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Post-genomic characterization of Nitrospina, a major marine nitrite oxidizer

 

Supervisor: Holger Daims

PhD student: Anna Mueller

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

 

 

 

Elucidating the role of versatile metabolisms in Nitrospinae and other marine nitrite oxidizers

The aim of my PhD project is to study alternative energy metabolisms and further characterize the ecophysiology of Nitrospinae, a marine nitrite oxidizing bacteria (NOB). Thus far, little is known about the Nitrospinae, with the exception of their chemolithoautotrophic ability to fix COthrough aerobic nitrite oxidation. Given their dependence on oxygen for carrying out nitrite oxidation, I hypothesize that the Nitrospinae can utilize various alternate energy metabolisms in low oxygen environments. However, only two highly related Nitrospinae have been isolated to date, which are distantly related to the environmentally dominant marine Nitrospinae clades.

In the first stage of my PhD, I established nitrite-oxidizing enrichment cultures from various marine samples to obtain strains that are representative of the dominant Nitrospinae. Using metagenomes acquired from these enrichments, I then identified two that contained representatives of abundant Nitrospinae. Due to the laboriousness of the isolation process, I developed a method using single-cell sorting via fluorescent activated cell sorting (FACS) paired with nitrification activity screens. This method has allowed me to culture two additional novel Nitrospinae strains that are distantly related to each other and those previously isolated. Subsequently, using Nanopore and Illumina metagenomic sequencing, I obtained two complete genomes and generated metabolic hypothesis by comparative analysis. We are also currently exploring their physiology via nitrite oxidation kinetics (microrespirometry).

 

In the second stage of my PhD, I have been collaborating with microfluidics experts from the Stocker lab at the ETH Zurich, to develop a Raman activated single-cell sorting method to further discover alternative metabolisms of NOB in the environment. Recently, it was shown that single metabolically active cells can be labelled using a substrate of interest and heavy water, deuterium oxide (D2O), which upon incorporation, can be detected by Raman microspectroscopy. I am adapting this method to marine NOB using nitrite, D2O, and defined incubation conditions (e.g., different dissolved O2 levels). Moreover, we are exploiting Raman signals originating from NOB-intrinsic cell characteristics, such as high-level resonance c-type cytochromes, to identify NOB in the environment. Positively identified cells can then be sorted using a custom made microfluidic device followed by single-cell DNA amplification and sequencing. This newly developed method allows us to study the ecophysiology of Nitrospinae in a targeted, activity-based, cultivation-independent manner. The optimal environmental systems to apply this method are oxygen minimum zones (OMZs). Here, insight into the unexplored metabolic diversity of NOB is urgently needed to explain the high abundance of Nitrospinae in O2 depleted regions and to further elucidate their ecological roles within and beyond the N cycle. We have therefore started a collaboration with experts in OMZ research from the Kuypers lab at the Max Planck Institute in Bremen, where I also spent my secondment. During this time, I was fortunate enough to join a sampling trip to a Peruvian OMZ with researchers from the Kuypers lab.  Using the sample from various depths and in situ O2 concentrations, which I have brought back to Vienna, I will use our Raman-activated cell sorting method to identity novel Nitrospinae. These will then be sequenced and analyzed to elucidate the habitat distribution and niche differentiation of marine NOB in OMZs with respect to alternative metabolisms and lifestyles.

The project will primarily be based at the University of Vienna, and the experiments with fresh marine samples will be conducted during an extended research stay in the lab of Prof. Andreas Schramm, University of Aarhus, Denmark.

 

Please find a list of publications here. 

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