The Repetitive DNA Elements Called CRISPRs and Their Associated Genes: Evidence of Horizontal Transfer Among Prokaryotes

We have found direct DNA repeats 21–47 bp in length interspersed with nonrepetitive sequences of similar length, or clustered regularly interspaced short palindromic repeats (CRISPRs) in a wide range of diverse prokaryotes, including many Archaeal and Eubacterial species. A number of cas, CRISPR-associated genes have also been characterized in many of the same organisms. Phylogenetic analysis of these cas genes suggests that the CRISPR loci have been propagated via HGT, horizontal gene transfer. We suggest a mechanism by which this HGT has occurred, namely, that the CRISPR loci can be carried between cells on megaplasmids ≥40 kb in length. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Stuart Newfeld]     

Molecular Evolution of Peptide Methionine Sulfoxide Reductases (MsrA and MsrB): On the Early Development of a Mechanism That Protects Against Oxidative Damage

Methionine sulfoxide reductases, enzymes that reverse the oxidation of methionine residues, have been described in a wide range of species. The reduction of the diastereoisomers of oxidized methionine is catalyzed by two different monomeric methionine sulfoxide reductases (MsrA and MsrB) and is best understood as an evolutionary response to high levels of oxygen either in the Earth’s atmosphere or possibly in more localized environments. Phylogenetic analyses of these proteins suggest that their distribution is the outcome of a complex history including many paralogy and lateral gene transfer events. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Martin Kreitman]     

Retrotransposon Sequence Variation in Four Asexual Plant Species

Transposable elements (TEs) can be viewed as genetic parasites that persist in populations due to their capacity for increase in copy number and the inefficacy of selection against them. A corollary of this hypothesis is that TEs are more likely to spread within sexual populations and be eliminated or inactivated within asexual populations. While previous work with animals has shown that asexual taxa may contain less TE diversity than sexual taxa, comparable work with plants has been lacking. Here we report the results of a study of Ty1/copia, Ty3/gypsy, and LINE-like retroelement diversity in four asexual plant species. Retroelement-like sequences, with a high degree of conservation both within and between species, were isolated from all four species. The sequences correspond to several previously annotated retroelement subfamilies. They also exhibit a pattern of nucleotide substitution characterized by an excess of synonymous substitutions, suggestive of a history of purifying selection. These findings were compared with retroelement sequence evolution in sexual plant taxa. One likely explanation for the discovery of conserved TE sequences in the genomes of these asexual taxa is simply that asexuality within these taxa evolved relatively recently, such that the loss and breakdown of TEs is not yet detectable through analysis of sequence diversity. This explanation is examined by conducting stochastic simulation of TE evolution and by using published information to infer rough estimates of the ages of asexual taxa. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Dmitri Petrov     

Lateral genetic transfer: open issues

Lateral genetic transfer (LGT) is an important adaptive force in evolution, contributing to metabolic, physiological and ecological innovation in most prokaryotes and some eukaryotes. Genomic sequences and other data have begun to illuminate the processes, mechanisms, quantitative extent and impact of LGT in diverse organisms, populations, taxa and environments; deep questions are being posed, and the provisional answers sometimes challenge existing paradigms. At the same time, there is an enhanced appreciation of the imperfections, biases and blind spots in the data and in analytical approaches. Here we identify and consider significant open questions concerning the role of LGT in genome evolution.     

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 <Emphasis Type="Italic">cadA</Emphasis> Gene in Cadmium-Resistant Bacteria from Cadmium-Polluted Soil in the Zhangshi Area of Northeast China

Cadmium-resistant bacteria were isolated from the farmland soil in Zhangshi Irrigation Area in Shenyang of Northeast China, an area has been polluted by heavy metals, especially cadmium, for more than 40 years. The cadA gene was detected in 4 Bacillus strains and for the first time in one Flavobacterium strain. The high sequence identity (93%–99%) of cadA gene, shared indels in different bacterial species and genera, and the phylogenetic incongruence between 16S rDNA gene tree and cadA gene tree suggested that lateral gene transfer (LGT) occurred among Bacillus and Flavobacterium spp. The LGT of cadA gene might play a vital role in promoting the spread of cadmium-resistant phenotypes throughout soil microbial communities. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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.     

