Using the pan-genomic framework for the discovery of genomic islands in the haloarchaeon Halorubrum ezzemoulense.

In this study, we use pan-genomics to characterize the genomic variability of the widely dispersed halophilic archaeal species Halorubrum ezzemoulense (Hez). We include a multi-regional sampling of newly sequenced, high-quality draft genomes. The pan-genome graph of the species reveals 50 genomic islands that represent rare accessory genetic capabilities available to members. Most notably, we observe rearrangements that have led to the insertion/recombination/replacement of mutually exclusive genomic islands in equivalent genome positions ("homeocassettes"). These conflicting islands encode for similar functions, but homologs from islands located between the same core genes exhibit high divergence on the amino acid level, while the neighboring core genes are nearly identical. Both islands of a homeocassette often coexist in the same geographic location, suggesting that either island may be beyond the reach of selective sweeps and that these loci of divergence between Hez members are maintained and persist long term. This implies that subsections of the population have different niche preferences and rare metabolic capabilities. After an evaluation of the gene content in the homeocassettes, we speculate that these islands may play a role in the speciation, niche adaptability, and group selection dynamics in Hez. Though homeocassettes are first described in this study, similar replacements and divergence of genes on genomic islands have been previously reported in other Haloarchaea and distantly related Archaea, suggesting that homeocassettes may be a feature in a wide range of organisms outside of Hez.IMPORTANCEThis study catalogs the rare genes discovered in strains of the species Halorubrum ezzemoulense (Hez), an obligate halophilic archaeon, through the perspective of its pan-genome. These rare genes are often found to be arranged on islands that confer metabolic and transport functions and contain genes that have eluded previous studies. The discovery of divergent, but homologous islands occupying equivalent genome positions ("homeocassettes") in different genomes, reveals significant new information on genome evolution in Hez. Homeocassette pairs encode for similar functions, but their dissimilarity and distribution imply high rates of recombination, different specializations, and niche preferences in Hez. The coexistence of both islands of a homeocassette pair in multiple environments demonstrates that both islands are beyond the reach of selective sweeps and that these genome content differences between strains persist long term. The switch between islands through recombination under different environmental conditions may lead to a greater range of niche adaptability in Hez.

Neighboring inteins interfere with one another's homing capacity.

Inteins are mobile genetic elements that invade conserved genes across all domains of life and viruses. In some instances, a single gene will have several intein insertion sites. In Haloarchaea, the minichromosome maintenance (MCM) protein at the core of replicative DNA helicase contains four intein insertion sites within close proximity, where two of these sites (MCM-a and MCM-d) are more likely to be invaded. A haloarchaeon that harbors both MCM-a and MCM-d inteins, Haloferax mediterranei, was studied in vivo to determine intein invasion dynamics and the interactions between neighboring inteins. Additionally, invasion frequencies and the conservation of insertion site sequences in 129 Haloferacales mcm homologs were analyzed to assess intein distribution across the order. We show that the inteins at MCM-a and MCM-d recognize and cleave their respective target sites and, in the event that only one empty intein invasion site is present, readily initiate homing (i.e. single homing). However, when two inteins are present co-homing into an intein-free target sequence is much less effective. The two inteins are more effective when invading alleles that already contain an intein at one of the two sites. Our in vivo and computational studies also support that having a proline in place of a serine as the first C-terminal extein residue of the MCM-d insertion site prevents successful intein splicing, but does not stop recognition of the insertion site by the intein's homing endonuclease.

Archaeal Tubulin-like Proteins Modify Cell Shape in Haloferax volcanii during Early Biofilm Development.

Tubulin, an extensively studied self-assembling protein, forms filaments in eukaryotic cells that affect cell shape, among other functions. The model archaeon Haloferax volcanii uses two tubulin-like proteins (FtsZ1/FtsZ2) for cell division, similar to bacteria, but has an additional six related tubulins called CetZ. One of them, CetZ1, was shown to play a role in cell shape. Typically, discoid and rod shapes are observed in planktonic growth, but under biofilm formation conditions (i.e., attached to a substratum), H. volcanii can grow filamentously. Here, we show that the deletion mutants of all eight tubulin-like genes significantly impacted morphology when cells were allowed to form a biofilm. ΔftsZ1, ΔcetZ2, and ΔcetZ4-6 created longer, less round cells than the parental and a higher percentage of filaments. ΔcetZ1 and ΔcetZ3 were significantly rounder than the parental, and ΔftsZ2 generated larger, flat, amorphic cells. The results show all tubulin homologs affect morphology at most timepoints, which therefore suggests these genes indeed have a function.

