Selenium (Se)-rich plants may be used to provide dietary Se to humans and livestock, and also to clean up Se-polluted soils or waters. This study focused on endophytic bacteria of plants that hyperaccumulate selenium (Se) to 0.5-1% of dry weight. Terminal restriction fragment length polymorphism (T-RFLP) analysis was used to compare the diversity of endophytic bacteria of hyperaccumulators Stanleya pinnata (Brassicaceae) and Astragalus bisulcatus (Fabaceae) with those from related non-accumulators Physaria bellii (Brassicaceae) and Medicago sativa (Fabaceae) collected on the same, seleniferous site. Hyperaccumulators and non-accumulators showed equal T-RF diversity. Parsimony analysis showed that T-RFs from individuals of the same species were more similar to each other than to those from other species, regardless of plant Se content or spatial proximity. Cultivable endophytes from hyperaccumulators S. pinnata and A. bisulcatus were further identified and characterized. The 66 bacterial morphotypes were shown by MS MALDI-TOF Biotyper analysis and 16S rRNA gene sequencing to include strains of Bacillus, Pseudomonas, Pantoea, Staphylococcus, Paenibacillus, Advenella, Arthrobacter, and Variovorax. Most isolates were highly resistant to selenate and selenite (up to 200 mM) and all could reduce selenite to red elemental Se, reduce nitrite and produce siderophores. Seven isolates were selected for plant inoculation and found to have plant growth promoting properties, both in pure culture and when co-cultivated with crop species Brassica juncea (Brassicaceae) or M. sativa. There were no effects on plant Se accumulation. We conclude that Se hyperaccumulators harbor an endophytic bacterial community in their natural seleniferous habitat that is equally diverse to that of comparable non-accumulators. The hyperaccumulator endophytes are characterized by high Se resistance, capacity to produce elemental Se and plant growth promoting properties.

• Keywords
• T-RFLP, bacteria, endophyte, hyperaccumulator, microbial diversity, phytoremediation, selenium,
• Publication type
• Journal Article MeSH

Bacteria were identified associated with biodegradation of aromatic pollutants biphenyl, benzoate, and naphthalene in a long-term polychlorinated biphenyl- and polyaromatic hydrocarbon-contaminated soil. In order to avoid biases of culture-based approaches, stable isotope probing was applied in combination with sequence analysis of 16 S rRNA gene pyrotags amplified from (13)C-enriched DNA fractions. Special attention was paid to pyrosequencing data analysis in order to eliminate the errors caused by either generation of amplicons (random errors caused by DNA polymerase, formation of chimeric sequences) or sequencing itself. Therefore, sample DNA was amplified, sequenced, and analyzed along with the DNA of a mock community constructed out of 8 bacterial strains. This warranted that appropriate tools and parameters were chosen for sequence data processing. (13)C-labeled metagenomes isolated after the incubation of soil samples with all three studied aromatics were largely dominated by Proteobacteria, namely sequences clustering with the genera Rhodanobacter Burkholderia, Pandoraea, Dyella as well as some Rudaea- and Skermanella-related ones. Pseudomonads were mostly labeled by (13)C from naphthalene and benzoate. The results of this study show that many biphenyl/benzoate-assimilating bacteria derive carbon also from naphthalene, pointing out broader biodegradation abilities of some soil microbiota. The results also demonstrate that, in addition to traditionally isolated genera of degradative bacteria, yet-to-be cultured bacteria are important players in bioremediation. Overall, the study contributes to our understanding of biodegradation processes in contaminated soil. At the same time our results show the importance of sequencing and analyzing a mock community in order to more correctly process and analyze sequence data.

• MeSH
• Bacteria genetics   isolation & purification   metabolism   MeSH
• Benzoates metabolism   MeSH
• Biphenyl Compounds metabolism   MeSH
• Biodegradation, Environmental MeSH
• DNA, Bacterial metabolism   MeSH
• Phylogeny MeSH
• Isotope Labeling MeSH
• Carbon Isotopes MeSH
• Soil Pollutants analysis   MeSH
• Naphthalenes metabolism   MeSH
• Soil Microbiology * MeSH
• Base Sequence MeSH
• Sequence Analysis, DNA MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Benzoates MeSH
• Biphenyl Compounds MeSH
• biphenyl MeSH Browser
• DNA, Bacterial MeSH
• Carbon Isotopes MeSH
• Soil Pollutants MeSH
• Naphthalenes MeSH
• naphthalene MeSH Browser

