Abstract With the rapid accumulation of genomic information from various eukaryotes in the last decade, genes proposed to have been derived from recent horizontal gene transfer (HGT) events have been reported even in non-phagotrophic unicellular and multicellular organisms, but the molecular pathways underlying HGT remain to be explained. The development of in vitro HGT detection systems, which permit the molecular and genetic analyses of donor and recipient organisms and quantify HGT, are helpful in order to gain insight into mechanisms that may contribute to contemporary HGT events or may have contributed to past HGT events. We applied a horizontal DNA transfer system model based on conjugal gene transfer called trans-kingdom conjugation (TKC) from the prokaryote Escherichia coli to the eukaryote Saccharomyces cerevisiae, and assessed whether and to what extent genetic variations in the eukaryotic recipient affect its receptivity to TKC. Strains from a collection of 4,823 knock-out mutants of S. cerevisiae MAT-α haploids were tested for their individual TKC receptivity. Two types of mutants, an ssd1 mutant and respiratory mutants, which are also found in experimental strains and in nature widely, were identified as highly receptive mutants. The TKC efficiency for spontaneously accrued petite (rho−/0) mutants of the functional allele (SSD1-V) strain showed increased receptivity. The TKC efficiency of the ssd1Δ mutant was 36% for bacterial conjugation, while that of the petite/ssd1Δ double mutants was even higher (220% in average) compared to bacterial conjugation. This increased TKC receptivity was also observed when other conjugal transfer systems were applied and the donor bacterium was changed to Agrobacterium tumefaciens. These results support the idea that the genomes of certain eukaryotes have been exposed to exogenous DNA more frequently and continuously than previously thought.

Citation: Moriguchi K, Yamamoto S, Tanaka K, Kurata N, Suzuki K (2013) Trans-Kingdom Horizontal DNA Transfer from Bacteria to Yeast Is Highly Plastic Due to Natural Polymorphisms in Auxiliary Nonessential Recipient Genes. PLoS ONE 8(9): e74590. https://doi.org/10.1371/journal.pone.0074590 Editor: John McCutcheon, University Of Montana - Missoula, United States of America Received: June 11, 2013; Accepted: August 5, 2013; Published: September 13, 2013 Copyright: © 2013 Moriguchi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported partly by the NIG Cooperative Research Program (2006-A48) and JSPS KAKENHI (20570221 and 21510209). 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.

Introduction The transfer of genes beyond mating barriers, termed Horizontal Gene Transfer (HGT) or Lateral Gene Transfer (LGT), is now widely recognized as an important factor in bacterial evolution [1], [2]. In contrast, in eukaryotes, HGT is considered a rather limited event that mainly occurred in ancestral phagotrophic unicellular eukaryotes [3]. Previously, a cursory examination of some of the first fully sequenced eukaryotic genomes, such as the human genome, indicated presence of few, if any, genes of bacterial origin that could have been acquired by HGT [4]. However, with the rapid accumulation of genomic information from various organisms in the last decade, genes proposed to have been acquired from HGT events occurring after evolutional loss of phagotrophy have been reported even in non-phagotrophic unicellular and multicellular organisms, such as yeasts, diatoms, higher plants, and bdelloids [5]–[8]. Many of the genes predicted to have arisen by HGT in eukaryotes are considered to have bacterial origins [9]. While evidence of HGT events throughout various stages of eukaryotic evolution has accumulated, the mechanisms underlying HGT remains to be explained [5]–[9]. In vitro HGT detection systems have been developed for molecular and genetic analyses of donor and recipient organisms and quantification of HGT. These systems are helpful in gaining insight on mechanisms that may contribute to HGT events, both contemporary and ancient. The type IV secretion system (T4SS) is a bacterial secretion system that transfers large DNA molecules and/or proteins. It is widely found among gram-positive and gram-negative bacteria, and its transfer capabilities extend from genetic transfer between bacterial phylums to transfer from bacteria to eukaryotes. Examples of trans-kingdom transfer by T4SS include the CagA protein-based transfer system observed in Helicobacter pylori, and the T-DNA transfer system of Agrobacterium tumefaciens that is used for gene introduction into plants [10], [11]. A bacterial conjugal transfer system, which is a type of T4SS, is encoded in the IncP-type plasmids. It has been demonstrated to be capable of transferring bacterial DNA to yeasts and mammalian cells in culture by a process referred to as trans-kingdom conjugation (TKC) [12]–[14]. In addition, the bacterial host range of this type of plasmid is promiscuous [15], which indicates that it endows donor competence on various bacteria. Based on the observed ability to facilitate DNA transfer across kingdoms and the promiscuous host range, it is conceivable that T4SS-based TKC might represent a potential driving force behind HGT from bacteria to eukaryotes. In this study, we attempted to identify the genetic features of a recipient that enable high receptivity, especially those that are spontaneously distributed in various strains. We examined efficiency of DNA transfer from E. coli to various genetically distinct strains of S. cerevisiae by TKC carried on a common IncP1α type plasmid, RK2 (RP4). S. cerevisiae was chosen as the eukaryotic model for testing our hypothesis for the following reasons: (a) yeast genes predicted to have arisen from bacteria via HGT have been previously reported [7], [16], and (b) a complete collection of yeast knock-out mutants is available, which allowed systematic and comprehensive analysis of the impact of the genetic variations in the eukaryotic recipient on its receptivity in TKC.

Materials and Methods Yeast and Bacterial Strains and Media The complete set of Yeast Deletion Clones (MAT-α haploids complete set) was purchased from Invitrogen (Carlsbad, CA). All other yeast and bacterial strains used in this study are listed in Table 1, including those provided by the National Bio-Resource Project (NBRP) of MEXT, Japan. The media used included YPD (1% yeast extract, 2% polypeptone, 2% glucose), YPG (1% yeast extract, 2% polypeptone, 2% glycerol), and synthetic defined medium (SD, containing 0.67% yeast nitrogen base w/o amino acids, 2% glucose, and addition of appropriate individual amino acids and/or uracil) for S. cerevisiae, and Luria Bertani broth (LB: 1% tryptone, 0.5% yeast extract, 1% NaCl) for E. coli and A. tumefaciens. TNB (40 mM Tris-HCl (pH 7.5) and 0.5% NaCl) was used for the standard TKC reaction. Alternatively, TNB at pH 6.5, MNB (40 mM MES (pH 5.5) and 0.5% NaCl), and PBS (purchased from Becton, Dickinson and Company; adjusted to pH 7.5) were used for analyzing the adaptability of TKC under various buffers. The freshwater was collected from Yamanaka-Ike pond in Hiroshima University, and sterilized by microfiltration using DISMIC-25AS (0.2-µm pore; ADVANTEC, Tokyo, Japan). All media ingredients or media except polypeptone were purchased from Becton, Dickinson and Company (Franklin Lakes, NJ). Polypeptone and all chemicals were purchased from Wako Pure Chemical Ind. Ltd. (Osaka, Japan). PPT PowerPoint slide

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larger image TIFF original image Download: Table 1. Strains and plasmids used in this study. https://doi.org/10.1371/journal.pone.0074590.t001 Plasmids The plasmids used in this study are shown in Table 1. The pRS313 vector, which was used for complementation analysis, was provided by the National Bio-Resource Project (NBRP) of MEXT, Japan. The SSD1-V and ssd1-d constructs were provided by Dr. T. Kokubo [17]. The helper plasmid pDPT51 was provided by Dr. Y. Fujita (Graduate School of Bioagricultural Science, Nagoya University, Nagoya, Japan). Screening for High-receptivity Mutants Cultures of the donor bacterium, E. coli HB101 or A. tumefaciens C58C1, carrying appropriate plasmids, grown in liquid medium containing appropriate antibiotics were collected, and resuspended in TNB at a concentration of 1.5×108 cfu/ml. E. coli suspension (25 µl) containing 3.8×106 cfu was used for each TKC reaction. Cultures of recipient yeast strains (parental or mutant strains) from a 96-well frozen stock plate were replica plated on YPD plates, and incubated for 48 h at 28°C. The yeast cells were picked up with toothpicks from the YPD plate, each approximately 1×106 cfu, and directly suspended in the E. coli suspension, to obtain a donor:recipient ratio of approximately 3.8∶1. The mixed suspension was then incubated for 1 hour at 28°C (TKC reaction). This incubation was critical for achieving stable and efficient TKC because the synthetic defined medium blocked TKC. Aliquots of the suspension were plated on a yeast growth medium to select for transconjugants, and incubated for 72 h at 28°C. The selection medium used lacked uracil, but contained 30 µg/ml