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Bacterial Growth Mechanisms and Their Role in Cell Size Homeostasis and Senescence

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Bacterial Growth Mechanisms and Their Role in Cell Size Homeostasis and Senescence Title Page Bacterial growth mechanisms and their role in cell size homeostasis and senescence by Maryam Kohram B.S., University of Tehran, 2012 M.Sc., University of Akron, 2015 Submitted to the Graduate Faculty of the Dietrich School of Arts and Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Pittsburgh 2021 Committee Page UNIVERSITY OF PITTSBURGH DIETRICH SCHOOL OF ARTS AND SCIENCES This dissertation was presented by Maryam Kohram It was defended on April 1, 2021 and approved by Zoltan N. Oltvai, Professor, Department of Pathology and Laboratory Medicine, University of Rochester Xiao-Lun Wu, Professor, Department of Physics and Astronomy Ayres Freitas, Associate Professor, Department of Physics and Astronomy Erik S. Wright, Assistant Professor, Department of Biomedical Informatics Thesis Advisor/Dissertation Director: Dr. Hanna Salman, Associate Professor, Department of Physics and Astronomy ii At Bacterial growth mechanisms and their role in cell size homeostasis and senescence Maryam Kohram, PhD University of Pittsburgh, 2021 Growth is a fundamental feature of living organisms, which plays an important role in maintaining cellular characteristics such as cell size and contributes to cellular fitness. It is determined by the rate of biochemical reactions, their efficiency, and their collective organization in a cell, and it is strongly influenced by the environment and the nutrients available to the cell. This complexity can lead to significant fluctuations in the cell’s growth rate. To avoid the accumulation of fluctuations over time and prevent processes from diverging, cells utilize control mechanisms to ensure the stability and accuracy of growth. The aim of this study is to better understand the dynamical processes and control mechanisms of cellular growth in the simple model organism E. coli bacterium, and how they contribute to cell size homeostasis and cellular senescence. We use an experimental setup consisting of a microfluidic device designed to trap single cells while continuously growing, to acquire long-term single cell measurements of cell- size and protein content. We apply new regression analyses methods to the measured growth dynamics, which are not motivated by any preconceived growth control model. Our results reveal dependencies among measured cellular variables that were not considered before, and which point to new growth control mechanisms. By further investigating these dependencies, we find that DNA concentration plays an important role in determining bacterial growth rate, which works to compensate for size differences acquired during cell division and thus allows cells to maintain size homeostasis. We then turn our attention to the effect of metabolism efficiency on growth rate and we study how various metabolic defects affect the ability of cells to grow in different iii environments. We focus on how these defects affect cellular senescence and contribute to cellular death. Our results uncover aging effects and distinguish different phenotypes of cell death. iv Table of contents Preface ....................................................................................................................................... xxix 1.0 Introduction ............................................................................................................................. 1 2.0 Design and fabrication of microfluidic machines .............................................................. 12 2.1 Fabrication of permanent mold .................................................................................. 14 2.1.1 Mask aligner .......................................................................................................14 2.1.1.1 First layer preparation .......................................................................... 18 2.1.1.2 Second layer preparation ...................................................................... 20 2.1.2 3D lithography printing .....................................................................................21 2.1.2.1 First layer fabrication using the nanoscribe ....................................... 24 2.1.2.2 Second layer fabrication procedure using maskless aligner .............. 24 2.1.3 Silanization .........................................................................................................25 2.2 PDMS fabrication ......................................................................................................... 25 3.0 Correlation among measured properties during cellular growth .................................... 27 3.1 Experimental methods ................................................................................................. 28 3.2 Cell cycle variables are correlated across generations .............................................. 29 3.3 Dynamics of cell cycle variables is approximately Markovian ................................ 33 3.4 Cell size dynamics can be described by just a few parameters ................................ 39 3.5 Parameter values vary between experiments ............................................................. 42 3.6 Effective parameters imply mechanisms of cell size regulation ............................... 45 3.7 Sister cells comparison confirms growth rate dependence on size fraction ............ 47 3.8 Comparison of sister cells reveals additional correlation in their growth .............. 50 v 3.9 Sister-cells correlation predicts correlation between niece and cousin cells ........... 54 4.0 Immediate bacterial growth correlation with cell content ................................................ 56 4.1 Materials and methods ................................................................................................. 58 4.1.1 Bacterial strains and plasmids ..........................................................................58 4.1.2 Experimental setup and data acquisition .........................................................59 4.1.3 DNA concentration estimation ..........................................................................60 4.1.4 Fluorescence calibration experiments ..............................................................65 4.2 Division of E. coli is symmetric ................................................................................... 67 4.3 Smaller sister has a higher growth rate ...................................................................... 70 4.4 Growth rate varies in one cycle ................................................................................... 72 4.5 DNA concentration is unequally distributed in sisters ............................................. 74 4.6 Lower DNA concentration causes lower growth rate ............................................... 80 4.7 Inhibition of cell division by overexpression of sulA confirms the results of division inhibition by cephalexin ..................................................................................................... 87 5.0 Aging as a consequence of ecosystem-level bacterial adaptability ................................... 92 5.1 Materials and methods ................................................................................................. 93 5.1.1 Bacterial strains, growth conditions and reagents ..........................................93 5.1.2 Microbatch culture experiments .......................................................................94 5.1.3 Microbatch culture data analysis .....................................................................94 5.1.4 Data reduction and clustering ...........................................................................97 5.1.5 Oxygen consumption rate and extracellular acidification rate......................99 5.1.6 Single-cell experiments in mother machine ...................................................101 vi 5.2 E. coli single gene deletion mutants display media-dependent growth in microbatch cultures .............................................................................................................................. 102 5.3 Deletion of the ATP synthase atpA subunit increases cellular respiration in E. coli ............................................................................................................................................ 109 5.4 Deletion of the ATP synthase atpA subunit increases death rate in single E. coli cells ............................................................................................................................................ 111 5.5 Mutant cells exhibit a higher post-replicative lifetime compared to wild type cells ............................................................................................................................................ 115 5.6 E. coli cells exhibit three death phenotypes .............................................................. 117 5.7 Single cells switch their phenotype to adapt to new conditions.............................. 120 5.8 Conclusions ................................................................................................................. 124 6.0 Discussions and future directions ...................................................................................... 126 Appendix A Fitting growth curves .......................................................................................... 132 Appendix B Constrained multivariate regression coefficients ............................................. 136 Appendix C Simulating
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