Whole Genome Shotgun Sequencing-Based Insights into the Benzene and Xylene Degrading Potentials of Bacteria

MUCCEE, FATIMA; MOHIUDDIN, FARHAN; SHAHAB, AANSA; ALMAJWAL, ALI; AFSAR, TAYYABA; AMOR, HOUDA; RAZAK, SUHAIL

doi:10.33073/pjm-2025-020

Figures & Tables

Total bases, GC and AT contents, and Q20 and Q30 ratios of total reads calculated during WGS analysis.

Krona image of taxonomic assignments to metagenomic contigs by Kraken.

Pathways associated with aromatic compound metabolism, with their respective abundances, explored in a bacterial consortium using whole genome sequencing.
BD – benzoate degradation, ABD – aminobenzoate degradation, FBD – fluorobenzoate degradation, CLAD – chloroalkane and chloroalkene degradation, CLHD – chlorocyclohexane and chlorobenzene degradation, TD – toluene degradation, XD – xylene degradation, SD – styrene degradation, AD – atrazine degradation, CD – caprolactam degradation, DD – dioxin degradation, ND – naphthalene degradation, PAHD – polycyclic aromatic hydrocarbon degradation, SD – steroid degradation, MP450 – metabolism of xenobiotics by cytochrome P450 — Pathways associated with aromatic compound metabolism, with their respective abundances, explored in a bacterial consortium using whole genome sequencing. BD – benzoate degradation, ABD – aminobenzoate degradation, FBD – fluorobenzoate degradation, CLAD – chloroalkane and chloroalkene degradation, CLHD – chlorocyclohexane and chlorobenzene degradation, TD – toluene degradation, XD – xylene degradation, SD – styrene degradation, AD – atrazine degradation, CD – caprolactam degradation, DD – dioxin degradation, ND – naphthalene degradation, PAHD – polycyclic aromatic hydrocarbon degradation, SD – steroid degradation, MP450 – metabolism of xenobiotics by cytochrome P450

Reconstruction of ortho, meta and para-xylene degradation pathways based on whole genome shotgun functional annotation.

Reconstruction of benzene, 3-aminobenzene sulfonate, toluene and 2-, 3- and 4-fluorobenzoate degradation pathways based on whole genome shotgun functional annotation.

Reactions of benzene and xylene metabolism catalyzed by bacterial enzymes identified in the current study using whole genome shotgun functional annotation.

Statistics of contigs produced by the metaSPAdes program_

No.	Quast quality parameters	Statistics (bp)
1	Contigs > 10,000 bp	4,992
2	Contigs ≥ 5,000 bp	940
3	Contigs ≥ 10,000 bp	431
4	Contigs ≥ 25,000 bp	169
5	Contigs ≥ 50,000 bp	76
6	Largest contig	469,435
7	Total length	30,570,959
8	Total length ≥ 1,000 bp	27,559,880
9	Total length ≥ 5,000 bp	19,294,370
10	Total length ≥ 10,000 bp	15,759,083
11	Total length ≥ 25,000 bp	11,859,173
12	Total length ≥ 50,000 bp	8,511,336
13	N50	10,938

Stoichiometry of reactions involved in benzene, xylene and toluene degradation identified in the current study bacteria based on WGS_

