Ancient Horizontal Gene Transfer from Bacteria Enhances Biosynthetic Capabilities of Fungi

Background

Polyketides are natural products with a wide range of biological functions and pharmaceutical applications. Discovery and utilization of polyketides can be facilitated by understanding the evolutionary processes that gave rise to the biosynthetic machinery and the natural product potential of extant organisms. Gene duplication and subfunctionalization, as well as horizontal gene transfer are proposed mechanisms in the evolution of biosynthetic gene clusters. To explain the amount of homology in some polyketide synthases in unrelated organisms such as bacteria and fungi, interkingdom horizontal gene transfer has been evoked as the most likely evolutionary scenario. However, the origin of the genes and the direction of the transfer remained elusive.

Methodology/Principal Findings

We used comparative phylogenetics to infer the ancestor of a group of polyketide synthase genes involved in antibiotic and mycotoxin production. We aligned keto synthase domain sequences of all available fungal 6-methylsalicylic acid (6-MSA)-type PKSs and their closest bacterial relatives. To assess the role of symbiotic fungi in the evolution of this gene we generated 24 6-MSA synthase sequence tags from lichen-forming fungi. Our results support an ancient horizontal gene transfer event from an actinobacterial source into ascomycete fungi, followed by gene duplication.

Conclusions/Significance

Given that actinobacteria are unrivaled producers of biologically active compounds, such as antibiotics, it appears particularly promising to study biosynthetic genes of actinobacterial origin in fungi. The large number of 6-MSA-type PKS sequences found in lichen-forming fungi leads us hypothesize that the evolution of typical lichen compounds, such as orsellinic acid derivatives, was facilitated by the gain of this bacterial polyketide synthase.     

A fungal phylogeny based on 42 complete genomes derived from supertree and combined gene analysis

Background  

To date, most fungal phylogenies have been derived from single gene comparisons, or from concatenated alignments of a small number of genes. The increase in fungal genome sequencing presents an opportunity to reconstruct evolutionary events using entire genomes. As a tool for future comparative, phylogenomic and phylogenetic studies, we used both supertrees and concatenated alignments to infer relationships between 42 species of fungi for which complete genome sequences are available.     

Sequences of isopenicillin N synthetase genes suggest horizontal gene transfer from prokaryotes to eukaryotes

Evolutionary distances between bacterial and fungal isopenicillin N synthetase (IPNS) genes have been compared to distances between the corresponding 5S rRNA genes. The presence of sequences homologous to the IPNS gene has been examined in DNAs from representative prokaryotic organisms and Ascomycotina. The results of both analyses strongly support two different events of horizontal transfer of the IPNS gene from bacteria to filamentous fungi. This is the first example of such a type of transfer from prokaryotes to eukaryotes.     

Origin and distribution of epipolythiodioxopiperazine (ETP) gene clusters in filamentous ascomycetes

Background  

Genes responsible for biosynthesis of fungal secondary metabolites are usually tightly clustered in the genome and co-regulated with metabolite production. Epipolythiodioxopiperazines (ETPs) are a class of secondary metabolite toxins produced by disparate ascomycete fungi and implicated in several animal and plant diseases. Gene clusters responsible for their production have previously been defined in only two fungi. Fungal genome sequence data have been surveyed for the presence of putative ETP clusters and cluster data have been generated from several fungal taxa where genome sequences are not available. Phylogenetic analysis of cluster genes has been used to investigate the assembly and heredity of these gene clusters.     

A novel mode of chromosomal evolution peculiar to filamentous Ascomycete fungi

Background  

Gene loss, inversions, translocations, and other chromosomal rearrangements vary among species, resulting in different rates of structural genome evolution. Major chromosomal rearrangements are rare in most eukaryotes, giving large regions with the same genes in the same order and orientation across species. These regions of macrosynteny have been very useful for locating homologous genes in different species and to guide the assembly of genome sequences. Previous analyses in the fungi have indicated that macrosynteny is rare; instead, comparisons across species show no synteny or only microsyntenic regions encompassing usually five or fewer genes. To test the hypothesis that chromosomal evolution is different in the fungi compared to other eukaryotes, synteny was compared between species of the major fungal taxa.     

