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Microbe Genes Could Curb Livestock Burps

Ruminant methane alone accounts for 31% of New Zealand's greenhouse gas emissions.

Ruminant methane alone accounts for 31% of New Zealand's greenhouse gas emissions.

By Graeme Attwood

The DNA sequence of a microbe that produces methane in ruminants provides a target for vaccines and other drugs to reduce greenhouse gas emissions from livestock.

Since their first domestication about 10,000 years ago, cattle, sheep, deer and goats have provided meat, milk and fibre for human use. Products derived from these ruminants are more commonly used than most people realise, with proteins derived from ruminants found in thousands of items ranging from sports drinks and processed foods to products used in oriental remedies.

However, the multi-chambered stomachs that ruminants have evolved to digest plant material produce large amounts of methane – a greenhouse gas that is an important agent of climate change. Our research is focused on understanding the microbes in the rumen that produce methane by sequencing the genomes of the most significant species of these “methanogens” in order to identify common features that can be targeted to knock-out methanogen activity. We have recently published the genome sequence of Methanobrevibacter ruminantium (Fig. 1), which is one of the main methanogens found in the rumen, and have begun to identify genes as targets for methane mitigation technologies.

Ruminant Digestion
Ruminants evolved around 54 million years ago, and their subsequent success as a group is based on their unique digestive systems. The rumen is adapted to hold large amounts of plant material and to digest it via the action of symbiotic microbes that live there. The microbes produce the enzymes needed for the breakdown of plant material, and grow on the sugars that are released. They produce fermentation products, most of which are absorbed and used by the ruminant for energy and growth.

However, some of these fermentation products, such as hydrogen, cannot be used by the ruminant and are further metabolised to form methane. Methane formation is carried out by a specialised group of microbes called methanogens . The scavenging of hydrogen by methanogens is an important function as it prevents its accumulation and keeps the normal digestive processes going in the rumen.

Methane formed from the action of methanogens is eventually released into the atmosphere by the animal through burping. Over time, methane is converted back to carbon dioxide, but this conversion is very slow.

During its lifetime in the atmosphere, methane can absorb and re-emit infrared radiation, thereby contributing to global warming. In fact, methane has 25 times the warming effect of carbon dioxide over a 100-year period. When its atmospheric concentration and half-life are considered, methane is thought to contribute 4–9% of the global greenhouse effect.

Ruminants in New Zealand
Farming of ruminants is the main agricultural activity in New Zealand. Around 10 million cattle, 39 million sheep and almost two million deer and goats are farmed, and their meat, milk and fibre products account for almost half of the country’s commodity exports, currently earning around NZ$17 billion each year. Because of the large numbers of ruminants and relatively small human population, New Zealand has an unusual greenhouse gas emission profile, with agricultural emissions accounting for almost half of the country’s total greenhouse gases. Ruminant methane alone accounts for 31% of greenhouse gas emissions.

The worldwide demand for more food and the trend for increased meat and milk consumption are expected to continue into the future and it is predicted that ruminants will remain important contributors to New Zealand’s greenhouse gas emissions. It is also likely in the future that taxes will be placed on agricultural emissions and that there will be consumer resistance to products from animals with large carbon “footprints”.

Methane Mitigation
To ensure the sustainability of ruminant-based agriculture in New Zealand there is a need to investigate the process of methane formation in ruminants with a view to reducing the country’s future greenhouse gas emissions.

Any intervention against ruminant methane must target all the different types of methanogens in the rumen, as any unaffected methanogens will proliferate and continue to produce methane.

Any intervention must be specific to methanogens and not affect any other microbes, as otherwise the digestive function of the rumen will be compromised. These requirements necessitate a detailed knowledge of both the range of methanogen types present in the rumen and of their physiology, metabolism and genetics so that their common features can be defined and targeted and precisely inhibited.

One of the best ways to obtain detailed knowledge of a methanogen is to sequence its genome. This involves randomly fragmenting methanogen DNA into many small pieces, sequencing each piece, and using overlapping regions of DNA to reassemble the sequence into a single completed molecule. The sequence is then computer-analysed to identify individual genes and, by comparison with sequences in databases, genes are assigned possible functions.

A complete genome sequence identifies all of the genes present within an organism, and is useful because it can be used to confirm previous observations, fill in missing pieces of knowledge relating to the organism itself or its metabolism, and can also discover completely new features that would otherwise not have been found via conventional experimentation.

For M. ruminantium, the genome sequence confirmed the presence of genes that encode the enzymatic steps required for growth using hydrogen, carbon dioxide and formate, and revealed the pathway for the conversion of these substrates to methane. It also explained the methanogen’s growth requirement for acetate (needed for cell carbon biosynthesis), the volatile fatty acid 2-methylbutyrate (a precursor for making the amino acid isoleucine), and the cofactor CoM (two genes involved in the pathway that usually make it are missing from the genome).

But most importantly in relation to developing methane mitigation technologies, the genome sequence has allowed us to fill in the knowledge gaps concerning the metabolism of this organism and begin to identify shared features of rumen methanogens that might serve as useful targets for their inhibition. The types of genes we are looking for fall into two categories:

• genes encoding key enzymes that are suitable as targets for small molecule inhibitors via an approach known as “chemogenomics”; and

• genes encoding cell surface proteins that may be suitable as vaccine targets.

Chemogenomics of Rumen Methanogens
The chemogenomics approach is widely used in the pharmaceutical industry as a way of using genome sequence information to identify specific inhibitors of key enzymes involved in human metabolic disorders or microorganisms that cause disease. In most human health cases there is a single enzyme or pathogen target, but for the rumen there are a range of methanogens present and their specific metabolic targets have not been clearly defined.

