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1.  Deciphering a pathway of Halobacterium salinarum N-glycosylation 
MicrobiologyOpen  2014;4(1):28-40.
Genomic analysis points to N-glycosylation as being a common posttranslational modification in Archaea. To date, however, pathways of archaeal N-glycosylation have only been described for few species. With this in mind, the similarities of N-linked glycans decorating glycoproteins in the haloarchaea Haloferax volcanii and Halobacterium salinarum directed a series of bioinformatics, genetic, and biochemical experiments designed to describe that Hbt. salinarum pathway responsible for biogenesis of one of the two N-linked oligosaccharides described in this species. As in Hfx. volcanii, where agl (archaeal glycosylation) genes that encode proteins responsible for the assembly and attachment of a pentasaccharide to target protein Asn residues are clustered in the genome, Hbt. salinarum also contains a group of clustered homologous genes (VNG1048G-VNG1068G). Introduction of these Hbt. salinarum genes into Hfx. volcanii mutant strains deleted of the homologous sequence restored the lost activity. Moreover, transcription of the Hbt. salinarum genes in the native host, as well as in vitro biochemical confirmation of the predicted functions of several of the products of these genes provided further support for assignments made following bioinformatics and genetic experiments. Based on the results obtained in this study, the first description of an N-glycosylation pathway in Hbt. salinarum is offered.
PMCID: PMC4335974  PMID: 25461760
Archaea; halophile; N-glycosylation; posttranslational modification
2.  Towards Glycoengineering in Archaea: Replacement of Haloferax volcanii AglD with Homologous Glycosyltransferases from Other Halophilic Archaea ▿  
Applied and Environmental Microbiology  2010;76(17):5684-5692.
Like eukarya and bacteria, archaea also perform N-glycosylation. However, the N-linked glycans of archaeal glycoproteins present a variety not seen elsewhere. Archaea accordingly rely on N-glycosylation pathways likely involving a broad range of species-specific enzymes. To harness the enormous applied potential of such diversity for the generation of glycoproteins bearing tailored N-linked glycans, the development of an appropriate archaeal glycoengineering platform is required. With a sequenced genome, a relatively well-defined N-glycosylation pathway, and molecular tools for gene manipulation, the haloarchaeon Haloferax volcanii (Hfx. volcanii) represents a promising candidate. Accordingly, cells lacking AglD, a glycosyltransferase involved in adding the final hexose of a pentasaccharide N-linked to the surface (S)-layer glycoprotein, were transformed to express AglD homologues from other haloarchaea. The introduction of nonnative versions of AglD led to the appearance of an S-layer glycoprotein similar to the protein from the native strain. Indeed, mass spectrometry confirmed that AglD and its homologues introduce the final hexose to the N-linked S-layer glycoprotein pentasaccharide. Heterologously expressed haloarchaeal AglD homologues contributed to N-glycosylation in Hfx. volcanii despite an apparent lack of AglD function in those haloarchaea from where the introduced homologues came. For example, although functional in Hfx. volcanii, no transcription of the Halobacterium salinarum aglD homologue, OE1482, was detected in cells of the native host grown under various conditions. Thus, at least one AglD homologue works more readily in Hfx. volcanii than in the native host. These results warrant the continued assessment of Hfx. volcanii as a glycosylation “workshop.”
PMCID: PMC2935037  PMID: 20601508
3.  N-Glycosylation of Haloferax volcanii Flagellins Requires Known Agl Proteins and Is Essential for Biosynthesis of Stable Flagella 
Journal of Bacteriology  2012;194(18):4876-4887.
N-glycosylation, a posttranslational modification required for the accurate folding and stability of many proteins, has been observed in organisms of all domains of life. Although the haloarchaeal S-layer glycoprotein was the first prokaryotic glycoprotein identified, little is known about the glycosylation of other haloarchaeal proteins. We demonstrate here that the glycosylation of Haloferax volcanii flagellins requires archaeal glycosylation (Agl) components involved in S-layer glycosylation and that the deletion of any Hfx. volcanii agl gene impairs its swimming motility to various extents. A comparison of proteins in CsCl density gradient centrifugation fractions from supernatants of wild-type Hfx. volcanii and deletion mutants lacking the oligosaccharyltransferase AglB suggests that when the Agl glycosylation pathway is disrupted, cells lack stable flagella, which purification studies indicate consist of a major flagellin, FlgA1, and a minor flagellin, FlgA2. Mass spectrometric analyses of FlgA1 confirm that its three predicted N-glycosylation sites are modified with covalently linked pentasaccharides having the same mass as that modifying its S-layer glycoprotein. Finally, the replacement of any of three predicted N-glycosylated asparagines of FlgA1 renders cells nonmotile, providing direct evidence for the first time that the N-glycosylation of archaeal flagellins is critical for motility. These results provide insight into the role that glycosylation plays in the assembly and function of Hfx. volcanii flagella and demonstrate that Hfx. volcanii flagellins are excellent reporter proteins for the study of haloarchaeal glycosylation processes.
PMCID: PMC3430349  PMID: 22730124
4.  Chromatin is an ancient innovation conserved between Archaea and Eukarya 
eLife  2012;1:e00078.
