Abstract
Polycyclic aromatic hydrocarbon (PAH)-degrading bacteria isolated from PAH-contaminated soils were analyzed genotypically and phenotypically for their capacity for metabolism of naphthalene and other PAH substrates. The methods used for the analyses were DNA hybridization using NAH7-derived gene probes, PAH spray plate assays, 14C-PAH mineralization assays, and dioxygenase activity assays. The results of the analyses showed a dominant number of PAH-degrading bacteria with a NAH7-like genotype. The results support the continued use of the nahA probe for contaminated soils to monitor the genetic potential of indigenous microorganisms to degrade PAHs. However, the finding of non-it nahA-hybridizing PAH-degrading bacteria show the limitation of NAH7-derived gene probes. Fifteen percent (13/89) of PAH-degrading bacteria isolated were not detected with the nahA gene probe. Four isolates (designated A5PH1, A8AN3, B1PH2, and B10AN1) did not hybridize with any of the NAH7-derived gene probes ( nahA, nahG, nahH, and nahR) used in this study. Considering the numerous unculturable microorganisms in nature and their potential genotypes, NAH7-derived gene probes may underestimate the microbial potential to catabolize PAHs. This necessitates development of new gene probes for enumeration and isolation of PAH-degrading bacteria to better understand the in situ microbial potential to degrade PAHs.
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Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA & Struhl K (1989) Current protocols in molecular biology. John Wiley & Sons, New York
Bagdasarian MRL, Ruckert B, Franklin FCH, Bagdasarian MM, Frey J & Timmis KN (1981) Specific-purpose plasmid cloning vectors. II. Broad host range, high copy number, RSF1010-derived vectors, and a host-vector system for gene cloning in Pseudomonas. Gene 16: 237–247
Bagdasarian MM, Amann E, Lurz R, Ruckert B & Bagdasarian M (1983) Activity of the hybrid trp-lac (tac) promoter of Escherichia coli in Pseudomonas putida. Construction of broad-hostrange, controlled-expression vectors. Gene 26: 273–282
Church GM & Gilbert W (1984) Genomic sequencing. Proc. Natl. Acad. Sci. USA 81: 1991–1995
Cushey MA & Morgan DJ (1990) Biological treatment of soils containing Manufactured Gas Plant residues. In: Gas Research Institute Topical Report No. GRI-90/0117, Gas Research Institute, Chicago, Ill
Davies JI & Evans WC (1964) Oxidative metabolism of naphthalene by soil pseudomonads. Biochem. J. 91: 251–261
Denome SA, Stanley DC, Olson ED & Young KD (1993) Metabolism of dibenzothiophene and naphthalene in Pseudomonas strains: complete DNA sequence of an upper naphthalene catabolic pathway. J. Bacteriol. 175: 6890–6901
Dunn NW & Gunsalus IC (1973) Transmissible plasmid coding early enzymes of naphthalene oxidation in Pseudomonas putida. J. Bacteriol. 114: 974–979
Eaton RW & Timmis KN (1986) Characterization of a plasmid-specified pathway for catabolism of isopropylbenzene in Pseudomonas putida RE204. J. Bacteriol. 168: 123–131
Eaton RW & Chapman PJ (1992) Bacterial metabolism: construction and use of recombinant bacteria to study ring cleavage of 1,2-dihydroxynaphthalene and subsequent reactions. J. Bacteriol. 174: 7542–7554
Ensley BD, Ratzkin BJ, Osslund TD, Simon MJ, Wackett LP & Gibson DT (1983) Expression of naphthalene oxidation genes in Escherichia coli results in the biosynthesis of indigo. Science 222: 167–169
Festl HWL & Schleifer KH (1986) DNA hybridization probe for the Pseudomonas fluorescens group. Appl. Environ. Microbiol. 52: 1190–1194