Therefore, the first step in identifying chemogenomics targets for rumen methanogens is to compare their metabolic pathways and identify the genes encoding the key steps. These genes are then compared across other microbial genomes to ensure that they are universally conserved within methanogens yet also differ from genes in non-methanogens.

This type of analysis of the M. ruminantium genome has revealed 33 conserved and methanogen-specific genes. Most of these genes encode enzymes involved in the methanogenesis pathway (Fig. 2). These genes are being investigated via a chemogenomics “pipeline” in which gene candidates are subcloned, their encoded enzyme produced in the bacterium Escherichia coli, and the enzyme screened against large collections of chemical compounds for specific inhibition of its enzymatic activity.

In some cases the chances of finding a useful inhibitor can be increased dramatically by predicting (from comparison with closely related structures) or determining (from X-ray diffraction studies of the crystallised protein) the enzyme’s structure. This can define the shape of the enzyme’s catalytic site (where the reaction occurs) and allow the design of molecules that fit exactly into the reaction site and hence interfere with the enzyme’s function. This approach identifies inhibitory molecules that, if they look promising, can be tested for their effectiveness in animal trials.

A Methanogen Vaccine
An alternative methane mitigation approach is to develop a vaccine against rumen methanogens. Vaccines are commonly used on farms to counter a variety of animal diseases, and offer the benefits of prolonged action, easy administration and cost-effectiveness.

Vaccines induce the ruminant immune system to produce antibodies that bind to disease-causing cells. These are then recognised by the immune system and cleared from the body.

The rumen itself is not immunologically active, so an anti-methanogen vaccine would rely on antibodies secreted into ruminant saliva. Ruminants produce large amounts of saliva each day (~180 litres in cattle, and 15 litres in sheep), so we believe that sufficient salivary antibodies will be produced against rumen methanogens.

As there is no mechanism for clearing cell–antibody complexes in the rumen, antibodies would be most effective if they are directed towards essential features of the methanogens so that antibody binding has a direct inhibitory effect.

Alternatively, surface proteins that mediate interactions between methanogens and other microorganisms in the rumen are also viable vaccine targets. Antibodies that bind to these proteins would prevent these associations from forming.

Our group is using a combined approach in which whole methanogen cells, fractions of cells and individual proteins identified from methanogen genome sequences are being tested. The M. ruminantium genome sequencing has identified 71 genes encoding surface proteins that are potentially useful as vaccine targets. Some of these are involved in energy metabolism, and are therefore prime candidates for vaccine development.

Of particular interest are membrane-embedded enzyme complexes that generate energy (e.g. ATP synthase [Aha in Fig. 2]) and catalyse the second last step in the methanogenesis pathway (e.g. H4MPT methyl transferase [Mtr in Fig. 2]), both essential functions in methanogens. Protein subunits of both of these enzyme complexes are being produced in E. coli and will be tested in sheep as anti-methanogen vaccines.

Unexpected Findings
An unanticipated outcome from the M. ruminantium sequencing was the discovery of a phage (a type of virus that infects microbes) integrated into the genome. Around 70 genes encode all of the proteins and regulatory switches needed to produce a functional phage.

Like viruses that infect humans, this phage injects its DNA into M. ruminantium, where it either inserts itself into the methanogen DNA and remains dormant or begins to replicate itself and produce more phage offspring.

In order for the phage to leave the methanogen cell, it produces a lytic enzyme that breaks open the cell and releases all of the cell’s contents, including the newly made phage. The methanogen cell is killed in this process, and therefore the lytic enzyme can potentially be used as an agent to kill methanogens directly.

We have found that this phage enzyme does indeed break open M. ruminantium cells, and the enzyme will be tested for its effectiveness against a wider range of rumen methanogens in simulated rumen conditions.

Another novel discovery was two genes coding for non-ribosomal peptide synthetases (NRPS), which are large enzyme complexes that make peptide-based molecules that can be used as anti­biotics, iron-binding molecules, immune system suppressors or anti-cancer drugs. The occurrence of NRPS genes in a methanogen is very unusual and, although their function is not known, they may have some cell-signalling role in mediating interactions between methanogens and other rumen microbes.

Conclusions
The reduction of methane emissions from ruminant animals is not an easy task. Methane is the thermodynamically favoured end-product in the rumen, and altering this process requires a fundamental shift to an alternative end point.

There also remains the question of what happens to hydrogen in the rumen if methanogens are inhibited? Hydrogen will continue to be produced from the fermentation of plant material and must be diverted or else it will accumulate and inhibit digestion.

Other rumen microbes called acetogens are able to use hydrogen and carbon dioxide to produce acetate, an end product that can be used by the animal. However, whether these organisms can effectively replace methanogens as scavengers of hydrogen in the rumen has not been determined.

Our research is beginning to provide new information about the methane-formation process in the rumen through the detailed study of methanogen genomes. Several promising leads for chemogenomic and vaccine targets are being investigated as possible intervention points for the inhibition of rumen methanogens, and will hopefully lead to methane-reducing technologies for ruminant animals.

The multitude of methanogen types present in the rumen means that more genome sequences will be required to ensure that our current targets are shared by all these methanogens and that any interventions developed will be generally effective. We hope that this work will contribute to the international effort to mitigate ruminant methane emissions and, in the long-term, help reduce global greenhouse gas emissions.

Dr Graeme Attwood is Program Leader of the Rumen Microbial Genomics team within AgResearch, which is sequencing and analysing the genomes of rumen methanogens for the New Zealand Pastoral Greenhouse Gas Research Consortium.