The Evolutionary Origins of Extreme Halophilic Archaeal Lineages.

Interest and controversy surrounding the evolutionary origins of extremely halophilic Archaea has increased in recent years, due to the discovery and characterization of the Nanohaloarchaea and the Methanonatronarchaeia. Initial attempts in explaining the evolutionary placement of the two new lineages in relation to the classical Halobacteria (also referred to as Haloarchaea) resulted in hypotheses that imply the new groups share a common ancestor with the Haloarchaea. However, more recent analyses have led to a shift: the Nanohaloarchaea have been largely accepted as being a member of the DPANN superphylum, outside of the euryarchaeota; whereas the Methanonatronarchaeia have been placed near the base of the Methanotecta (composed of the class II methanogens, the Halobacteriales, and Archaeoglobales). These opposing hypotheses have far-reaching implications on the concepts of convergent evolution (distantly related groups evolve similar strategies for survival), genome reduction, and gene transfer. In this work, we attempt to resolve these conflicts with phylogenetic and phylogenomic data. We provide a robust taxonomic sampling of Archaeal genomes that spans the Asgardarchaea, TACK Group, euryarchaeota, and the DPANN superphylum. In addition, we assembled draft genomes from seven new representatives of the Nanohaloarchaea from distinct geographic locations. Phylogenies derived from these data imply that the highly conserved ATP synthase catalytic/noncatalytic subunits of Nanohaloarchaea share a sisterhood relationship with the Haloarchaea. We also employ a novel gene family distance clustering strategy which shows this sisterhood relationship is not likely the result of a recent gene transfer. In addition, we present and evaluate data that argue for and against the monophyly of the DPANN superphylum, in particular, the inclusion of the Nanohaloarchaea in DPANN.

Nutrient supplementation experiments with saltern microbial communities implicate utilization of DNA as a source of phosphorus.

All environments including hypersaline ones harbor measurable concentrations of dissolved extracellular DNA (eDNA) that can be utilized by microbes as a nutrient. However, it remains poorly understood which eDNA components are used, and who in a community utilizes it. For this study, we incubated a saltern microbial community with combinations of carbon, nitrogen, phosphorus, and DNA, and tracked the community response in each microcosm treatment via 16S rRNA and rpoB gene sequencing. We show that microbial communities used DNA only as a phosphorus source, and provision of other sources of carbon and nitrogen was needed to exhibit a substantial growth. The taxonomic composition of eDNA in the water column changed with the availability of inorganic phosphorus or supplied DNA, hinting at preferential uptake of eDNA from specific organismal sources. Especially favored for growth was eDNA from the most abundant taxa, suggesting some haloarchaea prefer eDNA from closely related taxa. The preferential eDNA consumption and differential growth under various nutrient availability regimes were associated with substantial shifts in the taxonomic composition and diversity of microcosm communities. Therefore, we conjecture that in salterns the microbial community assembly is driven by the available resources, including eDNA.

Adaptation reduces competitive dominance and alters community assembly.

A growing body of theory predicts that evolution of an early-arriving species in a new environment can produce a competitive advantage against later arriving species, therefore altering community assembly (i.e. the community monopolization hypothesis). Applications of the community monopolization hypothesis are increasing. However, experimental tests of the hypothesis are rare. Here, we provide a rare experimental demonstration of the community monopolization hypothesis using two archaeal species. We first expose one species to low- and high-temperature environments for 135 days. Populations in the high-temperature treatment evolved a 20% higher median performance when grown at high temperature. We then demonstrate that early arrival and adaptation reduce the abundance of a late-arriving species in the high-temperature environment by 63% relative to when both species arrive simultaneously and neither species is adapted to high temperature. These results are consistent with the community monopolization hypothesis and suggest that adaptation can reduce competitive dominance to alter community assembly. Hence, community monopolization might be much more common in nature than previously assumed. Our results strongly support the idea that patterns of biodiversity might often stem from a race between local adaptation and colonization of pre-adapted species.

Insights into gene expression changes under conditions that facilitate horizontal gene transfer (mating) of a model archaeon.

