Horizontal gene transfer: More than 250 genes transferred

_{Denyse O'Leary

May 19, 2014

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– In the paste of Roquefort and on the surface of Camembert, the microscopic filamentous fungi Penicillium roqueforti and Penicillium camembertii are responsible for the formation of a greenish-blue mould in the former case and a so-called “bloom” in the latter. Sequencing of the genomes of these two fungi has recently, and surprisingly, shown that these genomes contain more than 250 strictly identical genes, thus indicating a transfer of genes between these two species. This type of event, until now little described in eukaryotes, may therefore be much more common than previously thought.

So-called horizontal gene transfers between species are well documented in bacteria, but much less so in fungi and other eukaryote organisms, although examples of such transfers in wine-making yeasts have been described in recent years. It therefore appeared that these transfers were rare, sporadic and had little impact in eukaroytes. However, the sequencing of several Penicillium species has suggested that the contrary might be true. Indeed, a comparison of the genomes of two important cheese fungi, Penicillium roqueforti and Penicillium camembertii has revealed the presence of numerous totally identical sequence fragments, while other genomic regions were only 85-90% similar. More.

Unlike Darwinian natural selection, HGT, also called gene swapping, is a demonstrable method of evolution.

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Comments

tjguy: "For me, gene swapping really doesn’t solve the evolution conumdrum because the new genes are not being created, simply swapped from one organism to another. What evolution needs to be able to explain is how this information was created to begin with. At some point in the evolutionary process, these genes did not exist and then they came into existence. How does that happen?" You are perfectly right: HGT does not explain functional information, it only redistributes it. Many forms of antibiotic resistance are based on gene swapping. No new genes are created in that process.gpuccio_{May 20, 2014
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Thank you Phoodoo for your response. But I still can't see Natural.Selection as a result - seems too circular a definition. Most definitions call it a "process". So the term "method" used in the OP seems apt. Ok, maybe "Natural Selection" is the result of a fertile imagination? I could grasp that definition. Cow based fertilizer fertile.ppolish_{May 20, 2014
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ppolish, Natural selection is the RESULT caused by variation in the fitness levels of individuals in a population. Some are (supposedly) more fit than others, and thus are naturally selected (more like not selected for) for through attrition. Now can this actually do anything to increase the fitness of any organism, certainly not. The only thing that increases an individuals fitness or that creates a new functioning trait is change, from one generation to another. How that change occurs, what makes it happen in a sustainable order, the pre-meditation of that change, the teleology of that change, the pre-existence of the ability to change within an organism-these are all challenges to Darwinism-challenges that Darwinism frankly has virtually no ability to adequately address. I was listening to a science talk about skin today. They were detailing all the levels of skin in a human, the amazing amounts of properties of skin, how some layers must be water permeable, while others must have sealing abilities, its thermal regulation properties, sensation functions, endocrine functioning, ability to stretch, grow and repair....is there any possible way an uncontrolled random mutation process could ever neatly create this multifaceted necessary part of life, with some organisms having useful skin, and others not so useful? Its preposterous. It doesn't really matter which aspect of change one wants to emphasize, HGT, random beneficial mutations, neutral mutations, or any other combination of unguided theories, none of them could ever make skin the way it is, without a plan. Imagine all of the random results that would have to be continually occurring and failing, before you would ever get to even the most rudimentary of a stretchy covering, that would somehow enable an organism to breathe and move. And then let that horrible covering be subjected to every possible experiment of mutation, waiting for it to somehow improve before destroying 99.9 percent of the existing population. Natural selection, spurred only by a chaos of random experiments is such a laughable concept, that it should provide anyone who doubts it with extreme confidence in their position.phoodoo_{May 20, 2014
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Thanks. That helps. For me, gene swapping really doesn't solve the evolution conumdrum because the new genes are not being created, simply swapped from one organism to another. What evolution needs to be able to explain is how this information was created to begin with. At some point in the evolutionary process, these genes did not exist and then they came into existence. How does that happen? Even creationists have no problem with gene swapping, but this is a limited solution because you have to have something to swap. If evolution could produce the genes that the organisms swapped, why didn't it recreate those same genes again in the new species? Am I misunderstanding Gene swapping? Gene swapping causes havoc for Darwin's elusive and probably imaginary tree of life as well I would think.tjguy_{May 20, 2014
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Ehm, it was not my intention t paste all of that. Something must have gone wrong. :)gpuccio_{May 20, 2014
