Dihydroxyacetone (DHA) is a ketose sugar that can be produced by oxidizing glycerol. DHA in the environment is taken up and phosphorylated to DHA-phosphate by glycerol kinase or DHA kinase. In hypersaline environments, it is hypothesized that DHA is produced as an overflow product from glycerol utilization by organisms such as Salinibacter ruber. Previous research has demonstrated that the halobacterial species Haloquadratum walsbyi can use DHA as a carbon source, and putative DHA kinase genes were hypothesized to be involved in this process. However, DHA metabolism has not been demonstrated in other halobacterial species, and the role of the DHA kinase genes was not confirmed. In this study, we examined the metabolism of DHA in Haloferax volcanii because putative DHA kinase genes were annotated in its genome, and it has an established genetic system to assay growth of mutant knockouts. Experiments in which Hfx. volcanii was grown on DHA as the sole carbon source demonstrated growth, and that it is concentration dependent. Three annotated DHA kinase genes (HVO_1544, HVO_1545, and HVO_1546), which are homologous to the putative DHA kinase genes present in Hqm. walsbyi, as well as the glycerol kinase gene (HVO_1541), were deleted to examine the effect of these genes on the growth of Hfx. volcanii on DHA. Experiments demonstrated that the DHA kinase deletion mutant exhibited diminished, but not absence of growth on DHA compared to the parent strain. Deletion of the glycerol kinase gene also reduced growth on DHA, and did so more than deletion of the DHA kinase. The results indicate that Hfx. volcanii can metabolize DHA and that DHA kinase plays a role in this metabolism. However, the glycerol kinase appears to be the primary enzyme involved in this process. BLASTp analyses demonstrate that the DHA kinase genes are patchily distributed among the Halobacteria, whereas the glycerol kinase gene is widely distributed, suggesting a widespread capability for DHA metabolism.

Extracellular DNA is found in all environments and is a dynamic component of the microbial ecosystem. Microbial cells produce and interact with extracellular DNA through many endogenous mechanisms. Extracellular DNA is processed and internalized for use as genetic information and as a major source of macronutrients, and plays several key roles within prokaryotic biofilms. Hypersaline sites contain some of the highest extracellular DNA concentrations measured in nature-a potential rich source of carbon, nitrogen, and phosphorus for halophilic microorganisms. We conducted DNA growth studies for the halophilic archaeon Haloferax volcanii DS2 and show that this model Halobacteriales strain is capable of using exogenous double-stranded DNA as a nutrient. Further experiments with varying medium composition, DNA concentration, and DNA types revealed that DNA is utilized primarily as a phosphorus source, that growth on DNA is concentration-dependent, and that DNA isolated from different sources is metabolized selectively, with a bias against highly divergent methylated DNA. Additionally, fluorescence microscopy showed that labeled DNA co-localized with H. volcanii cells. The gene Hvo_1477 was also identified using a comparative genomic approach as a factor likely to be involved in DNA processing at the cell surface, and deletion of Hvo_1477 created a strain deficient in the ability to grow on extracellular DNA. Widespread distribution of Hvo_1477 homologs in archaea suggests metabolism of extracellular DNA may be of broad ecological and physiological relevance in this domain of life.

The basal transcription factor TFE enhances transcription initiation by catalysing DNA strand-separation, a process that varies with temperature and ionic strength. Canonical TFE forms a heterodimeric complex whose integrity and function critically relies on a cubane iron-sulphur cluster residing in the TFEβ subunit. Halophilic archaea such as Haloferax volcanii have highly divergent putative TFEβ homologues with unknown properties. Here, we demonstrate that Haloferax TFEβ lacks the prototypical iron-sulphur cluster yet still forms a stable complex with TFEα. A second metal cluster contained in the zinc ribbon domain in TFEα is highly degenerate but retains low binding affinity for zinc, which contributes to protein folding and stability. The deletion of the tfeB gene in H. volcanii results in the aberrant expression of approximately one third of all genes, consistent with its function as a basal transcription initiation factor. Interestingly, tfeB deletion particularly affects foreign genes including a prophage region. Our results reveal the loss of metal centres in Hvo transcription factors, and confirm the dual function of TFE as basal factor and regulator of transcription.