The eukaryotic nucleosome is the fundamental unit of chromatin, comprising a protein octamer that wraps ∼147 bp of DNA and has essential roles in DNA compaction, replication and gene expression. Nucleosomes and chromatin have historically been considered to be unique to eukaryotes, yet studies of select archaea have identified homologs of histone proteins that assemble into tetrameric nucleosomes. Here we report the first archaeal genome-wide nucleosome occupancy map, as observed in the halophile Haloferax volcanii. Nucleosome occupancy was compared with gene expression by compiling a comprehensive transcriptome of Hfx. volcanii. We found that archaeal transcripts possess hallmarks of eukaryotic chromatin structure: nucleosome-depleted regions at transcriptional start sites and conserved −1 and +1 promoter nucleosomes. Our observations demonstrate that histones and chromatin architecture evolved before the divergence of Archaea and Eukarya, suggesting that the fundamental role of chromatin in the regulation of gene expression is ancient.
eLife digest
Single-celled microorganisms called archaea are one of the three domains of cellular life, along with bacteria and eukaryotes. Archaea are similar to bacteria in that they do not have nuclei, but genetically they have more in common with eukaryotes. Archaea are found in a wide range of habitats including the human colon, marshlands, the ocean and extreme environments such as hot springs and salt lakes.
It has been known since the 1990s that the DNA of archaea is wrapped around histones to form complexes that closely resemble the nucleosomes found in eukaryotes, albeit with four rather than eight histone subunits. Nucleosomes are the fundamental units of chromatin, the highly-ordered and compact structure that all the DNA in a cell is packed into. Now we know exactly how many nucleosomes are present in a given cell for some eukaryotes, notably yeast, and to a good approximation we know the position of each nucleosome during a variety of metabolic states and physiological conditions. We can also quantify the nucleosome occupancy, which is measure of the length of time that the nucleosomes spend in contact with the DNA: this is a critical piece of information because it determines the level of access that other proteins, including those that regulate gene expression, have to the DNA. These advances have been driven in large part by advances in technology, notably high-density microarrays for genome wide-studies of nucleosome occupancy, and massively parallel sequencing for direct nucleosome sequencing.
Ammar et al. have used these techniques to explore how the DNA of Haloferax volcanii, a species of archaea that thrives in the hyper-salty waters of the Dead Sea, is organized on a genome-wide basis. Despite some clear differences between the genomes of archaea and eukaryotes—for example, genomic DNA is typically circular in archaea and linear in eukaryotes—they found that the genome of Hfx. volcanii is organized into chromatin in a way that is remarkably similar to that seen in all eukaryotic genomes studied to date. This is surprising given that the chromatin in eukaryotes is confined to the nucleus, whereas there are no such constraints in archaea. In particular, Ammar et al. found that those regions of the DNA near the ends of genes that mark where the transcription of the DNA into RNA should begin and end contain have lower nucleosome occupancy than other regions. Moreover, the overall level of occupancy in Hfx. volcanii was twice that of eukaryotes, which is what one would expect given that nucleosomes in archaea contain half as many histone subunits as nucleosomes in eukaryotes. Ammar et al. also confirmed that that the degree of nucleosome occupancy is correlated with gene expression.
These two findings—the similarities between the chromatin in archaea and eukaryotes, and the correlation between nucleosome occupancy and gene expression in archaea—raise an interesting evolutionary possibility: the initial function of nucleosomes and chromatin formation might have been for the regulation of gene expression rather than the packaging of DNA. This is consistent with two decades of research that has shown that there is an extraordinary and complex relationship between the structure of chromatin and the process of gene expression. It is possible, therefore, that as the early eukaryotes evolved, nucleosomes and chromatin started to package DNA into compact structures that, among other things, helped to prevent DNA damage, and that this subsequently enabled the early eukaryotes to flourish.
PMCID: PMC3510453  PMID: 23240084
Haloferax volcanii; Nucleosome; Chromatin; Transcriptome; RNA-seq; Archaea; Other
5.  Glyco-engineering in Archaea: differential N-glycosylation of the S-layer glycoprotein in a transformed Haloferax volcanii strain 
Microbial biotechnology  2011;4(4):461-470.
Archaeal glycoproteins present a variety of N-linked glycans not seen elsewhere. The ability to harness the agents responsible for this unparalleled diversity offers the possibility of generating glycoproteins bearing tailored glycans, optimized for specific functions. With a well-defined N-glycosylation pathway and available genetic tools, the haloarchaeon Haloferax volcanii represents a suitable platform for such glyco-engineering efforts. In Hfx. volcanii, the S-layer glycoprotein is modified by an N-linked pentasaccharide. In the following, S-layer glycoprotein N-glycosylation was considered in cells in which AglD, the dolichol phosphate mannose synthase involved in addition of the final residue of the pentasaccharide, was replaced by a haloarchaeal homologue of AglJ, the enzyme involved in addition of the first residue of the N-linked pentasaccharide. In the engineering strain, the S-layer glycoprotein is modified by a novel N-linked glycan not found on this reporter from the parent strain. Moreover, deletion of AglD and introduction of the AglJ homologue from Halobacterium salinarum, OE2528R, into the deletion strain resulted in increased biosynthesis of the novel 894 Da glycan concomitant with reduced biogenesis of the pentasaccharide normally N-linked to the S-layer glycoprotein. These findings justify efforts designed to transform Hfx. volcanii into a glyco-engineering ‘workshop’.
