Zinc chloride inhibition of Nitrosococcus mobilis

Abstract

Nitrosococcus mobilis, a halophilic nitrifier, plays an important role in global nitrogen cycling and in the removal of nitrogen from wastewater treatment plants. However, ammonia oxidation is sensitive to a wide variety of inhibitors, including the heavy metal, zinc. Using a metabolic-steady-state reactor, shotgun DNA microarrays, and quantitative polymerase chain reaction (qPCR), this research looked at the dynamic physiological and transcriptional responses of N. mobilis to 1 and 10 microM ZnCl2. By oxygen uptake rate measurements, zinc was determined to act directly on the ammonia monooxygenase (AMO) enzyme. The addition of excess copper prevented the inhibition of AMO by ZnCl2 suggesting that zinc and copper compete for placement in the metal active site in AMO. Shotgun DNA microarrays identified four previously unsequenced genes that were up- or down-regulated in response to 10 microM ZnCl2. Genes up-regulated in response to zinc inhibition include methionine synthase I, UbiA prenyltransferase and a recG-like helicase. RuBisCO was the lone down-regulated gene identified. qPCR was used to track the gene expression of the identified genes over the course of the 4-h experiment for both ZnCl2 concentrations. Because of their physiological importance, the expressions of AMO and hydroxylamine oxidoreductase (HAO) were also monitored via qPCR. The qPCR results showed general agreement with the shotgun DNA microarray results for metH, UbiA, recG and RuBisCO, and revealed that AMO and HAO expression levels were maintained or modestly up-regulated during ZnCl2 inhibition.