Whole-genome sequencing, genome mining, metabolic reconstruction and evolution of pentachlorophenol and other xenobiotic degradation pathways in Bacillus tropicus strain AOA-CPS1.

A pentachlorophenol degrading bacterium was isolated from effluent of a wastewater treatment plant in Durban, South Africa, and identified as Bacillus tropicus strain AOA-CPS1 (BtAOA). The isolate degraded 29% of pentachlorophenol (PCP) within 9 days at an initial PCP concentration of 100 mg L-1 and 62% of PCP when the initial concentration was set at 350 mg L-1. The whole-genome of BtAOA was sequenced using Pacific Biosciences RS II sequencer with the Single Molecule, Real-Time (SMRT) Link (version 7.0.1.66975) and analysed using the HGAP4-de-novo assembly application. The contigs were annotated at NCBI, RASTtk and PROKKA prokaryotic genome annotation pipelines. The BtAOA genome is comprised of a 5,246,860-bp chromosome and a 58,449-bp plasmid with a GC content of 35.4%. The metabolic reconstruction for BtAOA showed that the organism has been naturally exposed to various chlorophenolic compounds including PCP and other xenobiotics. The chromosome encodes genes for core processes, stress response and PCP catabolic genes. Analogues of PCP catabolic gene (cpsBDCAE, and p450) sequences were identified from the NCBI annotation data, PCR-amplified from the whole genome of BtAOA, cloned into pET15b expression vector, overexpressed in E. coli BL21 (DE3) expression host, purified and characterized. Sequence mining and comparative analysis of the metabolic reconstruction of the BtAOA genome with closely related strains suggests that the operon encoding the first two enzymes in the PCP degradation pathway were acquired from a pre-existing pterin-carbinolamine dehydratase subsystem. The other two enzymes were recruited via horizontal gene transfer (HGT) from the pool of hypothetical proteins with no previous specific function, while the last enzyme was recruited from pre-existing enzymes from the TCA or serine-glyoxalase cycle via HGT events. This study provides a comprehensive understanding of the role of BtAOA in PCP degradation and its potential exploitation for bioremediation of other xenobiotic compounds.

Biotransformation of pentachlorophenol by an indigenous Bacillus cereus AOA-CPS1 isolated from wastewater effluent in Durban, South Africa.

Pentachlorophenol (PCP) is a recalcitrant biocide that bioaccumulates in the environment due to its persistent nature and has been listed as a priority pollutant due to its toxicological and health effects. In this study, a novel PCP-degrading Bacillus cereus strain AOA-CPS1 (BcAOA) was isolated from wastewater and characterized for PCP biotransformation in a batch reactor. The degradation kinetics were elucidated via substrate inhibition models, while PCP biotransformation was established by spectrophotometric and GC-MS analysis. BcAOA shared 95% sequence homology with Bacillus cereus strain XS2 and is closely related to some B. cereus strains which are previously reported to degrade PCP and other related pollutants. BcAOA degraded 74% of 350 mg l-1 of PCP within 9 days in a batch culture. The biotransformation of PCP by BcAOA followed the first and zero-order kinetics at low and high PCP concentration, respectively, with biokinetic constants: maximum biotransformation rate (0.0996 mg l-1 h-1); substrate inhibition constant (723.75 mg l-1); half-saturation constant (171.198 mg l-1) and R2 (0.98). The genes (pcpABCDE, cytochrome P450) encoding the enzymes involved in the biodegradation of PCP were amplified from the genomic DNA of BcAOA. Further, depending upon the genes amplified and identified metabolites using GC-MS, two different PCP biotransformation pathways were proposed in this study. Cloning and expression of the catabolic genes are underway to map out the concise pathway for PCP biotransformation by BcAOA.

Classic Pentachlorophenol Hydroxylating Phenylalanine 4-Monooxygenase from Indigenous Bacillus tropicus Strain AOA-CPS1: Cloning, Overexpression, Purification, Characterization and Structural Homology Modelling.

