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provided by Elsevier - Publisher Connector Current Biology 22, 1444–1448, August 7, 2012 ª2012 Elsevier Ltd All rights reserved DOI 10.1016/j.cub.2012.05.056 Report Low Barriers in Halophilic and the Formation of Recombinant Hybrids

Adit Naor,1 Pascal Lapierre,2 Moshe Mevarech,1 have 98.4% sequence identity in their 16S ribosomal R. Thane Papke,2 and Uri Gophna1,* RNA (rRNA) sequence and an average nucleotide sequence 1Department of Molecular Microbiology and Biotechnology, identity of 86.6% in shared protein coding genes (based George S. Wise Faculty of Life Sciences, Tel Aviv University, on the sequences of H. volcanii strain DS2 [17] Ramat Aviv 69978, Israel and H. mediterranei R4 strain: http://edwards.sdsu.edu/ 2Department of Molecular and Cell Biology, University of ). Previous studies showed that the two species Connecticut, Storrs 06269, CT, USA are able to exchange selectable plasmids [13]; however, their ability to fuse and form heterodiploid cells was never tested. We used H. mediterranei strain WR646 (DpyrE DtrpA hdrB+) Summary and H. volcanii strain H729 (DhdrB pyrE+ trpA+, kindly provided by T. Allers, personal communication) (see Table 1) to test for Speciation of sexually reproducing organisms requires coexistence of genetic markers from these two species reproductive barriers. Prokaryotes reproduce asexually through cell fusion. By mating the two species and plating but often exchange DNA by lateral gene transfer mecha- the mating culture on a medium lacking thymidine and trypto- nisms and recombination [1], yet distinct lineages are still phan, we selected for cells that possess both hdrB+ (from observed. Thus, barriers to gene flow such as geographic H. mediterranei) and trpA+ (from H. volcanii). Evidence for isolation, genetic incompatibility or a physiological inability the formation of such heterodiploids was obtained by exam- to transfer DNA represent potential underlying mechanisms ining surviving postselection cells via PCR (see Figure S1 avail- behind preferred exchange groups observed in prokaryotes able online). We used the polB gene as a species-specific [2–6]. In Bacteria, experimental evidence showed that molecular marker: it was previously shown that H. volcanii sequence divergence impedes homologous recombination and H. mediterranei contain differentially located intein inser- between bacterial species [7–11]. Here we study interspecies tions [18] (see Figure S2A), thus allowing easy distinction gene exchange in halophilic archaea that possess a para- between the two species and their . sexual mechanism of genetic exchange that is functional between species [12, 13]. In this process, cells fuse forming Inter- and Intraspecies Mating Efficiencies a diploid state containing the full genetic repertoire of both The ability of cells to recognize self and thus display fusion- parental cells, which facilitates genetic exchange and preference toward conspecific partners may present a genuine recombination. Later, cells separate, occasionally resulting barrier to gene exchange. We first tested mating efficiency in hybrids of the parental strains [14]. We show high recom- between H. volcanii and H. mediterranei, comparing the inter- bination frequencies between volcanii and and intraspecies cell fusion events of the two species, using Haloferax mediterranei, two species that have an average both plasmid-located and chromosomal selection markers. nucleotide sequence identity of 86.6%. Whole genome As shown above, the measured frequency of gene exchange sequencing of Haloferax interspecies hybrids revealed the by fusion of two H. volcanii strains using plasmids was 3 3 exchange of chromosomal fragments ranging from 310Kb 1024. When performing the selectable plasmid-based assay to 530Kb. These results show that recombination barriers for interspecies fusion, the efficiency dropped less than one may be more permissive in halophilic archaea than they order of magnitude, from 3.0 3 1024 to about 4.2 3 1025 are in bacteria. (see Figure 1A). The -based fusion assay showed a similar decline in fusion frequency, from 1.0 3 1024 to 4.2 3 25 Results and Discussion 10 , using the same genetic markers for both experiments. Thus, the drop in interspecies cell fusion efficiencies was Mating H. volcanii and H. mediterranei small. The mechanism for genetic exchange in the archaeal species Haloferax volcanii (Order ), involves cell fusion Inter- and Intraspecies Recombination Efficiencies and is symmetric and bidirectional in nature, resulting in a The fusing of cells represents a ‘‘premating’’ barrier to gene heterodiploid cell (containing two different chromosomes in transfer, because cells that are unable to fuse cannot physi- one cell) [12, 14]. Accordingly, when we performed mating cally move DNA from one cell to another. However, a ‘‘post- assays in H. volcanii and selected for heterodiploid colonies, mating’’ barrier, that of gene integration by homologous we obtained similar mating frequencies irrespective of recombination [1, 2] can also operate and be measured. To whether the selected traits were located on selectable estimate the effect of postmating barriers in Haloferax,we plasmids or chromosomes. The average efficiency of plasmid quantified the frequency of recombination within and between and whole chromosome sharing was estimated at 3 3 1024 species. Recombination efficiency was estimated by calcu- and 1 3 1024, respectively (see Figure 1A). lating the ratio of recombinants generated by spontaneous H. volcanii and Haloferax mediterranei were originally iso- crossover events in the heterodiploids, resulting in cells lated from the and from a saltern near Alicante, carrying a single chromosome type with both the selectable Spain, respectively [15, 16]. These two recognized species markers trpA and hdrB. Thus, the frequency measured is an underestimate, because many more unsuccessful recombina- tion events surely occurred but were selected against. We *Correspondence: [email protected] screened colonies (obtained when selecting for ‘‘diploids’’ Efficient Interspecies Recombination in Archaea 1445

