The Venus flytrap (Dionaea muscipula J. Ellis) is a carnivorous plant able to synthesize large amounts of phenolic compounds, such as phenylpropanoids, flavonoids, phenolic acids, and 1,4-naphtoquinones. In this study, the first genetic transformation of D. muscipula tissues is presented. Two wild-type Rhizobium rhizogenes strains (LBA 9402 and ATCC 15834) were suitable vector organisms in the transformation process. Transformation led to the formation of teratoma (transformed shoot) cultures with the bacterial rolB gene incorporated into the plant genome in a single copy. Using high-pressure liquid chromatography, we demonstrated that transgenic plants were characterized by an increased quantity of phenolic compounds, including 1,4-naphtoquinone derivative, plumbagin (up to 106.63 mg × g-1 DW), and phenolic acids (including salicylic, caffeic, and ellagic acid), in comparison to non-transformed plants. Moreover, Rhizobium-mediated transformation highly increased the bactericidal properties of teratoma-derived extracts. The antibacterial properties of transformed plants were increased up to 33% against Staphylococcus aureus, Enterococcus faecalis, and Escherichia coli and up to 7% against Pseudomonas aeruginosa. For the first time, we prove the possibility of D. muscipula transformation. Moreover, we propose that transformation may be a valuable tool for enhancing secondary metabolite production in D. muscipula tissue and to increase bactericidal properties against human antibiotic-resistant bacteria. KEY POINTS: • Rhizobium-mediated transformation created Dionaea muscipula teratomas. • Transformed plants had highly increased synthesis of phenolic compounds. • The MBC value was connected with plumbagin and phenolic acid concentrations.

• MeSH
• Agrobacterium genetics   MeSH
• Anti-Bacterial Agents pharmacology   MeSH
• Droseraceae * MeSH
• Phenols MeSH
• Humans MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Nanotechnology, new fascinating field of science, is bringing many application's options. However, it is necessary to understand their potential environmental risk and toxicity. Zinc selenide quantum dots (ZnSe QDs) are getting valuable due to wide industrial usage, mainly as cadmium free diodes or stabilizing ligand. Thanks to unique properties, they could also open the possibilities of application in the agriculture. Their effects on living organisms, including plants, are still unknown. Therefore, the attention of this work was given to antioxidant response of Arabidopsis thaliana to 100 and 250 μM ZnSe QDs foliar feeding. ZnSe QDs treatment had no statistically significant differences in morphology but led to increased antioxidant response in the leaves at the level of gene expression and production secondary antioxidant metabolites. Concurrently, analysis of growth properties of Agrobacterium tumefaciens was done. 250 μM ZnSe solution inhibited the Agrobacterium tumefaciens viability by 60%. This is the first mention about effect ZnSe QDs on the plants. Although QDs induced oxidative stress, the apply treatment dose of ZnSe QDs did not have significant toxic effect on the plants and even no morphological changes were observed. However, the same amount of ZnSe QD induced an inhibitory effect on Agrobacterium tumefaciens.

• MeSH
• Agrobacterium tumefaciens drug effects   growth & development   MeSH
• Antioxidants metabolism   MeSH
• Arabidopsis genetics   growth & development   metabolism   MeSH
• Gene Expression * MeSH
• Quantum Dots toxicity   MeSH
• Metabolomics * MeSH
• Selenium Compounds administration & dosage   toxicity   MeSH
• Zinc Compounds administration & dosage   toxicity   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Many Gram-negative bacteria use N-acyl-homoserine lactones (N-acyl-HSL) as quorum sensing signal molecules. N-acyl-HSL are the subject of investigation by many research groups. Simple methods have been designed to detect N-acyl-HSL using bacterial strains that doesn't produce its own signal molecules, but the reporter gene is expressed when exogenous N-acyl-HSL are present. This article reviews and discusses the most common and most currently obtainable systems using bacteria such as Agrobacterium tumefaciens, Chromobacterium violaceum, Escherichia coli or Vibrio fischeri. These biological sensing elements can be used to detect and study a wide range of N-acyl-HSL and related molecules interfering with quorum sensing systems.

