ABSTRACT
The adaptation of gene deletion methods based on the CRISPR-Cas9 system has facilitated the genetic manipulation of the pathogenic yeast Candida albicans, because homozygous mutants of this diploid fungus can now be generated in a single step, allowing the rapid screening of candidate genes for their involvement in a phenotype of interest. However, the Cas9-mediated double-strand breaks at the target site may result in an undesired loss of heterozygosity (LOH) on the affected chromosome and cause phenotypic alterations that are not related to the function of the investigated gene. In our present study, we harnessed Cas9-facilitated gene deletion to probe a set of genes that are constitutively overexpressed in strains containing hyperactive forms of the transcription factor Mrr1 for a possible contribution to the fluconazole resistance of such strains. To this aim, we used gene deletion cassettes containing two different dominant selection markers, caSAT1 and HygB, which confer resistance to nourseothricin and hygromycin, respectively, for simultaneous genomic integration in a single step, hypothesizing that this would minimize undesired LOH events at the target locus. We found that selection for resistance to both nourseothricin and hygromycin strongly increased the proportion of homozygous deletion mutants among the transformants compared with selection on media containing only one of the antibiotics, but it did not avoid undesired LOH events. Our results demonstrate that LOH on the target chromosome is a significant problem when using Cas9 for the generation of C. albicans gene deletion mutants, which demands a thorough examination of recombination events at the target site. IMPORTANCE: Candida albicans is one of the medically most important fungi and a model organism to study fungal pathogenicity. Investigating gene function in this diploid yeast has been facilitated by the adaptation of gene deletion methods based on the bacterial CRISPR-Cas9 system, because they enable the generation of homozygous mutants in a single step. We found that, in addition to increasing the efficiency of gene replacement by selection markers, the Cas9-mediated double-strand breaks also result in frequent loss of heterozygosity on the same chromosome, even when two different selection markers were independently integrated into the two alleles of the target gene. Since loss of heterozygosity for other genes can result in phenotypic alterations that are not caused by the absence of the target gene, these findings show that it is important to thoroughly analyze recombination events at the target locus when using Cas9 to generate gene deletion mutants in C. albicans.
Subject(s)
CRISPR-Cas Systems , Candida albicans , Loss of Heterozygosity , Recombination, Genetic , Candida albicans/genetics , Candida albicans/drug effects , Gene Deletion , Drug Resistance, Fungal/genetics , Antifungal Agents/pharmacology , Fluconazole/pharmacology , Hygromycin B/pharmacology , CRISPR-Associated Protein 9/genetics , Gene Editing/methods , Streptothricins/pharmacology , Genetic MarkersABSTRACT
Functional gene and protein characterizations in parasitic protists are often limited by their genetic tractability. Despite the development of CRISPR-Cas9-derived or inspired approaches for a handful of protist parasites, the overall genetic tractability of these organisms remains limited. The intestinal parasite Giardia lamblia is one such species, with the added challenge of a paucity of reliable selection markers. To address this limitation, we tested the feasibility of using Nourseothricin as an effective selection agent in Giardia. Here, we report that axenically-grown WB Giardia cells are sensitive to Nourseothricin and that engineering expression of the streptothricin acetyltransferase (SAT-1) gene from Streptomyces rochei in transgenic parasites confers resistance to this antibiotic. Furthermore, we determine that SAT-1-expressing parasites are cross-resistant neither to Neomycin nor Puromycin, which are widely used to select for transgenic parasites. Consequently, we show that Nourseothricin can be used in sequential combination with both Neomycin and Puromycin to select for dual transfection events. This work increases the number of reliable selection agents and markers for Giardia genetic manipulation, expanding the limited molecular toolbox for this species of global medical importance.