PMCID: PMC3413378  PMID: 21338478
6.  Glyco‐engineering in Archaea: differential N‐glycosylation of the S‐layer glycoprotein in a transformed Haloferax volcanii strain 
Microbial biotechnology  2011;4(4):461-470.
Archaeal glycoproteins present a variety of N‐linked glycans not seen elsewhere. The ability to harness the agents responsible for this unparalleled diversity offers the possibility of generating glycoproteins bearing tailored glycans, optimized for specific functions. With a well‐defined N‐glycosylation pathway and available genetic tools, the haloarchaeon Haloferax volcanii represents a suitable platform for such glyco‐engineering efforts. In Hfx. volcanii, the S‐layer glycoprotein is modified by an N‐linked pentasaccharide. In the following, S‐layer glycoprotein N‐glycosylation was considered in cells in which AglD, the dolichol phosphate mannose synthase involved in addition of the final residue of the pentasaccharide, was replaced by a haloarchaeal homologue of AglJ, the enzyme involved in addition of the first residue of the N‐linked pentasaccharide. In the engineering strain, the S‐layer glycoprotein is modified by a novel N‐linked glycan not found on this reporter from the parent strain. Moreover, deletion of AglD alone and introduction of the AglJ homologue from Halobacterium salinarum, OE2528R, into the deletion strain resulted in increased biosynthesis of the novel 894 Da glycan concomitant with reduced biogenesis of the pentasaccharide normally N‐linked to the S‐layer glycoprotein. These findings justify efforts designed to transform Hfx. volcanii into a glyco‐engineering ‘workshop’.
PMCID: PMC3413378  PMID: 21338478
7.  Substrate Promiscuity: AglB, the Archaeal Oligosaccharyltransferase, Can Process a Variety of Lipid-Linked Glycans 
Across evolution, N-glycosylation involves oligosaccharyltransferases that transfer lipid-linked glycans to selected Asn residues of target proteins. While these enzymes catalyze similar reactions in each domain, differences exist in terms of the chemical composition, length and degree of phosphorylation of the lipid glycan carrier, the sugar linking the glycan to the lipid carrier, and the composition and structure of the transferred glycan. To gain insight into how oligosaccharyltransferases cope with such substrate diversity, the present study analyzed the archaeal oligosaccharyltransferase AglB from four haloarchaeal species. Accordingly, it was shown that despite processing distinct lipid-linked glycans in their native hosts, AglB from Haloarcula marismortui, Halobacterium salinarum, and Haloferax mediterranei could readily replace their counterpart from Haloferax volcanii when introduced into Hfx. volcanii cells deleted of aglB. As the four enzymes show significant sequence and apparently structural homology, it appears that the functional similarity of the four AglB proteins reflects the relaxed substrate specificity of these enzymes. Such demonstration of AglB substrate promiscuity is important not only for better understanding of N-glycosylation in Archaea and elsewhere but also for efforts aimed at transforming Hfx. volcanii into a glycoengineering platform.
PMCID: PMC3911113  PMID: 24212570
8.  S-Layer Glycoproteins and Flagellins: Reporters of Archaeal Posttranslational Modifications 
Archaea  2010;2010:612948.
Many archaeal proteins undergo posttranslational modifications. S-layer proteins and flagellins have been used successfully to study a variety of these modifications, including N-linked glycosylation, signal peptide removal and lipid modification. Use of these well-characterized reporter proteins in the genetically tractable model organisms, Haloferax volcanii, Methanococcus voltae and Methanococcus maripaludis, has allowed dissection of the pathways and characterization of many of the enzymes responsible for these modifications. Such studies have identified archaeal-specific variations in signal peptidase activity not found in the other domains of life, as well as the enzymes responsible for assembly and biosynthesis of novel N-linked glycans. In vitro assays for some of these enzymes have already been developed. N-linked glycosylation is not essential for either Hfx. volcanii or the Methanococcus species, an observation that allowed researchers to analyze the role played by glycosylation in the function of both S-layers and flagellins, by generating mutants possessing these reporters with only partial attached glycans or lacking glycan altogether. In future studies, it will be possible to consider questions related to the heterogeneity associated with given modifications, such as differential or modulated glycosylation.
PMCID: PMC2913515  PMID: 20721273
9.  Conserved active site cysteine residue of archaeal THI4 homolog is essential for thiamine biosynthesis in Haloferax volcanii 
BMC Microbiology  2014;14:260.
Thiamine (vitamin B1) is synthesized de novo by certain yeast, fungi, plants, protozoans, bacteria and archaea. The pathway of thiamine biosynthesis by archaea is poorly understood, particularly the route of sulfur relay to form the thiazole ring. Archaea harbor structural homologs of both the bacterial (ThiS-ThiF) and eukaryotic (THI4) proteins that mobilize sulfur to thiazole ring precursors by distinct mechanisms.
