A systematic analysis of affinity tags in the haloarchaeal expression system, Haloferax volcanii for protein purification.

Extremophilic proteins are valuable in various fields, but their expression can be challenging in traditional hosts like Escherichia coli due to misfolding and aggregation. Haloferax volcanii (H. volcanii), a halophilic expression system, offers a solution. This study examined cleavable and non-cleavable purification tags at both the N- and C-termini when fused with the superfolder green fluorescent protein (sfGFP) in H. volcanii. Our findings reveal that an N-terminal 8xHis-tag or Strep-tag®II significantly enhances protein production, purity, and yield in H. volcanii. Further experiments with mCherry and halophilic alcohol dehydrogenase (ADH) showed improved expression and purification yields when the 8xHis-tag or Strep-tag®II was positioned at the C-terminus for mCherry and at the N-terminus for ADH. Co-positioning 8xHis-tag and Twin-Strep-tag® at the N-terminus of sfGFP, mCherry, and ADH yielded significantly enhanced results. These findings highlight the importance of thoughtful purification tag design and selection in H. volcanii, providing valuable insights for improving protein production and purification with the potential to advance biotechnological applications.

RecJ3/4-aRNase J form a Ubl-associated nuclease complex functioning in survival against DNA damage in Haloferax volcanii.

Nucleases are strictly regulated and often localized in the cell to avoid the uncontrolled degradation of DNA and RNA. Here, a new type of nuclease complex, composed of RecJ3, RecJ4, and aRNase J, was identified through its ATP-dependent association with the ubiquitin-like SAMP1 and AAA-ATPase Cdc48a. The complex was discovered in Haloferax volcanii, an archaeon lacking an RNA exosome. Genetic analysis revealed aRNase J to be essential and RecJ3, RecJ4, and Cdc48a to function in the recovery from DNA damage including genotoxic agents that generate double-strand breaks. The RecJ3:RecJ4:aRNase J complex (isolated in 2:2:1 stoichiometry) functioned primarily as a 3'-5' exonuclease in hydrolyzing RNA and ssDNA, with the mechanism non-processive for ssDNA. aRNase J could also be purified as a homodimer that catalyzed endoribonuclease activity and, thus, was not restricted to the 5'-3' exonuclease activity typical of aRNase J homologs. Moreover, RecJ3 and RecJ4 could be purified as a 560-kDa subcomplex in equimolar subunit ratio with nuclease activities mirroring the full RecJ3/4-aRNase J complex. These findings prompted reconstitution assays that suggested RecJ3/4 could suppress, alter, and/or outcompete the nuclease activities of aRNase J. Based on the phenotypic results, this control mechanism of aRNase J by RecJ3/4 is not necessary for cell growth but instead appears important for DNA repair. IMPORTANCE Nucleases are critical for various cellular processes including DNA replication and repair. Here, a dynamic type of nuclease complex is newly identified in the archaeon Haloferax volcanii, which is missing the canonical RNA exosome. The complex, composed of RecJ3, RecJ4, and aRNase J, functions primarily as a 3'-5' exonuclease and was discovered through its ATP-dependent association with the ubiquitin-like SAMP1 and Cdc48a. aRNase J alone forms a homodimer that has endonuclease function and, thus, is not restricted to 5'-3' exonuclease activity typical of other aRNase J enzymes. RecJ3/4 appears to suppress, alter, and/or outcompete the nuclease activities of aRNase J. While aRNase J is essential for growth, RecJ3/4, Cdc48a, and SAMPs are important for recovery against DNA damage. These biological distinctions may correlate with the regulated nuclease activity of aRNase J in the RecJ3/4-aRNaseJ complex.

Archaeal Hel308 suppresses recombination through a catalytic switch that controls DNA annealing.

