A candidate gene cluster for the bioactive natural product gyrophoric acid in lichen-forming fungi

Natural products of lichen-forming fungi are structurally diverse and have a variety of medicinal properties. Yet they a have limited implementation in industry as for most of the natural products, the corresponding genes remain unknown. Here we implement a long-read sequencing and bioinformatic approach to identify the biosynthetic gene cluster of the bioactive natural product gyrophoric acid (GA). Using 15 high-quality genomes representing nine GA-producing species of the lichen-forming fungal genus Umbilicaria, we identify the most likely GA cluster and investigate cluster gene organization and composition across the nine species. Our results show that GA clusters are promiscuous within Umbilicaria with only three genes that are conserved across species, including the PKS gene. In addition, our results suggest that the same cluster codes for different but structurally similar NPs, i.e., GA, umbilicaric acid and hiascic acid, bringing new evidence that lichen metabolite diversity is also generated through regulatory mechanisms at the molecular level. Ours is the first study to identify the most likely GA cluster. This information is essential for opening up avenues for biotechnological approaches to producing and modifying GA, and possibly other lichen compounds. We show that bioinformatics approaches are useful in linking genes and potentially associated natural products. Genome analyses help unlocking the pharmaceutical potential of organisms such as lichens, which are biosynthetically diverse, but slow growing, and usually uncultivable due to their symbiotic nature.

The Evolution of Hox Genes in Spiralia

The decoding of genomes of a larger number of animal species have provided further insights into the genomic Hox gene organization and with this indicated the evolutionary changes during the radiation of several clades. The expansion of gene expression studies during development and life history stages of more species, complete the picture of the relationship between cluster organisation and temporal and spatial correlation of the Hox activity. Now these results open the opportunity to look deeper into the regulatory pathways that form these patterns and identify what exact changes caused the evolution of the application of this iconical gene set for the evolution of new larval forms and new structures. Here we review recent progress of Hox gene related research in the large clade Spiralia, that comprises Annelida, Mollusca, Lophophorata, Platyhelminthes, Nemertea and others. Albeit their relationship to each other is not resolved yet, there are emerging patterns that indicate that Hox genes are mainly used for patterning late, adult body parts and that Hox genes are often not expressed on the larval stages. Hox genes seem also often recruited for the formation of morphological novelties. Together with the emerging genomic information Hox genes show a much more dynamic evolutionary history than previously assumed.

Species and Metabolic Pathways Involved in Bioremediation of Vietnamese Soil From Bien Hoa Airbase Contaminated With Herbicides

Four bacterial strains were isolated from enrichment cultures inoculated with soil from Bien Hoa military base in Vietnam contaminated with the herbicides 2,4-dichlorophenoxyacetate (2,4-D) and 2,4,5-trichlorophenoxyacetate (2,4,5-T). They were classified as Pseudomonas aeruginosa BT1 2.2, Sphingomonas histidinilytica BT1 5.2, Bordetella petrii BT1 9.2, and Achromobacter xylosoxidans BT1 10.2. All four were able to degrade 2,4-D and 2,4,5-T, but only the last three species used them as the sole sources of carbon and energy. Mass balance analyses suggest that between 33 and 46% of the carbon in the herbicides is incorporated into dry weight (DW). We obtained insight into their degradation pathways by the genomic analysis of these strains. A tfdCDEF gene cluster was found in A. xylosoxidans BT1 10.2 with amino acid sequences of their gene products showing high identity to those in B. petrii DSM12804. Bordetella petrii BT1 9.2 has a full complement of the tfdABCDEF genes. Surprisingly, the gene organization along with the amino acid sequences of the gene products are virtually identical to those of Cupriavidus pinatubonensis JMP134, referred to as type I tfd genes, and different from those of A. xylosoxidans BT1 10.2 and B. petrii DSM12804. We hypothesize that some of the genetic potential to degrade the herbicides has been recruited in recent mating events between these species and other members of the proteobacteria. This is the first report showing that B. petrii BT1 9.2 emerges as a key player in the degradation of 2,4-D.