Horizontal gene transfer is a means by which bacteria, archaea, and eukaryotes are able to trade DNA within and between species. While there are a variety of mechanisms through which this genetic exchange can take place, one means prevalent in the archaeon Haloferax volcanii involves the transient formation of cytoplasmic bridges between cells and is referred to as mating. This process can result in the exchange of very large fragments of DNA between the participating cells. Genes governing the process of mating, including triggers to initiate mating, mechanisms of cell fusion, and DNA exchange, have yet to be characterized. We used a transcriptomic approach to gain a more detailed knowledge of how mating might transpire. By examining the differential expression of genes expressed in cells harvested from mating conditions on a filter over time and comparing them to those expressed in a shaking culture, we were able to identify genes and pathways potentially associated with mating. These analyses provide new insights into both the mechanisms and barriers of mating in Hfx. volcanii.

Roadmap for naming uncultivated Archaea and Bacteria.

The assembly of single-amplified genomes (SAGs) and metagenome-assembled genomes (MAGs) has led to a surge in genome-based discoveries of members affiliated with Archaea and Bacteria, bringing with it a need to develop guidelines for nomenclature of uncultivated microorganisms. The International Code of Nomenclature of Prokaryotes (ICNP) only recognizes cultures as 'type material', thereby preventing the naming of uncultivated organisms. In this Consensus Statement, we propose two potential paths to solve this nomenclatural conundrum. One option is the adoption of previously proposed modifications to the ICNP to recognize DNA sequences as acceptable type material; the other option creates a nomenclatural code for uncultivated Archaea and Bacteria that could eventually be merged with the ICNP in the future. Regardless of the path taken, we believe that action is needed now within the scientific community to develop consistent rules for nomenclature of uncultivated taxa in order to provide clarity and stability, and to effectively communicate microbial diversity.

Interaction of two strongly divergent archaellins stabilizes the structure of the Halorubrum archaellum.

Halophilic archaea from the genus Halorubrum possess two extraordinarily diverged archaellin genes, flaB1 and flaB2. To clarify roles for each archaellin, we compared two natural Halorubrum lacusprofundi strains: One of them contains both archaellin genes, and the other has the flaB2 gene only. Both strains synthesize functional archaella; however, the strain, where both archaellins are present, is more motile. In addition, we expressed these archaellins in a Haloferax volcanii strain from which the endogenous archaellin genes were deleted. Three Hfx. volcanii strains expressing Hrr. lacusprofundi archaellins produced functional filaments consisting of only one (FlaB1 or FlaB2) or both (FlaB1/FlaB2) archaellins. All three strains were motile, although there were profound differences in the efficiency of motility. Both native and recombinant FlaB1/FlaB2 filaments have greater thermal stability and resistance to low salinity stress than single-component filaments. Functional supercoiled Hrr. lacusprofundi archaella can be composed of either single archaellin: FlaB2 or FlaB1; however, the two divergent archaellin subunits provide additional stabilization to the archaellum structure and thus adaptation to a wider range of external conditions. Comparative genomic analysis suggests that the described combination of divergent archaellins is not restricted to Hrr. lacusprofundi, but is occurring also in organisms from other haloarchaeal genera.

Complete Genome Sequence of Halorubrum ezzemoulense Strain Fb21.

Isolated from Aran-Bidgol Lake in Iran, and reported here, Halorubrum ezzemoulense strain Fb21 represents the first complete genome from this archaeal species. Local recombination in this genome is in stark contrast to equidistant recombination events in bacteria. The genome's GC bias, however, points to a genome architecture and origin that resemble those of a bacterium. Its availability, genome signatures, and frequent intragenomic recombination mean that Fb21 presents an attractive model organism for this species.

The Patchy Distribution of Restriction⁻Modification System Genes and the Conservation of Orphan Methyltransferases in Halobacteria.

Restriction⁻modification (RM) systems in bacteria are implicated in multiple biological roles ranging from defense against parasitic genetic elements, to selfish addiction cassettes, and barriers to gene transfer and lineage homogenization. In bacteria, DNA-methylation without cognate restriction also plays important roles in DNA replication, mismatch repair, protein expression, and in biasing DNA uptake. Little is known about archaeal RM systems and DNA methylation. To elucidate further understanding for the role of RM systems and DNA methylation in Archaea, we undertook a survey of the presence of RM system genes and related genes, including orphan DNA methylases, in the halophilic archaeal class Halobacteria. Our results reveal that some orphan DNA methyltransferase genes were highly conserved among lineages indicating an important functional constraint, whereas RM systems demonstrated patchy patterns of presence and absence. This irregular distribution is due to frequent horizontal gene transfer and gene loss, a finding suggesting that the evolution and life cycle of RM systems may be best described as that of a selfish genetic element. A putative target motif (CTAG) of one of the orphan methylases was underrepresented in all of the analyzed genomes, whereas another motif (GATC) was overrepresented in most of the haloarchaeal genomes, particularly in those that encoded the cognate orphan methylase.

