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1.  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
2.  Biofilms formed by the archaeon Haloferax volcanii exhibit cellular differentiation and social motility, and facilitate horizontal gene transfer 
BMC Biology  2014;12:65.
Archaea share a similar microbial lifestyle with bacteria, and not surprisingly then, also exist within matrix-enclosed communities known as biofilms. Advances in biofilm biology have been made over decades for model bacterial species, and include characterizations of social behaviors and cellular differentiation during biofilm development. Like bacteria, archaea impact ecological and biogeochemical systems. However, the biology of archaeal biofilms is only now being explored. Here, we investigated the development, composition and dynamics of biofilms formed by the haloarchaeon Haloferax volcanii DS2.
Biofilms were cultured in static liquid and visualized with fluorescent cell membrane dyes and by engineering cells to express green fluorescent protein (GFP). Analysis by confocal scanning laser microscopy showed that H. volcanii cells formed microcolonies within 24 h, which developed into larger clusters by 48 h and matured into flake-like towers often greater than 100 μm in height after 7 days. To visualize the extracellular matrix, biofilms formed by GFP-expressing cells were stained with concanavalin A, DAPI, Congo red and thioflavin T. Stains colocalized with larger cellular structures and indicated that the extracellular matrix may contain a combination of polysaccharides, extracellular DNA and amyloid protein. Following a switch to biofilm growth conditions, a sub-population of cells differentiated into chains of long rods sometimes exceeding 25 μm in length, compared to their planktonic disk-shaped morphology. Time-lapse photography of static liquid biofilms also revealed wave-like social motility. Finally, we quantified gene exchange between biofilm cells, and found that it was equivalent to the mating frequency of a classic filter-based experimental method.
The developmental processes, functional properties and dynamics of H. volcanii biofilms provide insight on how haloarchaeal species might persist, interact and exchange DNA in natural communities. H. volcanii demonstrates some biofilm phenotypes similar to bacterial biofilms, but also has interesting phenotypes that may be unique to this organism or to this class of organisms, including changes in cellular morphology and an unusual form of social motility. Because H. volcanii has one of the most advanced genetic systems for any archaeon, the phenotypes reported here may promote the study of genetic and developmental processes in archaeal biofilms.
Electronic supplementary material
The online version of this article (doi:10.1186/s12915-014-0065-5) contains supplementary material, which is available to authorized users.
PMCID: PMC4180959  PMID: 25124934
biofilm; Haloferax volcanii; microbial development; archaeal genetics; archaeal biofilm; horizontal gene transfer; amyloid; collective behavior; swarming; haloarchaea
3.  Functional Genomic and Advanced Genetic Studies Reveal Novel Insights into the Metabolism, Regulation, and Biology of Haloferax volcanii 
Archaea  2011;2011:602408.
The genome sequence of Haloferax volcanii is available and several comparative genomic in silico studies were performed that yielded novel insight for example into protein export, RNA modifications, small non-coding RNAs, and ubiquitin-like Small Archaeal Modifier Proteins. The full range of functional genomic methods has been established and results from transcriptomic, proteomic and metabolomic studies are discussed. Notably, Hfx. volcanii is together with Halobacterium salinarum the only prokaryotic species for which a translatome analysis has been performed. The results revealed that the fraction of translationally-regulated genes in haloarchaea is as high as in eukaryotes. A highly efficient genetic system has been established that enables the application of libraries as well as the parallel generation of genomic deletion mutants. Facile mutant generation is complemented by the possibility to culture Hfx. volcanii in microtiter plates, allowing the phenotyping of mutant collections. Genetic approaches are currently used to study diverse biological questions–from replication to posttranslational modification—and selected results are discussed. Taken together, the wealth of functional genomic and genetic tools make Hfx. volcanii a bona fide archaeal model species, which has enabled the generation of important results in recent years and will most likely generate further breakthroughs in the future.
PMCID: PMC3235422  PMID: 22190865
4.  A Gateway platform for functional genomics in Haloferax volcanii: deletion of three tRNA modification genes 
Archaea  2009;2(4):211-219.
