hydrogen during hypoxia had a 100-fold reduction in long-term surviva.

In this way M. smegmatis exploits the unusual properties of hydrogen for its long-term survival. By oxidising trace quantities of this high-energy fuel, the cell is able to stay energised during periods of starvation. And by producing hydrogen, the cell is also able to dissipate excess fuel as a diffusible gas during hypoxia.

Further studies of these phenomena may have wide-reaching consequences, from understanding how soil communities drive the composition of the atmosphere to appreciating the chemistry needed to produce efficient hydrogen fuel cells.

Chris Greening is a postdoctoral fellow at CSIRO's Biosecurity Flagship and The University of Otago’s Department of Microbiology and Immunology.