Based on comparative genome analysis, halophilic archaea are predicted to synthesize the pyrimidine moiety of thiamine by the bacterial pathway, initially suggesting that also a bacterial ThiS-ThiF type mechanism for synthesis of the thiazole ring is used in which the sulfur carrier ThiS is first activated by ThiF-catalyzed adenylation. The only ThiF homolog of Haloferax volcanii (UbaA) was deleted but this had no effect on growth in the absence of thiamine. Usage of the eukaryotic THI4-type sulfur relay was initially considered less likely for thiamine biosynthesis in archaea, since the active-site cysteine residue of yeast THI4p that donates the sulfur to the thiazole ring by a suicide mechanism is replaced by a histidine residue in many archaeal THI4 homologs and these are described as D-ribose-1,5-bisphosphate isomerases. The THI4 homolog of the halophilic archaea, including Hfx. volcanii (HVO_0665, HvThi4) was found to differ from that of methanogens and thermococci by having a cysteine residue (Cys165) corresponding to the conserved active site cysteine of yeast THI4p (Cys205). Deletion of HVO_0665 generated a thiamine auxotroph that was trans-complemented by a wild-type copy of HVO_0665, but not the modified gene encoding an HvThi4 C165A variant.
Based on our results, we conclude that the archaeon Hfx. volcanii uses a yeast THI4-type mechanism for sulfur relay to form the thiazole ring of thiamine. We extend this finding to a relatively large group of archaea, including haloarchaea, ammonium oxidizing archaea, and some methanogen and Pyrococcus species, by observing that these organisms code for THI4 homologs that have a conserved active site cysteine residue which is likely used in thiamine biosynthesis. Thus, archaeal members of IPR002922 THI4 family that have a conserved cysteine active site should be reexamined for a function in thiamine biosynthesis.
Electronic supplementary material
The online version of this article (doi:10.1186/s12866-014-0260-0) contains supplementary material, which is available to authorized users.
PMCID: PMC4215014  PMID: 25348237
Vitamin B1; Coenzyme biosynthesis; Thiamine; Sulfur relay; Archaea
10.  The haloarchaeal MCM proteins: bioinformatic analysis and targeted mutagenesis of the β7-β8 and β9-β10 hairpin loops and conserved zinc binding domain cysteines 
The hexameric MCM complex is the catalytic core of the replicative helicase in eukaryotic and archaeal cells. Here we describe the first in vivo analysis of archaeal MCM protein structure and function relationships using the genetically tractable haloarchaeon Haloferax volcanii as a model system. Hfx. volcanii encodes a single MCM protein that is part of the previously identified core group of haloarchaeal MCM proteins. Three structural features of the N-terminal domain of the Hfx. volcanii MCM protein were targeted for mutagenesis: the β7-β8 and β9-β10 β-hairpin loops and putative zinc binding domain. Five strains carrying single point mutations in the β7-β8 β-hairpin loop were constructed, none of which displayed impaired cell growth under normal conditions or when treated with the DNA damaging agent mitomycin C. However, short sequence deletions within the β7-β8 β-hairpin were not tolerated and neither was replacement of the highly conserved residue glutamate 187 with alanine. Six strains carrying paired alanine substitutions within the β9-β10 β-hairpin loop were constructed, leading to the conclusion that no individual amino acid within that hairpin loop is absolutely required for MCM function, although one of the mutant strains displays greatly enhanced sensitivity to mitomycin C. Deletions of two or four amino acids from the β9-β10 β-hairpin were tolerated but mutants carrying larger deletions were inviable. Similarly, it was not possible to construct mutants in which any of the conserved zinc binding cysteines was replaced with alanine, underlining the likely importance of zinc binding for MCM function. The results of these studies demonstrate the feasibility of using Hfx. volcanii as a model system for reverse genetic analysis of archaeal MCM protein function and provide important confirmation of the in vivo importance of conserved structural features identified by previous bioinformatic, biochemical and structural studies.
PMCID: PMC3972481  PMID: 24723920
Haloferax volcanii; archaea; Haloarchaea; MCM helicase; DNA replication; reverse genetics; zinc binding domain
11.  Haloferax volcanii cells lacking the flagellin FlgA2 are hypermotile 
Microbiology  2013;159(Pt 11):2249-2258.
Motility driven by rotational movement of flagella allows bacteria and archaea to seek favourable conditions and escape toxic ones. However, archaeal flagella share structural similarities with bacterial type IV pili rather than bacterial flagella. The Haloferax volcanii genome contains two flagellin genes, flgA1 and flgA2. While FlgA1 has been shown to be a major flagellin, the function of FlgA2 is elusive. In this study, it was determined that although FlgA2 by itself does not confer motility to non-motile ΔflgA1 Hfx. volcanii, a subset of these mutant cells contains a flagellum. Consistent with FlgA2 being assembled into functional flagella, FlgA1 expressed from a plasmid can only complement a ΔflgA1 strain when co-expressed with chromosomal or plasmid-encoded FlgA2. Surprisingly, a mutant strain lacking FlgA2, but expressing chromosomally encoded FlgA1, is hypermotile, a phenotype that is accompanied by an increased number of flagella per cell, as well as an increased flagellum length. Site-directed mutagenesis resulting in early translational termination of flgA2 suggests that the hypermotility of the ΔflgA2 strain is not due to transcriptional regulation. This, and the fact that plasmid-encoded FlgA2 expression in a ΔflgA2 strain does not reduce its hypermotility, suggests a possible regulatory role for FlgA2 that depends on the relative abundance of FlgA1. Taken together, our results indicate that FlgA2 plays both structural and regulatory roles in Hfx. volcanii flagella-dependent motility. Future studies will build upon the data presented here to elucidate the significance of the hypermotility of this ΔflgA2 mutant, and will illuminate the regulation and function of archaeal flagella.
