The Pharus latifolius genome bridges the gap of early grass evolution

The grass family (Poaceae) includes all commercial cereal crops and is a major contributor to biomass in various terrestrial ecosystems. The ancestry of all grass genomes includes a shared whole-genome duplication (WGD), named rho (ρ) WGD, but the evolutionary significance of ρ-WGD remains elusive. We sequenced the genome of Pharus latifolius, a grass species (producing a true spikelet) in the subfamily Pharoideae, a sister lineage to the core Poaceae including the (Panicoideae, Arundinoideae, Chloridoideae, Micrairoideae, Aristidoideae, and Danthonioideae (PACMAD) and Bambusoideae, Oryzoideae, and Pooideae (BOP) clades. Our results indicate that the P. latifolius genome has evolved slowly relative to cereal grass genomes, as reflected by moderate rates of molecular evolution, limited chromosome rearrangements and a low rate of gene loss for duplicated genes. We show that the ρ-WGD event occurred approximately 98.2 million years ago (Ma) in a common ancestor of the Pharoideae and the PACMAD and BOP grasses. This was followed by contrasting patterns of diploidization in the Pharus and core Poaceae lineages. The presence of two FRIZZY PANICLE-like genes in P. latifolius, and duplicated MADS-box genes, support the hypothesis that the ρ-WGD may have played a role in the origin and functional diversification of the spikelet, an adaptation in grasses related directly to cereal yields. The P. latifolius genome sheds light on the origin and early evolution of grasses underpinning the biology and breeding of cereals.

The Pharus genome fills an important genomic gap, providing numerous insights into how whole-genome duplication contributed to the origin and diversification of the grass family.     

Horizontal Gene Transfer and Gene Duplication of β-Fructofuranosidase Confer Lepidopteran Insects Metabolic Benefits

Horizontal gene transfer (HGT) is a potentially critical source of material for ecological adaptation and the evolution of novel genetic traits. However, reports on posttransfer duplication in organism genomes are lacking, and the evolutionary advantages conferred on the recipient are generally poorly understood. Sucrase plays an important role in insect physiological growth and development. Here, we performed a comprehensive analysis of the evolution of insect β-fructofuranosidase transferred from bacteria via HGT. We found that posttransfer duplications of β-fructofuranosidase were widespread in Lepidoptera and sporadic occurrences of β-fructofuranosidase were found in Coleoptera and Hymenoptera. β-fructofuranosidase genes often undergo modifications, such as gene duplication, differential gene loss, and changes in mutation rates. Lepidopteran β-fructofuranosidase gene (SUC) clusters showed marked divergence in gene expression patterns and enzymatic properties in Bombyx mori (moth) and Papilio xuthus (butterfly). We generated SUC1 mutations in B. mori using CRISPR/Cas9 to thoroughly examine the physiological function of SUC. BmSUC1 mutant larvae were viable but displayed delayed growth and reduced sucrase activities that included susceptibility to the sugar mimic alkaloid found in high concentrations in mulberry. BmSUC1 served as a critical sucrase and supported metabolic homeostasis in the larval midgut and silk gland, suggesting that gene transfer of β-fructofuranosidase enhanced the digestive and metabolic adaptation of lepidopteran insects. These findings highlight not only the universal function of β-fructofuranosidase with a link to the maintenance of carbohydrate metabolism but also an underexplored function in the silk gland. This study expands our knowledge of posttransfer duplication and subsequent functional diversification in the adaptive evolution and lineage-specific adaptation of organisms.     