chloramphenicol. In the first and second rounds of screening, a volume equivalent to 20% of the TKC reaction suspension was plated on the selection plate, while in the third round of screening, 50% was plated. TKC efficiency was measured by counting the colonies on the selection plates, and compared between the parental and mutant strains. In the first round of screening mutants showing a ≥4 fold for the ratio of TKC efficiency between the mutant and its parental strain were selected as enhanced receptivity mutants. These were subjected to repeat TKC reaction and a second round of screening, where mutants showing ≥8 fold were selected. At the third screening, the TKC reaction suspension was plated on both, the selection medium and the complete medium (YPD containing 30 µg/mL chloramphenicol), and the colonies were counted. TKC efficiency was expressed as the ratio of no. of colonies on selection medium to that on complete medium, and compared between various mutants and the parental strain. Mutants showing ≥16 fold either at the second or the third screening were defined as the high-receptivity mutants. Standard TKC Reaction The standard TKC reaction on this study was performed following the method used for the third screening except that each 12.5-µl volume of E. coli HB101 or A. tumefaciens C58C1, carrying appropriate plasmids, and yeast suspension in TNB, containing 3.8×106 and 1.0×106 cfu respectively, which were measured and adjusted by using a spectrometer, were mixed. The scale of the reaction was increased up to 3-fold to detect transconjugants when a reaction condition was stringent for TKC. TKC efficiency determined based on recovery of uracil prototrophic transconjugants and was expressed as number of colonies on selection plate divided by the number of colonies on complete plate (YPD with 30 µg/ml chloramphenicol), adjusted by dilution ratios. Yeast Genetic Methods Elimination of cytoplasmic petite mutants was accomplished by crossing the 22 candidate high-TKC-receptivity mutants of a MATα strain were crossed with a MATa strain derived from BY4741 strain that had a homologous nuclear genotype, but a rho0 mitochondrial genotype. The resultant diploids were examined for their ability to respire (Figure S3). In other experiments, a chemical transformation method using Lithium acetate [18] was applied for checking transformation efficiency in high TKC receptivity mutants, or S. cerevisiae. Direct Transformation Kit from Wako Pure Chemical Ind. Ltd. (Osaka, Japan) was used for complementation with plasmid encoded genes and for generating knock-out strains. Knock-out mutants were generated by integrating deletion cassettes containing kanMX4. The deletion cassette targeting each gene was prepared by PCR using a specific primer set and appropriate genomic DNA derived from the respective yeast deletion clones as the template. The primer sets used for the generation of knock-out mutants are listed in Table S3. Bacterial Conjugation Standard protocols for conjugation between two bacterial strains followed the standard TKC reaction, and the transconjugants were selected on LB solid medium containing the appropriate antibiotics rifampicin (30 µg/ml) and kanamycin (50 µg/ml). Mitochondrial Inhibitor Treatment Yeast strains were incubated overnight at 28°C on YPD plates containing either antimycin or erythromycin at various concentrations according to previous literature [19], [20] and shown in Figure 1. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 1. Effect of mitochondrial functional integrity and SSD1 mutation on efficiency of DNA transfer from Escherichia coli HB101 to Saccharomyces cerevisiae strains (BY4742 in (A), (B) and (C); EGY48 in (D)) by trans-kingdom conjugation (TKC). Enhanced TKC efficiency was observed in (A) the pretreatment of the recipient yeast with a mitochondrial translational inhibitor, erythromycin; (B) the pretreatment of the recipient yeast with a mitochondrial respiration inhibitor, antimycin; (C) a rho0 strain, lacking mitochondrial genome; and (D) an SSD1-knock-out mutant. The vertical axis “Log (TKCeffi)” represents the value of TKC efficiency (no. of transconjugant colonies/no. of recipient cell) converted to Log 10 . Data are represented as mean ± SD (n = 9 in A, B and D, 17 in C). Asterisks indicate a statistically significant difference: ***p<0.001 (two-tailed t-test). HB101 (pRH210, pAY205) was used as the donor. https://doi.org/10.1371/journal.pone.0074590.g001 Statistical Analysis All statistical analyses were performed using either Microsoft Excel or the public domain R program (http://www.R-project.org/).

