Analysis of the relationship between glycogen turnover and cell size in Escherichia coli

Thesis (MScAgric)--Stellenbosch University, 2020.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.AFRIKAANSE OPSOMMING: Glikogeen verteenwoordig ʼn belangrike voorraad van koolstofenergie in organismes van alle lewensdomeine. Onder toelaatbare omstandighede word oortollige glukose van die omgewing deur die Gram-negatiewe bakterium Escherichia coli omgeskakel na glikogeen om die sel met ʼn endogene koolstofvoorraad te voorsien. Dit kan spoedig gemobiliseer word om die sel met energie vir onafgebroke lewensvatbaarheid te voorsien, wanneer voedingstoestande agteruit gaan. Die beskikbaarheid van ekstrasellulêre voedingstowwe het ʼn positiewe impak op selgrootte en groeikoers in verskeie organismes. Bakterieë wat in voedingsryke middele gekweek word, wys beduidende verhogings in groeikoers en selgrootte, in vergelyking met stadig-groeiende bakterieë wat onder voedingstofarm omstandighede gekweek word. Sulke voedingstof-afhanklike verhogings in grootte en groei word vergesel deur ewe drastiese versnellings in die tempo van makromolekulêre biosintese (DNA/RNA/proteïen). Hoe bakterieë op sekere omgewingskodes reageer deur hul grootte waar te neem en om ewekansige fluktuasies wat van ‘normaal’ afwyk, reg te stel was die onderwerp van beduidende ondersoeke oor die afgelope dekades. Dit is nie verbasend nie aangesien die beheer en homeostase van selgrootte fundamenteel is vir selbiologie en, natuurlik, die oorlewing van eensellige bakterieë soos E. coli. Navorsing het tot dusver op selgrootte en die verloop van die bakteriële selsiklus binne die konteks van die beskikbaarheid van ekstrasellulêre voedingstowwe gefokus, terwyl daar min inligting bestaan oor die impak van endogene metabolisme op hierdie aspekte van bakteriële fisiologie. Die doel van hierdie ondersoek was om te verduidelik hoe glikogeenomset selgrootte en vordering van sekere stappe van die selsiklus beïnvloed deur gebruik te maak van ʼn reeks E. coli mutante wat kombinasies van drie glikogeenkataboliese ensieme kortkom, naamlik glikogeenfosforilase (GlgP), glikogeen-onvertakkingsensiem (GlgX) en maltodekstrien-fosforilase (MalP). Ontwrigting van malP het ʼn diepgaande effek op selgrootte omdat ΔmalP-mutante blykbaar nie die selsiklusprogressie tydens eksponensiële groei korrek kan koördineer nie en dit lei tot aansienlike heterogeniteit in selgrootte. Dit manifesteer as subpopulasies van langwerpige en filamentagtige selle. Terwyl sulke mutante nie noodwendig minder Z-ringe per sel vorm nie, word selverdeling duidelik vertraag, en die onderliggende rede dat selle filamente vorm, blyk ʼn abnormale werking van DNA-replikasie te wees. Mutasies in óf glgP óf glgX affekteer DNA-replikasie en selgrootte op verskillende maniere en mutante met ʼn letsel in die laasgenoemde alleel bevat saamvallende glikogeen-en proteïen-insluitingsliggame, veral merkbaar tydens eksponensiële groei. Die aard van die gebrekke in selgroottebeheer en DNA-replikasie in ΔmalP-mutante, veral die ΔmalP/ΔglgP/ΔglgX trippel-mutant, is verder bestudeer deur gene te verwyder wat by verskeie interaktiewe prosesse betrokke is. Mutasies in gene wat geassosieer is met glikogeenakkumulasie, pirovaatkinase-aktiwiteit, en SOS-bemiddelde of UDP-glukose-afhanklike vertraging van verdeling het gelei tot die vorming van mutante selle wat óf kleiner óf dieselfde grootte soos die wilde tipe is. Gedeeltelike onderdrukkings van die groottedefekte van die trippel-mutant is waargeneem by viervoudige mutante met addisionele uitwissings in gene wat betrokke is by aminosuurmetabolisme, ppGpp-biosintese, UDP-glukose-voortbrenging, DNA-replikasie en nukleoïed-strukturering. DNA-replikasie word duidelik met verskeie fisiologiese prosesse gekoördineer wat gesamentlik werk om duplisering van die genoom aan selgrootte, groeikoers en omgewingstoestande te koppel.Master