Hel308 helicases promote genome stability in archaea and are conserved in metazoans, where they are known as HELQ. Their helicase mechanism is well characterised, but it is unclear how they specifically contribute to genome stability in archaea. We show here that a highly conserved motif of Hel308/HELQ helicases (motif IVa, F/YHHAGL) modulates both DNA unwinding and a newly identified strand annealing function of archaeal Hel308. A single amino acid substitution in motif IVa results in hyper-active DNA helicase and annealase activities of purified Hel308 in vitro. All-atom molecular dynamics simulations using Hel308 crystal structures provided a molecular basis for these differences between mutant and wild type Hel308. In archaeal cells, the same mutation results in 160000-fold increased recombination, exclusively as gene conversion (non-crossover) events. However, crossover recombination is unaffected by the motif IVa mutation, as is cell viability or DNA damage sensitivity. By contrast, cells lacking Hel308 show impaired growth, increased sensitivity to DNA cross-linking agents, and only moderately increased recombination. Our data reveal that archaeal Hel308 suppresses recombination and promotes DNA repair, and that motif IVa in the RecA2 domain acts as a catalytic switch to modulate the separable recombination and repair activities of Hel308.

An archaeal Cas3 protein facilitates rapid recovery from DNA damage.

CRISPR-Cas systems provide heritable acquired immunity against viruses to archaea and bacteria. Cas3 is a CRISPR-associated protein that is common to all Type I systems, possesses both nuclease and helicase activities, and is responsible for degradation of invading DNA. Involvement of Cas3 in DNA repair had been suggested in the past, but then set aside when the role of CRISPR-Cas as an adaptive immune system was realized. Here we show that in the model archaeon Haloferax volcanii a cas3 deletion mutant exhibits increased resistance to DNA damaging agents compared with the wild-type strain, but its ability to recover quickly from such damage is reduced. Analysis of cas3 point mutants revealed that the helicase domain of the protein is responsible for the DNA damage sensitivity phenotype. Epistasis analysis indicated that cas3 operates with mre11 and rad50 in restraining the homologous recombination pathway of DNA repair. Mutants deleted for Cas3 or deficient in its helicase activity showed higher rates of homologous recombination, as measured in pop-in assays using non-replicating plasmids. These results demonstrate that Cas proteins act in DNA repair, in addition to their role in defense against selfish elements and are an integral part of the cellular response to DNA damage.

Bioengineering of air-filled protein nanoparticles by genetic and chemical functionalization.

BACKGROUND: Various bacteria and archaea, including halophilic archaeon Halobacterium sp. NRC-1 produce gas vesicle nanoparticles (GVNPs), a unique class of stable, air-filled intracellular proteinaceous nanostructures. GVNPs are an attractive tool for biotechnological applications due to their readily production, purification, and unique physical properties. GVNPs are spindle- or cylinder-shaped, typically with a length of 100 nm to 1.5 µm and a width of 30-250 nm. Multiple monomeric subunits of GvpA and GvpC proteins form the GVNP shell, and several additional proteins are required as minor structural or assembly proteins. The haloarchaeal genetic system has been successfully used to produce and bioengineer GVNPs by fusing several foreign proteins with GvpC and has shown various applications, such as biocatalysis, diagnostics, bioimaging, drug delivery, and vaccine development. RESULTS: We demonstrated that native GvpC can be removed in a low salt buffer during the GVNP purification, leaving the GvpA-based GVNP's shell intact and stable under physiological conditions. Here, we report a genetic engineering and chemical modification approach for functionalizing the major GVNP protein, GvpA. This novel approach is based on combinatorial cysteine mutagenesis within GvpA and genetic expansion of the N-terminal and C-terminal regions. Consequently, we generated GvpA single, double, and triple cysteine variant libraries and investigated the impact of mutations on the structure and physical shape of the GVNPs formed. We used a thiol-maleimide chemistry strategy to introduce the biotechnological relevant activity by maleimide-activated streptavidin-biotin and maleimide-activated SpyTag003-SpyCatcher003 mediated functionalization of GVNPs. CONCLUSION: The merger of these genetic and chemical functionalization approaches significantly extends these novel protein nanomaterials' bioengineering and functionalization potential to assemble catalytically active proteins, biomaterials, and vaccines onto one nanoparticle in a modular fashion.

Differences in homologous recombination and maintenance of heteropolyploidy between Haloferax volcanii and Haloferax mediterranei.

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

Progress and Challenges in Archaeal Genetic Manipulation.