In part due to the existence of simple methods for its cultivation and genetic manipulation, Haloferax volcanii is a major archaeal model organism. It is the only archaeon for which the whole set of post-transcriptionally modified tRNAs has been sequenced, allowing for an in silico prediction of all RNA modification genes present in the organism. One approach to check these predictions experimentally is via the construction of targeted gene deletion mutants. Toward this goal, an integrative “Gateway vector” that allows gene deletion in H. volcanii uracil auxotrophs was constructed. The vector was used to delete three predicted tRNA modification genes: HVO_2001 (encoding an archaeal transglycosyl tranferase or arcTGT), which is involved in archeosine biosynthesis; HVO_2348 (encoding a newly discovered GTP cyclohydrolase I), which catalyzes the first step common to archaeosine and folate biosynthesis; and HVO_2736 (encoding a member of the COG1444 family), which is involved in N4-acetylcytidine (ac4C) formation. Preliminary phenotypic analysis of the deletion mutants was conducted, and confirmed all three predictions.
PMCID: PMC2686393  PMID: 19478918
Archaea; GTP-cyclohydrolase I; halophile; tRNA-modification
5.  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
6.  Small regulatory RNAs in Archaea 
RNA Biology  2014;11(5):484-493.
Small regulatory RNAs (sRNAs) are universally distributed in all three domains of life, Archaea, Bacteria, and Eukaryotes. In bacteria, sRNAs typically function by binding near the translation start site of their target mRNAs and thereby inhibit or activate translation. In eukaryotes, miRNAs and siRNAs typically bind to the 3′-untranslated region (3′-UTR) of their target mRNAs and influence translation efficiency and/or mRNA stability. In archaea, sRNAs have been identified in all species investigated using bioinformatic approaches, RNomics, and RNA-Seq. Their size can vary significantly between less than 50 to more than 500 nucleotides. Differential expression of sRNA genes has been studied using northern blot analysis, microarrays, and RNA-Seq. In addition, biological functions have been unraveled by genetic approaches, i.e., by characterization of designed mutants. As in bacteria, it was revealed that archaeal sRNAs are involved in many biological processes, including metabolic regulation, adaptation to extreme conditions, stress responses, and even in regulation of morphology and cellular behavior. Recently, the first target mRNAs were identified in archaea, including one sRNA that binds to the 5′-region of two mRNAs in Methanosarcina mazei Gö1 and a few sRNAs that bind to 3′-UTRs in Sulfolobus solfataricus, three Pyrobaculum species, and Haloferax volcanii, indicating that archaeal sRNAs appear to be able to target both the 5′-UTR or the 3′-UTRs of their respective target mRNAs. In addition, archaea contain tRNA-derived fragments (tRFs), and one tRF has been identified as a major ribosome-binding sRNA in H. volcanii, which downregulates translation in response to stress. Besides regulatory sRNAs, archaea contain further classes of sRNAs, e.g., CRISPR RNAs (crRNAs) and snoRNAs.
PMCID: PMC4152357  PMID: 24755959
archaea; small regulatory RNAs; tRNA-derived fragments; translation; Methanosarcina mazei; Haloferax volcanii; Sulfolobus solfataricus; Nanoarchaeum equitans
7.  Mre11-Rad50 Promotes Rapid Repair of DNA Damage in the Polyploid Archaeon Haloferax volcanii by Restraining Homologous Recombination 
PLoS Genetics  2009;5(7):e1000552.
Polyploidy is frequent in nature and is a hallmark of cancer cells, but little is known about the strategy of DNA repair in polyploid organisms. We have studied DNA repair in the polyploid archaeon Haloferax volcanii, which contains up to 20 genome copies. We have focused on the role of Mre11 and Rad50 proteins, which are found in all domains of life and which form a complex that binds to and coordinates the repair of DNA double-strand breaks (DSBs). Surprisingly, mre11 rad50 mutants are more resistant to DNA damage than the wild-type. However, wild-type cells recover faster from DNA damage, and pulsed-field gel electrophoresis shows that DNA double-strand breaks are repaired more slowly in mre11 rad50 mutants. Using a plasmid repair assay, we show that wild-type and mre11 rad50 cells use different strategies of DSB repair. In the wild-type, Mre11-Rad50 appears to prevent the repair of DSBs by homologous recombination (HR), allowing microhomology-mediated end-joining to act as the primary repair pathway. However, genetic analysis of recombination-defective radA mutants suggests that DNA repair in wild-type cells ultimately requires HR, therefore Mre11-Rad50 merely delays this mode of repair. In polyploid organisms, DSB repair by HR is potentially hazardous, since each DNA end will have multiple partners. We show that in the polyploid archaeon H. volcanii the repair of DSBs by HR is restrained by Mre11-Rad50. The unrestrained use of HR in mre11 rad50 mutants enhances cell survival but leads to slow recovery from DNA damage, presumably due to difficulties in the resolution of DNA repair intermediates. Our results suggest that recombination might be similarly repressed in other polyploid organisms and at repetitive sequences in haploid and diploid species.
