FLUORESCENCE IMAGING AND MOLECULAR DYNAMICS SIMULATION OF THE INTRACYTOPLASMIC MEMBRANES OF METHANOTROPHIC BACTERIA A Dissertation Presented to The Graduate Faculty of The University of Akron In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy Kyle Whiddon December, 2018 FLUORESCENCE IMAGING AND MOLECULAR DYNAMICS SIMULATION OF THE INTRACYTOPLASMIC MEMBRANES OF METHANOTROPHIC BACTERIA Kyle Whiddon Dissertation Approved: Accepted: ______________________________ ______________________________ Advisor Department Chair Dr. Michael Konopka Dr. Christopher Ziegler ______________________________ ______________________________ Committee Member Dean of the College Dr. Leah Shriver Dr. Linda Subich ______________________________ ______________________________ Committee Member Dean of the Graduate School Dr. David Modarelli Dr. Chand Midha ______________________________ ______________________________ Committee Member Date Dr. Sailaja Paruchuri ______________________________ Committee Member Dr. Hazel Barton ii ABSTRACT Methanotrophs are a group of bacteria that are able to utilize methane as their sole carbon and energy source. The vast majority of these bacteria make extensive intracytoplasmic membrane (ICM) structures which house the particulate methane monooxygenase enzyme that converts methane to methanol. Methanotrophs are divided into two classes based on metabolic preferences and ICM architecture, with type I methanotrophs producing ICMs resembling stacked disks, and those of type II methanotrophs resembling concentric rings. Because these ICMs and other useful biomolecules, such as polyhydroxybutyrate (PHB), are synthesized from an inexpensive starting material they are of interest as a cost-effective source of biofuels. Thus far these membranes have been studied using primarily transmission electron microscopy which prevents in vivo analysis of these membranes and limits throughput. Here we demonstrate an alternative analysis for these ICMs using fluorescence microscopy and lipophilic dyes. Using confocal microscopy, ICMs can be quantified on a single cell level by measuring internal fluorescent area. We show that these measurements are sensitive to changes in ICM quantity and can be performed on live cells to monitor membrane dynamics. Furthermore, PHB can be imaged and quantified in a similar way. During imaging differences in ICM staining between type I and type II methanotrophs can be seen. These differences lead us to believe that ICMs in type I methanotrophs exist primarily as invaginations of the cytoplasmic membrane, while ICMs in type II iii methanotrophs exist as fully isolated structures. We also implement MARTINI coarse- grained molecular dynamics simulations to simulate interactions between model lipid bilayers and methane molecules in solution. These simulations show us that methane inserts itself into lipid bilayers with high efficiency and is able to passively diffuse into fully internalized membrane regions. The fluorescence methods detailed here can be used to supplement currently implemented techniques such as electron microscopy and gas chromatography in order to give researchers options for rapidly investigating the extent and dynamics of ICM production between strains or growth conditions. This will allow researchers to quickly optimize and upscale cultures for biofuel production. In addition, molecular dynamics simulations lay the groundwork for future studies. iv TABLE OF CONTENTS Page LIST OF FIGURES ......................................................................................................... viii CHAPTER I. INTRODUCTION ..........................................................................................................1 Methane Metabolism ...............................................................................................2 Intracytoplasmic Membranes ...................................................................................6 Metabolic Regulation ...............................................................................................8 Current Techniques and Project Goals ..................................................................10 II. MATERIALS AND METHODS .................................................................................16 Bacterial Strains and Growth Conditions ..............................................................16 Cloning ...................................................................................................................18 Imaging Sample Preparation ..................................................................................19 Confocal Imaging...................................................................................................21 TEM Imaging .........................................................................................................22 FRAP......................................................................................................................23 STED Imaging .......................................................................................................23 Image Data Analysis ..............................................................................................24 Real Time PCR ......................................................................................................26 Computational Input Generation............................................................................27 Computational Data Analysis ................................................................................28 v III. QUANTIFICATION OF TYPE I METHANOTROPH INTRACYTOPLASMIC MEMBRANES VIA FLUORESCENCE MICROSCOPY ......................................30 Introduction ............................................................................................................30 Fluorescence Imaging of ICMs ..............................................................................31 Analysis of Staining Times ....................................................................................33 Alternative Membrane Probes ...............................................................................35 Specificity of FM Dyes Towards ICMs .................................................................36 Quantification of ICMs ..........................................................................................37 In Vivo Imaging......................................................................................................42 STED Microscopy .................................................................................................45 pMMO Relation to ICMs .......................................................................................47 IV. DETERMINING INTRACYTOPLAMSIC MEMBRANE CONNECTIVITY TO THE CYTOPLAMSIC MEMBRANE ...............................................................50 Introduction ............................................................................................................50 ICM Imaging in Type II Methanotrophs ...............................................................51 Type I ICM Connectivity .......................................................................................52 Type II ICM Connectivity .....................................................................................57 Structure of Type II ICMs......................................................................................62 V. VISUALIZATION AND QUANTIFICATION OF POLYHYDROXYBUTYRATE IN TYPE II METHANOTROPHS.......................65 Introduction ............................................................................................................65 Nile Blue Staining of PHB .....................................................................................66 Quantifying PHB Fluorescence .............................................................................69 vi VI. COARSE-GRAINED MOLECULAR DYNAMICS SIMULATIONS OF METHANE IN MEMBRANES ...............................................................................72 Introduction ............................................................................................................72 Methane-Lipids Interaction ....................................................................................73 Methane Interaction with pMMO ..........................................................................79 Bilayer Penetration by Methane.............................................................................81 VII. CONCLUSON...........................................................................................................85 Summary of Work..................................................................................................85 Ongoing and Future Work .....................................................................................86 REFERENCES ..................................................................................................................89 vii LIST OF FIGURES Figure Page 1 Basic methane metabolism ......................................................................................4 2 Simplified Serine cycle outline ................................................................................5 3 Simplified RuMP cycle outline ................................................................................5 4 TEM images of type I (M. alcaliphilum 20Z, top) and type II (M. trichosporium OB3b, bottom) methanotrophs ..................................................7 5 Table of PCR primers used in