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. IMPORTANCE Denitrification, 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.

The nitrogen cycle is one of the most important biogeochemical cycles in nature.A great deal of effort has been put into understanding it, mainly because of their relationship with the efficiency of fertilization in soils used for agricultural purposes as well as with the emission of greenhouse gases such as nitrous and nitric oxide (1,9,10).Generally, hypersaline environments are dominated by haloarchaea, which are extremophilic microorganisms that belong to the Halobacteria class (Archaea domain) (6,11,12).These microorganisms are usually cataloged as partial denitrifiers, given the fact that they lack some genes of the complete denitrification pathway (1,9).This translates into potential greenhouse gas emissions to the atmosphere in saline environments and therefore the study of the denitrification pathway in haloarchaea is an important topic for the scientific community (9).
Haloferax mediterranei is the model organism for denitrification studies in haloarch aea, as it is a complete denitrifier that can reduce nitrate to dinitrogen (13).The first studies with this microorganism deepened on the purification and biochemical characterization of the respiratory nitrate (14)(15)(16) and nitrite reductase (17,18) and were followed by studies of the gas emission kinetics, transcription of the four genes that encode the main denitrification enzymes (narG, nirK, nor, and nosZ) and proteomics (13,19,20).These studies have shed light on some of the fundamental aspects of the denitrification pathway in haloarchaea, allowing the scientific community to understand better this pathway when driven by haloarchaea.However, the complete set of proteins in the electron transport chain and regulators that are involved in this process remained unknown until today.The only described transcriptional regulator is the oxygen sensor NarO identified in the haloarchaeon Haloferax volcanii (21).Recently, a proteomic approach was used to study the proteins that may be associated with denitrification in H. mediterranei, finding interesting proteins such as NarC that could be a component of the quinol oxidizing nitrate reductase complex, being the membrane attachment for NarGH.Also, the copper proteins, halocyanins, and azurins were associated as possible electron donors for NirK and NosZ, this being the first time that putative accessory elements of denitrification electron transport chains have been described in haloarchaea (20).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 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.
This lack of information on transcriptional regulators leaves a large knowledge gap in understanding this pathway.Due to this and to the advances in Next-Generation Sequencing (NGS), a comparative analysis of RNA-Seq in H. mediterranei was performed comparing oxic and denitrifying conditions.The differentially activated and suppressed pathways under denitrifying conditions have been analyzed, providing information about the processes that are changing in the cell metabolism of this haloarchaea when denitrification occurs.In addition, the differentially expressed regulators have been studied and discussed in this analysis together with other interesting proteins that could be related to the pathway under study.This is the first detailed analysis of denitrification in haloarchaea, and it should be the starting point for future research in this field to decipher the regulatory machinery that controls the denitrification response in haloarchaea.

