Gene loss through pseudogenization contributes to the ecological diversification of a generalist Roseobacter lineage

Ecologically relevant genes generally show patchy distributions among related bacterial genomes. This is commonly attributed to lateral gene transfer, whereas the opposite mechanism—gene loss—has rarely been explored. Pseudogenization is a major mechanism underlying gene loss, and pseudogenes are best characterized by comparing closely related genomes because of their short life spans. To explore the role of pseudogenization in microbial ecological diversification, we apply rigorous methods to characterize pseudogenes in the 279 newly sequenced Ruegeria isolates of the globally abundant Roseobacter group collected from two typical coastal habitats in Hong Kong, the coral Platygyra acuta and the macroalga Sargassum hemiphyllum. Pseudogenes contribute to ~16% of the accessory genomes of these strains. Ancestral state reconstruction reveals that many pseudogenization events are correlated with ancestral niche shifts. Specifically, genes related to resource scavenging and energy acquisition were often pseudogenized when roseobacters inhabiting carbon-limited and energy-poor coral skeleton switched to other resource-richer niches. For roseobacters inhabiting the macroalgal niches, genes for nitrogen regulation and carbohydrate utilization were important but became dispensable upon shift to coral skeleton where nitrate is abundant but carbohydrates are less available. Whereas low-energy-demanding secondary transporters are more favorable in coral skeleton, ATP-driven primary transporters are preferentially kept in the energy-replete macroalgal niches. Moreover, a large proportion of these families mediate organismal interactions, suggesting their rapid losses by pseudogenization as a potential response to host and niche shift. These findings illustrate an important role of pseudogenization in shaping genome content and driving ecological diversification of marine roseobacters.Subject terms: Ecology, Evolution, Microbiology     

The Genomes of the Fungal Plant Pathogens Cladosporium fulvum and Dothistroma septosporum Reveal Adaptation to Different Hosts and Lifestyles But Also Signatures of Common Ancestry

We sequenced and compared the genomes of the Dothideomycete fungal plant pathogens Cladosporium fulvum (Cfu) (syn. Passalora fulva) and Dothistroma septosporum (Dse) that are closely related phylogenetically, but have different lifestyles and hosts. Although both fungi grow extracellularly in close contact with host mesophyll cells, Cfu is a biotroph infecting tomato, while Dse is a hemibiotroph infecting pine. The genomes of these fungi have a similar set of genes (70% of gene content in both genomes are homologs), but differ significantly in size (Cfu >61.1-Mb; Dse 31.2-Mb), which is mainly due to the difference in repeat content (47.2% in Cfu versus 3.2% in Dse). Recent adaptation to different lifestyles and hosts is suggested by diverged sets of genes. Cfu contains an α-tomatinase gene that we predict might be required for detoxification of tomatine, while this gene is absent in Dse. Many genes encoding secreted proteins are unique to each species and the repeat-rich areas in Cfu are enriched for these species-specific genes. In contrast, conserved genes suggest common host ancestry. Homologs of Cfu effector genes, including Ecp2 and Avr4, are present in Dse and induce a Cf-Ecp2- and Cf-4-mediated hypersensitive response, respectively. Strikingly, genes involved in production of the toxin dothistromin, a likely virulence factor for Dse, are conserved in Cfu, but their expression differs markedly with essentially no expression by Cfu in planta. Likewise, Cfu has a carbohydrate-degrading enzyme catalog that is more similar to that of necrotrophs or hemibiotrophs and a larger pectinolytic gene arsenal than Dse, but many of these genes are not expressed in planta or are pseudogenized. Overall, comparison of their genomes suggests that these closely related plant pathogens had a common ancestral host but since adapted to different hosts and lifestyles by a combination of differentiated gene content, pseudogenization, and gene regulation.     

