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The Enterococcus hirae R40 penicillin-binding protein 5 and the methicillin-resistant Staphylococcus aureus
A el Kharroubi1, P Jacques, G Piras
1Centre d'Ingénierie des Protéines, Université de Liège, Belgium.
Abstract:
The penicillin-resistant Enterococcus hirae R40 has a typical profile of membrane-bound penicillin-binding proteins (PBPs) except that the 71 kDa PBP5 of low penicillin affinity represents about 50% of all the PBPs present. Water-soluble tryptic-digest peptides were selectively produced from PBP5, their N-terminal regions were sequenced and synthetic oligonucleotides were used as primers to generate a 476 bp DNA fragment by polymerase chain reaction. On the basis of these data, the PBP5-encoding gene was cloned in Escherichia coli by using pBR322 as vector. The gene, included in a 7.1 kb insert, had the information for a 678-amino acid-residue protein. PBP5 shows similarity, in the primary structure, with the high-molecular-mass PBPs of class B. In particular, amino acid alignment of the enterococcal PBP5 and the methicillin-resistant staphylococcal PBP2' generates scores that are 30, for the N-terminal domains, and 53, for the C-terminal domains, standard deviations above that expected for a run of 20 randomized pairs of proteins having the same amino acid compositions as the two proteins under consideration.
Insights
Penicillin-resistant Enterococcus hirae R40 features a unique PBP5 protein, comprising 50% of its penicillin-binding proteins (PBPs). This study cloned and sequenced the PBP5 gene, revealing its structural similarity to other high-molecular-mass PBPs.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Enterococcus hirae R40 exhibits penicillin resistance, with Penicillin-Binding Protein 5 (PBP5) constituting a significant portion (50%) of its total PBPs.
- PBP5 is characterized by its low affinity for penicillin, distinguishing it from typical PBP profiles.
Purpose of the Study:
- To investigate the genetic basis of penicillin resistance in Enterococcus hirae R40 by characterizing PBP5.
- To determine the primary structure and evolutionary relationships of PBP5.
Main Methods:
- Selective production and N-terminal sequencing of water-soluble tryptic-digest peptides from PBP5.
- Polymerase chain reaction (PCR) amplification using synthetic oligonucleotide primers to generate a DNA fragment.
- Cloning of the PBP5-encoding gene into Escherichia coli using the pBR322 vector.
Main Results:
- A 476 bp DNA fragment was generated, and the PBP5-encoding gene was successfully cloned.
- The cloned gene encoded a protein of 678 amino acid residues.
- PBP5 demonstrated significant primary structure similarity to high-molecular-mass PBPs of class B, particularly PBP2' from methicillin-resistant Staphylococcus aureus.
Conclusions:
- The PBP5 protein from Enterococcus hirae R40 shares structural homology with other class B PBPs, suggesting conserved functional domains.
- The characterization of PBP5 provides insights into the molecular mechanisms underlying penicillin resistance in this bacterium.