Global and gene set enrichment analysis of H. mediterranei transcriptome under denitrifying conditions
Denitrifying conditions imply a massive adaptation for H. mediterranei cells.The low oxygen concentration together with the need to reduce nitrate leads to important metabolic changes, in which de novo synthesis of some proteins and the repression of others requires a massive adjustment in transcription levels of different genes (20).Comparison between oxic and denitrifying conditions showed that among the 3,907 detected expressed genes (out of a total of 3,920 annotated genes), 416 were differentially expressed (log 2 FC < −2 or log 2 FC > +2 and padj <0.05).In all, 296 of these genes were upregulated under denitrifying conditions and 120 were underregulated (Fig. 1).
Analysis of differentially expressed genes by GSEA (Gene Set Enrichment Analysis) is usually done with Gene Ontology (GO) or KEGG databases (24)(25)(26).However, GO analysis was not possible with this data due to the lack of information about haloarchaea in this database.Only the KEGG database was used instead.KEGG showed the enriched pathways in the H. mediterranei transcriptome, considering the differentially expressed genes and the number of genes expressed in each pathway present.GSEA results are displayed in Fig. 2 and Table S1.
These results revealed that the statistically enriched pathways are associated with changes in the energy metabolism of H. mediterranei.Concerning the overall global changes, oxidative phosphorylation, replication, transcription, and translation are suppressed, which is consistent with the lower growth rates observed in H. mediterra nei in previous studies under denitrifying conditions, where the energy yield is lower than in aerobic respiration (9,27).On the other hand, nitrogen metabolism and fatty acid degradation were upregulated.In haloarchaea, fatty acid degradation is linked to the mobilization of polyhydroxyalkanoate (PHA) (28).H. mediterranei can produce PHA granules as carbon and energy storage (29).In fact, the E6P09_15675 and the E6P09_15680 genes encoding the PHA depolymerase PhaZh1 and the putative 3HB dehydrogenase BdhA showed a positive differential expression (log 2 FC = 3.62 and 3.66, respectively), and the genes encoding the main polymerase PhaEC (encoded by E6P09_17990 and E6P09_17995 genes) did not show significant changes (log 2 FC = 0.62 and 0.54, respectively).This may indicate that the cell could be using these reserves under denitrifying conditions since the function of PhaZh1 and BdhA in H. mediterranei is described as an in vivo PHA degradation pathway that produces acetoacetate as a final product (29).
Furthermore, the phosphotransferase system (PTS) was also activated.This system uses phosphoenolpyruvate as the phosphoryl donor to phosphorylate sugars for transport into the cell.In H. mediterranei the PTS gene cluster is located just adjacent to the glpR-fruK genes which encode the GlpR transcriptional regulator and the enzyme phosphofructokinase, which are also overexpressed in denitrifying conditions.The PTS gene cluster induction by fructose has been determined under the control of GlpR (30).The function of this system in the absence of fructose and denitrifying conditions is unclear.It could be related to the attempts of the cell to obtain energy or, as has been described in bacteria, it could not only function as a carbohydrate transporter but also regulate cellular processes by phosphorylating target proteins as transcriptional regulators (30).Two-component system genes were also upregulated under this condition.This system usually allows the cell to sense and respond to the changes in the environ ment and has been related to denitrification in bacteria (31,32).NarXL in bacteria is a two-component system, where NarX is a transmembrane sensor protein of nitrate and nitrite, whereas NarL is the protein that acts directly on the promoter regions of some denitrification-related genes (31,32).Nevertheless, there are no homologous genes to narX and narL in H. mediterranei genome and there is no information about how H. mediterranei can sense the oxygen concentration and the presence of N-oxides, but it is worth exploring the possibility that these systems can have a role in it.
Moreover, it should be noted that quorum-sensing-related genes were also activated under this condition.Up to date, there are no reports about whether haloarchaeal denitrification is related to quorum sensing as in other organisms, but there is no doubt that these findings open new questions and ways to explore haloarchaeal denitrification (33,34).
Intriguingly, the biosynthesis of cofactors was repressed, and this is an unexpected feature since several cofactors are needed for the new respiratory enzymes (Fe-S clusters, MoCo cofactor, etc.) (35).This repression could be due to an adaptation to an energeti cally less favorable situation in which non-essential biosynthetic pathways are repressed.However, the levels of repression are not high, and only three genes show a fold change greater than |−2|.