Evolution of mosaic operons by horizontal gene transfer and gene displacement <Emphasis Type="Italic">in situ</Emphasis>

Background

Shuffling and disruption of operons and horizontal gene transfer are major contributions to the new, dynamic view of prokaryotic evolution. Under the 'selfish operon' hypothesis, operons are viewed as mobile genetic entities that are constantly disseminated via horizontal gene transfer, although their retention could be favored by the advantage of coregulation of functionally linked genes. Here we apply comparative genomics and phylogenetic analysis to examine horizontal transfer of entire operons versus displacement of individual genes within operons by horizontally acquired orthologs and independent assembly of the same or similar operons from genes with different phylogenetic affinities.

Results

Since a substantial number of operons have been identified experimentally in only a few model bacteria, evolutionarily conserved gene strings were analyzed as surrogates of operons. The phylogenetic affinities within these predicted operons were assessed first by sequence similarity analysis and then by phylogenetic analysis, including statistical tests of tree topology. Numerous cases of apparent horizontal transfer of entire operons were detected. However, it was shown that apparent horizontal transfer of individual genes or arrays of genes within operons is not uncommon either and results in xenologous gene displacement in situ, that is, displacement of an ancestral gene by a horizontally transferred ortholog from a taxonomically distant organism without change of the local gene organization. On rarer occasions, operons might have evolved via independent assembly, in part from horizontally acquired genes.

Conclusions

The discovery of in situ gene displacement shows that combination of rampant horizontal gene transfer with selection for preservation of operon structure provides for events in prokaryotic evolution that, a priori, seem improbable. These findings also emphasize that not all aspects of operon evolution are selfish, with operon integrity maintained by purifying selection at the organism level.

     

Multiple losses of sex within a single genus of Microsporidia

Background  

Most asexual eukaryotic lineages have arisen recently from sexual ancestors and contain few ecologically distinct species, providing evidence for long-term advantages of sex. Ancient asexual lineages provide rare exceptions to this rule and so can yield valuable information relating to the evolutionary forces underlying the maintenance of sex. Microsporidia are parasitic, unicellular fungi. They include many asexual species which have traditionally been grouped together into large, presumably ancient taxonomic groups. However, these putative ancient asexual lineages have been identified on the basis of morphology, life cycles and small subunit ribosomal RNA (16S rRNA) gene sequences, all of which hold questionable value in accurately inferring phylogenetic relationships among microsporidia.     

Evidence of recent interkingdom horizontal gene transfer between bacteria and <Emphasis Type="Italic">Candida parapsilosis</Emphasis>

Background  

To date very few incidences of interdomain gene transfer into fungi have been identified. Here, we used the emerging genome sequences of Candida albicans WO-1, Candida tropicalis, Candida parapsilosis, Clavispora lusitaniae, Pichia guilliermondii, and Lodderomyces elongisporus to identify recent interdomain HGT events. We refer to these as CTG species because they translate the CTG codon as serine rather than leucine, and share a recent common ancestor.     

Genomic and evolutionary comparisons of diazotrophic and pathogenic bacteria of the order Rhizobiales

Background

Species belonging to the Rhizobiales are intriguing and extensively researched for including both bacteria with the ability to fix nitrogen when in symbiosis with leguminous plants and pathogenic bacteria to animals and plants. Similarities between the strategies adopted by pathogenic and symbiotic Rhizobiales have been described, as well as high variability related to events of horizontal gene transfer. Although it is well known that chromosomal rearrangements, mutations and horizontal gene transfer influence the dynamics of bacterial genomes, in Rhizobiales, the scenario that determine pathogenic or symbiotic lifestyle are not clear and there are very few studies of comparative genomic between these classes of prokaryotic microorganisms trying to delineate the evolutionary characterization of symbiosis and pathogenesis.