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tjguy:
In the P. roqueforti FM164 strain, all the sequences found identical to both P. rubens and P. camemberti clustered together within a single 575 kb region accounting for 2% of the genome, which we called ‘Wallaby' (Figs 1 and ?and2).2). This region lies within a 7.8 Mb chromosome (Supplementary Fig. S3). The genomes of additional strains of P. roqueforti were examined by SOLiD resequencing. Three lacked the entire Wallaby region, whereas the fourth carried the very same Wallaby sequence as FM164. These sequences were aligned for precise mapping of the Wallaby insertion point (Fig. 3). Some of the regions flanking Wallaby could be characterized in P. rubens and P. camemberti and were found to share 85–90% identity in the three species. However, these sequences were located on other scaffolds than those carrying the Wallaby fragments in the two later species (Fig. 2), indicating non-homologous locations of Wallaby between all three species. The possibility of misassembly yielding these different locations was excluded by the successful PCR amplification of fragments overlapping the edges of the identical sequences in the three genomes. Furthermore, rearrangement events appeared to have occurred after the transfers in all three species. Indeed, the Wallaby sequences of P. rubens and P. camemberti lacked various fragments present in P. roqueforti, and both contained a 86-kb fragment absent from P. roqueforti (Fig. 2). Wallaby displayed little similarity to sequences from public databases, even those of the well-studied Aspergillus genus, the closest relative of Penicillium18. When Blast hits could be obtained, they matched fungal genomes (Supplementary Fig. S4), indicating a fungal origin for Wallaby. A etranucleotide composition analysis of Wallaby and its comparison with several fungal genomes revealed a nucleotide composition of Wallaby that seemed to be different from the rest of the genomes of P. rubens, P. camemberti and P. roqueforti, but still closer from these genomes than any other (Table 2 and Supplementary Fig. S5). The nucleotide composition of Wallaby also differed from that of other available fungal genomes, but was nevertheless closest to Aspergillus and Penicillium strains, suggesting that the donor species lie within this clade (Table 2, Supplementary Fig. S5). Overall, these results indicate that Wallaby has been recently and independently acquired, in at least some of these Penicillium species, via horizontal transfers. Other alternative explanations, such as introgression, can be excluded because of the non-homologous locations of the identical sequences in P. rubens and P. camemberti, precluding a parsimonious hybridization hypothesis. The perfect identity of the sequences also argues for very recent transfer events. No synonymous mutations were found in Wallaby, except for some repeat induced point (RIP) mutation footprints in P. roqueforti, indicating that the presence of Wallaby is not an ancestral character. Pairwise comparison of the genome-wide distribution of 100% identical sequences between P. roqueforti and the three other genomes revealed no long stretches of sequences Nature Communications Nature Publishing Group Multiple recent horizontal transfers of a large genomic region in cheese making fungi Kevin Cheeseman, Jeanne Ropars, [...], and Yves Brygoo Additional article information Abstract While the extent and impact of horizontal transfers in prokaryotes are widely acknowledged, their importance to the eukaryotic kingdom is unclear and thought by many to be anecdotal. Here we report multiple recent transfers of a huge genomic island between Penicillium spp. found in the food environment. Sequencing of the two leading filamentous fungi used in cheese making, P. roqueforti and P. camemberti, and comparison with the penicillin producer P. rubens reveals a 575 kb long genomic island in P. roqueforti—called Wallaby—present as identical fragments at non-homologous loci in P. camemberti and P. rubens. Wallaby is detected in Penicillium collections exclusively in strains from food environments. Wallaby encompasses about 250 predicted genes, some of which are probably involved in competition with microorganisms. The occurrence of multiple recent eukaryotic transfers in the food environment provides strong evidence for the importance of this understudied and probably underestimated phenomenon in eukaryotes. Humans have created many novel, nutrient-rich and homogeneous environments exerting strong selection pressures and inducing the rapid adaptation of microorganisms, such as fungal pathogens of crops, food spoilers and domesticated fungi used for fermentation in beverage or food (for example, Saccharomyces for bread, beer and wine, Aspergillus for traditional fermented Asian foods, such as sake, soy sauce and miso, and Penicillium for cheese and cured or fermented meat). These rapid adaptations in fungi provide excellent models for studying general processes of eukaryotic genome evolution, including the functional and ecological impact of horizontal gene transfer1 and changes in metabolism2. Prokaryote-to-prokaryote transfers have been recognized as common and their associated impact important enough to raise questions about the possibility of reconstructing prokaryotic history through a tree of life or to change practices relating to antibiotic use in medicine. By contrast, horizontal transfers in eukaryotic species are still perceived by many to be isolated, sporadic events with a limited impact3,4,5. However, over the last 20 years or so, a number of cases of gene transfer in eukaryotes have been described. These documented cases include transfers of genetic material of prokaryotic origin into a eukaryote host6 and transfers in man-made environments, for example, between