Penicillium Arizonense as a Novel Producer Strain for Mycophenolic Acid and Expression Analysis of Biosynthesizing Gene Clusters

A novel potent mycophenolic acid (MPA) producer strain of the genus Penicillium was isolated from refrigerated Mozzarella cheese and identified as P. arizonenseHEWt1. The molecular mechanism of MPA production by this new isolate was our main target. To achieve this objective, we first isolated three MPA overproducer mutants by exposing the wild type to different doses of gamma-rays, and the fermentation conditions for the highest production of MPA by both the wild type and mutants were optimized. Then, orthologs of MPA gene clusters in P. brevicompactum were cloned and predicted from the genome of P. arizonense. Sequencing and bioinformatic analysis proved the presence of a cluster containing five putative genes in the P. arizonense HEWt1 genome ortholog to the MPA cluster, mpaA, mpaC, mpaF, mpaG, and mpaH. All predicted genes displayed 96-97% similarity with the related hypothetical protein of P. arizonense. The genes, mpaG, mpaC, and mpaF. represented 69%, 82%, 84%, respectively, similarity with their orthologous genes in P. brevicompactum, whereas mpaG and mpaA represented 75% and 79%, respectively, similarity to their orthologous genes in P. roqueforti. Gene expression analysis through quantitative rPCR indicated an increase in the transcription value of all annotated genes in the three mutants over the wild type. A highly significant increase in the gene expression of mpaC, mpaF, and mpaH was observed, with 8.4561±1.02, 5.6569±0.87, and 4.6268±0.18-fold increases, respectively, in P. arizonense-MT1 compared with wild-type. These results confirmed the potential participation of these genes in MPA biosynthesis and are the first report regarding the molecular mechanism of MPA production by P. arizonense.

NetREx – Network-based Rice Expression Analysis Server for Abiotic Stress Conditions

Recent focus on transcriptomic studies in food crops like rice, wheat and maize provide new opportunities to address issues related to agriculture and climate change. Re-analysis of such data available in public domain supplemented with annotations across molecular hierarchy can be of immense help to the plant research community, particularly co-expression networks representing transcriptionally coordinated genes that are often part of the same biological process. With this objective we have developed NetREx, a Network based Rice Expression Analysis Server, that hosts ranked co-expression networks of Oryza sativa using publicly available mRNA-seq data across uniform experimental conditions. It provides a range of interactable data viewers and modules for analysing user queried genes across different stress conditions (drought, flood, cold and osmosis) and hormonal treatments (abscisic and jasmonic acid) and tissues (root and shoot). Subnetworks of user-defined genes can be queried in preconstructed tissue-specific networks, allowing users to view the fold-change, module memberships, gene annotations and analysis of their neighborhood genes and associated pathways. The webserver also allows querying of orthologous genes from Arabidopsis, wheat, maize, barley, and sorghum. Here we demonstrate that NetREx can be used to identify novel candidate genes and tissue-specific interactions under stress conditions and can aid in the analysis and understanding of complex phenotypes linked to stress response in rice. Available at: https://bioinf.iiit.ac.in/netrex/index.html

Identification of upstream transcription factor binding sites in orthologous genes using mixed Student's t-test statistics

Background: Transcription factor (TF) regulates the transcription of DNA to messenger RNA by binding to upstream sequence motifs. Identifying the locations of known motifs in whole genomes is computationally intensive. Methodology/Principal Findings: This study presents a computational tool, named "Grit", for screening TF-binding sites (TFBS) by coordinating transcription factors to their promoter sequences in orthologous genes. This tool employs a newly developed mixed Student's t-test statistical method that detects high-scoring conserved and non-conserved binding sites among species. The program performs sequence scanning at a rate of 3.2 Mb/s on a quad-core Amazon server and has been benchmarked by the well-established ChIP-Seq datasets, putting Grit amongst the top-ranked TFBS predictors. It marginally outperforms the well-known transcription factor motif scanning tools, Pscan (4.8%) and FIMO (17.8%), in analyzing well-documented ChIP-Atlas human genome Chip-Seq datasets. Significance: Grit is a good alternative to current available motif scanning tools and is publicly available at http://www.thua45.cn/grit under an academic free license.

BOPAL 2.0 and a study of tRNA and rRNA gene evolution in Clostridium

We present BOPAL 2.0, an improved version of the BOPAL algorithm for the evolutionary history inference of tRNA and rRNA genes in bacterial genomes. Our approach can infer complete evolutionary scenarios and ancestral gene orders on a phylogeny and considers a wide range of events such as duplications, deletions, substitutions, inversions and transpositions. It is based on the fact that tRNA and rRNA genes are often organized in operons/clusters in bacteria, and this information is used to help identify orthologous genes for each genome comparison. BOPAL 2.0 introduces new features, such as a triple-wise alignment step, context-aware singleton matching and a second pass of the algorithm. Evaluation on simulated datasets shows that BOPAL 2.0 outperforms the original BOPAL in terms of the accuracy of inferred events and ancestral genomes. We also present a study of the tRNA/rRNA gene evolution in the Clostridium genus, in which the organization of these genes is very divergent. Our results indicate that tRNA and rRNA genes in Clostridium have evolved through numerous duplications, losses, transpositions and substitutions, but very few inversions were inferred.