Author Summary
Most organisms contain only one or two copies of their genome, but in some species multiple copies are found. The presence of multiple genome copies (polyploidy) has profound implications for DNA repair and is frequently seen in cancer cells. We have studied DNA repair in the archaeon Haloferax volcanii, which contains up to 20 genome copies. Archaea are a third form of life distinct from bacteria and eukaryotes. We have focused on the DNA repair proteins Mre11 and Rad50, which are found in virtually all organisms and which in humans act to prevent cancer. Surprisingly, we have found that H. volcanii cells deficient in Mre11-Rad50 are more resistant to DNA damage than wild-type cells. The DNA damage resistance of mre11 rad50 mutant cells appears to be due to the exclusive use of homologous recombination, a DNA repair mechanism that is accurate but has the potential to generate genome rearrangements that require time to resolve. Correspondingly, we have found repair of DNA damage in mre11 rad50 mutants takes longer than in wild-type cells. Our results suggest that polyploid organisms employ a program of DNA repair that minimizes their reliance on homologous recombination.
PMCID: PMC2700283  PMID: 19593371
8.  A Comprehensive Analysis of the Importance of Translation Initiation Factors for Haloferax volcanii Applying Deletion and Conditional Depletion Mutants 
PLoS ONE  2013;8(11):e77188.
Translation is an important step in gene expression. The initiation of translation is phylogenetically diverse, since currently five different initiation mechanisms are known. For bacteria the three initiation factors IF1 – IF3 are described in contrast to archaea and eukaryotes, which contain a considerably higher number of initiation factor genes. As eukaryotes and archaea use a non-overlapping set of initiation mechanisms, orthologous proteins of both domains do not necessarily fulfill the same function. The genome of Haloferax volcanii contains 14 annotated genes that encode (subunits of) initiation factors. To gain a comprehensive overview of the importance of these genes, it was attempted to construct single gene deletion mutants of all genes. In 9 cases single deletion mutants were successfully constructed, showing that the respective genes are not essential. In contrast, the genes encoding initiation factors aIF1, aIF2γ, aIF5A, aIF5B, and aIF6 were found to be essential. Factors aIF1A and aIF2β are encoded by two orthologous genes in H. volcanii. Attempts to generate double mutants failed in both cases, indicating that also these factors are essential. A translatome analysis of one of the single aIF2β deletion mutants revealed that the translational efficiency of the second ortholog was enhanced tenfold and thus the two proteins can replace one another. The phenotypes of the single deletion mutants also revealed that the two aIF1As and aIF2βs have redundant but not identical functions. Remarkably, the gene encoding aIF2α, a subunit of aIF2 involved in initiator tRNA binding, could be deleted. However, the mutant had a severe growth defect under all tested conditions. Conditional depletion mutants were generated for the five essential genes. The phenotypes of deletion mutants and conditional depletion mutants were compared to that of the wild-type under various conditions, and growth characteristics are discussed.
PMCID: PMC3828320  PMID: 24244275
9.  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
10.  Development of Additional Selectable Markers for the Halophilic Archaeon Haloferax volcanii Based on the leuB and trpA Genes 
Since most archaea are extremophilic and difficult to cultivate, our current knowledge of their biology is confined largely to comparative genomics and biochemistry. Haloferax volcanii offers great promise as a model organism for archaeal genetics, but until now there has been a lack of a wide variety of selectable markers for this organism. We describe here isolation of H. volcanii leuB and trpA genes encoding 3-isopropylmalate dehydrogenase and tryptophan synthase, respectively, and development of these genes as a positive selection system. ΔleuB and ΔtrpA mutants were constructed in a variety of genetic backgrounds and were shown to be auxotrophic for leucine and tryptophan, respectively. We constructed both integrative and replicative plasmids carrying the leuB or trpA gene under control of a constitutive promoter. The use of these selectable markers in deletion of the lhr gene of H. volcanii is described.
