Nuclease-free precise genome editing corrects MECP2 mutations associated with Rett syndrome.

Rett syndrome is an acquired progressive neurodevelopmental disorder caused by de novo mutations in the X-linked MECP2 gene which encodes a pleiotropic protein that functions as a global transcriptional regulator and a chromatin modifier. Rett syndrome predominantly affects heterozygous females while affected male hemizygotes rarely survive. Gene therapy of Rett syndrome has proven challenging due to a requirement for stringent regulation of expression with either over- or under-expression being toxic. Ectopic expression of MECP2 in conjunction with regulatory miRNA target sequences has achieved some success, but the durability of this approach remains unknown. Here we evaluated a nuclease-free homologous recombination (HR)-based genome editing strategy to correct mutations in the MECP2 gene. The stem cell-derived AAVHSCs have previously been shown to mediate seamless and precise HR-based genome editing. We tested the ability of HR-based genome editing to correct pathogenic mutations in Exons 3 and 4 of the MECP2 gene and restore the wild type sequence while preserving all native genomic regulatory elements associated with MECP2 expression, thus potentially addressing a significant issue in gene therapy for Rett syndrome. Moreover, since the mutations are edited directly at the level of the genome, the corrections are expected to be durable with progeny cells inheriting the edited gene. The AAVHSC MECP2 editing vector was designed to be fully homologous to the target MECP2 region and to insert a promoterless Venus reporter at the end of Exon 4. Evaluation of AAVHSC editing in a panel of Rett cell lines bearing mutations in Exons 3 and 4 demonstrated successful correction and rescue of expression of the edited MECP2 gene. Sequence analysis of edited Rett cells revealed successful and accurate correction of mutations in both Exons 3 and 4 and permitted mapping of HR crossover events. Successful correction was observed only when the mutations were flanked at both the 5' and 3' ends by crossover events, but not when both crossovers occurred either exclusively upstream or downstream of the mutation. Importantly, we concluded that pathogenic mutations were successfully corrected in every Rett line analyzed, demonstrating the therapeutic potential of HR-based genome editing.

Conversion of Polyethylenes into Fungal Secondary Metabolites.

Waste plastics represent major environmental and economic burdens due to their ubiquity, slow breakdown rates, and inadequacy of current recycling routes. Polyethylenes are particularly problematic, because they lack robust recycling approaches despite being the most abundant plastics in use today. We report a novel chemical and biological approach for the rapid conversion of polyethylenes into structurally complex and pharmacologically active compounds. We present conditions for aerobic, catalytic digestion of polyethylenes collected from post-consumer and oceanic waste streams, creating carboxylic diacids that can then be used as a carbon source by the fungus Aspergillus nidulans. As a proof of principle, we have engineered strains of A. nidulans to synthesize the fungal secondary metabolites asperbenzaldehyde, citreoviridin, and mutilin when grown on these digestion products. This hybrid approach considerably expands the range of products to which polyethylenes can be upcycled.

Fold-change of chromatin condensation in yeast is a conserved property.

During mitosis, chromatin is condensed and organized into mitotic chromosomes. Condensation is critical for genome stability and dynamics, yet the degree of condensation is significantly different between multicellular and single-cell eukaryotes. What is less clear is whether there is a minimum degree of chromosome condensation in unicellular eukaryotes. Here, we exploited two-photon microscopy to analyze chromatin condensation in live and fixed cells, enabling studies of some organisms that are not readily amenable to genetic modification. This includes the yeasts Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, and Candida albicans, as well as a protist Trypanosoma brucei. We found that mitotic chromosomes in this range of species are condensed about 1.5-fold relative to interphase chromatin. In addition, we used two-photon microscopy to reveal that chromatin reorganization in interphase human hepatoma cells infected by the hepatitis C virus is decondensed compared to uninfected cells, which correlates with the previously reported viral-induced changes in chromatin dynamics. This work demonstrates the power of two-photon microscopy to analyze chromatin in a broad range of cell types and conditions, including non-model single-cell eukaryotes. We suggest that similar condensation levels are an evolutionarily conserved property in unicellular eukaryotes and important for proper chromosome segregation. Furthermore, this provides new insights into the process of chromatin condensation during mitosis in unicellular organisms as well as the response of human cells to viral infection.