Genome-wide haploinsufficiency screen reveals a novel role for γ-TuSC in spindle organization and genome stability

How subunit dosage contributes to the assembly and function of multimeric complexes is an important question with implications in understanding biochemical, evolutionary, and disease mechanisms. Toward identifying pathways that are susceptible to decreased gene dosage, we performed a genome-wide screen for haploinsufficient (HI) genes that guard against genome instability in Saccharomyces cerevisiae. This led to the identification of all three genes (SPC97, SPC98, and TUB4) encoding the evolutionarily conserved γ-tubulin small complex (γ-TuSC), which nucleates microtubule assembly. We found that hemizygous γ-TuSC mutants exhibit higher rates of chromosome loss and increases in anaphase spindle length and elongation velocities. Fluorescence microscopy, fluorescence recovery after photobleaching, electron tomography, and model convolution simulation of spc98/+ mutants revealed improper regulation of interpolar (iMT) and kinetochore (kMT) microtubules in anaphase. The underlying cause is likely due to reduced levels of Tub4, as overexpression of TUB4 suppressed the spindle and chromosome segregation defects in spc98/+ mutants. We propose that γ-TuSC is crucial for balanced assembly between iMTs and kMTs for spindle organization and accurate chromosome segregation. Taken together, the results show how gene dosage studies provide critical insights into the assembly and function of multisubunit complexes that may not be revealed by using traditional studies with haploid gene deletion or conditional alleles.     

Molecular Evolution of the Primate α-/θ-Defensin Multigene Family

The primate α-/θ-defensin multigene family encodes versatile endogenous cationic and amphipathic peptides that have broad-spectrum antibacterial, antifungal and antiviral activity. Although previous studies have reported that α-/θ-defensin (DEFA/DEFT) genes are under birth-and-death evolution with frequent duplication and rapid evolution, the phylogenetic relationships of the primate DEFA/DEFT genes; the genetic bases for the existence of similar antimicrobial spectra among closely related species; and the evolutionary processes involved in the emergence of cyclic θ-defensins in Old World monkeys and their subsequent loss of function in humans, chimpanzees and gorillas require further investigation. In this study, the DEFA/DEFT gene repertoires from primate and treeshrew were collected, followed by detailed phylogenetic, sequence and structure, selection pressure and comparative genomics analyses. All treeshrew, prosimian and simian DEFA/DEFT genes are grouped into two major clades, which are tissue-specific for enteric and myeloid defensins in simians. The simian enteric and myeloid α-defensins are classified into six functional gene clusters with diverged sequences, variable structures, altered functional constraints and different selection pressures, which likely reflect the antimicrobial spectra among closely related species. Species-specific duplication or pseudogenization within each simian cluster implies that the antimicrobial spectrum is ever-shifting, most likely challenged by the ever-changing pathogen environment. The DEFT evolved from the myeloid DEFA8. The prosegment of θ-defensin is detected with adaptive changes coevolving with the new protein fold of mature peptide, coincident with the importance of the prosegment for the correct folding of the mature peptide. Lastly, a less-is-hitchhiking hypothesis was proposed as a possible explanation for the expansion of pseudogene DEFTP and the loss of functional DEFT, where the gain or loss of the hitchhiker is determined by its adjacent driver gene during the birth-and-death evolutionary process.     

Distinctive Patterns of Evolution of the δ-Globin Gene (HBD) in Primates

In most vertebrates, hemoglobin (Hb) is a heterotetramer composed of two dissimilar globin chains, which change during development according to the patterns of expression of α- and β-globin family members. In placental mammals, the β-globin cluster includes three early-expressed genes, ε(HBE)-γ(HBG)-ψβ(HBBP1), and the late expressed genes, δ (HBD) and β (HBB). While HBB encodes the major adult β-globin chain, HBD is weakly expressed or totally silent. Paradoxically, in human populations HBD shows high levels of conservation typical of genes under strong evolutionary constraints, possibly due to a regulatory role in the fetal-to-adult switch unique of Anthropoid primates. In this study, we have performed a comprehensive phylogenetic and comparative analysis of the two adult β-like globin genes in a set of diverse mammalian taxa, focusing on the evolution and functional divergence of HBD in primates. Our analysis revealed that anthropoids are an exception to a general pattern of concerted evolution in placental mammals, showing a high level of sequence conservation at HBD, less frequent and shorter gene conversion events. Moreover, this lineage is unique in the retention of a functional GATA-1 motif, known to be involved in the control of the developmental expression of the β-like globin genes. We further show that not only the mode but also the rate of evolution of the δ-globin gene in higher primates are strictly associated with the fetal/adult β-cluster developmental switch. To gain further insight into the possible functional constraints that have been shaping the evolutionary history of HBD in primates, we calculated dN/dS (ω) ratios under alternative models of gene evolution. Although our results indicate that HBD might have experienced different selective pressures throughout primate evolution, as shown by different ω values between apes and Old World Monkeys + New World Monkeys (0.06 versus 0.43, respectively), these estimates corroborated a constrained evolution for HBD in Anthropoid lineages, which is unlikely to be related to protein function. Collectively, these findings suggest that sequence change at the δ-globin gene has been under strong selective constraints over 65 Myr of primate evolution, likely due to a regulatory role in ontogenic switches of gene expression.     