Subject(s)
Giardia lamblia , Streptothricins , Giardia lamblia/genetics , Giardia lamblia/drug effects , Streptothricins/pharmacology , Acetyltransferases/genetics , Drug Resistance/genetics , Streptomyces/genetics , Streptomyces/drug effects , Antiprotozoal Agents/pharmacology , Organisms, Genetically Modified , CRISPR-Cas SystemsABSTRACT
Pseudomonas syringae pv. actinidiae (Psa) is a significant pathogenic bacterium affecting the kiwifruit industry. This study investigated the target sites of streptothricin-F (ST-F), produced by Streptomyces lavendulae gCLA4. The inhibition of ST-F on Psa was examined by the microscopic structural differences of Psa before and after treatment with ST-F, as well as the interaction between ST-F and cell division-related proteins. The results revealed filamentation of Psa after ST-F treatment, and fluorescence microscopy showed that ST-F inhibited the formation of the Z-ring composed of FtsZ protein. In vitro experiments and molecular docking demonstrated that ST-F can bind to FtsZ with a binding energy of 0.4 µM and inhibit FtsZ's GTP-dependent polymerization reaction. In addition, ST-F does not exert inhibitory effects on cell division in Psa strains overexpressing ftsZ. In conclusion, FtsZ is one of the target sites for ST-F inhibition of Psa, highlighting its potential as a therapeutic target for controlling Psa-induced kiwifruit bacterial canker.
Subject(s)
Actinidia , Streptothricins , Streptothricins/pharmacology , Pseudomonas syringae , Molecular Docking Simulation , Plant Diseases/microbiology , Actinidia/microbiologyABSTRACT
Nitzschia is one of the largest genera of diatoms found in a range of aquatic environments, from freshwater to seawater. This genus contains evolutionarily and ecologically unique species, such as those that have lost photosynthetic capacity or those that live symbiotically in dinoflagellates. Several Nitzschia species have been used as indicators of water pollution. Recently, Nitzschia species have attracted considerable attention in the field of biotechnology. In this study, a transformation method for the marine pennate diatom Nitzschia sp. strain NIES-4635, isolated from the coastal Seto Inland Sea, was established. Plasmids containing the promoter/terminator of the fucoxanthin chlorophyll a/c binding protein gene (fcp, or Lhcf) derived from Nitzschia palea were constructed and introduced into cells by multi-pulse electroporation, resulting in 500 µg/mL nourseothricin-resistant transformants with transformation frequencies of up to 365 colonies per 108 cells. In addition, when transformation was performed using a new plasmid containing a promoter derived from a diatom-infecting virus upstream of the green fluorescent protein gene (gfp), 44% of the nourseothricin-resistant clones exhibited GFP fluorescence. The integration of the genes introduced into the genomes of the transformants was confirmed by Southern blotting. The Nitzschia transformation method established in this study will enable the transformation this species, thus allowing the functional analysis of genes from the genus Nitzschia, which are important species for environmental and biotechnological development.
Subject(s)
Diatoms , Streptothricins , Diatoms/genetics , Diatoms/metabolism , Streptothricins/metabolism , Chlorophyll A/metabolism , Electroporation/methods , Plasmids/geneticsABSTRACT
The streptothricin natural product mixture (also known as nourseothricin) was discovered in the early 1940s, generating intense initial interest because of excellent gram-negative activity. Here, we establish the activity spectrum of nourseothricin and its main components, streptothricin F (S-F, 1 lysine) and streptothricin D (S-D, 3 lysines), purified to homogeneity, against highly drug-resistant, carbapenem-resistant Enterobacterales (CRE) and Acinetobacter baumannii. For CRE, the MIC50 and MIC90 for S-F and S-D were 2 and 4 µM, and 0.25 and 0.5 µM, respectively. S-F and nourseothricin showed rapid, bactericidal activity. S-F and S-D both showed approximately 40-fold greater selectivity for prokaryotic than eukaryotic ribosomes in in vitro translation assays. In vivo, delayed renal toxicity occurred at >10-fold higher doses of S-F compared with S-D. Substantial treatment effect of S-F in the murine thigh model was observed against the otherwise pandrug-resistant, NDM-1-expressing Klebsiella pneumoniae Nevada strain with minimal or no toxicity. Cryo-EM characterization of S-F bound to the A. baumannii 70S ribosome defines extensive hydrogen bonding of the S-F steptolidine moiety, as a guanine mimetic, to the 16S rRNA C1054 nucleobase (Escherichia coli numbering) in helix 34, and the carbamoylated gulosamine moiety of S-F with A1196, explaining the high-level resistance conferred by corresponding mutations at the residues identified in single rrn operon E. coli. Structural analysis suggests that S-F probes the A-decoding site, which potentially may account for its miscoding activity. Based on unique and promising activity, we suggest that the streptothricin scaffold deserves further preclinical exploration as a potential therapeutic for drug-resistant, gram-negative pathogens.