• MeSH
• Acyl-Butyrolactones * analysis   MeSH
• Agrobacterium tumefaciens metabolism   MeSH
• Biosensing Techniques * methods   MeSH
• Chromobacterium metabolism   MeSH
• Escherichia coli metabolism   MeSH
• Quorum Sensing MeSH
• Plant Tumor-Inducing Plasmids MeSH
• Vibrio metabolism   MeSH
• Publication type
• Research Support, Non-U.S. Gov't MeSH

Organophosphorus compounds have been widely employed to the development of warfare nerve agents and pesticides, resulting in a huge number of people intoxicated annually, being a serious problem of public health. Efforts worldwide have been done in order to design new technologies that are capable of combating or even reversing the poisoning caused by these OP nerve agents. In this line, the bioremediation arises as a promising and efficient alternative for this purpose. As an example of degrading enzymes, there is the organophosphate-degrading (OpdA) enzyme from Agrobacterium radiobacter, which has been quite investigated experimentally due to its high performance in the degradation of neurotoxic nerve agents. This work aims to look into the structural and electronic details that govern the interaction modes of these compounds in the OpdA active site, with the posterior hydrolysis reaction prediction. Our findings have brought about data about the OpdA performance towards different nerve agents, and among them, we may realize that the degradation efficiency strongly depends on the nerve agent structure and its stereochemistry, being in this case the compound Tabun the one more effectively hydrolyzed. By means of the chemical bonds (AIM) and orbitals (FERMO) analysis, it is suggested that the initial reactivity of the OP nerve agents in the OpdA active site does not necessarily dictate the reactivity and interaction modes over the reaction coordinate.

• MeSH
• Agrobacterium tumefaciens enzymology   MeSH
• Bacterial Proteins chemistry   metabolism   MeSH
• Biodegradation, Environmental * MeSH
• Biocatalysis MeSH
• Phosphoric Monoester Hydrolases chemistry   metabolism   MeSH
• Catalytic Domain MeSH
• Quantum Theory MeSH
• Humans MeSH
• Nerve Agents chemistry   metabolism   MeSH
• Sarin chemistry   metabolism   MeSH
• Molecular Docking Simulation MeSH
• Binding Sites MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Silene latifolia serves as a model species to study dioecy, the evolution of sex chromosomes, dosage compensation and sex-determination systems in plants. Currently, no protocol for genetic transformation is available for this species, mainly because S. latifolia is considered recalcitrant to in vitro regeneration and infection with Agrobacterium tumefaciens. Using cytokinins and their synthetic derivatives, we markedly improved the efficiency of regeneration. Several agrobacterial strains were tested for their ability to deliver DNA into S. latifolia tissues leading to transient and stable expression of the GUS reporter. The use of Agrobacterium rhizogenes strains resulted in the highest transformation efficiency (up to 4.7% of stable transformants) in hairy root cultures. Phenotypic and genotypic analyses of the T1 generation suggested that the majority of transformation events contain a small number of independent T-DNA insertions and the transgenes are transmitted to the progeny in a Mendelian pattern of inheritance. In short, we report an efficient and reproducible protocol for leaf disc transformation and subsequent plant regeneration in S. latifolia, based on the unique combination of infection with A. rhizogenes and plant regeneration from hairy root cultures using synthetic cytokinins. A protocol for the transient transformation of S.latifolia protoplasts was also developed and applied to demonstrate the possibility of targeted mutagenesis of the sex linked gene SlAP3 by TALENs and CRISPR/Cas9.

• MeSH
• Agrobacterium genetics   MeSH
• Chromosomes, Plant genetics   MeSH
• CRISPR-Cas Systems MeSH
• DNA, Bacterial genetics   MeSH
• Gene Expression MeSH
• Genetic Techniques MeSH
• Plants, Genetically Modified MeSH
• Models, Genetic MeSH
• Evolution, Molecular MeSH
• Regeneration genetics   MeSH
• Genes, Reporter MeSH
• Silene genetics   microbiology   physiology   MeSH
• Transcription Activator-Like Effector Nucleases MeSH
• Transformation, Genetic * MeSH
• Publication type
• Journal Article MeSH