Results

Non-symbiotic nitrogen-fixing bacteria and bacteria involved in bioremediation closer to symbionts and pathogens in study may assist in the origin and ancestry genes and the gene flow occurring in Rhizobiales. The genomic comparisons of 19 species of Rhizobiales, including nitrogen-fixing, bioremediators and pathogens resulted in 33 common clusters to biological nitrogen fixation and pathogenesis, 15 clusters exclusive to all nitrogen-fixing bacteria and bacteria involved in bioremediation, 13 clusters found in only some nitrogen-fixing and bioremediation bacteria, 01 cluster exclusive to some symbionts, and 01 cluster found only in some pathogens analyzed. In BBH performed to all strains studied, 77 common genes were obtained, 17 of which were related to biological nitrogen fixation and pathogenesis. Phylogenetic reconstructions for Fix, Nif, Nod, Vir, and Trb showed possible horizontal gene transfer events, grouping species of different phenotypes.

Conclusions

The presence of symbiotic and virulence genes in both pathogens and symbionts does not seem to be the only determinant factor for lifestyle evolution in these microorganisms, although they may act in common stages of host infection. The phylogenetic analysis for many distinct operons involved in these processes emphasizes the relevance of horizontal gene transfer events in the symbiotic and pathogenic similarity.     

A new experimental approach for studying bacterial genomic island evolution identifies island genes with bacterial host-specific expression patterns

Background  

Genomic islands are regions of bacterial genomes that have been acquired by horizontal transfer and often contain blocks of genes that function together for specific processes. Recently, it has become clear that the impact of genomic islands on the evolution of different bacterial species is significant and represents a major force in establishing bacterial genomic variation. However, the study of genomic island evolution has been mostly performed at the sequence level using computer software or hybridization analysis to compare different bacterial genomic sequences. We describe here a novel experimental approach to study the evolution of species-specific bacterial genomic islands that identifies island genes that have evolved in such a way that they are differentially-expressed depending on the bacterial host background into which they are transferred.     

Identification of multiple independent horizontal gene transfers into poxviruses using a comparative genomics approach

Background  

Poxviruses are important pathogens of humans, livestock and wild animals. These large dsDNA viruses have a set of core orthologs whose gene order is extremely well conserved throughout poxvirus genera. They also contain many genes with sequence and functional similarity to host genes which were probably acquired by horizontal gene transfer.     

Doxycycline-regulated gene expression in the opportunistic fungal pathogen Aspergillus fumigatus

Background  

Although Aspergillus fumigatus is an important human fungal pathogen there are few expression systems available to study the contribution of specific genes to the growth and virulence of this opportunistic mould. Regulatable promoter systems based upon prokaryotic regulatory elements in the E. coli tetracycline-resistance operon have been successfully used to manipulate gene expression in several organisms, including mice, flies, plants, and yeast. However, the system has not yet been adapted for Aspergillus spp.     

Analysis of plasmid genes by phylogenetic profiling and visualization of homology relationships using Blast2Network

Background  

Phylogenetic methods are well-established bioinformatic tools for sequence analysis, allowing to describe the non-independencies of sequences because of their common ancestor. However, the evolutionary profiles of bacterial genes are often complicated by hidden paralogy and extensive and/or (multiple) horizontal gene transfer (HGT) events which make bifurcating trees often inappropriate. In this context, plasmid sequences are paradigms of network-like relationships characterizing the evolution of prokaryotes. Actually, they can be transferred among different organisms allowing the dissemination of novel functions, thus playing a pivotal role in prokaryotic evolution. However, the study of their evolutionary dynamics is complicated by the absence of universally shared genes, a prerequisite for phylogenetic analyses.     

Two different secondary metabolism gene clusters occupied the same ancestral locus in fungal dermatophytes of the arthrodermataceae

Background

Dermatophyte fungi of the family Arthrodermataceae (Eurotiomycetes) colonize keratinized tissue, such as skin, frequently causing superficial mycoses in humans and other mammals, reptiles, and birds. Competition with native microflora likely underlies the propensity of these dermatophytes to produce a diversity of antibiotics and compounds for scavenging iron, which is extremely scarce, as well as the presence of an unusually large number of putative secondary metabolism gene clusters, most of which contain non-ribosomal peptide synthetases (NRPS), in their genomes. To better understand the historical origins and diversification of NRPS-containing gene clusters we examined the evolution of a variable locus (VL) that exists in one of three alternative conformations among the genomes of seven dermatophyte species.

