Tessa Fowler University Number
Tessa Fowler University Number: 149011032
Formative Writing Assignment:
Ampicillin is a broad spectrum cytolytic antibiotic (Sharma, Singh and Singh, 2013) that induces bacterial cell death through the prevention of peptidoglycan cell wall formation, leading to cell lysis (Yao, Kahne and Kishony, 2012). Ampicillin comprises of a central ?-lactam ring, with a thiazolidine ring attached to one side and an acyl-side chain with an additional amino group attached to the other (Raynor, 1997). Due to this amino group ampicillin is known as an aminopenicillin (Sharma, Singh and Singh, 2013) and therefore, unlike most penicillins which can mainly cause cell death in gram positive bacteria, ampicillin can pass through the lipopolysaccharide and outer membrane of gram negative bacteria, to reach the peptidoglycan layer in the periplasmic space (Raynor, 1997), inducing bacterial cell death.
The bacterial peptidoglycan cell wall is formed from peptide and polysaccharide molecules crosslinked by glycine residues (Kapoor, Saigal and Elongavan, 2017) and the enzyme which catalyses this reaction is known as transpeptidase (Nelson and Cox, 2013). On the introduction of ampicillin to bacterial cells, ampicillins ?-lactam ring binds to ?-lactam binding proteins such as transpeptidase (Nelson and Cox, 2013). This is because the ?-lactam ring mimics the structure of a precursor segment of peptidoglycan known as D-alanyl-D-Alanine (Kapoor, Saigal and Elongavan, 2017), the substrate of transpeptidase, and so competitively binds to the active site of transpeptidase (Nelson and Cox, 2013). On binding, a serine molecule in the active site of transpeptidase, reacts with the carbonyl group located on the ?-lactam ring of ampicillin. This forms a covalent bond between the two molecules, permanently inactivating transpeptidase and inhibiting cell wall synthesis (Nelson and Cox, 2013). The inhibition of cell wall synthesis causes an increase in osmotic pressure within the cell, leading to cell lysis of the inner membrane and in turn bacterial cell death (Yao, Kahne and Kishony, 2012).
To overcome the action of ?-lactam antibiotics, many pathogenic strains of bacteria have genetically evolved to become resistant (Munita, Bayer and Arias, 2015). Whereas the majority of gram positive bacterial strains have acquired mutations in the coding sequence for penicillin binding proteins, creating a structural change and preventing penicillins from binding (Munita, Bayer and Arias, 2015), gram negative bacteria have developed genes which encode proteins known as ?-lactamases to induce antibiotic resistance (Raynor, 1997). ?-lactamase enzymes work by hydrolysing the ?-lactam ring within ampicillin, thwarting the binding of ampicillin to ?-lactam binding proteins and therefore preventing the inhibitory action of ?-lactam antibiotics (Bush and Bradford, 2016).
Tessa Fowler University Number: 149011032
Through understanding the mechanism of ?-lactamases in antibiotic resistance, scientists have been able to exploit ?-lactamase encoding genes for use as selective markers following transformation of a plasmid vector into a bacterial cell such as E. coli. The pUC19 plasmid is 2686bp in length (Serban, Benevides and Thomas, 2002) and contains a gene that encodes a ?-lactamase which induces ampicillin resistance (Rivas et al., 2013). Following successful
transformation of a pUC19 plasmid into an E. coli strain such as DH5?, a strain that does not
carry an ampicillin resistance gene, the pUC19 vector will replicate and the ampicillin
resistance gene transcribed and translated to produce ?-lactamase, rendering the DH5? cell
ampicillin resistant (Rivas et al., 2013). This provides a selection marker as if, following transformation the DH5? cells are grown in the presence of ampicillin, only cells which have undergone successful transformation will be ampicillin resistant and form colonies. Studies to test the transformation of recombinant pUC19 vectors containing a gene of interest, or testing the efficiency of different transformation methods (Rivas et al., 2013), are some of the ways this technology of using pUC19 vectors containing an ampicillin resistance gene as a selection marker for successful transformation can be utilised.
Bush, K. and Bradford, P. A. (2016) ‘b -Lactams and b -Lactamase Inhibitors: An Overview’,
Perspectives in Medicine, 6(8), pp. 1–23.
Kapoor, G., Saigal, S. and Elongavan, A. (2017) ‘Action and resistance mechanisms of antibiotics: A guide for clinicians’, Journal of Anaethesiology and Clinical Pharmacology, 33(3), pp. 300–305.
Munita, J. M., Bayer, A. S. and Arias, C. A. (2015) ‘Evolving Resistance Among Gram-positive Pathogens’, Clinical Infectious Diseases, 61(Suppl 2), pp. 48–57.
Nelson, D. L. and Cox, M. M. (2013) Principles of Biochemistry 6th edition, pp. 224-225
Raynor, B. D. (1997) ‘PENICILLIN AND AMPICILLIN’, Infectious Disease Update, 4(4), pp. 147–152.
Rivas, A., Pina-perez, M. C., Rodriguez-Vargas, S., Zuniga, M., Martinez, A. and Rodrigo, D. (2013) ‘Sublethally damaged cells of Escherichia coli by Pulsed Electric Fields: The chance of transformation and proteomic assays’, Food Research International, 54, pp. 1120–1127.
Serban, D., Benevides, J. M. and Thomas, G. J. (2002) ‘DNA Secondary Structure and Raman Markers of Supercoiling in Escherichia coli’, Biochemistry, 41, pp. 847–853.
Sharma, S. K., Singh, L. and Singh, S. (2013) ‘Comparative Study between Penicillin and Ampicillin’, Journal of Applied Medial Sciences, 1(4), pp. 291–294.
Yao, Z., Kahne, D. and Kishony, R. (2012) ‘Distinct Single-Cell Morphological Dynamics under Beta-Lactam Antibiotics’, Molecular Cell. Elsevier Inc., 48(5), pp. 705–712.
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