BACKGROUND: Plant transformation via Agrobacterium tumefaciens is characterized by integration of commonly low number of T-DNAs at random positions in the genome. When integrated into an active gene region, promoterless reporter genes placed near the T-DNA border sequence are frequently transcribed and even translated to reporter proteins, which is the principle of promoter- and gene-trap lines. RESULTS: Here we show that even internal promotorless regions of T-DNAs are often transcribed. Such spontaneous transcription was observed in the majority of independently transformed tobacco BY-2 lines (over 65%) and it could effectively induce silencing if an inverted repeat was present within the T-DNA. We documented that the transcription often occurred in both directions. It was not directly connected with any regulatory elements present within the T-DNAs and at least some of the transcripts were initiated outside of the T-DNA. The likeliness of this read-through transcription seemed to increase in lines with higher T-DNA copy number. Splicing and presence of a polyA tail in the transcripts indicated involvement of Pol II, but surprisingly, the transcription was able to run across two transcription terminators present within the T-DNA. Such pervasive transcription was observed with three different T-DNAs in BY-2 cells and with lower frequency was also detected in Arabidopsis thaliana. CONCLUSIONS: Our results demonstrate unexpected pervasive read-through transcription of T-DNAs. We hypothesize that it was connected with a specific chromatin state of newly integrated DNA, possibly affected by the adjacent genomic region. Although this phenomenon can be easily overlooked, it can have significant consequences when working with highly sensitive systems like RNAi induction using an inverted repeat construct, so it should be generally considered when interpreting results obtained with the transgenic technology.

• MeSH
• Agrobacterium tumefaciens genetics   MeSH
• Arabidopsis genetics   MeSH
• Cell Line MeSH
• DNA, Bacterial genetics   MeSH
• Transcription, Genetic * MeSH
• Plants, Genetically Modified MeSH
• RNA, Messenger genetics   MeSH
• Inverted Repeat Sequences genetics   MeSH
• Promoter Regions, Genetic genetics   MeSH
• Genes, Reporter MeSH
• Nicotiana genetics   MeSH
• Publication type
• Journal Article MeSH

Old Yellow Enzymes (OYEs) are NAD(P)H dehydrogenases of not fully resolved physiological roles that are widespread among bacteria, plants, and fungi and have a great potential for biotechnological applications. We determined the apo form crystal structure of a member of the OYE class, glycerol trinitrate reductase XdpB, from Agrobacterium bohemicum R89-1 at 2.1 Å resolution. In agreement with the structures of the related bacterial OYEs, the structure revealed the TIM barrel fold with an N-terminal β-hairpin lid, but surprisingly, the structure did not contain its cofactor FMN. Its putative binding site was occupied by a pentapeptide TTSDN from the C-terminus of a symmetry related molecule. Biochemical experiments confirmed a specific concentration-dependent oligomerization and a low FMN content. The blocking of the FMN binding site can exist in vivo and regulates enzyme activity. Our bioinformatic analysis indicated that a similar self-inhibition could be expected in more OYEs which we designated as subgroup OYE C1. This subgroup is widespread among G-bacteria and can be recognized by the conserved sequence GxxDYP in proximity of the C termini. In proteobacteria, the C1 subgroup OYEs are typically coded in one operon with short-chain dehydrogenase. This operon is controlled by the tetR-like transcriptional regulator. OYEs coded in these operons are unlikely to be involved in the oxidative stress response as the other known members of the OYE family because no upregulation of XdpB was observed after exposing A. bohemicum R89-1 to oxidative stress.

• MeSH
• Agrobacterium enzymology   genetics   MeSH
• Genes, Bacterial MeSH
• Bacterial Proteins chemistry   genetics   metabolism   MeSH
• Flavin Mononucleotide metabolism   MeSH
• Catalytic Domain MeSH
• Kinetics MeSH
• Crystallography, X-Ray MeSH
• Protein Structure, Quaternary MeSH
• Models, Molecular MeSH
• NADPH Dehydrogenase chemistry   genetics   metabolism   MeSH
• Operon MeSH
• Oxidative Stress MeSH
• Oxidoreductases chemistry   genetics   metabolism   MeSH
• Computational Biology MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Two non-pathogenic strains R89-1 and R90T isolated from poppy seed (Papaver somniferum L.) wastes were phenotypically and genotypically characterized. Multilocus sequence analysis (MLSA) was conducted with six genes (atpD, glnA, gyrB, recA, rpoB, 16S rRNA). The strains represented a new species which clustered with Agrobacterium rubi NBRC 13261T and Agrobacterium skierniewicense Ch11T type strains. MLSA was further accompanied by whole-genome phylogeny, in silico DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) analyses for both strains. ANI and dDDH values were deep below the species delineation threshold. Phenotypic features of the novel strains unequivocally allowed their differentiation from all other Agrobacterium species. Unlike other agrobacteria, the strains were salt sensitive and were able to biotransform morphine alkaloids. The dominant cellular fatty acids are 18:1 w7c, 16:0 and 12:0 aldehyde/16:1 iso I/14:0 3OH summed in feature 2 and the major respiratory quinine is Q-10 (87%). The DNA G+C content is 56mol%. Microbial community analysis indicated probable association with P. somniferum plant material. Altogether, these characteristics showed that strains R90T and R89-1 represent a new species of the genus Agrobacterium which we propose to name Agrobacterium bohemicum. The type strain of A. bohemicum is R90T (=CCM 8736T=DSM 104667T).