A fragmented aflatoxin-like gene cluster in the forest pathogen Dothistroma septosporum

The polyketide toxin dothistromin is very similar in structure to the aflatoxin precursor, versicolorin B. Dothistromin is made by a pine needle pathogen, Dothistroma septosporum, both in culture and in planta. Orthologs of aflatoxin biosynthetic genes have been identified that are required for dothistromin biosynthesis in D. septosporum. In contrast to the situation in aflatoxin-producing fungi where 25 aflatoxin biosynthetic and regulatory genes are tightly clustered in one region of the genome, the dothistromin gene cluster is fragmented. Three mini-clusters of dothistromin genes have been identified, each located on a 1.3-Mb chromosome and each grouped with non-dothistromin genes. There are no obvious patterns of repeated sequences or transposon relics to suggest recent recombination events. Most dothistromin genes within the mini-clusters are co-regulated, suggesting that coordinate control of gene expression is achieved despite this unusual arrangement of secondary metabolite biosynthetic genes.     

Evolutionary relics dominate the small number of secondary metabolism genes in the hemibiotrophic fungus Dothistroma septosporum

Fungal secondary metabolites have important functions for the fungi that produce them, such as roles in virulence and competition. The hemibiotrophic pine needle pathogen Dothistroma septosporum has one of the lowest complements of secondary metabolite (SM) backbone genes of plant pathogenic fungi, indicating that this fungus produces a limited range of SMs. Amongst these SMs is dothistromin, a well-characterised polyketide toxin and virulence factor that is required for expansion of disease lesions in Dothistroma needle blight disease. Dothistromin genes are dispersed across six loci on one chromosome, rather than being clustered as for most SM genes. We explored other D. septosporum SM genes to determine if they are associated with gene clusters, and to predict what their likely products and functions might be. Of nine functional SM backbone genes in the D. septosporum genome, only four were expressed under a range of in planta and in culture conditions, one of which was the dothistromin PKS backbone gene. Of the other three expressed genes, gene knockout studies suggested that DsPks1 and DsPks2 are not required for virulence and attempts to determine a functional squalestatin-like SM product for DsPks2 were not successful. However preliminary evidence suggested that DsNps3, the only SM backbone gene to be most highly expressed in the early stage of disease, appears to be a virulence factor. Thus, despite the small number of SM backbone genes in D. septosporum, most of them appear to be poorly expressed or dispensable for virulence in planta. This work contributes to a growing body of evidence that many fungal secondary metabolite gene clusters might be non-functional and may be evolutionary relics.     

Comparative genomics analyses of alpha-keratins reveal insights into evolutionary adaptation of marine mammals

Background

Diversity of hair in marine mammals was suggested as an evolutionary innovation to adapt aquatic environment, yet its genetic basis remained poorly explored. We scanned α-keratin genes, one major structural components of hair, in 16 genomes of mammalian species, including seven cetaceans, two pinnipeds, polar bear, manatee and five terrestrial species.

Results

Extensive gene loss and high pseudogenization rate of α-keratin genes were identified in cetaceans when compared to terrestrial artiodactylans (average number of α-keratins 37.29 vs. 58.33; pseudogenization rate 29.89% vs. 8.00%), especially of hair follicle-specific keratin genes (average pseudogenization rate in cetaceans of 43.88% relative to 3.80% artiodactylian average). Compared to toothed whale, the much more number of intact functional α-keratin genes was examined in the baleen whale that had specific keratinized baleen. In contrast, the number of keratin genes in pinnipeds, polar bear and manatee were comparable to those of their respective terrestrial relatives. Additionally, four keratin genes (K39, K9, K42, and K74) were found to be pseudogenes or lost uniquely in cetaceans and manatees.

Conclusions

Species-specific evolution of α-keratin gene family identified in the marine mammals might be responsible for their different hair characteristics. Increased gene loss and pseudogenization rate identified in cetacean lineages was likely to contribute to hair-less phenotype to adaptation for complete aquatic environment. However, the fully aquatic manatee still remained the comparable number of intact genes to its terrestrial relative, probably due to its perioral bristles and bristle-like hairs on the oral disk. By contrast, similar evolution pattern of α-keratin gene repertoire in the pinnipeds, polar bear and their terrestrial relatives was likely due to abundant hair to keep warm when they went ashore. Interestingly, some keratin genes were exclusively lost in cetaceans and manatees, likely as a result of convergent hair-loss phenotype to inhabit completely aquatic environment in both groups.

