Effect of nitrate on microbial perchlorate reduction

Over the last decade perchlorate has been recognized as an important emerging water contaminant that poses a significant public health threat. Because of its chemical stability, low ionic charge density, and significant water solubility microbial remediation has been identified as the most feasible method for its in situ attenuation. Our previous studies have demonstrated that dissimilatory perchlorate reducing bacteria (DPRB) capable of the respiratory reduction of perchlorate into innocuous chloride are ubiquitous in soil and sedimentary environments. As part of their metabolism these organisms reduce perchlorate to chlorite which is subsequently dismutated into chloride and molecular oxygen. These initial steps are mediated by the perchlorate reductase and chlorite dismutase enzymes respectively. Previously we found that the activity of these organisms is dependent on the presence of molybdenum and is inhibited by the presence of oxygen and to different extents nitrate. However, to date, there is little understanding of the mechanisms involved in the regulation of perchlorate reduction by oxygen and nitrate. As a continuation of our studies into the factors that control DPRB activity we investigated these regulatory mechanisms in more detail as a model organism, Dechloromonas aromatica strain RCB, transitions from aerobic metabolism through nitrate reduction to perchlorate reduction. In series of growth transition studies where both nitrate and perchlorate were present, preference for nitrate to perchlorate was observed regardless of the nitrate to perchlorate ratio. Even when the organism was pre-grown anaerobically in perchlorate, nitrate was reduced prior to perchlorate. Using non-growth washed cell suspension, perchlorate- grown D. aromatica was capable of reducing both perchlorate and nitrate concomitantly suggesting the preferentially utilization of nitrate was not a result of enzyme functionality. To elucidate the mechanism for preferential utilization of nitrate, transcripts of perchlorate reductase and chlorite dismutase were analyzed to determine possible transcriptional regulation from nitrate. During growth transition studies, increase in the level of transcripts necessary for nitrate reduction and perchlorate reduction was observed concomitantly with decrease in the concentration of nitrate and perchlorate respectively suggesting transcriptional regulation was involved in the preferential utilization of nitrate and that nitrate might be a transcriptional inhibitor of perchlorate reduction. Again, using non-growth washed cell suspensions of perchlorate grown D. aromatica, a decrease of transcript level of the perchlorate reductase but not the chlorite dismutase was observed after incubation with nitrate. In conclusion, from physiological and molecular evidence, nitrate negatively regulates transcription of perchlorate reductase thus inhibiting perchlorate reduction. This result is unexpected as it is in contrast to the accepted dogma that microorganisms regulate their metabolisms to utilize electron acceptors in a sequential manner based on thermodynamic optimization which would imply that perchlorate should be used preferentially to nitrate.