The metabolically promiscuous pentachlorophenol (PCP) hydroxylating Phe4MO (represented as CpsB) was detected, amplified (from the genome of Bacillus tropicus strain AOA-CPS1), cloned, overexpressed, purified and characterized here. The 1.755-kb gene cloned in the pET15b vector expressed a ≅ 64 kDa monomeric protein which was purified to homogeneity by single-step affinity chromatography, with a total yield of 82.1%. The optimum temperature and pH of the enzyme were found to be 30 °C and 7.0, respectively. CpsB showed functional stability between pH 6.0-7.5 and temperature 25-30 °C. The enzyme-substrate reaction kinetic studies showed the allosteric nature of the enzyme and followed pre-steady state using NADH as a co-substrate with apparent vmax, Km, kcat and kcat/Km values of 0.465 µM.s-1, 140 µM, 0.099 s-1 and 7.07 × 10-4 µM-1.s-1, respectively, for the substrate PCP. The in-gel trypsin digestion experiments and bioinformatic tools confirmed that the reported enzyme is a Phe4MO with multiple putative conserved domains and metal ion-binding site. Though Phe4MO has been reported to have a diverse catalytic function, here we report, for the first time, that it functions as a PCP dehalogenase or PCP-4-monooxygenase by hydroxylating PCP. Hence, the use of this enzyme may be further explored in the bioremediation of PCP and other related xenobiotics.

Role of tetrachloro-1,4-benzoquinone reductase in phenylalanine hydroxylation system and pentachlorophenol degradation in Bacillus cereus AOA-CPS1.

This study reports a ≅12.5 kDa protein tetrachloro-1,4-benzoquinone reductase (CpsD) from Bacillus cereus strain AOA-CPS1 (BcAOA). CpsD is purified to homogeneity with a total yield of 35% and specific activity of 160 U·mg-1 of protein. CpsD showed optimal activity at pH 7.5 and 40 °C. The enzyme was found to be functionally stable between pH 7.0-7.5 and temperature between 30 °C and 35 °C. CpsD activity was enhanced by Fe2+ and inhibited by sodium azide and SDS. CpsD followed Michaelis-Menten kinetic exhibiting an apparent vmax, Km, kcat and kcat/Km values of 0.071 µmol·s-1, 94 µmol, 0.029 s-1 and 3.13 × 10-4 s-1·µmol-1, respectively, for substrate tetrachloro-1,4-benzoquinone. The bioinformatics analysis indicated that CpsD belongs to the PCD/DCoH superfamily, with specific conserved protein domains of pterin-4α-carbinolamine  dehydratase (PCD). This study proposed that CpsD catalysed the reduction of tetrachloro-1,4-benzoquinone to tetrachloro-p-hydroquinone and released the products found in phenylalanine hydroxylation system (PheOHS) via a Ping-Pong or atypical ternary mechanism; and regulate expression of phenylalanine 4-monooxygenase by blocking reverse flux in BcAOA PheOHS using a probable Yin-Yang mechanism. The study also concluded that CpsD may play a catalytic and regulatory role in BcAOA PheOHS and pentachlorophenol degradation pathway.

Cloning, overexpression, purification, characterization and structural modelling of a metabolically active Fe2+ dependent 2,6-dichloro-p-hydroquinone 1,2-dioxygenase (CpsA) from Bacillus cereus strain AOA-CPS_1.

2,6-Dichloro-p-hydroquinone (DiCHQ) aromatic-ring cleavage by DiCHQ 1,2-dioxygenase (CpsA) is very crucial for complete transformation of pentachlorophenol (PCP) to 2-chloromaleylacetate in Bacillus cereus AOA-CPS_1 (BcAOA). The 978 bp gene (cpsA) was detected and amplified in the genome of BcAOA; cloned, overexpressed and purified to homogeneity. CpsA showed a single ≅36.9 kDa protein band on SDS-PAGE and exhibited optimum activity at 30 °C and pH 9.0. CpsA was stable between 20 °C and 40 °C, and also retained about 90% of its activity at 60 °C for 120 min. The enzyme retained about 90% activity between pH 9.0 and 11.5 and 60% activity at pH 13.0. CpsA was found to be Fe2+ dependent as about 90% increased activity was observed in the presence of FeSO4. CpsA showed apparent vmax, Km, kcat and kcat/Km of 27.77 ± 0.9 µMs-1, 0.990 ± 0.03 mM, 4.20 ± 0.04 s-1 and 4.24 ± 0.03 s-1 mM-1, respectively at pH 9.0. Analysis of the reaction products via GC-MS confirmed 2-chloromaleylacetate as the ring-cleavage product. CpsA 3D structure revealed a conserved 2-His-1-carboxylate facial triad motif (His 9, His 244 and Thr 11), with Fe3+ at the centre. Findings from this study provide new insights into the involvement of this enzyme in PCP degradation and suggests alternate possible mechanism of ring-cleavage by dioxygenases.