PMCID: PMC348920  PMID: 14766575
11.  Data in support of global role of the membrane protease LonB in Archaea: Potential protease targets revealed by quantitative proteome analysis of a lonB mutant in Haloferax volcanii 
Data in Brief  2015;4:50-53.
This data article provides information in support of the research article “Global role of the membrane protease LonB in Archaea: Potential protease targets revealed by quantitative proteome analysis of a lonB mutant in Haloferax volcanii” [1]. The proteome composition of a wt and a LonB protease mutant strain (suboptimal expression) in the archaeon Haloferax volcanii was assessed by a quantitative shotgun proteomic approach. Membrane and cytosol fractions of H. volcanii strains were examined at two different growth stages (exponential and stationary phase). Data is supplied in the present article. This study represents the first proteome examination of a Lon-deficient cell of the Archaea Domain.
PMCID: PMC4510384  PMID: 26217762
12.  Structural insights into the adaptation of proliferating cell nuclear antigen (PCNA) from Haloferax volcanii to a high-salt environment 
The crystal structure of PCNA from the halophilic archaeon H. volcanii reveals specific features of the charge distribution on the protein surface that reflect adaptation to a high-salt environment and suggests a different type of interaction with DNA in halophilic PCNAs.
The sliding clamp proliferating cell nuclear antigen (PCNA) plays vital roles in many aspects of DNA replication and repair in eukaryotic cells and in archaea. Realising the full potential of archaea as a model for PCNA function requires a combination of biochemical and genetic approaches. In order to provide a platform for subsequent reverse genetic analysis, PCNA from the halophilic archaeon Haloferax volcanii was subjected to crystallographic analysis. The gene was cloned and expressed in Escherichia coli and the protein was purified by affinity chromatography and crystallized by the vapour-diffusion technique. The structure was determined by molecular replacement and refined at 3.5 Å resolution to a final R factor of 23.7% (R free = 25%). PCNA from H. volcanii was found to be homotrimeric and to resemble other homotrimeric PCNA clamps but with several differences that appear to be associated with adaptation of the protein to the high intracellular salt concentrations found in H. volcanii cells.
PMCID: PMC2756170  PMID: 19770505
PCNA–DNA interactions; sliding clamps; halophilic environment
13.  Improved Strains and Plasmid Vectors for Conditional Overexpression of His-Tagged Proteins in Haloferax volcanii▿  
Research into archaea will not achieve its full potential until systems are in place to carry out genetics and biochemistry in the same species. Haloferax volcanii is widely regarded as the best-equipped organism for archaeal genetics, but the development of tools for the expression and purification of H. volcanii proteins has been neglected. We have developed a series of plasmid vectors and host strains for conditional overexpression of halophilic proteins in H. volcanii. The plasmids feature the tryptophan-inducible p.tnaA promoter and a 6×His tag for protein purification by metal affinity chromatography. Purification is facilitated by host strains, where pitA is replaced by the ortholog from Natronomonas pharaonis. The latter lacks the histidine-rich linker region found in H. volcanii PitA and does not copurify with His-tagged recombinant proteins. We also deleted the mrr restriction endonuclease gene, thereby allowing direct transformation without the need to passage DNA through an Escherichia coli dam mutant.
PMCID: PMC2838008  PMID: 20097827
14.  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
15.  Genetic and Proteomic Analyses of a Proteasome-Activating Nucleotidase A Mutant of the Haloarchaeon Haloferax volcanii▿ †  
Journal of Bacteriology  2007;190(1):193-205.