Chromosome Dynamics Visualized with an Anti-Centromeric Histone H3 Antibody in Allium

Due to the ease with which chromosomes can be observed, the Allium species, and onion in particular, have been familiar materials employed in cytogenetic experiments in biology. In this study, centromeric histone H3 (CENH3)-coding cDNAs were identified in four Allium species (onion, welsh onion, garlic and garlic chives) and cloned. Anti-CENH3 antibody was then raised against a deduced amino acid sequence of CENH3 of welsh onion. The antibody recognized all CENH3 orthologs of the Allium species tested. Immunostaining with the antibody enabled clear visualization of chromosome behavior during mitosis in the species. Furthermore, three-dimensional (3D) observation of mitotic cell division was achieved by subjecting root sections to immunohistochemical techniques. The 3D dynamics of the cells and position of cell-cycle marker proteins (CENH3 and α-tubulin) were clearly revealed by immunohistochemical staining with the antibodies. The immunohistochemical analysis made it possible to establish an overview of the location of dividing cells in the root tissues. This breakthrough in technique, in addition to the two centromeric DNA sequences isolated from welsh onion by chromatin immuno-precipitation using the antibody, should lead to a better understanding of plant cell division. A phylogenetic analysis of Allium CENH3s together with the previously reported plant CENH3s showed two separate clades for monocot species tested. One clade was made from CENH3s of the Allium species with those of Poaceae species, and the other from CENH3s of a holocentric species (Luzula nivea). These data may imply functional differences of CENH3s between holocentric and monocentric species. Centromeric localization of DNA sequences isolated from welsh onion by chromatin immuno-precipitation (ChIP) using the antibody was confirmed by fluorescence in situ hybridization and ChIP-quantitative PCR.     

Operon for Biosynthesis of Lipstatin,the Beta-Lactone Inhibitor of Human Pancreatic Lipase

Lipstatin, isolated from Streptomyces toxytricini as a potent and selective inhibitor of human pancreatic lipase, is a precursor for tetrahydrolipstatin (also known as orlistat, Xenical, and Alli), the only FDA-approved antiobesity medication for long-term use. Lipstatin features a 2-hexyl-3,5-dihydroxy-7,10-hexadecadienoic-β-lactone structure with an N-formyl-l-leucine group attached as an ester to the 5-hydroxy group. It has been suggested that the α-branched 3,5-dihydroxy fatty acid β-lactone moiety of lipstatin in S. toxytricini is derived from Claisen condensation between two fatty acid substrates, which are derived from incomplete oxidative degradation of linoleic acid based on feeding experiments. In this study, we identified a six-gene operon (lst) that was essential for the biosynthesis of lipstatin by large-deletion, complementation, and single-gene knockout experiments. lstA, lstB, and lstC, which encode two β-ketoacyl–acyl carrier protein synthase III homologues and an acyl coenzyme A (acyl-CoA) synthetase homologue, were indicated to be responsible for the generation of the α-branched 3,5-dihydroxy fatty acid backbone. Subsequently, the nonribosomal peptide synthetase (NRPS) gene lstE and the putative formyltransferase gene lstF were involved in decoration of the α-branched 3,5-dihydroxy fatty acid chain with an N-formylated leucine residue. Finally, the 3β-hydroxysteroid dehydrogenase-homologous gene lstD might be responsible for the reduction of the β-keto group of the biosynthetic intermediate, thereby facilitating the formation of the unique β-lactone ring.     