Genomic Characterization of the Titan-like Cell Producing Naganishia tulchinskyi, the First Novel Eukaryote Isolated from the International Space Station.

Multiple strains of a novel yeast belonging to genus Naganishia were isolated from environmental surfaces aboard the International Space Station (ISS). These strains exhibited a phenotype similar to Titan cell (~10 µm diameter) morphology when grown under a combination of simulated microgravity and 5% CO2 conditions. Confocal, scanning, and transmission electron microscopy revealed distinct morphological differences between the microgravity-grown cells and the standard Earth gravity-grown cells, including larger cells and thicker cell walls, altered intracellular morphology, modifications to extracellular fimbriae, budding, and the shedding of bud scars. Phylogenetic analyses via multi-locus sequence typing indicated that these ISS strains represented a single species in the genus Naganishia and were clustered with Naganishia diffluens. The name Naganishia tulchinskyi is proposed to accommodate these strains, with IF6SW-B1T as the holotype. The gene ontologies were assigned to the cell morphogenesis, microtubule-based response, and response to UV light, suggesting a variety of phenotypes that are well suited to respond to microgravity and radiation. Genomic analyses also indicated that the extracellular region, outer membrane, and cell wall were among the highest cellular component results, thus implying a set of genes associated with Titan-like cell plasticity. Finally, the highest molecular function matches included cytoskeletal motor activity, microtubule motor activity, and nuclear export signal receptor activity.

Genomic Characterization of Potential Plant Growth-Promoting Features of Sphingomonas Strains Isolated from the International Space Station.

In an ongoing microbial tracking investigation of the International Space Station (ISS), several Sphingomonas strains were isolated. Based on the 16S rRNA gene sequence, phylogenetic analysis identified the ISS strains as Sphingomonas sanguinis (n = 2) and one strain isolated from the Kennedy Space Center cleanroom (used to assemble various Mars mission spacecraft components) as Sphingomonas paucimobilis. Metagenomic sequence analyses of different ISS locations identified 23 Sphingomonas species. An abundance of shotgun metagenomic reads were detected for S. sanguinis in the location from where the ISS strains were isolated. A complete metagenome-assembled genome was generated from the shotgun reads metagenome, and its comparison with the whole-genome sequences (WGS) of the ISS S. sanguinis isolates revealed that they were highly similar. In addition to the phylogeny, the WGS of these Sphingomonas strains were compared with the WGS of the type strains to elucidate genes that can potentially aid in plant growth promotion. Furthermore, the WGS comparison of these strains with the well-characterized Sphingomonas sp. LK11, an arid desert strain, identified several genes responsible for the production of phytohormones and for stress tolerance. Production of one of the phytohormones, indole-3-acetic acid, was further confirmed in the ISS strains using liquid chromatography-mass spectrometry. Pathways associated with phosphate uptake, metabolism, and solubilization in soil were conserved across all the S. sanguinis and S. paucimobilis strains tested. Furthermore, genes thought to promote plant resistance to abiotic stress, including heat/cold shock response, heavy metal resistance, and oxidative and osmotic stress resistance, appear to be present in these space-related S. sanguinis and S. paucimobilis strains. Characterizing these biotechnologically important microorganisms found on the ISS and harnessing their key features will aid in the development of self-sustainable long-term space missions in the future. IMPORTANCESphingomonas is ubiquitous in nature, including the anthropogenically contaminated extreme environments. Members of the Sphingomonas genus have been identified as potential candidates for space biomining beyond earth. This study describes the isolation and identification of Sphingomonas members from the ISS, which are capable of producing the phytohormone indole-3-acetic acid. Microbial production of phytohormones will help future in situ studies, grow plants beyond low earth orbit, and establish self-sustainable life support systems. Beyond phytohormone production, stable genomic elements of abiotic stress resistance, heavy metal resistance, and oxidative and osmotic stress resistance were identified, rendering the ISS Sphingomonas isolate a strong candidate for biotechnology-related applications.

Identification of the pigment and its role in UV resistance in Paecilomyces variotii, a Chernobyl isolate, using genetic manipulation strategies.

