Transfer of energy pathway genes in microbial enhanced biological phosphorus removal communities

Background

Lateral gene transfer (LGT) is an important evolutionary process in microbial evolution. In sewage treatment plants, LGT of antibiotic resistance and xenobiotic degradation-related proteins has been suggested, but the role of LGT outside these processes is unknown. Microbial communities involved in Enhanced Biological Phosphorus Removal (EBPR) have been used to treat wastewater in the last 50 years and may provide insights into adaptation to an engineered environment. We introduce two different types of analysis to identify LGT in EBPR sewage communities, based on identifying assembled sequences with more than one strong taxonomic match, and on unusual phylogenetic patterns. We applied these methods to investigate the role of LGT in six energy-related metabolic pathways.

Results

The analyses identified overlapping but non-identical sets of transferred enzymes. All of these were homologous with sequences from known mobile genetic elements, and many were also in close proximity to transposases and integrases in the EBPR data set. The taxonomic method had higher sensitivity than the phylogenetic method, identifying more potential LGTs. Both analyses identified the putative transfer of five enzymes within an Australian community, two in a Danish community, and none in a US-derived culture.

Conclusions

Our methods were able to identify sequences with unusual phylogenetic or compositional properties as candidate LGT events. The association of these candidates with known mobile elements supports the hypothesis of transfer. The results of our analysis strongly suggest that LGT has influenced the development of functionally important energy-related pathways in EBPR systems, but transfers may be unique to each community due to different operating conditions or taxonomic composition.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1752-5) contains supplementary material, which is available to authorized users.     

Laterally transferred genomic islands in Xanthomonadales related to pathogenicity and primary metabolism.

Lateral gene transfer (LGT) is considered as one of the drivers in bacterial genome evolution, usually associated with increased fitness and/or changes in behavior, especially if one considers pathogenic vs. non-pathogenic bacterial groups. The genomes of two phytopathogens, Xanthomonas campestris pv. campestris and Xanthomonas axonopodis pv. citri, were previously inspected for genome islands originating from LGT events, and, in this work, potentially early and late LGT events were identified according to their altered nucleotide composition. The biological role of the islands was also assessed, and pathogenicity, virulence and secondary metabolism pathways were functions highly represented, especially in islands that were found to be recently transferred. However, old islands are composed of a high proportion of genes related to cell primary metabolic functions. These old islands, normally undetected by traditional atypical composition analysis, but confirmed as product of LGT by atypical phylogenetic reconstruction, reveal the role of LGT events by replacing core metabolic genes normally inherited by vertical processes.     

Assessing the effects of a sequestered germline on interdomain lateral gene transfer in Metazoa

A sequestered germline in Metazoa has been argued to be an obstacle to lateral gene transfer (LGT), though few studies have specifically assessed this claim. Here, we test the hypothesis that the origin of a sequestered germline reduced LGT events in Bilateria (i.e., triploblast lineages) as compared to early‐diverging Metazoa (i.e., Ctenophora, Cnidaria, Porifera, and Placozoa). We analyze single‐gene phylogenies generated with over 900 species sampled from among Bacteria, Archaea, and Eukaryota to identify well‐supported interdomain LGTs. We focus on ancient interdomain LGT (i.e., those between prokaryotes and multiple lineages of Metazoa) as systematic errors in single‐gene tree reconstruction create uncertainties for interpreting eukaryote‐to‐eukaryote transfer. The breadth of the sampled Metazoa enables us to estimate the timing of LGTs, and to examine the pattern before versus after the evolution of a sequestered germline. We identified 58 LGTs found only in Metazoa and prokaryotes (i.e., bacteria and/or archaea), and seven genes transferred from prokaryotes into Metazoa plus one other eukaryotic clade. Our analyses indicate that more interdomain transfers occurred before the development of a sequestered germline, consistent with the hypothesis that this feature is an obstacle to LGT.     

WhatEntamoeba histolytica andGiardia lamblia tell us about the evolution of eukaryotic diversity

Entamoeba histolytica andGiardia lamblia are microaerophilic protists, which have long been considered models of ancient pre-mitochondriate eukaryotes. As transitional eukaryotes, amoebae and giardia appeared to lack organelles of higher eukaryotes and to depend upon energy metabolism appropriate for anaerobic conditions early in the history of the planet. However, our studies have shown that amoebae and giardia contain splicoeosomal introns, ras-family signal-transduction proteins, ATP-binding casettes (ABC)-family drug transporters, Golgi, and a mitochondrion-derived organelle (amoebae only). These results suggest that most of the organelles of higher eukaryotes were present in the common ancestor of all eukaryotes, and so dispute the notion of transitional eukaryotic forms. In addition, phylogenetic studies suggest many of the genes encoding the fermentation enzymes of amoebae and giardia derive from prokaryotes by lateral gene transfer (LGT). While LGT has recently been shown to be an important determinant of prokaryotic evolution, this is the first time that LGT has been shown to be an important determinant of eukaryotic evolution. Further, amoebae contain cyst wall-associated lectins, which resemble, but are distinct from lectins in the walls of insects (convergent evolution). Giardia have a novel microtubule-associated structure which tethers together pairs of nuclei during cell division. It appears then that amoebae and giardia tell us less about the origins of eukaryotes and more about the origins of eukaryotic diversity.     