Halorubrum chaoviator Mancinelli et al. 2009 is a later, heterotypic synonym of Halorubrum ezzemoulense Kharroub et al. 2006. Emended description of Halorubrum ezzemoulense Kharroub et al. 2006.

A polyphasic comparative taxonomic study of Halorubrum ezzemoulense Kharroub et al. 2006, Halorubrum chaoviator Mancinelli et al. 2009 and eight new Halorubrum strains related to these haloarchaeal species was carried out. Multilocus sequence analysis using the five concatenated housekeeping genes atpB, EF-2, glnA, ppsA and rpoB', and phylogenetic analysis based on the 757 core protein sequences obtained from their genomes showed that Hrr. ezzemoulense DSM 17463T, Hrr. chaoviator Halo-G*T (=DSM 19316T) and the eight Halorubrum strains formed a robust cluster, clearly separated from the remaining species of the genus Halorubrum. The orthoANI value and digital DNA-DNA hybridization value, calculated by the Genome-to-Genome Distance Calculator (GGDC), showed percentages among Hrr. ezzemoulense DSM 17463T, Hrr. chaoviator DSM 19316T and the eight Halorubrum strains ranging from 99.4 to 97.9 %, and from 95.0 to 74.2 %, respectively, while these values for those strains and the type strains of the most closely related species of Halorubrum were 88.7-77.4 % and 36.1-22.3 %, respectively. Although some differences were observed, the phenotypic and polar lipid profiles were quite similar for all the strains studied. Overall, these data show that Hrr. ezzemoulense, Hrr. chaoviator and the eight new Halorubrum isolates constitute a single species. Thus, Hrr. chaoviator should be considered as a later, heterotypic synonym of Hrr. ezzemoulense. We propose an emended description of Hrr. ezzemoulense, including the features of Hrr. chaoviator and those of the eight new isolates.

Hundreds of novel composite genes and chimeric genes with bacterial origins contributed to haloarchaeal evolution.

BACKGROUND: Haloarchaea, a major group of archaea, are able to metabolize sugars and to live in oxygenated salty environments. Their physiology and lifestyle strongly contrast with that of their archaeal ancestors. Amino acid optimizations, which lowered the isoelectric point of haloarchaeal proteins, and abundant lateral gene transfers from bacteria have been invoked to explain this deep evolutionary transition. We use network analyses to show that the evolution of novel genes exclusive to Haloarchaea also contributed to the evolution of this group. RESULTS: We report the creation of 320 novel composite genes, both early in the evolution of Haloarchaea during haloarchaeal genesis and later in diverged haloarchaeal groups. One hundred and twenty-six of these novel composite genes derived from genetic material from bacterial genomes. These latter genes, largely involved in metabolic functions but also in oxygenic lifestyle, constitute a different gene pool from the laterally acquired bacterial genes formerly identified. These novel composite genes were likely advantageous for their hosts, since they show significant residence times in haloarchaeal genomes-consistent with a long phylogenetic history involving vertical descent and lateral gene transfer-and encode proteins with optimized isoelectric points. CONCLUSIONS: Overall, our work encourages a systematic search for composite genes across all archaeal major groups, in order to better understand the origins of novel prokaryotic genes, and in order to test to what extent archaea might have adjusted their lifestyles by incorporating and recycling laterally acquired bacterial genetic fragments into new archaeal genes.

Genotypic and Lipid Analyses of Strains From the Archaeal Genus Halorubrum Reveal Insights Into Their Taxonomy, Divergence, and Population Structure.