Figure 1. Barriers to Gene Exchange between Species of Haloferax (A) A Comparison of cell fusion efficiencies between inter- and intraspecies mating (see Experimental Proce- dures). The experiment was performed allowing 48 hr for mating and selecting for markers located either on the main chromosome or on exogenic plasmids. (B) Recombination frequencies within and between species. Frequencies were calculated as described in Experimental Procedures. The results shown are an average of three independent experiments; error bars represent SEM.

using the tryptophan and thymidine) by PCR to establish the trpA+ allele, which was the selectable marker present in whether they were heterodiploid (no recombination) or had this genome (the exact points of crossovers are specified in a single chromosome type, which indicates a recombination Supplemental Data). Of the ten hybrids sequenced, no two event. For the intraspecies mating, we examined the unse- recombined fragments were exactly alike in terms of size lected pyrE alleles, which differ between the two strains. For and insertion sites; however, the sites of recombination the interspecies mating, we PCR amplified the polB as appeared nonrandom. Most notable were insertions at locus described in the Experimental Procedures (see Figure S2B). coordinates 840933 (7/10 strains) and 329465 (2/10 strains) Notably, PCR amplification showing a single amplicon for of the H. volcanii genome. Sequence identity in the 400 bp polB or pyrE alleles indicates that recombination of those surrounding the points of recombination ranged between loci occurred in the interrogated colony. Intraspecies analysis 88%–93%, slightly higher than the intergenomic average. revealed that of the successful cell-fusion events, 62% re- However, coordinate 840933 is located in a transfer RNA sulted in recombinants, whereas interspecies experiments (tRNA) gene with 98.5% sequence identity along 73 bp yielded a postfusion recombination frequency of 8%; less between the two parental strains and another recombination than an order of magnitude difference (Figure 2B). This is in point in one hybrid also occurred in a tRNA gene (100% iden- stark contrast to studied Gram-positive and Gram-negative tity along 73 bp). These trends are similar to observations Bacteria, where comparable levels of interspecies sequence made by Boucher et al. who documented a natural interspe- divergence resulted in a frequency drop of two to three orders cies recombination event that occurred in the rRNA gene, of magnitudes [19]. which has regions with high sequence conservation across species in the halophilic archaeon Natrinema sp. strain Creation of Interspecies Hybrids XA3-1 [1]. Furthermore, recent metagenomic analysis of To further characterize interspecies recombination events, we interspecies recombination in acidophilic archaea, showed selected for specific recombinants (see Figure S3). In this recombination events clustered around areas of unusually protocol, the heterodiploid state was eliminated by growing high interspecies sequence similarity [21]. cells on solid medium containing uracil and 5-fluoroorotic Both H. volcanii and H. mediterranei harbor three natural acid (5FOA). This process prevents growth of any cell carrying endogenous plasmids each [17, 22], some of which were an H. volcanii pyrE+ chromosome. Recombinant colonies that also present in the hybrids. All hybrids contained pHV1, grew in the absence of thymidine and tryptophan were ob- pHV4, and pNM100. Plasmids pHV3 or pHM300 were also tained. Analysis of 25 colonies that grew under these selective found in the hybrids, but never both in the same strain (see conditions using PCR amplification confirmed the presence of Table S3). recombinant hybrid containing H. mediterranei Whether prokaryotes can be divided into cohesive clusters hdrB+ and DpyrE alleles and the H. volcanii trpA+ allele (one that one may call ‘‘species,’’ and which species concept such example is shown in Figure S1). is appropriate for them, if any, remains a debated issue We further analyzed ten hybrids by genome sequencing. [23, 24]. Previous studies showed that in Bacteria, the major Comparison of hybrid chromosomes to those of the parental barrier to gene exchange between species is recombination strains revealed in each hybrid a single large H. volcanii chro- [7, 9–11], with a log-linear decline in recombination with mosomal fragment ranging in size from 310 Kb to 530 Kb sequence divergence (reviewed in [19]). Nevertheless, other (mean: 475 Kb) in size recombined into the H. mediterranei mechanisms such as restriction-modification systems, DNA chromosome (Figure 2). These recombinant fragments re- uptake signal sequences, and quorum sensing molecules placed their H. mediterranei counterparts while maintaining can also play a role [25–27]. Here we show that in halophilic gene synteny between H. volcanii and H. mediterranei [20]. archaea recombination appears only weakly restricted once Predictably, all these H. volcanii genome fragments contained cell fusion has occurred, because the differences in recom- bination rates within and between species is quite small. Compared to previously characterized bacteria, overall sexual Table 1. Archael Strains and Plasmids Used in This Work isolation appears to be moderately lower in Haloferax species Strain/plasmid Description Source (Figure 3, gray), but unlike the former in which the isolation is WR644 H. mediterranei DpyrE2 DtrpA this study primarily due to reduced recombination efficiency, in the latter H729 H. volcanii DhdrB T. Allers both recombination and mating barriers are of comparable H53 H. volcanii DpyrE2 DtrpA [44] magnitude, and both are relatively weak (Figure 3, black). It Hybrids H729- and WR646-derived strains this study appears that not only do some archaea recombine efficiently pWL102 Contains mevinolin resistance [46] within species [21, 28–30], but our findings suggest that their pMDS1 Contains novobiocin resistance [47] ability to both exchange DNA (e.g., cell fusion) and recombine Current Biology Vol 22 No 15 1446

