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FRIMEDBIO-Fri prosj.st. med.,helse,biol

Phenotypic diversification in denitrification: mechanisms, prevalence and implications

Alternative title: Fenotypisk diversifisering ved denitrifikasjon: mekanismer, utbredelse og implikasjoner

Awarded: NOK 8.3 mill.

Microbes rule our planet, and they colonize every habitat imaginable. Their ubiquity is due to their impressive range of lifestyles and their sheer numbers make them important drivers of Earth's biogeochemical cycles, such as the carbon- and nitrogen cycle. If a substance can be eaten or breathed (respired), there will be microbes (i.e., bacteria, archaea, fungi) specialized to do just that. Like all other living organisms, bacteria require elements such as carbon and nitrogen to build their macromolecules (e.g., DNA and proteins) and they need energy to drive their metabolism. Respiration is a very efficient strategy to harvest energy. Whereas animals are limited to breathing oxygen (aerobic respiration), many bacteria can use other substances instead (anaerobic respiration). Denitrification, the stepwise reduction of nitrate (NO3-), via nitrite (NO2-), nitric oxide (NO) and nitrous oxide (N2O) to nitrogen gas (N2), is one type of anaerobic respiration which is found in a wide range of bacteria. These organisms usually prefer aerobic respiration but will switch to denitrification when necessary. In order to do this, they need to sense that oxygen is scarce, express the relevant genes and build myriad new proteins, including four core enzymes for each of the reduction steps. It is in the organisms' interest to keep this transition under stringent control because 1) aerobic respiration is energetically more profitable than denitrification and 2) building new proteins is costly. If the organism responds too quickly, while aerobic respiration is still possible, it will waste resources building an unnecessary machinery; if it is too slow, it risks becoming trapped in anoxia with no means of harvesting energy for life and growth. Bacteria have developed many ways of regulating denitrification, but a common trait is that low oxygen and the presence of nitrate/nitrite/NO induce gene expression. Until recently, the underlying consensus has been that in a population of cells belonging to the same denitrifying species, all would express their entire denitrification apparatus when facing anoxia. We have found that this is not true. Within the project, we have shown that some bacteria, such as the model organism Paracoccus denitrificans, hedge their bets. Instead of going all in and expressing the entire set of denitrification proteins in all cells, cells within the same population will differentiate. All cells will produce the N2O reductase, thus reducing N2O to N2, but only a fraction will carry out the entire four-step process. Hence, P. denitrificans becomes a net N2O sink when facing anoxia. Moreover, we have shown that P. denitrificans also hedges its bets when oxygen returns. It conserves the ability to denitrify in a sub-population of non-growing persister cells. Within the project, we have studied the regulatory mechanisms behind these phenomena as well as enquiring about the prevalence of bet-hedging among denitrifying bacteria. This has implications for our understanding of N2O emissions from natural systems. Another important result of this project is that our progress in understanding the physiology of denitrifying bacteria has put us un a position to invent new biotechnological processes. This will be further explored within the recently initiated project "AnaPro" (https://www.nmbu.no/prosjekter/node/43227), funded by the Novo Nordic Foundation.

The work within this project has equipped us with detailed knowledge of denitrifier physiology, particularly in terms of bet-hedging. This has implications for our understanding of soil N2O emissions, thus climate forcing, and it has unveiled further fundamental questions. But it has also pointed us towards new biotechnological opportunities, one example being the development of a new process (patent pending: #GB2100105.2) for high density culturing of denitrifying bacteria. The project leader has gained experience with project administration and with taking the lead in supervision of Phd students, reaching out to collaborators and planning new projects. These skills were important when applying for funding for the AnaPro project (https://www.nmbu.no/prosjekter/node/43227), at the Novo Nordic Foundation (NNF), where we will explore the imperatives and further develop high density cultivation by denitrification. With this, we have ventured into a more applied direction, with new collaborators within Europe (Technical University, Delft and Vrije University, Amsterdam). We have also strengthened local connections and are collaborating with colleagues at NMBU for greater bandwidth (cross disciplinarity). This is important within the Anapro project, but also as we continue our basic research on the regulatory biology and physiology of denitrifiers, which requires advanced skills within microbiology, biochemistry and chemistry. The university (NMBU) routinely awards YRT grant holders with an extra PhD scholarship, which was also the case here. Thus, the project has spurred the production of two PhD theses: The RCN-financed candidate is expected to defend in late 2022, whereas the NMBU financed candidate will defend in 2024. The career of the project leader has evolved considerably during-, and due to the project: From being a researcher within a group, with limited responsibilities, to becoming part of the leader group and building her own team. Moreover, the NRC YRT project not only facilitated the NNF grant, but the career development over recent years played an integral part when the project leader recently secured a permanent position as Associate Professor at NMBU.

The aim of Dephend is to understand phenotypic diversification (PD) in the regulation of anoxic respiration in denitrifying prokaryotes, and explore the implications for their contribution to the emission of gaseous nitrogen oxides to the atmosphere. The point of departure is our discovery of PD in the model organism Paracoccus denitrificans, and circumstantial evidence for similar regulatory traits among other denitrifying bacteria. This opens a new avenue for research on the regulatory biology of denitrification. The project will improve our understanding of the mechanisms involved, assess how widespread such regulatory traits are among denitrifying prokaryotes, and clarify the implications for the emissions of NO, N2O and HONO in the field. In short, the PD phenomena involve early expression of N2O reductase in all cells, while only a fraction of the cells express nitrite reductase (NirS), ascribed to stochastic initial transcription of the gene, but with a positive feedback via NO. Dephend will further explore this NO signaling; if different NO-responsive factors or post-transcriptional phenomena are involved; and the possible fitness value of cell diversification. The project will combine molecular manipulations, refined physiological experiments and mathematical modelling. P. denitrificans expressing NirS with an mCherry fusion tag will be used as a model. Moreover, the prevalence and diversity of PD phenomena will be explored in a range of other soil bacteria. Dephend will be conducted in a setting where findings can rapidly be taken into a more applied context. This includes the exploration of strategies to mitigate annually increasing N2O emissions resulting from modern agricultural practice. Thus, the scope of Dephend encompasses basic knowledge generation relevant across disciplines in microbiology, and increased understanding of the processes driving the emission of N-oxides which affect atmospheric chemistry and contribute to climate forcing.

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FRIMEDBIO-Fri prosj.st. med.,helse,biol

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