Browsing by Author "Mushfieldt, Kristeena"

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    Mutational analysis of E. coli maltodextrin metabolism
    (Stellenbosch : Stellenbosch University, 2017-03) Mushfieldt, Kristeena; Lloyd, James Richard; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics. Institute for Plant Biotechnology.
    ENGLISH ABSTRACT: Escherichia coli glycogen and maltodextrin metabolism has been extensively studied; however, the roles of the enzymes involved in the mobilization of these polyglucans remains unclear. The mal enzymes; maltodextrin phosphorylase (MalP), amylomaltase (MalQ) and maltodextrin glucosidase (MalZ), which are responsible for mobilization of maltodextrins in the cytoplasm, are also examined for their effects on glycogen metabolism. Since the glycogen phosphorylase (GlgP), is structurally and functionally similar to MalP, the role of this enzyme was examined in relation to maltodextrin metabolism. To elucidate their functions, a reverse genetics approach was used, where genes which encode them were mutated in E. coli. Double mutant combinations of most of the mutated genes were manufactured. One double mutant combination (ΔmalQ/ΔmalP) proved, however, impossible to isolate. The double mutants, single mutants and wild-type strains were grown in media supplemented with either maltose or glucose and analysed for a number of parameters, including polyglucan content, growth and cell morphology. When the strains were grown with glucose supplementation, there was no effect on the growth rate of strains. Increased glycogen accumulation was observed in ΔmalP/ΔglgP, ΔmalQ/ΔglgP and ΔmalZ/ΔglgP double mutants when compared to the ΔglgP single mutant indicating that mutations in the mal enzymes have an effect on glycogen accumulation. Scanning electron microscopy (SEM) of ΔmalP, ΔmalP/ΔglgP and ΔmalQ strains revealed the accumulation of a few elongated cells. Since these strains also showed increased glycogen accumulation in liquid and on solid media, it may be that its accumulation is a factor which could explain the elongation of cells. Growth of strains with maltose supplementation caused variations in the growth rate of strains, however, the mechanism whereby this is affected is not evident. Polyglucan accumulation in some strains indicated an additional role for MalZ in maltodextrin as well as glycogen metabolism based on the observations made for ΔmalZ/ΔglgP and ΔmalZ/ΔmalQ mutants. Scanning electron microscopy indicated that growth in maltose has some impact on the morphology of cultures since strains appeared to either have invaginations in the cell wall (ΔmalP, ΔmalZ/ΔmalP, ΔmalP/ΔglgP) or have lysed (ΔmalQ ,ΔmalQ/glgP). Overall, the data presented here demonstrates that MalP, MalQ, MalZ and GlgP may have additional functional roles in E. coli polyglucan metabolism that have not been previously elucidated.