     

A Polyketide Synthase Gene Required for Biosynthesis of the Aflatoxin-like Toxin, Dothistromin

Dothistromin is a polyketide toxin, produced by a fungal forest pathogen, with structural similarity to the aflatoxin precursor versicolorin B. Biochemical and genetic studies suggested that there are common steps in the biosynthetic pathways for these metabolites and showed similarities between some of the genes. A polyketide synthase gene (pksA) was isolated from dothistromin-producing Dothistroma septosporum by hybridization with an aflatoxin ortholog from Aspergillus parasiticus. Inactivation of this gene in D. septosporum resulted in mutants that could not produce dothistromin but that could convert exogenous aflatoxin precursors, including norsolorinic acid, into dothistromin. The mutants also had reduced asexual sporulation compared to the wild type. So far four other genes are known to be clustered immediately alongside pksA. Three of these (cypA, moxA, avfA) are predicted to be orthologs of aflatoxin biosynthetic genes. The other gene (epoA), located between avfA and moxA, is predicted to encode an epoxide hydrolase, for which there is no homolog in either the aflatoxin or sterigmatocystin gene clusters. The pksA gene is located on a small chromosome of ~1.3 Mb in size, along with the dothistromin ketoreductase (dotA) gene.     

Complete Chloroplast Genome of Sedum sarmentosum and Chloroplast Genome Evolution in Saxifragales

Comparative chloroplast genome analyses are mostly carried out at lower taxonomic levels, such as the family and genus levels. At higher taxonomic levels, chloroplast genomes are generally used to reconstruct phylogenies. However, little attention has been paid to chloroplast genome evolution within orders. Here, we present the chloroplast genome of Sedum sarmentosum and take advantage of several available (or elucidated) chloroplast genomes to examine the evolution of chloroplast genomes in Saxifragales. The chloroplast genome of S. sarmentosum is 150,448 bp long and includes 82,212 bp of a large single-copy (LSC) region, 16.670 bp of a small single-copy (SSC) region, and a pair of 25,783 bp sequences of inverted repeats (IRs).The genome contains 131 unique genes, 18 of which are duplicated within the IRs. Based on a comparative analysis of chloroplast genomes from four representative Saxifragales families, we observed two gene losses and two pseudogenes in Paeonia obovata, and the loss of an intron was detected in the rps16 gene of Penthorum chinense. Comparisons among the 72 common protein-coding genes confirmed that the chloroplast genomes of S. sarmentosum and Paeonia obovata exhibit accelerated sequence evolution. Furthermore, a strong correlation was observed between the rates of genome evolution and genome size. The detected genome size variations are predominantly caused by the length of intergenic spacers, rather than losses of genes and introns, gene pseudogenization or IR expansion or contraction. The genome sizes of these species are negatively correlated with nucleotide substitution rates. Species with shorter duration of the life cycle tend to exhibit shorter chloroplast genomes than those with longer life cycles.     

Epigenetic silencing may aid evolution by gene duplication

Gene duplication is commonly regarded as the main evolutionary path toward the gain of a new function. However, even with gene duplication, there is a loss-versus-gain dilemma: most newly born duplicates degrade to pseudogenes, since degenerative mutations are much more frequent than advantageous ones. Thus, something additional seems to be needed to shift the loss versus gain equilibrium toward functional divergence. We suggest that epigenetic silencing of duplicates might play this role in evolution. This study began when we noticed in a previous publication (Lynch M, Conery JS [2000] Science 291:1151–1155) that the frequency of functional young gene duplicates is higher in organisms that have cytosine methylation (H. sapiens, M. musculus, and A. thaliana) than in organisms that do not have methylated genomes (S. cerevisiae, D. melanogaster, and C. elegans). We find that genome data analysis confirms the likelihood of much more efficient functional divergence of gene duplicates in mammals and plants than in yeast, nematode, and fly. We have also extended the classic model of gene duplication, in which newly duplicated genes have exactly the same expression pattern, to the case when they are epigenetically silenced in a tissue- and/or developmental stage-complementary manner. This exposes each of the duplicates to negative selection, thus protecting from pseudogenization. Our analysis indicates that this kind of silencing (i) enhances evolution of duplicated genes to new functions, particularly in small populations, (ii) is quite consistent with the subfunctionalization model when degenerative but complementary mutations affect different subfunctions of the gene, and (iii) furthermore, may actually cooperate with the DDC (duplication– degeneration–complementation) process. Dedicated to the memory of Susumu Ohno     

