The role of highly phosphorylated nucleotides in the development of bacterial resistance to antibiotics

  • Jane Leitch

    Student thesis: Doctoral Thesis


    The term ‘Magic Spot’ (MS) defines a small group of highly phosphorylated purines (HPNs; MSI, ppGpp; MSII, pppGpp) produced by bacterial cells during the late exponential and early stationary phases of growth, in response to adverse nutritional conditions. These compounds are involved in the phenomena of endosporulation and stringent response which both elicit an increase in observed antibiotic resistance in many bacterial species. It is thought that HPNs indicate the onset of and/or initiate bacterial resistance to the presence of antibiotics. The effects of nutrient limitation (carbon, nitrogen, magnesium and phosphate) upon the two bacterial species, Escherichiacoli NCIMB 10000 and Bacillus subtilis NCIMB 12900, were observed. The appearance and disappearance of various nucleotides (AMP, GMP, ADP, GDP, ATP and GTP) involved in HPN biosynthesis in E. coli and B. subtilis grown in nutrient-limited media were observed via fluorescence 2D-TLC. Cellular extracts of both E. coli and B. subtilis exhibited a reduction in the apparent content of ATP, GTP, ADP and GDP when observed by 2D-TLC. A fluorescent spot was observed at the origin which was assumed to correspond to ‘Magic Spot’. Results indicate that ATP is the pyrophosphate donor in HPN synthesis and that GDP and GTP are the acceptor molecules. A new rapid isocratic HPLC protocol was developed in this study, in order to quantify the 2D-TLC results. Cellular extracts taken from batch cultures of E. coli and B. subtilis grown under conditions of nutrient limitation, both challenged and unchallenged with sub-minimal inhibitory concentrations (MIC) of erythromycin, exhibited a reduction in the concentration of AMP, GMP, ADP, GDP, ATP and GTP with the concurrent synthesis of HPNs. The HPNs exhibited peaks with Rt values of 3.8 mins (ppGpp, MSI) and 2.5 mins (pppGpp, MSII) respectively. Further experiments involving the fluorimetric analysis of ATP concentration in growing bacterial cultures revealed a marked decrease in ATP concentration per cell at the onset of stationary phase and support the previous observations regarding the role of this compound in the synthesis of HPNs. The new isocratic HPLC protocol was utilised for the observation of the kinetics of nucleotide and HPN concentrations in cellular extracts taken from batch cultures of both microorganisms. These results suggest a close relationship between the reduction and disappearance of certain nucleotides and the synthesis and appearance of HPNs. The detection of HPNs in the cultures challenged with sub-MIC erythromycin, at either the onset of or during stationary phase of growth, indicates that the onset of sporulation and/or stringent response is closely associated with the onset of bacterial resistance to this antibiotic.
    Date of AwardJun 1998
    Original languageEnglish

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