The halophilic archaeon Haloferax volcanii encodes two related proteasome-activating nucleotidase proteins, PanA and PanB, with PanA levels predominant during all phases of growth. In this study, an isogenic panA mutant strain of H. volcanii was generated. The growth rate and cell yield of this mutant strain were lower than those of its parent and plasmid-complemented derivatives. In addition, a consistent and discernible 2.1-fold increase in the number of phosphorylated proteins was detected when the panA gene was disrupted, based on phosphospecific fluorescent staining of proteins separated by 2-dimensional gel electrophoresis. Subsequent enrichment of phosphoproteins by immobilized metal ion and metal oxide affinity chromatography (in parallel and sequentially) followed by tandem mass spectrometry was employed to identify key differences in the proteomes of these strains as well as to add to the restricted numbers of known phosphoproteins within the Archaea. In total, 625 proteins (approximately 15% of the deduced proteome) and 9 phosphosites were identified by these approaches, and 31% (195) of the proteins were identified by multiple phosphoanalytical methods. In agreement with the phosphostaining results, the number of identified proteins that were reproducibly exclusive or notably more abundant in one strain was nearly twofold greater for the panA mutant than for the parental strain. Enriched proteins exclusive to or more abundant in the panA mutant (versus the wild type) included cell division (FtsZ, Cdc48), dihydroxyacetone kinase-linked phosphoenolpyruvate phosphotransferase system (EI, DhaK), and oxidoreductase homologs. Differences in transcriptional regulation and signal transduction proteins were also observed, including those differences (e.g., OsmC and BolA) which suggest that proteasome deficiency caused an up-regulation of stress responses (e.g., OsmC versus BolA). Consistent with this, components of the Fe-S cluster assembly, protein-folding, DNA binding and repair, oxidative and osmotic stress, phosphorus assimilation, and polyphosphate synthesis systems were enriched and identified as unique to the panA mutant. The cumulative proteomic data not only furthered our understanding of the archaeal proteasome system but also facilitated the assembly of the first subproteome map of H. volcanii.
PMCID: PMC2223738  PMID: 17965165
16.  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
17.  Biosynthesis and Role of N-Linked Glycosylation in Cell Surface Structures of Archaea with a Focus on Flagella and S Layers 
The genetics and biochemistry of the N-linked glycosylation system of Archaea have been investigated over the past 5 years using flagellins and S layers as reporter proteins in the model organisms, Methanococcus voltae, Methanococcus maripaludis, and Haloferax volcanii. Structures of archaeal N-linked glycans have indicated a variety of linking sugars as well as unique sugar components. In M. voltae, M. maripaludis, and H. volcanii, a number of archaeal glycosylation genes (agl) have been identified by deletion and complementation studies. These include many of the glycosyltransferases and the oligosaccharyltransferase needed to assemble the glycans as well as some of the genes encoding enzymes required for the biosynthesis of the sugars themselves. The N-linked glycosylation system is not essential for any of M. voltae, M. maripaludis, or H. volcanii, as demonstrated by the successful isolation of mutants carrying deletions in the oligosaccharyltransferase gene aglB (a homologue of the eukaryotic Stt3 subunit of the oligosaccharyltransferase complex). However, mutations that affect the glycan structure have serious effects on both flagellation and S layer function.
PMCID: PMC2952790  PMID: 20976295
18.  Construction and use of halobacterial shuttle vectors and further studies on Haloferax DNA gyrase. 
Journal of Bacteriology  1991;173(12):3807-3813.
We report here on advances made in the construction of plasmid shuttle vectors suitable for genetic manipulations in both Escherichia coli and halobacteria. Starting with a 20.4-kb construct, pMDS1, new vectors were engineered which were considerably smaller yet retained several alternative cloning sites. A restriction barrier observed when plasmid DNA was transferred into Haloferax volcanii cells was found to operate via adenine methylation, resulting in a 10(3) drop in transformation efficiency and the loss of most constructs by incorporation of the resistance marker into the chromosome. Passing shuttle vectors through E. coli dam mutants effectively avoided this barrier. Deletion analysis revealed that the gene(s) for autonomous replication of pHK2 (the plasmid endogenous to Haloferax strain Aa2.2 and used in the construction of pMDS1) was located within a 4.2-kb SmaI-KpnI fragment. Convenient restriction sites were identified near the termini of the novobiocin resistance determinant (gyrB), allowing the removal of flanking sequences (including gyrA). These deletions did not appear to significantly affect transformation efficiencies or the novobiocin resistance phenotype of halobacterial transformants. Northern blot hybridization with strand- and gene-specific probes identified a single gyrB-gyrA transcript of 4.7 kb. This is the first demonstration in prokaryotes that the two subunits of DNA gyrase may be cotranscribed.