Gene-rich plastid genomes of two parasitic red algal species,Laurencia australis and L. verruciformis (Rhodomelaceae,Ceramiales), and a taxonomic revision of Janczewskia

Parasitic red algae are an interesting system for investigating the genetic changes that occur in parasites. These parasites have evolved independently multiple times within the red algae. The functional loss of plastid genomes can be investigated in these multiple independent examples, and fine-scale patterns may be discerned. The only plastid genomes from red algal parasites known so far are highly reduced and missing almost all photosynthetic genes. Our study assembled and annotated plastid genomes from the parasites Janczewskia tasmanica and its two Laurencia host species (Laurencia elata and one unidentified Laurencia sp. A25) from Australia and Janczewskia verruciformis, its host species (Laurencia catarinensis), and the closest known free-living relative (Laurencia obtusa) from the Canary Islands (Spain). For the first time we show parasitic red algal plastid genomes that are similar in size and gene content to free-living host species without any gene loss or genome reduction. The only exception was two pseudogenes (moeB and ycf46) found in the plastid genome of both isolates of J. tasmanica, indicating potential for future loss of these genes. Further comparative analyses with the three highly reduced plastid genomes showed possible gene loss patterns, in which photosynthetic gene categories were lost followed by other gene categories. Phylogenetic analyses did not confirm monophyly of Janczewskia, and the genus was subsumed into Laurencia. Further investigations will determine if any convergent small-scale patterns of gene loss exist in parasitic red algae and how these are applicable to other parasitic systems.     

Extensive Rearrangements in the Chloroplast Genome of <Emphasis Type="Italic">Trachelium caeruleum</Emphasis> Are Associated with Repeats and tRNA Genes

Chloroplast genome organization, gene order, and content are highly conserved among land plants. We sequenced the chloroplast genome of Trachelium caeruleum L. (Campanulaceae), a member of an angiosperm family known for highly rearranged genomes. The total genome size is 162,321 bp, with an inverted repeat (IR) of 27,273 bp, large single-copy (LSC) region of 100,114 bp, and small single-copy (SSC) region of 7,661 bp. The genome encodes 112 different genes, with 17 duplicated in the IR, a tRNA gene (trnI-cau) duplicated once in the LSC region, and a protein-coding gene (psbJ) with two duplicate copies, for a total of 132 putatively intact genes. ndhK may be a pseudogene with internal stop codons, and clpP, ycf1, and ycf2 are so highly diverged that they also may be pseudogenes. ycf15, rpl23, infA, and accD are truncated and likely nonfunctional. The most conspicuous feature of the Trachelium genome is the presence of 18 internally unrearranged blocks of genes inverted or relocated within the genome relative to the ancestral gene order of angiosperm chloroplast genomes. Recombination between repeats or tRNA genes has been suggested as a mechanism of chloroplast genome rearrangements. The Trachelium chloroplast genome shares with Pelargonium and Jasminum both a higher number of repeats and larger repeated sequences in comparison to eight other angiosperm chloroplast genomes, and these are concentrated near rearrangement endpoints. Genes for tRNAs occur at many but not all inversion endpoints, so some combination of repeats and tRNA genes may have mediated these rearrangements.     