Polyploidy, the phenomenon of having more than one copy of the genome in an organism, is common among haloarchaea. While providing short-term benefits for DNA repair, polyploidy is generally regarded as an "evolutionary trap" that by the notion of the Muller's ratchet will inevitably conclude in the species' decline or even extinction due to a gradual reduction in fitness. In most reported cases of polyploidy in archaea, the genetic state of the organism is considered as homoploidy i.e. all copies of the genome are identical. Here we demonstrate that while this is indeed the prevalent genetic status in the halophilic archaeon Haloferax volcanii, its close relative H. mediterranei maintains a prolonged heteroploidy state in a nonselective environment once a second allele is introduced. Moreover, a strong genetic linkage was observed between two distant loci in H. mediterranei indicating a low rate of homologous recombination while almost no such linkage was shown in H. volcanii indicating a high rate of recombination in the latter species. We suggest that H. volcanii escapes Muller's ratchet by means of an effective chromosome-equalizing gene-conversion mechanism facilitated by highly active homologous recombination, whereas H. mediterranei must elude the ratchet via a different, yet to be elucidated mechanism.

Iron is part of many redox and other enzymes and, thus, it is essential for all living beings. Many oxic environments have extremely low concentrations of free iron. Therefore, many prokaryotic species evolved siderophores, i.e., small organic molecules that complex Fe3+ with very high affinity. Siderophores of bacteria are intensely studied, in contrast to those of archaea. The haloarchaeon Haloferax volcanii contains a gene cluster that putatively encodes siderophore biosynthesis genes, including four iron uptake chelate (iuc) genes. Underscoring this hypothesis, Northern blot analyses revealed that a hexacistronic transcript is generated that is highly induced under iron starvation. A quadruple iuc deletion mutant was generated, which had a growth defect solely at very low concentrations of Fe3+, not Fe2+. Two experimental approaches showed that the wild type produced and exported an Fe3+-specific siderophore under low iron concentrations, in contrast to the iuc deletion mutant. Bioinformatic analyses revealed that haloarchaea obtained the gene cluster by lateral transfer from bacteria and enabled the prediction of enzymatic functions of all six gene products. Notably, a biosynthetic pathway is proposed that starts with aspartic acid, uses several group donors and citrate, and leads to the hydroxamate siderophore Schizokinen.

S-layers commonly cover archaeal cell envelopes and are composed of proteins that self-assemble into a paracrystalline surface structure. Despite their detection in almost all archaea, there are few reports investigating the structural properties of these proteins, with no reports exploring this topic for halophilic S-layers. The objective of the present study was to investigate the secondary and tertiary organization of the Haloferax volcanii S-layer protein. Such investigations were performed using circular dichroism, fluorescence spectroscopy, dynamic light scattering and transmission electron microscopy. The protein secondary structure is centered on β-sheets and is affected by environmental pH, with higher disorder in more alkaline conditions. The pH can also affect the protein's tertiary structure, with higher tryptophan side-chain exposure to the medium under the same conditions. The concentrations of Na, Mg and Ca ions in the environment also affect the protein structures, with small changes in α-helix and β-sheet content, as well as changes in tryptophan side chain exposure. These changes in turn influence the protein's functional properties, with cell envelope preparations revealing striking differences when in different salt conditions. Thermal denaturation assays revealed that the protein is stable. It has been reported that the S-layer protein N-glycosylation process is affected by external factors and the present study indicates for the first time changes in the protein structure.

Haloferax volcanii is an easily culturable moderate halophile that grows on simple defined media, is readily transformable, and has a relatively stable genome. This, in combination with its biochemical and genetic tractability, has made Hfx. volcanii a key model organism, not only for the study of halophilicity, but also for archaeal biology in general.