Subject(s)
Anti-Bacterial Agents , Streptothricins , Animals , Mice , Anti-Bacterial Agents/pharmacology , Streptothricins/chemistry , Streptothricins/pharmacology , Escherichia coli/genetics , RNA, Ribosomal, 16S/genetics , Gram-Negative Bacteria , Carbapenems/pharmacology , Ribosomes , Microbial Sensitivity TestsABSTRACT
Particulate-bound mercury (PBM) is a global environmental concern owing to its large dry deposition velocities and scavenging coefficients, both of which drive Hg into terrestrial and marine ecosystems. PBM observation studies have been widely conducted over East Asia, but comparable studies in Peninsular Southeast Asia (PSEA) remain scarce. This is the first study reporting PBM concentrations for Ho Chi Minh City (HCMC), the biggest metropolitan area in Vietnam. A total of 222 samples were collected in 2018 and contained an average PBM10 (particulate matter - PM with diameter ≤10 µm) concentration and Hg mass fraction (i.e. PBM/PM) of 67.3 ± 45.9 pg m-3 and 1.18 ± 1.12 µg g-1, respectively. Although PBM concentration was lower than those reported in Chinese megacities, the Hg mass fraction was similar to those in China, suggesting strong enrichment from anthropogenic Hg emissions in HCMC. Traffic-induced particulate emission and deposition processes were major factors governing PBM temporal variation at our site. In addition, the prevailing southwest monsoon winds brought air masses that passed through industrial areas and were associated with a higher Hg mass fraction. Statistically significant positive correlations (R2 = 0.11-0.52, p < 0.01) were observed for PBM with PM and the Hg mass fraction, indicating similar PM and Hg sources or oxidized Hg adsorption onto PM via gas-particle partitioning. Moreover, PCA results revealed a higher contribution of primary sources than secondary sources to PBM concentration variability in HCMC. A health risk assessment indicated that the PBM concentrations at HCMC posed minimal non-carcinogenic risks (HI < 1) for children and adults, but dermal contact may act as an important exposure route since lightweight clothing is common among residents. This PBM dataset over PSEA, a region with high atmospheric Hg emissions, provides a valuable resource for the Hg scientific community to improve our understanding of Hg biogeochemical cycle.
Subject(s)
Air Pollutants , Mercury , Air Pollutants/analysis , Asia, Southeastern , Child , Dust/analysis , Ecosystem , Environmental Monitoring/methods , Humans , Mercury/analysis , Particulate Matter/analysis , Risk Assessment , StreptothricinsABSTRACT
This brief commentary aims to provide an overview of the available and relatively new precision management of reward deficiencies manifested as substance and behavioral disorders. Current and future advances, concepts, and the substantial evidential basis of this potential therapeutic and prophylactic treatment modality are presented. Precision Behavioral Management (PBM), conceptualized initially as Precision Addiction Management (PAM), certainly deserves consideration as an important modality for the treatment of impaired cognitive control in reward processing as manifested in people with neurobiologically expressed Reward Deficiency Syndrome (RDS).
Subject(s)
Behavior, Addictive , Reward , Behavior, Addictive/prevention & control , Behavior, Addictive/psychology , Cognition , Humans , Streptothricins , SyndromeABSTRACT
Recently, microorganisms and their metabolites in the Antarctic marine environment have attracted attention as useful sources for novel therapeutics, including anticancer drugs. Here, we investigated the effects of citromycin, isolated from the Antarctic marine-derived fungus, Sporothrix sp., on human ovarian cancer cells. Citromycin inhibited the migration and invasion of human ovarian cancer SKOV3 and A2780 cells, but had no cytotoxic activity against them. Additionally, it inhibited the expression of epithelial-mesenchymal transition (EMT) markers and the activation of matrix metalloproteinase (MMP)-2 and MMP9. Moreover, extracellular signal-regulated kinase (ERK)-1/2 signaling was inhibited after citromycin treatment, and the ectopic expression of ERK negated the anti-invasive activity of citromycin. Our findings suggest that citromycin inhibits the migration and invasion of human ovarian cancer cells by downregulating the expression levels of EMT markers and MMP-2/9 via inhibition of the ERK1/2 pathway.
