AbstractsBiology & Animal Science

An Investigation into Carbon Flow through the Metabolic Networks of Rhodobacter sphaeroides

by Michael Steven Carter




Institution: The Ohio State University
Department: Microbiology
Degree: PhD
Year: 2014
Keywords: Microbiology; Acetate Metabolism; Propionate Metabolism; Transcription Regulation; Metabolic Regulation; Rhodobacter sphaeroides; Metabolism; Regulation
Record ID: 2042949
Full text PDF: http://rave.ohiolink.edu/etdc/view?acc_num=osu1403873922


Abstract

Predicting carbon flow within an organism requires a complete knowledge of the pathways that compose the organism’s metabolic networks. Even in pathways that have been characterized, an incomplete knowledge of the regulation of the responsible genes and enzymes disallows prediction of carbon flow through the network. By investigating the interplay of the pathways that compose the networks, this work intends to offer insights into the strategies employed by the model organism Rhodobacter sphaeroides for metabolic regulation. Chapter 2 investigate which enzymes participate in carbon flow through the C4/C3 node of central metabolism. The growth of strains with mutations in two different malic enzyme genes were compromised in their ability to grow with some substrates that require C4 to C3 conversion (succinate and (S)-malate), but their growth was unimpaired during growth with acetate, which is assimilated via succinate and malate. A pyruvate phosphate dikinase mutant was unable to grow on acetate, suggesting that gluconeogenesis occurs exclusively from pyruvate during acetate growth. Growth results with a pyruvate carboxylase mutant indicated that pyruvate carboxylase is responsible for C3 to C4 conversion on substrates that are to be assimilated through C3 intermediates. Chapters 3 and 4 examine the role of carbon flow through intermediary pathways of short chain acyl-CoA assimilation in R. sphaeroides. R. sphaeroides employs the ethylmalonyl-CoA pathway for acetyl-CoA assimilation, which shares reactions with polyhydroxybutyrate biosynthesis, and the first committed reaction is catalyzed by crotonyl-CoA carboxylase/reductase (Ccr). Transcript levels of ccr were 30-fold higher during acetate growth than succinate growth, and ccr promoter-reporter fusions were likewise regulated. Mutating the gene that encodes PhaR, a transcriptional regulator of polyhydroxybutyrate synthesis, did not affect regulation. PccR was identified as a regulator of pccB, the gene for the Beta-subunit of propionyl-CoA carboxylase of the methylmalonyl-CoA pathway for propionyl-CoA assimilation. Transcript levels of pccB in R. sphaeroides were 11-fold higher during propionate/HCO3 growth compared to succinate growth. When pccR was mutated, pccB expression was deregulated. Mutation analyses with pccB promoter-reporter fusions revealed regulation by PccR requires two TTTGCAAA motifs. Despite a TTGCAA site upstream of ccr, ccr expression was unaffected by mutation of pccR. PccR-like proteins form a family of regulators of assimilation of short chain fatty acyl-CoA molecules (the ScfR family) that can be subdivided into four classes. Genes for members of each class clustered with genes of the glyoxylate bypass (RamB), the methylcitrate cycle (MccR), the methylmalonyl-CoA pathway (PccR), or genes for the assimilation of isobutyryl-CoA (IbcR). The sum of the evidence presented herein continues the characterization of modes by which carbon flows through metabolic networks. The work in Chapter 2 highlights the obscurity that remains regarding the control…