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dc.contributor.advisorGoldie, Hughesen_US
dc.creatorMadhavan, Seemaen_US
dc.date.accessioned2008-10-27T11:54:54Zen_US
dc.date.accessioned2013-01-04T05:07:03Z
dc.date.available2009-10-27T08:00:00Zen_US
dc.date.available2013-01-04T05:07:03Z
dc.date.created2002-09en_US
dc.date.issued2002-09-01en_US
dc.date.submittedSeptember 2002en_US
dc.identifier.urihttp://hdl.handle.net/10388/etd-10272008-115454en_US
dc.description.abstractPhosphoenolpyruvate carboxykinase [E.C.4.1.1.49] (Pck) catalyses a key reaction of gluconeogeneis in Escherichia coli. It converts the Krebs cycle intermediate oxaloacetate (OAA) to phophoenolpyruvate (PEP), which is a part of glycolysis. Transcription of pckA, the structural gene for Pck is regulated by catabolite repression and the transcription increases 100 fold in early stationary phase, by an unknown mechanism. This study used mini Tn10-ATS (KanR) to isolate mutants that affect the expression of pckA in stationary phase. Using penicillin selection, Succinate⁻, KanR mutants defective in Pck were isolated. The mutants had lower growth yields and lower Pck specific activity than the wild type. Experiments with a slow growing recA strain and a pckA::Tn10 mutant indicated that the lowered Pck levels were not correlated with low growth yields. Four independent mutants were isolated which had KanR tightly linked to the Suc⁻ phenotype but not to pckA as determined by P1 transduction. These mutants fermented maltose and arabinose, which indicated that the mutations were not in the cya or crp genes. PCR with IS10 and REP (Repetitive Extragenic Palindrome) primers was done to amplify presumptive mini Tn10 insertions. However, no homology to the transposon could be found and it was hypothesised that the mutants contained spontaneous KanR mutations, which resulted in Pck⁻ (Succinate⁻) phenotype. Since it was known that mutations in the atp genes, encoding ATP synthase had Suc⁻ KanR phenotypes, P1 transduction was used to test for the linkage to genes close to the atp operon. Three of the mutations were tightly linked to ilvD and to rbs, while the other appeared to have multiple KanR and could not be mapped further. The mutants were found to have low levels of ATP synthase as well as Pck and transductants regained both enzyme activities and kanamycin sensitivity. Plasmids containing all or just the F₁ portion of the atp operon complemented the phenotypes (Suc⁻, Pck⁻, KanR and ATP synthase⁻) of all the mutants. DNA fragments encoding the F₁ region of ATP synthase of the mutants were sequenced after PCR amplification. In two mutants there was a two base pair "GC" deletion in atpG resulting in a truncation of 28 amino acids at the carboxyl terminus end of the γ subunit of ATP synthase. The other mutant had a "T" deletion in atpG, which led to a 40 amino acid truncation at the carboxyl terminus of the γ subunit of ATP synthase. Complementation of the Suc⁻ phenotype with plasmid pBWG15 expressing the γ subunit confirmed that the mutations were in atpG in the mutant HG205. This study led us to identify a role of the atpG gene in pckA expression in stationary phase. The atpG mutations could affect expression of pckA in different ways. First, gluconeogenesis is an energy consuming process. Low ATP levels (low energy state) in the mutants could lead to low Pck levels. Second, atpG might affect pckA at a genetic level. Third, the γ subunit is a gate for proton flow and links ATP synthesis to proton translocation. The presence of the faulty γ subunit could make ATP synthase a proton pore, which results in collapse of the pH gradient. This could have some effect on the expression of pckA. Fourth, intracellular pH might also affect the synthesis or activity of Pck. There are a number of genes, e.g. ompF, lamB, mar operon, whose expression changes as a function of pH. Lastly, ATP synthase might interact with a protein kinase or is a protein kinase itself and affects the phosphorylation of a protein that activates the expression of pckA. From the results of this work, atp genes seem to play an important role in the expression of Pck activity. The atp genes could regulate Pck activity at the transcriptional, translational or protein levels. This opens up a new area of investigation of the stationary phase regulation of pckA.en_US
dc.language.isoen_USen_US
dc.titleMutations in atpG, encoding the γ subunit of ATP synthase cause lowered expression of pckA in Escherichia colien_US
thesis.degree.departmentMicrobiology and Immunologyen_US
thesis.degree.disciplineMicrobiology and Immunologyen_US
thesis.degree.grantorUniversity of Saskatchewanen_US
thesis.degree.levelDoctoralen_US
thesis.degree.nameDoctor of Philosophy (Ph.D.)en_US
dc.type.materialtexten_US
dc.type.genreThesisen_US
dc.contributor.committeeMemberRoesler, William J.en_US
dc.contributor.committeeMemberHayes, Sidneyen_US
dc.contributor.committeeMemberDeneer, Harryen_US
dc.contributor.committeeMemberBull, Harolden_US
dc.contributor.committeeMemberXiao, Weien_US


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