Denitrification-related genes and more
RNAseq analysis revealed that the transcription of operons and genes encoding the main denitrification enzymes as well as some previously identified accessory proteins (electron donors) were induced under denitrifying conditions versus oxic conditions (Fig. 3; Tables S2 to S4) (20).Intriguingly, other proteins that have not previously been linked with denitrification in H. mediterranei showed an increase in their transcription levels.These are DUF2249 proteins and cyanoglobin which could have a potential role in haloarchaeal denitrification.
DUF2249 domain-containing proteins: DUF2249 (Domain of unknown function 2249) is a protein domain widespread among proteins from all domains of life.Its function is unknown and the information about it is scarce, but this domain has been related to two different protein superfamilies.Proteins of these superfamilies present the DDUF2249 domain, or the gene encoding these enzymes is close to other genes encoding DUF2249-containing proteins in the genome (36,37).DUF2249 has been related to the hemerythrin-like domain superfamily, which is a family mainly characterized by its ability to reversibly bind oxygen through its binuclear nonheme iron centers (37).However, this family is also involved in other interesting processes such as the repair of iron centers, the reduction of nitric oxide to nitrous oxide, or the binding of cations (37).DUF2249 has also been associated with the heme-copper oxidase superfamily (36).These enzymes are principally involved in the last step of aerobic respiration, reducing dioxygen to water.However, the nitric oxide reductases also belong to this superfamily and other hemecopper oxidases have been related to detoxification and exporting of N-compounds, maintenance of cellular iron homeostasis, and being part of the electron transference in denitrification (36).Moreover, two DUF2249-containing proteins from Thermus thermo philus called DrpA and DrpB have been linked directly with denitrification, being proposed as nitrate sensors (38).
The genome of H. mediterranei encodes four DUF2249-containing proteins, all of them differentially activated under denitrifying conditions.E6P09_00795 and E6P09_00770 genes (encoding one DUF2249 domain-containing protein each) are close to nirK and nor gene positions, E6P09_17395 (encoding one DUF2249 domain-containing protein) is between the nar and nos operons and E6P09_17415 is located in the nar operon (Fig. 3; Table 1; Table S2 to S4).
E6P09_00795 and E6P09_00770 were analyzed by STRING to look for possible interactions with other proteins (Fig. 4) (E6P09_17395 and E6P09_17415 STRING analysis were not possible because the proteins encoded by these genes did not appear in the STRING database).STRING network showed that these proteins present direct interactions with nirK and genes that encode proteins related to electron transference, such as halocyanins (E6P09_00760), and to the biosynthesis of the PQQ coenzyme (cmo_1).Indirectly, they also present interaction with nor, assimilatory nitrate reduc tase (nas), assimilatory nitrite reductase (nirA), and proteins involved in archaeal heme biosynthesis (nirD and nirJ) (39,40).Regarding this data, it seems that the DUF2249-con taining proteins could have a direct or indirect role in the nitrate metabolism in H. mediterranei.
To deepen this topic, an alignment and a phylogenetic tree were built using the nitrate sensor DrpA from T. thermophilus and the H. mediterranei proteins (Fig. 5) (38).The alignment showed that E6P09_17415 presents differences in length with the other proteins because it also carries other domains apart from the DUF2249 (perhaps due to a gene fusion) (Fig. 5A).E6P09_00795 gene product is the most similar (42.9% of identity) to DrpA and is the only one that conserves the histidine residues that are present in DrpA and other homologs found in nitrate respiration gene clusters of different Thermus spp.and other genera (Fig. 5A and B) (38).Hence, this gene product could have a sensor function like DrpA.
Cyanoglobin: The gene E6P09_11805 of H. mediterranei encodes a cyanoglobin whose expression is increased under denitrifying conditions (log 2 FC = 4.16).Hemoglobins are well-known proteins because they are essential for oxygen transport (41).In the three domains of life, there are different types of hemoglobins, cyanoglobins being one of them (42,43).Specifically, these belong to the group of truncated hemoglobins that have shown high binding capacities for oxygen (43,44).However, this is not the only ligand they can bind, they also can bind other gases such as nitric oxide with high affinity (43,44).This capacity of nitric oxide binding is related in some truncated hemoglobins with the potential dioxygenase activity that is present in some globins, which can efficiently convert nitric oxide to nitrate as a protective mechanism against nitrosative stress under conditions of low oxygen (43,45,46).Another function of globins is oxygen signaling, but this activity is not present in truncated hemoglobins like cyanoglobin (47).However, any of these functions has been proven in archaeal globins.
Reviewing, the exploration of the potential connection of DUF2249-containing proteins and cyanoglobins with denitrification showed interesting results that are very promising.Further investigation of these proteins using a genetic manipulation approach to obtain deletion/overexpression mutant strains would greatly help in understanding the role of these proteins in haloarchaeal denitrification if any.