To gain a better understanding of how divergence occurs, and how taxonomy can benefit from studying natural populations, we isolated and examined 25 closely related Halorubrum strains obtained from different hypersaline communities and compared them to validly named species and other reference strains using five taxonomic study approaches: phylogenetic analysis using the 16S rRNA gene and multilocus sequencing analysis (MLSA), polar lipid profiles (PLP), average nucleotide identity (ANI) and DNA-DNA hybridization (DDH). 16S rRNA gene sequence could not differentiate the newly isolated strains from described species, while MLSA grouped strains into three major clusters. Two of those MLSA clusters distinguished candidates for new species. The third cluster with concatenated sequence identity equal to or greater than 97.5% was comprised of strains from Aran-Bidgol Lake (Iran) and solar salterns in Namibia and Spain, and two previously described species isolated from Mexico and Algeria. PLP and DDH analyses showed that Aran-Bidgol strains formed uniform populations, and that strains isolated from other geographic locations were heterogeneous and divergent, indicating that they may constitute different species. Therefore, applying only sequencing approaches and similarity cutoffs for circumscribing species may be too conservative, lumping concealed diversity into a single taxon. Further, our data support the interpretation that local populations experience unique evolutionary homogenization pressures, and once relieved of insular constraints (e.g., through migration) are free to diverge.

Comparative Analysis of Surface Layer Glycoproteins and Genes Involved in Protein Glycosylation in the Genus Haloferax.

Within the Haloferax genus, both the surface (S)-layer protein, and the glycans that can decorate it, vary between species, which can potentially result in many different surface types, analogous to bacterial serotypes. This variation may mediate phenotypes, such as sensitivity to different viruses and mating preferences. Here, we describe S-layer glycoproteins found in multiple Haloferax strains and perform comparative genomics analyses of major and alternative glycosylation clusters of isolates from two coastal sites. We analyze the phylogeny of individual glycosylation genes and demonstrate that while the major glycosylation cluster tends to be conserved among closely related strains, the alternative cluster is highly variable. Thus, geographically- and genetically-related strains may exhibit diverse surface structures to such an extent that no two isolates present an identical surface profile.

Characterizing the DNA Methyltransferases of Haloferax volcanii via Bioinformatics, Gene Deletion, and SMRT Sequencing.

DNA methyltransferases (MTases), which catalyze the methylation of adenine and cytosine bases in DNA, can occur in bacteria and archaea alongside cognate restriction endonucleases (REases) in restriction-modification (RM) systems or independently as orphan MTases. Although DNA methylation and MTases have been well-characterized in bacteria, research into archaeal MTases has been limited. A previous study examined the genomic DNA methylation patterns (methylome) of the halophilic archaeon Haloferax volcanii, a model archaeal system which can be easily manipulated in laboratory settings, via single-molecule real-time (SMRT) sequencing and deletion of a putative MTase gene (HVO_A0006). In this follow-up study, we deleted other putative MTase genes in H. volcanii and sequenced the methylomes of the resulting deletion mutants via SMRT sequencing to characterize the genes responsible for DNA methylation. The results indicate that deletion of putative RM genes HVO_0794, HVO_A0006, and HVO_A0237 in a single strain abolished methylation of the sole cytosine motif in the genome (Cm4TAG). Amino acid alignments demonstrated that HVO_0794 shares homology with characterized cytosine CTAG MTases in other organisms, indicating that this MTase is responsible for Cm4TAG methylation in H. volcanii. The CTAG motif has high density at only one of the origins of replication, and there is no relative increase in CTAG motif frequency in the genome of H. volcanii, indicating that CTAG methylation might not have effectively taken over the role of regulating DNA replication and mismatch repair in the organism as previously predicted. Deletion of the putative Type I RM operon rmeRMS (HVO_2269-2271) resulted in abolished methylation of the adenine motif in the genome (GCAm6BN6VTGC). Alignments of the MTase (HVO_2270) and site specificity subunit (HVO_2271) demonstrate homology with other characterized Type I MTases and site specificity subunits, indicating that the rmeRMS operon is responsible for adenine methylation in H. volcanii. Together with HVO_0794, these genes appear to be responsible for all detected methylation in H. volcanii, even though other putative MTases (HVO_C0040, HVO_A0079) share homology with characterized MTases in other organisms. We also report the construction of a multi-RM deletion mutant (ΔRM), with multiple RM genes deleted and with no methylation detected via SMRT sequencing, which we anticipate will be useful for future studies on DNA methylation in H. volcanii.

Assessment of MultiLocus Sequence Analysis As a Valuable Tool for the Classification of the Genus Salinivibrio.

