Researchers develop long-sought genetic system for unlocking mysteries of special deep-sea methanogen

A team of Virginia Tech researchers has broken new ground on early Earth metabolism and high temperature bio-catalysis research. They have developed a genetic system for Methanocaldococcus jannaschii, an ancient single-celled organism that belongs to a group called archaea and lives in deep-sea volcanoes or hydrothermal vents.

Their findings are published in the journal Frontiers in Microbiology on July 3.

“This is an important breakthrough in evolutionary biology research, bringing important tools to the field,” said Biswarup Mukhopadhyay, a researcher with the College of Agriculture and Life Sciences Department of Biochemistry, the lead for the study. “M. jannaschii performs a respiratory metabolism that is about 3.5 billion years old and lives in a habitat that mimics the environmental conditions of early Earth.”  

Since the beginning of the whole genome sequencing era, scientists have attempted to genetically manipulate this organism’s chromosome with the goal of learning the genetic basis for M. jannaschii’s unique capabilities. Mukhopadhyay’s team has now made this possible. As a result, scientists can manipulate the genome – add and remove genes – to learn more about what makes this deep-sea volcano dweller so resilient and what remnants of early metabolisms it harbors. This knowledge will have broad applications.

Because M. jannaschii produces methane, it is known as a methanogen. The organism grows in the absence of light and oxygen at temperatures nearly hot enough to boil water. The environmental conditions it faces inside hydrothermal vents, including toxic compounds in addition to scorching temperatures, are similar to those that existed billions of years ago. The methanogen survives in this inhospitable undersea pressure cooker by consuming hydrogen and carbon dioxide. 

The Mukhopadhyay laboratory’s breakthrough provides a new avenue for scientists to investigate how cellular metabolisms developed. Yet, while organism’s origin is scientifically enticing, there are more contemporary reasons for studying this ancient process. 

“Early methanogens were likely Lone Rangers, creating entire cells from simple salt and gasses such as hydrogen on their own,” said Mukhopadhyay. “However, their appetite for hydrogen helped them morph to become key members of numerous anaerobic microbial communities – communities with implications for humans and cows.”

During the human digestion process, hydrogen is a byproduct of the microbial degradation of food in the large intestine. Likewise, the gas is also produced when cows digest feed in their rumen. Continual removal of hydrogen during digestion is essential, since accumulation of the gas brings the degradation process to a halt.

“This is where methanogens go into action,” said Mukhopadhyay. “By consuming hydrogen, the organisms free our gut bacteria to continue digesting. This process enables both humans and cows to receive nutrients.”

When hydrogen production prohibits the digestion of foods, we receive fewer nutrients. Conversely, if hydrogen is removed too rapidly, the production of excessive nutrients can lead to obesity and diabetes.  

“For the cattle industry, the goal is to produce supplements that will optimize the methanogen-bacteria collaboration in the rumen towards high efficiency feed utilization,” said Mukhopadhyay. “This will enable more economical production of meat and dairy. For humans, similar supplements will prevent excessive nutrient production, helping to alleviate obesity and diabetes.”

On a larger scale, the same process also facilities municipal and industrial waste treatment, the recycling of plant material in freshwater sediment, and the production of natural gas, or methane, from renewable resources or agricultural waste. In each of these instances, anaerobic microbes degrade organic materials, and in the process, generate hydrogen. Unless this gas is removed, hydrogen will impede the degradation process. In these systems, improvement in methanogen-bacteria collaboration would be beneficial.

In addition, many of these collaborative microbial processes lead to the emission of methane, a potent greenhouse gas, to the atmosphere. Therefore, there is a major emphasis in learning the mechanisms of methanogenesis.

The Mukhopadhyay laboratory has another motivation for research on M. jannaschii that has to do with biocatalysis at high temperatures. Methane is an important fuel, as it is the principal component of natural gas. The mechanistic details for microbiological production of methane have been studied intensely for decades, but with organisms that grow at lower temperatures. This is a major gap, since the temperatures at which M. jannaschii grows could provide high reaction rates and efficiencies with few chances of unwanted microbial contamination.

To make advancements in these application areas, it is important to understand how methanogens developed the complex hydrogen consumption machineries to help microbes digest food and degrade complex organic matter. Greater knowledge of how the process of methane production from hydrogen and carbon dioxide began in the ancient M. jannaschii would provide an effective starting point. This has been another important motivation in manipulating the organism’s chromosome.

The team’s first major breakthrough took place in 2017. Dwi Susanti, a former research scientist in the researcher’s lab, and Mary Frazier, a former microbiology undergraduate student, and Mukhopadhyay succeeded in deleting a resistance gene from the genome of M. jannaschii, making it sensitive to a toxic material that the organism faces in volcanoes. They used a metabolic trick that allowed the delivery of foreign DNA into the organism. While M. jannaschii thrives at 85 ^oC, it can grow at 65 ^oC, albeit slowly. At such a reduced temperature, the organism does not build a complicated, protective membrane. The complicated membrane, which is found when the organism grows at higher temperatures, is believed to prohibit DNA transfer.

With this lead, the team has developed tools that now allow the generation of mutants of M. jannaschii for physiological studies and for easy purification of the organism’s proteins and protein complexes. This capability, and a reactor-based cultivation system that Mukhopadhyay and his colleagues developed previously, provide an opportunity to discover the metabolic mechanisms that have made M. jannaschii so resilient in the harsh conditions it faces in volcanoes. They also hope to better understand where today’s methanogenesis originated.

“M. jannaschii is one of the most important model organisms for the archaea,” said William Whitman, a renowned microbiologist and professor of microbiology at the University of Georgia. “So far, it has provided invaluable insights into ancient life and life at extreme temperatures. The development of the genetic system for this organism in the Mukhopadhyay laboratory enables further discoveries about this wonderful organism.”

This research was funded by the National Aeronautics and Space Administration’s Astrobiology: Exobiology and Evolutionary Biology program and the Virginia Tech Agricultural Experiment Station Hatch Program.