PMCID: PMC208012  PMID: 1711028
19.  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
20.  Essential requirements for the detection and degradation of invaders by the Haloferax volcanii CRISPR/Cas system I-B 
RNA Biology  2013;10(5):865-874.
To fend off foreign genetic elements, prokaryotes have developed several defense systems. The most recently discovered defense system, CRISPR/Cas, is sequence-specific, adaptive and heritable. The two central components of this system are the Cas proteins and the CRISPR RNA. The latter consists of repeat sequences that are interspersed with spacer sequences. The CRISPR locus is transcribed into a precursor RNA that is subsequently processed into short crRNAs. CRISPR/Cas systems have been identified in bacteria and archaea, and data show that many variations of this system exist. We analyzed the requirements for a successful defense reaction in the halophilic archaeon Haloferax volcanii. Haloferax encodes a CRISPR/Cas system of the I-B subtype, about which very little is known. Analysis of the mature crRNAs revealed that they contain a spacer as their central element, which is preceded by an eight-nucleotide-long 5′ handle that originates from the upstream repeat. The repeat sequences have the potential to fold into a minimal stem loop. Sequencing of the crRNA population indicated that not all of the spacers that are encoded by the three CRISPR loci are present in the same abundance. By challenging Haloferax with an invader plasmid, we demonstrated that the interaction of the crRNA with the invader DNA requires a 10-nucleotide-long seed sequence. In addition, we found that not all of the crRNAs from the three CRISPR loci are effective at triggering the degradation of invader plasmids. The interference does not seem to be influenced by the copy number of the invader plasmid.
PMCID: PMC3737343  PMID: 23594992
archaea; Haloferax volcanii; CRISPR/Cas; crRNA; PAM; seed sequence
21.  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
22.  In Vivo Analysis of an Essential Archaeal Signal Recognition Particle in Its Native Host 
Journal of Bacteriology  2002;184(12):3260-3267.
The evolutionarily conserved signal recognition particle (SRP) plays an integral role in Sec-mediated cotranslational protein translocation and membrane protein insertion, as it has been shown to target nascent secretory and membrane proteins to the bacterial and eukaryotic translocation pores. However, little is known about its function in archaea, since characterization of the SRP in this domain of life has thus far been limited to in vitro reconstitution studies of heterologously expressed archaeal SRP components identified by sequence comparisons. In the present study, the genes encoding the SRP54, SRP19, and 7S RNA homologs (hv54h, hv19h, and hv7Sh, respectively) of the genetically and biochemically tractable archaeon Haloferax volcanii were cloned, providing the tools to analyze the SRP in its native host. As part of this analysis, an hv54h knockout strain was created. In vivo characterization of this strain revealed that the archaeal SRP is required for viability, suggesting that cotranslational protein translocation is an essential process in archaea. Furthermore, a method for the purification of this SRP employing nickel chromatography was developed in H. volcanii, allowing the successful copurification of (i) Hv7Sh with a histidine-tagged Hv54h, as well as (ii) Hv54h and Hv7Sh with a histidine-tagged Hv19h. These results provide the first in vivo evidence that these components interact in archaea. Such copurification studies will provide insight into the significance of the similarities and differences of the protein-targeting systems of the three domains of life, thereby increasing knowledge about the recognition of translocated proteins in general.
PMCID: PMC135113  PMID: 12029042
23.  An Archaeal Immune System Can Detect Multiple Protospacer Adjacent Motifs (PAMs) to Target Invader DNA* 
The Journal of Biological Chemistry  2012;287(40):33351-33363.
Background: CRISPR/Cas systems allow archaea and bacteria to resist invasion by foreign nucleic acids.
Results: The CRISPR/Cas system in Haloferax recognized six different PAM sequences that could trigger a defense response.
Conclusion: The PAM sequence specificity of the defense response in type I CRISPR systems is more relaxed than previously thought.
