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   In this study, we have demonstrated the applicability of electroporation for the stable nuclear transformation of Coccomyxa solorinae-saccatae . An antibiogram revealed that Hygromycin B and G418 are the most effective selective agents among eight different antibiotics tested. We have shown that a plasmid vector containing the hptII gene, coding for hygromycin B phosphotransferase, with expression driven by the strong cauliflower mosaic virus CaMV35S promoter ensures sufficient protection of transformed algal cells against high concentrations of Hygromycin B. The ability to drive transgene expression in the alga C. solorinae-saccatae offers unique opportunities to study the physiology of lichenic algae, as it is one of the symbiotic strains of the Coccomyxa simplex group. Furthermore, our findings demonstrate that electroporation is a convenient and effective technique for the transformation of algae in the Coccomyxa genus.

Ulva prolifera is a fast-growing green seaweed that has garnered considerable interest in both fundamental and applied research. Here, we established a molecular tool by employing a selectable marker gene that allowed the isolation of U. prolifera cells integrating exogenous DNA. We developed a modular plasmid for expressing exogenous genes in U. prolifera based on the bacterial antibiotic-resistance marker, aminoglycoside phosphotransferase gene ( aph7” ). Integration of aph7” in macroalgae can generate transformants resistant to hygromycin B. In addition, we characterized the promoter region of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase gene (pUpRbcS) to drive the expression of aph7” . The transcripts were consistently confirmed from antibiotic-selected transformants, stably retaining the exogenous gene in the succeeding generations. Subsequently, a CRISPR-based knock-in system was established, facilitating the integration of aph7” cassette in the endogenous selection gene encoding for adenine phosphoribosyltransferase ( UpAPT ). APT gene can serve as an endogenous marker in algae that exhibits a lethal phenotype under cultivation with 2-fluoroadenine. The resulting knock-in mutants could resist the co-selection of the antibiotic hygromycin B and 2-fluoroadenine. Our results advance U. prolifera as a genetic platform, enabling functional research to elucidate Ulva biology, and to bring forth biotechnological utilization of algal resources.

Callus browning, a common trait derived from the indica rice cultivar ( Oryza sativa L.), is a challenge to transformation regeneration. Here, we report the map-based cloning of BROWNING OF CALLUS1 ( BOC1 ) using a population derived from crossing Teqing, an elite indica subspecies exhibiting callus browning, and Yuanjiang, a common wild rice accession ( Oryza rufipogon Griff.) that is less susceptible to callus browning. We show that BOC1 encodes a SIMILAR TO RADICAL-INDUCED CELL DEATH ONE (SRO) protein. Callus browning can be reduced by appropriate upregulation of BOC1 , which consequently improves the genetic transformation efficiency. The presence of a Tourist -like miniature inverted-repeat transposable element ( Tourist MITE) specific to wild rice in the promoter of BOC1 increases the expression of BOC1 in callus. BOC1 may decrease cell senescence and death caused by oxidative stress. Our study provides a gene target for improving tissue culturability and genetic transformation. Callus browning heavily affects indica rice transformation regeneration. Here, the authors show transposon insertion in the promoter of BOC1 gene, encoding a SIMILAR TO RADICAL-INDUCED CELL DEATH ONE protein, can upregulate its expression and decrease callus browning in cultivated rice by releasing oxidative stress.

The ever-growing rise of antibiotic resistance among bacterial pathogens is one of the top healthcare threats today. Although combination antibiotic therapies represent a potential approach to more efficiently combat infections caused by susceptible and drug-resistant bacteria, only a few known drug pairs exhibit synergy/cooperativity in killing bacteria. Here, we discover that well-known ribosomal antibiotics, hygromycin A (HygA) and macrolides, which target peptidyl transferase center and peptide exit tunnel, respectively, can act cooperatively against susceptible and drug-resistant bacteria. Remarkably, HygA slows down macrolide dissociation from the ribosome by 60-fold and enhances the otherwise weak antimicrobial activity of the newest-generation macrolide drugs known as ketolides against macrolide-resistant bacteria. By determining a set of high-resolution X-ray crystal structures of drug-sensitive wild-type and macrolide-resistant Erm-methylated 70S ribosomes in complex with three HygA-macrolide pairs, we provide a structural rationale for the binding cooperativity of these drugs and also uncover the molecular mechanism of overcoming Erm-type resistance by macrolides acting together with hygromycin A. Altogether our structural, biochemical, and microbiological findings lay the foundation for the subsequent development of synergistic antibiotic tandems with improved bactericidal properties against drug-resistant pathogens, including those expressing erm genes. The authors discovered that hygromycin A not only enhances the cell-killing properties of macrolides but also renders them active against resistant bacteria. The provided structures of antibiotic pairs in complex with WT and macrolide-resistant ribosomes rationalize binding cooperativity of these drugs.