No. of reaction	Stoichiometry of reactions
No. of reaction	Reactants	Products
Benzene degradation pathway
I	C₆H₆ + H₂ + CO₂ benzene	C₇H₈O + [O^-]benzyl alcohol
II	C₇H₈O	C₇H₆O + H₂benzaldehyde
III	C₇H₆O + [O^-]	C₇H₆O₂benzoate
IV	C₇H₆O₂ + [O^-] + H₂O	C₇H₈O₄cis-1,2-dihydroxycyclohexa-3,5-diene-1-carboxylate
V	C₇H₈O₄	C₆H₆O₂catechol
VI	C₆H₆O₂ + 2 [H⁺]	C₆H₅O₄^- + 3 [H⁺]cis, cis-muconate
VII	C₆H₅O₄^-	C₆H₆O₄muconolactone
VIII	C₆H₆O₄	C₆H₅O₄+ [H⁺]3-oxoadipate enol lactone
IX	C₆H₅O₄	C₆H₅O₅^2-3-oxoadipate
X	C₆H₅O₅^2- + [H⁺] + C₂₁H₃₆N₇O₁₆P₃S	C₂₇H₄₂N₇O₂₀P₃S + [O]3-oxoadipyl-CoA
XI	C₂₇H₄₂N₇O₂₀P₃S + 5H₂O	C₂₃H₃₈N₇O₁₇P₃S + 4CO₂+ 7H₂Oacetyl-CoA
p-Xylene degradation pathway
I	C₈H₁₀ + [O^-] p-xylene	C₈H₁₀O4-methylbenzyl alcohol
II	C₈H₁₀O + CO₂	C₉H₁₀O₂ + [O^-]p-methylbenzoate
III	C₉H₁₀O₂ + 2O₂	C₈H₉O₄ + CO₂ + [H⁺]cis-1-2-dihydroxy-4-methylcyclohexa-3,5-diene-1-carboxylate
IV	C₈H₉O₄	C₇H₈O₂ + CO₂ + [H⁺]4-methylcatechol
V	C₇H₈O₂ + O₂	C₇H₈O₄2-hydroxyl-5-methyl-cis, cis-muconic semialdehyde
VI	C₇H₈O₄ + [O]	C₇H₈O₅2-hydroxyl-5-methyl-cis, cis-muconate
VII	C₇H₈O₅	C₇H₈O₅2-oxo-5-methyl-cis-muconate
VIII	C₇H₈O₅	C₆H₈O₃ + CO₂2-hydroxyl-cis-hexa-2,4-dienoate
IX	C₆H₈O₃ + H₂O	C₆H₁₀O₄4-hydroxyl-2-oxohexanoate
X	C₆H₁₀O₄ + 2O₂ + H₂O	3CO₂+ C₃H₃O₃^- + H₂Opyruvate
m-Xylene degradation pathway
I	C₈H₁₀+ [O] m-xylene	C₈H₁₀O3-methylbenzyl alcohol
II	C₈H₁₀O	C₈H₈O + H₂3-methylbenzaldehyde
III	C₈H₈O + CO₂+ H₂	C₉H₁₀O₂ + H₂Om-methylbenzoate
IV	C₉H₁₀O₂ + CO₂	C₈H₈O₄ + H₂1,2-dihydroxy-3-methylcyclohexa-3,5-dienecarboxylate
o-Xylene degradation pathway
I	C₈H₁₀ + [O^-] o-xylene	C₈H₁₀O2-methylbenzyl alcohol
II	C₈H₁₀O	C₈H₈O + H₂2-methylbenzaldehyde
III	C₈H₈O + CO₂ + H₂	C₉H₁₀O₂ + [O]o-methylbenzoate
IV	C₉H₁₀O + CO₂	C₈H₁₀O₄1,2-dihydroxy-6-methylcyclohexa-3,5-dienecarboxylate
V	C₈H₁₀O₄	C₇H₈O₂ + CO₂ + H₂3-methylcatechol
VI	C₇H₈O₂ + O₂	C₇H₈O₄cis, cis-2-hydroxyl-6-oxohept-2,4-dienoate
VII	C₇H₈O₄ + [O^-]	C₅H₆O₃ + 2CO₂ + H₂cis-2-hydroxy penta-2,4-dienoate
VIII	C₅H₆O₃ + H₂O	C₅H₇O₄^- + [H⁺]4-hydroxyl-2-oxopentanoate
IX	C₅H₇O₄^- + [O^-]	C₃H₃O₃^- + 2CO₂ + 2H₂ pyruvate
Toluene degradation pathway
I	C₆H₅CH₃ + [O] toluene	C₆H₅CH₂OHbenzyl alcohol
II	C₆H₅CH₂OH benzyl alcohol	C₆H₅CHO + H₂benzaldehyde
III	C₆H₅CHO + [O] benzaldehyde	C₇H₅O₂ + [H]benzoate

Functional annotation of genes associated with aromatic compound degradation via the KEGG database_