Fleming JT, Sanseverino J & Sayler GS (1993) Quantitative relationship between naphthalene catabolic gene frequency and expression in predicting PAH degradation in soils at town gas manufacturing sites. Environ. Sci. Tech. 27: 1068–1074
Foght JM & Westlake DW (1988) Degradation of polycyclic aromatic hydrocarbons and aromatic heterocycles by Pseudomonas species. Can. J. Microbiol. 34: 1135–1141
Fredrickson JK, Brockman FJ, Workman DJ, Li SW & Stevens TO (1991) Isolation and characterization of a subsurface bacterium capable of growth on toluene, naphthalene, and other aromatic compounds. Appl. Environ. Microbiol. 57: 796–803
Ghosal D, You LS & Gunsalus IC (1987) Nucleotide sequence and expression of gene nahH of plasmid NAH7 and homology with gene xylE of TOL pWWO. Gene 55: 19–28
Gibson DT, Donald VM, Jerina M, Yagi H & Yeh HJC (1975) Oxidation of the carcinogens benzo[a]pyrene and benz[a]anthracene to dihydrodiols by a bacterium. Science 189: 295–297
Goyal AK & Zylstra GJ (1996) Molecular cloning of novel genes for polycyclic aromatic hydrocarbon degradation from Comamonas testosteroni GZ39. Appl. Environ. Microbiol. 62: 230–236
Grund AD & Gunsalus IC (1983) Cloning of genes for naphthalene metabolism in Pseudomonas putida. J. Bacteriol. 156: 89–94
Harvey RG (1991) PAHs, Chemistry and Carcinogenicity. Cambridge Univ. Press
Heitkamp MA & Cerniglia CA (1987) Effects of chemical structure and exposure on the microbial degradation of polycyclic aromatic hydrocarbons in freshwater and estuarine ecosystems. Env. Tox. Chem. 6(7): 535–546
Heitkamp MA, Freeman JP, Miller DW & Cerniglia CA (1988) Pyrene degradation by Mycobacterium sp.: Identification of ring oxidation and ring fission products. Appl. Environ. Microbiol. 54: 2556–2565
Kastner M, Breuer-Jammali M & Mahro B (1994) Enumeration and characterization of the soil microflora from hydrocarbon-contaminated soil sites able to mineralize polycyclic aromatic hydrocarbons (PAH). Appl. Microbiol. Biotech. 41: 267–273
King JMH, Sanseverino J, Digrazia PM, Applegate BM, Sayler GS & Blackburn JW (1991) Molecular bioanalytical methods for monitoring PAH biodegradation in manufactured gas plant soils, Final report, Vol. II, Gas Research Institute, Chicago
Kiyohara H, Nagao K & Yano K (1982) Rapid screen for bacteria degrading water-insoluble, solid hydrocarbons on agar plates. Appl. Environ. Microbiol. 43: 454–457
Kiyohara H, Takizawa N & Nagao K (1992) Natural distribution of bacteria metabolizing many kinds of polycyclic aromatic hydrocarbons. J. Ferment. Bioeng. 74: 49–51
Leblond J, Applegate BM, Menn F-M & Sayler GS (1995). NAH7-mediated metabolism of methyl-substituted naphthalenes. Abstr. Q-412, In Abstracts of the 95th General Meeting of the American Society of Microbiology. American Society of Microbiology, Washington, DC
Mackay D & Shiu WY (1977) Aqueous solubility of PAHs. J. Chem. Eng. Data 22: 399–402
Means JR, Ward SG, Hassett JJ & Banwart WL (1980) Sorption of polynuclear aromatic hydrocarbons by sediments and soils. Environ. Sci. Technol. 14: 1524–1528
Mueller JG, Lantz SE, Devereux R, Berg JD & Pritchard PH (1994) Studies on the microbial ecology of polycyclic aromatic hydrocarbon biodegradation. In: Hinchee RE, Leeson A, Semprini L & Ong SK (Eds) Bioremediation of Chlorinated and Polycyclic Aromatic Hydrocarbon Compounds (pp. 218–230). Lewis Publishers, Boca Raton, Florida
Sambrook J, Fritsch EF & Maniatis T (1989) Molecular Cloning: A Laboratory Manual (2nd ed.). Cold Spring Harbor Lab. Press, New York