Fungi produce secondary metabolites that are not directly involved in their growth, but often contribute to their adaptation to extreme environmental stimuli and enable their survival. Conidial pigment or melanin is one of the secondary metabolites produced naturally by a polyketide synthesis (PKS) gene cluster in several filamentous fungi and is known to protect these fungi from extreme radiation conditions. Several pigmented or melanized fungi have been shown to grow under extreme radiation conditions at the Chernobyl nuclear accident site. Some of these fungi, including Paecilomyces variotii, were observed to grow towards the source of radiation. Therefore, in this study, we wanted to identify if the pigment produced by P. variotii, contributes to providing protection against radiation condition. We first identified the PKS gene responsible for synthesis of pigment in P. variotii and confirmed its role in providing protection against UV irradiation through CRISPR-Cas9 mediated gene deletion. This is the first report that describes the use of CRISPR methodology to create gene deletions in P. variotii. Further, we showed that the pigment produced by this fungus, was not inhibited by DHN-melanin pathway inhibitors, indicating that the fungus does not produce melanin. We then identified the pigment synthesized by the PKS gene of P. variotii, as a naptho-pyrone Ywa1, by heterologously expressing the gene in Aspergillus nidulans. The results obtained will further aid in understanding the mechanistic basis of radiation resistance.

Advances in space microbiology.

Microbial research in space is being conducted for almost 50 years now. The closed system of the International Space Station (ISS) has acted as a microbial observatory for the past 10 years, conducting research on adaptation and survivability of microorganisms exposed to space conditions. This adaptation can be either beneficial or detrimental to crew members and spacecraft. Therefore, it becomes crucial to identify the impact of two primary stress conditions, namely, radiation and microgravity, on microbial life aboard the ISS. Elucidating the mechanistic basis of microbial adaptation to space conditions aids in the development of countermeasures against their potentially detrimental effects and allows us to harness their biotechnologically important properties. Several microbial processes have been studied, either in spaceflight or using devices that can simulate space conditions. However, at present, research is limited to only a few microorganisms, and extensive research on biotechnologically important microorganisms is required to make long-term space missions self-sustainable.

Methylobacterium ajmalii sp. nov., Isolated From the International Space Station.

Four strains belonging to the family of Methylobacteriaceae were isolated from different locations on the International Space Station (ISS) across two consecutive flights. Of these, three were identified as Gram-negative, rod-shaped, catalase-positive, oxidase-positive, motile bacteria, designated as IF7SW-B2T, IIF1SW-B5, and IIF4SW-B5, whereas the fourth was identified as Methylorubrum rhodesianum. The sequence similarity of these three ISS strains, designated as IF7SW-B2T, IIF1SW-B5, and IIF4SW-B5, was <99.4% for 16S rRNA genes and <97.3% for gyrB gene, with the closest being Methylobacterium indicum SE2.11T. Furthermore, the multi-locus sequence analysis placed these three ISS strains in the same clade of M. indicum. The average nucleotide identity (ANI) values of these three ISS strains were <93% and digital DNA-DNA hybridization (dDDH) values were <46.4% with any described Methylobacterium species. Based on the ANI and dDDH analyses, these three ISS strains were considered as novel species belonging to the genus Methylobacterium. The three ISS strains showed 100% ANI similarity and dDDH values with each other, indicating that these three ISS strains, isolated during various flights and from different locations, belong to the same species. These three ISS strains were found to grow optimally at temperatures from 25 to 30°C, pH 6.0 to 8.0, and NaCl 0 to 1%. Phenotypically, these three ISS strains resemble M. aquaticum and M. terrae since they assimilate similar sugars as sole carbon substrate when compared to other Methylobacterium species. Fatty acid analysis showed that the major fatty acid produced by the ISS strains are C18 : 1-ω7c and C18 : 1-ω6c. The predominant quinone was ubiquinone 10, and the major polar lipids were diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, and an unidentified lipid. Therefore, based on genomic, phylogenetic, biochemical, and fatty acid analyses, strains IF7SW-B2T, IIF1SW-B5, and IIF4SW-B5, are assigned to a novel species within the genus Methylobacterium, and the name Methylobacterium ajmalii sp. nov. is proposed. The type strain is IF7SW-B2T (NRRL B-65601T and LMG 32165T).