PMCID: PMC3836490  PMID: 23989184
12.  The Complete Genome Sequence of Haloferax volcanii DS2, a Model Archaeon 
PLoS ONE  2010;5(3):e9605.
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.
Methodology/Principal Findings
We report here the sequencing and analysis of the genome of Hfx. volcanii DS2, the type strain of this species. The genome contains a main 2.848 Mb chromosome, three smaller chromosomes pHV1, 3, 4 (85, 438, 636 kb, respectively) and the pHV2 plasmid (6.4 kb).
The completed genome sequence, presented here, provides an invaluable tool for further in vivo and in vitro studies of Hfx. volcanii.
PMCID: PMC2841640  PMID: 20333302
13.  AglQ Is a Novel Component of the Haloferax volcanii N-Glycosylation Pathway 
PLoS ONE  2013;8(11):e81782.
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.
PMCID: PMC3827465  PMID: 24236216
14.  Biochemical characterisation of LigN, an NAD+-dependent DNA ligase from the halophilic euryarchaeon Haloferax volcanii that displays maximal in vitro activity at high salt concentrations 
DNA ligases are required for DNA strand joining in all forms of cellular life. NAD+-dependent DNA ligases are found primarily in eubacteria but also in some eukaryotic viruses, bacteriophage and archaea. Among the archaeal NAD+-dependent DNA ligases is the LigN enzyme of the halophilic euryarchaeon Haloferax volcanii, the gene for which was apparently acquired by Hfx.volcanii through lateral gene transfer (LGT) from a halophilic eubacterium. Genetic studies show that the LGT-acquired LigN enzyme shares an essential function with the native Hfx.volcanii ATP-dependent DNA ligase protein LigA.
To characterise the enzymatic properties of the LigN protein, wild-type and three mutant forms of the LigN protein were separately expressed in recombinant form in E.coli and purified to apparent homogeneity by immobilised metal ion affinity chromatography (IMAC). Non-isotopic DNA ligase activity assays using λ DNA restriction fragments with 12 bp cos cohesive ends were used to show that LigN activity was dependent on addition of divalent cations and salt. No activity was detected in the absence of KCl, whereas maximum activity could be detected at 3.2 M KCl, close to the intracellular KCl concentration of Hfx.volcanii cells.
LigN is unique amongst characterised DNA ligase enzymes in displaying maximal DNA strand joining activity at high (> 3 M) salt levels. As such the LigN enzyme has potential both as a novel tool for biotechnology and as a model enzyme for studying the adaptation of proteins to high intracellular salt levels.
PMCID: PMC1684257  PMID: 17132163
15.  Polyploidy in haloarchaea: advantages for growth and survival 
The investigated haloarchaeal species, Halobacterium salinarum, Haloferax mediterranei, and H. volcanii, have all been shown to be polyploid. They contain several replicons that have independent copy number regulation, and most have a higher copy number during exponential growth phase than in stationary phase. The possible evolutionary advantages of polyploidy for haloarchaea, most of which have experimental support for at least one species, are discussed. These advantages include a low mutation rate and high resistance toward X-ray irradiation and desiccation, which depend on homologous recombination. For H. volcanii, it has been shown that gene conversion operates in the absence of selection, which leads to the equalization of genome copies. On the other hand, selective forces might lead to heterozygous cells, which have been verified in the laboratory. Additional advantages of polyploidy are survival over geological times in halite deposits as well as at extreme conditions on earth and at simulated Mars conditions. Recently, it was found that H. volcanii uses genomic DNA as genetic material and as a storage polymer for phosphate. In the absence of phosphate, H. volcanii dramatically decreases its genome copy number, thereby enabling cell multiplication, but diminishing the genetic advantages of polyploidy. Stable storage of phosphate is proposed as an alternative driving force for the emergence of DNA in early evolution. Several additional potential advantages of polyploidy are discussed that have not been addressed experimentally for haloarchaea. An outlook summarizes selected current trends and possible future developments.
PMCID: PMC4056108  PMID: 24982654
Haloferax volcanii; archaea; polyploidy; gene conversion; desiccation; survival
16.  Dihydroxyacetone metabolism in Haloferax volcanii 
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.
PMCID: PMC3863723  PMID: 24379808
dihydroxyacetone metabolism; dihydroxyacetone kinase; glycerol kinase; archaea; Halobacteria; Haloarchaea
17.  Genome-wide identification of transcriptional start sites in the haloarchaeon Haloferax volcanii based on differential RNA-Seq (dRNA-Seq) 
BMC Genomics  2016;17:629.
Differential RNA-Seq (dRNA-Seq) is a recently developed method of performing primary transcriptome analyses that allows for the genome-wide mapping of transcriptional start sites (TSSs) and the identification of novel transcripts. Although the transcriptomes of diverse bacterial species have been characterized by dRNA-Seq, the transcriptome analysis of archaeal species is still rather limited. Therefore, we used dRNA-Seq to characterize the primary transcriptome of the model archaeon Haloferax volcanii.
