Quantification of Naphthalene Dioxygenase (NahAC) and Catechol Dioxygenase (C23O) Catabolic Genes Produced by Phenanthrene-Degrading Pseudomonas fluorescens AH-40

Author(s): Asmaa M.M. Mawad, Wael S. Abdel-Mageed, Abd El-Latif Hesham*

Journal Name: Current Genomics

Volume 21 , Issue 2 , 2020

Become EABM
Become Reviewer
Call for Editor

Graphical Abstract:


Background: Petroleum polycyclic aromatic hydrocarbons (PAHs) are known to be toxic and carcinogenic for humans and their contamination of soils and water is of great environmental concern. Identification of the key microorganisms that play a role in pollutant degradation processes is relevant to the development of optimal in situ bioremediation strategies.

Objective: Detection of the ability of Pseudomonas fluorescens AH-40 to consume phenanthrene as a sole carbon source and determining the variation in the concentration of both nahAC and C23O catabolic genes during 15 days of the incubation period.

Methods: In the current study, a bacterial strain AH-40 was isolated from crude oil polluted soil by enrichment technique in mineral basal salts (MBS) medium supplemented with phenanthrene (PAH) as a sole carbon and energy source. The isolated strain was genetically identified based on 16S rDNA sequence analysis. The degradation of PAHs by this strain was confirmed by HPLC analysis. The detection and quantification of naphthalene dioxygenase (nahAc) and catechol 2,3-dioxygenase (C23O) genes, which play a critical role during the mineralization of PAHs in the liquid bacterial culture were achieved by quantitative PCR.

Results: Strain AH-40 was identified as pseudomonas fluorescens. It degraded 97% of 150 mg phenanthrene L-1 within 15 days, which is faster than previously reported pure cultures. The copy numbers of chromosomal encoding catabolic genes nahAc and C23O increased during the process of phenanthrene degradation.

Conclusion: nahAc and C23O genes are the main marker genes for phenanthrene degradation by strain AH-40. P. fluorescence AH-40 could be recommended for bioremediation of phenanthrene contaminated site.

Keywords: Bacteria, catabolic genes, isolation, PAHs pollutant, 16S rDNA, Pseudomonas fluorescens.