Halophilic archaea thrive in hypersaline conditions associated with desiccation, ultraviolet (UV) irradiation and redox active compounds, and thus are naturally tolerant to a variety of stresses. Here, we identified mutations that promote enhanced tolerance of halophilic archaea to redox-active compounds using Haloferax volcanii as a model organism. The strains were isolated from a library of random transposon mutants for growth on high doses of sodium hypochlorite (NaOCl), an agent that forms hypochlorous acid (HOCl) and other redox acid compounds common to aqueous environments of high concentrations of chloride. The transposon insertion site in each of twenty isolated clones was mapped using the following: (i) inverse nested two-step PCR (INT-PCR) and (ii) semi-random two-step PCR (ST-PCR). Genes that were found to be disrupted in hypertolerant strains were associated with lysine deacetylation, proteasomes, transporters, polyamine biosynthesis, electron transfer, and other cellular processes. Further analysis revealed a ΔpsmA1 (α1) markerless deletion strain that produces only the α2 and β proteins of 20S proteasomes was hypertolerant to hypochlorite stress compared with wild type, which produces α1, α2, and β proteins. The results of this study provide new insights into archaeal tolerance of redox active compounds such as hypochlorite.

The function of membrane proteases range from general house-keeping to regulation of cellular processes. Although the biological role of these enzymes in archaea is poorly understood, some of them are implicated in the biogenesis of the archaeal cell envelope and surface structures. The membrane-bound ATP-dependent Lon protease is essential for cell viability and affects membrane carotenoid content in Haloferax volcanii. At least two different proteases are needed in this archaeon to accomplish the posttranslational modifications of the S-layer glycoprotein. The rhomboid protease RhoII is involved in the N-glycosylation of the S-layer protein with a sulfoquinovose-containing oligosaccharide while archaeosortase ArtA mediates the proteolytic processing coupled-lipid modification of this glycoprotein facilitating its attachment to the archaeal cell surface. Interestingly, two different signal peptidase I homologs exist in H. volcanii, Sec11a and Sec11b, which likely play distinct physiological roles. Type IV prepilin peptidase PibD processes flagellin/pilin precursors, being essential for the biogenesis and function of the archaellum and other cell surface structures in H. volcanii.

The halophilic archaeon Haloferax volcanii has a multireplicon genome, consisting of a main chromosome, three secondary chromosomes, and a plasmid. Genes for the initiator protein Cdc6/Orc1, which are commonly located adjacent to archaeal origins of DNA replication, are found on all replicons except plasmid pHV2. However, prediction of DNA replication origins in H. volcanii is complicated by the fact that this species has no less than 14 cdc6/orc1 genes. We have used a combination of genetic, biochemical, and bioinformatic approaches to map DNA replication origins in H. volcanii. Five autonomously replicating sequences were found adjacent to cdc6/orc1 genes and replication initiation point mapping was used to confirm that these sequences function as bidirectional DNA replication origins in vivo. Pulsed field gel analyses revealed that cdc6/orc1-associated replication origins are distributed not only on the main chromosome (2.9 Mb) but also on pHV1 (86 kb), pHV3 (442 kb), and pHV4 (690 kb) replicons. Gene inactivation studies indicate that linkage of the initiator gene to the origin is not required for replication initiation, and genetic tests with autonomously replicating plasmids suggest that the origin located on pHV1 and pHV4 may be dominant to the principal chromosomal origin. The replication origins we have identified appear to show a functional hierarchy or differential usage, which might reflect the different replication requirements of their respective chromosomes. We propose that duplication of H. volcanii replication origins was a prerequisite for the multireplicon structure of this genome, and that this might provide a means for chromosome-specific replication control under certain growth conditions. Our observations also suggest that H. volcanii is an ideal organism for studying how replication of four replicons is regulated in the context of the archaeal cell cycle.

Enzyme-mediated synthesis of pharmaceutical compounds is a 'green' alternative to traditional synthetic chemistry, and microbial engineering opens up the possibility of using whole cells as mini-factories. Whole-cell biocatalysis reduces cost by eliminating expensive enzyme purification and cofactor addition steps, as well as resulting in increased enzyme stability. Haloferax volcanii is a model halophilic archaeon encoding highly salt and organic solvent tolerant enzymes such as alcohol dehydrogenase (HvADH2), which catalyses the reduction of aldehydes and ketone in the presence of NADPH/NADH cofactor. A H. volcanii strain for constitutive HvADH2 expression was generated using a strong synthetic promoter (p.syn). The strain was immobilised in calcium alginate beads and repeatedly used as a whole-cell biocatalyst. The reduction of acetophenone, used as test substrate, was very successful and high yields were detected from immobilised whole cells over repeated biotransformation cycles. The immobilised H. volcanii retained stability and high product yields after 1 month of storage at room temperature. This newly developed system offers halophilic enzyme expression in its native environment, high product yield, stability and reusability without the addition of any expensive NADPH/NADH cofactor. This is the first report of whole cell-mediated biocatalysis by the halophilic archaeon H. volcanii.

