Anaerobic Growth of Haloarchaeon Haloferax volcanii by Denitrification Is Controlled by the Transcription Regulator NarO

Abstract

The extremely halophilic archaeon Haloferax volcanii grows anaerobically by denitrification. A putative DNA-binding protein, NarO, is encoded upstream of the respiratory nitrate reductase gene of H. volcanii. Disruption of the narO gene resulted in a loss of denitrifying growth of H. volcanii, and the expression of the recombinant NarO recovered the denitrification capacity. A novel CX n CXCX 7 C motif showing no remarkable similarities with known sequences was conserved in the N terminus of the NarO homologous proteins found in the haloarchaea. Restoration of the denitrifying growth was not achieved by expression of any mutant NarO in which any one of the four conserved cysteines was individually replaced by serine. A promoter assay experiment indicated that the narO gene was usually transcribed, regardless of whether it was cultivated under aerobic or anaerobic conditions. Transcription of the genes encoding the denitrifying enzymes nitrate reductase and nitrite reductase was activated under anaerobic conditions. A putative cis element was identified in the promoter sequence of haloarchaeal denitrifying genes. These results demonstrated a significant effect of NarO, probably due to its oxygen-sensing function, on the transcriptional activation of haloarchaeal denitrifying genes. IMPORTANCEH. volcanii is an extremely halophilic archaeon capable of anaerobic growth by denitrification. The regulatory mechanism of denitrification has been well understood in bacteria but remains unknown in archaea. In this work, we show that the helix-turnhelix (HTH)-type regulator NarO activates transcription of the denitrifying genes of H. volcanii under anaerobic conditions. A novel cysteine-rich motif, which is critical for transcriptional regulation, is present in NarO. A putative cis element was also identified in the promoter sequence of the haloarchaeal denitrifying genes.

Haloferax mediterranei, an Archaeal Model for Denitrification in Saline Systems, Characterized Through Integrated Physiological and Transcriptional Analyses

“…The negative regulation of NAR by O 2 via O 2 -sensing transcription factors is common in denitrifying bacteria (Spiro, 2012) and has also been observed for N 2 OR (Bergaust et al, 2010(Bergaust et al, , 2012. In H. mediterranei, the AcrR-like transcriptional regulator occupies the same position as the NarO regulator in Haloferax volcanii, recently identified as a putative O 2 sensor in this strain (Hattori et al, 2016). The "core" reductases, NIR and NOR, were not induced by hypoxia alone, but apparently required the presence of NO (and possibly nitrite).…”

Denitrifying haloarchaea within the genus Haloferax display divergent respiratory phenotypes, with implications for their release of nitrogenous gases

“…H. volcanii is genetically tractable and has been used as a model for studies of haloarchaea. It has been identified as a denitrifier capable of anaerobic growth under microoxic conditions using nitrate as alternative electron acceptor (Hattori et al, ). In contrast to findings by Hattori et al ., H. volcanii did not reduce nitrate to nitrite in our hands.…”

“…Currently, little is known about the regulation of denitrification in Haloferax species. However, Hattori and colleagues () recently identified a putative O 2 sensor, NarO, regulating nar and nir transcription in H. volcanii . A similar sequence is found in H. denitrificans , but in H. mediterranei , the corresponding position is occupied by an AcrR‐like transcriptional regulator (Fig.…”

“…In a third species, H. volcanii, the results appear conflicting, most likely because different strains have been used for denitrification studies. Even so, it is generally considered a denitrifying organism (Bickel-Sandkötter and Ufer, 1995;Hattori et al, 2016).…”

Microbial Mat Functional and Compositional Sensitivity to Environmental Disturbance

“…A range of taxonomically diverse heterotrophic and autotrophic microbes are capable of denitrification, making phylogenetic inference difficult (Zumft, 1997;Bowen et al, 2014). However, it is known that halophilic archaea within the phyla Euryarchaeota and 640 some taxa within the alphaproteobacterial orders, Rhodospirllales and Rhodobacterales, play a role in denitrification (Tomlinson et al, 1986;Yoshimatsu et al, 2000;Hattori et al, 2016). While taxonomic links to microbial mat denitrification are speculative, OTUs classified within the Halobacteriaceae, Rhodospirllales, and Rhodobacterales were active across years but increased activity following salinity reduction, and may play a role in denitrification 645 within this ecosystem.…”