Zhu, X.; Fan, Z.T.; Wu, X.; Jung, K.H.; Ohman-Strickland, P.; Bonanno, L.J.; Lioy, P.J. Ambient concentrations and personal exposure to polycyclic aromatic hydrocarbons (PAH) in an urban community with mixed sources of air pollution. J. Expo. Sci. Environ. Epidemiol., 2011, 21(5), 437-449.
[http://dx.doi.org/10.1038/jes.2011.2] [PMID: 21364704]
Li, C.; Zhang, C.; Song, G.; Liu, H.; Sheng, G.; Ding, Z. Characterization of a protocatechuate catabolic gene cluster in Rhodococcus ruber OA1 involved in naphthalene degradation. Ann. Microbiol., 2016, 66, 469-478.
Park, J-W.; Crowley, D.E. Dynamic changes in nahAc gene copy numbers during degradation of naphthalene in PAH-contaminated soils. Appl. Microbiol. Biotechnol., 2006, 72(6), 1322-1329.
[http://dx.doi.org/10.1007/s00253-006-0423-5] [PMID: 16804694]
Ferrarese, E.; Andreottola, G.; Oprea, I.A. Remediation of PAH-contaminated sediments by chemical oxidation. J. Hazard. Mater., 2008, 152(1), 128-139.
[http://dx.doi.org/10.1016/j.jhazmat.2007.06.080] [PMID: 17689010]
Vogelsang, C.; Grung, M.; Jantsch, T.G.; Tollefsen, K.E.; Liltved, H. Occurrence and removal of selected organic micropollutants at mechanical, chemical and advanced wastewater treatment plants in Norway. Water Res., 2006, 40(19), 3559-3570.
[http://dx.doi.org/10.1016/j.watres.2006.07.022] [PMID: 16996560]
Hesham, A.E-L.; Mohamed, E.A.; Mawad, A.M.; Elfarash, A.; Abd El-Fattah, B.S.; El-Rawy, M.A. Molecular characterization of fusarium solani degrades a mixture of low and high molecular weight polycyclic aromatic hydrocarbons. Open Biotechnol. J., 2017, 11, 27-35.
Mawad, A.M.; Hesham, A.E-L.; Mostafa, Y.M.; Shoriet, A. Pyrene degrading Achromobacter denitrificans ASU-035: growth rate, enzymes activity, and cell surface properties. Rendiconti Lincei., 2016, 27, 557-563.
Kamyabi, A.; Nouri, H.; Moghimi, H. Characterization of pyrene degradation and metabolite identification by Basidioascus persicus and mineralization enhancement with bacterial-yeast co-culture. Ecotoxicol. Environ. Saf., 2018, 163, 471-477.
[http://dx.doi.org/10.1016/j.ecoenv.2018.07.098] [PMID: 30075450]
Chen, M.; Xu, P.; Zeng, G.; Yang, C.; Huang, D.; Zhang, J. Bioremediation of soils contaminated with polycyclic aromatic hydrocarbons, petroleum, pesticides, chlorophenols and heavy metals by composting: Applications, microbes and future research needs. Biotechnol. Adv., 2015, 33(6 Pt 1), 745-755.
[http://dx.doi.org/10.1016/j.biotechadv.2015.05.003] [PMID: 26008965]
Hesham, Ael-L.; Mawad, A.M.; Mostafa, Y.M.; Shoreit, A. Biodegradation ability and catabolic genes of petroleum-degrading Sphingomonas koreensis strain ASU-06 isolated from Egyptian oily soil. BioMed Res. Int., 2014, 2014 127674
Xia, W.; Du, Z.; Cui, Q.; Dong, H.; Wang, F.; He, P.; Tang, Y. Biosurfactant produced by novel Pseudomonas sp. WJ6 with biodegradation of n-alkanes and polycyclic aromatic hydrocarbons. J. Hazard. Mater., 2014, 276, 489-498.
[http://dx.doi.org/10.1016/j.jhazmat.2014.05.062] [PMID: 24929788]
Hennessee, C.T.; Li, Q.X. Effects of PAH mixtures on degradation, gene expression, and metabolite production in four Mycobacterium species. Appl. Environ. Microbiol., 2016, 82(11), AEM.00100-16.
Luo, A.; Wu, Y-R.; Xu, Y.; Kan, J.; Qiao, J.; Liang, L. Characterization of a cytochrome P450 monooxygenase capable of high molecular weight PAHs oxidization from Rhodococcus sp. P14. Process Biochem., 2016, 51, 2127-2133.
Zeng, J.; Zhu, Q.; Wu, Y.; Lin, X. Oxidation of polycyclic aromatic hydrocarbons using Bacillus subtilis CotA with high laccase activity and copper independence. Chemosphere, 2016, 148, 1-7.
[http://dx.doi.org/10.1016/j.chemosphere.2016.01.019] [PMID: 26784443]