Discussion Alterations in the genetic makeup are integral to evolution of species. Our results reveal two important features regarding the contribution of HGT to eukaryotic evolution: (1) the receptivity of a recipient eukaryotic organism to DNA transfer from a bacterium may be altered drastically by the presence of different alleles of certain loci and by the polymorphisms in non-essential genes, which are not primarily coded for blocking HGT in the recipient eukaryote, and (2) T4SS-mediated TKC may represent the mechanistic means behind HGT from bacteria to eukaryotes (Figure 6). PPT PowerPoint slide

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larger image TIFF original image Download: Figure 6. A schematic diagram of the gene and plasmid flow from bacteria to eukaryotes by T4SS. In bacteria X and Y, genes are transferred from genomic DNA to various plasmids depicted as A, B, and C by using transposons or another method (i). A conjugative plasmid A (such as IncP plasmids) can move from bacterium X to another bacterium Y or to a eukaryote A by its own T4SS system, i.e., T4SS A (ii). A mobilizable plasmid B (such as IncQ plasmids) transfers from bacterium X to Y and eukaryote A with the help of its own mob genes and T4SS A (iii). The plasmids A and B can also transfer from bacterium Y to eukaryote A by T4SS A (ii and iii). The eukaryote A has a subpopulation that accepts exogenous DNA efficiently transferred by T4SS A. The transferred bacterial genes spread and are maintained in the entire population, if they take on available roles, for its survival. In bacterium Y, a plasmid C transfers to eukaryote B by T4SS C (such as vir genes in Ti and Ri plasmids; iv). The plasmid B is also able to transfer to eukaryote B by using mob genes and T4SS C (v). https://doi.org/10.1371/journal.pone.0074590.g006 Yeast mutants such as the petite (rho−/0) mutants studied here that exhibit defects in mitochondrial function through a partial or complete lack of mitochondrial DNA comprise approximately 1–2% of natural yeast populations [22]. Petite mutant accumulation can be increased even further, e.g., by other mutations such as a single nucleotide polymorphism (SNP) in the so-called MIP1 gene that can increase petite mutant accumulation by 4-fold [33]. SNPs in SSD1 have also been found in various wild and clinically isolated yeast strains used in the Saccharomyces Genome Resequencing Project (SGRP; 34). Eighteen non-synonymous SNPs in SSD1 have been found, with two of them being nonsense SNPs [34]. Although the growth or survival advantage of the existence of various SNPs, including defective mutations, in these genes is unclear, it is clear that mutants receptive to DNA via TKC emerge frequently and are widely distributed in nature. In addition, the TKC efficiency of high-receptivity strains was comparable with the conjugation efficiency from E. coli to A. tumefaciens or E. coli under certain conditions (Figures 4 and S4C). TKC may be one of the major driving forces behind gene transfer from bacteria to eukaryotes under natural conditions. The identification of mutants in nonessential genes with higher TKC efficiency indicates the possibility that various subpopulations within recipient species can accept exogenous DNA at higher rates (Figure 6). The existence of such a subpopulation may be applied not only to TKC but also to other DNA transfer or HGT mechanisms that might exist. Due to the lack of experimental approaches, past