Computational protocol: Strain-specific bioaccumulation and intracellular distribution of Cd2+ in bacteria isolated from the rhizosphere, ectomycorrhizae, and fruitbodies of ectomycorrhizal fungi

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Protocol publication

[…] A total of 15 bacterial strains were isolated from the rhizosphere, ectomycorrhizae, and fruitbodies of ectomycorrhizal fungi associated with willows (Salix viminalis L.) growing at anthropogenic degraded sites. The examined strains represented three phyla (Firmicutes, Proteobacteria, and Bacteroidetes) and were characterized by different cell wall compositions, both Gram-negative and Gram-positive. Four strains from the class Bacilli (Bacillus cereus B1, B2, B3, B4) had been previously identified and described by Hrynkiewicz et al. (). Six strains from the class Gammaproteobacteria (Pseudomonas sp. IV-111-14, Luteibacter sp. II-116-7, Serratia entomophila I-111-21), Betaproteobacteria (Massilia sp. III-116-18), Bacteroidetes sp. I-116-1 and Flavobacterium sp. I-111-11 were selected from a group of 50 investigated bacterial strains on the basis of physiological properties by Hrynkiewicz et al. (). The strain Pseudomonas fulva was isolated from the interior tissues of the roots of Scots pine trees growing in sand dunes at the Baltic Sea of Poland (Strzelczyk and Li ), a noncontaminated site. The remaining four strains, Bacillus sp. ML1-2, Luteibacter rhizovicinus ML3-1, Variovorax sp. ML3-12, and Pseudomonas fluorescens LIC 1, were isolated from ectomycorrhizae or fruitbodies collected at heav- metal-contaminated test sites (Hrynkiewicz et al. ) and identified and described for the first time (based on 16S rDNA) in this work. The identification procedure was performed according to Hrynkiewicz et al. (, ) with small modifications. The DNA of bacterial strains was isolated using Dneasy® Blood & Tissue Kit (Qiagen, Hilden, Germany). The amplification of the 16S ribosomal RNA (16S rRNA) gene was performed using Taq PCR Master Mix (Qiagen, Hilden, Germany) and primers F1 (5′-GAG TTT GAT CCT GGC TCA G-3′) and R12 (5′-ACG GCT ACC TTG TTA CGA CTT–3′) (Dorsch and Stackebrandt ). The thermal program comprised an initial denaturation step of 2 min at 95 °C, followed by 30 cycles of 1-min denaturation at 94 °C, 1-min annealing at 55 °C, and 2-min extension at 72 °C, and a final elongation step of 5 min at 72 °C. The PCR products were purified using the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany). Direct sequencing of PCR products was performed using the PCR primers as sequencing primers. Sequences were aligned manually aided by the Sequencher system (TW Version 5.1, Gene Codes, Ann Arbor, MI, USA). The BLAST database (Altschul et al. ) of the National Center for Biotechnology Information (NCBI) was used to compare obtained sequences of all isolates with known 16S rRNA gene sequences of bacterial strains. The DNA sequences determined in this study were deposited in GenBank under accession numbers [KM411501], [KM411502], [KM411503], [KM411504] (Table ). The results of molecular identification of newly identified bacterial strains were presented in the form of phylogenetic tree and presented in the supplementary data (Fig. ). [...] The differences in elemental composition between the control cells and the Cd-treated cells were analyzed by Student’s t test using Statistica software (Statistica ver. 7, Statsoft). The linear dependence of the Cd2+ content on the quantities of other elements within the bacterial cells was expressed by Pearson’s correlation coefficient, and the significance of this correlation was verified by Student’s t test. […]

Pipeline specifications

Software tools Sequencher, Statistica
Applications Miscellaneous, Phylogenetics