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.

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.

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.

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.

Diversity of DNA Replication in the Archaea.

DNA replication is arguably the most fundamental biological process. On account of their shared evolutionary ancestry, the replication machinery found in archaea is similar to that found in eukaryotes. DNA replication is initiated at origins and is highly conserved in eukaryotes, but our limited understanding of archaea has uncovered a wide diversity of replication initiation mechanisms. Archaeal origins are sequence-based, as in bacteria, but are bound by initiator proteins that share homology with the eukaryotic origin recognition complex subunit Orc1 and helicase loader Cdc6). Unlike bacteria, archaea may have multiple origins per chromosome and multiple Orc1/Cdc6 initiator proteins. There is no consensus on how these archaeal origins are recognised- some are bound by a single Orc1/Cdc6 protein while others require a multi- Orc1/Cdc6 complex. Many archaeal genomes consist of multiple parts-the main chromosome plus several megaplasmids-and in polyploid species these parts are present in multiple copies. This poses a challenge to the regulation of DNA replication. However, one archaeal species (Haloferax volcanii) can survive without replication origins; instead, it uses homologous recombination as an alternative mechanism of initiation. This diversity in DNA replication initiation is all the more remarkable for having been discovered in only three groups of archaea where in vivo studies are possible.

A genetic investigation of the KEOPS complex in halophilic Archaea.

KEOPS is an important cellular complex conserved in Eukarya, with some subunits conserved in Archaea and Bacteria. This complex was recently found to play an essential role in formation of the tRNA modification threonylcarbamoyladenosine (t(6)A), and was previously associated with telomere length maintenance and transcription. KEOPS subunits are conserved in Archaea, especially in the Euryarchaea, where they had been studied in vitro. Here we attempted to delete the genes encoding the four conserved subunits of the KEOPS complex in the euryarchaeote Haloferax volcanii and study their phenotypes in vivo. The fused kae1-bud32 gene was shown to be essential as was cgi121, which is dispensable in yeast. In contrast, pcc1 (encoding the putative dimerizing unit of KEOPS) was not essential in H. volcanii. Deletion of pcc1 led to pleiotropic phenotypes, including decreased growth rate, reduced levels of t(6)A modification, and elevated levels of intra-cellular glycation products.

Mre11-Rad50 promotes rapid repair of DNA damage in the polyploid archaeon Haloferax volcanii by restraining homologous recombination.

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.

Regulated polyploidy in halophilic archaea.

Polyploidy is common in higher eukaryotes, especially in plants, but it is generally assumed that most prokaryotes contain a single copy of a circular chromosome and are therefore monoploid. We have used two independent methods to determine the genome copy number in halophilic archaea, 1) cell lysis in agarose blocks and Southern blot analysis, and 2) Real-Time quantitative PCR. Fast growing H. salinarum cells contain on average about 25 copies of the chromosome in exponential phase, and their ploidy is downregulated to 15 copies in early stationary phase. The chromosome copy number is identical in cultures with a twofold lower growth rate, in contrast to the results reported for several other prokaryotic species. Of three additional replicons of H. salinarum, two have a low copy number that is not growth-phase regulated, while one replicon even shows a higher degree of growth phase-dependent regulation than the main replicon. The genome copy number of H. volcanii is similarly high during exponential phase (on average 18 copies/cell), and it is also downregulated (to 10 copies) as the cells enter stationary phase. The variation of genome copy numbers in the population was addressed by fluorescence microscopy and by FACS analysis. These methods allowed us to verify the growth phase-dependent regulation of ploidy in H. salinarum, and they revealed that there is a wide variation in genome copy numbers in individual cells that is much larger in exponential than in stationary phase. Our results indicate that polyploidy might be more widespread in archaea (or even prokaryotes in general) than previously assumed. Moreover, the presence of so many genome copies in a prokaryote raises questions about the evolutionary significance of this strategy.

Interactions of RadB, a DNA repair protein in archaea, with DNA and ATP.

The RecA family of recombinases (RecA, Rad51, RadA and UvsX) catalyse strand-exchange between homologous DNA molecules by utilising conserved DNA-binding modules and a common core ATPase domain. RadB was identified in archaea as a Rad51-like protein on the basis of conserved ATPase sequences. However, RadB does not catalyse strand exchange and does not turn over ATP efficiently. RadB does bind DNA, and here we report a triplet of residues (Lys-His-Arg) that is highly conserved at the RadB C terminus, and is crucial for DNA binding. This is consistent with the motif forming a "basic patch" of highly conserved residues identified in an atomic structure of RadB from Thermococcus kodakaraensis. As the triplet motif is conserved at the C terminus of XRCC2 also, a mammalian Rad51-paralogue, we present a phylogenetic analysis that clarifies the relationship between RadB, Rad51-paralogues and recombinases. We investigate interactions between RadB and ATP using genetics and biochemistry; ATP binding by RadB is needed to promote survival of Haloferax volcanii after UV irradiation, and ATP, but not other NTPs, induces pronounced conformational change in RadB. This is the first genetic analysis of radB, and establishes its importance for maintaining genome stability in archaea. ATP-induced conformational change in RadB may explain previous reports that RadB controls Holliday junction resolution by Hjc, depending on the presence or the absence of ATP.