Isorenieratene biosynthesis in green sulfur bacteria requires the cooperative actions of two carotenoid cyclases

The cyclization of lycopene to γ- or β-carotene is a major branch point in the biosynthesis of carotenoids in photosynthetic bacteria. Four families of carotenoid cyclases are known, and each family includes both mono- and dicyclases, which catalyze the formation of γ- and β-carotene, respectively. Green sulfur bacteria (GSB) synthesize aromatic carotenoids, of which the most commonly occurring types are the monocyclic chlorobactene and the dicyclic isorenieratene. Recently, the cruA gene, encoding a conserved hypothetical protein found in the genomes of all GSB and some cyanobacteria, was identified as a lycopene cyclase. Further genomic analyses have found that all available fully sequenced genomes of GSB encode an ortholog of cruA. Additionally, the genomes of all isorenieratene-producing species of GSB encode a cruA paralog, now named cruB. The cruA gene from the chlorobactene-producing GSB species Chlorobaculum tepidum and both cruA and cruB from the brown-colored, isorenieratene-producing GSB species Chlorobium phaeobacteroides strain DSM 266^T were heterologously expressed in lycopene- and neurosporene-producing strains of Escherichia coli, and the cruB gene of Chlorobium clathratiforme strain DSM 5477^T was also heterologously expressed in C. tepidum by inserting the gene at the bchU locus. The results show that CruA is probably a lycopene monocyclase in all GSB and that CruB is a γ-carotene cyclase in isorenieratene-producing species. Consequently, the branch point for the synthesis of mono- and dicyclic carotenoids in GSB seems to be the modification of γ-carotene, rather than the cyclization of lycopene as occurs in cyanobacteria.     

Habitat-Associated Phylogenetic Community Patterns of Microbial Ammonia Oxidizers

Microorganisms mediating ammonia oxidation play a fundamental role in the connection between biological nitrogen fixation and anaerobic nitrogen losses. Bacteria and Archaea ammonia oxidizers (AOB and AOA, respectively) have colonized similar habitats worldwide. Ammonia oxidation is the rate-limiting step in nitrification, and the ammonia monooxygenase (Amo) is the key enzyme involved. The molecular ecology of this process has been extensively explored by surveying the gene of the subunit A of the Amo (amoA gene). In the present study, we explored the phylogenetic community ecology of AOB and AOA, analyzing 5776 amoA gene sequences from >300 isolation sources, and clustering habitats by environmental ontologies. As a whole, phylogenetic richness was larger in AOA than in AOB, and sediments contained the highest phylogenetic richness whereas marine plankton the lowest. We also observed that freshwater ammonia oxidizers were phylogenetically richer than their marine counterparts. AOA communities were more dissimilar to each other than those of AOB, and consistent monophyletic lineages were observed for sediments, soils, and marine plankton in AOA but not in AOB. The diversification patterns showed a more constant cladogenesis through time for AOB whereas AOA apparently experienced two fast diversification events separated by a long steady-state episode. The diversification rate (γ statistic) for most of the habitats indicated γ_AOA > γ_AOB. Soil and sediment experienced earlier bursts of diversification whereas habitats usually eutrophic and rich in ammonium such as wastewater and sludge showed accelerated diversification rates towards the present. Overall, this work shows for the first time a global picture of the phylogenetic community structure of both AOB and AOA assemblages following the strictest analytical standards, and provides an ecological view on the differential evolutionary paths experienced by widespread ammonia-oxidizing microorganisms. The emerged picture of AOB and AOA distribution in different habitats provides a new view to understand the ecophysiology of ammonia oxidizers on Earth.     