Extensive sequence divergence of non-coding regions between Aspergillus fumigatus, a major fungal pathogen of humans, and its relatives

Invasive aspergillosis is a deadly fungal disease; more than 400,000 patients are infected worldwide each year and the mortality rate can be as high as 50-95%. Of the ~450 species in the genus Aspergillus only a few are known to be clinically relevant, with the major pathogen Aspergillus fumigatus being responsible for ~50% of all invasive mold infections. Genomic comparisons of A. fumigatu to other Aspergillus species have historically focused on protein-coding regions. However, most A. fumigatus genes, including those that modulate its virulence, are also present in non-pathogenic close relatives of A. fumigatus. Our hypothesis is that differential gene regulation - mediated through the non-coding regions upstream of genes' transcription start sites - contributes to A. fumigatus pathogenicity. To begin testing this, we compared non-coding regions up to 500 base pairs upstream of the first codon of single-copy orthologous genes from the two A. fumigatus reference strains Af293 and A1163 and eight closely related Aspergillus section Fumigati species. We found that non-coding regions showed extensive sequence variation and lack of homology across species. By examining the evolutionary rates of both protein-coding and non-coding regions in a subset of orthologous genes with highly conserved non-coding regions across the phylogeny, we identified 418 genes, including 25 genes known to modulate A. fumigatus virulence, whose non-coding regions exhibit a different rate of evolution in A. fumigatus. Examination of sequence alignments of these non-coding regions revealed numerous instances of insertions, deletions, and other types of mutations of at least a few nucleotides in A. fumigatus compared to its close relatives. These results show that closely related Aspergillus species that vary greatly in their pathogenicity exhibit extensive non-coding sequence variation and identify numerous changes in non-coding regions of A. fumigatus genes known to contribute to virulence.

Phylogenomics of Haloarchaea: The Controversy of the Genera Natrinema-Haloterrigena

The haloarchaeal genera Natrinema and Haloterrigena were described almost simultaneously by two different research groups and some strains studied separately were described as different species of these genera. Furthermore, the description of additional species were assigned to either Natrinema or Haloterrigena, mainly on the basis of the phylogenetic comparative analysis of single genes (16S rRNA gene and more recently rpoB’ gene), but these species were not adequately separated or assigned to the corresponding genus. Some studies suggested that the species of these two genera should be unified into a single genus, while other studies indicated that the genera should remain but some of the species should be reassigned. In this study, we have sequenced or collected the genomes of the type strains of species of Natrinema and Haloterrigena and we have carried out a comparative genomic analysis in order to clarify the controversy related to these two genera. The phylogenomic analysis based on the comparison of 525 translated single-copy orthologous genes and the Overall Genome Relatedness Indexes (i.e., AAI, POCP, ANI, and dDDH) clearly indicate that the species Haloterrigena hispanica, Haloterrigena limicola, Haloterrigena longa, Haloterrigena mahii, Haloterrigena saccharevitans, Haloterrigena thermotolerans, and Halopiger salifodinae should be transferred to the genus Natrinema, as Natrinema hispanicum, Natrinema limicola, Natrinema longum, Natrinema mahii, Natrinema saccharevitans, Natrinema thermotolerans, and Natrinema salifodinae, respectively. On the contrary, the species Haloterrigena turkmenica, Haloterrigena salifodinae, and Haloterrigena salina will remain as the only representative species of the genus Haloterrigena. Besides, the species Haloterrigena daqingensis should be reclassified as a member of the genus Natronorubrum, as Natronorubrum daqingense. At the species level, Haloterrigena jeotgali and Natrinema ejinorense should be considered as a later heterotypic synonyms of the species Haloterrigena (Natrinema) thermotolerans and Haloterrigena (Natrinema) longa, respectively. Synteny analysis and phenotypic features also supported those proposals.