Habe, H.; Omori, T. Genetics of polycyclic aromatic hydrocarbon metabolism in diverse aerobic bacteria. Biosci. Biotechnol. Biochem., 2003, 67(2), 225-243.
[http://dx.doi.org/10.1271/bbb.67.225] [PMID: 12728980]
Cerniglia, C.E. Biodegradation of polycyclic aromatic hydrocarbons. Microorganisms to combat pollution; Springer, 1992, pp. 227-244.
Duarte, M.; Nielsen, A.; Camarinha-Silva, A.; Vilchez-Vargas, R.; Bruls, T.; Wos-Oxley, M.L.; Jauregui, R.; Pieper, D.H. Functional soil metagenomics: elucidation of polycyclic aromatic hydrocarbon degradation potential following 12 years of in situ bioremediation. Environ. Microbiol., 2017, 19(8), 2992-3011.
[http://dx.doi.org/10.1111/1462-2920.13756] [PMID: 28401633]
Ahn, C.K.; Woo, S.H.; Park, J.M. Surface solubilization of phenanthrene by surfactant sorbed on soils with different organic matter contents. J. Hazard. Mater., 2010, 177(1-3), 799-806.
[http://dx.doi.org/10.1016/j.jhazmat.2009.12.104] [PMID: 20096994]
Herrick, J.B.; Stuart-Keil, K.G.; Ghiorse, W.C.; Madsen, E.L. Natural horizontal transfer of a naphthalene dioxygenase gene between bacteria native to a coal tar-contaminated field site. Appl. Environ. Microbiol., 1997, 63(6), 2330-2337.
[http://dx.doi.org/10.1128/AEM.63.6.2330-2337.1997] [PMID: 9172352]
McLellan, S.L.; Warshawsky, D.; Shann, J.R. The effect of polycyclic aromatic hydrocarbons on the degradation of benzo[a]pyrene by Mycobacterium sp. strain RJGII-135. Environ. Toxicol. Chem., 2002, 21(2), 253-259.
[http://dx.doi.org/10.1002/etc.5620210205] [PMID: 11833792]
Fuchs, G.; Boll, M.; Heider, J. Microbial degradation of aromatic compounds - from one strategy to four. Nat. Rev. Microbiol., 2011, 9(11), 803-816.
[http://dx.doi.org/10.1038/nrmicro2652] [PMID: 21963803]
Kim, Y-H.; Freeman, J.P.; Moody, J.D.; Engesser, K-H.; Cerniglia, C.E. Effects of pH on the degradation of phenanthrene and pyrene by Mycobacterium vanbaalenii PYR-1. Appl. Microbiol. Biotechnol., 2005, 67(2), 275-285.
[http://dx.doi.org/10.1007/s00253-004-1796-y] [PMID: 15592827]
Hesham, A.E-L.; Mawad, A.M.; Mostafa, Y.M.; Shoreit, A. Study of enhancement and inhibition phenomena and genes relating to degradation of petroleum polycyclic aromatic hydrocarbons in isolated bacteria. Microbiology, 2014, 83, 599-607.
Arulazhagan, P.; Vasudevan, N. Biodegradation of polycyclic aromatic hydrocarbons by a halotolerant bacterial strain Ochrobactrum sp. VA1. Mar. Pollut. Bull., 2011, 62(2), 388-394.
[http://dx.doi.org/10.1016/j.marpolbul.2010.09.020] [PMID: 20934193]
Hesham, A.E-L. New safety and rapid method for extraction of genomic DNA from bacteria and yeast strains suitable for PCR amplifications. J. Pure Appl. Microbiol., 2014, 8(1), 383-388.
Mawad, A.; Helmy, Y.A.; Shalkami, A-G.; Kathayat, D.; Rajashekara, G. E. coli Nissle microencapsulation in alginate-chitosan nanoparticles and its effect on Campylobacter jejuni in vitro. Appl. Microbiol. Biotechnol., 2018, 102(24), 10675-10690.
[http://dx.doi.org/10.1007/s00253-018-9417-3] [PMID: 30302522]
Patagundi, B.I.; Shivasharan, C.; Kaliwal, B. Isolation and characterization of cellulase producing bacteria from soil. Int. J. Curr. Microbiol. Appl. Sci., 2014, 3, 59-69.
Kumar, M.A.; Zamana, P.A.; Kumar, V.V.; Baskaralingam, P.; Thiruvengadaravi, K.V.; Amudha, T. Achromobacter xylosoxidans strain APZ for phthalocyanine dye degradation: chemo-metric optimization and canonical correlation analyses. J. Water Process Eng., 2017, 18, 73-82.
Elfarash, A.; Mawad, A.M.; Yousef, N.M.; Shoreit, A.A. Azoreductase kinetics and gene expression in the synthetic dyes-degrading Pseudomonas. Egyptian J. Basic and Appl. Sci., 2017, 4, 315-322.