No.	Associated biochemical pathway	Genes identified
1	Xylene degradation pathway	toluene methyl-monooxygenase [EC:1.14.15.26],aryl-alcohol dehydrogenase [EC:1.1.1.90],benzaldehyde dehydrogenase (NAD) [EC:1.2.1.28],benzoate/toluate 1,2-dioxygenase subunit α [EC:1.14.12.-],dihydroxycyclohexadiene carboxylate dehydrogenase [EC:1.3.1.-, 1.3.1.67, 1.3.1.68],catechol 2,3-dioxygenase [EC:1.13.11.2],2-hydroxymuconate-semialdehyde hydrolase [EC:3.7.1.9],2-oxopent-4-enoate/cis-2-oxohex-4-enoate hydratase [EC:4.2.1.80, 4.2.1.132],4-hydroxy-2-oxovalerate/4-hydroxy-2-oxohexanoate aldolase [EC:4.1.3.39 4.1.3.43],acetaldehyde/propanal dehydrogenase [EC:1.2.1.10, 1.2.1.87],aminomuconate-semialdehyde/2-hydroxymuconate-6-semialdehydedehydrogenase [EC:1.2.1.32 1.2.1.85],4-oxalocrotonate tautomerase [EC:5.3.2.6],2-oxo-3-hexenedioate decarboxylase [EC:4.1.1.77],2-keto-4-pentenoate hydratase [EC:4.2.1.80],4-hydroxy 2-oxovalerate aldolase [EC:4.1.3.39],acetaldehyde dehydrogenase [EC:1.2.1.10]
2	Toluene degradation pathway	toluene methyl-monooxygenase [EC:1.14.15.26],aryl-alcohol dehydrogenase [EC:1.1.1.90],benzaldehyde dehydrogenase (NAD) [EC:1.2.1.28]
3	Benzene	benzoate/toluate 1,2-dioxygenase subunit alpha [EC:1.14.12.10], dihydroxycyclohexadiene carboxylate dehydrogenase [EC:1.3.1.25],catechol 1,2-dioxygenase [EC:1.13.11.1],muconate cycloisomerase [EC:5.5.1.1],muconolactone D-isomerase [EC:5.3.3.4],3-oxoadipate enol-lactonase [EC:3.1.1.24],3-oxoadipate CoA-transferase, alpha subunit [EC:2.8.3.6],acetyl-CoA acyltransferase [EC:2.3.1.16],3-oxoadipyl-CoA thiolase [EC:2.3.1.174],catechol 2,3-dioxygenase [EC:1.13.11.2],2-hydroxymuconate-semialdehyde hydrolase [EC:3.7.1.9],2-keto-4-pentenoate hydratase [EC:4.2.1.80],4-hydroxy 2-oxovalerate aldolase [EC:4.1.3.39],acetaldehyde dehydrogenase [EC:1.2.1.10],protocatechuate 3,4-dioxygenase, alpha subunit [EC:1.13.11.3],3-carboxy-cis, cis-muconate cycloisomerase [EC:5.5.1.2],4-carboxymuconolactone decarboxylase [EC:4.1.1.44],3-hydroxybenzoate 6-monooxygenase [EC:1.14.13.24]
3	Aminobenzoate degradation pathway	benzaldehyde dehydrogenase (NAD) [EC:1.2.1.28],amidase [EC:3.5.1.4],4-hydroxybenzoate decarboxylase subunit C [EC:4.1.1.61]
4	Fluorobenzoate degradation pathway	benzoate/toluate 1,2-dioxygenase subunit alpha [EC:1.14.12.10],dihydroxycyclohexadiene carboxylate dehydrogenase [EC:1.3.1.25],catechol 1,2-dioxygenase [EC:1.13.11.1],muconate cycloisomerase [EC:5.5.1.1]

Whole Genome Shotgun Sequencing-Based Insights into the Benzene and Xylene Degrading Potentials of Bacteria

Figures & Tables

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Statistics of contigs produced by the metaSPAdes program_

Stoichiometry of reactions involved in benzene, xylene and toluene degradation identified in the current study bacteria based on WGS_

Functional annotation of genes associated with aromatic compound degradation via the KEGG database_

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