| Popis: |
1,1,1-trichloro-2,2-bis(4-chlorophenyl)-ethane (DDT) residues (DDTr) are widespread and persistent environmental contaminants, and have been classed as priority pollutants by the United Nations Environment Programme (UNEP). DDTr are potent endocrine disrupting molecules, and have been associated with reproductive abnormalities in juvenile alligators and rats. Microorganisms that metabolise DDTr both aerobically and anaerobically have been isolated and characterised. Bacteria that degrade DDTr aerobically typically utilise a dioxygenase to initiate degradative reactions through ring-hydroxylation, and convert DDTr to 4-chlorobenzoate without further degradation. Terrabacter sp. strain DDE-1 was isolated from DDTr-contaminated soil from Canterbury, New Zealand, and aerobically degrades 1,1-dichloro-2,2-bis-(4-chlorophenyl)-ethylene (DDE) to 4-chlorobenzoate, when grown in the presence of biphenyl (BP). The intermediates of degradation were inferred to be the end products of dioxygenase activity. Sequencing of a large linear plasmid, pBPH-1, from strain DDE-1 identified a cluster of genes with high levels of sequence similarity to BP-degradation genes from Rhodococcus spp. and Pseudomonas spp. This plasmid is lost at high frequency producing the plasmid-cured strain MJ-2, which has lost the ability to degrade BP or DDE. The aim of this study was to confirm that DDE-degradation in strain DDE-1 is encoded by the bph operon located on pBPH-1. No genetic systems to study gene function in either DDE-1 or MJ-2 could be developed using an array of broad-host range vectors. However, heterologous expression of the bph genes in Rhodococcus erythropolis strain TA422 was successful, with the recombinant strain TA425, obtaining the ability to utilise BP and DDE as a sole source of carbon and energy. DDE-1 was shown to convert indole to indigo, but MJ-2 could not, indicating that the biphenyl dioxygenase located on pBPH-1 is responsible for this activity. The bph genes from strain DDE-1 also conferred the ability to produce indigo from indole on strain TA425, confirming successful expression of the functional biphenyl dioxygenase in this strain. Despite several attempts to show quantitative degradation in strain TA425 using gas chromatography, the results were inconclusive Further analysis is needed to provide unequivocal evidence of DDE-degradation by strain TA425. Attempts to express the bph genes in rhizosphere-colonising bacteria, such a Rhizobium spp. or Pseudomonas spp., were unsuccessful, as evidenced by the inability to produce indigo, hence the lack of a functional biphenyl dioxygenase. However, RT-PCR did indeed indicate that P. aeruginosa strain Fin1 produced a bphA1 transcript, indicating that an error is occurring post-transcriptionally in these strains, to prevent production of the functional enzyme. New Zealand has recently been shown to contain hotspots of DDTr-contamination. The second aim of this study was to determine the prevalence of DDTr-degrading bacteria and to gain insight into the types of bacteria that inhabit sites contaminated with DDTr. To investigate this, culture-dependent and culture-independent techniques were employed. Enrichment for DDTr-degrading bacteria yielded species of Rhodococcus and Ralstonia using DDTr-overlayer plate assays. The polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) were used to amplify and analyse the 16S rDNA and 16S rRNA for the identification of dominant and active bacteria in soil samples. The results of this analysis identified bacteria such as Williamsia spp. and Gordonia spp. that degrade other types of pollutants. This analysis did not identify a predominance of Rhodococcus or Ralstonia spp., or other bacteria that have been shown to degrade DDTr. To identify ecologically relevant members of the bacterial communities in DDTr-contaminated soils, and potentially important metabolic pathways, identification of ring-hydroxylating dioxygenase (RHD) genes was performed. PCR and restriction fragment length polymorphism (RFLP) analysis were employed together with phylogenetic analyses. The results showed that the RHD genes identified, clustered separately to those genes previously characterised from cultivated bacteria. Among these genes, one phylogenetic group was most closely related to the dioxygenase genes from Ralstonia eutropha H850, which is potent PCB-degrading bacterium that possesses a dioxygenase with a wide substrate range for many types of heavily chlorinated, PCB congeners. The identification of a predominance of genes with similarity to phenyl-propionate dioxygenases has been not been recognised previously in soil studies. |