Transcriptional regulators with differential expression under denitrifying conditions
The regulatory network under denitrification has been studied deeply in several bacteria from different genera but in H. mediterranei is completely unknown.This analysis has delved into this, and all the identified differentially expressed transcriptional regulators were selected for further analysis of their different domains to know in which processes they might be involved (Fig. 6).Metal-dependent transcriptional regulator.This regulator had the higher positive log 2 FC among all the identified regulators (Fig. 6).It belongs to the DtxR family of transcriptional repressors, that are well studied in pathogenic bacteria.These regulators were first studied in Corynebacterium diphtheriae because they control the expression of the tox operon involved in the biosynthesis of toxins (48,49).This control is mediated by the presence of iron, which acts as a ligand molecule (48,49).However, in other studies has been discovered that DtxR regulators not only control pathogenesis genes but also metal homeostasis in other organisms, including haloarchaea (50)(51)(52).The regulator found in this analysis possesses three domains: the HTH domain with DNA binding capacity, a ligand binding together with a dimerization domain, and a FeoA domain.These characteristics allow us to assign this regulator as a SirR transcriptional repressor.SirR usually responds to changes in iron concentration, as has been shown in other haloarchaea such as Halobacterium salinarum, but in this case, the oxic and denitrifying cultures had the same composition (therefore same iron concentration) (50,52).We also observed that a set of differentially expressed genes related to iron uptake are repressed under denitrifying conditions.These genes are listed in Table 2.This is a very interesting behavior because it opens new questions about how iron homeostasis and denitrification are connected.Thus, this SirR repressor could be a possible regulator of this set of genes.
PadR transcriptional regulator-GvpE Gas vesicle regulator: The production of gas vesicles is a strategy that allows some haloarchaea to float in the water column migrating to regions with optimal conditions (53).GvpE and GvpD are the known regulatory proteins that control the production of gas vesicles and GvpA and GvpC are the major components of them (54).In previous studies carried out in H. mediterranei, it has been found that the transcript levels of gvpA and gvpD genes are lower under denitrifying conditions compared to oxic conditions, and therefore the formation of gas vesicles is suppressed (55).Intriguingly, the data contrast with what has been found in the results of the present study, where the two gas vesicle gene clusters (gvpACNO and gvpDEF GHIJKLM) were differentially activated under anoxic conditions (Fig. 6 shows the log 2 FC of gvpE gene).In contrast with the growth conditions described in this work, Hechler and Pfeifer (55) grew H. mediterranei in a different media, and the anoxic conditions were set up by flushing gas into the media (55).The differences observed could be due to a more gradual transition to anoxia in the present study (and probably more similar to what happens in its natural environments).Maybe this gradual oxygen depletion could be the trigger for the gas vesicle synthesis as preparation for anoxia, being an alert signal for the cell to be adapted to a new condition in which these genes are expressed meanwhile oxygen is present.However, a fast transference to oxygen-restrictive conditions does not allow the cell to produce the whole gas vesicle machinery.
Bacterioopsin activators.Bacterioopsin activators are named due to their relationship to the activation of the bop gene cluster in H. salinarum (56,57).This cluster encodes the genes of the bacterioopsin biosynthesis, the bacteriorhodopsin proton pump, and its expression is induced in low oxygen concentration and availability of light conditions by the bat activator (56,58).The bat activator is characterized by a PAS domain, a redox sensory motif, a GAF domain, a light-responsive cyclic GMP-binding motif, an HTH domain, and an associated domain between the HTH and the GAF domain (21,59).In H. mediterranei, two genes that encode for transcriptional regulators similar to bacter ioopsin activators with differential expression, E6P09_08650 and E6P09_00735, have been found (Fig. 6).Intriguingly, both gene products lack the PAS and GAF domain and the E6P09_08650 also lacks the associated domain.However, other well-characterized bacterioopsin activators related to anaerobic metabolisms from different haloarchaea showed the lack of these domains too, as the NarO regulator related to the activation of denitrifying genes in H. volcanii, and the DmsR regulator that activates the DMSO respiration in H. salinarum, (21,60).Furthermore, as the bacterioopsin regulators found in other microorganisms, the genes found in H. mediterranei are also classified as dimethyl sulfoxide reductase transcriptional activators by the PANTHER classification system (61).
Regarding these characteristics, it can be concluded that is worth further studying these two regulators, as they may be the main regulators of the denitrification pathway in H. mediterranei due to their similarities with NarO and DmsR regulators.Especially, the E6P09_00735 gene due to its genomic location, since it is part of the nirK and nor genetic cluster, as it is discussed in the next section (Fig. 3; Table S2 to S4).Nitrogen assimilation proteins: Some genes related to ammonium assimilation showed differential expression levels.The genes encoding the regulatory proteins GlnK1 and GlnK2, and the high-affinity ammonium transporters Amt1 and Amt2, are overexpressed in anoxic conditions (Table 3).GlnK proteins (PII proteins) are signaling proteins involved in the sensing of cellular C/N and ATP/ADP ratios and are widely distributed among Bacteria and Archaea and they have also been found in plastids of plants (62,63).They regulate the intake of ammonium through the Amt transporter as well as the glutamine synthetase activity (64).In H. mediterranei, glnk1 is co-transcribed with amt1 and glnk2 with amt2, and their transcription is enhanced in the absence of ammonium (65).The increase in the expression of these genes in the present study could be due to the shift to anoxic conditions or the decrease in ammonium levels during the growth, although under the anoxic conditions, ammonium was still the nitrogen source, as no increase in the transcription of nitrate assimilation genes was detected.The decrease in ammonium concentration may also explain the increase in the transcription of the gene encoding glutamate synthase, gltS.The glutamine synthetase (GlnA)-glutamate synthase (GltS) pathway is preferential for ammonium assimilation when ammonium concentration is low, while in ammonium abundance, assimilation occurs preferentially via glutamate dehydrogenase.However, there is no increase in glnA transcription and no decrease in glutamate dehydrogenase, gdh1, transcription (Table 3), which is highly repressed under ammonium starvation (65).Probably, these changes in gene expression occur at lower ammonium concentrations than those obtained in the present study, but what is most noteworthy is the high transcriptional repression of glnA2 and glnA3 genes.These genes show a high homology with glutamine synthetase (GlnA), but it has been proposed that their role was different (66).Although the role of GlnA3 has not yet been described, GlnA2 is known to have glutamate-putrescine ligase activity and could be responsible for the growth of H. mediterranei in the presence of putrescine as the sole source of nitrogen and carbon (67).The transcriptional repression of both genes does not appear to be related to the decrease in ammonium concentration, as previous analyses of H. mediterranei's response to ammonium starvation did not reveal a reduction in the transcription of glnaA2 or glnA3.Therefore, the role of these two genes in the response to anoxia is unclear but could be due to a possible role of polyamines, such as putrescine, as metabolic regulators.It is known that polyamines stimulate the synthesis of some proteins and increase the fidelity of translation (68).In Escherichia coli, the effect of putrescine in gene transcription was studied, showing that some genes up-regulated were involved in iron uptake and energy metabolism, whereas down-regulated genes were related to amino acid metabolism or biosynthesis of cofactors and carriers (68).Similar effects could be given in H. mediterranei.ArsR transcriptional regulators: The ArsR family of transcriptional regulators is widely distributed not only in Bacteria but also in the Archaea domain, representing the third more abundant family of transcriptional regulators (69).Despite its abundance and contrary to the bacterial counterpart, the ArsR family in Archaea is not well studied and the information about it is limited.Mainly, these regulators are involved in heavy metal stress response, but in Archaea, they have also acquired other functions (70,71).However, up to date, there is no direct relationship between these regulators and the nitrogen cycle.In gram-positive bacteria, an ArsR repressor called SufR has been related to iron-sulfur cluster assembly but not in Archaea (72).The results of this study show that there were five differentially expressed regulators from this family, suggesting that some of these regulators are directly or indirectly involved in the anoxic/denitrifying response (Fig. 6).Therefore, more studies are needed to understand the specific role of these regulators and discover which genes are under their regulation.
Other transcriptional regulators: In this study, other regulators from different families have shown differential expression in denitrifying conditions concerning oxic condi tions.These are AsnC/Lrp, IclR, and PhoU regulators.AsnC/Lrp regulators are the most abundant in Archaea, they are considered global regulators that can act as activators or repressors and are involved in the regulation of many functions (69,73).They are well studied, and even in H. mediterranei there are studies of specific AsnC/Lrp regu lators which has been linked with the assimilation of nitrogen, but no relationship with denitrification has been established (74,75).IclR regulators are also involved in different processes and present a dual function acting not only as activators but also as repressors.As the AsnC/Lrp regulators, they are involved in different processes, such as carbon metabolisms, amino acid, and antibiotic production or quorum sensing, but no direct relationship with denitrification has been found (76)(77)(78)(79).Finally, PhoU is a regulator related to phosphate uptake, it has been studied in bacteria and the haloarchaeon H. salinarum, showing an increase in its expression (together with other phosphate metabolisms related genes called the PHO stimulon) when cells are grown under phosphate starvation (80,81).This is not the case in our study because phosphate concentration in the media is the same in all conditions.However, we have not measured the intracellular concentration and maybe during anoxic/denitrifying conditions could suffer some changes derived from the repression of this gene.