The Role of Recombinant AAV in Precise Genome Editing.

The replication-defective, non-pathogenic, nearly ubiquitous single-stranded adeno-associated viruses (AAVs) have gained importance since their discovery about 50 years ago. Their unique life cycle and virus-cell interactions have led to the development of recombinant AAVs as ideal genetic medicine tools that have evolved into effective commercialized gene therapies. A distinctive property of AAVs is their ability to edit the genome precisely. In contrast to all current genome editing platforms, AAV exclusively utilizes the high-fidelity homologous recombination (HR) pathway and does not require exogenous nucleases for prior cleavage of genomic DNA. Together, this leads to a highly precise editing outcome that preserves genomic integrity without incorporation of indel mutations or viral sequences at the target site while also obviating the possibility of off-target genotoxicity. The stem cell-derived AAV (AAVHSCs) were found to mediate precise and efficient HR with high on-target accuracy and at high efficiencies. AAVHSC editing occurs efficiently in post-mitotic cells and tissues in vivo. Additionally, AAV also has the advantage of an intrinsic delivery mechanism. Thus, this distinctive genome editing platform holds tremendous promise for the correction of disease-associated mutations without adding to the mutational burden. This review will focus on the unique properties of direct AAV-mediated genome editing and their potential mechanisms of action.

Draft Genome Sequences of Tremellomycetes Strains Isolated from the International Space Station.

The draft genome sequences of six eukaryotic microbial strains belonging to the class Tremellomycetes isolated from the International Space Station were assembled. Further characterization of these sequences will aid in the understanding of the influence of microgravity conditions on these organisms' potential pathogenicity.

Draft Genome Sequences of Sphingomonas Species Associated with the International Space Station.

The draft genome sequences of three Sphingomonas strains isolated from the International Space Station (ISS) were assembled. These genomic sequences will help in understanding the influence of microgravity conditions on their potential bioactive compound production and other important characteristics compared to their Earth counterparts.

Autonomously Replicating Linear Plasmids That Facilitate the Analysis of Replication Origin Function in Candida albicans.

The ability to generate autonomously replicating plasmids has been elusive in Candida albicans, a prevalent human fungal commensal and pathogen. Instead, plasmids generally integrate into the genome. Here, we assessed plasmid and transformant properties, including plasmid geometry, transformant colony size, four selectable markers, and potential origins of replication, for their ability to drive autonomous plasmid maintenance. Importantly, linear plasmids with terminal telomere repeats yielded many more autonomous transformants than circular plasmids with the identical sequences. Furthermore, we could distinguish (by colony size) transient, autonomously replicating, and chromosomally integrated transformants (tiny, medium, and large, respectively). Candida albicansURA3 and a heterologous marker, ARG4, yielded many transient transformants indicative of weak origin activity; the replication of the plasmid carrying the heterologous LEU2 marker was highly dependent upon the addition of a bona fide origin sequence. Several bona fide chromosomal origins, with an origin fragment of ∼100 bp as well as a heterologous origin, panARS, from Kluyveromyces lactis, drove autonomous replication, yielding moderate transformation efficiency and plasmid stability. Thus, C. albicans maintains linear plasmids that yield high transformation efficiency and are maintained autonomously in an origin-dependent manner.IMPORTANCE Circular plasmids are important tools for molecular manipulation in model fungi such as baker's yeast, yet, in Candida albicans, an important yeast pathogen of humans, prior studies were not able to generate circular plasmids that were autonomous (duplicated without inserting themselves into the chromosome). Here, we found that linearizing circular plasmids with sequences from telomeres, the chromosome ends, allows the plasmids to duplicate and segregate in C. albicans We used this system to identify chromosomal sequences that facilitate the initiation of plasmid replication (origins) and to show that an ∼100-bp fragment of a C. albicans origin and an origin sequence from a distantly related yeast can both function as origins in C. albicans Thus, the requirements for plasmid geometry, but not necessarily for origin sequences, differ between C. albicans and baker's yeast.

Sophorolipid exhibits antifungal activity by ROS mediated endoplasmic reticulum stress and mitochondrial dysfunction pathways in Candida albicans.