Horizontal Gene Transfer in Aminoacyl-tRNA Synthetases Including Leucine-Specific Subtypes

Aminoacyl-tRNA synthetases catalyze a fundamental reaction for the flow of genetic information from RNA to protein. Their presence in all organisms known today highlights their important role in the early evolution of life. We investigated the evolutionary history of aminoacyl-tRNA synthetases on the basis of sequence data from more than 200 Archaea, Bacteria, and Eukaryota. Phylogenetic profiles are in agreement with previous observations that many genes for aminoacyl-tRNA synthetases were transferred horizontally between species from all domains of life. We extended these findings by a detailed analysis of the history of leucyl-tRNA synthetases. Thereby, we identified a previously undetected case of horizontal gene transfer from Bacteria to Archaea based on phylogenetic profiles, trees, and networks. This means that, finally, the last subfamily of aminoacyl-tRNA synthetases has lost its exceptional position as the sole subfamily that is devoid of horizontal gene transfer. Furthermore, the leucyl-tRNA synthetase phylogenetic tree suggests a dichotomy of the archaeal/eukaryotic-cytosolic and bacterial/eukaryotic-mitochondrial proteins. We argue that the traditional division of life into Prokaryota (non-chimeric) and Eukaryota (chimeric) is favorable compared to Woese’s trichotomy into Archaea/Bacteria/Eukaryota. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Yves Van de Peer]     

The use of chemical profiling for monitoring metabolic changes in artificial soil slurries caused by horizontal gene transfer

This study explores the utility of Fourier transform infra-red spectroscopy (FT-IR) as a metabolomic tool to detect changes in water-extractable chemical profile resulting from horizontal gene transfer (HGT) events in artificial soil slurries. A GFP–Km (Green fluorescent protein–kanamycin) cassette tagged HGT recipient Acinetobacter strain ADPWH67 with the salicylate hydroxylase gene (salA) disrupted was introduced to slurries containing either sterile or non-sterile soil. The subsequent addition of naked salA DNA allowed the specific monitoring of HGT events by enumerating GFP-expressing colonies on minimal media with salicylate as a sole carbon source. DNA sequencing confirmed that salA was restored in these transformants. Gene transformation frequencies of around 10⁻⁶ were achieved in the presence of sterile and non-sterile soils. Aqueous extracts of the soil slurries were then analyzed using FT-IR in order to ascertain whether any shifts in chemical profile could be detected. We found that following HGT events FT-IR chemical profiles were clearly altered when analyzed with multivariate statistics. Furthermore, these changes could be explained by differences in key chemical signatures including salicylate as well as other biomolecules found in soils. The slurry extracts were also subjected to GC-MS which confirmed the results of FT-IR analyses. FT-IR was therefore demonstrated to have utility for the rapid screening of metabolomic changes in soils following effective HGT events. In addition, this approach could potentially link specific metabolite changes with corresponding catabolic genes.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Assessing genetic diversity and population structure in a citrus germplasm collection utilizing simple sequence repeat markers (SSRs)