Subject(s)
Ovarian Neoplasms , Sporothrix , Antarctic Regions , Cell Line, Tumor , Cell Movement , Female , Fungi , Humans , Neoplasm Invasiveness/prevention & control , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/metabolism , StreptothricinsABSTRACT
Whole-genome (WG) transformation (WGT) with DNA from the same or another species has been used to obtain strains with superior traits. Very few examples have been reported in eukaryotes-most apparently involving integration of large fragments of foreign DNA into the host genome. We show that WGT of a haploid acetic acid-sensitive Saccharomyces cerevisiae strain with DNA from a tolerant strain, but not from nontolerant strains, generated many tolerant transformants, some of which were stable upon subculturing under nonselective conditions. The most tolerant stable transformant contained no foreign DNA but only seven nonsynonymous single nucleotide polymorphisms (SNPs), of which none was present in the donor genome. The SNF4 mutation c.[805GâT], generating Snf4E269*, was the main causative SNP. Allele exchange of SNF4E269* or snf4Δ in industrial strains with unrelated genetic backgrounds enhanced acetic acid tolerance during fermentation under industrially relevant conditions. Our work reveals a surprisingly small number of mutations introduced by WGT, which do not bear any sequence relatedness to the genomic DNA (gDNA) of the donor organism, including the causative mutation. Spontaneous mutagenesis under protection of a transient donor gDNA fragment, maintained as extrachromosomal circular DNA (eccDNA), might provide an explanation. Support for this mechanism was obtained by transformation with genomic DNA of a yeast strain containing NatMX and selection on medium with nourseothricin. Seven transformants were obtained that gradually lost their nourseothricin resistance upon subculturing in nonselective medium. Our work shows that WGT is an efficient strategy for rapidly generating and identifying superior alleles capable of improving selectable traits of interest in industrial yeast strains.
Subject(s)
Saccharomyces cerevisiae Proteins , Streptothricins , AMP-Activated Protein Kinases/genetics , Acetic Acid , Alleles , Mutation/genetics , Polymorphism, Single Nucleotide/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Transcription Factors/geneticsABSTRACT
PURPOSE: Anaemia is common in patients presenting with aneurysmal subarachnoid (aSAH) and intracerebral haemorrhage (ICH). In surgical patients, anaemia was identified as an idenpendent risk factor for postoperative mortality, prolonged hospital length of stay (LOS) and increased risk of red blood cell (RBC) transfusion. This multicentre cohort observation study describes the incidence and effects of preoperative anaemia in this critical patient collective for a 10-year period. METHODS: This multicentre observational study included adult in-hospital surgical patients diagnosed with aSAH or ICH of 21 German hospitals (discharged from 1 January 2010 to 30 September 2020). Descriptive, univariate and multivariate analyses were performed to investigate the incidence and association of preoperative anaemia with RBC transfusion, in-hospital mortality and postoperative complications in patients with aSAH and ICH. RESULTS: A total of n = 9081 patients were analysed (aSAH n = 5008; ICH n = 4073). Preoperative anaemia was present at 28.3% in aSAH and 40.9% in ICH. RBC transfusion rates were 29.9% in aSAH and 29.3% in ICH. Multivariate analysis revealed that preoperative anaemia is associated with a higher risk for RBC transfusion (OR = 3.25 in aSAH, OR = 4.16 in ICH, p < 0.001), for in-hospital mortality (OR = 1.48 in aSAH, OR = 1.53 in ICH, p < 0.001) and for several postoperative complications. CONCLUSIONS: Preoperative anaemia is associated with increased RBC transfusion rates, in-hospital mortality and postoperative complications in patients with aSAH and ICH. TRIAL REGISTRATION: ClinicalTrials.gov , NCT02147795, https://clinicaltrials.gov/ct2/show/NCT02147795.