Similar numbers but different repertoires of olfactory receptor genes in humans and chimpanzees

Animals recognize their external world through the detection of tens of thousands of chemical odorants. Olfactory receptor (OR) genes encode proteins for detecting odorant molecules and form the largest multigene family in mammals. It is known that humans have fewer OR genes and a higher fraction of OR pseudogenes than mice or dogs. To investigate whether these features are human specific or common to all higher primates, we identified nearly complete sets of OR genes from the chimpanzee and macaque genomes and compared them with the human OR genes. In contrast to previous studies, here we show that the number of OR genes ( approximately 810) and the fraction of pseudogenes (51%) in chimpanzees are very similar to those in humans, though macaques have considerably fewer OR genes. The pseudogenization rates and the numbers of genes affected by positive selection are also similar between humans and chimpanzees. Moreover, the most recent common ancestor between humans and chimpanzees had a larger number of functional OR genes (>500) and a lower fraction of pseudogenes (41%) than its descendents, suggesting that the OR gene repertoires are in a phase of deterioration in both lineages. Interestingly, despite the close evolutionary relationship between the 2 species, approximately 25% of their functional gene repertoires are species specific due to massive gene losses. These findings suggest that the tempo of evolution of OR genes is similar between humans and chimpanzees, but the OR gene repertoires are quite different between them. This difference might be responsible for the species-specific ability of odor perception.     

Secondary Metabolism and Biotrophic Lifestyle in the Tomato Pathogen Cladosporium fulvum

Cladosporium fulvum is a biotrophic fungal pathogen that causes leaf mould of tomato. Analysis of its genome suggested a high potential for production of secondary metabolites (SM), which might be harmful to plants and animals. Here, we have analysed in detail the predicted SM gene clusters of C. fulvum employing phylogenetic and comparative genomic approaches. Expression of the SM core genes was measured by RT-qrtPCR and produced SMs were determined by LC-MS and NMR analyses. The genome of C. fulvum contains six gene clusters that are conserved in other fungal species, which have undergone rearrangements and gene losses associated with the presence of transposable elements. Although being a biotroph, C. fulvum has the potential to produce elsinochrome and cercosporin toxins. However, the corresponding core genes are not expressed during infection of tomato. Only two core genes, PKS6 and NPS9, show high expression in planta, but both are significantly down regulated during colonization of the mesophyll tissue. In vitro SM profiling detected only one major compound that was identified as cladofulvin. PKS6 is likely involved in the production of this pigment because it is the only core gene significantly expressed under these conditions. Cladofulvin does not cause necrosis on Solanaceae plants and does not show any antimicrobial activity. In contrast to other biotrophic fungi that have a reduced SM production capacity, our studies on C. fulvum suggest that down-regulation of SM biosynthetic pathways might represent another mechanism associated with a biotrophic lifestyle.     

Gene losses during human origins

Pseudogenization is a widespread phenomenon in genome evolution, and it has been proposed to serve as an engine of evolutionary change, especially during human origins (the “less-is-more” hypothesis). However, there has been no comprehensive analysis of human-specific pseudogenes. Furthermore, it is unclear whether pseudogenization itself can be selectively favored and thus play an active role in human evolution. Here we conduct a comparative genomic analysis and a literature survey to identify 80 nonprocessed pseudogenes that were inactivated in the human lineage after its separation from the chimpanzee lineage. Many functions are involved among these genes, with chemoreception and immune response being outstandingly overrepresented, suggesting potential species-specific features in these aspects of human physiology. To explore the possibility of adaptive pseudogenization, we focus on CASPASE12, a cysteinyl aspartate proteinase participating in inflammatory and innate immune response to endotoxins. We provide population genetic evidence that the nearly complete fixation of a null allele at CASPASE12 has been driven by positive selection, probably because the null allele confers protection from severe sepsis. We estimate that the selective advantage of the null allele is about 0.9% and the pseudogenization started shortly before the out-of-Africa migration of modern humans. Interestingly, two other genes related to sepsis were also pseudogenized in humans, possibly by selection. These adaptive gene losses might have occurred because of changes in our environment or genetic background that altered the threat from or response to sepsis. The identification and analysis of human-specific pseudogenes open the door for understanding the roles of gene losses in human origins, and the demonstration that gene loss itself can be adaptive supports and extends the “less-is-more” hypothesis.     