In the halophilic archaea Haloferax volcanii, the surface (S)-layer glycoprotein can be modified by two distinct N-linked glycans. The tetrasaccharide attached to S-layer glycoprotein Asn-498 comprises a sulfated hexose, two hexoses and a rhamnose. While Agl11-14 have been implicated in the appearance of the terminal rhamnose subunit, the precise roles of these proteins have yet to be defined. Accordingly, a series of in vitro assays conducted with purified Agl11-Agl14 showed these proteins to catalyze the stepwise conversion of glucose-1-phosphate to dTDP-rhamnose, the final sugar of the tetrasaccharide glycan. Specifically, Agl11 is a glucose-1-phosphate thymidylyltransferase, Agl12 is a dTDP-glucose-4,6-dehydratase and Agl13 is a dTDP-4-dehydro-6-deoxy-glucose-3,5-epimerase, while Agl14 is a dTDP-4-dehydrorhamnose reductase. Archaea thus synthesize nucleotide-activated rhamnose by a pathway similar to that employed by Bacteria and distinct from that used by Eukarya and viruses. Moreover, a bioinformatics screen identified homologues of agl11-14 clustered in other archaeal genomes, often as part of an extended gene cluster also containing aglB, encoding the archaeal oligosaccharyltransferase. This points to rhamnose as being a component of N-linked glycans in Archaea other than Hfx. volcanii.

Archaeal genomes are densely packed; thus, correct transcription termination is an important factor for orchestrated gene expression. A systematic analysis of RNA 3´ termini, to identify transcription termination sites (TTS) using RNAseq data has hitherto only been performed in two archaea, Methanosarcina mazei and Sulfolobus acidocaldarius. In this study, only regions directly downstream of annotated genes were analysed, and thus, only part of the genome had been investigated. Here, we developed a novel algorithm (Internal Enrichment-Peak Calling) that allows an unbiased, genome-wide identification of RNA 3´ termini independent of annotation. In an RNA fraction enriched for primary transcripts by terminator exonuclease (TEX) treatment we identified 1,543 RNA 3´ termini. Approximately half of these were located in intergenic regions, and the remainder were found in coding regions. A strong sequence signature consistent with known termination events at intergenic loci indicates a clear enrichment for native TTS among them. Using these data we determined distinct putative termination motifs for intergenic (a T stretch) and coding regions (AGATC). In vivo reporter gene tests of selected TTS confirmed termination at these sites, which exemplify the different motifs. For several genes, more than one termination site was detected, resulting in transcripts with different lengths of the 3´ untranslated region (3´ UTR).

Archaea play indispensable roles in global biogeochemical cycles, yet many crucial cellular processes, including cell-shape determination, are poorly understood. Haloferax volcanii, a model haloarchaeon, forms rods and disks, depending on growth conditions. Here, we used a combination of iterative proteomics, genetics, and live-cell imaging to identify mutants that only form rods or disks. We compared the proteomes of the mutants with wild-type cells across growth phases, thereby distinguishing between protein abundance changes specific to cell shape and those related to growth phases. The results identified a diverse set of proteins, including predicted transporters, transducers, signaling components, and transcriptional regulators, as important for cell-shape determination. Through phenotypic characterization of deletion strains, we established that rod-determining factor A (RdfA) and disk-determining factor A (DdfA) are required for the formation of rods and disks, respectively. We also identified structural proteins, including an actin homolog that plays a role in disk-shape morphogenesis, which we named volactin. Using live-cell imaging, we determined volactin's cellular localization and showed its dynamic polymerization and depolymerization. Our results provide insights into archaeal cell-shape determination, with possible implications for understanding the evolution of cell morphology regulation across domains.

