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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 this 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, it is thought that the Nitrospinae must utilize various alternate energy metabolisms in low oxygen environments, where they are often observed. However, only two highly related Nitrospinae have been isolated to date, which are distantly related to the environmentally dominant marine Nitrospinae clades, limiting the ability to predict and test alternate metabolisms.

In the first stage of this PhD project, nitrite-oxidizing enrichment cultures from various marine samples were established to obtain strains that are representative of the dominant Nitrospinae. Using metagenomes acquired from these enrichments, two enrichments were identified that contained representatives of abundant Nitrospinae. Due to the laboriousness of the isolation process, a new isolation method was developed that uses single-cell sorting via fluorescent activated cell sorting (FACS) paired with nitrification activity screens. This method has enabled the cultivation of two additional novel Nitrospinae strains that are distantly related to each other and those previously isolated. Subsequently, using Nanopore and Illumina metagenomic sequencing, two complete genomes were obtained and metabolic hypothesis were generated by comparative analysis. Metabolic strategies were further explored via nitrite oxidation kinetics (microrespirometry).

The second stage of this project involved a collaboration 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. This method will be adapted to marine NOB using nitrite, D2O, and defined incubation conditions (e.g., different dissolved O2 levels). Moreover, Raman signals originating from NOB-intrinsic cell characteristics will be exploited, 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 will enable studies of 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. Therefore, a collaboration has been established with experts in OMZ research from the Kuypers lab at the Max Planck Institute in Bremen, which included a 3.5 month research stay in Bremen. During this time, sampling of the Peruvian OMZ with researchers from the Kuypers lab was conducted. The sample from various depths and in situ O2 concentrations that were collected in Peru, are going to be used with 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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