Genetic material is inherited from parents to offspring and this process is known as vertical transmission. However genetic material can be transferred form one organism to another in a non-genealogical fashion. Such type of transmission is defined as horizontal transmission or gene transfer (HGT) (1). Although mechanisms for the transfer of genetic material between organisms were known from the early years of molecular biology and genetics research, and the theoretical potential of cross-species gene transfer in evolution was proposed in the 1980s, the concept of HGT emerged in the 1990s (2). It was invoked as an alternative explanation for rarely observed incongruent phylogenetic relationships between species (2). However, the recent availability of genome sequence information and the thorough study of multiple pro- and eukaryotic genomes has revealed that HGT is pervasive and powerful among microbes (1,2,3). Additionally, more recent studies have shown that HGT is also evident between animals and bacteria, with the bacteria being the donor species (4,5). In plants, HGT has been relatively well documented, and in most cases involves the transfer of genetic material from a parasite to its host plant. Yet, HGTs with the plant species being the donor have rarely been documented.
Recently Nikolaidis et al (6) reported a rare case of HGT from plants to multiple plant parasites or free living microorganisms. Specifically, they found that members of the plant expansin gene family, which code for plant cell-wall loosening proteins and are comprised of two distinct protein domains D1 and D2, were transferred from plants to bacteria, fungi, and unicellular eukaryotes (amoebozoa).
Having previously established that the bacterial protein EXLX1 from Bacillus subtilis is structurally and functionally very similar to plant expansins (7,8,9), Nikolaidis et al investigated the evolution of the expansin family in-depth. To do so they used the bacterial EXLX1 sequence as their primary-sequence database interrogator. Like expansins, EXLX1 protein contains two domains, D1 and D2, which are tightly packed structurally with a conserved open surface spanning both of them (Figure 1a,b). To ensure that the resulted raw sequence alignments of their exhaustive similarity searches are not random hits, the authors employed a set of established strict search criteria. Remarkably, they identified numerous sequences from bacteria, fungi, and amoebozoa that align to both EXLX1 domains and therefore may share ancestry with it. If so, the identified sequences are EXLX1 homologs. By employing proven phylogenetic tools and methods, as well as protein domain and fold recognition programs, the authors confirmed that all identified proteins contain both expansin domains, and showed that the predicted protein structures are very similar to both B. subtilis EXLX1 and plant expansins, further supporting the homology inference (Figures 1 and 2).
|Figure 1. The EXLX1 homologs are predicted to contain two domains, fold similarly to the Zea mays EXPB1 (a) and the B. subtilis EXLX1 (b), and contain a conserved long hydrophobic surface. (c, d) structural alignments of the three-dimensional models of the EXLX1 homologs from Ralstonia and Erwinia with the EXLX1structure. Surface (e) and ribbon (f) representations of the EXLX1 structure are colored according to conservation in 70 EXP domain sequences from bacteria and fungi (blue to red with increasing conservation). From Nikolaidis et al. (2014) (6).|
|Figure 2. The Bacillus subtilis EXLX1protein has many homologs in bacteria, fungi, and amoebozoa. (a) Phylogenetic relationships of representative B. subtilisEXLX1 homologs. Two different phylogenetic methods (NJ and ML) were used with gamma-distributed distances from the WAG substitution model with α = 1.72. Alignment gaps were excluded and the total number of sites used to construct the trees was 176. The numbers at the nodes are bootstrap values (NJ/ML). The biology of each species is shown with different symbols next to the species name. Species names abbreviations are given in supplementary table S1, Supplementary Material online. Only sequences producing BLAST hits with E-values lower than 10−4 and query coverage higher than 80% were used for the construction of these trees (b). Many EXLX1homologs contain additional domains. The domain organization of the EXLX1 homologs was identified using the Conserved Domains Database (CDD) database from NCBI coupled to fold recognition analysis. We define as expansin the domain that contains both D1 and D2 domains according to the EXLX1structure (Kerff et al. 2008; Georgelis et al. 2013). From Nikolaidis et al. (2014) (6).|
However, sequence similarity is not sufficient for showing the genetic-material-transmission type—vertical or horizontal. Five significant observations led the authors to support the HGT type: a) the sporadic distribution of organisms harboring expansin homologs, b) the biological features of these organisms –plant pathogens, soil inhabitants, or cellulose producers, c) the incongruence between the phylogenetic tree derived from EXLX1 and its homologs and the established bacterial or fungal species tree, d) the fusion of additional and shared protein domains (cellulase GH5 or carbohydrate-binding modules) in several EXLX1 homologs, and e) the functional similarities between microbial and plant expansins, especially the lack of catalytic activity. The latter observation argues against convergence (independent fusion of D1 and D2 domains) because such a scenario would require the biochemically and evolutionarily improbable independent loss and gain of the same amino acid residues in multiple distant phyla.