yeasts used for wine fermentation1 or pathogenic fungi on crops6,7,8,9. Both the sizes and number of genes involved vary widely. Transfers of a single gene, a complete metabolic pathway10, whole chromosomes11 or even cases of the integration of almost complete genomes from bacterial endosymbionts into their eukaryotic hosts12 have been described. Notable impacts of recently described horizontal transfers include key roles in land colonization by plants13, pigment production in spider mites through the acquisition of fungal carotenoid biosynthesis genes14 and the emergence of plant diseases through transfers in fungi15. Despite these examples, the lack of specific evolutionary trends in reported cases of lateral gene transfer in eukaryotes has led to the view of ancient, sporadic and isolated events with relatively little global impact on eukaryotic kingdoms, rather than a more frequently and widely occurring phenomenon. The frequency and importance of eukaryote-to-eukaryote gene transfer may, however, be underestimated. Penicillium species are ubiquitous filamentous ascomycetes important to the biotechnology, biomedical and food industries. They commonly occur as food spoilage agents and opportunistic pathogens and are widely used as versatile cell factories. P. camemberti and P. roqueforti are used as starter cultures for cheeses. P. camemberti, used for the maturation of soft cheeses, such as Camembert, is the result of many selection programs aiming to improve the texture and colour of the conidia or physiological characteristics. P. camemberti has never been isolated from substrates other than dairy products. P. roqueforti is widespread in food and also occurs in silage and natural environments. It is used as a starter culture in the production of most blue-veined cheeses (including Roquefort, Gorgonzola, Stilton and Danish Blue) and its abilities to tolerate cold temperatures, low oxygen concentrations, alkaline and weak acid preservatives, make it a common spoilage agent in refrigerated stored foods, meat products, rye bread and silage16,17. To our knowledge, despite the importance of the filamentous fungi used in cheese making, no genome sequence has yet been published for any of these species. The availability of these first two genome sequences will therefore provide a useful resource for improving our knowledge of edible cheese moulds and for comparative genomics. Here we sequence and assemble the genomes of P. roqueforti and P. camemberti and compare them with two other available Penicillium genomes, those of the penicillin producer and food spoilage agent P. rubens, previously known as P. chrysogenum18, and of the Citrus pathogen P. digitatum19. We report a case of multiple, present-day horizontal transfers of a very large (over 500 kb) genomic island between several cheese fungi. This genomic island harbours about 250 genes, some of which are probably involved in competition with other microorganisms. Beyond the potential conceptual and applied implications of recurrent horizontal transfers occurring in food, this finding indicates that horizontal gene transfer (HGT) may be more widespread and important than previously thought in eukaryotes. Results Detection of a horizontally transferred genomic island The global characteristics and comparisons of the genomes of P. camemberti, P. roqueforti, P. rubens and P. digitatum are given in Table 1. The genomes have similar sizes and number of genes, with the exception of P. digitatum, which has fewer genes than the other three genomes. This smaller number of genes is thought to be the result of a streamlining process affecting the genome of P. digitatum due to its specialized plant pathogenic lifestyle19. Assembly quality, as shown by the N50 metric and the number of scaffolds, is high for P. roqueforti and P. rubens. The initial in silico assembly for P. roqueforti has been experimentally validated and further improved (see below). The genome assemblies for P. camemberti and P. digitatum appear more fragmented. Table 1 Table 1 Accession numbers and global characteristics of the Penicillium genomes compared in this study. Surprisingly, several scaffolds from P. roqueforti, P. camemberti and P. rubens had stretches of more than 5 kb displaying 100% identity in common (Supplementary Fig. S1), whereas mean pairwise identity was, otherwise, only 85–90% between genomes. P. digitatum completely lacked these regions that were identical in the other Penicillium species. We investigated the nature of these shared sequences, by locating these regions accurately in the genome of P. roqueforti by improving assembly quality. The availability of a high-quality genome sequence in this clade will also improve the resolving power of comparative genome analysis in subsequent studies. For this purpose, we used a combination of polymerase chain reaction (PCR) and molecular combing, a powerful fluorescent in situ hybridization-based technique for the direct visualization of single-DNA molecules20,21. Molecular combing resulted in the successful mapping of scaffolds, including some separated by more than a 100 kb, onto single-DNA molecules, thus constituting a new means of improving or experimentally validating de novo genome assemblies (Supplementary Fig. S2). It yielded an assembly in which 92% of the P. roqueforti genome was clustered into six super scaffolds of chromosomal sizes (Supplementary Fig. S3). In the P. roqueforti FM164 strain, all the sequences found identical to both P. rubens and P. camemberti clustered together within a single 575 kb region accounting for 2% of the genome, which we called ‘Wallaby' (Figs 1 and ?and2).2). This region lies within a 7.8 Mb chromosome (Supplementary Fig. S3). The genomes of additional strains of P. roqueforti were examined by SOLiD resequencing. Three lacked the entire Wallaby region, whereas