Significance: The PAM sequence requirements for interference and adaptation appear to differ markedly.
The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system provides adaptive and heritable immunity against foreign genetic elements in most archaea and many bacteria. Although this system is widespread and diverse with many subtypes, only a few species have been investigated to elucidate the precise mechanisms for the defense of viruses or plasmids. Approximately 90% of all sequenced archaea encode CRISPR/Cas systems, but their molecular details have so far only been examined in three archaeal species: Sulfolobus solfataricus, Sulfolobus islandicus, and Pyrococcus furiosus. Here, we analyzed the CRISPR/Cas system of Haloferax volcanii using a plasmid-based invader assay. Haloferax encodes a type I-B CRISPR/Cas system with eight Cas proteins and three CRISPR loci for which the identity of protospacer adjacent motifs (PAMs) was unknown until now. We identified six different PAM sequences that are required upstream of the protospacer to permit target DNA recognition. This is only the second archaeon for which PAM sequences have been determined, and the first CRISPR group with such a high number of PAM sequences. Cells could survive the plasmid challenge if their CRISPR/Cas system was altered or defective, e.g. by deletion of the cas gene cassette. Experimental PAM data were supplemented with bioinformatics data on Haloferax and Haloquadratum.
PMCID: PMC3460438  PMID: 22767603
Archaea; Microbiology; RNA; RNA Metabolism; RNA Processing; CRISPR/Cas; Haloferax volcanii; PAM
24.  Proteasomal Components Required for Cell Growth and Stress Responses in the Haloarchaeon Haloferax volcanii▿ †  
Journal of Bacteriology  2008;190(24):8096-8105.
Little is known regarding the biological roles of archaeal proteases. The haloarchaeon Haloferax volcanii is an ideal model for understanding these enzymes, as it is one of few archaea with an established genetic system. In this report, a series of H. volcanii mutant strains with markerless and/or conditional knockouts in each known proteasome gene was systematically generated and characterized. This included single and double knockouts of genes encoding the 20S core α1 (psmA), β (psmB), and α2 (psmC) subunits as well as genes (panA and panB) encoding proteasome-activating nucleotidase (PAN) proteins closely related to the regulatory particle triple-A ATPases (Rpt) of eukaryotic 26S proteasomes. Our results demonstrate that 20S proteasomes are required for growth. Although synthesis of 20S proteasomes containing either α1 or α2 could be separately abolished via gene knockout with little to no impact on growth, conditional depletion of either β alone or α1 and α2 together rendered the cells inviable. In contrast, the PAN proteins were not essential based on the robust growth of the panA panB double knockout strain. Deletion of genes encoding either α1 or PanA did, however, render cells more sensitive to growth on organic versus inorganic nitrogen sources and hypo-osmotic stress and limited growth in the presence of l-canavanine. Abolishment of α1 synthesis also had a severe impact on the ability of cells to withstand thermal stress. This contrasted with what was seen for panA knockouts, which displayed enhanced thermotolerance. Together, these results provide new and important insight into the biological role of proteasomes in archaea.
PMCID: PMC2593203  PMID: 18931121
25.  Development of a Gene Knockout System for the Halophilic Archaeon Haloferax volcanii by Use of the pyrE Gene 
Journal of Bacteriology  2003;185(3):772-778.
So far, the extremely halophilic archaeon Haloferax volcanii has the best genetic tools among the archaea. However, the lack of an efficient gene knockout system for this organism has hampered further genetic studies. In this paper we describe the development of pyrE-based positive selection and counterselection systems to generate an efficient gene knockout system. The H. volacanii pyrE1 and pyrE2 genes were isolated, and the pyrE2 gene was shown to code for the physiological enzyme orotate phosphoribosyl transferase. A ΔpyrE2 strain was constructed and used to isolate deletion mutants by the following two steps: (i) integration of a nonreplicative plasmid carrying both the pyrE2 wild-type gene, as a selectable marker, and a cloned chromosomal DNA fragment containing a deletion in the desired gene; and (ii) excision of the integrated plasmid after selection with 5-fluoroorotic acid. Application of this gene knockout system is described.
PMCID: PMC142808  PMID: 12533452

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