The genus Salinivibrio includes obligatory halophilic bacteria and is commonly isolated from hypersaline habitats and salted food products. They grow optimally between 7.5 and 10% salts and are facultative anaerobes. Currently, this genus comprises four species, one of them, S. costicola, with three subspecies. In this study we isolated and characterized an additional 70 strains from solar salterns located in different locations. Comparative 16S rRNA gene sequence analysis identified these strains as belonging to the genus Salinivibrio but could not differentiate strains into species-like groups. To achieve finer phylogenetic resolution, we carried out a MultiLocus Sequence Analysis (MLSA) of the new isolates and the type strains of the species of Salinivibrio based on the individual as well as concatenated sequences of four housekeeping genes: gyrB, recA, rpoA, and rpoD. The strains formed four clearly differentiated species-like clusters called phylogroups. All of the known type and subspecies strains were associated with one of these clusters except S. sharmensis. One phylogroup had no previously described species coupled to it. Further DNA-DNA hybridization (DDH) experiments with selected representative strains from these phylogroups permitted us to validate the MLSA study, correlating the species level defined by the DDH (70%) with a 97% cut-off for the concatenated MLSA gene sequences. Based on these criteria, the novel strains forming phylogroup 1 could constitute a new species while strains constructing the other three phylogroups are members of previously recognized Salinivibrio species. S. costicola subsp. vallismortis co-occurs with S. proteolyticus in phylogroup 4, and separately from other S. costicola strains, indicating its need for reclassification. On the other hand, genome fingerprinting analysis showed that the environmental strains do not form clonal populations and did not cluster according to their site of cultivation. In future studies regarding the classification and identification of new Salinivibrio strains we recommend the following strategy: (i) initial partial sequencing of the 16S rRNA gene for genus-level identification; (ii) sequencing and concatenation of the four before mentioned housekeeping genes for species-level discrimination; (iii) DDH experiments, only required when the concatenated MLSA similarity values among a new isolate and other Salinivibrio strains are above the 97% cut-off.

Analysis of the bacteriorhodopsin-producing haloarchaea reveals a core community that is stable over time in the salt crystallizers of Eilat, Israel.

Stability of microbial communities can impact the ability of dispersed cells to colonize a new habitat. Saturated brines and their halophile communities are presumed to be steady state systems due to limited environmental perturbations. In this study, the bacteriorhodopsin-containing fraction of the haloarchaeal community from Eilat salt crystallizer ponds was sampled five times over 3 years. Analyses revealed the existence of a constant core as several OTUs were found repeatedly over the length of the study: OTUs comprising 52 % of the total cloned and sequenced PCR amplicons were found in every sample, and OTUs comprising 89 % of the total sequences were found in more than one, and often more than two samples. LIBSHUFF and UNIFRAC analyses showed statistical similarity between samples and Spearman's coefficient denoted significant correlations between OTU pairs, indicating non-random patterns in abundance and co-occurrence of detected OTUs. Further, changes in the detected OTUs were statistically linked to deviations in salinity. We interpret these results as indicating the existence of an ever-present core bacteriorhodopsin-containing Eilat crystallizer community that fluctuates in population densities, which are controlled by salinity rather than the extinction of some OTUs and their replacement through immigration and colonization.

Deciphering the Translation Initiation Factor 5A Modification Pathway in Halophilic Archaea.

Translation initiation factor 5A (IF5A) is essential and highly conserved in Eukarya (eIF5A) and Archaea (aIF5A). The activity of IF5A requires hypusine, a posttranslational modification synthesized in Eukarya from the polyamine precursor spermidine. Intracellular polyamine analyses revealed that agmatine and cadaverine were the main polyamines produced in Haloferax volcanii in minimal medium, raising the question of how hypusine is synthesized in this halophilic Archaea. Metabolic reconstruction led to a tentative picture of polyamine metabolism and aIF5A modification in Hfx. volcanii that was experimentally tested. Analysis of aIF5A from Hfx. volcanii by LC-MS/MS revealed it was exclusively deoxyhypusinylated. Genetic studies confirmed the role of the predicted arginine decarboxylase gene (HVO_1958) in agmatine synthesis. The agmatinase-like gene (HVO_2299) was found to be essential, consistent with a role in aIF5A modification predicted by physical clustering evidence. Recombinant deoxyhypusine synthase (DHS) from S. cerevisiae was shown to transfer 4-aminobutyl moiety from spermidine to aIF5A from Hfx. volcanii in vitro. However, at least under conditions tested, this transfer was not observed with the Hfx. volcanii DHS. Furthermore, the growth of Hfx. volcanii was not inhibited by the classical DHS inhibitor GC7. We propose a model of deoxyhypusine synthesis in Hfx. volcanii that differs from the canonical eukaryotic pathway, paving the way for further studies.