Catabolism of naphthalene and phenanthrene by Burkholderia sp. strain RP007
Permanent link to Research Commons versionhttps://hdl.handle.net/10289/15348
Burkholderia sp. strain RP007 was isolated for its ability to utilise phenanthrene as a sole source of carbon and energy. This strain was identified as a Burkholderia sp. by biochemical tests, fatty acid analysis, and nucleotide sequencing of the 16S rRNA gene. The ability of Burkholderia sp. strain RP007 to mineralise [9-¹⁴C]phenanthrene and [1-¹⁴C]naphthalene was confirmed. A gene cluster (phn) which represents a new group of upper pathway genes for polycyclic aromatic hydrocarbon (PAH) degradation was cloned from Burkholderia sp. strain RP007. Nucleotide sequencing of an 11.5 kb HindIII fragment cloned from RP007 revealed the presence of nine open reading frames (ORFѕ). Escherichia coli carrying the recombinant phn genes was able to transform naphthalene to salicylic acid. The phn genes are significantly different in sequence and gene order from the previously described nah, ndo, pah and dox genes for PAH degradation. The phn locus encodes iron-sulfur protein α and β subunits of a PAH initial dioxygenase, but not the ferredoxin and reductase components. The dihydrodiol dehydrogenase of the RP007 pathway, PhnB, shows greater similarity to analogous dehydrogenases from described biphenyl pathways than to those characterised from naphthalene/phenanthrene pathways. Furthermore, the RP007 extradiol dioxygenase, PhnC, shows no similarity to other extradiol dioxygenases for naphthalene or biphenyl oxidation, but is a member of the recently proposed Class III extradiol dioxygenases. Upstream of the phn catabolic genes are two putative regulatory genes phnR and phnS. phnR is divergently transcribed with respect to the other genes and is similar to the σ⁵⁴-dependent family of positive transcriptional regulators. phnS is a LysR-type transcriptional activator. Expression experiments suggest the phn operon is under regulatory control which may involve PhnR and PhnS. Two loci encoding aromatic meta cleavage pathways were also cloned from Burkholderia sp. strain RP007. The nucleotide sequence of the catechol 2,3-dioxygenase genes, and adjacent open reading frames, suggest these are meta pathways of different classes which specify catabolism of different aromatic substrates. One of these may be the lower pathway for phenanthrene and naphthalene degradation. The presence of catabolic genes homologous to the phnAc gene of Burkholderia sp. strain RP007, relative to the archetypal nahAc gene of Pseudomonas putida G7, was screened for in both culturable and nonculturable microbial communities. Of 77 environmental isolates cultured for their ability to degrade naphthalene or phenanthrene, 28 showed homology to nahAc, whilst none of the strains possessed catabolic genes similar to phnAc. However, using phnAc-specific primers, PCR products homologous to phnAc were amplified from DNA extracts of eight hydrocarbon-contaminated soils, demonstrating the widespread distribution of this genotype. Analysis of these phnAc PCR products by nucleotide sequencing of selected cloned products revealed very high (98.5-100%) homology to the phnAc gene of RP007, suggesting the phn genotype is highly conserved. PCR products were also amplified from soil DNA using nahAc-specific primers, and sequencing of these products revealed a diversity similar to that amongst previously described nah-like genes.
The University of Waikato
All items in Research Commons are provided for private study and research purposes and are protected by copyright with all rights reserved unless otherwise indicated.
- Higher Degree Theses