Madurella mycetomatis is the main cause of mycetoma, a chronic granulomatous infection for which currently no adequate therapy is available. To improve therapy, more knowledge on a molecular level is required to understand how M . mycetomatis is able to cause this disease. However, the genetic toolbox for M . mycetomatis is limited. To date, no method is available to genetically modify M . mycetomatis . In this paper, a protoplast-mediated transformation protocol was successfully developed for this fungal species, using hygromycin as a selection marker. Furthermore, using this method, a cytoplasmic-GFP-expressing M . mycetomatis strain was created. The reported methodology will be invaluable to explore the pathogenicity of M . mycetomatis and to develop reporter strains which can be useful in drug discovery as well as in genetic studies.

We previously reported Agrobacterium-mediated transformation methods for the liverwort Marchantia polymorpha using the hygromycin phosphotransferase gene as a marker for selection with hygromycin. In this study, we developed three additional markers for M. polymorpha transformation: the gentamicin 3'-acetyltransferase gene for selection with gentamicin; a mutated acetolactate synthase gene for selection with chlorsulfuron; and the neomycin phosphotransferase II gene for selection with G418. Based on these four marker genes, we have constructed a series of Gateway binary vectors designed for transgenic experiments on M. polymorpha. The 35S promoter from cauliflower mosaic virus and endogenous promoters for constitutive and heat-inducible expression were used to create these vectors. The reporters and tags used were Citrine, 3×Citrine, Citrine-NLS, TagRFP, tdTomato, tdTomato-NLS, GR, SRDX, SRDX-GR, GUS, ELuc(PEST), and 3×FLAG. These vectors, designated as the pMpGWB series, will facilitate molecular genetic analyses of the emerging model plant M. polymorpha.

On the basis of the one strain-many compounds (OSMAC) strategy, two new hygromycin A derivatives (3, 4), together with six known compounds were isolated from a medicinal plant inter rhizospheric Streptomyces in Pulsatilla chinensis. The structures of 3 and 4 were elucidated using NMR and HRESIMS analyses. A plausible biosynthetic pathway for these compounds was discussed. All the compounds were evaluated for their antimicrobial and cytotoxic activities. Compound 5 exhibited potent inhibitory activity against S. aureus and B. subtilis with the MICs of 16 and 8 μg ml−1, while 4 showed weak inhibitory activity against S. aureus.

In this study, we have shown the applicability of electroporation and hygromycin B as a convenient selectable marker for stable nuclear transformation of Coccomyxa subellipsoidea C-169. Since it is the first sequenced eukaryotic microorganism from polar environment, this offers unique opportunities to study adaptation mechanisms to cold.

The transportation of proteins encoded by nuclear genes from plant cytosol to chloroplast is essential for chloroplast functions. Proteins that have a chloroplast transit peptide (cTP) are imported into chloroplasts via translocases on the outer and inner chloroplast envelope. How proteins lacking transit sequence are imported into chloroplast remains largely unknown. During screening of an Arabidopsis population transformed with a hairpin RNA gene-silencing library, we identified some transgenic plants that had active expression of the selectable marker gene, hygromycin phosphotransferase (HPT), but were sensitive to the selection agent, hygromycin B (HyB). Mutant and complementation analysis showed that this HyB sensitivity of transgenic plants was due to silencing of the HS1 (Hygromycin-Sensitive 1) gene. HS1 is localized in the chloroplast and interacts physically with HPT in yeast cells and in planta . Fluorescence and immunoblotting analysis showed that HPT could not be transported effectively into chloroplasts in Aths1 , which resulted in Aths1 is sensitivity to hygromycin on higher HyB-containing medium. These data revealed that HS1 is involved in HyB resistance in transgenic Arabidopsis through facilitating cytosol-chloroplast transportation of HPT. Our findings provide novel insights on transportation of chloroplast cTP-less proteins.

Background Cutaneotrichosporon oleaginosus is an oleaginous yeast that can produce up to 80% lipid per dry weight. Its high capacity for the biosynthesis of single cell oil makes it highly interesting for the production of engineered lipids or oleochemicals for industrial applications. However, the genetic toolbox for metabolic engineering of this non-conventional yeast has not yet been systematically expanded. Only three long endogenous promoter sequences have been used for heterologous gene expression, further three dominant and one auxotrophic marker have been established. Results In this study, the structure of putative endogenous promoter sequences was analyzed based on more than 280 highly expressed genes. The identified motifs of regulatory elements and translational initiation sites were used to annotate the four endogenous putative promoter sequences D9FADp, UBIp, PPIp, and 60Sp. The promoter sequences were tested in a construct regulating the known dominant marker hygromycin B phosphotransferase. The four newly described promoters and the previously established GAPDHp successfully initiated expression of the resistance gene and PPIp was selected for further marker development. The geneticin G418 resistance (aminoglycoside 3’-phosphotransferase, APH) and the nourseothricin resistance gene N-acetyl transferase (NAT) were tested for applicability in C. oleaginosus . Both markers showed high transformation efficiency, positive rate, and were compatible for combined use in a successive and simultaneous manner. Conclusions The implementation of four endogenous promoters and one novel dominant resistance markers for C. oleaginosus opens up new opportunities for genetic engineering and strain development. In combination with recently developed methods for targeted genomic integration, the established toolbox allows a wide spectrum of new strategies for genetic and metabolic engineering of the industrially highly relevant yeast.