Adit Naor

Native homing endonucleases can target conserved genes in humans and in animal models

In recent years, both homing endonucleases (HEases) and zinc-finger nucleases (ZFNs) have been engineered and selected for the targeting of desired human loci for gene therapy. However, enzyme engineering is lengthy and expensive and the off-target effect of the manufactured endonucleases is difficult to predict. Moreover, enzymes selected to cleave a human DNA locus may not cleave the homologous locus in the genome of animal models because of sequence divergence, thus hampering attempts to assess the in vivo efficacy and safety of any engineered enzyme prior to its application in human trials. Here, we show that naturally occurring HEases can be found, that cleave desirable human targets. Some of these enzymes are also shown to cleave the homologous sequence in the genome of animal models. In addition, the distribution of off-target effects may be more predictable for native HEases. Based on our experimental observations, we present the HomeBase algorithm, database and web server that allow a high-throughput computational search and assignment of HEases for the targeting of specific loci in the human and other genomes. We validate experimentally the predicted target specificity of candidate fungal, bacterial and archaeal HEases using cell free, yeast and archaeal assays.

Cell fusion and hybrids in Archaea: prospects for genome shuffling and accelerated strain development for biotechnology.

The ability to exchange DNA between cells is a molecular process that exists in different species in the domain Archaea. Such horizontal gene transfer events were shown to take place between distant species of archaea and to result in the transfer of large genomic regions. Here we describe recent progress in this field, discuss the potential use of natural gene exchange processes to perform genome shuffling and argue its possible biotechnological applications.

Homing endonucleases residing within inteins: evolutionary puzzles awaiting genetic solutions

Inteins are selfish genetic elements that disrupt the sequence of protein-coding genes and are excised post-translationally. Most inteins also contain a HEN (homing endonuclease) domain, which is important for their horizontal transmission. The present review focuses on the evolution of inteins and their nested HENs, and highlights several unsolved questions that could benefit from molecular genetic approaches. Such approaches can be well carried out in halophilic archaea, which are naturally intein-rich and have highly developed genetic tools for their study. In particular, the fitness effects of harbouring an intein/HEN can be tested in direct competition assays, providing additional insights that will improve current evolutionary models.

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 (t6A), 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 t6A modification, and elevated levels of intra-cellular glycation products.

Evidence from phylogenetic and genome fingerprinting analyses suggests rapidly changing variation in Halorubrum and Haloarcula populations

Halobacteria require high NaCl concentrations for growth and are the dominant inhabitants of hypersaline environments above 15% NaCl. They are well-documented to be highly recombinogenic, both in frequency and in the range of exchange partners. In this study, we examine the genetic and genomic variation of cultured, naturally co-occurring environmental populations of Halobacteria. Sequence data from multiple loci (~2500 bp) identified many closely and more distantly related strains belonging to the genera Halorubrum and Haloarcula. Genome fingerprinting using a random priming PCR amplification method to analyze these isolates revealed diverse banding patterns across each of the genera and surprisingly even for isolates that are identical at the nucleotide level for five protein coding sequenced loci. This variance in genome structure even between identical multilocus sequence analysis (MLSA) haplotypes indicates that accumulation of genomic variation is rapid: faster than the rate of third codon substitutions.

Extracellular DNA metabolism in Haloferax volcanii

Extracellular DNA is found in all environments and is a dynamic component of the microbial ecosystem. Microbial cells produce and interact with extracellular DNA through many endogenous mechanisms. Extracellular DNA is processed and internalized for use as genetic information and as a major source of macronutrients, and plays several key roles within prokaryotic biofilms. Hypersaline sites contain some of the highest extracellular DNA concentrations measured in nature–a potential rich source of carbon, nitrogen, and phosphorus for halophilic microorganisms. We conducted DNA growth studies for the halophilic archaeon Haloferax volcanii DS2 and show that this model Halobacteriales strain is capable of using exogenous double-stranded DNA as a nutrient. Further experiments with varying medium composition, DNA concentration, and DNA types revealed that DNA is utilized primarily as a phosphorus source, that growth on DNA is concentration-dependent, and that DNA isolated from different sources is metabolized selectively, with a bias against highly divergent methylated DNA. Additionally, fluorescence microscopy showed that labeled DNA co-localized with H. volcanii cells. The gene Hvo_1477 was also identified using a comparative genomic approach as a factor likely to be involved in DNA processing at the cell surface, and deletion of Hvo_1477 created a strain deficient in the ability to grow on extracellular DNA. Widespread distribution of Hvo_1477 homologs in archaea suggests metabolism of extracellular DNA may be of broad ecological and physiological relevance in this domain of life.

In Vivo Characterization of the Homing Endonuclease within the polB Gene in the Halophilic Archaeon Haloferax volcanii

Inteins are parasitic genetic elements, analogous to introns that excise themselves at the protein level by self-splicing, allowing the formation of functional non-disrupted proteins. Many inteins contain a homing endonuclease (HEN) gene, and rely on its activity for horizontal propagation. In the halophilic archaeon, Haloferax volcanii, the gene encoding DNA polymerase B (polB) contains an intein with an annotated but uncharacterized HEN. Here we examine the activity of the polB HEN in vivo, within its natural archaeal host. We show that this HEN is highly active, and able to insert the intein into both a chromosomal target and an extra-chromosomal plasmid target, by gene conversion. We also demonstrate that the frequency of its incorporation depends on the length of the flanking homologous sequences around the target site, reflecting its dependence on the homologous recombination machinery. Although several evolutionary models predict that the presence of an intein involves a change in the fitness of the host organism, our results show that a strain deleted for the intein sequence shows no significant changes in growth rate compared to the wild type.

