Investigating organized complexity in multicellular magnetotactic bacteria using culture independent techniques

Abstract

The multiple independent emergences of multicellularity significantly altered the course of evolution on Earth, leading to the complex life forms inhabiting the planet today. However, the transition from a unicellular ancestor to a multicellular organism remains poorly understood. Because of the relative scarcity of multicellularity in the domains Bacteria and Archaea, research on the evolution of multicellularity has predominantly focused on eukaryotic model organisms. To help resolve how microbial life shifts from single-cellular to multicellular, this thesis investigates cellular differentiation of individual cells within multicellular magnetotactic bacteria (MMB), the only known example of obligate multicellular bacteria. MMB have been shown to lack a unicellular life stage, and instead grow as symmetrical, single-species consortia that orient themselves along Earth's geomagnetic field using a specialized organelle called the magnetosome. Because MMB have remained recalcitrance to cultivation, this dissertation necessitated the use of multiple culture-independent approaches capable of addressing the genomic and physiological underpinnings of the MMB lifecycle. A correlative microscopy workflow was developed to allow for species specific analysis of MMB morphology, biochemistry, and physiology. This was accomplished by performing stable isotope probing (SIP) on samples followed by fluorescence in situ hybridization to identify specific species. Next, electron microscopy was used followed by nano-scale secondary ion mass spectrometry (NanoSIMS) and Raman microspectroscopy. Because Raman and NanoSIMS are becoming increasingly common for microbial SIP studies, the comparability of these techniques was explored, yielding an optimized approach for SIP-Raman- NanoSIMS studies. In addition to these methods, bioorthogonal non-canonical amino acid tagging was used to study the in situ activity as well as variation of protein synthesis within cells. Furthermore, single-cell metagenomics was performed on individual MMB allowing for a detailed analysis of MMB metabolic potential and clonality. The findings presented in this thesis expand our understanding of the mechanisms underpinning the multicellular nature of MMB.