Molecular detection and analysis of the diversity of bacterial dehalogenases
The identification and isolation of bacteria capable of dehalogenating highly chlorinated aromatic compounds is currently a time consuming process, therefore the ability to predict the potential of a site to naturally remediate contamination is limited. This work has assessed a selection of modern molecular biological techniques to detect the presence of specific dehalogenase enzymes or the genes encoding them, as an indicator of a contaminated site's potential to perform remediation naturally. DNA and protein based detection strategies were tested in a variety of formats. DNA detection of dehalogenase genes was assessed by varieties of hybridisation probing and PCR detection. Protein based detection utilised specific antibody based detection of dehalogenases from bacterial proteomes. A combined technique exploiting the specificity of antibody detection and the sensitivity of PCR amplification was assessed by ribosomal display. DNA based detection techniques demonstrated a high sensitivity but lacked the required level of specificity for use in routine testing, with the exception of single specific primer PCR which was demonstrated to detect specific changes in a bacterial population following contamination. Protein based detection lacked the sensitivity necessary for a field based detection system but the potential for specifically fingerprinting bacterial species was observed. The ribosomal display technique, although combining sensitivity and specificity, could not be fully evaluated during the course of this work.