Table_1_Enriching the endophytic bacterial microbiota of Ginkgo roots.xlsx

Bacterial endophytes of Ginkgo roots take part in the secondary metabolic processes of the fossil tree and contribute to plant growth, nutrient uptake, and systemic resistance. However, the diversity of bacterial endophytes in Ginkgo roots is highly underestimated due to the lack of successful isolates and enrichment collections. The resulting culture collection contains 455 unique bacterial isolates representing 8 classes, 20 orders, 42 families, and 67 genera from five phyla: Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria, and Deinococcus-Thermus, using simply modified media (a mixed medium without any additional carbon sources [MM)] and two other mixed media with separately added starch [GM] and supplemented glucose [MSM]). A series of plant growth-promoting endophytes had multiple representatives within the culture collection. Moreover, we investigated the impact of refilling carbon sources on enrichment outcomes. Approximately 77% of the natural community of root-associated endophytes were predicted to have successfully cultivated the possibility based on a comparison of the 16S rRNA gene sequences between the enrichment collections and the Ginkgo root endophyte community. The rare or recalcitrant taxa in the root endosphere were mainly associated with Actinobacteria, Alphaproteobacteria, Blastocatellia, and Ktedonobacteria. By contrast, more operational taxonomic units (OTUs) (0.6% in the root endosphere) became significantly enriched in MM than in GM and MSM. We further found that the bacterial taxa of the root endosphere had strong metabolisms with the representative of aerobic chemoheterotrophy, while the functions of the enrichment collections were represented by the sulfur metabolism. In addition, the co-occurrence network analysis suggested that the substrate supplement could significantly impact bacterial interactions within the enrichment collections. Our results support the fact that it is better to use the enrichment to assess the cultivable potential and the interspecies interaction as well as to increase the detection/isolation of certain bacterial taxa. Taken together, this study will deepen our knowledge of the indoor endophytic culture and provide important insights into the substrate-driven enrichment.</p

Table_2_Enriching the endophytic bacterial microbiota of Ginkgo roots.xlsx

Bacterial endophytes of Ginkgo roots take part in the secondary metabolic processes of the fossil tree and contribute to plant growth, nutrient uptake, and systemic resistance. However, the diversity of bacterial endophytes in Ginkgo roots is highly underestimated due to the lack of successful isolates and enrichment collections. The resulting culture collection contains 455 unique bacterial isolates representing 8 classes, 20 orders, 42 families, and 67 genera from five phyla: Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria, and Deinococcus-Thermus, using simply modified media (a mixed medium without any additional carbon sources [MM)] and two other mixed media with separately added starch [GM] and supplemented glucose [MSM]). A series of plant growth-promoting endophytes had multiple representatives within the culture collection. Moreover, we investigated the impact of refilling carbon sources on enrichment outcomes. Approximately 77% of the natural community of root-associated endophytes were predicted to have successfully cultivated the possibility based on a comparison of the 16S rRNA gene sequences between the enrichment collections and the Ginkgo root endophyte community. The rare or recalcitrant taxa in the root endosphere were mainly associated with Actinobacteria, Alphaproteobacteria, Blastocatellia, and Ktedonobacteria. By contrast, more operational taxonomic units (OTUs) (0.6% in the root endosphere) became significantly enriched in MM than in GM and MSM. We further found that the bacterial taxa of the root endosphere had strong metabolisms with the representative of aerobic chemoheterotrophy, while the functions of the enrichment collections were represented by the sulfur metabolism. In addition, the co-occurrence network analysis suggested that the substrate supplement could significantly impact bacterial interactions within the enrichment collections. Our results support the fact that it is better to use the enrichment to assess the cultivable potential and the interspecies interaction as well as to increase the detection/isolation of certain bacterial taxa. Taken together, this study will deepen our knowledge of the indoor endophytic culture and provide important insights into the substrate-driven enrichment.</p

