sRNAs: Critical yet overlooked regulators of bacterial denitrification
Lead Research Organisation:
University of East Anglia
Department Name: Biological Sciences
Abstract
Faced with a shortage of oxygen in their environment many bacterial species are capable of switching to using nitrate, rather than oxygen to support life. Denitrification, allows bacteria to convert nitrate to gaseous products, nitric oxide (NO), nitrous oxide (N2O) and finally to dinitrogen (N2) that can be released into the atmosphere. This denitrification process can takes place in agricultural soils where nitrogen-rich fertilisers added to stimulate plant growth can also stimulate bacterial life. However, this bacterial survival strategy has significant environmental consequences as sometimes N2O a potent greenhouse gas that can damage the ozone layer is released into the atmosphere rather than harmless N2.
When discussing greenhouse gas emissions, the general public are acutely aware of the problems posed by carbon dioxide and methane. However, emissions of N2O, perhaps best known as the dental anaesthetic "laughing gas", should also be a cause for public and political concern. Over the last 100 years, N2O in the atmosphere has increased by more than 20% and this atmospheric loading is increasing each year. N2O emissions are expected to continue to increase year on year as a result of the growing population and demand for food, animal feed, and energy, as well as an increase in sources from industrial processes and wastewater treatment. Although the atmospheric levels of N2O are only a fraction of that of CO2, N2O has a 300-fold greater global warming potential. Since it has an atmospheric lifetime of more than 100 years the N2O produced today will influence the climate experienced by our great-great grandchildren therefore it is important to devise strategies to mitigate these releases now.
The pathways by which denitrifying bacteria produce N2O or N2 from nitrate are understood from a molecular level with structures of the proteins that convert nitrate to nitrite (nitrate reductases) and nitrite to nitric oxide (nitrite reductases) being known. The enzyme that breaks down N2O to inert N2 is a protein called nitrous oxide reductase or NosZ. It is the major enzyme on the planet that is responsible for the destruction of the potent N2O greenhouse gas. Without NosZ the atmospheric levels of N2O would be even greater than they currently are. However, as sometimes in the environment N2O is being emitted by denitrifying bacteria, NosZ is not being produced and we want to know why.
The understanding of how these denitrification proteins are switched on/off in the environment is not well understood and our team have been focusing on addressing this important global challenge. This requires fundamental studies on model organisms as it allows us to manipulate them in controlled laboratory environments, addressing the mechanisms by which environmental factors and genetic switches regulate N2O emissions in model denitrifying bacteria. Our model organism is the bacteria Paracoccus denitrificans, which is found in marine and terrestrial environments, is biochemically and genetically tractable and grows well under denitrifying conditions in the laboratory. In particular, this study will focus on new pathways of regulation by sRNA. sRNAs are small switches produced by bacteria that allow them to quickly respond to changes in their environments and switch genes on/off. Our team have recently discovered sRNAs are critical for controlling denitrification and N2O emissions and have characterised the mechanism of how one of these sRNA work. We now need to understand how other sRNAs that regulate production and consumption of N2O function using our established techniques which will allow us to resolve the core sRNAs that control bacterial N2O emissions. This information can then subsequently be used to control N2O in the environment through manipulation of these switches, perhaps through the composition of fertilizers applied to soils.
When discussing greenhouse gas emissions, the general public are acutely aware of the problems posed by carbon dioxide and methane. However, emissions of N2O, perhaps best known as the dental anaesthetic "laughing gas", should also be a cause for public and political concern. Over the last 100 years, N2O in the atmosphere has increased by more than 20% and this atmospheric loading is increasing each year. N2O emissions are expected to continue to increase year on year as a result of the growing population and demand for food, animal feed, and energy, as well as an increase in sources from industrial processes and wastewater treatment. Although the atmospheric levels of N2O are only a fraction of that of CO2, N2O has a 300-fold greater global warming potential. Since it has an atmospheric lifetime of more than 100 years the N2O produced today will influence the climate experienced by our great-great grandchildren therefore it is important to devise strategies to mitigate these releases now.