N-glycosylation is a post-translational modification performed by members of all three domains of life. Studies on the halophile Haloferax volcanii have offered insight into the archaeal version of this universal protein-processing event. In the present study, AglQ was identified as a novel component of the pathway responsible for the assembly and addition of a pentasaccharide to select Asn residues of Hfx. volcanii glycoproteins, such as the S-layer glycoprotein. In cells deleted of aglQ, both dolichol phosphate, the lipid carrier used in Hfx. volcanii N-glycosylation, and modified S-layer glycoprotein Asn residues only presented the first three pentasaccharide subunits, pointing to a role for AglQ in either preparing the third sugar for attachment of the fourth pentasaccharide subunit or processing the fourth sugar prior to its addition to the lipid-linked trisaccharide. To better define the precise role of AglQ, shown to be a soluble protein, bioinformatics tools were recruited to identify sequence or structural homologs of known function. Site-directed mutagenesis experiments guided by these predictions identified residues important for AglQ function. The results obtained point to AglQ acting as an isomerase in Hfx. volcanii N-glycosylation.

In most bacteria, cell division depends on the tubulin homolog FtsZ and other proteins, such as SepF, that form a complex termed the divisome. Cell division also depends on FtsZ in many archaea, but other components of the divisome are unknown. Here, we demonstrate that a SepF homolog plays important roles in cell division in Haloferax volcanii, a halophilic archaeon that is known to have two FtsZ homologs with slightly different functions (FtsZ1 and FtsZ2). SepF co-localizes with both FtsZ1 and FtsZ2 at midcell. Attempts to generate a sepF deletion mutant were unsuccessful, suggesting an essential role. Indeed, SepF depletion leads to severe cell division defects and formation of large cells. Overexpression of FtsZ1-GFP or FtsZ2-GFP in SepF-depleted cells results in formation of filamentous cells with a high number of FtsZ1 rings, while the number of FtsZ2 rings is not affected. Pull-down assays support that SepF interacts with FtsZ2 but not with FtsZ1, although SepF appears delocalized in the absence of FtsZ1. Archaeal SepF homologs lack a glycine residue known to be important for polymerization and function in bacteria, and purified H. volcanii SepF forms dimers, suggesting that polymerization might not be important for the function of archaeal SepF.

Type IV pili are evolutionarily conserved cell surface filaments that promote surface adhesion and cell aggregation providing bacteria and archaea protection from a variety of stress conditions. In fact, prokaryotic genomes frequently contain several copies of the core biosynthesis genes, pilB and pilC, encoding an ATPase and membrane anchor, respectively. Recent phylogenetic analyses suggest that in haloarchaea, a subset of pilB-C paralogs, such as the Haloferax volcanii pilB1-C1, were gained via horizontal transfer from the crenarchaea, while the co-regulated type IV pilus subunits, the pilins, evolved by duplication, followed by diversification of the ancestral euryarchaeal pilins. Here, we report the identification of an H. volcanii pilB1 transposon mutant that exhibits an adhesion defect in defined media. A similar defect observed in an H. volcanii ∆pilB1-C1 strain can be rescued by expressing pilB1-C1 in trans. However, these proteins cannot rescue the severe adhesion defect of a previously reported ∆pilB3-C3 strain. Conversely, pilB3-C3, which are not predicted to have been laterally transferred, expressed in trans can rescue the adhesion defect of a ∆pilB1-C1 strain. This cross-complementation supports the proposed hybrid origin of the operon containing pilB1-C1 and shows that at least certain euryarchaeal PilB paralogs can work with different pilin sets. Efficient recognition of the euryarchaeal pilins by the crenarchaeal PilB1-C1 may have required some degree of pilin modification, but perhaps the modifications were minor enough that PilB3 recognition of these pilins was not precluded, resulting in modular evolution and an extensive combinatorial diversity that allows for adaptation to a variety of stress conditions and attachment to varied surfaces.

