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Nucleotide sequences of the pbpX genes encoding the penicillin-binding proteins 2x from Streptococcus pneumoniae R6
G Laible1, R Hakenbeck, M A Sicard
1Max-Planck Institut für Molekulare Genetik, Berlin, FRG.
Abstract:
Development of penicillin resistance in Streptococcus pneumoniae is due to successive mutations in penicillin-binding proteins (PBPs) which reduce their affinity for beta-lactam antibiotics. PBP2x is one of the high-Mr PBPs which appears to be altered both in resistant clinical isolates, and in cefotaxime-resistant laboratory mutants. In this study, we have sequenced a 2564 base-pair chromosomal fragment from the penicillin-sensitive S. pneumoniae strain R6, which contains the PBP2x gene. Within this fragment, a 2250 base-pair open reading frame was found which coded for a protein having an Mr of 82.35kD, a value which is in good agreement with the Mr of 80-85 kD measured by SDS-gel electrophoresis of the PBP2x protein itself. The N-terminal region resembled an unprocessed signal peptide and was followed by a hydrophobic sequence that may be responsible for membrane attachment of PBP2x. The corresponding nucleotide sequence of the PBP2x gene from C504, a cefotaxime-resistant laboratory mutant obtained after five selection steps, contained three nucleotide substitutions, causing three amino acid alterations within the beta-lactam binding domain of the PBP2x protein. Alterations affecting similar regions of Escherichia coli PBP3 and Neisseria gonorrhoeae PBP2 from beta-lactam-resistant strains are known. The penicillin-binding domain of PBP2x shows highest homology with these two PBPs and S. pneumoniae PBP2b. In contrast, the N-terminal extension of PBP2x has the highest homology with E. coli PBP2 and methicillin-resistant Staphylococcus aureus PBP2'. No significant homology was detected with PBP1a or PBP1b of Escherichia coli, or with the low-Mr PBPs.
Insights
Penicillin resistance in Streptococcus pneumoniae stems from mutations in penicillin-binding proteins (PBPs). Sequencing revealed three key mutations in PBP2x from a resistant strain, altering its beta-lactam binding domain.
Area of Science:
- Microbiology
- Molecular Biology
- Antibiotic Resistance
Background:
- Penicillin resistance in Streptococcus pneumoniae is a growing public health concern.
- This resistance is primarily mediated by alterations in penicillin-binding proteins (PBPs), which are targets for beta-lactam antibiotics.
- PBP2x, a high-molecular-weight PBP, is frequently implicated in resistance, particularly in clinical isolates and laboratory mutants.
Purpose of the Study:
- To characterize the PBP2x gene and its protein product in Streptococcus pneumoniae.
- To identify specific genetic alterations in PBP2x associated with cefotaxime resistance.
- To compare the PBP2x sequence with homologous proteins from other bacterial species.
Main Methods:
- DNA sequencing of a 2564 base-pair fragment containing the PBP2x gene from the sensitive strain R6.
- Analysis of the open reading frame and predicted protein sequence of PBP2x.
- Sequencing of the PBP2x gene from the cefotaxime-resistant mutant C504.
- Comparative sequence analysis of PBP2x with other known PBPs.
Main Results:
- A 2250 base-pair open reading frame coding for an 82.35 kD protein was identified for PBP2x.
- The N-terminal region of PBP2x contains a putative signal peptide and a hydrophobic membrane-anchoring sequence.
- Three nucleotide substitutions were found in the PBP2x gene of the resistant mutant C504, leading to amino acid changes in the beta-lactam binding domain.
- PBP2x shares homology in its binding domain with PBP3 of E. coli and PBP2 of N. gonorrhoeae, and in its N-terminal region with PBP2 of E. coli and PBP2' of S. aureus.
Conclusions:
- The identified PBP2x sequence and predicted protein structure are consistent with its role in Streptococcus pneumoniae.
- Specific mutations within the beta-lactam binding domain of PBP2x are directly linked to cefotaxime resistance.
- Comparative analysis highlights conserved regions and potential functional domains across different bacterial PBPs involved in beta-lactam resistance.