Studies on 3-Hydroxypropionate Metabolism in Rhodobacter sphaeroides Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Steven Joseph Carlson Graduate Program in Microbiology The Ohio State University 2018 Dissertation Committee Dr. Birgit E. Alber, Advisor Dr. F. Robert Tabita Dr. Venkat Gopalan Dr. Joseph A. Krzycki 1 Copyrighted by Steven Joseph Carlson 2018 2 Abstract In this work, the involvement of multiple biochemical pathways used by the metabolically versatile Rhodobacter sphaeroides to assimilate 3-hydroxypropionate was investigated. In Chapter 2, evidence of a 3-hydroxypropionate oxidative path is presented. The mutant RspdhAa2SJC was isolated which lacks pyruvate dehydrogenase activity and is unable to grow with pyruvate. Robust 3-hydropropionate growth with RspdhAa2SJC indicated an alternative mechanism exists to maintain the acetyl-CoA pool. Further, RsdddCMA4, lacking the gene encoding a possible malonate semialdehyde dehydrogenase, was inhibited for growth with 3-hydroxypropionate providing support for a 3-hydroxypropionate oxidative pathway which involves conversion of malonate semialdehyde to acetyl-CoA. We propose that the 3- hydroxypropionate growth of RspdhAa2SJC is due to the oxidative conversion of 3- hydroxypropionate to acetyl-CoA. In Chapter 3, the involvement of the ethylmalonyl-CoA pathway (EMCP) during growth with 3-hydroxypropionate was studied. Phenotypic analysis of mutants of the EMCP resulted in varying degrees of 3-hydroxypropionate growth. Specifically, a mutant lacking crotonyl-CoA carboxylase/reductase grew similar to wild type with 3- hydroxypropionate. However, mutants lacking subsequent enzymes in the EMCP exhibited 3-hydroxypropionate growth defects that became progressively more severe the ii later the enzyme participated in the EMCP. To resolve this finding, a late blockage EMCP strain has 3-hydroxypropionate growth restored by introducing an early blockage to the EMCP. Furthermore, the introduction of thioesterase YciA to inhibited mutant strains restored 3-hydroxypropionate growth with concomitant excretion of EMCP- derived metabolites showing a CoA-thioester intermediate accumulation most likely causes a decrease in free coenzyme A levels and the growth inhibition. The work confirms the EMCP is not essential for 3-hydroxypropionate growth. However, flux through the EMCP occurs. In Chapter 4, a novel way to alter flux through the EMCP was discovered. Late blockage EMCP mutants were inhibited for 3-hydroxypropionate growth, but spontaneously began growing after 100 hours. Whole genome sequencing of suppressor isolates identified a common mutation in the prkB gene, encoding phosphoribulokinase B of the Calvin-Benson-Bassham (CBB) cycle. The prkB mutation requirement for suppression was verified by introducing mutated alleles to the inhibited strains where 3- hydroxypropionate growth was restored. Finally, introduction of thioesterase YciA did not cause excretion of EMCP-derived metabolites during 3-hydroxypropionate growth in a suppressor strain indicating the prkB mutation decreases flux through the EMCP. In Chapter 5, the role of propionyl-CoA carboxylase during 3-hydroxypropionate, propionate, and acetate assimilation was investigated. Propionyl-CoA carboxylase (PccBA) catalyzes the conversion of propionyl-CoA to (2S)-methylmalonyl-CoA in the methylmalonyl-CoA pathway (MMCP) used for propionyl-CoA assimilation. The assimilation of acetyl-CoA and 3-hydroxypropionate also leads to formation of iii propionyl-CoA whereby the MMCP would be required. A pccB mutant strain could not - grow with propionate/HCO3 confirming the requirement of the MMCP for propionyl- CoA assimilation. However, the same mutant could still grow with acetate and 3- hydroxypropionate. For acetate growth, metabolite analysis showed that propionate was excreted indicating a mechanism to prevent accumulation of propionyl-CoA formed during flux through the EMCP. For 3-hydroxypropionate growth, redirection of the carbon toward acetyl-CoA via the 3-hydroxypropionate oxidative pathway and entry into the EMCP was shown to allow growth when the 3-hydroxypropionate reductive pathway is blocked in R. sphaeroides. iv Dedication To those I adore most – Jamie, Henry, and Theodore. v Acknowledgments Many thanks to all the past and present members of the Alber laboratory. To Dr. Birgit Alber, for the training, support, and example you set in the lab. You gave me the freedom to explore through research and I am very grateful. To Dr. Marie Asao for teaching me my very first enzyme assay and your willingness (and patience) to answer all my questions. To Dr. Michael Carter for his sage wisdom, intriguing commentary on life, and continued support since his departure from the lab. To Daniel Ortiz, for your friendship. Un abrazo. Many thanks to Dr. Tabita and the members of his laboratory. Though I wasn’t an official member, I was treated as such and am grateful for their generosity, expertise, and friendship. Much of the work would not have been possible without their equipment or help. To my committee for the insight, suggestions, and time that was given to help throughout this process. I am forever grateful to my family for their unconditional love and support throughout my time as a graduate student. I am indebted to them for all that they sacrificed to see me through to the end. I love and cherish you all. vi Vita 2004………... ……………..