Denitrification in haloarchaea: from genes to climate change

The main characteristic of hypersaline environments is the high concentration of salts present in waters and soils, which is considerably higher than that found in seawater (0.6 M or 3.5% (w/v)). When the salt concentration exceeds 16% (w/v), the predominant group of microorganisms is the haloarchaea. These microorganisms, as a class within the Archaea domain, are facultative aerobes, heterotrophic/phototrophic, characterised mainly by being obligate halophiles. They require high salt concentrations to live, usually between 1.5 and 4 M NaCl, which corresponds to 9-30% salts (w/v). In hypersaline environments, aerobic respiration becomes difficult because the availability of oxygen is very low due to the presence of high salt concentrations. Because of all these factors, microorganisms are pushed towards the use of alternative electron acceptors. Apart from O2 respiration, denitrification is the most energetically profitable pathway. In this sense, the genus Haloferax has been used as model to study denitrification since some of the members are the best-known haloarchaea in terms of microbial ecology, biochemistry and molecular biology. The hypothesis of the present doctoral thesis is that there are some species of haloarchaea with the ability to reduce NO3-/NO2- to N2 because they have the complete and active machinery of denitrification (complete denitrifiers), while others, because the route is truncated at some point, would generate as products NO and N2O (incomplete denitrifiers). The first group could act as sink of nitrogenous gases and toxic N anions in hypersaline environments, while the latter would be a source of greenhouse gases, thus contributing to global warming and climate change. In fact, nowadays, the anthropogenic activities are favouring the accumulation of N-sources as NO3-, NO2- and NH4+ in media (including saline/hypersaline media). In this sense, the doctoral thesis means a multidisciplinary advance in the knowledge of denitrification in the genus Haloferax, serving as a model to elucidate the characteristics of this respiratory route in extreme environments such as hypersaline ecosystems. The development and the main conclusions are set out below: 1. Using bioinformatics, an in silico analysis of the organization of the gene clusters related to denitrification in different species has been carried out. Based on the haloarchaeal genomes analysed, the genes involved in denitrification are grouped into three gene clusters (nar, nir-nor, nos) coding for denitrification enzymes NarGHI, NirK, qNor and NosZ. In case of incomplete denitrifiers, some of the genes or clusters are absent. 2. Also, analysing both, complete and draft haloarchaeal genomes, the vast majority reveal the presence of a single norZ orthologue. The evidences show that Nor-coding genes in haloarcheae are incorrectly annotated. 3. A semi-automatic incubation system was used to describe the phenotypic profile of denitrification in H. mediterranei, H. denitrificans and H. volcanii and their relationship with the emission of nitrogenous gases. Out of the species tested, only H. mediterranei was able to consistently reduce all available N-oxyanions to N2, while the other two released significant amounts of NO and N2O. 4. Also, H. mediterranei was used for the subsequent study about the transcriptional analysis of the main genes involved in denitrification (narG, nirK, norZ and nosZ) coupled with real-time measurements of all the intermediates (NO2-, NO, N2O and N2). It reflected a well-orchestrated system of gene expression during denitrification, being Nar and Nos, both transcriptionally activated by hypoxia (and probably nitrate), while Nir and Nor expression require the presence of nitric oxide (and possibly nitrite) as well as Nos. 5. Finally, using a proteomic approach, the components of the machinery of dentrification in H. mediterranei were described at protein level: the four N-reductases, but also other proteins related to electron flow and proton motive force (some of them not described at protein level so far).