Twenty-four simple sequence repeat (SSR) markers were used to detect molecular polymorphisms among 370 mostly sexually derived Citrus accessions from the collection of citrus germplasm maintained at the University of California, Riverside. A total of 275 alleles were detected with an average of 11.5 alleles per locus and an average polymorphism information content of 0.625. Genetic diversity statistics were calculated for each individual SSR marker, the entire population, and for specified Citrus groups. Phylogenetic relationships among all citrus accessions and putative non-hybrid Citrus accessions were determined by constructing neighbor-joining trees. There was strong support for monophyly at the species level when hybrid taxa were removed from the data set. Both of these trees indicate that Fortunella clusters within the genus Citrus but Poncirus is a sister genus to Citrus. Additionally, Citrus accessions were probabilistically assigned to populations or multiple populations if their genotype indicated an admixture by a model-based clustering approach. This approach identified five populations in this data set. These separate analyses (distance and model based) both support the hypothesis that there are only a few naturally occurring species of Citrus and most other types of Citrus arose through various hybridization events between these naturally occurring forms.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Fine-Scale Crossover Rate Heterogeneity in <Emphasis Type="Italic">Drosophila pseudoobscura</Emphasis>

Broad-scale differences in crossover rate across the genome have been characterized in most genomes studied. Fine-scale differences, however, have only been examined in a few taxa, such as Arabidopsis, yeast, humans, and mice. No prior studies have directly looked for fine-scale recombination rate heterogeneity in Drosophila. We produced 370 Drosophila pseudoobscura containing a crossover event within the 2-megabase (MB) region between the genes yellow and white. We then examined 19 intervals within this region and determined where the crossovers occurred. We found that recombination events occur nonrandomly on a small scale and that mild “hotspots“ of a few kilobases exist in Drosophila. Among the regions studied, recombination rates varied from 1.4 to 52 cM/MB. We also observed a trend toward high codon bias in regions of high recombination. Finally, we identified a significantly positive correlation between recombination rate and simple repeats, as well as the motif CACAC. These sequence features may contribute to broad-scale variation in crossover rate and, thus, shed light on features associated with crossover rate heterogeneity at a genome-wide scale. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Dmitri Petrov]     

A Multistep Process Gave Rise to RNA Polymerase IV of Land Plants

Since their discovery in Metazoa, the three nuclear RNA polymerases (RNAPs) have been found in fungi, plants, and diverse protists. In all eukaryotes studied to date, RNAPs I, II, and III collectively transcribe all major RNAs made in the nucleus. We have found genes for the largest subunit (RPD1/RPE1) of a new DNA-dependent RNAP, RNAP IV, in all major land plant taxa and in closely related green algae. Genes for the second-largest subunit (RPD2) of this enzyme were found in all land plants. Phylogenetic study indicates that RNAP IV genes are sister to the corresponding RNAP II genes. Our results show the genesis of RNAP IV to be a multistep process in which the largest and second-largest subunit genes evolved by independent duplication events in the ancestors of Charales and land plants. These findings provide insights into evolutionary mechanisms that can explain the origin of multiple RNAPs in the eukaryotic nucleus. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Patrick Keeling]     

The Evolutionary History of the Transposable Element Penelope in the Drosophila virilis Group of Species

We have used phylogenetic techniques to study the evolutionary history of the Penelope transposable element in the Drosophila virilis species group. Two divergent types of Penelope have been detected, one previously described, clade I, and a new one which we have termed clade III. The phylogeny of some copies of the Penelope clade I element was partially consistent with the species phylogeny of the D. montana subphylad, suggesting cospeciation and allowing the estimation of the evolutionary rate of Penelope. Divergence times of elements found in different species are younger than the age of the species, suggesting horizontal transfer events. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Dmitri Petrov]     

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.     

Nonrandom Representation of Sex-Biased Genes on Chicken Z Chromosome

Several lines of evidence suggest that the X chromosome of various animal species has an unusual complement of genes with sex-biased or sex-specific expression. However, the study of the X chromosome gene content in different organisms provided conflicting results. The most striking contrast concerns the male-biased genes, which were reported to be almost depleted from the X chromosome in Drosophila but overrepresented on the X chromosome in mammals. To elucidate the reason for these discrepancies, we analysed the gene content of the Z chromosome in chicken. Our analysis of the publicly available expressed sequence tags (EST) data and genome draft sequence revealed a significant underrepresentation of ovary-specific genes on the chicken Z chromosome. For the brain-expressed genes, we found a significant enrichment of male-biased genes but an indication of underrepresentation of female-biased genes on the Z chromosome. This is the first report on the nonrandom gene content in a homogametic sex chromosome of a species with heterogametic female individuals. Further comparison of gene contents of the independently evolved X and Z sex chromosomes may offer new insight into the evolutionary processes leading to the nonrandom genomic distribution of sex-biased and sex-specific genes. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Manyuan Long]     