Impact of a homing intein on recombination frequency and organismal fitness

Significance Parasitic interactions can result in changes to the host’s behavior in a way that promotes the distribution or life cycle of the parasite. Inteins are molecular parasites found in all three domains of life. Here we look at the influence of an intein in the DNA polymerase on a population of halophilic archaea in simulations, in experiments, and in the wild. This intein has a fitness cost that is higher than expected for a self-splicing genetic element. In these populations, where mating is independent of host replication, the intein increases the recombination rate between cells with and without inteins. This modification may contribute to the long-term persistence of these genetic parasites, despite the fitness burden they impart on their host. Inteins are parasitic genetic elements that excise themselves at the protein level by self-splicing, allowing the formation of functional, nondisrupted proteins. Many inteins contain a homing endonuclease (HEN) domain and rely on its activity for horizontal propagation. However, successful invasion of an entire population will make this activity redundant, and the HEN domain is expected to degenerate quickly under these conditions. Several theories have been proposed for the continued existence of the both active HEN and noninvaded alleles within a population. However, to date, these models were not directly tested experimentally. Using the natural cell fusion ability of the halophilic archaeon Haloferax volcanii we were able to examine this question in vivo, by mating polB intein-positive [insertion site c in the gene encoding DNA polymerase B (polB-c)] and intein-negative cells and examining the dispersal efficiency of this intein in a natural, polyploid population. Through competition between otherwise isogenic intein-positive and intein-negative strains we determined a surprisingly high fitness cost of over 7% for the polB-c intein. Our laboratory culture experiments and samples taken from Israel’s Mediterranean coastline show that the polB-c inteins do not efficiently take over an inteinless population through mating, even under ideal conditions. The presence of the HEN/intein promoted recombination when intein-positive and intein-negative cells were mated. Increased recombination due to HEN activity contributes not only to intein dissemination but also to variation at the population level because recombination tracts during repair extend substantially from the homing site.

A halocin-H4 mutant Haloferax mediterranei strain retains the ability to inhibit growth of other halophilic archaea

Many members of the Halobacteriaceae were found to produce halocins, molecules that inhibit the growth of other halophilic archaea. Halocin H4 that is produced by Haloferax mediterranei and inhibits the growth of Halobacterium salinarum is one of the best studied halocins to date. The gene encoding this halocin had been previously identified as halH4, located on one of Hfx. mediterranei megaplasmids. We generated a mutant of the halH4 gene and examined the killing ability of the Haloferax mediterranei halH4 mutant with respect to both Halobacterium salinarum and Haloferax volcanii. We showed that both wild-type Hfx. mediterranei and the halH4 mutant strain efficiently inhibited the growth of both species, indicating halocin redundancy. Surprisingly, the halH4 deletion mutant exhibited faster growth in standard medium than the wild type, and is likely to have a better response to several nucleotides, which could explain this phenotype.

Identification of a novel protein complex essential for effector translocation across the parasitophorous vacuole membrane of Toxoplasma gondii

Toxoplasma gondii is an obligate intracellular parasite that can infect virtually all nucleated cells in warm-blooded animals. The ability of Toxoplasma tachyzoites to infect and successfully manipulate its host is dependent on its ability to transport “GRA” proteins that originate in unique secretory organelles called dense granules into the host cell in which they reside. GRAs have diverse roles in Toxoplasma’s intracellular lifecycle, including co-opting crucial host cell functions and proteins, such as the cell cycle, c-Myc and p38 MAP kinase. Some of these GRA proteins, such as GRA16 and GRA24, are secreted into the parasitophorous vacuole (PV) within which Toxoplasma replicates and are transported across the PV membrane (PVM) into the host cell, but the translocation process and its machinery are not well understood. We previously showed that TgMYR1, which is cleaved by TgASP5 into two fragments, localizes to the PVM and is essential for GRA transport into the host cell. To identify additional proteins necessary for effector transport, we screened Toxoplasma mutants defective in c-Myc up-regulation for their ability to export GRA16 and GRA24 to the host cell nucleus. Here we report that novel proteins MYR2 and MYR3 play a crucial role in translocation of a subset of GRAs into the host cell. MYR2 and MYR3 are secreted into the PV space and co-localize with PV membranes and MYR1. Consistent with their predicted transmembrane domains, all three proteins are membrane-associated, and MYR3, but not MYR2, stably associates with MYR1, whose N- and C-terminal fragments are disulfide-linked. We further show that fusing intrinsically disordered effectors to a structured DHFR domain blocks the transport of other effectors, consistent with a translocon-based model of effector transport. Overall, these results reveal a novel complex at the PVM that is essential for effector translocation into the host cell.