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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.

Hygromycin B (HYG-B) is a 5-glycosylated 2-dexoystreptamine- aminoglycoside antibiotic-(2DOS-AGA) produced by Streptomyces hygroscopicus subspecies hygroscopicus NRRL ISP-5578 with broad-spectrum activity against many pathogenic bacteria, fungi and helminths. In the literature, limited studies are concerned with the biosynthetic pathway and different cultural conditions affecting the production of HYG-B. This study aimed to optimize key environmental conditions influencing its production as one-factor-at-a-time (OFAT) and as a statistical model of response surface D-optimal design (DOD). Moreover, the biosynthetic pathway of HYG-B in light of the identified genes/proteins of the HYG-B gene cluster was proposed and elucidated. The effect of culture media composition and incubation time were studied OFAT, and the results showed that both culture media 6 (CM6) and CM4 gave the highest specific productivity, 5.88 and 3.99 µg/mg, respectively, and 7 days as incubation time. So, using CM6 and 7 days incubation resulted in a sevenfold increase (190 µg/mL) compared to the original unoptimized condition (CM1 and 6 days incubation; 26.9 µg/mL). Three important factors—initial pH, incubation temperature, and agitation—were tested using a DOD quadratic model generating 20 experimental runs. An initial pH of 6.4, an incubation temperature, of 28 ℃, and agitation. of 295 rpm were predicted and experimentally verified, resulting in a 13-fold increase (371.5 µg/mL) compared to the unoptimized condition and a sevenfold increase compared to that obtained as OFAT. In conclusion, DOD design is an efficient tool for optimizing HYG-B. However, the optimized conditions should be scaled up in a bioreactor for industrial production of HYG-B by S. hygroscopicus NRRL ISP-5578.

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.

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.

Abiotic stress tolerance of plants is a very complex trait and involves multiple physiological and biochemical processes. Thus, the improvement of plant stress tolerance should involve pyramiding of multiple genes. In the present study, we report the construction and application of a bicistronic system, involving the internal ribosome entry site (IRES) sequence from the 5′UTR of the heat-shock protein of tobacco gene NtHSF - 1 , to the improvement of salt tolerance in transgenic tobacco plants. Two genes from wheat encoding two important vacuolar ion transporters, Na + /H + antiporter ( TNHXS1 ) and H + -pyrophosphatase ( TVP1 ), were linked via IRES to generate the bicistronic construct TNHXS1 -IRES- TVP1 . Molecular analysis of transgenic tobacco plants revealed the correct integration of the TNHXS1 -IRES- TVP1 construct into tobacco genome and the production of the full-length bicistronic mRNA from the 35S promoter. Ion transport analyses with tonoplast vesicles isolated from transgenic lines confirmed that single-transgenic lines TVP1cl19 and TNHXS1cl7 had greater H + -PPiase and Na + /H + antiport activity, respectively, than the WT. Interestingly, the co-expression of TVP1 and TNHXS1 increased both Na + /H + antiport and H + -PPiase activities and induced the H + pumping activity of the endogenous V-ATPase. Transgenic tobacco plants expressing TNHXS1 -IRES- TVP1 showed a better performance than either of the single gene-transformed lines and the wild type plants when subjected to salt treatment. In addition, the TNHXS1 -IRES- TVP1 transgenic plants accumulated less Na + and more K + in their leaf tissue than did the wild type and the single gene-transformed lines. These results demonstrate that IRES system, described herein, can co-ordinate the expression of two important abiotic stress-tolerance genes and that this expression system is a valuable tool for obtaining transgenic plants with improved salt tolerance.

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.

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.

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.

Secondary metabolites play important roles in microbial communities, but their natural functions are often unknown and may be more complex than appreciated. While compounds with antibiotic activity are often assumed to underlie microbial competition, they may alternatively act as signal molecules. In either scenario, microorganisms might evolve responses to sublethal concentrations of these metabolites, either to protect themselves from inhibition or to change certain behaviors in response to the local abundance of another species. Here, we report that violacein production by C. violaceum ATCC 31532 is induced in response to hygromycin A from Streptomyces sp. 2AW, and we show that this response is dependent on inhibition of translational polypeptide elongation and a previously uncharacterized two-component regulatory system. The breadth of the transcriptional response beyond violacein induction suggests a surprisingly complex metabolite-mediated microbe-microbe interaction and supports the hypothesis that antibiotics evolved as signal molecules. These novel insights will inform predictive models of soil community dynamics and the unintended effects of clinical antibiotic administration. Antibiotics produced by bacteria play important roles in microbial interactions and competition Antibiosis can induce resistance mechanisms in target organisms, and at sublethal doses, antibiotics have been shown to globally alter gene expression patterns. Here, we show that hygromycin A from Streptomyces sp. strain 2AW. induces Chromobacterium violaceum ATCC 31532 to produce the purple antibiotic violacein. Sublethal doses of other antibiotics that similarly target the polypeptide elongation step of translation likewise induced violacein production, unlike antibiotics with different targets. C. violaceum biofilm formation and virulence against Drosophila melanogaster were also induced by translation-inhibiting antibiotics, and we identified an a ntibiotic- i nduced r esponse ( air ) two-component regulatory system that is required for these responses. Genetic analyses indicated a connection between the Air system, quorum-dependent signaling, and the negative regulator VioS, leading us to propose a model for induction of violacein production. This work suggests a novel mechanism of interspecies interaction in which a bacterium produces an antibiotic in response to inhibition by another bacterium and supports the role of antibiotics as signal molecules. IMPORTANCE Secondary metabolites play important roles in microbial communities, but their natural functions are often unknown and may be more complex than appreciated. While compounds with antibiotic activity are often assumed to underlie microbial competition, they may alternatively act as signal molecules. In either scenario, microorganisms might evolve responses to sublethal concentrations of these metabolites, either to protect themselves from inhibition or to change certain behaviors in response to the local abundance of another species. Here, we report that violacein production by C. violaceum ATCC 31532 is induced in response to hygromycin A from Streptomyces sp. 2AW, and we show that this response is dependent on inhibition of translational polypeptide elongation and a previously uncharacterized two-component regulatory system. The breadth of the transcriptional response beyond violacein induction suggests a surprisingly complex metabolite-mediated microbe-microbe interaction and supports the hypothesis that antibiotics evolved as signal molecules. These novel insights will inform predictive models of soil community dynamics and the unintended effects of clinical antibiotic administration.