Diversification of Paralogous α-Isopropylmalate Synthases by Modulation of Feedback Control and Hetero-Oligomerization in Saccharomyces cerevisiae

Production of α-isopropylmalate (α-IPM) is critical for leucine biosynthesis and for the global control of metabolism. The budding yeast Saccharomyces cerevisiae has two paralogous genes, LEU4 and LEU9, that encode α-IPM synthase (α-IPMS) isozymes. Little is known about the biochemical differences between these two α-IPMS isoenzymes. Here, we show that the Leu4 homodimer is a leucine-sensitive isoform, while the Leu9 homodimer is resistant to such feedback inhibition. The leu4Δ mutant, which expresses only the feedback-resistant Leu9 homodimer, grows slowly with either glucose or ethanol and accumulates elevated pools of leucine; this phenotype is alleviated by the addition of leucine. Transformation of the leu4Δ mutant with a centromeric plasmid carrying LEU4 restored the wild-type phenotype. Bimolecular fluorescent complementation analysis showed that Leu4-Leu9 heterodimeric isozymes are formed in vivo. Purification and kinetic analysis showed that the hetero-oligomeric isozyme has a distinct leucine sensitivity behavior. Determination of α-IPMS activity in ethanol-grown cultures showed that α-IPM biosynthesis and growth under these respiratory conditions depend on the feedback-sensitive Leu4 homodimer. We conclude that retention and further diversification of two yeast α-IPMSs have resulted in a specific regulatory system that controls the leucine–α-IPM biosynthetic pathway by selective feedback sensitivity of homomeric and heterodimeric isoforms.     

MFalpha1, the gene encoding the alpha mating pheromone of Candida albicans

Candida albicans, the single most frequently isolated human fungal pathogen, was thought to be asexual until the recent discovery of the mating-type-like locus (MTL). Homozygous MTL strains were constructed and shown to mate. Furthermore, it has been demonstrated that opaque-phase cells are more efficient in mating than white-phase cells. The similarity of the genes involved in the mating pathway in Saccharomyces cerevisiae and C. albicans includes at least one gene (KEX2) that is involved in the processing of the α mating pheromone in the two yeasts. Taking into account this similarity, we searched the C. albicans genome for sequences that would encode the α pheromone gene. Here we report the isolation and characterization of the gene MFα1, which codes for the precursor of the α mating pheromone in C. albicans. Two active α-peptides, 13 and 14 amino acids long, would be generated after the precursor molecule is processed in C. albicans. To examine the role of this gene in mating, we constructed an mfα1 null mutant of C. albicans. The mfα1 null mutant fails to mate as MTLα, while MTLa mfα1 cells are still mating competent. Experiments performed with the synthetic α-peptides show that they are capable of inducing growth arrest, as demonstrated by halo tests, and also induce shmooing in MTLa cells of C. albicans. These peptides are also able to complement the mating defect of an MTLα kex2 mutant strain when added exogenously, thereby confirming their roles as α mating pheromones.     

Genetic Analysis of Resistance to Benzimidazoles in Physarum: Differential Expression of β-Tubulin Genes

Physarum displays two vegetative cell types, uninucleate myxamoebae and multinucleate plasmodia. Mutant myxamoebae of Physarum resistant to the antitubulin drug methylbenzimidazole-2-yl-carbamate (MBC) were isolated. All mutants tested were cross-resistant to other benzimidazoles but not to cycloheximide or emetine. Genetic analysis showed that mutation to MBC resistance can occur at any one of four unlinked loci, benA, benB, benC or benD. MBC resistance of benB and benD mutants was expressed in plasmodia, but benA and benC mutant plasmodia were MBC sensitive, suggesting that benA and benC encode myxamoeba-specific products. Myxamoebae carrying the recessive benD210 mutation express a β-tubulin with noval electrophoretic mobility, in addition to a β-tubulin with wild-type mobility. This and other evidence indicates that benD is a structural gene for β-tubulin, and that at least two β-tubulin genes are expressed in myxamoebae. Comparisons of the β-tubulins of wildtype and benD210 strains by gel electrophoresis revealed that, of the three (or more) β-tubulin genes expressed in Physarum, one, benD, is expressed in both myxamoebae and plasmodia, one is expressed specifically in myxamoebae and one is expressed specifically in plasmodia. However, mutation in only one gene, benD, is sufficient to confer MBC resistance on both myxamoebae and plasmodia.     