Bacteria that are able to utilize biphenyl as a sole source of carbon were extracted and isolated from polychlorinated biphenyl (PCB)-contaminated soil vegetated by horseradish. Isolates were identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The usage of MALDI Biotyper for the classification of isolates was evaluated and compared to 16S rRNA gene sequence analysis. A wide spectrum of bacteria was isolated, with Arthrobacter, Serratia, Rhodococcus, and Rhizobium being predominant. Arthrobacter isolates also represented the most diverse group. The use of MALDI Biotyper in many cases permitted the identification at the level of species, which was not achieved by 16S rRNA gene sequence analyses. However, some isolates had to be identified by 16S rRNA gene analyses if MALDI Biotyper-based identification was at the level of probable or not reliable identification, usually due to a lack of reference spectra included in the database. Overall, this study shows the possibility of using MALDI-TOF MS and MALDI Biotyper for the fast and relatively nonlaborious identification/classification of soil isolates. At the same time, it demonstrates the dominant role of employing 16S rRNA gene analyses for the identification of recently isolated strains that can later fill the gaps in the protein-based identification databases.

• MeSH
• Armoracia MeSH
• Bacteria chemistry   classification   genetics   isolation & purification   MeSH
• Biphenyl Compounds metabolism   MeSH
• Biodiversity MeSH
• DNA, Bacterial chemistry   genetics   MeSH
• Soil Pollutants metabolism   MeSH
• Molecular Sequence Data MeSH
• Soil Microbiology * MeSH
• Rhizosphere MeSH
• DNA, Ribosomal chemistry   genetics   MeSH
• RNA, Ribosomal, 16S genetics   MeSH
• Sequence Analysis, DNA MeSH
• Cluster Analysis MeSH
• Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization methods   MeSH
• Bacterial Typing Techniques methods   MeSH
• Publication type
• Journal Article MeSH
• Evaluation Study MeSH
• Research Support, Non-U.S. Gov't MeSH
• Comparative Study MeSH
• Names of Substances
• Biphenyl Compounds MeSH
• biphenyl MeSH Browser
• DNA, Bacterial MeSH
• Soil Pollutants MeSH
• DNA, Ribosomal MeSH
• RNA, Ribosomal, 16S MeSH

The aim of this work was to construct transgenic plants with increased capabilities to degrade organic pollutants, such as polychlorinated biphenyls. The environmentally important gene of bacterial dioxygenase, the bphC gene, was chosen to clone into a plant of Nicotiana tabacum. The chosen bphC gene encodes 2,3-dihydroxybiphenyl-1,2-dioxygenase, which cleaves the aromatic ring of dihydroxybiphenyl, and we cloned it in fusion with the gene for β-glucuronidase (GUS), luciferase (LUC) or with a histidine tail. Several genetic constructs were designed and prepared and the possible expression of desired proteins in tobacco plants was studied by transient expression. We used genetic constructs successfully expressing dioxygenase's genes we used for preparation of transgenic tobacco plants by agrobacterial infection. The presence of transgenic DNA , mRNA and protein was determined in parental and the first filial generation of transgenic plants with the bphC gene. Properties of prepared transgenic plants will be further studied.

• Keywords
• 2,3-dihydroxybiphenyl-1,2-dioxygenase, Nicotiana tabacum, bphC, phytoremediation, transgenic plant,
• MeSH
• Bacterial Proteins genetics   metabolism   MeSH
• Biodegradation, Environmental MeSH
• Burkholderiaceae enzymology   genetics   MeSH
• Dioxygenases genetics   metabolism   MeSH
• Plants, Genetically Modified enzymology   genetics   metabolism   MeSH
• Glucuronidase genetics   metabolism   MeSH
• Cloning, Molecular MeSH
• Luciferases genetics   metabolism   MeSH
• Polychlorinated Biphenyls metabolism   MeSH
• Recombinant Fusion Proteins genetics   metabolism   MeSH
• Nicotiana enzymology   genetics   metabolism   MeSH
• Green Fluorescent Proteins genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Evaluation Study MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 2,3-dihydroxybiphenyl oxygenase MeSH Browser
• Bacterial Proteins MeSH
• Dioxygenases MeSH
• Glucuronidase MeSH
• Luciferases MeSH
• Polychlorinated Biphenyls MeSH
• Recombinant Fusion Proteins MeSH
• Green Fluorescent Proteins MeSH