Comparative Genome Analysis of ‘Candidatus Phytoplasma luffae’ Reveals the Influential Roles of Potential Mobile Units in Phytoplasma Evolution

Phytoplasmas are insect-transmitted plant pathogens that cause substantial losses in agriculture. In addition to economic impact, phytoplasmas induce distinct disease symptoms in infected plants, thus attracting attention for research on molecular plant-microbe interactions and plant developmental processes. Due to the difficulty of establishing an axenic culture of these bacteria, culture-independent genome characterization is a crucial tool for phytoplasma research. However, phytoplasma genomes have strong nucleotide composition biases and are repetitive, which make it challenging to produce complete assemblies. In this study, we utilized Illumina and Oxford Nanopore sequencing technologies to obtain the complete genome sequence of ‘Candidatus Phytoplasma luffae’ strain NCHU2019 that is associated with witches’ broom disease of loofah (Luffa aegyptiaca) in Taiwan. The fully assembled circular chromosome is 769 kb in size and is the first representative genome sequence of group 16SrVIII phytoplasmas. Comparative analysis with other phytoplasmas revealed that NCHU2019 has an exceptionally repetitive genome, possessing a pair of 75 kb repeats and at least 13 potential mobile units (PMUs) that account for ∼25% of its chromosome. This level of genome repetitiveness is exceptional for bacteria, particularly among obligate pathogens with reduced genomes. Our genus-level analysis of PMUs demonstrated that these phytoplasma-specific mobile genetic elements can be classified into three major types that differ in gene organization and phylogenetic distribution. Notably, PMU abundance explains nearly 80% of the variance in phytoplasma genome sizes, a finding that provides a quantitative estimate for the importance of PMUs in phytoplasma genome variability. Finally, our investigation found that in addition to horizontal gene transfer, PMUs also contribute to intra-genomic duplications of effector genes, which may provide redundancy for neofunctionalization or subfunctionalization. Taken together, this work improves the taxon sampling for phytoplasma genome research and provides novel information regarding the roles of mobile genetic elements in phytoplasma evolution.

Insights Into the Molecular Evolution of AT-Hook Motif Nuclear Localization Genes in Brassica napus

AT-hook motif nuclear localization (AHL) proteins belong to a family of transcription factors, and play important roles in plant growth and development and response to various stresses through protein-DNA and protein-protein interactions. To better understand the Brassica napus AHL gene family, AHL genes in B. napus and related species were analyzed. Using Arabidopsis as a reference, 122 AHL gene family members were first identified in B. napus. According to the phylogenetic tree and gene organization, the BnaAHLs were classified into two clades (Clade-A and Clade-B) and three types (Type-I, Type-II, and Type-III). Gene organization and motif distribution analysis suggested that the AHL gene family is relatively conserved during evolution. These BnaAHLs are unevenly distributed on 38 chromosomes and expanded by whole-genome duplication (WGD) or segmental duplication. And large-scale loss events have also occurred in evolution. All types of BnaAHLs are subject to purification or neutral selection, while some positive selection sites are also identified in Type-II and Type-III groups. At the same time, the purification effect of Type-I members are stronger than that of the others. In addition, RNA-seq data and cis-acting element analysis also suggested that the BnaAHLs play important roles in B. napus growth and development, as well as in response to some abiotic and biotic stresses. Protein-protein interaction analysis identified some important BnaAHL-binding proteins, which also play key roles in plant growth and development. This study is helpful to fully understand the origin and evolution of the AHL gene in B. napus, and lays the foundation for their functional studies.

Characterization of promoters in archaeal genomes based on DNA structural parameters

The transcription machinery of archaea can be roughly classified as a simplified version of eukaryotic organisms. The basal transcription factor machinery binds to the TATA-box found around 28 nucleotides upstream of the transcription start site; however, some transcription units lack a clear TATA-box and still have TBP/TFB binding over them. This apparent absence of conserved sequences could be a consequence of sequence divergence associated with the upstream region, operonic and gene organization. Furthermore, earlier studies have found that a structural analysis gains more information compared to a simple sequence inspection. In this work, we evaluated and coded 3630 archaeal promoter sequences of three organisms, Haloferax volcanii, Thermococcus kodakarensis, and Sulfolobus solfataricus into DNA duplex stability, enthalpy, curvature, and bendability parameters. We also split our dataset into conserved TATA and degenerated TATA promoters in order to identify differences among these two classes of promoters. The structural analysis reveals variations in archaeal promoters’ architecture, i.e., a distinctive signal is observed in the TFB, TBP, and TFE binding sites independently of these being TATA-conserved or TATA-degenerated. In addition, the promoter encountering method was validated with upstream regions of 13 other archaea, suggesting that there might be promoter sequences among them. Therefore, we suggest a novel method for locating promoters within the genome of archaea based on energetic/structural features.

