Differential detection of key enzymes of polyaromatic-hydrocarbon-degrading bacteria using PCR and gene probes

Abstract

Bacteria with ability to degrade polyaromatic hydrocarbons (PAHs), isolated from wastewater and soil samples, were investigated for their taxonomic, physiological and genetic diversity. Eighteen isolates able to metabolize naphthalene or phenanthrene as sole carbon source were taxonomically affiliated to different subclasses of the Proteobacteria (Sphingomonas spp., Acidovorax spp., Comamonas spp. and Pseudomonas spp.) and to phyla of Gram-positive bacteria with low and high DNA G+C content (Paenibacillus sp. and Rhodococcus spp., respectively). Representatives of the genera Pseudomonas and Sphingomonas formed a remarkably high fraction of these isolates; 9 out of 18 strains belonged to these groups. Tests for enzyme activities showed that the majority of the isolates growing with PAHs as sole sources of carbon and energy had an active catechol2,3-dioxygenase (C230). C230 specific activities were very diverse, ranging from 0-1 to 650 mU (mg protein)-'. Pseudomonas and Sphingomonas strains showed considerably higher activities than the other isolates. All PAH degraders were examined for the presence of an initial PAH dioxygenase and C230, which catalyse key steps of PAH degradation, by PCR amplification of gene fragments and subsequent hybridization. PCR primers and internal oligonucleotide probes were developed for the specific detection of the genes of Pseudomonas and Sphingomonas strains. (1996). The aim of the present investigation was to isolate bacterial PAH degraders from aquatic a n d terrestrial sites, to determine their taxonomic affiliation and to characterize them physiologically a n d genetically with respect to their ability to degrade PAHs. al., 1996) are specific for most genuine pseudornonads, distinct P. putida strains, and the genera Comamonas and Acidovorax, respectively. Keywords METHODSFurther examination of the isolates was done by physiological tests as reported previously (Kampfer et al., 1991). Fatty acid methyl ester (FAME) patterns were used as a further marker for identification. Preparation of FAME extracts, gas chromatographic analysis and numerical identification by the MIDI system (Sherlock 2.11) were performed as described previously (Kampfer et al., 1996).Determination of naphthalene and phenanthrene mineralization potential. The potential of the isolated bacteria for mineralization of PAHs was examined by gas chromatographically quantifying remaining PAH and sirnultaneously monitoring bacterial growth in batch cultures.For each tested strain, five airtight flasks were each filled with 10 rnl basal medium containing either 005 ' / o naphthalene or phenanthrene and were inoculated with approximately 10' cells. Cell number was estimated by measuring the optical density at 600nm, Remaining PAH in the cultures was extracted by vigorously shaking (30 rnin) with 5 rnl n-hexane for gas chromatographic analysis. The gas chromatograph HP 6890 was equipped with a flame-ionization detector and a 25 m capillary column (Ultra 2, cross-linked 5 % phenyl methyl siloxan ; Hewlett Packard) ....

Isolation and Characterization of Polycyclic Aromatic Hydrocarbon-Degrading Bacteria Associated with the Rhizosphere of Salt Marsh Plants

“…nov., later to be emended as P. validus (22), which was able to utilize p-hydroxybenzoate, phthalate, isophthalate, protocatechuate, trimellitate, quinate, phenol, p-cresol, and naphthalene. Recently, Meyer et al (33) reported the isolation of a PAHdegrading tentative Paenibacillus sp. from tar oil-contaminated soil.…”

High aromatic ring-cleavage diversity in birch rhizosphere: PAH treatment-specific changes of I.E.3 group extradiol dioxygenases and 16S rRNA bacterial communities in soil

“…Fingerprinting of functional genes for key enzymes in catabolic pathways offers a powerful tool for assessing biodegradation potential in the environment. Previous studies on assessing the genetic potential for PAH degradation have been hampered by the relatively narrow scope of the PCR primers that are mainly targeted to the lower-pathway 1.2.A group extradiol dioxygenases, which catalyses ring cleavage of the catechols (Meyer et al, 1999;Widada et al, 2002;Junca and Pieper, 2004), a common intermediate of aromatic degradation. The targeted upper-pathway I.E.3 dioxygenases in our study are functionally different because the substrate is a double-hydroxylated aromatic compound including polyaromatic, substituted polyaromatic and biphenyl structures, which demand different substrate specificities for assorted structures (Eltis and Bolin, 1996; Bulk-1200 Number of OTUs Figure 5 OTU richness curves of extradiol dioxygenase gene libraries.…”

Biodegradation of High-Molecular-Weight Polycyclic Aromatic Hydrocarbons by Bacteria

“…Fresh insights into HMW PAH biodegradation, such as the observation that fluoranthene is anaerobically oxidized to carbon dioxide under sulfate-reducing conditions in ocean sediments (22), show that knowledge in the field of PAH biodeg- radation is expanding in new directions. Advances in molecular biology are aiding in the detection of PAH-degrading organisms from environmental samples (1,37,76) or in the differential detection of enzymes (73). Increases in our understanding of the microbial ecology of HMW PAH-degrading communities and the mechanisms by which HMW PAH biodegradation occur will prove helpful for predicting the environmental fate of these compounds and for developing practical PAH bioremediation strategies in the future.…”