Subject(s)
Anemia , Subarachnoid Hemorrhage , Adult , Anemia/complications , Anemia/epidemiology , Anemia/therapy , Cerebral Hemorrhage/complications , Cerebral Hemorrhage/therapy , Erythrocyte Transfusion/adverse effects , Humans , Registries , Streptothricins , Subarachnoid Hemorrhage/complications , Subarachnoid Hemorrhage/diagnosis , Subarachnoid Hemorrhage/therapyABSTRACT
OBJECTIVES: The effect of photobiomodulation (PBM) treatment on wound healing and macrophage polarization was investigated in vivo. Animal models of oral ulcers were simulated through chemically induced oral ulcers in rats. MATERIALS AND METHODS: PBM treatment using an infrared pulsed laser was used to treat oral ulcers in the animal models. Twelve Sprague-Dawley rats were randomly divided into four groups depending on set absorbed energy: Group 1 (control), Group 2 (30 J), Group 3 (60 J), and Group 4 (100 J). Laser treatment was performed every other day for 8 days after ulcer confirmation. Parameters used were as follows: wavelength 808 nm, power output 50 mW, spot size 10 mm, frequency 10 Hz, and pulse duration 1 millisecond. Ulcers were measured to determine the effect of the treatments over time. Histology, immunostaining, and real-time polymerase chain reaction analyses were performed to evaluate the effect of PBM treatment on macrophage-related (IL-6/IL-10) and wound-healing-related (TNF-α/TGF-ß/MMP-2) cytokine expression. RESULTS: Histological examinations indicate that the PBM treatment stimulated a higher level of wound recovery after 8 days of treatment at 60 J absorbed energy compared to other treatment groups. Analyses of relative gene expression of proinflammatory, anti-inflammatory, and tissue remodeling cytokines indicate that the macrophages in the tissue samples were predominantly characterized as M2 subtypes (alternatively activated), which possibly accounts for the accelerated tissue repair in the animal model of oral ulcer. CONCLUSION: This preliminary study stands as a proof of concept regarding the potential use of infrared laser PBM treatment for oral ulcers which have not been previously investigated upon. PBM treatment affects macrophage polarization and enhances wound healing. Further experimentation will be conducted to expand the understanding of how PBM treatment affects the healing mechanism of ulcers.
Subject(s)
Low-Level Light Therapy , Oral Ulcer , Animals , Cytokines/metabolism , Macrophages/metabolism , Oral Ulcer/radiotherapy , Rats , Rats, Sprague-Dawley , Rats, Wistar , Streptothricins , Ulcer , Wound HealingABSTRACT
Streptomyces have been reported as a remarkable source for bioactive secondary metabolites with complex structural and functional diversity. In this study, 35 isolates of genus Streptomyces were purified from rhizospheric and marine soils collected from previously unexplored habitats and screened for antimicrobial activities. One of these isolates, G1, when tested in vitro, was found highly active against wide range of microbes including Gram-positive, Gram-negative bacteria, and different fungal pathogens. It was identified as mesophilic, alkaliphilic, and moderately halotolerant as it showed optimum growth at temperature 30 °C, pH 8.0 in casein-starch-peptone-yeast extract-malt extract medium supplemented with 5% NaCl. Sequence analysis of the 16S rRNA gene indicated 100% identity of this isolate to Streptomyces fimbriatus. Moreover, maximum antimicrobial activity was achieved in starch nitrate medium supplemented with 1% glycerol as carbon and 0.03% soy meal as nitrogen source. The antimicrobial compounds produced by this isolate were extracted in methanol. Bioassay-guided fractionation through thin layer chromatography of methanolic extract resulted in the separation of a most active fraction with an Rf value of 0.46. This active fraction was characterized by FTIR and LCMS analysis and found similar to streptothricin D like antibiotic with m/z 758.42.
Subject(s)
Geologic Sediments , Streptothricins , Anti-Bacterial Agents/pharmacology , Bacteria/drug effects , Geologic Sediments/microbiology , RNA, Ribosomal, 16S/genetics , Streptomyces/chemistry , Streptothricins/chemistry , Streptothricins/isolation & purification , Streptothricins/metabolism , Streptothricins/pharmacologyABSTRACT
Neurospora crassa is an excellent model fungus for studies on molecular genetics, biochemistry, physiology, and molecular cell biology. Along with the rapid progress of Neurospora research, new tools facilitating more efficient and accurate genetic analysis are in high demand. Here, we tested whether the dominant selective makers widely used in yeasts are applicable in N. crassa. Among them, we found that the strains of N. crassa are sensitive to the aminoglycoside antibiotics, G418 and nourseothricin. 1000 µg/mL of G418 or 50 µg/mL of nourseothricin is sufficient to inhibit Neurospora growth completely. When the neomycin phosphotransferase gene (neo) used in mammalian cells is expressed, N. crassa shows potent resistance to G418. This establishes G418-resistant marker as a dominant selectable marker to use in N. crassa. Similarly, when the nourseothricin acetyltransferase gene (nat) from Streptomyces noursei is induced by qa-2 promoter in the presence of quinic acid (QA), N. crassa shows potent resistance to nourseothricin. When nat is constitutively expressed by full-length or truncated versions of the promoter from the N. crassa cfp gene (NCU02193), or by the trpC promoter of Aspergillus nidulans, the growth of N. crassa in the presence of nourseothricin is proportional to the expression levels of Nat. Finally, these two markers are used to knock-out wc-2 or al-1 gene from the N. crassa genome. The successful development of these two markers in this study expands the toolbox for N. crassa and very likely for other filamentous fungi as well.