Recognizing the pseudogenes in bacterial genomes

Pseudogenes are now known to be a regular feature of bacterial genomes and are found in particularly high numbers within the genomes of recently emerged bacterial pathogens. As most pseudogenes are recognized by sequence alignments, we use newly available genomic sequences to identify the pseudogenes in 11 genomes from 4 bacterial genera, each of which contains at least 1 human pathogen. The numbers of pseudogenes range from 27 in Staphylococcus aureus MW2 to 337 in Yersinia pestis CO92 (e.g. 1–8% of the annotated genes in the genome). Most pseudogenes are formed by small frameshifting indels, but because stop codons are A + T-rich, the two low-G + C Gram-positive taxa (Streptococcus and Staphylococcus) have relatively high fractions of pseudogenes generated by nonsense mutations when compared with more G + C-rich genomes. Over half of the pseudogenes are produced from genes whose original functions were annotated as ‘hypothetical’ or ‘unknown’; however, several broadly distributed genes involved in nucleotide processing, repair or replication have become pseudogenes in one of the sequenced Vibrio vulnificus genomes. Although many of our comparisons involved closely related strains with broadly overlapping gene inventories, each genome contains a largely unique set of pseudogenes, suggesting that pseudogenes are formed and eliminated relatively rapidly from most bacterial genomes.     

Extensive Reorganization of the Plastid Genome of <Emphasis Type="Italic">Trifolium subterraneum</Emphasis> (Fabaceae) Is Associated with Numerous Repeated Sequences and Novel DNA Insertions

The plastid genome of Trifolium subterraneum is 144,763 bp, about 20 kb longer than those of closely related legumes, which also lost one copy of the large inverted repeat (IR). The genome has undergone extensive genomic reconfiguration, including the loss of six genes (accD, infA, rpl22, rps16, rps18, and ycf1) and two introns (clpP and rps12) and numerous gene order changes, attributable to 14–18 inversions. All endpoints of rearranged gene clusters are flanked by repeated sequences, tRNAs, or pseudogenes. One unusual feature of the Trifolium subterraneum genome is the large number of dispersed repeats, which comprise 19.5% (ca. 28 kb) of the genome (versus about 4% for other angiosperms) and account for part of the increase in genome size. Nine genes (psbT, rbcL, clpP, rps3, rpl23, atpB, psbN, trnI-cau, and ycf3) have also been duplicated either partially or completely. rpl23 is the most highly duplicated gene, with portions of this gene duplicated six times. Comparisons of the Trifolium plastid genome with the Plant Repeat Database and searches for flanking inverted repeats suggest that the high incidence of dispersed repeats and rearrangements is not likely the result of transposition. Trifolium has 19.5 kb of unique DNA distributed among 160 fragments ranging in size from 30 to 494 bp, greatly surpassing the other five sequenced legume plastid genomes in novel DNA content. At least some of this unique DNA may represent horizontal transfer from bacterial genomes. These unusual features provide direction for the development of more complex models of plastid genome evolution. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Age and Detection of Retroprocessed Pseudogenes in Murine Rodents

Retroprocessed pseudogenes, calmodulin II (ψ₁, ψ₂, and ψ₃ CALMII), ψα-tubulin, π-glutathione S-transferase (ψπ-GST) from rat, lactic acid dehydrogenase (ψ LDH) from mouse, and heat shock protein 60 chaperonin (ψ HSP60) from Chinese hamster, were examined for their presence in these species by polymerase chain reaction (PCR). Pseudogenes of these murine rodents were detected by PCR only in those species in which the genes were originally identified, suggesting that the selected pseudogene of one species arose too recently to be detected in the genomes of the other rodent species. The calculated ages of the rodent pseudogenes ranged from 1.7 Myr (ψα-tubulin) to 7.5 Myr (ψ₃ CALMII) when employing a homologous functional gene of the taxon as a reference in the relative rate test with the mouse or rat as the outgroup. Given the high rate of divergence of the genes of rodents relative to other species, selection of an outgroup with similar mutation rates seems warranted. To justify further the conclusion that the selected pseudogenes were indeed retroprocessed after these three taxa diverged, the presence of the pseudogenes in the genome of different rat species was examined. The existence of ψ₃ CALMII and ψα-tubulin pseudogenes of Rattus norvegicus among species belonging to Rattus sensu stricto is evidence for the common ancestry of this group. Received: 23 May 2001 / Accepted: 31 May 2001