HGT events have thus far been deduced mainly from in silico analyses. Even in the case of T-DNA transfer, which continuously occurs in nature and fields, the past T-DNA transfer events have only been found in genomes of one genus (Nicotiana) [35]. Thus, it is very difficult to identify direct evidence that can prove the pathway of ancient HGT events. For these reasons, the establishment of in vitro HGT detection systems that enable molecular and genetic analyses of donor and recipient organisms as well as the quantification of HGT–such as a TKC model system–would be helpful for elucidating the mechanisms that contribute to HGT events both contemporary and past events. In the model HGT system for DNA transfer from bacteria to yeast designed and presented here, we emphasize that the TKC process was carried out in a suspension of donor and recipient cells under ambient conditions (Figures 3 and S4B), in the absence of heat shock step and/or membrane destabilizing agents, i.e. conditions that are in contrast to those applied in other methods of gene transfer in yeast [21], [36]. Such a method should be considered for its biosafety in biotechnological applications, as has been considered for bacterial conjugal transfer, which occurs in the natural environment. Although we have shown that T4SS mediated HGT could be a convincing driving force, T4SS is not omnipotent for explaining HGT in Eukaryotes. HGT-derived genes found in bdelloid rotifers appear to have originated from various organisms such as bacteria, yeasts and plants [6], and some of the HGT-derived genes reported in higher plants are likely to have originated from fungi [8]. Again, these data strongly indicate the importance of experimental approaches that focuses on the driving force of HGT in addition to phagotrophy and TKC. A recent study showed that TKC may also be useful and applicable as a method for introducing bacterial genes into eukaryotes [37]. Its main advantage is the simplicity of execution. The only requirement is of generating an E. coli strain with a helper plasmid and a plasmid encoding the gene of interest to be transferred. Such a strain would be allowed to interact with the eukaryotic recipient to achieve HGT. The method avoids DNA extraction or having to transfer it into agrobacteria. As for S. cerevisiae, the treatment with antibiotics, which inhibit mitochondrial functional integrity, could easily increase TKC efficiency (Figure 1A and 1B). In addition, the EGY48 strain, which is used for the yeast two-hybrid system, was successfully modified as a high receptivity strain by introducing a mutation into SSD1 (Figure 1D). These data show that this has potential for use as a gene introduction method. Three TKC vectors, pRS313::oriTP, pRS315::oriTP, and pRS316::oriTP were designed in such a way that their respective multiple cloning sites as well as their parental yeast shuttle vectors could be used. These vectors are available at a public bioresource bank for universal use (Table 1). In this study, we focused on determining the potential of TKC as a driving force behind HGT and attempted to identify the genetic background that enables high receptivity in the recipient organism. Our results strongly support the idea that genomes of certain eukaryotes have been exposed to exogenous DNA more frequently and continuously than previously thought, with DNA and gene transfer frequencies from bacteria similar to those measured between prokaryotes.