Genes encoding accessory proteins could share their transcriptomic regula tion with the main denitrification genes
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).The list of the promoter regions with the consensus sequence was used to look for regions under its regulation that were differentially expressed under denitrifying conditions (log 2 FC < −2 or log 2 FC > +2 and padj <0.05).The screening showed that 25 promoter regions displayed differentially expressed genes under its control (Table S5).
As expected, among these differentially expressed genes are the ones encoding the main enzymes (nar operon, nirK, nor, and nos operon).However, other genes in this list encode proteins that have been mentioned and discussed for the first time in this article as possible accessory proteins to denitrification in haloarchaea.These genes are the bacterioopsin activator gene E6P09_00735, the cyanoglobin gene E6P09_11805 and two out of four genes encoding DUF2249 domain containing-proteins (E6P09_00770 and E6P09_17415 genes).This finding strengthens the hypothesis that these proteins are involved in denitrification metabolism.Intriguingly, this list also contains two genomic organizations in which the motif was repeated in different promoter regions and showed a high number of genes with a positive log 2 FC value.The first organization is located on the main chromosome is about 12.5 kb (coordinates 142,837 to 155,489) and covers the region between E6P09_00735 and E6P09_00790 genes.This region has eight repeats of the motif in different positions (Fig. 3; Table S2 to S4).In addition, this gene clus ter includes not only the nirK gene and nor gene, but also two genes encoding for proteins containing the DUF2249 domain (genes E6P09_00770 and E6P09_00795), the bacterioopsin activator E6P09_00735 and halocyanins that can act as electron donors (E6P09_00740 and E6P09_00760).The second organization is located on the pHME322 megaplasmid, is about 26.8 kb, and is found between the E6P09_17385 gene and the beginning of the megaplasmid (coordinates 1 to 2,079) and E6P09_17435 gene and the end of the megaplasmid.This region has five repetitions of the motif, and it includes the nar and nos operons and the other two genes encoding DUF2249 domain-containing proteins (E6P09_17415 and E6P09_17395 genes) (Fig. 3; Table S2 to S4).
Most of the genes present in these two regions showed positive differential expression under denitrification conditions and their genomic location links different proteins that have been proposed to have a role in denitrification, such as the DUF2249 domain-containing proteins and the bacterioopsin activator (E6P09_00735).Although an evolutionary analysis is outside the scope of this article, these two delimited regions can give more information about the evolution of denitrification in haloarchaea if they are analyzed in depth.However, it is worth noting that nir and nor genes have been linked in the past (9,83).The presence of "genomic denitrification islands" in different prokaryotic genomes has been hypothesized to explain the clustering of denitrification genes and it could be the explanation of why these genes are clustered in H. mediterranei (83).In addition, the analysis of these clusters can be used to identify new genes related to the denitrification pathway, as is the case in this research, exploiting the idea that gene clustering is due to the fact that they code for proteins that interact physically or functionally (83)(84)(85).

