In our previous study, Pseudomonas fluorescens Pf0-1L, harbouring the IncP-7 carbazole-degradative plasmid pCAR1 : : rfp, was shown to be undetectable within 5 days post-inoculation in carbazole-contaminated artificial freshwater microcosms containing several plasmid-free bacteria in addition to Pf0-1L(pCAR1 : : rfp). Fourteen days after the inoculation, carbazole degraders become detectable. Here, we revealed that these isolates were not pCAR1 transconjugants, but Pf0-1L(pCAR1 : : rfp) mutants, based on RFLP and BOX-A1R-based repetitive extragenic palindromic-PCR (BOX-PCR) analysis. Notably, the mutants displayed more rapid initiation of carbazole degradation than the parent strain Pf0-1L(pCAR1 : : rfp). The mutants were unable to degrade anthranilate due to a 163 bp deletion in the antA gene, which was overcome by their transformation with a wild-type antABC-expressing plasmid. Quantitative RT-PCR analysis indicated that the transcriptional induction of carbazole-, anthranilate- and catechol-degradative genes was comparable in both parent and mutant strains. The deletion mutants became dominant in the artificial water microcosm. The mutation caused anthranilate to accumulate instead of catechol, a toxic compound for the parent strain, and may be beneficial to host survival in artificial microcosms.

Four bacterial strains isolated from hydrocarbon-contaminated soils in Lagos, Nigeria, displayed extensive degradation abilities on carbazole, an N-heterocyclic aromatic hydrocarbon. Physicochemical analyses of the sampling sites (ACPP, MWO, NESU) indicate gross pollution of the soils with a high hydrocarbon content (157,067.9 mg/kg) and presence of heavy metals. Phylogenetic analysis of the four strains indicated that they were identified as Achromobacter sp. strain SL1, Pseudomonas sp. strain SL4, Microbacterium esteraromaticum strain SL6, and Stenotrophomonas maltophilia strain BA. The rates of degradation of carbazole by the four isolates during 30 days of incubation were 0.057, 0.062, 0.036, and 0.050 mg L(-1) h(-1) for strains SL1, SL4, SL6, and BA. Gas chromatographic (GC) analyses of residual carbazole after 30 days of incubation revealed that 81.3, 85, 64.4, and 76 % of 50 mg l(-1) carbazole were degraded by strains SL1, SL4, SL6, and BA, respectively. GC-mass spectrometry and high-performance liquid chromatographic analyses of the extracts from the growing and resting cells of strains SL1, SL4, and SL6 cultured on carbazole showed detection of anthranilic acid and catechol while these metabolites were not detected in strain BA under the same conditions. This study has established for the first time carbazole angular dioxygenation and mineralization by isolates from African environment.

e Carbazole 1,9a-dioxygenase (CARDO), a Rieske nonheme iron oxygenase (RO), is a three-component system composed of a terminal oxygenase (Oxy), ferredoxin, and a ferredoxin reductase. Oxy has angular dioxygenation activity against carbazole. Previously, site-directed mutagenesis of the Oxy-encoding gene from Janthinobacterium sp. strain J3 generated the I262V, F275W, Q282N, and Q282Y Oxy derivatives, which showed oxygenation capabilities different from those of the wild-type enzyme. To understand the structural features resulting in the different oxidation reactions, we determined the crystal structures of the derivatives, both free and complexed with substrates. The I262V, F275W, and Q282Y derivatives catalyze the lateral dioxygenation of carbazole with higher yields than the wild type. A previous study determined the crystal structure of Oxy complexed with carbazole and revealed that the carbonyl oxygen of Gly178 hydrogen bonds with the imino nitrogen of carbazole. In these derivatives, the carbazole was rotated approximately 15, 25, and 25°, respectively, compared to the wild type, creating space for a water molecule, which hydrogen bonds with the carbonyl oxygen of Gly178 and the imino nitrogen of carbazole. In the crystal structure of the F275W derivative complexed with fluorene, C-9 of fluorene, which corresponds to the imino nitrogen of carbazole, was oriented close to the mutated residue Trp275, which is on the opposite side of the binding pocket from the carbonyl oxygen of Gly178. Our structural analyses demonstrate that the fine-tuning of hydrophobic residues on the surface of the substratebinding pocket in ROs causes a slight shift in the substrate-binding position that, in turn, favors specific oxygenation reactions toward various substrates. Rieske nonheme iron oxygenases (ROs) catalyze the initial oxygenation reaction of aromatic compounds (1). ROs are of interest for the biodegradation of aromatic pollutants and for synthetic applications requiring enantio-and regiospecific reactions. Some ROs break down toxic heterocyclic compounds by a single oxidization reaction. ROs are generally composed of two or three components, a terminal oxygenase (Oxy) and one or two electron transfer components. The Oxy is activated by the electron transfer component(s) and then catalyzes the oxygenation of the substrate. Recent biochemical and structural studies have revealed that Oxys typically exhibit an ␣ 3 or (␣␤) 3 configuration (2). The ␣ subunit contains a Rieske [2Fe-2S] cluster and a mononuclear iron. The Rieske [2Fe-2S] cluster accepts electrons from the electron transfer component(s) and transfers electrons to the mononuclear iron. The mononuclear iron at the active site activates molecular oxygen, which attacks the substrate. The two oxygen atoms of the molecular oxygen bind to tandemly linked carbon atoms in an aromatic ring, creating two hydroxyl groups in the cis configuration (3). This process is called lateral dioxygenation. The amino acid residues that affect the substrate specificity of ROs have bee...

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