Norwegian title of the thesis:
Nitrat-ammonifiserende Bacillus vireti og Wollinella succinogenes; Produksjon og reduksjon av drivhusgassen N2O
Prescribed subject of the trial lecture:
Transport and assembly of redox proteins involved in nitrate and nitrite respiration
Time and place for the trial lecture and the public defence:
May 23rd, 2014, at Auditoriet, SU105, Sørhellinga kl. 12:15
Professor Åsa Frostegård, IKBM (main supervisor)
Dr. Linda Bergaust, (co-supervisor), NMBU
Professor Lars Bakken (co-supervisor), NMBU
Professor David Richardson, University of East Anglia, United Kingdom
Professor Simon de Vries, Delft University of Technology, The Netherlands
Professor Leiv Sigve Håvarstein, IKBM, NMBU, Norway
The doctoral thesis is available for public review at the UMB library.
Thesis number 2014:43, ISSN 1894-6402, ISBN 978-82-575-1207-1
It has been a common notion that, while denitrification leads to loss of reactive nitrogen, dissimilatory nitrate reduction to ammonium (DNRA) retains nitrogen in the ecosystem. Yet, an increasing number of studies indicate that DNRA might be responsible for a substantial part of nitrous oxide emissions from agricultural soils. The mechanisms involved in this emission from DNRA are not well understood. It is assumed that no intermediate compounds are released from any of the enzymes involved in DNRA, and that the putative intermediates nitric oxide and hydroxylamine remain enzyme bound until their reduction is completed. Nevertheless, production of nitric oxide and nitrous oxide has been observed in different DNRA performing bacteria, making this assumption questionable. The enzymatic routes leading to nitric oxide production are not clarified, although studies suggest that it may be formed during nitrate reduction by the nitrate reductase NarG. Different enzymes are known to detoxify nitric oxide through reduction to nitrous oxide, while the capacity to reduce nitrous oxide is uncommon to DNRA organisms and has so far only been detected in a few strains.
Most of the current knowledge on DNRA is based on investigations of a limited number of Gamma- or Epsilonproteobacteria, while Gram-positive bacteria are understudied in this respect. The present thesis presents a whole-genome analysis of the soil bacterium Bacillus vireti. Functional genes involved in the anaerobic metabolism of this organism were identified, as well as putative binding regions for regulating proteins. The latter include the two-component signal transduction system ResDE, the oxygen sensing global regulator Fnr, the two-component nitrate/nitrite sensing regulator NarX/NarL, the redox regulator Rex and the nitric oxide sensitive transcriptional repressor NsrR, similar to regulatory proteins reported from Escherichia coli and Bacillus subtilis. When grown in batch cultures in the presence of oxygen and nitrate, Bacillus vireti reduced these electron acceptors concomitantly, resulting in fast growth. Fermentation started when oxygen approached depletion, producing acetate, formate, lactate and succinate. In the treatment with low initial nitrate levels (5 mM), nitrite accumulated until nitrate depletion. At this point, transcription of the genes encoding the nitrite reductases NrfA and NirB started and led to complete reduction of the nitrite to ammonium. At nitrate concentrations higher than 20 mM, the transcription of those genes was lower but more long-lasting, and 50% of the initial nitrate was recovered as nitric oxide, nitrous oxide and di-nitrogen. The nitric oxide, produced from an unknown source, was reduced via nitrous oxide to N2 using two enzymes connected to denitrification; an unusual nitric oxide reductase, qCuANor encoded by cbaA, and a z-type nitrous oxide reductase, encoded by nosZ. The delayed expression of nosZ in the high nitrate treatments could explain the transient nitrous oxide in those samples.
The Epsilonproteobacterium Wolinella succinogenes is a model organism for electron transfer pathways and DNRA. It was found to possess an atypical c-type NosZ whose gene was interrupted by an insertion element in the type strain (ATCC 29543). The nosZ was restored and transformed back into the genome of Wolinella succinogenes. Gas analysis demonstrated that the restored nosZ yielded a functional nitrous oxide reductase. Under the experimental conditions, the wild type produced only little nitrous oxide, which was reduced to di-nitrogen in the nosZ complementation strain. Incubations with sterile medium showed the production of nitric oxide, but not of nitrous oxide, from chemical decomposition of nitrite. In cultures of W. succinogenes, nitrite accumulated when the electron donor formate was depleted in medium with excess nitrate. The resulting nitric oxide was apparently reduced to nitrous oxide by W. succinogenes, although no known gene encoding a nitric oxide reductase was found in the genome.
The combination of detailed gas kinetics and transcription analyses has brought us closer to understanding the regulation of the complex anaerobic metabolism of two very different organisms which may act as strong nitrite producers and may be net sources and sinks of nitric oxide and nitrous oxide when nitrate is available in ample amounts.