Subject(s)
Drug Resistance, Fungal/genetics , Genetic Markers , Neurospora crassa/drug effects , Neurospora crassa/genetics , Acetyltransferases/genetics , Anti-Bacterial Agents/pharmacology , DNA Transposable Elements , Drug Resistance, Fungal/drug effects , Gene Expression Regulation, Fungal , Gene Knockout Techniques , Genes, Dominant , Gentamicins/pharmacology , Kanamycin Kinase/genetics , Microorganisms, Genetically-Modified , Promoter Regions, Genetic , Quinic Acid/pharmacology , Streptothricins/pharmacologyABSTRACT
Many Candida species that cause infection have diploid genomes and do not undergo classical meiosis. The application of clustered regularly interspaced short palindromic repeat-Cas9 (CRISPR-Cas9) gene editing systems has therefore greatly facilitated the generation of gene disruptions and the introduction of specific polymorphisms. However, CRISPR methods are not yet available for all Candida species. We describe here an adaption of a previously developed CRISPR system in Candida parapsilosis that uses an autonomously replicating plasmid. Guide RNAs can be introduced in a single cloning step and are released by cleavage between a tRNA and a ribozyme. The plasmid also contains CAS9 and a selectable nourseothricin SAT1 marker. It can be used for markerless editing in C. parapsilosis, C. orthopsilosis, and C. metapsilosis We also show that CRISPR can easily be used to introduce molecular barcodes and to reintroduce wild-type sequences into edited strains. Heterozygous mutations can be generated, either by careful selection of the distance between the polymorphism and the Cas9 cut site or by providing two different repair templates at the same time. In addition, we have constructed a different autonomously replicating plasmid for CRISPR-Cas9 editing in Candida tropicalis We show that editing can easily be carried out in multiple C. tropicalis isolates. Nonhomologous end joining (NHEJ) repair occurs at a high level in C. metapsilosis and C. tropicalisIMPORTANCECandida species are a major cause of infection worldwide. The species associated with infection vary with geographical location and with patient population. Infection with Candida tropicalis is particularly common in South America and Asia, and Candida parapsilosis infections are more common in the very young. Molecular methods for manipulating the genomes of these species are still lacking. We describe a simple and efficient CRISPR-based gene editing system that can be applied in the C. parapsilosis species group, including the sister species Candida orthopsilosis and Candida metapsilosis We have also constructed a separate system for gene editing in C. tropicalis.
Subject(s)
CRISPR-Cas Systems , Candida/genetics , Plasmids/genetics , Candida tropicalis/genetics , DNA Barcoding, Taxonomic , DNA End-Joining Repair , Gene Editing/methods , Genetic Vectors , Genome, Fungal , Mutation , Streptothricins/pharmacologyABSTRACT
Microbial production of biofuels and bioproducts offers a sustainable and economic alternative to petroleum-based fuels and chemicals. The basidiomycete yeast Rhodosporidium toruloides is a promising platform organism for generating bioproducts due to its ability to consume a broad spectrum of carbon sources (including those derived from lignocellulosic biomass) and to naturally accumulate high levels of lipids and carotenoids, two biosynthetic pathways that can be leveraged to produce a wide range of bioproducts. While R. toruloides has great potential, it has a more limited set of tools for genetic engineering relative to more advanced yeast platform organisms such as Yarrowia lipolytica and Saccharomyces cerevisiae Significant advancements in the past few years have bolstered R. toruloides' engineering capacity. Here we expand this capacity by demonstrating the first use of CRISPR-Cas9-based gene disruption in R. toruloides Transforming a Cas9 expression cassette harboring nourseothricin resistance and selecting transformants on this antibiotic resulted in strains of R. toruloides exhibiting successful targeted disruption of the native URA3 gene. While editing efficiencies were initially low (0.002%), optimization of the cassette increased efficiencies 364-fold (to 0.6%). Applying these optimized design conditions enabled disruption of another native gene involved in carotenoid biosynthesis, CAR2, with much greater success; editing efficiencies of CAR2 deletion reached roughly 50%. Finally, we demonstrated efficient multiplexed genome editing by disrupting both CAR2 and URA3 in a single transformation. Together, our results provide a framework for applying CRISPR-Cas9 to R. toruloides that will facilitate rapid and high-throughput genome engineering in this industrially relevant organism.IMPORTANCE Microbial biofuel and bioproduct platforms provide access to clean and renewable carbon sources that are more sustainable and environmentally friendly than petroleum-based carbon sources. Furthermore, they can serve as useful conduits for the synthesis of advanced molecules that are difficult to produce through strictly chemical means. R. toruloides has emerged as a promising potential host for converting renewable lignocellulosic material into valuable fuels and chemicals. However, engineering efforts to improve the yeast's production capabilities have been impeded by a lack of advanced tools for genome engineering. While this is rapidly changing, one key tool remains unexplored in R. toruloides: CRISPR-Cas9. The results outlined here demonstrate for the first time how effective multiplexed CRISPR-Cas9 gene disruption provides a framework for other researchers to utilize this revolutionary genome-editing tool effectively in R. toruloides.
