Universal immunoprobe for (per)chlorate-reducing bacteria

Recent studies in our lab have demonstrated the ubiquity and diversity of microorganisms which couple growth to the reduction of chlorate or perchlorate [(per)chlorate] under anaerobic conditions. We identified two taxonomic groups, the Dechloromonas and the Dechlorosoma groups, which represent the dominant (per) chlorate-reducing bacteria (CIRB) in the environment. As part of these studies we demonstrated that chlorite dismutation is a central step in the reductive pathway of (per)chlorate that is common to all CIRB and which is mediated by the enzyme chlorite dismutase (CD). Initial studies on CD suggested that this enzyme is highly conserved among the CIRB, regardless of their phylogenetic affiliation. As such, this enzyme makes an ideal target for a probe specific for these organisms. Polyclonal antibodies were commercially raised against the purified CD from the CIRB Dechloromonas agitata strain CKB. The obtained antiserum was deproteinated by ammonium sulfate precipitation, and the antigen binding activity was assessed using dot blot analysis of a serial dilution of the antiserum. The titers obtained with purified CD indicated that the antiserum had a high affinity for the CD enzyme, and activity was observed in dilutions as low as 10(-6) of the original antiserum. The antiserum was active against both cell lysates and whole cells of D. agitata, but only if the cells were grown anaerobically with (per)chlorate. No response was obtained with aerobically grown cultures. In addition to D. agitata, dot blot analysis employed with both whole-cell suspensions and cell lysates of several diverse CIRB representing the alpha, beta, and gamma subclasses of Proteobacteria tested positive regardless of phylogenetic affiliation. Interestingly, the dot blot response obtained for each of the CIRB cell lysates was different, suggesting that there may be some differences in the antigenic sites of the CD protein produced in these organisms. In general, no reactions were observed with cells or cell lysates of the organisms closely related to the CIRB which could not grow by (per)chlorate reduction. These studies have resulted in the development of a highly specific and sensitive immunoprobe based on the commonality of the CD enzyme in CIRB which can be used to assess dissimilatory (per) chlorate-reducing populations in environmental samples regardless of their phylogenetic affiliations.