Transcriptional regulation of bacterial nitrous oxide emissions in complex ecosystems

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Project Aims and Objectives: If we are to mitigate bacterial N2O emissions then understanding how they are environmentally and transcriptionally regulated is an essential pre-requisite. This studentship will:1. Establish the role of bacterial sRNA in controlling denitrification.We have mapped the primary transcriptome of N2O producing versus consuming cultures of Paracoccus denitrificans (Pd) and identified 160 small RNAs (sRNA), 40% of which are differentially expressed between the conditions. Bacterial sRNA are important regulators of a range of physiological processes, but never before in denitrification. We have experimentally confirmed 2/5 tested sRNAs, which impact on Pd N2O emissions. We need to understand how sRNA impact on denitrification. The student will pulse express these sRNA in denitrifying cultures of Pd, extract total cellular RNA and measure the transcriptome using RNA-seq. Once these targets have been identified they are further validated by transcriptional /translational fusions and physiology experiments. The student has the scope to build on this to identify N2O inducible sRNA of representative species from clade I (N2O producers and consumers) and II (N2O consumers only) bacteria. This objective will identify a core sRNAome - conserved across denitrifying bacteria and environments, that may act as global targets for N2O mitigation, and establish a new dogma for regulation of this important biogeochemical cycle.2. Compare how environmental variables impact on N2O production in model soil ((Pd), Dyadobacter fermentans (Df)) and marine (Ruegeria pomeroyi (Rp)) denitrifiers.Under anaerobic, NO3--rich, Cu-limited conditions, N2O release by Pd (clade I) is ~4000 times higher than in Cu-replete cultures; this is due to down-regulation of nosZ in Cu-limited cells. Whether this is the case in other denitrifiers, will be tested here by repeating these experiments with Rp (marine) and Df (soil, clade II) and measuring the effect on transcription of the denitrification apparatus. This will demonstrate if the copper regulation of N2O emissions spans denitrifying species from other environments. We hypothesise that the ratio of carbon source to NO3- also impacts on nosZ transcription and we will examine the physiology and transcriptome of wild-type Pd, Df and Rp under Cu-replete and Cu-deplete conditions at a range of electron donor:acceptor ratios. This objective will determine the regulatory network which controls this switch and the impact of carbon:nitrate:copper mixes in fertilizer regimes and surface run off. 3. Determine the biochemistry and gene regulation used for N2O production /consumption in complex ecosytems. N2O in soil and aquatic environments is generated by phylogenetically diverse bacterial and archaeal ammonia oxidisers and denitrifiers. Community composition and active components are driven by environmental conditions and determine which organisms dominate nitrous oxide production. Comparative metatranscriptomics of soil from an upland grassland peaty podzol soil at Glensaugh (in collaboration with University of Edinburgh) and aquatic (Wensum and Yare) samples, with different emission characteristics, will be used to identify functional and regulatory genes associated with these environmental variations. Dominant active strains will be isolated and characterised. This objective will increase knowledge of the diversity, and commonality, of regulatory networks of N2O producing organisms beyond traditional 'laboratory-rats'