Mutational analysis of E. coli maltodextrin metabolism

Mushfieldt, Kristeena (2017-03)

Thesis (MSc)--Stellenbosch University, 2017.

Thesis

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.

AFRKAANSE OPSOMMING: Escherichia coli (E. coli) glikogeen en maltodextrin metabolisme is breedvoerig bestudeer; in teen deel is die rol van hierdie poliglukane met betrekking tot mobilisering steeds onbekend. Die mal ensieme naamlik ; maltodekstrin fosforylase (MalP), amylomaltase (MalQ) en maltodekstrin glucosidase (MalZ), wat verantwoordelik is vir die mobilisering van maltodektrins in die sitoplasma, ook gebestudeer vir hul rol in glikogeen metabolisme. Aangesien glikogeenfosforilase (GlgP), struktureel en funksioneel ooreenstemming toon aan MalP, word die rol van hierdie ensiem met betrekking tot maltodekstrin metabolisme ondersoek. Dus, was 'n omgekeerde genetiese benadering gebruik om die funksie aan hierdie ensieme toe te ken d.m.v die gene wat vir dit enkodeer in E. coli te muteer. In meeste gevalle was dubbele gemuteerde kombinasies van die gemuteerde gene vervaardig,behalwe, een dubbele gemuteerde kombinasie (ΔmalQ/ΔmalP) was egter onmoontlik om te isoleer. Dubbel en enkele mutante asook wilde-tipe-stamme was opgegroei in groei media aangevul met óf maltose of glukose; waarna verskeie parameters geanaliseer was, insluitend poliglukaan inhoud, groei asook sel morfologie, gemeet was. Wanneer die groei media aangevul was met glukose, was daar geen effek op die groeikoers van stamme waargeneem nie. Verhoogde glikogeen akkumulasie was waargeneem in ΔmalP/ΔglgP, ΔmalQ/ΔglgP en ΔmalZ/ΔglgP dubbel mutante in vergelyking met die ΔglgP enkele mutante wat aandui dat mutasies in die mal ensieme dalk 'n uitwerking mag hê op glikogeen akkumulasie. Skandering elektroon mikroskopie (SEM) het getoon dat die ΔmalP, ΔmalP/ΔglgP en ΔmalQ stamme ‘n paar verlengde selle gevorm het. Aangesien die bogenoemde stamme ook glikogeen akkumulasie in beide vloeibare en op soliede media getoon het kan dit wees dat die akkumulasie van hierdie glikogeen moontlik die verlenging van die selle kan verduidelik. Die groei van die kulture met maltose aanvulling veroorsaak verskille in die groeikoers van stamme, maar die meganisme waardeur dit beïnvloed word, is nog steeds onbekend. Poliglukaan akkumulasie was slegs geobserveer in sekere stamme, dus mag dit 'n bykomende funksie vir MalZ in maltodekstrin en glikogeen metabolisme aandui, gebaseer op die waarnemings vir ΔmalZ/ΔglgP en ΔmalZ/ΔmalQ mutante. Skandering electron mikroskopie het ook aangedui dat die groeikoers in die teenwoordigheid van maltose ook 'n impak mag hê op die morfologie van kulture aangesien stamme naamlik invouings van die selwand (ΔmalP, ΔmalZ/ΔmalP, ΔmalP/ΔglgP) óf liseering (ΔmalQ, Δ malQ/glgP). In samevatting, het die data getoon dat MalP, MalQ, MalZ en GlgP dalk addisionele funksionele rolle mag hê in E. coli poliglukaan metabolisme wat nie voorheen beskryf is nie.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/100798
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