Effects of nitrogen sources on the nitrate assimilation in Haloferax mediterranei: growth kinetics and transcriptomic analysis

The haloarchaeon Haloferax mediterranei is able to grow in a defined culture media not only in the presence of inorganic nitrogen salt but also with amino acid as the sole nitrogen source. Assimilatory nitrate and nitrite reductases, respectively, catalyze the first and second reactions. The genes involved in this process are nasA, which encodes nitrate reductase and is found within the operon nasABC, and nasD, which encodes nitrite reductase. These genes are subjected to transcriptional regulation, being repressed in the presence of ammonium and induced with either nitrate or nitrite. This type of regulation has also been described when the amino acids are used as nitrogen source in the minimal media. Furthermore, it has been observed that the microorganism growth depends on nitrogen source, obtaining the lowest growth rate in the presence of nitrate and aspartate. In this paper, we present the results of a comparative study of microorganism growth and transcriptomic analysis of the operon nasABC and gene nasD in different nitrogen sources. The results are the first ever produced in relation to amino acids as nitrogen sources within the Halobacteriaceae family.

Nitrogen metabolism in haloarchaea

The nitrogen cycle (N-cycle), principally supported by prokaryotes, involves different redox reactions mainly focused on assimilatory purposes or respiratory processes for energy conservation. As the N-cycle has important environmental implications, this biogeochemical cycle has become a major research topic during the last few years. However, although N-cycle metabolic pathways have been studied extensively in Bacteria or Eukarya, relatively little is known in the Archaea. Halophilic Archaea are the predominant microorganisms in hot and hypersaline environments such as salted lakes, hot springs or salted ponds. Consequently, the denitrifying haloarchaea that sustain the nitrogen cycle under these conditions have emerged as an important target for research aimed at understanding microbial life in these extreme environments. The haloarchaeon Haloferax mediterranei was isolated 20 years ago from Santa Pola salted ponds (Alicante, Spain). It was described as a denitrifier and it is also able to grow using NO3-, NO2- or NH4+ as inorganic nitrogen sources. This review summarizes the advances that have been made in understanding the N-cycle in halophilic archaea using Hfx mediterranei as a haloarchaeal model. The results obtained show that this microorganism could be very attractive for bioremediation applications in those areas where high salt, nitrate and nitrite concentrations are found in ground waters and soils.

Transcriptional profiles of Haloferax mediterranei based on nitrogen availability

The haloarchaeon Haloferax mediterranei is able to grow in the presence of different inorganic and organic nitrogen sources by means of the assimilatory pathway under aerobic conditions. In order to identify genes of potential importance in nitrogen metabolism and its regulation in the halophilic microorganism, we have analysed its global gene expression in three culture media with different nitrogen sources: (a) cells were grown stationary and exponentially in ammonium, (b) cells were grown exponentially in nitrate, and (c) cells were shifted to nitrogen starvation conditions. The main differences in the transcriptional profiles have been identified between the cultures with ammonium as nitrogen source and the cultures with nitrate or nitrogen starvation, supporting previous results which indicate the absence of ammonium as the factor responsible for the expression of genes involved in nitrate assimilation pathway. The results have also permitted the identification of transcriptional regulators and changes in metabolic pathways related to the catabolism and anabolism of amino acids or nucleotides. The microarray data was validated by real-time quantitative PCR on 4 selected genes involved in nitrogen metabolism. This work represents the first transcriptional profiles study related to nitrogen assimilation metabolism in extreme halophilic microorganisms using microarray technology.

Role of the denitrifying Haloarchaea in the treatment of nitrite-brines

Haloferax mediterranei is a denitrifying halophilic archaeon able to reduce nitrate and nitrite under oxic and anoxic conditions. In the presence of oxygen, nitrate and nitrite are used as nitrogen sources for growth. Under oxygen scarcity,this haloarchaeon uses both ions as electron acceptors via a denitrification pathway. In the present work, the maximal nitriteconcentration tolerated by this organism was determined by studying the growth of H. mediterranei in minimal medium containing30, 40 and 50 mM nitrite as sole nitrogen source and under initial oxic conditions at 42 °C. The results showed theability of H. mediterranei to withstand nitrite concentrations up to 50 mM. At the beginning of the incubation, nitrate wasdetected in the medium, probably due to the spontaneous oxidation of nitrite under the initial oxic conditions. The completeremoval of nitrite and nitrate was accomplished in most of the tested conditions, except in culture medium containing 50 mMnitrite, suggesting that this concentration compromised the denitrification capacity of the cells. Nitrite and nitrate reductases activities were analyzed at different growth stages of H. mediterranei. In all cases, the activities of the respiratory enzymeswere higher than their assimilative counterparts; this was especially the case for NirK. The denitrifying and possibly detoxifyingrole of this enzyme might explain the high nitrite tolerance of H. mediterranei. This archaeon was also able to remove60 % of the nitrate and 75 % of the nitrite initially present in brine samples collected from a wastewater treatment facility.These results suggest that H. mediterranei, and probably other halophilic denitrifying Archaea, are suitable candidates for thebioremediation of brines with high nitrite and nitrate concentrations.