Three independent cultures of Hfx. volcanii grown under optimal conditions to the mid-exponential growth phase were used to determine the primary transcriptome and map the 5′-ends of the transcripts. In total, 4749 potential TSSs were detected. A position weight matrix (PWM) was derived for the promoter predictions, and the results showed that 64 % of the TSSs were preceded by stringent or relaxed basal promoters. Of the identified TSSs, 1851 belonged to protein-coding genes. Thus, fewer than half (46 %) of the 4040 protein-coding genes were expressed under optimal growth conditions. Seventy-two percent of all protein-coding transcripts were leaderless, which emphasized that this pathway is the major pathway for translation initiation in haloarchaea. A total of 2898 of the TSSs belonged to potential non-coding RNAs, which accounted for an unexpectedly high fraction (61 %) of all transcripts. Most of the non-coding TSSs had not been previously described (2792) and represented novel sequences (59 % of all TSSs). A large fraction of the potential novel non-coding transcripts were cis-antisense RNAs (1244 aTSSs). A strong negative correlation between the levels of antisense transcripts and cognate sense mRNAs was found, which suggested that the negative regulation of gene expression via antisense RNAs may play an important role in haloarchaea. The other types of novel non-coding transcripts corresponded to internal transcripts overlapping with mRNAs (1153 iTSSs) and intergenic small RNA (sRNA) candidates (395 TSSs).
This study provides a comprehensive map of the primary transcriptome of Hfx. volcanii grown under optimal conditions. Fewer than half of all protein-coding genes have been transcribed under these conditions. Unexpectedly, more than half of the detected TSSs belonged to several classes of non-coding RNAs. Thus, RNA-based regulation appears to play a more important role in haloarchaea than previously anticipated.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-016-2920-y) contains supplementary material, which is available to authorized users.
PMCID: PMC4983044  PMID: 27519343
Archaea; Haloferax volcanii; dRNA-Seq; Transcriptome; Promoter; Leaderless transcript; Non-coding RNA; sRNA; Antisense RNA
18.  Genetic and Physical Mapping of DNA Replication Origins in Haloferax volcanii 
PLoS Genetics  2007;3(5):e77.
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.
Author Summary
Haloferax volcanii is a member of the archaea, which are renowned for thriving in extreme environments. Archaea have circular chromosomes like bacteria but use enzymes similar to those found in eukaryotes to replicate their DNA. Few archaeal species have systems for genetics, and this has limited our understanding of DNA replication. We used genetics to map the chromosomal sites (origins) at which DNA replication initiates in H. volcanii. This species has a multipart genome comprising one main chromosome, three secondary chromosomes, and a plasmid. Five DNA replication origins were found and confirmed to function in vivo. All are adjacent to genes for the initiator protein Cdc6/Orc1, a common feature of archaeal replication origins. Two of the sequences are located on the main chromosome, confirming that multiple origins are often used to replicate circular chromosomes in archaea. Intriguingly, one of the origins from a secondary chromosome appears “dominant” to the principal chromosomal origin, suggesting either a hierarchy or differential usage of origins. This might reflect the different replication requirements of their respective chromosomes. Given the ease of genetic manipulation, H. volcanii holds great promise for studying how replication of four chromosomes is regulated in the context of the archaeal cell cycle.
PMCID: PMC1868953  PMID: 17511521
19.  A Manual Curation Strategy to Improve Genome Annotation: Application to a Set of Haloarchael Genomes 
Life  2015;5(2):1427-1444.
Genome annotation errors are a persistent problem that impede research in the biosciences. A manual curation effort is described that attempts to produce high-quality genome annotations for a set of haloarchaeal genomes (Halobacterium salinarum and Hbt. hubeiense, Haloferax volcanii and Hfx. mediterranei, Natronomonas pharaonis and Nmn. moolapensis, Haloquadratum walsbyi strains HBSQ001 and C23, Natrialba magadii, Haloarcula marismortui and Har. hispanica, and Halohasta litchfieldiae). Genomes are checked for missing genes, start codon misassignments, and disrupted genes. Assignments of a specific function are preferably based on experimentally characterized homologs (Gold Standard Proteins). To avoid overannotation, which is a major source of database errors, we restrict annotation to only general function assignments when support for a specific substrate assignment is insufficient. This strategy results in annotations that are resistant to the plethora of errors that compromise public databases. Annotation consistency is rigorously validated for ortholog pairs from the genomes surveyed. The annotation is regularly crosschecked against the UniProt database to further improve annotations and increase the level of standardization. Enhanced genome annotations are submitted to public databases (EMBL/GenBank, UniProt), to the benefit of the scientific community. The enhanced annotations are also publically available via HaloLex.
PMCID: PMC4500146  PMID: 26042526
genome annotation; Gold Standard Protein; Halobacteria; halophilic archaea; manual curation
20.  Towards a Systems Approach in the Genetic Analysis of Archaea: Accelerating Mutant Construction and Phenotypic Analysis in Haloferax volcanii 
Archaea  2010;2010:426239.
With the availability of a genome sequence and increasingly sophisticated genetic tools, Haloferax volcanii is becoming a model for both Archaea and halophiles. In order for H. volcanii to reach a status equivalent to Escherichia coli, Bacillus subtilis, or Saccharomyces cerevisiae, a gene knockout collection needs to be constructed in order to identify the archaeal essential gene set and enable systematic phenotype screens. A streamlined gene-deletion protocol adapted for potential automation was implemented and used to generate 22 H. volcanii deletion strains and identify several potentially essential genes. These gene deletion mutants, generated in this and previous studies, were then analyzed in a high-throughput fashion to measure growth rates in different media and temperature conditions. We conclude that these high-throughput methods are suitable for a rapid investigation of an H. volcanii mutant library and suggest that they should form the basis of a larger genome-wide experiment.
