Analysis of the external signals driving the transcriptional regulation of the main genes involved in denitrification in Haloferax mediterranei

Abstract

Haloferax mediterranei is the model microorganism for the study of the nitrogen cycle in haloarchaea. This archaeon not only assimilate N-species such as nitrate, nitrite, or ammonia, but also it can perform denitrification under low oxygen conditions, using nitrate or nitrite as alternative electron acceptors. However, the information currently available on the regulation of this alternative respiration in this kind of microorganism is scarce. Therefore, in this research, the study of haloarchaeal denitrification using H. mediterranei has been addressed by analyzing the promoter regions of the four main genes of denitrification (narGH, nirK, nor, and nosZ) through bioinformatics, reporter gene assays under oxic and anoxic conditions and by site-directed mutagenesis of the promoter regions. The results have shown that these four promoter regions share a common semi-palindromic motif that plays a role in the control of the expression levels of nor and nosZ (and probably nirK) genes. Regarding the regulation of the genes under study, it has been concluded that nirK, nor, and nosZ genes share some expression patterns, and therefore their transcription could be under the control of the same regulator whereas nar operon expression displays differences, such as the activation by dimethyl sulfoxide with respect to the expression in the absence of an electron acceptor, which is almost null under anoxic conditions. Finally, the study with different electron acceptors demonstrated that this haloarchaea does not need complete anoxia to perform denitrification. Oxygen concentrations around 100 μM trigger the activation of the four promoters. However, a low oxygen concentration per se is not a strong signal to activate the promoters of the main genes involved in this pathway; high activation also requires the presence of nitrate or nitrite as final electron acceptors.

Transcriptomic profiling of haloarchaeal denitrification through RNA-Seq analysis

“…Previous studies found a regulatory motif (CGAAYATDKTYG) in the promoter regions of the main denitrification genes and other genes in the H. mediterranei genome, using the bioinformatic tool FIMO ( 21 , 23 , 82 ). These other genes include previously identified accessory proteins and a number of other proteins that have not been formally linked to the denitrification process ( 20 ).…”

“…Denitrification-related genes found in the genome of H. mediterranei in a previous study were compared with the transcriptomic analyses of the current study ( 23 ). From that study, a set of 82 promoter regions that shared the regulatory motif CGAAYATDKTYG was used for further analyses.…”

“…These copper proteins are postulated to replace the role of c-type cytochromes, which traditionally act as electron donors in other microbial organisms ( 22 ). Another recent study identified the external signals that can activate this alternative respiration in H. mediterranei ( 23 ). In this study, the activation of the promoters of the genes that encode the main denitrification enzymes was analyzed and it was found that the promoter of the respiratory nitrate reductase is regulated differently from the promoters of the genes that encode the three other enzymes.…”

“…In this study, the activation of the promoters of the genes that encode the main denitrification enzymes was analyzed and it was found that the promoter of the respiratory nitrate reductase is regulated differently from the promoters of the genes that encode the three other enzymes. In addition, regulatory motifs were identified in all the promoter regions, except for that of the nitrate reductase ( 23 ). Notably, the identity of the regulator capable of binding to this regulatory sequence remains undisclosed.…”