In the present study, we investigated the mechanism of cell death in C. albicans due to treatment with sophorolipid (SL). SL is an extracellular glycolipid biosurfactant produced by various species of non-pathogenic yeasts and is known to inhibit the growth and biofilm formation of C. albicans. This study revealed that treatment of C. albicans cells with SL increases the ROS production and expression of oxidative stress-related genes significantly (SOD1, CAT1). Increased ROS level within the cells causes ER stress and release of Ca2+ in the cytoplasm and alteration of the mitochondrial membrane potential (MMP). Quantitative real time-polymerase chain reaction (qRT-PCR) data showed that SL also upregulates the Endoplasmic Reticulum (ER) stress marker HAC1. Flow cytometric analysis (AnnexinV/PI) indicated that the cell death may have occurred due to necrosis which was further confirmed by LDH release assay and transmission electron microscopy (TEM). Further experiments with the null mutant Δ hog1 strain of C. albicans SC5314 indicated the activation of the osmotic stress response pathway (HOG-MAPK) and SAP9. This study gave an insight into the mechanism of cell death initiation by glycolipids and indicated that further modification of these molecules can lead to the development of new therapeutic agent against C. albicans.

Improved Tet-On and Tet-Off systems for tetracycline-regulated expression of genes in Candida.

Tetracycline-regulated expression of genes is often used for functional analysis of Candida albicans genes. However, the widely used Tet-On system has certain limitations such as prolonged lag time (up to 8 h) for induction and non-uniform expression among the cells. We speculated that poor expression of tetracycline-controlled transactivator (Tet-transactivator) from CaADH1 promoter could be responsible for this, and thus compared the effect of expressing this protein under the control of CaADH1, CaTDH3 and CaRP10 promoters on the expression of GFP from the TET promoter. Only CaRP10 promoter facilitated a more uniform and rapid induction of GFP. However, a high concentration of doxycycline was needed for induction, which is not desirable for assessing certain phenotypes. Tet-Off systems are known to require a low concentration of doxycycline, but a limitation of the widely used Tet-Off system for C. albicans is the use of CaENO1 promoter, which is known to be repressed in the presence of gluconeogenic carbon source, for expression of transactivator. Thus, we have converted the above-mentioned Tet-On systems to Tet-Off systems by site-directed mutagenesis of the Tet-transactivator. Compared to the Tet-On systems, the Tet-Off systems required about 200-fold less concentration of doxycycline for modulation of gene expression. Only the Tet-Off system with CaRP10 promoter driving the expression of transactivator allowed rapid and high level expression of GFP compared to those with CaADH1 or CaTDH3 promoters. The utility of CaRP10 based Tet-On and Tet-Off systems was further validated by the conditional expression of the CaTUP1 gene. We have also adapted these systems for use with Candida tropicalis and find that the Tet-Off system is functional in this species. The Tet systems reported here will be useful for conditional expression of genes in C. albicans as well as C. tropicalis.

Critical role for CaFEN1 and CaFEN12 of Candida albicans in cell wall integrity and biofilm formation.

Sphingolipids are involved in several cellular functions, including maintenance of cell wall integrity. To gain insight into the role of individual genes of sphingolipid biosynthetic pathway, we have screened Saccharomyces cerevisiae strains deleted in these genes for sensitivity to cell wall perturbing agents calcofluor white and congo red. Only deletants of FEN1 and SUR4 genes were found to be sensitive to both these agents. Candida albicans strains deleted in their orthologs, CaFEN1 and CaFEN12, respectively, also showed comparable phenotypes, and a strain deleted for both these genes was extremely sensitive to cell wall perturbing agents. Deletion of these genes was reported earlier to sensitise cells to amphotericin B (AmB), which is a polyene drug that kills the cells mainly by binding and sequestering ergosterol from the plasma membrane. Here we show that their AmB sensitivity is likely due to their cell wall defect. Further, we show that double deletant of C. albicans is defective in hyphae formation as well as biofilm development. Together this study reveals that deletion of FEN1 and SUR4 orthologs of C. albicans leads to impaired cell wall integrity and biofilm formation, which in turn sensitise cells to AmB.