Subject(s)
Basidiomycota/genetics , CRISPR-Cas Systems , Gene Editing/methods , Genome, Fungal , Basidiomycota/drug effects , Drug Resistance, Fungal , Fungal Proteins/genetics , Streptothricins/pharmacologyABSTRACT
Acylation of epsilon amino groups of lysyl side chains is a widespread modification of proteins and small molecules in cells of all three domains of life. Recently, we showed that Bacillus subtilis and Bacillus anthracis encode the GCN5-related N-acetyltransferase (GNAT) SatA that can acetylate and inactivate streptothricin, which is a broad-spectrum antibiotic produced by actinomycetes in the soil. To determine functionally relevant residues of B. subtilis SatA (BsSatA), a mutational screen was performed, highlighting the importance of a conserved area near the C terminus. Upon inspection of the crystal structure of the B. anthracis Ames SatA (BaSatA; PDB entry 3PP9), this area appears to form a pocket with multiple conserved aromatic residues; we hypothesized this region contains the streptothricin-binding site. Chemical and site-directed mutagenesis was used to introduce missense mutations into satA, and the functionality of the variants was assessed using a heterologous host (Salmonella enterica). Results of isothermal titration calorimetry experiments showed that residue Y164 of BaSatA was important for binding streptothricin. Results of size exclusion chromatography analyses showed that residue D160 was important for dimerization. Together, these data advance our understanding of how SatA interacts with streptothricin.IMPORTANCE This work provides insights into how an abundant antibiotic found in soil is bound to the enzyme that inactivates it. This work identifies residues for the binding of the antibiotic and probes the contributions of substituting side chains for those in the native protein, providing information regarding hydrophobicity, size, and flexibility of the antibiotic binding site.
Subject(s)
Acetyltransferases/metabolism , Anti-Bacterial Agents/metabolism , Bacillus anthracis/enzymology , Bacillus subtilis/enzymology , Bacterial Proteins/metabolism , Streptothricins/metabolism , Acetylation , Acetyltransferases/chemistry , Acetyltransferases/genetics , Anti-Bacterial Agents/chemistry , Bacillus anthracis/genetics , Bacillus anthracis/metabolism , Bacillus subtilis/chemistry , Bacillus subtilis/genetics , Bacillus subtilis/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Binding Sites , Streptothricins/chemistryABSTRACT
Streptomyces griseus S4-7 is representative of strains responsible for the specific soil suppressiveness of Fusarium wilt of strawberry caused by Fusarium oxysporum f. sp. fragariae. Members of the genus Streptomyces secrete diverse secondary metabolites including lantipeptides, heat-stable lanthionine-containing compounds that can exhibit antibiotic activity. In this study, a class II lantipeptide provisionally named grisin, of previously unknown biological function, was shown to inhibit F. oxysporum. The inhibitory activity of grisin distinguishes it from other class II lantipeptides from Streptomyces spp. Results of quantitative reverse transcription-polymerase chain reaction with lanM-specific primers showed that the density of grisin-producing Streptomyces spp. in the rhizosphere of strawberry was positively correlated with the number of years of monoculture and a minimum of seven years was required for development of specific soil suppressiveness to Fusarium wilt disease. We suggest that lanM can be used as a diagnostic marker of whether a soil is conducive or suppressive to the disease.