The mitochondrial genome of the grape powdery mildew pathogen Erysiphe necator is intron rich and exhibits a distinct gene organization

Powdery mildews are notorious fungal plant pathogens but only limited information exists on their genomes. Here we present the mitochondrial genome of the grape powdery mildew fungus Erysiphe necator and a high-quality mitochondrial gene annotation generated through cloning and Sanger sequencing of full-length cDNA clones. The E. necator mitochondrial genome consists of a circular DNA sequence of 188,577 bp that harbors a core set of 14 protein-coding genes that are typically present in fungal mitochondrial genomes, along with genes encoding the small and large ribosomal subunits, a ribosomal protein S3, and 25 mitochondrial-encoded transfer RNAs (mt-tRNAs). Interestingly, it also exhibits a distinct gene organization with atypical bicistronic-like expression of the nad4L/nad5 and atp6/nad3 gene pairs, and contains a large number of 70 introns, making it one of the richest in introns mitochondrial genomes among fungi. Sixty-four intronic ORFs were also found, most of which encoded homing endonucleases of the LAGLIDADG or GIY-YIG families. Further comparative analysis of five E. necator isolates revealed 203 polymorphic sites, but only five were located within exons of the core mitochondrial genes. These results provide insights into the organization of mitochondrial genomes of powdery mildews and represent valuable resources for population genetic and evolutionary studies.

The Conservation of Chloroplast Genome Structure and Improved Resolution of Infrafamilial Relationships of Crassulaceae

Crassulaceae are the largest family in the angiosperm order Saxifragales. Species of this family are characterized by succulent leaves and a unique photosynthetic pathway known as Crassulacean acid metabolism (CAM). Although the inter- and intrageneric relationships have been extensively studied over the last few decades, the infrafamilial relationships of Crassulaceae remain partially obscured. Here, we report nine newly sequenced chloroplast genomes, which comprise several key lineages of Crassulaceae. Our comparative analyses and positive selection analyses of Crassulaceae species indicate that the overall gene organization and function of the chloroplast genome are highly conserved across the family. No positively selected gene was statistically supported in Crassulaceae lineage using likelihood ratio test (LRT) based on branch-site models. Among the three subfamilies of Crassulaceae, our phylogenetic analyses of chloroplast protein-coding genes support Crassuloideae as sister to Kalanchoideae plus Sempervivoideae. Furthermore, within Sempervivoideae, our analyses unambiguously resolved five clades that are successively sister lineages, i.e., Telephium clade, Sempervivum clade, Aeonium clade, Leucosedum clade, and Acre clade. Overall, this study enhances our understanding of the infrafamilial relationships and the conservation of chloroplast genomes within Crassulaceae.

New Volvovirus Isolate From Domesticated House Cricket, Acheta Domesticus (Orthoptera: Gryllidae)

BackgroundDNA viruses have a broad variety of genetic material. Most viral DNA experiments focused on those with recognized pathogenic characteristics. Herein, we employed hybrid approach to systematically identify viral DNA from the Acheta domesticus genome and also curated primer library to reconfirm the infection of Acheta domesticus volvovirus (AdVVV) from A. domesticus samples obtained from breeding facility in Thailand. Methods and ResultsThe AdVVV nucleotide sequence was anchored and examined from genome sequence of A. domesticus. Subsequently, we sequenced the overlapping amplified DNA to assemble the whole genome of AdVVV isolate. The genome sequence began with the putative nonanucleotide origin of replication (1-TAGTATTAC), and had four open reading frames. The circular nature of AdVVV was confirmed as typical stemloop assembled by complementary initial and ending nucleotides sequences. The newly discovered volvovirus isolates from Thailand is also highly homologous (97.34 -98.77 %) with previously identified volvovirus sequences and having identical gene organization. This correlation is particularly surprising considering that the identified volvovirus considerably mutated compared to previously discovered volvovirus isolates. ConclusionsThis research appears to be the first to find volvovirus isolates from Thailand with an alternative approach to identify viral isolates. These results widen our knowledge on rapidly evolving volvovirus, and provide insights for better identification and the phylogenetic analysis based on different segments of viral genome. Also, the alternative method used in the present study will aid in the detection of DNA viruses and the assembly of their genomes.

Subtelomeric assembly of a multi-gene pathway for antimicrobial defense compounds in cereals

Non-random gene organization in eukaryotes plays a significant role in genome evolution. Here, we investigate the origin of a biosynthetic gene cluster for production of defence compounds in oat—the avenacin cluster. We elucidate the structure and organisation of this 12-gene cluster, characterise the last two missing pathway steps, and reconstitute the entire pathway in tobacco by transient expression. We show that the cluster has formed de novo since the divergence of oats in a subtelomeric region of the genome that lacks homology with other grasses, and that gene order is approximately colinear with the biosynthetic pathway. We speculate that the positioning of the late pathway genes furthest away from the telomere may mitigate against a ‘self-poisoning’ scenario in which toxic intermediates accumulate as a result of telomeric gene deletions. Our investigations reveal a striking example of adaptive evolution underpinned by remarkable genome plasticity.