……………………Logan High School 2008………………….................................…….B.S. Wildlife and Conservation Biology, Ohio University 2012-present ………………………...………….Graduate Teaching and Research Associate, Department of Microbiology, The Ohio State University Publications Carlson SJ, Fleig A, Baron MK, Berg IA, Alber BE. 2018. Barriers to 3- hydroxypropionate-dependent growth of Rhodobacter sphaeroides by distinct disruptions of the ethylmalonyl-Coenzyme A pathway. J. Bacteriol. (Published online November 19, 2018) Fields of Study Major Field: Microbiology vii Table of Contents Abstract .......................................................................................................................... ii Dedication .......................................................................................................................v Acknowledgments ......................................................................................................... vi Vita .............................................................................................................................. vii Table of Contents ........................................................................................................ viii List of Tables................................................................................................................xiv List of Figures ............................................................................................................... xv Chapter 1: Introduction ..................................................................................................1 1.1 Rhodobacter sphaeroides, a model organism for carbon assimilation .....................1 1.2 Carbon assimilation in R. sphaeroides during photoheterotrophic growth...............2 1.3 Using precursor metabolites to develop a metabolic scheme ...................................4 1.4 3-Hydroxypropionate, a tool to uncover to new physiological phenomenon ...........5 1.5 Assimilation of 3-hydroxypropionate – A tale of two pathways, the reductive path 6 1.6 Assimilation of 3-hydroxypropionate carbon beyond the reductive path: Propionyl- CoA assimilation using the methylmalonyl-CoA pathway.......................................... 11 1.7 Assimilation of 3-hydroxypropionate carbon beyond the reductive path: C4 to C3 conversion ................................................................................................................. 12 1.8 Assimilation of 3-hydroxypropionate carbon beyond the reductive path: Acetyl- CoA formation ........................................................................................................... 13 1.9 Assimilation of 3-hydroxypropionate – A tale of two pathways, the oxidative path .................................................................................................................................. 13 1.10 Assimilation of acetyl-CoA using the ethylmalonyl-CoA pathway ..................... 15 1.11 Early steps of the ethylmalonyl-CoA pathway and PHB metabolism .................. 16 1.12 The ethylmalonyl-CoA pathway ......................................................................... 17 Chapter 2: Evidence of a 3-hydroxypropionate oxidative pathway in Rhodobacter sphaeroides ................................................................................................................... 19 2.1 Introduction ......................................................................................................... 19 viii 2.2 Materials and Methods ......................................................................................... 25 2.2.1 Materials. ...................................................................................................... 25 2.2.2 Bacterial strains and growth conditions ......................................................... 25 2.2.3 NCBI Database search for enzymes capable of converting pyruvate to acetyl- CoA (or an intermediate requiring a second enzyme to form acetyl-CoA) .............. 25 2.2.4 Isolation