Relaxing the criteria of their sequence similarity searches, the authors also examined whether sequences similar to each one of the two expansin domains exist. Their results were positive for both domains. Applying their phylogenetic/protein fold recognition methodology to the sequences similar only to the second expansin domain (D2), the authors showed that the fungal swollenin protein family is homologous to expansins. Swollenin proteins are composed of two domains, too. Interestingly, although their N-terminal D1 domain contains many conserved insertions and therefore bears very low similarity with the expansin D1 domain, the folding patterns are very similar.
Regarding the timing of the expansin HGT, the lack of any differences in parametric measures such as GC-content, amino acid or nucleotide usage, etc., in the EXLX1 homologs allowed the authors to conclude it was not recent. Two additional observations augmented this conclusion. First, the phylogenetic patterns revealed that the HGT of expansins was followed by vertical transfers during certain fungal or amoebozoan species evolution. Second, several bacterial and fungal distant species contain expansin genes fused with cellulose GH5 and carbohydrate-binding domains, respectively. According to the authors’ phylogenetic analysis these extra domains were most likely acquired independently by convergence. Therefore the HGT of expansins from plants to other organisms preceded the long-lasting and slow events of convergence and speciation. Hence it must be ancient.
Regarding the origin of the expansin gene family, the authors, following a reductio ad absurdum argumentation, favor the scenario of a single origin in the common ancestor of plants and subsequent horizontal transmission to non-plant species. The patchy distribution of EXLX1 homologs in a small percentage of the tested bacterial (128/4,281 or 3%) and fungal (28 /543 or 5.2%) genomes argues against a single origin in bacteria and subsequent vertical transmission, since such a scenario demands the absurd assumption of multiple independent gene losses. Additionally, the authors’ previously reported functional similarities between plant and microbial expansins (8,9) argues against convergence, and therefore augments the single origin scenario.
Nikolaidis et al., offer a long list of logical and tightly woven arguments for the HGT scenario of non-plant expansins. However they do admit the difficulty of proving it beyond reasonable doubt. Another task—yet harder in the case of expansins—is to determine precisely the donor and recipient species, as well as its mechanism and timing. As more genomes are being sequenced we will probably be able to define at least a plant lineage that contributed its expansins to its intimately associated parasites. If so, then investigation for the potential mechanisms of HGT will be somehow eased. A current plausible such mechanism includes a plasmid-mediated transfer (10).
Besides the mechanism and timing of expansins’ HGT, their adaptive significance for the recipient species is of essence. Experimental studies on the physiological role of non-plant expansins have started to shed light on this topic. The authors report several such studies and propose that the HGT of expansin proteins in plant-interacting microbes contributed new or alternative tools for colonization or infection. The latter hypothesis implies an adaptive advantage for the plant-infecting organisms and together with the results of other reports on the role of HGT to the emergence of new diseases suggests that the observed rarity of HGT is not indicative of its importance in organismal evolution.
Contributed by: Zhenguo Zhang
Advantageous mutations may facilitate the adaptation of organisms to new environments. However, a single mutation which is advantageous in a given genetic background may be deleterious in another genetic background. This occurs when gene interaction or epistasis exists (1). An interesting case of epistatic interaction was recently observed in the hemoglobin of the deer mouse, Peromyscus maniculatus (2).