Serial Analysis of Gene Expression (SAGE) of<Emphasis Type="Italic"> Magnaporthe grisea</Emphasis>: genes involved in appressorium formation

Treatment with cyclic AMP (cAMP) induces appressorium formation in the phytopathogenic fungus Magnaporthe grisea, the causative agent of rice blast disease. In a search for the M. grisea genes responsible for appressorium formation and host invasion, SAGE (Serial Analysis of Gene Expression) was carried out using mRNA isolated from fungal conidia germinating in the presence and absence of cAMP. From cAMP-treated conidia 5087 tags including 2889 unique tags were isolated, whereas untreated conidia yielded 2342 unique tags out of total of 3938. cAMP treatment resulted in up- and down-regulation of genes corresponding to 57 and 53 unique tags, respectively. Upon consultation of EST/cDNA databases, 22 tags with higher representation in cAMP-treated conidia were annotated with putative gene names. Furthermore, 28 tags corresponding to cAMP-induced genes could be annotated with the help of the recently published genome sequence of M. grisea. cAMP-induced genes identified by SAGE included many genes that have not been described so far, as well as a number of genes known to be involved in pathogenicity, e.g. MPG1, MAS1 and MAC1. RT-PCR of 13 randomly selected genes confirmed the SAGE results, verifying the fidelity of the SAGE data.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by E. Cerdá-Olmedo     

Organisation and structural evolution of the rice glutathione <Emphasis Type="Italic">S</Emphasis>-transferase gene family

Glutathione S-transferases (GSTs) comprise a large family of key defence enzymes against xenobiotic toxicity. Here we describe the comprehensive characterisation of this important multigene family in the model monocot species rice [Oryza sativa (L.)]. Furthermore, we investigate the molecular evolution of the family based on the analysis of (1) the patterns of within-genome duplication, and (2) the phylogenetic relationships and evolutionary divergence among rice, Arabidopsis, maize and soybean GSTs. By in-silico screening of the EST and genome divisions of the Genbank/EMBL/DDBJ database we have isolated 59 putative genes and two pseudogenes, making this the largest plant GST family characterised to date. Of these, 38 (62%) are represented by genomic and EST sequences and 23 (38%) are known only from their genomic sequences. A preliminary survey of EST collections shows a large degree of variability in gene expression between different tissues and environmental conditions, with a small number of genes (13) accounting for 80% of all ESTs. Rice GSTs are organised in four main phylogenetic classes, with 91% of all rice genes belonging to the two plant-specific classes Tau (40 genes) and Phi (16 genes). Pairwise identity scores range between 17 and 98% for proteins of the same class, and 7 and 21% for interclass comparisons. Rapid evolution by gene duplication is suggested by the discovery of two large clusters of 7 and 23 closely related genes on chromosomes 1 and 10, respectively. A comparison of the complete GST families in two monocot and two dicot species suggests a monophyletic origin for all Theta and Zeta GSTs, and no more than three common ancestors for all Phi and Tau genes.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by M.-A. Grandbastien     

Intron-flanking EST–PCR markers: from genetic marker development to gene structure analysis in Rhododendron