Data_Sheet_1_Enriching the endophytic bacterial microbiota of Ginkgo roots.docx

Bacterial endophytes of Ginkgo roots take part in the secondary metabolic processes of the fossil tree and contribute to plant growth, nutrient uptake, and systemic resistance. However, the diversity of bacterial endophytes in Ginkgo roots is highly underestimated due to the lack of successful isolates and enrichment collections. The resulting culture collection contains 455 unique bacterial isolates representing 8 classes, 20 orders, 42 families, and 67 genera from five phyla: Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria, and Deinococcus-Thermus, using simply modified media (a mixed medium without any additional carbon sources [MM)] and two other mixed media with separately added starch [GM] and supplemented glucose [MSM]). A series of plant growth-promoting endophytes had multiple representatives within the culture collection. Moreover, we investigated the impact of refilling carbon sources on enrichment outcomes. Approximately 77% of the natural community of root-associated endophytes were predicted to have successfully cultivated the possibility based on a comparison of the 16S rRNA gene sequences between the enrichment collections and the Ginkgo root endophyte community. The rare or recalcitrant taxa in the root endosphere were mainly associated with Actinobacteria, Alphaproteobacteria, Blastocatellia, and Ktedonobacteria. By contrast, more operational taxonomic units (OTUs) (0.6% in the root endosphere) became significantly enriched in MM than in GM and MSM. We further found that the bacterial taxa of the root endosphere had strong metabolisms with the representative of aerobic chemoheterotrophy, while the functions of the enrichment collections were represented by the sulfur metabolism. In addition, the co-occurrence network analysis suggested that the substrate supplement could significantly impact bacterial interactions within the enrichment collections. Our results support the fact that it is better to use the enrichment to assess the cultivable potential and the interspecies interaction as well as to increase the detection/isolation of certain bacterial taxa. Taken together, this study will deepen our knowledge of the indoor endophytic culture and provide important insights into the substrate-driven enrichment.</p

Restrikční endonukleázy a genetické testy

This bachelor thesis Restriction endonucleases and genetic tests engages in the issue of restriction enzymes. In the literary review I describe the character of restriction endonucleases, their classification and mechanism of operation. The thesis furthermore touches a topic concerning the effect of the restriction reaction conditions and discusses techniques of utilization of these enzymes, where for example DNA cloning, analysis of variability or genome mapping belong. The practical part of the thesis includes some work with a software for an automatic search for restriction sites and for restriction DNA evaluation

Comparison of the physical organization of <i>nif</i>, <i>fix</i>, and associated genes from fosmid DX-1A-14 with those from three closest organisms.

<p>Organisms: <i>Azoarcus sp.</i>, <i>Herbaspirillum seropedicae</i>, <i>Acetobacter diazotrophicus</i>. The <i>nif</i> genes are highlighted in bold. <i>nrf</i>, <i>nif</i> associated genes; <i>orf</i>, hypothetical protein. The structure of <i>nif</i> gene cluster differs greatly from those of unknown microorganisms. The <i>nif</i> gene cluster does not contain <i>modABC</i> genes occurring in <i>H. seropedicae</i>, and RubisCO gene cluster can not be identified in the <i>nif</i> gene cluster region of <i>H. seropedicae</i>. <i>nifQ</i> gene is clustered together with <i>nifENX</i> and <i>fdxB</i> genes in <i>A. diazotrophicus</i>, while <i>nifV</i>, <i>nifW</i>, <i>fixABCX</i>, and <i>nifQ</i> genes constitute a single operon in fosmid DX-1A-14.</p

Phylogenetic tree of <i>nifK</i> genes.

<p>The phylogenetic tree was constructed by the neighbor-joining method using MEGA, version 4.0 with 1000 bootstrap repetitions. The sequences obtained from metagenomic sequencing of acid mine drainage are designated DX_SY_, followed by their number in library. These sequences are shown in bold. Only some representatives of 93 <i>nifK</i> sequences are shown here. The scale represents the number of amino acid substitutions per site.</p

Reverse transcription PCR of <i>fixX</i> and <i>nifQ</i> gene fragment.

<p>Lane M, 100; lane 1, PCR products with the template of total RNA extraction; lane 2, PCR products with the template of total RNA extracting digested by RNase.</p

List of ORFs from fosmid DX-1A-14, gene length, and similar genes in GenBank.

a<p>Nucleotide identity of fosmid DX-1A-14 gene to the gene of the organism to which it is most related.</p

Gene products in the <i>nif-fix</i> cluster of fosmid DX-1A-14.

a<p>Organism in which the gene products most similar to that of the nif-fix cluster was found. Organism: <i>Herbaspirillum seropedicae, Burkholderia vietnamiensis, Beijerinckia indica, Rubrivivax benzoatilyticus, Candidatus Accumulibacter phosphatis clade IIA, Ralstonia eutropha, Methylococcus capsulatus, Methylovorus sp., Nitrosomonas europaea, Rhodopseudomonas palustris, Acidovorax delafieldii.</i></p>b<p>Identity of the deduced anomic acid sequence of gene product to the gene product of the organism to which it is most related.</p

Classification of total <i>nif</i> genes obtained from metagenomic sequencing reads.

<p>742 <i>nif</i> sequences were classified into different phyla based on the similarities to the known sequences. The community is massively dominated by γ<i>-proteobacteria</i>, followed in smaller amounts by <i>α-, β-proteobacteria</i> and <i>Verrucomicrobia</i>.</p