Results

The first conformation of the VL (termed VLA) contains only 539 base pairs of sequence and lacks protein-coding genes, whereas the other two conformations (termed VLB and VLC) span 36 Kb and 27 Kb and contain 12 and 10 genes, respectively. Interestingly, both VLB and VLC appear to contain distinct secondary metabolism gene clusters; VLB contains a NRPS gene as well as four porphyrin metabolism genes never found to be physically linked in the genomes of 128 other fungal species, whereas VLC also contains a NRPS gene as well as several others typically found associated with secondary metabolism gene clusters. Phylogenetic evidence suggests that the VL locus was present in the ancestor of all seven species achieving its present distribution through subsequent differential losses or retentions of specific conformations.

Conclusions

We propose that the existence of variable loci, similar to the one we studied, in fungal genomes could potentially explain the dramatic differences in secondary metabolic diversity between closely related species of filamentous fungi, and contribute to host adaptation and the generation of metabolic diversity.     

Evolution of GHF5 endoglucanase gene structure in plant-parasitic nematodes: no evidence for an early domain shuffling event

Background  

Endo-1,4-beta-glucanases or cellulases from the glycosyl hydrolase family 5 (GHF5) have been found in numerous bacteria and fungi, and recently also in higher eukaryotes, particularly in plant-parasitic nematodes (PPN). The origin of these genes has been attributed to horizontal gene transfer from bacteria, although there still is a lot of uncertainty about the origin and structure of the ancestral GHF5 PPN endoglucanase. It is not clear whether this ancestral endoglucanase consisted of the whole gene cassette, containing a catalytic domain and a carbohydrate-binding module (CBM, type 2 in PPN and bacteria) or only of the catalytic domain while the CBM2 was retrieved by domain shuffling later in evolution. Previous studies on the evolution of these genes have focused primarily on data of sedentary nematodes, while in this study, extra data from migratory nematodes were included.     

RibAlign: a software tool and database for eubacterial phylogeny based on concatenated ribosomal protein subunits

Background  

Until today, analysis of 16S ribosomal RNA (rRNA) sequences has been the de-facto gold standard for the assessment of phylogenetic relationships among prokaryotes. However, the branching order of the individual phlya is not well-resolved in 16S rRNA-based trees. In search of an improvement, new phylogenetic methods have been developed alongside with the growing availability of complete genome sequences. Unfortunately, only a few genes in prokaryotic genomes qualify as universal phylogenetic markers and almost all of them have a lower information content than the 16S rRNA gene. Therefore, emphasis has been placed on methods that are based on multiple genes or even entire genomes. The concatenation of ribosomal protein sequences is one method which has been ascribed an improved resolution. Since there is neither a comprehensive database for ribosomal protein sequences nor a tool that assists in sequence retrieval and generation of respective input files for phylogenetic reconstruction programs, RibAlign has been developed to fill this gap.     

Genetic flux over time in the <Emphasis Type="Italic">Salmonella</Emphasis> lineage

Background  

DNA sequences that are shared between closely related organisms while being absent from their common ancestor and from sister lineages of that ancestor are likely to have been acquired by horizontal gene transfer. Over time, the composition of those sequences tends to become more similar to the compositional signature of their host (amelioration).     

Fungal-specific PCR primers developed for analysis of the ITS region of environmental DNA extracts

Background  

The Internal Transcribed Spacer (ITS) regions of fungal ribosomal DNA (rDNA) are highly variable sequences of great importance in distinguishing fungal species by PCR analysis. Previously published PCR primers available for amplifying these sequences from environmental samples provide varying degrees of success at discriminating against plant DNA while maintaining a broad range of compatibility. Typically, it has been necessary to use multiple primer sets to accommodate the range of fungi under study, potentially creating artificial distinctions for fungal sequences that amplify with more than one primer set.