Archaea inhabit a wide variety of habitats and are well-placed to provide insights into the origins of eukaryotes. In this primer, we examine the available model archaeal genetic systems. We consider the limitations and barriers involved in genetically modifying different archaeal species, the techniques and breakthroughs that have contributed to their tractability, and potential areas for future development.

Genetic Manipulation of Haloferax Species.

In this chapter, we describe the reverse genetics methodology behind generating a targeted gene deletion or replacement in archaeal species of the genus Haloferax, which are renowned for their ease of manipulation. Individual steps in the method include the design of a gene-targeting vector, its use in transforming Haloferax to yield "pop-in" and "pop-out" clones, and techniques for validating the genetically manipulated strain. The vector carries DNA fragments of 500-1000 bp that flank the gene of interest (or a mutant allele), in addition to the pyrE2 gene for uracil biosynthesis (Bitan-Banin et al. J Bacteriol 185:772-778, 2003). The latter is used as a selectable marker for the transformation of Haloferax, wherein the vector integrates by homologous recombination at the genomic locus to generate the "pop-in" strain; this is also known as allele-coupled exchange. Culturing of these transformants in nonselective broth and subsequent plating on 5-fluoroorotic acid (5-FOA)-containing media selects for excision of the vector, yielding either wild type or mutant "pop-out" clones. These 5-FOA-resistant clones are screened to confirm the desired mutation, using a combination of phenotypic assays, colony hybridization and Southern blotting. The pop-in/pop-out method allows for the recycling of the pyrE2 marker to enable multiple gene deletions to be carried out in a single strain, thereby providing insights into the function of multiple proteins and how they interact in their respective cellular pathways.

Cas1 and Fen1 Display Equivalent Functions During Archaeal DNA Repair.

CRISPR-Cas constitutes an adaptive prokaryotic defence system against invasive nucleic acids like viruses and plasmids. Beyond their role in immunity, CRISPR-Cas systems have been shown to closely interact with components of cellular DNA repair pathways, either by regulating their expression or via direct protein-protein contact and enzymatic activity. The integrase Cas1 is usually involved in the adaptation phase of CRISPR-Cas immunity but an additional role in cellular DNA repair pathways has been proposed previously. Here, we analysed the capacity of an archaeal Cas1 from Haloferax volcanii to act upon DNA damage induced by oxidative stress and found that a deletion of the cas1 gene led to reduced survival rates following stress induction. In addition, our results indicate that Cas1 is directly involved in DNA repair as the enzymatically active site of the protein is crucial for growth under oxidative conditions. Based on biochemical assays, we propose a mechanism by which Cas1 plays a similar function to DNA repair protein Fen1 by cleaving branched intermediate structures. The present study broadens our understanding of the functional link between CRISPR-Cas immunity and DNA repair by demonstrating that Cas1 and Fen1 display equivalent roles during archaeal DNA damage repair.

The lanthipeptide biosynthetic clusters of the domain Archaea.

Research on Archaea's secondary metabolites is still lagging behind that of Bacteria and Eukarya. Our goal was to contribute to this knowledge gap by analyzing the lanthipeptide's clusters in Archaea. As previously proposed, Archaea encodes only class II synthetases (LanMs), which we found to be confined to the class Halobacteria (also known as haloarchaea). In total, we analyzed the phylogeny and the domains of 42 LanMs. Four types were identified, and the majority of them belong to the CCG group due to their cyclization domain, which includes LanMs of Cyanobacteria. Putative cognate peptides were predicted for most of LanMs and are a very diverse group of molecules that share a Kx(Y/F)(D/E)xx(F/Y) motif in their leader peptides. According to their homology, some of them were categorized into subfamilies, including Halolancins, Haladacins, Haloferaxcins and Halobiforcins. Many LanM genes were associated with mobile genetic elements, and their vicinities mainly encode ABC and MFS transporters, tailoring enzymes and uncharacterized proteins. Our results suggest that the biosynthesis of lanthipeptides in haloarchaea can entail distinct enzymology that must lead to the production of peptides with novel structures and unpredicted biological and ecological roles. Finally, an Haloferax mediterranei knockout, lacking its three lanM genes, was generated, and it was concluded that its antimicrobial activity is not primarily related to the production of lanthipeptides.