Metal homeostasis is widely controlled under denitrifying conditions
Metals such as copper, iron, manganese, molybdenum, and nickel are micronutrients that act as redox centers or serve as a cofactor of different proteins in the cells (86)(87)(88)(89).However, other metals such as arsenic have no biological roles and are potentially toxic to cells (90).Consequently, metal homeostasis is an important process for maintaining cell integrity, and haloarchaea have developed different strategies for dealing with metallic compounds (87,88).
Expression analysis of different gene clusters under denitrifying conditions denoted that there is strict control of metal homeostasis in H. mediterranei cells.In the previous section, it was reported that a regulator of the DtxR family of transcriptional repress ors showed a high log 2 FC value under this condition, and it may be involved in the repression of the iron uptake of the cell.However, iron is not the only metal that seems to be regulated in this study.One of the two Zn uptake systems (ZnuABC) encoded in the H. mediterranei genome showed a highly positive expression change (Table 4).The Zn uptake system in haloarchaea is similar to the bacterial system.It is accomplished by a membrane permease (ZnuB), an ATPase (ZnuC), and a soluble periplasmic protein that captures Zn (II) and moves it to the membrane component ZnuB (91).This system in haloarchaea has been studied in the microorganisms H. volcanii, and it has been proposed that it is regulated by an sRNA (sRNA479) that negatively regulates its expression (92).sRNA479 does not have any homologous in H. mediterranei, but there are other sRNAs identified in the H. mediterranei genome that are in the same genetic position as sRNA479 (immediately downstream a CAS gene cassette).These are sRNA112, sRNA113, and sRNA114 (93).Other interesting gene clusters related to metals that showed highly positive differential expression are two operons that encode an electron donor (plastocyanin or halocyanin), a transmembrane protein of unknown function that showed similarity to metal transporters (data not shown) and a multi copper oxidase domain-containing protein (Table 4).These gene clusters probably are related to copper homeostasis, but multicopper oxidases have shown different roles and more experimental procedures would be needed to understand their role in this study (94).Another remarkable gene cluster with differential expression is encoding an SCO family protein together with a hypothetical protein, a thioredoxin family protein, and a cytochrome c biogenesis protein (Table 4).On the one hand, SCO proteins are involved in copper homeostasis, delivering Cu to other enzymes, or participating in protection against oxidative stress (95).On the other hand, it is known that NosZ possesses copper centers in its structure, and these centers need a maturation process to be active (96).One of the steps of this maturation process is the reduction of disulfide bonds present in the chaperone of the CuA center of NosZ and this reduction is carried out by an SCO protein (97,98).There is no evidence that this SCO protein is the protein in charge of this maturation of NosZ in H. mediterranei, but its gene is the only one encoding an SCO protein with high expression under denitrifying conditions.Furthermore, it is worth mentioning the acnA gene (E6P09_05600) encoding the aconitase enzyme from the tricarboxylic acid cycle is the only protein from this pathway that was differentially activated (Log 2 FC = 2.89) under denitrifying conditions (Table S6).In previous studies from mammals and some bacteria, it has been reported that this protein loses its aconitase activity by losing its [4Fe-4S] center under oxidative stress or iron deprivation (99).This event acts as a switch, enabling aconitase to bind RNA and regulate genes related to the iron response (100).This behavior has not been described in Archaea but looking at the changes in different iron genes during denitrification it would be interesting to explore the mRNA binding capacity of the archaeal aconitase to deep more in the metal homeostasis of this kind of microorganisms.Finally, an operon encoding molybdenum-related genes also showed upregulation under denitrifying conditions.This operon includes the molybdate transporter modA gene (E6P09_00610) and the mobA gene (E6P09_00615) involved in the biosynthesis of the molybdenum cofactor of some enzymes such as the respiratory nitrate reductase (89,101).In summary, metal homeostasis under denitrifying conditions is highly regulated in haloarchaea, especially the metabolism of iron, copper, molybdenum, and zinc showed differences in genes related to their metabolisms.This control is probably because cells need a balance between the requirement of cofactors to build all the key enzymes as well as accessory proteins necessary to perform denitrification (many of their structures involved MoCo cofactors, Fe-S clusters, and/or Cu centers) and the redox stress that a high concentration of metals could produce.
In conclusion, the transcriptomic analysis conducted in this study has provided comprehensive insights into the metabolic state of the microorganism under denitri fying conditions.The observed reduction in metabolic activity can be attributed to the inhibition of crucial pathways such as replication, transcription, and translation.Moreover, our investigation has established a link between denitrification and the regulation of metal homeostasis, as evidenced by substantial alterations in the expres sion of genes associated with metal-related processes.This phenomenon can be attributed to the fact that denitrification enzymes are metalloproteins, requiring optimal intracellular metal concentrations and redox balance for efficient functionality.
Furthermore, our analyses have unveiled several genes encoding proteins implicated in denitrification processes.Notably, bacterioopsin activators have emerged as potential key regulators of essential denitrification genes, while proteins harboring the DUF2249 domain and cyanoglobin appear to serve as putative sensors for denitrification-related nitrogen species, with the latter possibly functioning as an oxygen sensor as well.An additional intriguing aspect addressed in this study is the presence of regulatory motifs associated with denitrification in activated genes, reinforcing the notion of a coordinated and shared regulatory network governing these genes under denitrifying conditions.Moreover, up-regulation of genes encoding proteins related to gas vesicle production was observed, suggesting that cells employ this strategy to reach zones of higher oxygen in the water column.All these adaptations are summarized in Fig. 7.
The primary objective of this analysis is to serve as a valuable resource for future molecular studies, facilitating a deeper understanding of the genetic regulation and metabolic adaptations exhibited by haloarchaea in response to denitrification.The identification of potential targets for future investigations is explicitly delineated in this work, offering a roadmap for advancing our knowledge in this area of microbial physiol ogy.Aerobic cultures were grown in an Erlenmeyer flask, with shaking (170 rpm) and with a high air chamber (90% of flask volume) to ensure high oxygen exchange with the media.These cultures were grown to an optical density at 600 nm (OD 600 nm ) around 0.2 and then moved to denitrifying conditions.For setting up this condition, the cultures were transferred for 36 hours to closed flasks.These flasks were filled completely without leaving an air chamber in the head space to not allow gas exchange.All the cultures were conducted in triplicate.