The pathways by which denitrifying bacteria produce N2O or N2 from nitrate are understood from a molecular level with structures of the proteins that convert nitrate to nitrite (nitrate reductases) and nitrite to nitric oxide (nitrite reductases) being known. The enzyme that breaks down N2O to inert N2 is a protein called nitrous oxide reductase or NosZ. It is the major enzyme on the planet that is responsible for the destruction of the potent N2O greenhouse gas. Without NosZ the atmospheric levels of N2O would be even greater than they currently are. However, as sometimes in the environment N2O is being emitted by denitrifying bacteria, NosZ is not being produced and we want to know why.
The understanding of how these denitrification proteins are switched on/off in the environment is not well understood and our team have been focusing on addressing this important global challenge. This requires fundamental studies on model organisms as it allows us to manipulate them in controlled laboratory environments, addressing the mechanisms by which environmental factors and genetic switches regulate N2O emissions in model denitrifying bacteria. Our model organism is the bacteria Paracoccus denitrificans, which is found in marine and terrestrial environments, is biochemically and genetically tractable and grows well under denitrifying conditions in the laboratory. In particular, this study will focus on new pathways of regulation by sRNA. sRNAs are small switches produced by bacteria that allow them to quickly respond to changes in their environments and switch genes on/off. Our team have recently discovered sRNAs are critical for controlling denitrification and N2O emissions and have characterised the mechanism of how one of these sRNA work. We now need to understand how other sRNAs that regulate production and consumption of N2O function using our established techniques which will allow us to resolve the core sRNAs that control bacterial N2O emissions. This information can then subsequently be used to control N2O in the environment through manipulation of these switches, perhaps through the composition of fertilizers applied to soils.
Technical Summary
Nitrous oxide (N2O) has been described as the biggest single cause of ozone depletion over the arctic. Its atmospheric loading is increasing year on year, and it has a very long atmospheric lifetime of ~150 years. N2O is estimated to contribute up to ~10% of the global radiative forcing of greenhouse gas emissions. Agriculture accounts for a large portion of anthropogenic N2O. Understanding the environmental factors that control N2O production and consumption by microbes is a critical and major challenge if practical mitigation strategies for N2O emissions are to be developed. Denitrifying bacteria play an important role in the synthesis and consumption of N2O, but very little is known about the regulatory networks that modulate the activity of the biochemical apparatus that catalyses production and destruction of this greenhouse gas. Bacterial small RNAs (sRNAs) have important regulatory roles in a range of physiological processes, and we have shown, for the first time, a critical role for them in regulating this important biogeochemical process.
This proposal, building on our published and preliminary data, will comprehensively analyse the role of sRNAs in environmental regulation of denitrification and N2O emissions, a major step on the road to developing sRNA as novel mitigation targets. This project will continue to develop Paracoccus denitrificans (Pden) as a regulatory, as well as a biochemical model of bacterial denitrification, that can be accessed and added to by others in the field. Although Pden is an excellent biochemical and physiological model of bacterial denitrification, and the archetypal bacteria to map the regulatory network controlling the denitrification apparatus, this proposal will also demonstrate the broader significance of our findings in other model denitrifiers (O3). This fundamental research is critical so that we might in future have the potential to biostimulate complete denitrification in the environment.
This proposal, building on our published and preliminary data, will comprehensively analyse the role of sRNAs in environmental regulation of denitrification and N2O emissions, a major step on the road to developing sRNA as novel mitigation targets. This project will continue to develop Paracoccus denitrificans (Pden) as a regulatory, as well as a biochemical model of bacterial denitrification, that can be accessed and added to by others in the field. Although Pden is an excellent biochemical and physiological model of bacterial denitrification, and the archetypal bacteria to map the regulatory network controlling the denitrification apparatus, this proposal will also demonstrate the broader significance of our findings in other model denitrifiers (O3). This fundamental research is critical so that we might in future have the potential to biostimulate complete denitrification in the environment.
