Fig 1.
Molecular characterization of arsenic resistant strain.
(A). PCR amplification of the isolated strain AT-02 DNA (using different dilutions) via universal 16SrRNA primers 27F and 1492 R. Lane 1,2, and 3 shows the amplified band of 1500b.p by the universal primers. The amplified product was further subjected to sequencing. (B). Phylogenetic tree constructed via Neighbour Joining method using MEGA11 showing high similarity of the isolated AT-02 strain with Klebsiella oxytoca ADY16 strain.
Fig 2.
Determination of the AT-02 strain arsenic resistance.
(A).Growth curve of the AT-02 strain at different arsenate concentrations ranging from 1000–10,000 μg/mL showing that the isolated strain can survive till 10,000 μg/mL. Media without arsenate (shown in blue) is indicated as LB was used as a control. (B).Growth curve of the AT-02 strain at different arsenite concentrations ranging from 200–600 μg/mL showing that the isolated strain can survive till 600 μg/mL. Media without arsenite is indicated as LB (shown in blue) was used as a control. (C).Growth curve of the reference strain Klebsiella pneumoniae strain at different arsenate concentrations ranging from 1000–10,000 μg/mL showing arsenic sensitivity. The control strain can only grow easily in the absence of arsenate. (D).Growth curve of the Klebsiella pneumoniae strain at different arsenite concentrations ranging from 200–600 μg/mL μg/mL showing that the reference strain was not able to survive in the presence of arsenite. (E&F).Growth comparison of Klebsiella oxytoca (AT-02) with Klebsiella pneumoniae on LB broth bearing different concentrations of arsenate (1000–10,000 μg/mL) and arsenite (200 μg/mL). (G&H). Growth comparison of Klebsiella oxytoca (AT-02) with Klebsiella pneumoniae on LB agar plates bearing different concentrations of arsenate (1000–10,000 μg/mL) and arsenite (200–600 μg/mL). The comparison showed that the Klebsiella oxytoca (AT-02) can survive till 10,000ppm arsenate and 600ppm arsenite respectively.
Fig 3.
Amplification and sequence analysis of arsB.
(A). Phylogenetic tree based on Neighbour joining method using MEGA11 based on 27 amino acid sequences showed similarity between arsB of AT-02 and Klebsiella pneumoniae. (B).Phylogenetic tree between distantly similar species was constructed by Maximum Likelihood method showed similarity between arsB of AT-02 and E.coli. (C).Multiple sequence alignment based on 6 different amino acid sequences show presence of conserved arsenic binding residues (Histidine (H), Arginine (R), Arginine (R), Tryptophan (W), Valine (V)) highlighted in blue boxes. These residues are conserved in AT-02, E.coli, K. pneumoniae and P.putida, However, the in Lactobacillus these residues are replaced by Lysine (K), Valine (V), Phenylalanine (F),Leucine(L) and Serine (S).
Fig 4.
Protein structure prediction of arsB.
(A). Protein structure prediction of AT-02 (Klebsiella oxytoca) ArsB by trRosetta showing 79.8% alpha helix, 4.4% turn, 13.3%coils and 2.5% 3–10 helix. (B). Three-dimensional structure of Klebsiella oxytoca ArsB with arsenic binding residues highlighted in cyan (Histidine (H142), Arginine (R146), Arginine (R172), Tryptophan (W175), Valine (V183)).(C).Protein structure prediction of E.coli ArsB by trRosetta showing 81.2% alpha helix, 4.4% turn, 13.3%coils and 1.1% 3–10 helix. (D). Three-dimensional structure of E.coli ArsB with arsenic binding residues highlighted in yellow (Histidine (H124), Arginine (R132), Arginine (R158), Tryptophan (W161), Valine (V169)).(E).Protein structure prediction of Klebsiella pneumoniae ArsB by trRosetta showing 81.2% alpha helix, 4.4% turn, 13.3%coils and 1.1% 3–10 helix. (F).Three-dimensional structure of Klebsiella pneumoniae ArsB with arsenic binding residues highlighted in yellow (Histidine (H124), Arginine (R132), Arginine (R158), Tryptophan (W161), Valine (V169)). (G).Protein structure prediction of P.putida ArsB by trRosetta showing 72.2% alpha helix, 13.9% turn, 13.9%coils and 0% 3–10 helix.(H). Three-dimensional structure of P.putida ArsB with arsenic binding residues highlighted in yellow (Histidine (H17), Arginine (R21), Arginine (R47), Tryptophan (W50), Valine (V58)).
Fig 5.
(A).Structural superposition of ArsB Klebsiella oxytoca and E.coli using TM-align (https://zhanggroup.org/TM-align/)) showed 96%identical folding indicating high structural similarity with 1.26 RMSD (root mean square deviation) value. (B).Structural superposition of ArsB Klebsiella oxytoca and Klebsiella pneumoniae using TM-align (https://zhanggroup.org/TM-align/)) showed 96%identical folding indicating high structural similarity with 1.26 RMSD (root mean square deviation) value. (C). Structural superposition of ArsB Klebsiella oxytoca and P.putida using TM-align (https://zhanggroup.org/TM-align/)) showed 91.3%identical folding indicating comparatively less structural similarity with 2.06 RMSD (root mean square deviation) value.
Fig 6.
Mechanism of arsenic resistance and bioremediation.
(A). Real time PCR analysis showing dose dependent increase in arsB expression at RNA level. As compared to ¼ MIC, 1.2 and 3/4MIC showed an increase in arsB by 10 and 12 folds. (B). An increase in biofilm formation was observed when AT-02 was grown in the presence of 1000ppm arsenate. A two-fold significant increase in absorbance was observed due to more biofilm formation due to arsenate. (C). Biosorption of arsenic at different time intervals showing approximately 50% arsenic removal within 0.5hrs. A slow but increase in arsenic bioremediation was observed till 66% on increasing the incubation time till 2hours. (D).The bioremediation efficiency of bacterial biomass against arsenic showed a progressive increase in its removal with increasing incubation time.at different time intervals showing approximately 50% arsenic removal within 0.5hrs. (A-D).Error bars represent ±SD of three independent experiments i.e. biological replicates. P-values (*P<0.05, **P<0.01) were obtained by using Student ‘s t-test.
Fig 7.
Fourier–transform infrared spectroscopy (FTIR) analysis of arsenic adsorption.
FTIR analysis of arsenic adsorption on AT-02 biomass showing peak shifts in the finger print region and above 3000cm-.