the fourth carried the very same Wallaby sequence as FM164. These sequences were aligned for precise mapping of the Wallaby insertion point (Fig. 3). Some of the regions flanking Wallaby could be characterized in P. rubens and P. camemberti and were found to share 85–90% identity in the three species. However, these sequences were located on other scaffolds than those carrying the Wallaby fragments in the two later species (Fig. 2), indicating non-homologous locations of Wallaby between all three species. The possibility of misassembly yielding these different locations was excluded by the successful PCR amplification of fragments overlapping the edges of the identical sequences in the three genomes. Furthermore, rearrangement events appeared to have occurred after the transfers in all three species. Indeed, the Wallaby sequences of P. rubens and P. camemberti lacked various fragments present in P. roqueforti, and both contained a 86-kb fragment absent from P. roqueforti (Fig. 2). Figure 1 Figure 1 Structural characterization of the Wallaby region in P. roqueforti FM164. Figure 2 Figure 2 Comparative genomic structure of Wallaby. Figure 3 Figure 3 Read mapping of the genomes of two additional strains against the Wallaby locus of the P. roqueforti FM164. Wallaby displayed little similarity to sequences from public databases, even those of the well-studied Aspergillus genus, the closest relative of Penicillium18. When Blast hits could be obtained, they matched fungal genomes (Supplementary Fig. S4), indicating a fungal origin for Wallaby. A tetranucleotide composition analysis of Wallaby and its comparison with several fungal genomes revealed a nucleotide composition of Wallaby that seemed to be different from the rest of the genomes of P. rubens, P. camemberti and P. roqueforti, but still closer from these genomes than any other (Table 2 and Supplementary Fig. S5). The nucleotide composition of Wallaby also differed from that of other available fungal genomes, but was nevertheless closest to Aspergillus and Penicillium strains, suggesting that the donor species lie within this clade (Table 2, Supplementary Fig. S5). Table 2 Table 2 Pearson correlation coefficients for Z-score of tetranucleotide frequency. Overall, these results indicate that Wallaby has been recently and independently acquired, in at least some of these Penicillium species, via horizontal transfers. Other alternative explanations, such as introgression, can be excluded because of the non-homologous locations of the identical sequences in P. rubens and P. camemberti, precluding a parsimonious hybridization hypothesis. The perfect identity of the sequences also argues for very recent transfer events. No synonymous mutations were found in Wallaby, except for some repeat induced point (RIP) mutation footprints in P. roqueforti, indicating that the presence of Wallaby is not an ancestral character. Pairwise comparison of the genome-wide distribution of 100% identical sequences between P. roqueforti and the three other genomes revealed no long stretches of sequences identical in all three genomes other than those in Wallaby. The only variation within Wallaby thus corresponded to RIP mutation footprints in the P. roqueforti Wallaby region (Fig. 2; Supplementary Fig. S6). In fungi, the RIP mechanism induces multiple C:G to T:A substitutions in repeated sequences during sex events22,23. As a consequence, sequence identity in Wallaby fragments with RIP footprints dropped to 90–97% between P. roqueforti and the other two Penicillium sequences, but this concerned exclusively RIP C:G to T:A substitutions. This is consistent with the occurrence of RIP after the transfer event in P. roqueforti.
Emphasis mine.gpuccio_{May 20, 2014
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.... thus indicating a transfer of genes between these two species.
I don't understand this deduction. If one cheese fungi evolved from the other, couldn't that also explain the findings? I'm not a fan of molecules to man evolution, but this amount of change seems possible for even biblical creationists to accept. So maybe I'm missing something, but why hgt as opposed to simple speciation? When talking about sonar in bats and whales it is seen as convergent evolution rather than common descent. I understand the thinking even though I disagree with it, but how does one distinguish between HGT and simple speciation?tjguy_{May 19, 2014
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Phoodoo, natural selection (cheese fungi eg) is the result of what? The result of gene swapping? The result of Evolution in general. Sorry for dumb question.ppolish_{May 19, 2014
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I find myself agreeing with Phoodoo (please don't tell anyone). Darwin came up with his theory without any knowledge of genetics, DNA, mutations, meiosis, mitosis, transcription, etc. which is one of the reasons that it has stood the test of time. Each new discovery has been consistent, with some modification, to the original theory. For his theory to work, it needs two things. A source of variation and differential reproduction within a population. Early on, we thought that the source of variation came from mutations and various rearrangement of genes on chromosomes. We now know that there are other sources of variation, including HGT. So, if anything, HGT just adds more credibility to natural selection, not less.Acartia_bogart_{May 19, 2014
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Sorry to be contentious, but natural selection is not a method, it is a result. So the only apt comparison is between random mutations and horizontally altered genes. Therefore natural selection could result from either method of genome variation (but I doubt it does much).phoodoo_{May 19, 2014
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