Haloferax volcanii-a model archaeon for studying DNA replication and repair.

The tree of life shows the relationship between all organisms based on their common ancestry. Until 1977, it comprised two major branches: prokaryotes and eukaryotes. Work by Carl Woese and other microbiologists led to the recategorization of prokaryotes and the proposal of three primary domains: Eukarya, Bacteria and Archaea. Microbiological, genetic and biochemical techniques were then needed to study the third domain of life. Haloferax volcanii, a halophilic species belonging to the phylum Euryarchaeota, has provided many useful tools to study Archaea, including easy culturing methods, genetic manipulation and phenotypic screening. This review will focus on DNA replication and DNA repair pathways in H. volcanii, how this work has advanced our knowledge of archaeal cellular biology, and how it may deepen our understanding of bacterial and eukaryotic processes.

Adaptation induced by self-targeting in a type I-B CRISPR-Cas system.

Haloferax volcanii is, to our knowledge, the only prokaryote known to tolerate CRISPR-Cas-mediated damage to its genome in the WT background; the resulting cleavage of the genome is repaired by homologous recombination restoring the WT version. In mutant Haloferax strains with enhanced self-targeting, cell fitness decreases and microhomology-mediated end joining becomes active, generating deletions in the targeted gene. Here we use self-targeting to investigate adaptation in H. volcanii CRISPR-Cas type I-B. We show that self-targeting and genome breakage events that are induced by self-targeting, such as those catalyzed by active transposases, can generate DNA fragments that are used by the CRISPR-Cas adaptation machinery for integration into the CRISPR loci. Low cellular concentrations of self-targeting crRNAs resulted in acquisition of large numbers of spacers originating from the entire genomic DNA. In contrast, high concentrations of self-targeting crRNAs resulted in lower acquisition that was mostly centered on the targeting site. Furthermore, we observed naïve spacer acquisition at a low level in WT Haloferax cells and with higher efficiency upon overexpression of the Cas proteins Cas1, Cas2, and Cas4. Taken together, these findings indicate that naïve adaptation is a regulated process in H. volcanii that operates at low basal levels and is induced by DNA breaks.

SnapShot: Microbial Extremophiles.

Extremophiles are remarkable examples of life's resilience, thriving in hot springs at boiling temperatures, in brine lakes saturated with salt, and in the driest deserts. We review the biogeography, currently known limits of life, and molecular adaptations to extremes. See the online interactive map for additional detail on biogeography, environmental microbiology, and exemplary species. To view this SnapShot, open or download the PDF.

Characterisation of a solvent-tolerant haloarchaeal (R)-selective transaminase isolated from a Triassic period salt mine.

Transaminase enzymes (TAms) are becoming increasingly valuable in the chemist's toolbox as a biocatalytic route to chiral amines. Despite high profile successes, the lack of (R)-selective TAms and robustness under harsh industrial conditions continue to prove problematic. Herein, we report the isolation of the first haloarchaeal TAm (BC61-TAm) to be characterised for the purposes of pharmaceutical biocatalysis. BC61-TAm is an (R)-selective enzyme, cloned from an extremely halophilic archaeon, isolated from a Triassic period salt mine. Produced using a Haloferax volcanii-based expression model, the resulting protein displays a classic halophilic activity profile, as well as thermotolerance (optimum 50 °C) and organic solvent tolerance. Molecular modelling predicts the putative active site residues of haloarchaeal TAms, with molecular dynamics simulations providing insights on the basis of BC61-TAm's organic solvent tolerance. These results represent an exciting advance in the study of transaminases from extremophiles, providing a possible scaffold for future discovery of biocatalytic enzymes with robust properties.

Cyclic nucleotides in archaea: Cyclic di-AMP in the archaeon Haloferax volcanii and its putative role.