Old genes in new places: A taxon-rich analysis of interdomain lateral gene transfer events

Vertical inheritance is foundational to Darwinian evolution, but fails to explain major innovations such as the rapid spread of antibiotic resistance among bacteria and the origin of photosynthesis in eukaryotes. While lateral gene transfer (LGT) is recognized as an evolutionary force in prokaryotes, the role of LGT in eukaryotic evolution is less clear. With the exception of the transfer of genes from organelles to the nucleus, a process termed endosymbiotic gene transfer (EGT), the extent of interdomain transfer from prokaryotes to eukaryotes is highly debated. A common critique of studies of interdomain LGT is the reliance on the topology of single-gene trees that attempt to estimate more than one billion years of evolution. We take a more conservative approach by identifying cases in which a single clade of eukaryotes is found in an otherwise prokaryotic gene tree (i.e. exclusive presence). Starting with a taxon-rich dataset of over 13,600 gene families and passing data through several rounds of curation, we identify and categorize the function of 306 interdomain LGT events into diverse eukaryotes, including 189 putative EGTs, 52 LGTs into Opisthokonta (i.e. animals, fungi and their microbial relatives), and 42 LGTs nearly exclusive to anaerobic eukaryotes. To assess differential gene loss as an explanation for exclusive presence, we compare branch lengths within each LGT tree to a set of vertically-inherited genes subsampled to mimic gene loss (i.e. with the same taxonomic sampling) and consistently find shorter relative distance between eukaryotes and prokaryotes in LGT trees, a pattern inconsistent with gene loss. Our methods provide a framework for future studies of interdomain LGT and move the field closer to an understanding of how best to model the evolutionary history of eukaryotes.     

Functional replacement of a primary metabolic pathway via multiple independent eukaryote‐to‐eukaryote gene transfers and selective retention

Although lateral gene transfer (LGT) is now recognized as a major force in the evolution of prokaryotes, the contribution of LGT to the evolution and diversification of eukaryotes is less understood. Notably, transfers of complete pathways are believed to be less likely between eukaryotes, because the successful transfer of a pathway requires the physical clustering of functionally related genes. Here, we report that in one of the closest unicellular relatives of animals, the choanoflagellate, Monosiga, three genes whose products work together in the glutamate synthase cycle are of algal origin. The concerted retention of these three independently acquired genes is best explained as the consequence of a series of adaptive replacement events. More generally, this study argues that (i) eukaryote‐to‐eukaryote transfers of entire metabolic pathways are possible, (ii) adaptive functional replacements of primary pathways can occur, and (iii) functional replacements involving eukaryotic genes are likely to have also contributed to the evolution of eukaryotes. Lastly, these data underscore the potential contribution of algal genes to the evolution of nonphotosynthetic lineages.     

Characterization of an Ancient Lepidopteran Lateral Gene Transfer

Bacteria to eukaryote lateral gene transfers (LGT) are an important potential source of material for the evolution of novel genetic traits. The explosion in the number of newly sequenced genomes provides opportunities to identify and characterize examples of these lateral gene transfer events, and to assess their role in the evolution of new genes. In this paper, we describe an ancient lepidopteran LGT of a glycosyl hydrolase family 31 gene (GH31) from an Enterococcus bacteria. PCR amplification between the LGT and a flanking insect gene confirmed that the GH31 was integrated into the Bombyx mori genome and was not a result of an assembly error. Database searches in combination with degenerate PCR on a panel of 7 lepidopteran families confirmed that the GH31 LGT event occurred deep within the Order approximately 65–145 million years ago. The most basal species in which the LGT was found is Plutella xylostella (superfamily: Yponomeutoidea). Array data from Bombyx mori shows that GH31 is expressed, and low dN/dS ratios indicates the LGT coding sequence is under strong stabilizing selection. These findings provide further support for the proposition that bacterial LGTs are relatively common in insects and likely to be an underappreciated source of adaptive genetic material.     