The First Whole Genome Sequencing of Sanghuangporus sanghuang Provides Insights into Its Medicinal Application and Evolution

Sanghuangporus is a medicinal macrofungal genus typified by S. sanghuang, the very species utilized in traditional Chinese medicines by Chinese ancient people. To facilitate the medicinal application of S. sanghuang, we, for the first time, perform its genome sequencing and analyses from a monokaryon strain. A 33.34 Mb genome sequence was assembled to 26 contigs, which lead to the prediction of 8278 protein-coding genes. From these genes, the potential biosynthesis pathway of sesquiterpenoids was, for the first time, identified from Sanghuangporus, besides that of triterpenoids. While polysaccharides are the main medicinal metabolites in S. sanghuang, flavonoids are especially abundant medicinal metabolites comparing with other medicinal macrofungal groups. From the genomic perspective, S. sanghuang has a tetrapolar heterothallic mating system, and has its special nutritional strategy and advantageous medicinal properties compared with S. baumii and S. vaninii. A phylogenomics analysis indicates that Sanghuangporus emerged 15.39 million years ago and S. sanghuang has a closer phylogenetic relationship with S. baumii than S. vaninii. However, S. sanghuang shares a higher region of synteny and more orthologous genes, including carbohydrate-active enzymes with S. vaninii than S. baumii. A comparative genomics analysis with S. baumii and S. vaninii indicates that species diversification within Sanghuangporus may be driven by the translocation and translocation plus inversion of genome sequences, while the expansion and contraction of gene families may contribute to the host specificity of Sanghuangporus species. In general, the genome sequence of S. sanghuang provides insights into its medicinal application and evolution.

Circular RNA repertoires are associated with evolutionarily young transposable elements

Circular RNAs (circRNAs) are found across eukaryotes and can function in post-transcriptional gene regulation. Their biogenesis through a circle-forming backsplicing reaction is facilitated by reverse-complementary repetitive sequences promoting pre-mRNA folding. Orthologous genes from which circRNAs arise, overall contain more strongly conserved splice sites and exons than other genes, yet it remains unclear to what extent this conservation reflects purifying selection acting on the circRNAs themselves. Our analyses of circRNA repertoires from five species representing three mammalian lineages (marsupials, eutherians: rodents, primates) reveal that surprisingly few circRNAs arise from orthologous exonic loci across all species. Even the circRNAs from orthologous loci are associated with young, recently active and species-specific transposable elements, rather than with common, ancient transposon integration events. These observations suggest that many circRNAs emerged convergently during evolution - as a byproduct of splicing in orthologs prone to transposon insertion. Overall, our findings argue against widespread functional circRNA conservation.

Functional Activity of Recombinant Forms of Amh and Synergistic Action with Fsh in European Sea Bass Ovary

Although anti-Müllerian hormone (AMH) has classically been correlated with the regression of Müllerian ducts in male mammals, involvement of this growth factor in other reproductive processes only recently come to light. Teleost is the only gnathostomes that lack Müllerian ducts despite having amh orthologous genes. In adult teleost gonads, Amh exerts a role in the early stages of germ cell development in both males and females. Mechanisms involving the interaction of Amh with gonadotropin- and growth factor-induced functions have been proposed, but our overall knowledge regarding Amh function in fish gonads remains modest. In this study, we report on Amh actions in the European sea bass ovary. Amh and type 2 Amh receptor (Amhr2) are present in granulosa and theca cells of both early and late-vitellogenic follicles and cannot be detected in previtellogenic ovaries. Using the Pichia pastoris system a recombinant sea bass Amh has been produced that is endogenously processed to generate a 12–15 kDa bioactive mature protein. Contrary to previous evidence in lower vertebrates, in explants of previtellogenic sea bass ovaries, mature Amh has a synergistic effect on steroidogenesis induced by the follicle-stimulating hormone (Fsh), increasing E2 and cyp19a1a levels.

Myxospermy Evolution in Brassicaceae: A Highly Complex and Diverse Trait with Arabidopsis as an Uncommon Model

The ability to extrude mucilage upon seed imbibition (myxospermy) occurs in several Angiosperm taxonomic groups, but its ancestral nature or evolutionary convergence origin remains misunderstood. We investigated seed mucilage evolution in the Brassicaceae family with comparison to the knowledge accumulated in Arabidopsis thaliana. The myxospermy occurrence was evaluated in 27 Brassicaceae species. Phenotyping included mucilage secretory cell morphology and topochemistry to highlight subtle myxospermy traits. In parallel, computational biology was driven on the one hundred genes constituting the so-called A. thaliana mucilage secretory cell toolbox to confront their sequence conservation to the observed phenotypes. Mucilage secretory cells show high morphology diversity; the three studied Arabidopsis species had a specific extrusion modality compared to the other studied Brassicaceae species. Orthologous genes from the A. thaliana mucilage secretory cell toolbox were mostly found in all studied species without correlation with the occurrence of myxospermy or even more sub-cellular traits. Seed mucilage may be an ancestral feature of the Brassicaceae family. It consists of highly diverse subtle traits, probably underlined by several genes not yet characterized in A. thaliana or by species-specific genes. Therefore, A. thaliana is probably not a sufficient reference for future myxospermy evo–devo studies.