Supporting Information Figure S1. Schematic representation of TKC detection. The donor E. coli has a helper plasmid and a mobilizable plasmid. The helper plasmid contains T4SS genes derived from an IncP1α plasmid, called tra genes (gray box). The mobilizable plasmid contains the origin of transfer (oriT; black dot) of an IncQ plasmid and URA3 gene (black box). When the mobilizable plasmid transfers into the recipient yeast cell lacking the URA3 gene, the transconjugant survives on selection medium with no added uracil. https://doi.org/10.1371/journal.pone.0074590.s001 (TIF) Figure S2. An example of the TKC results at the third screening. (A) Template showing format of plating of the various mutants. Mutants for nuclear-encoded mitochondrial genes are underlined. * KO mutant of dubious ORF unlikely to encode a protein, and its overlapping gene is shown in parentheses. ** High-receptivity mutants included in Table S1. (B) Transconjugants grown on a selection plate; volume equivalent to 50% of each TKC reaction mix was plated. (C) Recipient yeast cells grown on a YPD+chloramphenicol plate; a volume equivalent to 1/5000 of each TKC reaction mix was plated. HB101-containing plasmids pRH210 and pAY205 were used as the donors. https://doi.org/10.1371/journal.pone.0074590.s002 (TIF) Figure S3. Confirmation of the mitochondrial integrity among the identified high-receptivity mutants. The high-receptivity mutants were mated with a MATa strain, carrying wild-type nuclear genome but a rho0 mitochondrial genome derived from the BY4741 strain. The resultant heterogeneous diploid strains were serially diluted and spotted on both rich glucose (YPD) and rich respiratory glycerol (YPG) media, and were incubated at 28°C for 48 h and 72 h, respectively. The 14 KO mutants for non-mitochondrial genes are underlined. https://doi.org/10.1371/journal.pone.0074590.s003 (TIF) Figure S4. Confirmation and characterization of the high-receptivity mutants. (A) Effect of knock-out mutation in the 5 candidate genes in the yeast strain BY4741 on TKC. Data are represented as mean ± SD (n = 3). Asterisks indicate a statistically significant difference: ***p<0.001 (two-tailed t-test). (B) Comparison between TKC and bacterial conjugation efficiency under TNB and filter-sterilized freshwater environments. Data are represented as mean ± SD (n = 5). Upper- and lowercase letters above the bars indicate significant differences at p<0.05 (Holm’s test) among yeast and bacterial strains under each condition. (C) Effect of cell wall digestion on TKC efficiency. Normal: the recipient cells were resuspended in TNB before testing for TKC. Mock: cells pretreated with TNB +1 M sorbitol, and TKC reaction performed in the same solution. Zymolyase: pretreatment with TNB +1 M sorbitol +0.5 mg/mL Zymolyase-100 T, and TKC reaction performed in TNB +1 M sorbitol. Each pretreatment was performed for 1 h at 28°C. Data are represented as mean ± SD (n = 7 in BY4742 with sorbitol, n = 4 in others). Lowercase letters above the bars indicate significant differences at p<0.05 (Holm’s test). HB101 (pRH210, pAY205) was used as the donor in all experiments. https://doi.org/10.1371/journal.pone.0074590.s004 (TIF) Figure S5. Confirmation of high TKC receptivity in ssd1Δ and rho0 mutants using other selection markers. (A) A TKC vector pRS313::oriTP, carrying HIS3 gene as a selection marker, was used and the transconjugants in parental and mutant strains were selected on a selection medium plate lacking leucine. (B) A TKC vector pRS315::oriTP, carrying HIS3 gene as a selection marker, was used. https://doi.org/10.1371/journal.pone.0074590.s005 (TIF) Table S1. List of high-receptivity mutants screened from the complete set of Yeast Deletion Clones (MATα haploids complete set). Efficiency of transfer of the URA3 marker gene from Escherichia coli to various yeast deletion strains was measured relative to transfer to the parental strain (fold increase vs. wt). https://doi.org/10.1371/journal.pone.0074590.s006 (DOC) Table S2. Results of the TKC experiment without the helper plasmid. https://doi.org/10.1371/journal.pone.0074590.s007 (DOC) Table S3. PCR primers used in this study. https://doi.org/10.1371/journal.pone.0074590.s008 (DOC)

Acknowledgments We express our gratitude to the following persons who kindly provided us with useful materials. The SSD1-V and ssd1-d constructs were provided by Dr. T. Kokubo (Graduate School of Nanobioscience, Yokohama City University). The helper plasmid, pDPT51, was provided by Dr. Y. Fujita (Graduate School of Bioagricultural Science, Nagoya University). We are grateful to Dr. Jon Y. Suzuki (USDA-ARS-Pacific Basin Agricultural Research Center) for helpful discussions and critical reading of the manuscript. We also thank Shin-ya Fujihara, Yosuke Ikegaya and Satoko Kagei for their excellent technical assistance.

Author Contributions Conceived and designed the experiments: KM SY KT KS. Performed the experiments: KM NK. Analyzed the data: KM NK. Contributed reagents/materials/analysis tools: KM KS. Wrote the paper: KM.