• MeSH
• Agrobacterium classification   genetics   isolation & purification   MeSH
• Genes, Bacterial MeSH
• Biotransformation MeSH
• DNA, Bacterial genetics   MeSH
• Phylogeny * MeSH
• Nucleic Acid Hybridization MeSH
• Fatty Acids chemistry   MeSH
• Multilocus Sequence Typing MeSH
• Papaver microbiology   MeSH
• RNA, Ribosomal, 16S genetics   MeSH
• Sequence Analysis, DNA MeSH
• Seeds microbiology   MeSH
• Ubiquinone analogs & derivatives   chemistry   MeSH
• Base Composition MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Czech Republic MeSH

A plant selection system based on the phosphomannose isomerase gene (pmi) as a selectable marker is often used to avoid selection using antibiotic resistance. Nevertheless, pmi gene is endogenous in several plant species and therefore difficult to use in such cases. Here we evaluated and compared Agrobacterium-mediated transformation of Linum usitatissimum breeding line AGT-952 (without endogenous pmi gene) and Nicotiana tabacum var. WSC-38 (with endogenous pmi gene). Transformation was evaluated for vectors bearing transgenes that have the potential to be involved in improved phytoremediation of contaminated environment. Tobacco regenerants selection resulted in 6.8% transformation efficiency when using a medium supplemented with 30 g/L mannose with stepwise decrease of the sucrose concentration. Similar transformation efficiency (5.3%) was achieved in transformation of flax. Relatively low selection efficiency was achieved (12.5% and 34.8%, respectively). The final detection of efficient pmi selection was conducted using PCR and the non-endogenous genes; pmi transgene for flax and todC2 transgene for tobacco plants.

• MeSH
• Agrobacterium genetics   MeSH
• Transformation, Bacterial genetics   MeSH
• Biodegradation, Environmental MeSH
• Plants, Genetically Modified genetics   MeSH
• Culture Media chemistry   MeSH
• Flax drug effects   genetics   MeSH
• Mannose-6-Phosphate Isomerase genetics   MeSH
• Mannose metabolism   pharmacology   MeSH
• Selection, Genetic MeSH
• Nicotiana drug effects   genetics   MeSH
• Publication type
• Journal Article MeSH

Intact, growing cells of strongly acidophilic fungi Acidea extrema and Acidothrix acidophila have been successfully transformed by introduction of heterologous DNA fragment (composed of the glyceraldehyde-phosphate-dehydrogenase gene promoter from Emericella nidulans, a metallothionein-coding gene AsMt1 from Amanita strobiliformis and glyceraldehyde-phosphate-dehydrogenase gene terminator from Colletotrichum gloeosporioides) with the length of 1690 bp. The transformation procedure was based on the DNA transfer mediated by Agrobacterium tumefaciens bearing disarmed helper plasmid pMP90 and binary vector pCambia1300 with inserted DNA fragment of interest. The transformants proved to be mitotically stable, and the introduced gene was expressed at least at the level of transcription. Our work confirms that metabolic adaptations of strongly acidophilic fungi do not represent an obstacle for genetic transformation using conventional methods and can be potentially used for production of heterologous proteins. A promising role of the fast growing A. acidophila as active biomass in biotechnological processes is suggested not only by the low susceptibility of the culture grown at low pH to contaminations but also by reduced risk of accidental leaks of genetically modified microorganisms into the environment because highly specialized extremophilic fungi can poorly compete with common microflora under moderate conditions.

• MeSH
• Agrobacterium tumefaciens genetics   MeSH
• Amanita genetics   MeSH
• Ascomycota genetics   MeSH
• Gene Expression MeSH
• Genetic Vectors MeSH
• Hydrogen-Ion Concentration MeSH
• Metallothionein genetics   MeSH
• Plasmids MeSH
• Promoter Regions, Genetic MeSH
• Transcription Termination, Genetic MeSH
• Transformation, Genetic * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH