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Copper Acquisition

Periodic Reporting for period 1 - CORA (Copper Acquisition)

Reporting period: 2016-06-01 to 2018-05-31

All organisms on Earth require certain types of trace metal co-factors like Fe, Mn and Cu for key enzymes in their metabolisms. Aerobic ammonia oxidation is one of the most important biological metabolic pathways of the nitrogen cycle on Earth and involves oxidation of ammonia to nitrite. Until 2005 it was believed that only bacteria contributed to this pathway. However, since 2005 it is now recognized that AOA are the main microorganisms controlling ammonia in terrestrial and aquatic systems. Ammonia oxidizing archaea (AOA) require copper (Cu) to carry out ammonia oxidation because it is a co-factor to many of the enzymes involved in this pathway. In contrast to bacteria, where various models of Cu acquisition and incorporation of Cu into enzymes exist, it is currently unknown how AOA acquire Cu in the environment. One hypothesis is that similar to bacteria, AOA secrete chalkophores—biological molecules that capture Cu and transport it back into the AOA cell. AOA might also take up Cu into the cell by an active transport mechanism or a cell pore that binds trace-metals. The key aim of project CORA was to determine how archaea take up Cu from the environment. This study was innovative because it is among the first to address this problem and will provide fundamental understanding to a key process that is of economic importance in agriculture and wastewater remediation. AOA provide crops with nitrogen for growth in soils and are used in remediating wastewater containing high levels of ammonia contamination. Results from this study will contribute to the fundamental understanding of Cu acquisition by archaea and will also be the first step to understanding an important factor that likely controls ammonia oxidation by AOA in terrestrial and aquatic environments.
As part of this project we carried out experiments in which N.viennensis was grown at various Cu concentrations ranging from 10-12 to 10-15 mol L-1. These concentrations are realistic for certain soil types including those high in calcium carbonates. By tracking the amount of nitrite produced by N.viennensis over time at various Cu concentrations we were able to determine the concentration of Cu that hindered N.viennensis ammonia oxidation. We also carried out experiments where these same cultures were re-exposed to Cu. In this case, these cultures regained their ability to oxidize ammonia. We also monitored how fast Cu-limited and Cu-replete cultures grew at various Cu concentrations. Similar to the nitrite results, N.viennensis cell growth slowed down when Cu became limited and resumed upon re-exposure to Cu. These combined results show that Cu plays an important role in regulating cell growth and ammonia oxidation in N.viennensis.

Another question our project attempted to answer was what types of proteins N.viennensis could be using to obtain Cu? To answer this question we grew N.viennensis under Cu-limited conditions and Cu-replete conditions and determined what types of genes were triggered in response to these conditions. We used two different methods, one method involved using quantitative polymerase chain reaction (qPCR) which can measure the amount of genes that are active in an organism. By comparing specific genes we hypothesized to be involved in Cu-uptake between the two conditions we could determine if these genes could potentially be involved in Cu-uptake. Another approach we used is transcriptomics. With this type of method, one can determine several hundred genes that could potentially be active under both conditions. We also carried out experiments where we screened for the presence of a Cu-binding chemical complex in cultures.
Results of the project in general will contribute to understanding how Cu can influence ammonia oxidation pathways of archaea. This has global implications as ammonia oxidation is a part of the nitrification step of the nitrogen cycle. This step is only made possible because of the help of microorganisms that oxidize ammonia in the environment. Studies of various terrestrial and aquatic systems have shown that AOA play a pivotal role in these systems.
Gene expression results from the project CORA are expected to contribute to advancing the current state of the art by identifying potential genes (and hence potential gene products) that could be involved in Cu uptake in the archaeon N.viennensis. This information will be the first of its kind and open the field to future studies whereby Cu acquisition can be studied in other types of archaea.
Methods used in to cultivate and analyze growth of N.viennensis
Nitrite production and growth of N.viennensis