Figure 1.
Predicted positions for the activity of pnpA and pnpB in p-nitrophenol (PNP) degradation pathway of strain SJ98.
Figure 2.
Comparison of p-nitrophenol (PNP) degradation gene clusters from different bacterial strains, schematic representation of the ORFs and distance between the gene clusters when arranged together as shown in pnpAB and pnpCDE1E2F
(A) schematic representation of distance between Contig 11 (NZ_AJHK02000011.1) and contigs 12 (NZ_AJHK02000012.1) (B) if contigs 12 and then contigs 11 placed in series together then distance between the gene clusters and (C) PNP degradation gene clusters found in the other PNP degrading bacteria Pseudomonas sp. NyZ402 (GU123925.1), Pseudomonas putida DLLE-4 (FJ376608), Pseudomonas sp. 1-7 (FJ821777) and Pseudomonas sp. WBC-3 (EF577044).
Similar genes have been assigned by the same colors.
Figure 3.
Representative SDS PAGE gel picture of
(A) His-6-PnpA purification by Ni-NTA chromatography, Lane M) Marker, Lane 1) uninduced, 2) induced whole cell lysate, 3) Induced supernatant, 4) pellet, 5) & 6) flow through, 7) wash and lane 8) Elution of His-6-PnpA is showing with a red arrow.(B) His-6-PnpB purification by Ni-NTA chromatography, Lane M) Marker, Lane1) uninduced , 2) induced whole cell lysate, 3) Induced supernatant, 4) pellet, 5) & 6) flow through, 7) wash and lane 8) Elution of His-6-PnpB is showing with a red arrow.
Figure 4.
Enzyme assay of p-nitrophenol 4-monooxygenase (PnpM)
(A) Spectrophotometric analysis PnpM activity as shown by decrease in spectral absorption at 420 and 340 nm corresponding to depletion of p-nitrophenol (PNP) and NADH in a time dependent manner. (B) Lack of PnpM activity in negative control sample (C) Enzymatic transformation of PNP to BQ was confirmed by HPLC analysis.
Figure 5.
Time course analysis of PNP transformation by purified His-6-PnpA.
The samples were collected at different time points and analyzed for the formation of BQ and HQ by HPLC analysis. Standard deviation values were calculated by taking the mean of triplicate experiments.
Figure 6.
Enzyme assay of p-benzoquinone reductase (BqR) (A) Spectrophotometric analysis of BqR activity of PnpB; depletion in the p-benzoquinone along with NAD(P)H (B) Negative control of BqR activity without protein.
Figure 7.
Phylogenetic tree of amino acid sequences (A) p-nitrophenol 4-monooxygenase (PnpM), Benzenetriol dioxygenase (BtD) from stain SJ98 was taken as outgroup for the phylogenetic tree for PNP monooxygenases and (B) p-benzoquinone reductase (BqR), nitrite reductase from E. coli was taken as an outgroup to create the phylogenetic tree for the benzoquinone reductases.
Neighbor joining method was applied at the bootstrap value of 1000. Accession number of the proteins is written in parentheses.