Quartet mapping and the extent of lateral transfer in bacterial genomes

Several recent analyses have used quartet-based methods to assess the congruence among phylogenies derived for large sets of genes from prokaryotic genomes. The principal conclusion from these studies is that lateral gene transfer (LGT) has blurred prokaryotic phylogenies to such a degree that the darwinian scheme of treelike evolution might be abandoned in favor of a net or web. Here, we focus on one of these methods, quartet mapping, and show that its application can lead to overestimation of the extent of inferred LGT in prokaryotes, particularly when applied to distantly related taxa.     

Networks of gene sharing among 329 proteobacterial genomes reveal differences in lateral gene transfer frequency at different phylogenetic depths

Lateral gene transfer (LGT) is an important mechanism of natural variation among prokaryotes. Over the full course of evolution, most or all of the genes resident in a given prokaryotic genome have been affected by LGT, yet the frequency of LGT can vary greatly across genes and across prokaryotic groups. The proteobacteria are among the most diverse of prokaryotic taxa. The prevalence of LGT in their genome evolution calls for the application of network-based methods instead of tree-based methods to investigate the relationships among these species. Here, we report networks that capture both vertical and horizontal components of evolutionary history among 1,207,272 proteins distributed across 329 sequenced proteobacterial genomes. The network of shared proteins reveals modularity structure that does not correspond to current classification schemes. On the basis of shared protein-coding genes, the five classes of proteobacteria fall into two main modules, one including the alpha-, delta-, and epsilonproteobacteria and the other including beta- and gammaproteobacteria. The first module is stable over different protein identity thresholds. The second shows more plasticity with regard to the sequence conservation of proteins sampled, with the gammaproteobacteria showing the most chameleon-like evolutionary characteristics within the present sample. Using a minimal lateral network approach, we compared LGT rates at different phylogenetic depths. In general, gene evolution by LGT within proteobacteria is very common. At least one LGT event was inferred to have occurred in at least 75% of the protein families. The average LGT rate at the species and class depth is about one LGT event per protein family, the rate doubling at the phylum level to an average of two LGT events per protein family. Hence, our results indicate that the rate of gene acquisition per protein family is similar at the level of species (by recombination) and at the level of classes (by LGT). The frequency of LGT per genome strongly depends on the species lifestyle, with endosymbionts showing far lower LGT frequencies than free-living species. Moreover, the nature of the transferred genes suggests that gene transfer in proteobacteria is frequently mediated by conjugation.     

The Frequency of Eubacterium-to-Eukaryote Lateral Gene Transfers Shows Significant Cross-Taxa Variation Within Amoebozoa

Single-celled bacterivorous eukaryotes offer excellent test cases for evaluation of the frequency of prey-to-predator lateral gene transfer (LGT). Here we use analysis of expressed sequence tag (EST) data sets to quantify the extent of LGT from eubacteria to two amoebae, Acanthamoeba castellanii and Hartmannella vermiformis. Stringent screening for LGT proceeded in several steps intended to enrich for authentic events while at the same time minimizing the incidence of false positives due to factors such as limitations in database coverage and ancient paralogy. The results were compared with data obtained when the same methodology was applied to EST libraries from a number of other eukaryotic taxa. Significant differences in the extent of apparent eubacterium-to-eukaryote LGT were found between taxa. Our results indicate that there may be substantial inter-taxon variation in the number of LGT events that become fixed even between amoebozoan species that have similar feeding modalities. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Martin Kreitman]     

Phylogenetic analyses of diplomonad genes reveal frequent lateral gene transfers affecting eukaryotes

BACKGROUND: Lateral gene transfer (LGT) is an important evolutionary mechanism among prokaryotes. The situation in eukaryotes is less clear; the human genome sequence failed to give strong support for any recent transfers from prokaryotes to vertebrates, yet a number of LGTs from prokaryotes to protists (unicellular eukaryotes) have been documented. Here, we perform a systematic analysis to investigate the impact of LGT on the evolution of diplomonads, a group of anaerobic protists.RESULTS: Phylogenetic analyses of 15 genes present in the genome of the Atlantic Salmon parasite Spironucleus barkhanus and/or the intestinal parasite Giardia lamblia show that most of these genes originated via LGT. Half of the genes are putatively involved in processes related to an anaerobic lifestyle, and this finding suggests that a common ancestor, which most probably was aerobic, of Spironucleus and Giardia adapted to an anaerobic environment in part by acquiring genes via LGT from prokaryotes. The sources of the transferred diplomonad genes are found among all three domains of life, including other eukaryotes. Many of the phylogenetic reconstructions show eukaryotes emerging in several distinct regions of the tree, strongly suggesting that LGT not only involved diplomonads, but also involved other eukaryotic groups.CONCLUSIONS: Our study shows that LGT is a significant evolutionary mechanism among diplomonads in particular and protists in general. These findings provide insights into the evolution of biochemical pathways in early eukaryote evolution and have important implications for studies of eukaryotic genome evolution and organismal relationships. Furthermore, "fusion" hypotheses for the origin of eukaryotes need to be rigorously reexamined in the light of these results.     

