Differential detection of key enzymes of polyaromatic-hydrocarbon-degrading bacteria using PCR and gene probes.
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 catechol 2,3-dioxygenase (C230). C230 specific activities were very diverse, ranging from 0.1 to 650 mU (mg protein)-1. 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.
Meyer
Svenja
S
Justus-Liebig-University Giessen, Institute of Applied Microbiology, Senckenbergstraße 3, D-35390 Giessen, Germany.
Moser
Ralf
R
University of Technology Berlin, Institute of Biotechnology, Department of Microbiology and Genetics, Gustav-Meyer-Allee 25, D-13355 Berlin, Germany.
Neef
Alexander
A
Justus-Liebig-University Giessen, Institute of Applied Microbiology, Senckenbergstraße 3, D-35390 Giessen, Germany.
Stahl
Ulf
U
University of Technology Berlin, Institute of Biotechnology, Department of Microbiology and Genetics, Gustav-Meyer-Allee 25, D-13355 Berlin, Germany.
Kämpfer
Peter
P
Justus-Liebig-University Giessen, Institute of Applied Microbiology, Senckenbergstraße 3, D-35390 Giessen, Germany.
D050557:Catechol 2,3-Dioxygenase; D015342:DNA Probes; D049308:Dioxygenases; D006090:Gram-Negative Bacteria / Q000145:classification / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D006094:Gram-Positive Bacteria / Q000145:classification / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D007220:Industrial Waste; D009281:Naphthalenes / Q000378:metabolism; D009693:Nucleic Acid Hybridization; D010105:Oxygenases / Q000378:metabolism; D010616:Phenanthrenes / Q000378:metabolism; D011084:Polycyclic Aromatic Hydrocarbons / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods; D012988:Soil Microbiology; D014871:Water Microbiology
D015342:DNA Probes; D007220:Industrial Waste; D009281:Naphthalenes; D010616:Phenanthrenes; D011084:Polycyclic Aromatic Hydrocarbons; C031721:naphthalene; C031181:phenanthrene; D010105:Oxygenases; D049308:Dioxygenases; D050557:Catechol 2,3-Dioxygenase