Post-transcriptional processing of messenger RNA is an important regulatory strategy that allows relatively fast responses to changes in environmental conditions. In halophile systems biology, the protein perspective of this problem (i.e., ribonucleases which implement the cleavages) is generally more studied than the RNA perspective (i.e., processing sites). In the present in silico work, we mapped genome-wide transcriptional processing sites (TPS) in two halophilic model organisms, Halobacterium salinarum NRC-1 and Haloferax volcanii DS2. TPS were established by reanalysis of publicly available differential RNA-seq (dRNA-seq) data, searching for non-primary (monophosphorylated RNAs) enrichment. We found 2093 TPS in 43% of H. salinarum genes and 3515 TPS in 49% of H. volcanii chromosomal genes. Of the 244 conserved TPS sites found, the majority were located around start and stop codons of orthologous genes. Specific genes are highlighted when discussing antisense, ribosome and insertion sequence associated TPS. Examples include the cell division gene ftsZ2, whose differential processing signal along growth was detected and correlated with post-transcriptional regulation, and biogenesis of sense overlapping transcripts associated with IS200/IS605. We hereby present the comparative, transcriptomics-based processing site maps with a companion browsing interface.

Persister cells are phenotypic variants within a microbial population, which are dormant and transiently tolerant to stress. Persistence has been studied extensively in bacteria, and in eukaryotes to a limited extent, however, it has never been observed in archaea. Using the model haloarchaeon, Haloferax volcanii DS2, we demonstrated persister cell formation in this domain, with time-kill curves exhibiting a characteristic biphasic pattern following starvation or exposure to lethal concentrations of various biocidal compounds. Repeated challenges of surviving cells showed that, as with bacteria, persister formation in H. volcanii was not heritable. Additionally, as previously shown with bacteria, persister formation in H. volcanii was suppressed by exogenous indole. The addition of spent culture media to assays conducted on planktonic cells showed that H. volcanii-conditioned media stimulated persistence, whereas conditioned media of other haloarchaea or halophilic bacteria did not, suggesting the involvement of a species-specific signal. Using a TLC overlay assay, the quorum sensing bioreporter Agrobacterium tumefaciens ATCC BAA-2240 detected the presence of C4 and C6 acyl homoserine lactone-like signal molecules in a H. volcanii culture extract. While synthetic bacterial AHLs did not induce persistence, this is potentially due to structural differences between bacterial and archaeal signals, and does not discount a quorum sensing component in haloarchaeal persister formation. The observation of persister cell formation by this haloarchaeon may provide some insights into the survival of these organisms in stressful or dynamic environments.

Transcriptional regulators that integrate cellular and environmental signals to control cell division are well known in bacteria and eukaryotes, but their existence is poorly understood in archaea. We identified a conserved gene (cdrS) that encodes a small protein and is highly transcribed in the model archaeon Haloferax volcanii. The cdrS gene could not be deleted, but CRISPR interference (CRISPRi)-mediated repression of the cdrS gene caused slow growth and cell division defects and changed the expression of multiple genes and their products associated with cell division, protein degradation, and metabolism. Consistent with this complex regulatory network, overexpression of cdrS inhibited cell division, whereas overexpression of the operon encoding both CdrS and a tubulin-like cell division protein (FtsZ2) stimulated division. Chromatin immunoprecipitation-DNA sequencing (ChIP-Seq) identified 18 DNA-binding sites of the CdrS protein, including one upstream of the promoter for a cell division gene, ftsZ1, and another upstream of the essential gene dacZ, encoding diadenylate cyclase involved in c-di-AMP signaling, which is implicated in the regulation of cell division. These findings suggest that CdrS is a transcription factor that plays a central role in a regulatory network coordinating metabolism and cell division. IMPORTANCE Cell division is a central mechanism of life and is essential for growth and development. Members of the Bacteria and Eukarya have different mechanisms for cell division, which have been studied in detail. In contrast, cell division in members of the Archaea is still understudied, and its regulation is poorly understood. Interestingly, different cell division machineries appear in members of the Archaea, with the Euryarchaeota using a cell division apparatus based on the tubulin-like cytoskeletal protein FtsZ, as in bacteria. Here, we identify the small protein CdrS as essential for survival and a central regulator of cell division in the euryarchaeon Haloferax volcanii. CdrS also appears to coordinate other cellular pathways, including synthesis of signaling molecules and protein degradation. Our results show that CdrS plays a sophisticated role in cell division, including regulation of numerous associated genes. These findings are expected to initiate investigations into conditional regulation of division in archaea.