Gene gain and gene loss in streptococcus: is it driven by habitat?

Bacterial genomes can evolve either by gene gain, gene loss, mutating existing genes, and/or by duplication of existing genes. Recent studies have clearly demonstrated that the acquisition of new genes by lateral gene transfer (LGT) is a predominant force in bacterial evolution. To better understand the significance of LGT, we employed a comparative genomics approach to model species-specific and intraspecies gene insertions/deletions (ins/del among 12 sequenced streptococcal genomes using a maximum likelihood method. This study indicates that the rate of gene ins/del is higher on the external branches and varies dramatically for each species. We have analyzed here some of the experimentally characterized species-specific genes that have been acquired by LGT and conclude that at least a portion of these genes have a role in adaptation.     

Defining the Core of Nontransferable Prokaryotic Genes: The Euryarchaeal Core

If lateral gene transfer (LGT) has affected all genes over the course of prokaryotic evolution, reconstruction of organismal phylogeny is compromised. However, if a core of genes is immune to transfer, then the evolutionary history of that core might be our most reliable guide to the evolution of organisms. Such a core should be preferentially included in the subset of genes shared by all organisms, but where universally conserved genes have been analyzed, there is too little phylogenetic signal to allow determination of whether or not they indeed have the same history (Hansmann and Martin 2000; Teichmann and Mitchison 1999). Here we look at a more restricted set, 521 homologous genes (COGs) simultaneously present in four sequenced euryarchaeal genomes. Although there is overall little robust phylogenetic signal in this data set, there is, among well-supported trees, strong representation of all three possible four-taxon topologies. ``Informational' genes seem no less subject to LGT than are ``operational genes,' within the euryarchaeotes. We conclude that (i) even in this collection of conserved genes there has been extensive LGT (orthologous gene replacement) and (ii) the notion that there is a core of nontransferable genes (the ``core hypothesis') has not been proven and may be unprovable. Received: 7 November 2000 / Accepted: 20 February 2001     

Replacement of the Arginine Biosynthesis Operon in Xanthomonadales by Lateral Gene Transfer

The role of lateral gene transfer (LGT) in prokaryotes has been shown to rapidly change the genome content, providing new gene tools for environmental adaptation. Features related to pathogenesis and resistance to strong selective conditions have been widely shown to be products of gene transfer between bacteria. The genomes of the γ-proteobacteria from the genus Xanthomonas, composed mainly of phytopathogens, have potential genomic islands that may represent imprints of such evolutionary processes. In this work, the evolution of genes involved in the pathway responsible for arginine biosynthesis in Xanthomonadales was investigated, and several lines of evidence point to the foreign origin of the arg genes clustered within a potential operon. Their presence inside a potential genomic island, bordered by a tRNA gene, the unusual ranking of sequence similarity, and the atypical phylogenies indicate that the metabolic pathway for arginine biosynthesis was acquired through LGT in the Xanthomonadales group. Moreover, although homologues were also found in Bacteroidetes (Flavobacteria group), for many of the genes analyzed close homologues are detected in different life domains (Eukarya and Archaea), indicating that the source of these arg genes may have been outside the Bacteria clade. The possibility of replacement of a complete primary metabolic pathway by LGT events supports the selfish operon hypothesis and may occur only under very special environmental conditions. Such rare events reveal part of the history of these interesting mosaic Xanthomonadales genomes, disclosing the importance of gene transfer modifying primary metabolism pathways and extending the scenario for bacterial genome evolution.     

The role of lateral gene transfer in the evolution of isoprenoid biosynthesis pathways

Lateral gene transfer (LGT) is a major force in microbial genome evolution. Here, we present an overview of lateral transfers affecting genes involved in isopentenyl diphosphate (IPP) synthesis. Two alternative metabolic pathways can synthesize this universal precursor of isoprenoids, the 1-deoxy-D-xylulose 5-phosphate (DOXP) pathway and the mevalonate (MVA) pathway. We have surveyed recent genomic data and the biochemical literature to determine the distribution of the genes composing these pathways within the bacterial domain. The scattered distribution observed is incompatible with a simple scheme of vertical transmission. LGT (among and between bacteria, archaea and eukaryotes) more parsimoniously explains many features of this pattern. This alternative scenario is supported by phylogenetic analyses, which unambiguously confirm several cases of lateral transfer. Available biochemical data allow the formulation of hypotheses about selective pressures favouring transfer. The phylogenetic diversity of the organisms involved and the range of possible causes and effects of these transfer events make the IPP biosynthetic pathways an ideal system for studying the evolutionary role of LGT.     

