1887

Abstract

sp. strain DBF63 is capable of degrading fluorene (FN) to tricarboxylic acid cycle intermediates via phthalate and protocatechuate. Genes were identified for the protocatechuate branch of the -ketoadipate pathway (, ) by sequence analysis of a 70 kb DNA region of the FN-catabolic linear plasmid pDBF1. RT-PCR analysis of RNA from DBF63 cells grown with FN, dibenzofuran, and protocatechuate indicated that the operon was expressed during both FN and protocatechuate degradation in strain DBF63. The gene encoding -ketoadipate enol-lactone hydrolase () was not fused to the next gene, which encodes -carboxymuconolactone decarboxylase (), in strain DBF63, even though the presence of the gene (the fusion of and ) within a gene cluster has been thought to be a Gram-positive trait. Quantitative RT-PCR analysis revealed that mRNA levels increased sharply in response to protocatechuate, and a biotransformation experiment with ,-muconate using carrying both and indicated that PcaD exhibited -ketoadipate enol-lactone hydrolase activity. The location of the gene cluster on the linear plasmid, and the insertion sequences around the gene cluster suggest that the ecologically important -ketoadipate pathway genes, usually located chromosomally, may be spread widely among bacterial species via horizontal transfer or transposition events.

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2005-11-01
2019-10-21
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Annotated ORFs of the analyzed DNA region of pDBF1.[ PDF file] (297 KB)

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Supplementary Fig. 1. (A) RT-PCR analysis of pcaR, pcaHG, pcaDC and pcaFIJ genes. Total RNA from DBF63 cells grown in FN, DF or protocatechuate (PCA) were used as the templates in RT-PCR. Complementary DNA samples were appropriately diluted prior to PCR. Primer sets (Table 2) and PCR conditions are described in the text. M5 and M6 represent DNA molecular mass marker 5 and 6 (Nippon Gene), respectively. Sizes of the standards (bp) are shown to the left of the image. (B) Expression of pcaD gene in DBF63 cells induced by PCA (left panel) and FN (right panel). RNA preparation and quantitative RT-PCR were performed as described in the text. Transcript levels were normalized using 16S rRNA as an internal control. Results from three measurements are shown with error bars. The mean values of the pcaD mRNA levels in the control samples, supplemented with 50 µl ethanol (EtOH; left panel) or dimethylformamide (DMF; right panel), were arbitrarily set at 1.0. The independent experiments were repeated twice, and a similar tendency was observed both times.[ PDF file] (525 KB)

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