PMCID: PMC3017900  PMID: 21234384
21.  Generation of comprehensive transposon insertion mutant library for the model archaeon, Haloferax volcanii, and its use for gene discovery 
BMC Biology  2014;12:103.
Archaea share fundamental properties with bacteria and eukaryotes. Yet, they also possess unique attributes, which largely remain poorly characterized. Haloferax volcanii is an aerobic, moderately halophilic archaeon that can be grown in defined media. It serves as an excellent archaeal model organism to study the molecular mechanisms of biological processes and cellular responses to changes in the environment. Studies on haloarchaea have been impeded by the lack of efficient genetic screens that would facilitate the identification of protein functions and respective metabolic pathways.
Here, we devised an insertion mutagenesis strategy that combined Mu in vitro DNA transposition and homologous-recombination-based gene targeting in H. volcanii. We generated an insertion mutant library, in which the clones contained a single genomic insertion. From the library, we isolated pigmentation-defective and auxotrophic mutants, and the respective insertions pinpointed a number of genes previously known to be involved in carotenoid and amino acid biosynthesis pathways, thus validating the performance of the methodologies used. We also identified mutants that had a transposon insertion in a gene encoding a protein of unknown or putative function, demonstrating that novel roles for non-annotated genes could be assigned.
We have generated, for the first time, a random genomic insertion mutant library for a halophilic archaeon and used it for efficient gene discovery. The library will facilitate the identification of non-essential genes behind any specific biochemical pathway. It represents a significant step towards achieving a more complete understanding of the unique characteristics of halophilic archaea.
Electronic supplementary material
The online version of this article (doi:10.1186/s12915-014-0103-3) contains supplementary material, which is available to authorized users.
PMCID: PMC4300041  PMID: 25488358
Haloferax volcanii; Halophilic archaea; Insertion mutant library; Mu transposition; MuA protein; Gene discovery
22.  2′-O-methylation of the wobble residue of elongator pre-tRNAMet in Haloferax volcanii is guided by a box C/D RNA containing unique features 
RNA Biology  2011;8(5):782-791.
The wobble residue C34 of Haloferax volcanii elongator tRNAMet is 2′-O-methylated. Neither a protein enzyme nor a guide RNA for this modification has been described. In this study, we show that this methylation is guided by a box C/D RNA targeting the intron-containing precursor of the tRNA. This guide RNA is starkly different from its homologs. This unique RNA of approximately 75 bases, named sR-tMet, is encoded in the genomes of H. volcanii and several other haloarchaea. A unique feature of sR-tMet is that the mature RNA in H. volcanii is substantially larger than its predicted size, whereas those in other haloarchaea are as predicted. While the 5′-ends of all tested haloarchaeal sR-tMets are equivalent, H. volcanii sR-tMet possesses an additional 51-base extension at its 3′ end. This extension is present in the precursor but not in the mature sR-tMet of Halobacterium sp, suggesting differential 3′-end processing of sR-tMet in these two closely related organisms. Archaeal box C/D RNAs mostly contain a K-loop at the C′/D′ motif. Another unique feature of sR-tMet is that its C′/D′ motif lacks either a conventional K-turn or a K-loop. Instead, it contains two tandem, sheared G•A base pairs and a pyrimidine-pyrimidine pair in the non-canonical stem; the latter may form an alternative K-turn. Gel shift assays indicate that the L7Ae protein can form a stable complex with this unusual C′/D′ motif, suggesting a novel RNA structure for L7Ae interaction.
PMCID: PMC3256356  PMID: 21654217
sRNA; snoRNA; Guide RNA; tRNA modification; RNA 2′-O-methylation; RNA-guided modification; Ribonucleoprotein; Box C/D RNA
23.  Different routes to the same ending: comparing the N-glycosylation processes of Haloferax volcanii and Haloarcula marismortui, two halophilic archaea from the Dead Sea 
Molecular microbiology  2011;81(5):1166-1177.
Recent insight into the N-glycosylation pathway of the haloarchaeon, Haloferax volcanii, is helping to bridge the gap between our limited understanding of the archaeal version of this universal post-translational modification and the better-described eukaryal and bacterial processes. To delineate as yet undefined steps of the Hfx. volcanii N-glycosylation pathway, a comparative approach was taken with the initial characterization of N-glycosylation in Haloarcula marismortui, a second haloarchaeon also originating from the Dead Sea. While both species decorate the reporter glycoprotein, the S-layer glycoprotein, with the same N-linked pentasaccharide and employ dolichol phosphate as lipid glycan carrier, species-specific differences in the two N-glycosylation pathways exist. Specifically, Har. marismortui first assembles the complete pentasaccharide on dolichol phosphate and only then transfers the glycan to the target protein, as in the bacterial N-glycosylation pathway. In contrast, Hfx. volcanii initially transfers the first four pentasaccharide subunits from a common dolichol phosphate carrier to the target protein and only then delivers the final pentasaccharide subunit from a distinct dolichol phosphate to the N-linked tetrasaccharide, reminiscent of what occurs in eukaryal N-glycosylation. This study further indicates the extraordinary diversity of N-glycosylation pathways in Archaea, as compared with the relatively conserved parallel processes in Eukarya and Bacteria.