Figure 3. Relative Frequency of Recombinants Obtained for Different Figure 2. A Comparison of Ten Hybrids to Their H. Mediterranei Parent Prokaryotic Species, as a Function of Sequence Divergence All hybrids have a 100% sequence identity to the H. mediterranei genome Black filled circle, Escherichia coli; black filled square, Streptococcus pneu- except for specific regions indicated for each hybrid. These regions show moniae; black upside-down triangle, Bacillus subtilis; unfilled triangle, a 100% sequence identity to H. volcanii. The size of the recombinant DNA Bacillus mojavensis; gray filled triangle, Haloferax volcanii mating and fragment for each hybrid is indicated. Numbers represent the different recombination; gray filled square, Haloferax volcanii recombination. Fig- hybrid strains. The figure is to scale. ure is inspired by [19]. Bacterial data is from references [9–11]. across species is much higher than that of Bacteria. Neverthe- populations, and phylogenetic analysis demonstrated that less, previous data support the notion that halophilic archaea less than one percent of the gene trees within the species are particularly promiscuous [31–33], and thus the ability of had the same topology [38]. Our in vitro evidence supports additional archaeal groups to undergo efficient genome frag- the conclusions drawn from naturally occurring haloarchaeal ment exchange across species remains to be determined. and other populations that recombination can act as a homog- In three natural populations of the , which enizing force for genes, species, and perhaps genera, and that is closely related to Haloferax, for each one the ratio of recom- barriers to recombination may speed up speciation. bination to mutation (r:m) was estimated at two per allele and What is evident from the Haloferax spp. experiments and nine per [34]. This r:m may be considered low measurements of recombinant and heterodiploid colonies compared to some bacteria [35], suggesting that recombina- presented in this study is that the molecular experiments are tion may not play too important of a role. Nevertheless, several in agreement with the analysis of natural haloarchaeal popula- important observations suggest that the relative recombina- tions: both demonstrate more gene flow within than between tion rate is highly significant in the evolution of haloarchaeal ‘‘species’’ and yet have quite leaky ‘‘species’’ barriers to the species. (1) Within Halorubrum spp. alleles at different loci transfer of DNA. This suggests that sympatric speciation are unlinked and in equilibrium [28, 34], which is the expected may not be the most likely mechanism for haloarchaeal speci- outcome of a sexually reproducing eukaryote, not that of an ation, because high levels of homogenization via recombina- asexual population. This is also seen in Bacteria, and in other tion occur even above the species level (e.g., see [28, 32, 33], Archaea [36, 37]. Unlinked loci by definition indicate that [34]). Such homogenization could counteract the accumula- sufficient recombination has happened in a population to tion of mutations, because a single adaptive allele at any locus randomize alleles within individuals, irrespective of the rate. could purge any accumulated diversity at that locus, unless Therefore, any higher recombination rate might be selected populations are divided between different microniches (e.g., against, as randomly distributed alleles cannot be more [38]). However, genetic isolation also operates at a larger scale randomly shuffled. (2) In each of two different Halorubrum and we think it more likely that geographic and migrational populations a single allele (but not the same in both) was found forces acting on halophilic archaeal communities may be aid- in all but two strains within phylogroups A and B [34]. During ing their speciation. These organisms require extremely high this fixation period (e.g., reduction of diversity at a single salinity for growth, resulting in island-like habitats isolated locus), all the other loci within each phylogroup remained from one another. Furthermore, other extremophiles have highly variable (e.g., up to 12 different alleles and up to 9.5 been noted for their geographic endemism [39–41]. Thus, times the number of neutral mutations), which would be migration followed by geographic isolation can restrict gene impossible if loci are physically linked as in a clonal population flow. This will have at least two effects: it will prevent recombi- [34]. Indeed, this observed outcome means that recombina- nation and thus homogenization between former exchange tion is unlinking genes so fast that a single allele was fixed in groups and generate new exchange groups found in the new the population before that allele could acquire a substitution locations allowing the acquisition of new alleles and loci in a neutral codon position. (3) Each Halorubrum locus exam- (thus forming a 1,000-organism chimera). As long as the rate ined had a different ‘‘species’’ tree topology indicating that of migration is slower than the rate of recombination and muta- recombination between multiple closely related trading part- tion, the homogenization forces keeping members of species ners generates diversity and that fixation of single alleles at looking alike will be subdued, divergences will accumulate, every locus occurs frequently if not continually through the and speciation will proceed. evolution of these populations. Recent genome-wide analysis of oceanic Vibrio populations with r:m similar to Halorubrum Experimental Procedures demonstrated similar patterns of variation: there were many reported instances of reduced variation at loci within Strains and plasmids used in this work are listed in Table 1. Efficient Interspecies Recombination in Archaea 1447

