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

Gophna U (2011) In Vivo Characterization of the Homing Endonuclease within the polB Gene in the Halophilic Archaeon Haloferax volcanii. PLoS ONE 6(1): e15833. doi:10.1371/journal.pone.0015833 In Vivo Characterization of the Homing Endonuclease within the polB Gene in the Halophilic Archaeon Haloferax volcanii Adit Naor 0 Rona Lazary 0 Adi Barzel 0 R. Thane Papke 0 Uri Gophna 0 Arthur J. Lustig, Tulane University Health Sciences Center, United States of America 0 1 Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University , Tel Aviv , Israel , 2 Department of Molecular Cell Biology, University of Connecticut , Storrs, Connecticut , United States of America 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. - Funding: This work was supported by a grant from the Biational Science Foundation (BSF) grant number 2007043. UG is supported by the James S. McDonnell Foundation and the Israeli Ministry of Health. RTP acknowledges National Science Foundation awards DEB-0919290, DEB-0830024 and the University of Connecticut Research Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Inteins are parasitic genetic elements within open reading frames able to perform self-splicing at the level of the protein. The intein is transcribed and translated along with the gene in which it resides, and is subsequently excised from the protein between its two bordering exteins by an autocatalytic process, in which the exteins are joined together [1,2]. Homing Endonucleases (HENs) are a diverse class of site-specific DNases found in archaea, bacteria and lower eukaryotes, and in some of their respective viruses [3,4]. HENs are selfish genetic elements that reside within self splicing introns and inteins, and promote the horizontal propagation of their respective intron/intein into intron-less or intein-less alleles by cleaving the vacant target site to induce homologous recombination or reverse transcription. HENs recognize relatively long target sequences (1440 bp), a fact that has made them a potential tool for gene therapy and genetic engineering [5]. The gene for DNA polymerase B is a known target for inteins in halophilic archaea [see InBase, the database of known inteins: http://tools.neb.com/inbase/index.php, [6]]. A multiple alignment of haloarchaeal polB homologs (Fig. 1A) revealed that three sites within these genes can contain intein insertions, and that at least one organism (Haloquadratum walsbyi) has inteins occupying all three locations. In Haloferax volcanii the polB gene contains a single 437 amino acid-long intein (Hvo PolB) inserted at amino acid position 1063 from the N-terminus, which has been annotated in InBase as having a putative HEN. It has been proposed that the presence of an intein involves a change in the fitness of the host organism [1], but this has not been tested experimentally. Here we assayed the in vivo endonuclease activity encoded by the HEN located in the Hfx. volcanii polB gene. We also generated a strain that was cured of the polB intein and tested its fitness. Results and Discussion The polB gene of Hfx. volcanii is annotated in InBase as containing a putative intein with an endonuclease of the DOD (dodecapeptide) family. However, the only selfish element motifs previously recognized in this gene are the ones defining the intein, namely blocks A, B (characterizing the N-terminal protein splicing region), F and G (characterizing the C-terminal protein splicing region). In contrast, the blocks indicating the conserved domains in the HEN were not annotated. By aligning the amino acid sequence of Hvo PolB to that of known DOD HENs in InBase, we identified motifs corresponding to DOD blocks C, D, E and H, (see Figure 1B and Figure S1). This demonstrated that the Hvo PolB contains a DOD HEN that may be studied in vivo. Curing the intein is hampered by HEN activity Although inteins are present in many essential genes in numerous organisms, their potential effect on host fitness has not been tested [1]. To determine whether the presence of an intein in the polB gene of Hfx. volcanii affects the fitness of this archaeon, we attempted to cure the Hfx. volcanii polB gene of its intein. By employing the pop-in/pop-out strategy for allele exchange, previously developed for Hfx. volcanii ([7], see materials and methods and figure 2), a plasmid construct was generated containing a polB gene fragment (approximately 1700bp out of about 4000bp) that includes the original stop codon at the 39 end but not the intein (Figure 2A#1). Thus, an intein-less polB allele was created lacking the first 1000 nucleotides of this gene. The intein-less construct was created by overlap PCR (see materials and methods), cloned into the pTA131 vector [8], and the resulting suicide plasmid (pAN9, see Table 1 and Figure 2A#2), was transformed into the uracil auxotroph Hfx. volcanii strain WR532 (DpyrE). Transformed colonies were selected for on a medium lacking uracil. The plasmid integration via homologous recombination occurs at either flanking region (Figure 2A#2), resulting in two possible different arrangements. In the first alternative, integration occurs through homologous recombination in the region 59 to the intein, (Figure 2A#3) resulting in an intact polB gene lacking the intein, and a second copy containing only two 850 bp sequences surrounding the intein sequence. The second alternative, is that integration occurs 39 to the intein (Figure 2A #4), and results in an intact, intein-containing, polB sequence, followed by a second sequence, containing only 850 bp flanking the intein. In both cases one intact polB gene will be expressed, but one versio (...truncated)