Cell growth, RNA extraction, and rRNA depletion
RNA extraction was performed using the QIAGEN RNeasy Kit.Samples were taken from three biological replicates in both conditions: just before the culture switches to the denitrifying condition (OD 600 nm = 0.20-0.24),and after 36 hours of growing under this condition (OD 600 nm = 0.64-0.70).RNA from each sample was treated with Invitrogen Kit TURBO DNA-free.11 µg of RNA sample was digested in a 30 µL reaction using 8 Units (U) of TURBO DNAse.First, the samples were incubated at 37°C for 1 hour with 4 U of TURBO DNAse, then four more units were added, and the sample was incubated for one additional hour.DNA digestion result was checked by 2% TAE agarose gel and by PCR using primer pairs for three different genomic regions of the H. mediterranei genome (regions were selected randomly; Table S7).Moreover, all samples were analyzed before and after DNAse treatment by Agilent Bioanalyzer with an RNA 6000 Pico Kit, High Sensitivity RNA ScreenTape assay (RIN for all samples before DNAse treatment was >9 to be validated).
Finally, rRNA from samples was depleted using riboPOOL-specific probes for H. volcanii (siTOOLS Biotech) following the manufacturer's instructions.10 ng of RNA from each sample was used for the depletion.rRNA depletion was checked by Agilent Bioanalyzer with an RNA 6000 Pico Kit, High Sensitivity RNA ScreenTape assay.