Genome Sequencing Supplemental Information Genome sequencing of hybrids was done by the Centre d’innovation Ge´ nome Que´ bec et Universite´ McGill. One hybrid (number 13) was Supplemental Information includes three figures, three tables, and Supple- sequenced using Illumina paired end sequencing, and the others were mental Data and can be found with this article online at doi:10.1016/j.cub. sequenced by HiSeq - Illumina Single reads 50 bp sequencing. 2012.05.056.

Mating Protocol Acknowledgments

Each culture was grown to an OD600nm of 1–1.1, and 2 ml samples were taken from both strains and applied to a 0.2 mm filter connected to a The authors would like to thank W. Ford Doolittle and J. Peter Gogarten for vacuum to eliminate excess medium. The filter was then placed on a petri critical reading of the manuscript, Martin Kupiec for excellent advice and dish containing a rich medium (HY medium + thymidine, see below) for discussion, T. Allers for providing H. volcanii strains and instructive 48 hr at 42C. The cells were washed then resuspended in casamino (CA) comments, and Jonathan A. Eisen for providing H. mediterranei genomic broth, washed twice more in the same medium, and plated on selective data. This work was funded by the U.S.–Israel Binational Science Founda- media. tion to U.G. and R.T.P. (award number 2007043) and the National Science Foundation (award numbers, 0919290 and 0830024) to R.T.P. 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