Hemoglobin is the oxygen transporter in red blood cells of all vertebrates. It can load oxygen from the respiratory organs (such as lungs and gills) and release it in other tissues (such as muscles), where oxygen is utilized for generating energy. Hemoglobin is a tetramer consisting of two α-chains and two β-chains, encoded by α- and β-globin genes, respectively (Fig. 1). It has been known that the hemoglobin of high-altitude deer mouse populations has a high hemoglobin-oxygen affinity, which enhances the physiological performance under hypoxia. However, the molecular mechanism of this high oxygen affinity was unknown. Natarajan et al. compared the hemoglobins of deer mice living in highland (Rocky Mountains) and lowland (Great Plains) populations and identified 12 key amino acid mutations, among which 8 mutations occurred in the α-globin and 4 in the β-globin (Fig. 2). These 12 mutations were separated into three regions of the genes based on the linkage disequilibrium information: 5 mutations in α-globin exon 2, 3 mutations in α-globin exon 3, and 4 mutations in β-globin (Fig. 2). The allelic group for each region was denoted by the letter ‘H’ or ‘L’, depending on whether it came from the highland (H) or lowland (L) population. The notation HH-H thus represents a combination of the highland allelic groups in the three regions (α-globin exon 2, α-globin exon 3, and β-globin). To test the epistasis among the mutations at these regions, Natarajan et al. constructed eight recombinant hemoglobins by permuting all (23 = 8) combinations of allelic group variants (Fig. 2), and tested the oxygen affinity of the recombinant proteins in vitro with or without allosteric effectors (Cl- and DPG).
The results of this experiment clearly showed that epistasis occurred among the allelic groups of the three regions (Table 1). For example, under the condition with the Cl- anion (the +KCl line in Table 1), changing from the L to H allelic group in any region in the LL-L background decreased oxygen affinity (corresponding to a higher P50 value), but the simultaneous changes in all three regions to the H allelic group (i.e., HH-H) increased oxygen affinity, contrary to the expectation from the additive effect model. As shown in the lower half of Table 1, the sensitivity of recombinant hemoglobins to the allosteric effectors (denoted by ΔlogP50) is also modulated by epistatic interactions of these three regions. 3-D structural analysis of different hemoglobin variants indicated no direct interactions among these mutational sites, but different sets of hydrogen bonds formed in each recombinant hemoglobin (2). This implies that the epistatic interactions of these mutations may be mediated by coordinated changes of protein topology.
This study (2) demonstrates that the effect of a mutation on the oxygen affinity depends on the genetic background. Since epistasis is prevalent in the genome (3), it is important to take into account the genetic background when one wants to know the evolution of a complex character with epistatic effect. In this case there are several possible ways of evolution from low-altitude hemoglobins to high-altitude hemoglobins or vice versa, as in the case of other proteins (4, 5). Genetic drift and environmental changes also may have played important roles.
Abstract of the original paper
Epistatic interactions between mutant sites in the same protein can exert a strong influence on pathways of molecular evolution. We performed protein engineering experiments that revealed pervasive epistasis among segregating amino acid variants that contribute to adaptive functional variation in deer mouse hemoglobin (Hb). Amino acid mutations increased or decreased Hb-O2 affinity depending on the allelic state of other sites. Structural analysis revealed that epistasis for Hb-O2 affinity and allosteric regulatory control is attributable to indirect interactions between structurally remote sites. The prevalence of sign epistasis for fitness-related biochemical phenotypes has important implications for the evolutionary dynamics of protein polymorphism in natural populations.
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2. Natarajan C, Inoguchi N, Weber RE, Fago A, Moriyama H, Storz JF: Epistasis among adaptive mutations in deer mouse hemoglobin. Science 2013, 340(6138):1324-1327.
3. Nei M, Ebooks Corporation: Mutation-driven evolution. In., 1st edn. Oxford: Oxford University Press; 2013: 1 online resource.
4. Salverda ML, Dellus E, Gorter FA, Debets AJ, van der Oost J, Hoekstra RF, Tawfik DS, de Visser JA: Initial mutations direct alternative pathways of protein evolution. PLoS Genet 2011, 7(3):e1001321.
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