Quantification of Aerobic Ammonia-Oxidizing Bacteria in Soil using Activity-Based Fluorescent Labeling of Ammonia Monooxygenase

Nitrification is a key step in the biological nitrogen cycle and has a large effect on the fate of nitrogen species in both wastewater treatment systems and agricultural soils. Aerobic ammonia-oxidizing bacteria (AOB) initiate nitrification by converting ammonia (NH3) to nitrite (NO2-) and are therefore pivotal to the process. AOB are ubiquitous in the environment but are difficult to quantify as they grow poorly on solid media. Other quantification methods like iquid most-probable number techniques are slow and error-prone, while modern molecular approaches involving polymerase chain reaction amplification are faster and more accurate but do not differentiate between active and inactive AOB. In this study, we explored using activity-based fluorescent mechanisms for rapidly quantifying metabolically active forms of AOB in soils. Initial experiments using Nitrosomonas europaea aimed to establish a relationship between NH3-dependent nitrite production and bacterial cell numbers. Active AMO was treated cells with 1,7-octadiyne (17OD) to inactivate the enzyme and a subsequent copper-dependent "click" reaction attached a fluor. The labeled protein was quantified by SDS-PAGE and IR scanning. In future experiments, AOB will be stimulated in soil microcosms by adding NH4Cl. AMO will again be inactivated by adding 17OD, and total bacteria will be separated from the soil samples using gradient centrifugation. After "click" conjugation with AlexaFluor 647 azide, the abundance of AMO will be determined with SDS-PAGE and IR analysis while metabolically active AOB will be measured via fluorescence-activating cell sorting.