PMCID: PMC3412612  PMID: 21815949
24.  AglR is required for addition of the final mannose residue of the N-linked glycan decorating the Haloferax volcanii S-layer glycoprotein 
Biochimica et biophysica acta  2012;1820(10):1664-1670.
Recent studies of Haloferax volcanii have begun to elucidate the steps of N-glycosylation in Archaea, where this universal post-translational modification remains poorly described. In Hfx. volcanii, a series of Agl proteins catalyzes the assembly and attachment of a N-linked pentasaccharide to the S-layer glycoprotein. Although roles have been assigned to the majority of Agl proteins, others await description. In the following, the contribution of AglR to N-glycosylation was addressed.
A combination of bioinformatics, gene deletion, mass spectrometry and metabolic radiolabeling served to show a role for AglR in archaeal N-glycosylation at both the dolichol phosphate and reporter glycoprotein levels.
The modified behavior of the S-layer glycoprotein isolated from cells lacking AglR points to an involvement of this protein in N-glycosylation. In cells lacking AglR, glycan-charged dolichol phosphate, including mannose-charged dolichol phosphate, accumulates. At the same time, the S-layer glycoprotein does not incorporate mannose, the final subunit of the N-linked pentasaccharide decorating this protein. AglR is a homologue of Wzx proteins, annotated as flippases responsible for delivering lipid-linked O-antigen precursor oligosaccharides across the bacterial plasma membrane during lipopolysaccharide biogenesis.
The effects resulting from aglR deletion are consistent with AglR interacting with dolichol phosphate-mannose, possibly acting as a dolichol phosphate-mannose flippase or contributing to such activity.
General significance
Little is known of how lipid-linked oligosaccharides are translocated across membrane during N-glycosylation. The possibility of Hfx. volcanii AglR mediating or contributing to flippase activity could help address this situation.
PMCID: PMC3572504  PMID: 22750201
Archaea; Dolichylphosphate-mannose; Haloferax volcanii; N-glycosylation; S-layer glycoprotein
25.  Experimental Characterization of Cis-Acting Elements Important for Translation and Transcription in Halophilic Archaea 
PLoS Genetics  2007;3(12):e229.
The basal transcription apparatus of archaea is well characterized. However, much less is known about the mechanisms of transcription termination and translation initation. Recently, experimental determination of the 5′-ends of ten transcripts from Pyrobaculum aerophilum revealed that these are devoid of a 5′-UTR. Bioinformatic analysis indicated that many transcripts of other archaeal species might also be leaderless. The 5′-ends and 3′-ends of 40 transcripts of two haloarchaeal species, Halobacterium salinarum and Haloferax volcanii, have been determined. They were used to characterize the lengths of 5′-UTRs and 3′-UTRs and to deduce consensus sequence-elements for transcription and translation. The experimental approach was complemented with a bioinformatics analysis of the H. salinarum genome sequence. Furthermore, the influence of selected 5′-UTRs and 3′-UTRs on transcript stability and translational efficiency in vivo was characterized using a newly established reporter gene system, gene fusions, and real-time PCR. Consensus sequences for basal promoter elements could be refined and a novel element was discovered. A consensus motif probably important for transcriptional termination was established. All 40 haloarchaeal transcripts analyzed had a 3′-UTR (average size 57 nt), and their 3′-ends were not posttranscriptionally modified. Experimental data and genome analyses revealed that the majority of haloarchaeal transcripts are leaderless, indicating that this is the predominant mode for translation initiation in haloarchaea. Surprisingly, the 5′-UTRs of most leadered transcripts did not contain a Shine-Dalgarno (SD) sequence. A genome analysis indicated that less than 10% of all genes are preceded by a SD sequence and even most proximal genes in operons lack a SD sequence. Seven different leadered transcripts devoid of a SD sequence were efficiently translated in vivo, including artificial 5′-UTRs of random sequences. Thus, an interaction of the 5′-UTRs of these leadered transcripts with the 16S rRNA could be excluded. Taken together, either a scanning mechanism similar to the mechanism of translation initiation operating in eukaryotes or a novel mechanism must operate on most leadered haloarchaeal transcripts.
Author Summary
Expression of the information encoded in the genome of an organism into its phenotype involves transcription of the DNA into messenger RNAs and translation of mRNAs into proteins. The textbook view is that an mRNA consists of an untranslated region (5′-UTR), an open reading frame encoding the protein, and another untranslated region (3′-UTR). We have determined the 5′-ends and the 3′-ends of 40 mRNAs of two haloarchaeal species and used this dataset to gain information about nucleotide elements important for transcription and translation. Two thirds of the mRNAs were devoid of a 5′-UTR, and therefore the major pathway for translation initiation in haloarchaea involves so-called leaderless transcripts. Very unexpectedly, most leadered mRNAs were found to be devoid of a sequence motif believed to be essential for translation initiation in bacteria and archaea (Shine-Dalgarno sequence). A bioinformatic genome analysis revealed that less than 10% of the genes contain a Shine-Dalgarno sequence. mRNAs lacking this motif were efficiently translated in vivo, including mRNAs with artificial 5′-UTRs of total random sequence. Thus, translation initiation on these mRNAs either involves a scanning mechanism similar to the mechanism operating in eukaryotes or a totally novel mechanism operating at least in haloarchaea.
PMCID: PMC2151090  PMID: 18159946

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