Resumen de Producción de óxidos de nitrógeno gaseosos en Thermus thermophilus
The denitrification is an energy coupled prokaryotic process that consists in the sequential reduction of the nitrogen oxides (nitrate, nitrite, nitric oxide and nitrous oxide) to produce N2 as final product. Although N2 is described as the major final product, partial denitrification is very common in ground and wastewater bacteria, leading to the production of high levels of nitrous oxide, one of the gases with higher greenhouse effects.
Several strains of the ancient bacterium Thermus thermophilus are able to carry out one or more of the denitrification steps under anaerobic conditions. Reduction of nitrate is encoded by an apparently conjugative element (NCE), which, depending on the isolate, contains the genes for a tetrameric nitrate reductase, one or two nitrate/nitrite transporters, a NADH dehydrogenase and sensory systems that signals the cell to switch between aerobic and anaerobic metabolism. Reduction of nitrite to nitric oxide was studied in a previous work, whereas this thesis has been dedicated to study the last steps of the denitrification pathway in this bacterium.
Nitric oxide reduction is genetically linked to nitrite reduction, being encoded by a cluster of 9 genes (nor-nir) located upstream of NCE, in what constitutes a denitrification supercluster. In the first chapter, we describe a three-genes operon that encodes homologues to the two subunits of the cytochrome c dependent nitric oxide reductases (NorC-NorB), and a small additional gene, norH, with homologues found only among Aquificales, and in other members of the Thermus phylum. In the second chapter, we show how the complete denitrification supercluster is transferable in a single step with high frequency by conjugation-like process, because of its linkage to a plastic and likely mobilizable megaplasmid. As part of this chapter, the sequence of one of the denitrificant transconjugant is analyzed, and hypotheses regarding denitrification mobility are concomitantly raised. In the third chapter we demonstrate that the final product of nitrate reduction by the denitrification supercluster is actually nitrous oxide, and not di-nitrogen as speculated. Chapters four and five are dedicated to the functional and biochemical analysis of the nitric oxide reductase. Major conclusion of these chapters is that the Nitric oxide reductase of T. thermophilus contains a third subunit (NorH) absent in the counterparts from modern Proteobacteria. This subunit affects the stability and in vivo efficiency of the enzyme. A final chapter is dedicated to study the denitrification in a T. thermophilus strain (B) that has no homologues to the NOR studied in this Thesis. A complete sequence of this strain is provided, and hypotheses are raised onthe nature of the enzyme that actually reduces NO in this strain.
