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Importance of Cyanate as Substrate for Nitrifiers

 

Supervisor: Michael Wagner

PhD student: Johanna Wiesinger

Group: Division of Microbial Ecology, Centre for Microbiology and Environmental Systems Science

 

 

 

Transcriptomics and regulation of gene expression of the thaumarchaeon Nitrososphaera gargensis in response to cyanate and of the complete ammonia oxidizer N. inopinata under quorum quenching

Nitrification is an important process of the biogeochemical nitrogen cycle. Ammonia-oxidizing bacteria (AOB) and archaea (AOA), as well as complete ammonia-oxidizing (comammox) and nitrite oxidizing bacteria (NOB) are the key players in nitrification. The ammonia-oxidizing archaeon Nitrososphaera gargensis is the first microorganism described to use cyanate as sole source of energy, reductant, and nitrogen. One of my projects focuses on understanding global gene regulation and expression in N. gargensis during growth on cyanate versus ammonia as sole substrate. To achieve this goal, extraction of sufficient amounts of high molecular weight RNA of N. gargensis is necessary. I achieved this goal via optimization of the growth medium of N. gargensis. While this sounds straightforward, it took me more than a year before I managed to find the optimal medium and to measure cyanate consumption of cells grown in this new medium. Subsequently, after some months of fine-tuning and biomass pooling, sufficient amounts of biomass of this slow growing organism for RNA sequencing was obtained.

Currently, I am analyzing in depth the transcriptomic data. One interesting finding of the transcriptome analysis is that an approximately 200 bp long intergenic region is about 70-fold upregulated under cyanate treatment. This region is predicted to encode a small RNA (sRNA), and surprisingly this sRNA represents over 30% of all recovered transcriptome reads, excluding ribosomal RNA. I am now investigating the function on this sRNA in N. gargensis. To identify transcriptional regulators involved in the metabolic switch to cyanate consumption, I performed a novel pull down assay to fish for potential transcriptional regulators that bind to the cyanase promotor region. This technique, that was established and optimized by Prof. Per H. Nielsen at Aalborg University, Denmark, has the potential to reveal important insights into various aspects of gene regulation in nitrifiers and other not yet genetically accessible organisms. During two 3 months secondments at Aalborg University, I performed several pulldown experiments with N. gargensis. A preliminary screening of the pulled-down proteins revealed various known DNA interactors, including the TATA-box-binding protein of N. gargensis. We will now compare the fished proteins with the transcriptome and proteome data under cyanate treatment to identify regulatory proteins of the cyanase gene.

In parallel to these projects I got involved in investigating the quorum sensing system (QS) of the comammox organism N. inopinata. We were able to demonstrate the activity of this communication system via RT-PCR targeting mRNA of the transcriptional regulator LuxR and the acyl-homoserine lactone (AHL) synthase LuxI. Further the activity of these genes in the comammox strain was demonstrated via LC/MS detection of AHLs in the supernatant. But hundreds of milliliter culture were necessary to detect the AHLs with a GFP based sensor strain and we struggled to quench the QS effectively with AHL competitors.

At the 5th ICON conference I met Brett Mellbye, who successfully quenched the QS in Nitrobacter winogradskyi via cleavage of the AHL molecules with a lactonase (AiiA). He provided us with a more sensitive AHL sensor strain, as well as the AiiA. The AiiA approach has led to a significant decrease of AHLs in N. inopinata cultures and based on RT-qPCR results in a significant downregulation of luxI and luxR compared to a control group and cultures treated with heat-inactivated lactonase. Using this quenching approach, I performed comparative transcriptomic analyses of quenched and untreated cultures and I am currently analyzing the sequencing data to reveal genes regulated in their expression by QS.

The project will primarily be based at the University of Vienna, and transcriptomic as well as proteomic experiments will be conducted during an extended research stay in the lab of Prof. Per Nielsen, University of Aalborg, Denmark.

 

Please find a list of publications here.

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