The role of cyclic nucleotides as second messengers for intracellular signal transduction has been well described in bacteria. One recently discovered bacterial second messenger is cyclic di-adenylate monophosphate (c-di-AMP), which has been demonstrated to be essential in bacteria. Compared to bacteria, significantly less is known about second messengers in archaea. This study presents the first evidence of in vivo presence of c-di-AMP in an archaeon. The model organism Haloferax volcanii was demonstrated to produce c-di-AMP. Its genome encodes one diadenylate cyclase (DacZ) which was shown to produce c-di-AMP in vitro. Similar to bacteria, the dacZ gene is essential and homologous overexpression of DacZ leads to cell death, suggesting the need for tight regulation of c-di-AMP levels. Such tight regulation often indicates the control of important regulatory processes. A central target of c-di-AMP signaling in bacteria is cellular osmohomeostasis. The results presented here suggest a comparable function in H. volcanii. A strain with decreased c-di-AMP levels exhibited an increased cell area in hypo-salt medium, implying impaired osmoregulation. In summary, this study expands the field of research on c-di-AMP and its physiological function to archaea and indicates that osmoregulation is likely to be a common function of c-di-AMP in bacteria and archaea.

DNA repair in the archaea-an emerging picture.

There has long been a fascination in the DNA repair pathways of archaea, for two main reasons. Firstly, many archaea inhabit extreme environments where the rate of physical damage to DNA is accelerated. These archaea might reasonably be expected to have particularly robust or novel DNA repair pathways to cope with this. Secondly, the archaea have long been understood to be a lineage distinct from the bacteria, and to share a close relationship with the eukarya, particularly in their information processing systems. Recent discoveries suggest the eukarya arose from within the archaeal domain, and in particular from lineages related to the TACK superphylum and Lokiarchaea. Thus, archaeal DNA repair proteins and pathways can represent a useful model system. This review focuses on recent advances in our understanding of archaeal DNA repair processes including base excision repair, nucleotide excision repair, mismatch repair and double-strand break repair. These advances are discussed in the context of the emerging picture of the evolution and relationship of the three domains of life.

Evolution of Genome Architecture in Archaea: Spontaneous Generation of a New Chromosome in Haloferax volcanii.

The common ancestry of archaea and eukaryotes is evident in their genome architecture. All eukaryotic and several archaeal genomes consist of multiple chromosomes, each replicated from multiple origins. Three scenarios have been proposed for the evolution of this genome architecture: 1) mutational diversification of a multi-copy chromosome; 2) capture of a new chromosome by horizontal transfer; 3) acquisition of new origins and splitting into two replication-competent chromosomes. We report an example of the third scenario: the multi-origin chromosome of the archaeon Haloferax volcanii has split into two elements via homologous recombination. The newly generated elements are bona fide chromosomes, because each bears "chromosomal" replication origins, rRNA loci, and essential genes. The new chromosomes were stable during routine growth but additional genetic manipulation, which involves selective bottlenecks, provoked further rearrangements. To the best of our knowledge, rearrangement of a naturally evolved prokaryotic genome to generate two new chromosomes has not been described previously.

Structural and functional adaptation of Haloferax volcanii TFEα/ß.

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

RadB acts in homologous recombination in the archaeon Haloferax volcanii, consistent with a role as recombination mediator.

Homologous recombination plays a central role in the repair of double-strand DNA breaks, the restart of stalled replication forks and the generation of genetic diversity. Regulation of recombination is essential since defects can lead to genome instability and chromosomal rearrangements. Strand exchange is a key step of recombination - it is catalysed by RecA in bacteria, Rad51/Dmc1 in eukaryotes and RadA in archaea. RadB, a paralogue of RadA, is present in many archaeal species. RadB has previously been proposed to function as a recombination mediator, assisting in RadA-mediated strand exchange. In this study, we use the archaeon Haloferax volcanii to provide evidence to support this hypothesis. We show that RadB is required for efficient recombination and survival following treatment with DNA-damaging agents, and we identify two point mutations in radA that suppress the ΔradB phenotype. Analysis of these point mutations leads us to propose that the role of RadB is to act as a recombination mediator, which it does by inducing a conformational change in RadA and thereby promoting its polymerisation on DNA.

Finally, Archaea Get Their CRISPR-Cas Toolbox.

The majority of archaea encode CRISPR-Cas systems but only a few CRISPR-Cas-based genetic tools have been developed for organisms from this domain. Nayak and Metcalf have harnessed a bacterial Cas9 protein for genome editing in Methanosarcina acetivorans, enabling efficient gene deletion and replacement.