Chlorophenoxyalkanoic acid herbicides, including 2,4-dichlorophenoxyacetic acid (2,4-D), are a group of structurally related compounds which have been extensively applied to the environment for control of broad-leaved weeds. Persistence in the environment presents a serious concern due to environmental toxicity and their impact on human health. Soil microbial communities are capable of total degradation .of these pollutants. Understanding the stability and diversity of the degradative microbial communities, and quantification of sp'ecific catabolic gene expression and dissemination in soil is necessary for the bioremediation of contaminated sites. Environmental soil samples were collected from various sites. A 2,4-D degrading isolate, Burkholderia hospita, was isolated from soil and demonstrated to be predominant in 2,4-D degrading communities using DGGE and clone library analyses of 168 rRNA. This study encompasses the first report of Burkholderia hospita capable of 2,4-D degradation. B. hospita MM1720 contained a large catabolic plasmid which harbours 2,4-D degradative genes. Phylogenetic analysis of tfdA, tfdB and tfdC from B. hospita provided evidence that the acquisition of these functional genes in B. hospita occurred through horizontal gene flow rather than from a common ancestor. In addition, tfdA from B. hospita belonged to the class III tfdA genes and clustered with other tfdA genes recovered from p- and r-Proteobacteria. . A novel GFP system was constructed to study an in vitro biofilm system for plasmid transfer. In vitro biofilms were found to enhance 2,4-D degradation compared to planktonic monocultures. Quantitative PCR (qPCR) was optimised and used to monitor the expression of tfdA transferred into different species within biofilms. Significant upregulation of tfdA gene expression was noted within biofilms compared to planktonic cells. A novel culture-independent method for the detection and quantification of horizontal gene flow among bacteria in soil was developed. All transconjugants acquired the broadhost pMM172::gfjJ, detected by flow cytometry and were determined to belong to a wide range of a-, p- and r-Proteobacteria with high homology to Pseudomonas, Burkholderia, Ralstonia, Variovorax and Sphingomonas genera. This approach shows the detection of a broad transconjugant diversity of a self-transmissible broad-host range plasmid. However, only Pseudomonas, Burkholderia and Ralstonia species were able to grow on 2,4-D as the sole carbon and energy source. Screening for detection of GFP fluorescing transconjugants .by flow cytometry showed greater efficiency compared to methods based on either selective media for detection of culturable transconjugants or DGGE for analysis of microbial community structure. Thus qPCR has been shown to be a suitable approach for assessment of dissemination of catabolic genes in environments and may be developed as a useful tool towards prediction ofthe rate ofxenobiotic degradation. This study provides new insights into the molecular genetics and control mechanisms involved in chlorophenoxy acid herbicide degradation and contribute towards the understanding of the role and potential of horizontal gene transfer for chlorophenoxy acid bioremediation. These new developments in microbial ecology approaches are essential for the assessment and selection of catabolic genes for bioaugmentation optimisation which .may be used for future bioremediation management.

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