Transcriptomic profiling of haloarchaeal denitrification through RNA-Seq analysis

Abstract

Denitrification, a crucial biochemical pathway prevalent among haloarchaea in hypersaline ecosystems, has garnered considerable attention in recent years due to its ecological implications. Nevertheless, the underlying molecular mechanisms and genetic regulation governing this respiration/detoxification process in haloarchaea remain largely unexplored. In this study, RNA-sequencing was used to compare the transcriptomes of the haloarchaeon Haloferax mediterranei under oxic and denitrifying conditions, shedding light on the intricate metabolic alterations occurring within the cell, such as the accurate control of the metal homeostasis. Furthermore, the investigation identifies several genes encoding transcriptional regulators and potential accessory proteins with putative roles in denitrification. Among these are bacterioopsin-like transcriptional activators, proteins harboring a domain of unknown function (DUF2249), and cyanoglobin. In addition, the study delves into the genetic regulation of denitrification, finding a regulatory motif within promoter regions that activates numerous denitrification-related genes. This research serves as a starting point for future molecular biology studies in haloarchaea, offering a promising avenue to unravel the intricate mechanisms governing haloarchaeal denitrification, a pathway of paramount ecological importance.IMPORTANCEDenitrification, a fundamental process within the nitrogen cycle, has been subject to extensive investigation due to its close association with anthropogenic activities, and its contribution to the global warming issue, mainly through the release of N₂O emissions. Although our comprehension of denitrification and its implications is generally well established, most studies have been conducted in non-extreme environments with mesophilic microorganisms. Consequently, there is a significant knowledge gap concerning extremophilic denitrifiers, particularly those inhabiting hypersaline environments. The significance of this research was to delve into the process of haloarchaeal denitrification, utilizing the complete denitrifier haloarchaeon Haloferax mediterranei as a model organism. This research led to the analysis of the metabolic state of this microorganism under denitrifying conditions and the identification of regulatory signals and genes encoding proteins potentially involved in this pathway, serving as a valuable resource for future molecular studies.

Keywords: Haloferax mediterranei; denitrification; haloarchaea; transcriptional regulation; transcriptomics.

Fig 1

Volcano plot of the transcriptomic profile of differential gene expression between oxic and denitrifying conditions.

Fig 2

Gene set enrichment analysis (GSEA). Differentially suppressed or activated pathways of H. mediterranei under denitrifying conditions.

Fig 3

Gene clusters of the genes encoding the main enzymes of denitrification. Bars indicate the log₂FC value of the genes (oxic vs denitrifying conditions). Green arrows indicate genes encoding the main denitrification enzymes or accessory proteins previously identified in other studies; orange arrows indicate the protein-coding genes discussed in this article in later sections; blue arrows indicate the rest of the genes. Small black arrows indicate promoter regions that present the regulatory motif found in different promoter regions (CGAAYATDKTYG) (further discussed in the next sections).

Fig 4

STRING network of the protein interaction of two DUF2249 related with nitrate metabolism enzymes.

Fig 5

Comparisons of DUF2249 proteins. (A) Sequence alignment of all the DUF2249 domain-containing proteins present in the H. mediterranei genome together with DrpA from T. thermophilus. Colors indicate conserved amino acids. pid, percent identity compared to DrpA. (B) Phylogenetic tree of the DUF2249 domain-containing proteins. DrpA has the closest relationship with E6P09_00795 protein from H. mediterranei.

Fig 6

Domain scheme of transcriptional regulators with differential expression. Yellow background, regulator with log₂FC > +2; red background, regulators with log₂FC < −2. HTH-domains, blue; ligand binding domains, gray; other domains, green. The length and location of the domains are an approximation.

Fig 7

Schematic representation of the regulation and the adaptation mechanisms identified by RNA-Seq analysis of H. mediterranei cells under denitrifying conditions. Gene products encoded by differentially upregulated (continuous figure shape) and downregulated genes (discontinuous figure shape) are represented. SirR and BopAct are transcriptional regulators with an assigned putative function. Abbreviations: Sider., siderophores; BopAct., bacterioopsin activator; DUF2249, Domain of Unknown Function 2249-containing protein.