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 ().
A schematic diagram of the gene and plasmid flow from bacteria to eukaryotes by T4SS.
Yeast mutants such as the petite
) 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 
. 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 
. 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 
. 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 ( 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 (). 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
. 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 ( 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 
. 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 
, and some of the HGT-derived genes reported in higher plants are likely to have originated from fungi 
. 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 
. 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 (). 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
(). These data show that this has potential for use as a gene introduction method. Three TKC vectors, pRS313::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 ().
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.