RNA-Seq protocol, data processing, mRNA differential expression, and gene enrichment analysis
Depleted samples were sent to Microomics Systems S.L (Barcelona, Spain) for library construction using NEBNext® Ultra II Directional RNA Library Prep Kit for Illumina, and sequencing using Illumina Hiseq 2500 2 × 125.
Sequencing data were quality-checked using FastQC v0.11.8 and adaptors were trimmed by Trimmomatic v0.39 (102,103).Pre-processed data were aligned to the reference genome using Bowtie2 v2.3.5.1 and SAM files were converted to BAM files and indexed using SAMtools (104).The quality of the alignment was checked by qualimap v.2.2.2, and the count table was built with featureCounts v1.6.4 (105,106).Differential expression analysis between aerobic and denitrifying conditions was performed using DESeq2 v1.24.0 and Gene Set Enrichment Analysis (GSEA) was used to detect which KEGG pathways were enriched in the gene list derived from DESeq2 analysis (26,107,108).R package clusterProfiler v3.12.0 was used for this purpose using a gene expression filter of padj <0.05 (109).The expression changes of the genes encoding the main denitrification genes (narG, nirK, nor, and nosZ) were used as direct markers for the validation of the study.Finally, RNA-Seq data visualization was done using Integrative Genomics Viewer (IGV) software v2.13 (110).

Denitrification-related genes search by FIMO
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.This motif had been found in the promoter regions of the nar operon, nirK gene, nor gene, and nos operon in the mentioned study.Subsequently, the genes under the regulation of these promoter regions were used to identify the log 2 FC (FC: Fold Change) of its expression under denitrifying conditions.Finally, the genes that showed differential expression were filtered for further discussion.

Sequence alignments
Protein sequences of the gene products of the DUF2249 containing proteins present in the H. mediterranei genome and DrpA of T. thermophilus were downloaded from the UniProt Database (111).Sequences were aligned (default parameters were used) and afterward, a phylogenetic neighbor-joining tree (Bootstrap 500) was built with these alignments using MEGA 11 software (112).

FIG 1
FIG1 Volcano plot of the transcriptomic profile of differential gene expression between oxic and denitrifying conditions.

FIG 2
FIG 2 Gene set enrichment analysis (GSEA).Differentially suppressed or activated pathways of H. mediterranei under denitrifying conditions.

FIG 3
FIG 3 Gene clusters of the genes encoding the main enzymes of denitrification.Bars indicate the log 2 FC 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 5
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
FIG 6 Domain scheme of transcriptional regulators with differential expression.Yellow background, regulator with log 2 FC > +2; red background, regulators with log 2 FC < −2.HTH-domains, blue; ligand binding domains, gray; other domains, green.The length and location of the domains are an approximation.

FIG 7
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.

TABLE 1
Expression changes of all the DUF2249-containing proteins encoded in the H. mediterranei genome under denitrifying conditions (oxic vs denitrifying conditions) 4IG4STRING network of the protein interaction of two DUF2249 related with nitrate metabolism enzymes.

TABLE 3
Expression changes of the genes related to nitrogen assimilation in H. mediterranei under denitrifying conditions (oxic vs denitrifying conditions) a amtB2 and glnK2 genes are misannotated as a single transcript for amtB2.