The molecular evolution of catalatic hydroperoxidases: evidence for multiple lateral transfer of genes between prokaryota and from bacteria into eukaryota

The past decade has produced an increasing number of reports on horizontal gene transfer between prokaryotic organisms. Only recently, with the flood of available whole genome sequence data and a renewed intensity of the debate about the universal tree of life, a very few reports on lateral gene transfer (LGT) from prokaryotes into the Eukaryota have been published. We have investigated and report here on the molecular evolution of the gene families that encode catalatic hydroperoxidases. We have found that this process included not only frequent horizontal gene transfer among prokaryotes but also several lateral gene transfer events between bacteria and fungi and between bacteria and the protistan ancestor of the alga/plant lineage.     

Detection of Lateral Gene Transfer Events in the Prokaryotic tRNA Synthetases by the Ratios of Evolutionary Distances Method

The availability of large numbers of genomic sequences has demonstrated the importance of lateral gene transfer (LGT) in prokaryotic evolution. However, considerable uncertainty remains concerning the frequency of LGT compared to other evolutionary processes. To examine LGTs in ancient lineages of prokaryotes a method was developed that utilizes the ratios of evolutionary distances (RED) to distinguish between alternative evolutionary histories. The advantages of this approach are that the variability inherent in comparing protein sequences is transparent, the direction of LGT and the relative rates of evolution are readily identified, and it is possible to detect other types of evolutionary events. This method was standardized using 35 genes encoding ribosomal proteins that were believed to share a vertical evolution. Using RED-T, an original computer program designed to implement the RED method, the evolution of the genes encoding the 20 aminoacyl-tRNA synthetases was examined. Although LGTs were common in the evolution of the aminoacyl-tRNA synthetases, they were not sufficient to obscure the organismal phylogeny. Moreover, much of the apparent complexity of the gene tree was consistent with the formation of the paralogs in the ancestors to the modern lineages followed by more recent loss of one paralog or the other.     

Trends and barriers to lateral gene transfer in prokaryotes

Gene acquisition by lateral gene transfer (LGT) is an important mechanism for natural variation among prokaryotes. Laboratory experiments show that protein-coding genes can be laterally transferred extremely fast among microbial cells, inherited to most of their descendants, and adapt to a new regulatory regime within a short time. Recent advance in the phylogenetic analysis of microbial genomes using networks approach reveals a substantial impact of LGT during microbial genome evolution. Phylogenomic networks of LGT among prokaryotes reconstructed from completely sequenced genomes uncover barriers to LGT in multiple levels. Here we discuss the kinds of barriers to gene acquisition in nature including physical barriers for gene transfer between cells, genomic barriers for the integration of acquired DNA, and functional barriers for the acquisition of new genes.     

Molecular evidence for the evolution of metal homeostasis genes by lateral gene transfer in bacteria from the deep terrestrial subsurface

Lateral gene transfer (LGT) plays a vital role in increasing the genetic diversity of microorganisms and promoting the spread of fitness-enhancing phenotypes throughout microbial communities. To date, LGT has been investigated in surface soils, natural waters, and biofilm communities but not in the deep terrestrial subsurface. Here we used a combination of molecular analyses to investigate the role of LGT in the evolution of metal homeostasis in lead-resistant subsurface bacteria. A nested PCR approach was employed to obtain DNA sequences encoding P(IB)-type ATPases, which are proteins that transport toxic or essential soft metals such as Zn(II), Cd(II), and Pb(II) through the cell wall. Phylogenetic incongruencies between a 16S rRNA gene tree and a tree based on 48 P(IB)-type ATPase amplicons and sequences available for complete bacterial genomes revealed an ancient transfer from a member of the beta subclass of the Proteobacteria (beta-proteobacterium) that may have predated the diversification of the genus PSEUDOMONAS: Four additional phylogenetic incongruencies indicate that LGT has occurred among groups of beta- and gamma-proteobacteria. Two of these transfers appeared to be recent, as indicated by an unusual G+C content of the P(IB)-type ATPase amplicons. This finding provides evidence that LGT plays a distinct role in the evolution of metal homeostasis in deep subsurface bacteria, and it shows that molecular evolutionary approaches may be used for investigation of this process in microbial communities in specific environments.