Isolation and characterization of a gene cluster for dibenzofuran degradation in a new dibenzofuran-utilizing bacterium, Paenibacillus sp. strain YK5

Abstract

Spore-forming bacterial strains capable of utilizing dibenzofuran (DF) as a sole source of carbon and energy were isolated. Characteristics of the isolates justified their classification into the genus Paenibacillus, and their closest relative was P. naphthalenovorans. Degenerate primers for aromatic hydrocarbon dioxygenase alpha subunit (AhDOa) genes and genomic DNA of the strain YK5 were used for gene isolation. The nucleotide sequences of clones of the PCR products revealed that the strain YK5 carries at least five different AhDOa genes. Northern hybridization analysis showed that one of the AhDOa genes was transcribed under DF-containing culture conditions. A gene cluster encoding the AhDOa was isolated. The genes predicted to encode extradiol dioxygenase (dbfB) and hydrolase (dbfC) were found to be an upstream of genes encoding the alpha and beta subunit of the AhDO (dbfA1 and dbfA2, respectively); the latter two gene products showed 60 and 53% identity to the amino acid sequences of DbfA1 and DbfA2 of Terrabacter sp. DBF63, respectively. Two Paenibacillus validus JCM 9077 strains transformed with the dbf gene clusters acquired the ability to convert DF to 2,2',3-trihydroxybiphenyl (THBP) and salicylic acid (SAL). These results suggest that the enzymes encoded by the gene cluster isolated in this study are involved in DF metabolism in YK5.

The GAF-Like-Domain-Containing Transcriptional Regulator DfdR Is a Sensor Protein for Dibenzofuran and Several Hydrophobic Aromatic Compounds

“…The procedures used for isolation of genomic DNA, plasmid DNA, and total RNA of bacterial strains and the procedures used for Southern and Northern hybridization analyses have been described previously (20)(21)(22). PCR amplification was carried out using ExTaq DNA polymerase (TAKARA BIO, Shiga, Japan) according to the manufacturer's protocol.…”

“…The presence of a transcriptional regulation system for genes derived from DBF63, YK3, and YK5 has been demonstrated. Transcription of the dfdA1 and dbfA1 YK5 genes was induced in strains YK3 and YK5, respectively, by cultivation with DF (20,22). In contrast, tran-scription of the dbfA1 DBF63 gene was induced not only by DF but also by fluorene (36), and it was subsequently demonstrated that the dbfA1A2 genes are linked to the fluorenephthalate catabolic gene cluster (16).…”

“…In addition, we recently reported isolation of a low-GϩC-content spore-forming DF-utilizing bacterium, Paenibacillus sp. strain YK5 (22).…”

“…Genes involved in the angular dioxygenation of DF have been found in strains RW1 (dxnA1A2, fdx1, and redA2) (5)(6)(7)(8)11), DBF63 (dbfA1A2) (25), YK3 (dfdA1 to dfdA4) (20), and YK5 (dbfA1A2 YK5 ) (22). The presence of a transcriptional regulation system for genes derived from DBF63, YK3, and YK5 has been demonstrated.…”

Functional Expression of Three Rieske Non-Heme Iron Oxygenases Derived from Actinomycetes inRhodococcusSpecies for Investigation of Their Degradation Capabilities of Dibenzofuran and Chlorinated Dioxins

“…Chlorinated dioxins were quantified in three individual extracts of each substrate with GC-MS, as described previously. 32) Substrates extracted from the resting cell cultures of RD2 transformed with vector plasmid pRK401 were used as negative control. The substrate depletion rates were calculated by comparing the amount of substrate remaining in the AhDO-expressed resting cell cultures, the mean being the amount of substrate remaining in the negative control.…”