Fluorescence In Situ Hybridization Fish For Microbial Cells

Author : Jan David Brüwer
Publisher :
ISBN 13 :
Total Pages : 0 pages
Book Rating : 4.:/5 (144 download)

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Download or read book Using Fluorescence in Situ Hybridization to Study Intracellular Properties written by Jan David Brüwer and published by . This book was released on 2024 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Microorganisms dominate the surface ocean and significantly shape biogeochemical cycles, particularly the carbon cycle. Through photosynthesis, primary producers fix inorganic carbon and produce organic matter. The phytoplankton-derived organic matter fuels differently specialized heterotrophic bacteria that remineralize large proportions of it. At the same time, phages, which are viruses that infect bacteria, shape the bacterial community composition through top-down controls. Environmental microbial ecology often uses microscopically derived changes in cell abundance or incubation experiments with labeled substrates to derive bacterial growth or activity. However, the former does not account for mortality, while the latter is prone to biases by bottle effects and substrate preferences. Nevertheless, marine microbial ecology requires a thorough understanding of bacterial growth and mortality to understand the effects on, e.g., the carbon cycle. Additionally, microbial ecologists have little understanding of the effect of phages on the heterotrophic community. For example, no studies have quantified the amount of phage-infected heterotrophs in complex marine samples to date. For this thesis, I studied the bacterial life cycle, from cell division to cell death, in environmental samples. To do so, I used fluorescence in situ hybridization (FISH) techniques and high-throughput image cytometry, complemented with metagenomic analyses. A significant focus was on bacteria of the SAR11 clade. SAR11 are specialized to grow in oligotrophic habitats that are nutrient and substrate-depleted. They are assumed to be outcompeted by specialized bacteria during high-substrate conditions, such as phytoplankton blooms. Additionally, there is an ongoing debate on whether phage infection has a considerable effect on SAR11 communities. In this thesis, I demonstrate rapidly dividing SAR11 communities during phytoplankton blooms with a concomitantly high phage infection rate. In Chapter 2, I used FISH in combination with a fluorescent DNA stain to study the frequency of dividing cells by visualizing the intracellular DNA distribution. During a cell replication cycle, the dublicated genomes need to be separated into the future daughter cells. The correlation with experimentally derived in situ cell division rates allowed the calculation of cell division across an entire phytoplankton bloom. Measurements revealed faster SAR11 cell division rates than anticipated by cell counts. Hence, calculated mortality rates were high during these times. As the mortality was taxon-specific, I hypothesized phage-induced lysis as a cause. In Chapter 3, I designed direct-geneFISH probes to visualize phage-infected SAR11 cells in the environment. I could thereby provide the missing link between cell division and mortality rates, as revealed in Chapter 2. The highest amounts of phage-infected SAR11 cells (up to 19 % of the SAR11 cells) were detected when taxon-specific mortality and cell division rates were highest. Additionally, I found a phenomenon of phage-infected, ribosome-depleted cells, which I dubbed 'zombie cells'. I thoroughly discuss possible explanations for their emergence and propose that nucleotides from ribosomal RNA are used as substrates for phage genome synthesis. Additionally, I show that both phage-infected and zombie cells occur globally. In Chapter 4, I assessed the influences of future ocean scenarios and a marine heatwave on the microbial community, including cell division and grazing rates. Anthropogenic influences shape the ocean as a habitat with unknown consequences for the microbial community. I found no significant influences of the mild marine heatwave, while the future ocean scenarios caused differences in bacterial abundances. Overall, the results indicate a stable and adaptable marine microbial community in the face of a changing ocean. In the general discussion (Chapter 5), I discuss the methodological approaches used in this thesis to study bacterial cell division rate and the viral community. I further summarize and discuss the insights into SAR11 ecology gained through this thesis, as well as the importance and emergence of zombie cells. The discussion is rounded off with an outlook, proposing directions for future research projects to understand further the interplay of bacterial hosts and their phages, as well as zombie cells.