Subject(s)
Fragaria , Fusarium , Soil Microbiology , Soil/chemistry , Anti-Bacterial Agents/metabolism , Fragaria/microbiology , Plant Diseases/prevention & control , Streptothricins/metabolismABSTRACT
Some members of the Sporothrix genus can cause sporotrichosis, a worldwide distributed mycosis that affects several mammalian species, including human beings. Sporothrix schenckii and Sporothrix brasiliensis are the fungal species frequently associated with this disease, and the latter has gained significant interest because of the increased number of cases associated with transmission by cats. Despite the relevance of these organisms in the medical field, limited strategies for their genetic manipulation have been explored. Thus far, gene silencing using the hygromycin B resistance cassette is the sole strategy currently available to study these organisms. Here, we report the generation of a cassette that confers resistance to nourseothricin, which was successfully transferred from Agrobacterium tumefaciens to Sporothrix cells. Therefore, this can be used as a second selective marker to manipulate the genome of these organisms.
Subject(s)
Genetic Engineering/methods , Genome, Fungal , Plasmids/chemistry , Sporothrix/genetics , Streptothricins/pharmacology , Agrobacterium tumefaciens/genetics , Anti-Bacterial Agents/pharmacology , Drug Resistance, Fungal/genetics , Plasmids/metabolism , Sporothrix/drug effects , Transformation, GeneticABSTRACT
Studies on Caenorhabditis elegans would benefit from the introduction of new selectable markers to allow more complex types of experiments to be conducted with this model animal. We established a new antibiotic selection marker for C. elegans transformation based on nourseothricin (NTC) and its resistance-encoding gene, streptothricin-acetyl transferase 2 (Sat2). NTC was able to efficiently prevent worm development at very low concentrations, and the worms expressing Sat2 were able to survive on the selection plates without any developmental defects. Using CRISPR/Cas9 and NTC selection, we were able to easily insert a 13-kb expression cassette into a defined locus in C. elegans. The structure and spectrum of NTC differs from other antibiotics like hygromycin B and geneticin, making it possible to use NTC alongside them. Indeed, we confirmed NTC-sat2 selection could work with the hygromycin B selection system simultaneously. Thus, the new NTC-Sat2 system can act as a useful dominant marker for gene transfer and genome editing in C. elegans.
Subject(s)
Acetyltransferases/genetics , Caenorhabditis elegans/genetics , Gene Editing/methods , Genetic Markers , Animals , CRISPR-Cas Systems/genetics , Culture Media/chemistry , Genetic Markers/genetics , Genome, Helminth , Hygromycin B/pharmacology , Streptothricins/pharmacologyABSTRACT
Candida glabrata is the second most common species causing candidiasis. C. glabrata can also readily acquire resistance to azole drugs, complicating its treatment. Here we add to the collection of disruption markers to aid in genetic analysis of this yeast. This new construct is marked with a nourseothricin resistance cassette that produces an estrogen-activated form of Cre recombinase in a methionine-regulated manner. This allows eviction and reuse of this cassette in a facile manner. Using this new disruption marker, we have constructed a series of strains lacking different members of the major facilitator superfamily (MFS) of membrane transporter proteins. The presence of 15 MFS proteins that may contribute to drug resistance in C. glabrata placed a premium on development of a marker that could easily be reused to construct multiple gene-disrupted strains. Employing this recyclable marker, we found that loss of the MFS transporter-encoding gene FLR1 caused a dramatic increase in diamide resistance (as seen before), and deletion of two other MFS-encoding genes did not influence this phenotype. Interestingly, loss of FLR1 led to an increase in levels of oxidized glutathione, suggesting a possible molecular explanation for this enhanced oxidant sensitivity. We also found that while overproduction of the transcription factor Yap1 could suppress the fluconazole sensitivity caused by loss of the important ATP-binding cassette transporter protein Cdr1, this required the presence of FLR1. IMPORTANCE Export of drugs is a problem for chemotherapy of infectious organisms. A class of membrane proteins called the major facilitator superfamily contains a large number of proteins that often elevate drug resistance when overproduced but do not impact this phenotype when the gene is removed. We wondered if this absence of a phenotype for a disruption allele might be due to the redundancy of this group of membrane proteins. We describe the production of an easy-to-use recyclable marker cassette that will allow construction of strains lacking multiple members of the MFS family of transporter proteins.