Expression and characterization of the ponA (ORF I) gene of Haemophilus influenzae: functional complementation in a heterologous system.

AUTOR(ES)

Sharma, U K

RESUMO

The coding sequence of the Haemophilus influenzae ORF I gene was amplified by PCR and cloned into different Escherichia coli expression vectors. The ORF I-encoded protein was approximately 90 kDa and bound 3H-benzyl-penicillin and 125I-cephradine. This high-molecular-weight penicillin-binding protein (PBP) was also shown to possess transglycosylase activity, indicating that the ORF I product is a bifunctional PBP. The ORF I protein was capable of maintaining the viability of E. coli delta ponA ponB::spcr cells in transcomplementation experiments, establishing the functional relevance of the significant amino acid homology seen between E. coli PBP 1A and 1B and the H. influenzae ORF I product. In addition, the physiological functioning of the H. influenzae ORF I (PBP 1A) product in a heterologous species established the ability of the enzyme not only to recognize the E. coli substrate but also to interact with heterologous cell division proteins. The affinity of the ORF I product for 3H-benzylpenicillin and 125I-cephradine, the MIC of beta-lactams for E. coli delta ponA ponB::spcr expressing the ORF I gene, and the amino acid alignment of the PBP 1 family of high-molecular-weight PBPs group the ORF I protein into the PBP 1A family of high-molecular-weight PBPs.

Documentos Relacionados

• Cloning, characterization and heterologous expression of the SmaI restriction-modification system.
• Cloning and characterization of the ponA gene encoding penicillin-binding protein 1 from Neisseria gonorrhoeae and Neisseria meningitidis.
• Haemophilus influenzae: antibiotic susceptibility.
• Laboratory identification of Haemophilus influenzae: effects of basal media on the results of the satellitism test and evaluation of the RapID NH system.
• IgA1 proteases of Haemophilus influenzae: cloning and characterization in Escherichia coli K-12.