Browsing by Author "Van der Walt, Felix"
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- ItemAnalysis of the relationship between glycogen turnover and cell size in Escherichia coli(Stellenbosch : Stellenbosch University, 2020-03) Van der Walt, Felix; Lloyd, James Richard; Stellenbosch University. Faculty of Agrisciences. Dept. of Genetics. Institute for Plant Biotechnology (IPB).ENGLISH ABSTRACT: Glycogen represents an important carbon energy store in organisms across all domains of life. Under permissible conditions, excess environmental glucose is incorporated into glycogen by the Gram-negative bacterium Escherichia coli to provide the cell with an endogenous carbon store. This can rapidly be mobilized to provide the cell with energy for sustained viability when nutritional conditions deteriorate. Extracellular nutrient availability positively impacts cell size and growth rate in a variety of organisms. Bacteria cultured in nutrient-rich media display significant increases in growth rate and cell size, compared to their slow-growing counterparts in nutrient-deprived conditions. Such nutrient-dependent increases in size and growth are accompanied by equally dramatic elevations in the rates of macromolecular biosynthesis (DNA/RNA/protein). How bacteria respond to environmental cues through their ability to sense size and correct random fluctuations that would deviate it from ‘normal’ has been the subject of substantial investigations over the last few decades. This is unsurprising as cell size control and homeostasis are fundamental to cell biology and, of course, to the survival of unicellular bacteria like E. coli. Research has historically focused on cell size and progression of the bacterial cell cycle within the context of extracellular nutrient availability, yet little is known about how endogenous metabolism affects these aspects of bacterial physiology. This investigation aimed to elucidate how glycogen turnover impacts cell size and progression of cell cycle events using E. coli mutants affecting three glycogen catabolic enzymes, glycogen phosphorylase (GlgP), glycogen debranching enzyme (GlgX) and maltodextrin phosphorylase (MalP). Disruption of malP resulted in a profound effect on cell size as ΔmalP mutants are unable to properly coordinate cell cycle progression during exponential growth, leading to substantial heterogeneity in size. This manifests as subpopulations of elongated and filamentous cells. Whilst such mutants do not necessarily form fewer Z-rings per cell, they clearly delay division and grow into filaments and the underlying reason for this appears to be a malfunction of DNA replication. Mutations in either glgP or glgX differently impact DNA replication and cell size and mutants with a lesion in the latter allele contain coinciding glycogen and protein inclusion bodies, particularly noticeable during exponential growth. The nature of the flaws to cell size control and DNA replication observed in ΔmalP mutant strains, specifically the ΔmalP/ΔglgP/ΔglgX triple mutant, was further scrutinized by introducing lesions to genes involved in several interacting processes. Mutating genes associated with glycogen accumulation, pyruvate kinase activity, and SOS-mediated or UDP-glucose-dependent division inhibition led to the formation of mutant cells either smaller or equal in size to the wild type. Partial suppressions to the size defects of the triple mutant were observed in quadruple mutant strains with disruptions to genes involved in amino acid metabolism, ppGpp biosynthesis, UDP-glucose generation, DNA replication and nucleoid structuring. DNA replication is clearly coordinated with diverse physiological processes acting in concert to link duplication of the genome with cell size, growth rate and environmental conditions.