The Tyrosine Aminomutase TAM1 Is Required for β-Tyrosine Biosynthesis in Rice

Non-protein amino acids, often isomers of the standard 20 protein amino acids, have defense-related functions in many plant species. A targeted search for jasmonate-induced metabolites in cultivated rice (Oryza sativa) identified (R)-β-tyrosine, an isomer of the common amino acid (S)-α-tyrosine in the seeds, leaves, roots, and root exudates of the Nipponbare cultivar. Assays with 119 diverse cultivars showed a distinct presence/absence polymorphism, with β-tyrosine being most prevalent in temperate japonica cultivars. Genetic mapping identified a candidate gene on chromosome 12, which was confirmed to encode a tyrosine aminomutase (TAM1) by transient expression in Nicotiana benthamiana and in vitro enzyme assays. A point mutation in TAM1 eliminated β-tyrosine production in Nipponbare. Rice cultivars that do not produce β-tyrosine have a chromosome 12 deletion that encompasses TAM1. Although β-tyrosine accumulation was induced by the plant defense signaling molecule jasmonic acid, bioassays with hemipteran and lepidopteran herbivores showed no negative effects at physiologically relevant β-tyrosine concentrations. In contrast, root growth of Arabidopsis thaliana and other tested dicot plants was inhibited by concentrations as low as 1 μM. As β-tyrosine is exuded into hydroponic medium at higher concentrations, it may contribute to the allelopathic potential of rice.     

DNA polymerases ν and θ are required for efficient immunoglobulin V gene diversification in chicken

The chicken DT40 B lymphocyte line diversifies its immunoglobulin (Ig) V genes through translesion DNA synthesis–dependent point mutations (Ig hypermutation) and homologous recombination (HR)–dependent Ig gene conversion. The error-prone biochemical characteristic of the A family DNA polymerases Polν and Polθ led us to explore the role of these polymerases in Ig gene diversification in DT40 cells. Disruption of both polymerases causes a significant decrease in Ig gene conversion events, although POLN^−/−/POLQ^−/− cells exhibit no prominent defect in HR-mediated DNA repair, as indicated by no increase in sensitivity to camptothecin. Polη has also been previously implicated in Ig gene conversion. We show that a POLH^−/−/POLN^−/−/POLQ^−/− triple mutant displays no Ig gene conversion and reduced Ig hypermutation. Together, these data define a role for Polν and Polθ in recombination and suggest that the DNA synthesis associated with Ig gene conversion is accounted for by three specialized DNA polymerases.     

Clostridium scindens: a human gut microbe with a high potential to convert glucocorticoids into androgens

Clostridium scindens American Type Culture Collection 35704 is capable of converting primary bile acids to toxic secondary bile acids, as well as converting glucocorticoids to androgens by side-chain cleavage. The molecular structure of the side-chain cleavage product of cortisol produced by C. scindens was determined to be 11β-hydroxyandrost-4-ene-3,17-dione (11β-OHA) by high-resolution mass spectrometry, ¹H and ¹³C NMR spectroscopy, and X-ray crystallography. Using RNA-Seq technology, we identified a cortisol-inducible (∼1,000-fold) operon (desABCD) encoding at least one enzyme involved in anaerobic side-chain cleavage. The desC gene was cloned, overexpressed, purified, and found to encode a 20α-hydroxysteroid dehydrogenase (HSDH). This operon also encodes a putative “transketolase” (desAB) hypothesized to have steroid-17,20-desmolase/oxidase activity, and a possible corticosteroid transporter (desD). RNA-Seq data suggests that the two-carbon side chain of glucocorticords may feed into the pentose-phosphate pathway and are used as a carbon source. The 20α-HSDH is hypothesized to function as a metabolic “rheostat” controlling rates of side-chain cleavage. Phylogenetic analysis suggests this operon is rare in nature and the desC gene evolved from a gene encoding threonine dehydrogenase. The physiological effect of 11β-OHAD on the host or other gut microbes is currently unknown.