The physiological role of AtpI and AtpE of F-type ATP synthases in bacterial energetics
|dc.contributor.advisor||Cook, Gregory M.|
|dc.contributor.author||Iglesias Cans, Marina|
|dc.identifier.citation||Iglesias Cans, M. (2014). The physiological role of AtpI and AtpE of F-type ATP synthases in bacterial energetics (Thesis, Doctor of Philosophy). University of Otago. Retrieved from http://hdl.handle.net/10523/4587||en|
|dc.description.abstract||Bacterial F-type ATP synthases are large molecular weight protein complexes composed of a membrane-embedded portion Fo, and a large cytoplasmic portion F1. The complete enzyme complex consists of eight subunits (α3, β3, γ, δ, ε, a, b2 and c10-15), whereas nine open reading frames are present in most bacterial atp operons (i.e. atpIBEFHAGDC). The first gene in most bacterial atp operons starts with atpI, yet the role of AtpI has remained enigmatic with roles in magnesium transport and c-ring assembly reported. No studies have assessed the role of AtpI on cell physiology at a metabolic and transcriptional level. For this purpose, a non-polar unmarked gene deletion of atpI in Escherichia coli was constructed and compared to the isogenic wild type parental strain. The ΔatpI mutant had a lower molar growth yield on succinate compared to the wild type. This lower yield was not attributable to lower levels of atp operon expression, but was correlated with reduced ATP synthase assembly, ATP synthase activity (hydrolysis and synthesis), and ATP levels in the cell. Whole genome expression comparison by RNA microarray analysis was performed between ΔatpI and wild type strains at both mid-exponential phase and early-stationary phase. At mid-exponential phase, 57 genes were significantly downregulated (p < 0.05; > 1.4 fold expression change) in ΔatpI mutant strain compared to the wild type strain. At early-stationary phase, 11 genes were downregulated and 4 upregulated in the mutant strain. Analysis of this data revealed that the lack of AtpI triggers a shift in central metabolism as a compensatory mechanism to balance ATP levels due to a non-fully active ATP synthase. ΔatpI mutants showed a decrease in the expression of the puu operon, a set of genes involved in the putrescine metabolic pathway, decreasing the amount of succinate going into the tricarboxylic acid cycle (TCA). Acetolactate dehydrogenase (ilv operon), responsible for the utilization of pyruvate to synthesize amino acids (valine and isoleucine), was also downregulated, increasing the amount of pyruvate entering the TCA and generating energy directly in the form of ATP via oxidative phosphorylation. Another important observation was the upregulation of a pH-dependent Na+/H+ antiporter, which shows an imbalance in the intracellular pH at high pH due to a lower H+ uptake activity of the ATP synthase in the absence of AtpI. These data demonstrate that deletion of atpI in E. coli causes changes in ATP synthase assembly and activity, central carbon metabolism and pH homeostasis. AtpI has been reported to be essential for the assembly of a c11-ring in a hybrid F1Fo (F1 from thermophilic Bacillus PS3 and Na+-translocating membrane-bound Fo from Propionigenium modestum). The c11 ring is unique to Na+-coupled ATP synthases found in many environmental bacteria. However, the role of Na+-coupling in bacterial pathogens has not been assessed. We report a new Na+-F1Fo ATP synthase with a novel Na+-binding signature in the atpE gene of the atp operon of the anaerobic bacterium Fusobacterium nucleatum, a Gram-negative bacterium implicated in the aetiology of periodontal diseases. F. nucleatum uses the free energy of glutamate decarboxylation (glutaconyl-CoA decarboxylase) to generate a sodium motive force (ΔμNa+) across the cytoplasmic membrane. This ΔμNa+ is directly coupled to ATP synthesis, via an F1Fo-ATP synthase with a novel Na+ recognition motif. Molecular modelling and free-energy simulations of the rotary element of the enzyme, the c-ring, indicated Na+ specificity in physiological settings. Consistently, activity measurements showed Na+ stimulation of the enzyme, either membrane-embedded or isolated, and ATP synthesis was sensitive to the Na+ ionophore monensin. Furthermore, Na+ has a protective effect against inhibitors targeting the ion-binding sites in the complete ATP synthase. Several new inhibitors of the F. nucleatum F1Fo-ATP synthase were identified and these inhibitors may be new antibacterial compounds, distinct from any existing antibiotics.|
|dc.publisher||University of Otago|
|dc.rights||All items in OUR Archive are provided for private study and research purposes and are protected by copyright with all rights reserved unless otherwise indicated.|
|dc.title||The physiological role of AtpI and AtpE of F-type ATP synthases in bacterial energetics|
|thesis.degree.discipline||Microbiology and Immunology|
|thesis.degree.name||Doctor of Philosophy|
|thesis.degree.grantor||University of Otago|
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