The Cloning Of Bacteria

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Enterococci are facultative anaerobic and Gram-positive bacteria often forming normal fecal flora of humans. However, these bacteria are increasingly appearing as primary pathogens among patients with compromised immune systems (Hemalatha, Bhaskaran, Sowmiya, & Anusheela Howlader, 2017). This manifestation is believed to be due to its resistance to various antibiotics along with its ability to form biofilms and acquiring some virulence factors (Santajit & Indrawattana, 2016).

Enterococci in hospitals lead to the urinary tract, soft tissue, and bacteremia infection in patients exposed to different antibiotic classes (Guzman Prieto et al., 2016; Santajit & Indrawattana, 2016). Unfortunately, enterococcal infection treatments are not well developed because of its resistance to the most used antibiotics. Except for one class of cephalosporin, they are resistant to all beta-lactam antibiotics including antistaphylococcal penicillins (Khan, Miller, & Arias, 2018).

Bacterial cell walls consist of a modified peptidoglycan layer containing proteins and polymers, which is necessary for the survival of the cell (Pazos & Peters, 2019). This layer of peptidoglycan is developed in bacteria by a synergic action of various proteins. These include several penicillin-binding proteins (PBP) (Pazos & Peters, 2019; Van Dross-Anderson & Ladin, 2018). These may be endopeptidases, transpeptidases, as well as carboxypeptidases, which can modify different peptidoglycans while synthesizing new peptides (Miyachiro, Contreras-Martel, & Dessen, 2019). PBP can be classified according to its enzymatic activity: class A is a multifunctional PBP with glycosyltransferase and transpeptidase activity; class B is a transpeptidase; class C is carboxypeptidase and endopeptidase (Moon et al., 2018).

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In enterococci, the reduction in β-lactam antibiotic susceptibility is due to the single low-affinity class PBP (class B) expression (Djorić, Little, & Kristich, 2020). Typically, Enterococcus hirae is often chosen as a model species to study PBPs (Montealegre et al., 2017). The protein is expressed in Escherichia coli and the kinetics of certain PBPs is analyzed to understand its role in providing antibiotic resistance to these species.

The catalytic serine interferes with the penultimate D-Ala carbonyl (of the donor stem peptide) in Class B PBP transpeptidases (Caveney et al., 2019). This releases the C-D-Ala and forms a covalent acyl-enzyme conjugation with the donor stem peptide. Then the D-Ala carbonyl group conjugation experiences a nucleophilic attack by a primary amine present at the end of the side chain of the acceptor stem peptide, creating a bridge between the peptides, thus connecting the glycan strands (Caveney et al., 2019; Maya-Martinez et al., 2019; Walter & Mayer, 2019).

The donor substrate D-Ala-D-Ala sequence is mimicked by beta-lactam antibiotics functioning as inhibitors of cell wall synthesis (Moon et al., 2018; Phillips-Jones & Harding, 2018). Following their discovery as a β-lactam antibiotics target, PBPs are extensively researched, particularly concerning its role in conferring antibiotic resistance to enterococci and staphylococci against β-lactams (Guzman Prieto et al., 2016).

The current study focuses on the kinetic properties of PBP4 isolated from Enterococcus hirae. Limited focus has been given to PBP4 from Enterococci to date. We have overexpressed E. hirae PBP4 in E.coli and checked binding efficiency against β-lactam antibiotics. In addition to SDS PAGE, the kinetic properties are studied by LABCHIP GXII and all the samplings are done using Microlab Hamilton Robotic arms for increased consistency and accuracy. We also investigated the cellular localization of the protein this investigation will provide an insight into the behavior and role of PBP4 in offering β-lactam antibiotic resistance to the species.

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The Cloning Of Bacteria. (2022, February 17). Edubirdie. Retrieved April 23, 2024, from
“The Cloning Of Bacteria.” Edubirdie, 17 Feb. 2022,
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