With a long-term goal of constructing a linkage map of Rhododendron enriched with gene-specific markers, we utilized Rhododendron catawbiense ESTs for the development of high-efficiency (in terms of generating polymorphism frequency) PCR-based markers. Using the gene-sequence alignment between Rhododendron ESTs and the genomic sequences of Arabidopsis homologs, we developed ‘intron-flanking‘ EST–PCR-based primers that would anneal in conserved exon regions and amplify across the more highly diverged introns. These primers resulted in increased efficiency (61% vs. 13%; 4.7-fold) of polymorphism-detection compared with conventional EST–PCR methods, supporting the assumption that intron regions are more diverged than exons. Significantly, this study demonstrates that Arabidopsis genome database can be useful in developing gene-specific PCR-based markers for other non-model plant species for which the EST data are available but genomic sequences are not. The comparative analysis of intron sizes between Rhododendron and Arabidopsis (made possible in this study by aligning of Rhododendron ESTs with Arabidopsis genomic sequences and the sequencing of Rhododendron genomic PCR products) provides the first insight into the gene structure of Rhododendron. Electronic Supplementary Material Supplementary material is available for this article at     

Catalytic activity,duplication and evolution of the CYP98 cytochrome P450 family in wheat

A burst of evolutionary duplication upon land colonization seems to have led to the large superfamily of cytochromes P450 in higher plants. Within this superfamily some clans and families are heavily duplicated. Others, such as genes involved in the phenylpropanoid pathway have led to fewer duplication events. Eight coding sequences belonging to the CYP98 family reported to catalyze the 3-hydroxylation step in this pathway were isolated from Triticum aestivum (wheat) and expressed in yeast. Comparison of the catalytic properties of the recombinant enzymes with those of CYP98s from other plant taxa was coupled to phylogenetic analyses. Our results indicate that the unusually high frequency of gene duplication in the wheat CYP98 family is a direct or indirect result from ploidization. While ancient duplication led to evolution of enzymes with different substrate preferences, most of recent duplicates underwent silencing via degenerative mutations. Three of the eight tested CYP98s from wheat have phenol meta-hydroxylase activity, with p-coumaroylshikimate being the primary substrate for all of these, as it is the case for CYP98s from sweet basil and Arabidopsis thaliana. However, CYP98s from divergent taxa have acquired different additional subsidiary activities. Some of them might be significant in the metabolism of various free or conjugated phenolics in different plant species. One of the most significant is meta-hydroxylation of p-coumaroyltyramine, predominantly by the wheat enzymes, for the synthesis of suberin phenolic monomers. Homology modeling, confirmed by directed mutagenesis, provides information on the protein regions and structural features important for some observed changes in substrate selectivity. They indicate that the metabolism of quinate ester and tyramine amide of p-coumaric acid rely on the same recognition site in the protein.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Evolutionary history and systematics of Acer section Acer – a case study of low-level phylogenetics

The phylogenetic and systematic position of all species of Acer section Acer from North America, East Asia, and western Eurasia are evaluated using various splits-based networks (distance networks, bipartition networks), ITS motif analysis, and morphology. Molecular analyses are based on 276 ITS clones obtained from 101 specimens collected mainly from natural stands. The large sample size ensures to cover sufficiently inter- and intraspecific ITS variability of this group. Formerly recognised species are generally supported by ITS data and morphology; the combination of molecular (ITS) and morphological criteria allows defining seven (supraspecific) taxonomic groups prior to a phylogenetic reconstruction. Phylogenetic signals captured in modern ITS sequences are partly incompatible but clearly suggest that Acer section Acer underwent three major radiations. Horizontal gene flow is indicated between ancestors of extant taxa that are isolated at present times. The level of ITS derivation can be estimated and corresponds to levels of morphological differentiation and (palaeo-) biogeographical patterns. Based on our results we question the potential of cladistic approaches to infer low-level evolution in an adequate manner and demonstrate that speciation in members of Acer section Acer is not generally linked to cladogenesis. The data and methodologies provided here allow to trace pathways of low-level evolution and to analyse such data sets with a less restricted (non-dichotomous) dynamic concept. Electronic Supplementary Material Supplementary material is available in the online version of this article at