Sanseverino J, Applegate B, K. Robinson & Sayler GS (1991) Analysis of plasmids isolated from polynuclear aromatic hydrocarbon-degrading bacteria. Abstract presented at the 91st Annual Meeting of the American Society for Microbiology, Dallas, Texas
Sanseverino J, Applegate BM, King JHM & Sayler GS (1993a) Plasmid-mediated mineralization of naphthalene, phenanthrene, and anthracene. Appl. Environ. Microbiol. 59: 1931–1937
Sanseverino J, Werner C, Fleming JT, Applegate BM, King JHM & Sayler GS (1993b) Molecular diagnostics of polycyclic aromatic hydrocarbon degradation in manufactured gas plant soils. Biodegradation 4: 303–321
Schneider J, Grosser R, Jayasimhulu K, Xue W & Warshawsky D (1996) Degradation of pyrene, benzo[a]anthracene, and benzo[a]pyrene by Mycobacterium sp. strain RJGII-135, isolated from a former coal gasification site. Appl. Environ. Microbiol. 62: 13–19
Selifonov, SA, Grifoll M, Eaton RW, & Chapman PJ. (1996) Oxidation of naphthenoaromatic and methyl-substituted aromatic compounds by naphthalene 1,2-dioxygenase. Appl. Environ. Microbiol. 62: 507–514
Shiaris MP (1989). Seasonal biotransformation of naphthalene, phenanthrene, and benzo[a]pyrene in surficial estuarine sediments. Appl. Environ. Microbiol. 55: 1391–1399
Simon MJ, Osslund TD, Saunder R, Ensley BTD, Suggs S, Harcourt A, Suen W, Cruden DL, Gibson DT & Zylstra GJ (1993) Sequence of genes encoding naphthalene dioxygenase in Pseudomonas putida strains G7 and NCIB 9816–4. Gene 127: 31–37
Starovoitov II, Nefedova MY, Yakovlev GI, Zyakun AM & Adanin VM (1975) Gentisic acid as a microbial oxidation product of naphthalene. Izv. Akad. Nauk. SSSR Ser. Khim. 9: 2091–2092
Tausson WO (1927) Naphthalin als kohlenstoffquelle fur bakterien. Planta 4: 214–256
Walter U, Beyer M, Klein J & Rehm H-J (1991) Degradation of pyrene by Rhodococcus sp. UW1. Appl. Microbiol. Biotech. 34: 671–676
Weissenfels WD, Beyer M & Klein J (1992) Adsorption of polycyclic aromatic hydrocarbons (PAHs) by soil particles: influence on biodegradability and biotoxicity. Appl. Microbiol. Biotechnol. 36: 689–696
Wubbolts MG, Reuvekamp P & Witholt B (1994) TOL plasmid-specified xylene oxygenase is a wide substrate range monooxygenase capable of olefin epoxidation. Enz Microbial Technol. 16(7): 608–615
Yang YY, Chen RF & Shiaris MP (1994) Metabolism of naphthalene, fluorene, and phenanthrene: preliminary characterization of a cloned gene cluster from Pseudomonas putida NCIB 9816. J. Bacteriol. 176: 2158–2164
Yen K-M & Gunsalus IC (1982) Plasmid gene organization: naphthalene/salicylate oxidation. Proc. Natl. Acad. Sci. USA 79: 874–878
Yen K-M, Karl MR, Blatt LM, Simon MJ, Winter RB, Fausset PR, Lu HS, Harcourt AA & Chen KK (1991) Cloning and characterization of a Pseudomonas mendocina KR1 gene cluster encoding toluene-4-monooxygenase. J. Bacteriol. 173: 5315–5327
You I-S, Ghosal D & Gunsalus IC (1988) Nucleotide sequence of plasmid NAH7 gene nahR and DNA binding of nahR product. J. Bacteriol. 170: 5409–5415
You I-S, Ghosal D & Gunsalus IC (1991) Nucleotide sequence analysis of the Pseudomonas putida PpG7 salicylate hydroxylase gene (nahG) and its 3′-flanking region. Biochem. 30: 1635–1641
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Ahn, Y., Sanseverino, J. & Sayler, G.S. Analyses of polycyclic aromatic hydrocarbon-degrading bacteria isolated from contaminated soils. Biodegradation 10, 149–157 (1999). https://doi.org/10.1023/A:1008369905161
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DOI: https://doi.org/10.1023/A:1008369905161