UBC Theses and Dissertations
Stringent regulation of peptidoglycan synthesis in Escherichia coli Ramey, William David
During amino acid deprivation, the amount of meso-diaminopimelic acid (Dap) incorporated into peptidoglycan by dap lys amino acid auxotrophs of Escherichia coli was found to be dependent on the activity of the relA gene product. In relA⁺ bacteria, the- incorporation was substantially reduced, whereas the incorporation in relA⁻ bacteria was essentially equal to that in the unstarved control. The inhibition of Dap incorporation in relA⁺ bacteria was readily overcome by restoration of the required amino acid or by addition of chloramphenicol (CAM). Guanosine 5'-diphosphate 3'-diphosphate (ppGpp) is the product of the reaction between the relA gene product and idling ribosomes in stringent cells. In vitro experiments indicated that physiological levels of ppGpp inhibited at least two steps in peptidoglycan biosynthesis. One was the phospho-N-acetylmuramoyl-pentapeptide transferase (EC18.104.22.168) reaction and the other inhibition was probably at the transfer of peptidoglycan precursors from the glycosyl carrier lipid (GCL) to the nascent peptidoglycan. Quantitation of the peptidoglycan precursors and the net peptidoglycan in relA⁺ control and amino acid-deprived bacteria indicated that peptidoglycan accumulation was inhibited. There was as much UDP-MurNAc-pentapeptide and GCL-linked intermediates in the amino acid-deprived bacteria as in the control bacteria. This suggests that the transfer of lipid-linked precursors to nascent acceptor is the site of inhibition of Dap incorporation. In addition, the pool of soluble nucleotide-linked precursors was found to accumulate when relA⁻ bacteria were deprived of required amino acids. This suggests that the size of the precursor pool is also regulated by the activity of the relA gene product.
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