Zeng, F.; Cui, K.; Li, X.; Fu, J.; Sheng, G. Biodegradation kinetics of phthalate esters by Pseudomonas fluoresences FS1. Process Biochem., 2004, 39, 1125-1129.
Ortega-Calvo, J-J.; Saiz-Jimenez, C. Effect of humic fractions and clay on biodegradation of phenanthrene by a Pseudomonas fluorescens strain isolated from soil. Appl. Environ. Microbiol., 1998, 64(8), 3123-3126.
[http://dx.doi.org/10.1128/AEM.64.8.3123-3126.1998] [PMID: 9687489]
Abbasnezhad, H.; Foght, J.M.; Gray, M.R. Adhesion to the hydrocarbon phase increases phenanthrene degradation by Pseudomonas fluorescens LP6a. Biodegradation, 2011, 22(3), 485-496.
[http://dx.doi.org/10.1007/s10532-010-9421-5] [PMID: 20886260]
Kumar, S.; Upadhayay, S.K.; Kumari, B.; Tiwari, S.; Singh, S.N.; Singh, P.K. In vitro degradation of fluoranthene by bacteria isolated from petroleum sludge. Bioresour. Technol., 2011, 102(4), 3709-3715.
[http://dx.doi.org/10.1016/j.biortech.2010.11.101] [PMID: 21177104]
Jin, D.; Jiang, X.; Jing, X.; Ou, Z. Effects of concentration, head group, and structure of surfactants on the degradation of phenanthrene. J. Hazard. Mater., 2007, 144(1-2), 215-221.
[http://dx.doi.org/10.1016/j.jhazmat.2006.10.012] [PMID: 17113708]
Han, M-J.; Choi, H-T.; Song, H-G. Degradation of phenanthrene by Trametes versicolor and its laccase. J. Microbiol., 2004, 42(2), 94-98.
[PMID: 15357301]
Lin, M.; Hu, X.; Chen, W.; Wang, H.; Wang, C. Biodegradation of phenanthrene by Pseudomonas sp. BZ-3, isolated from crude oil contaminated soil. Int. Biodeterior. Biodegradation, 2014, 94, 176-181.
Wang, B.; Xu, X.; Yao, X.; Tang, H.; Ji, F. Degradation of phenanthrene and fluoranthene in a slurry bioreactor using free and Ca-alginate-immobilized Sphingomonas pseudosanguinis and Pseudomonas stutzeri bacteria. J. Environ. Manage., 2019, 249 109388
[http://dx.doi.org/10.1016/j.jenvman.2019.109388] [PMID: 31466043]
Guo, C.; Dang, Z.; Wong, Y.; Tam, N.F. Biodegradation ability and dioxgenase genes of PAH-degrading Sphingomonas and Mycobacterium strains isolated from mangrove sediments. Int. Biodeterior. Biodegradation, 2010, 64, 419-426.
Ding, G-C.; Heuer, H.; Smalla, K. Dynamics of bacterial communities in two unpolluted soils after spiking with phenanthrene: soil type specific and common responders. Front. Microbiol., 2012, 3, 290.
[http://dx.doi.org/10.3389/fmicb.2012.00290] [PMID: 22934091]
Junca, H.; Pieper, D.H. Amplified functional DNA restriction analysis to determine catechol 2,3-dioxygenase gene diversity in soil bacteria. J. Microbiol. Methods, 2003, 55(3), 697-708.
[http://dx.doi.org/10.1016/S0167-7012(03)00214-8] [PMID: 14607412]
Nyyssönen, M.; Piskonen, R.; Itävaara, M. A targeted real-time PCR assay for studying naphthalene degradation in the environment. Microb. Ecol., 2006, 52(3), 533-543.
[http://dx.doi.org/10.1007/s00248-006-9082-4] [PMID: 17013553]
Tuomi, P.M.; Salminen, J.M.; Jørgensen, K.S. The abundance of nahAc genes correlates with the 14C-naphthalene mineralization potential in petroleum hydrocarbon-contaminated oxic soil layers. FEMS Microbiol. Ecol., 2004, 51(1), 99-107.
[http://dx.doi.org/10.1016/j.femsec.2004.07.011] [PMID: 16329859]
Okuta, A.; Ohnishi, K.; Harayama, S. PCR isolation of catechol 2,3-dioxygenase gene fragments from environmental samples and their assembly into functional genes. Gene, 1998, 212(2), 221-228.
[http://dx.doi.org/10.1016/S0378-1119(98)00153-X] [PMID: 9611265]

Rights & PermissionsPrintExport Cite as

Article Details

Year: 2020
Published on: 23 February, 2020
Page: [111 - 118]
Pages: 8
DOI: 10.2174/1389202921666200224101742
Price: $65

Article Metrics

PDF: 19
PRC: 1