Expression and characterization of the ponA (ORF I) gene of Haemophilus influenzae: functional complementation in a heterologous system.
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.
Sharma
U K
UK
Astra Research Centre India, Malleswaram, Bangalore, India.
Dwarakanath
P
P
Banerjee
N
N
Town
C
C
Balganesh
T S
TS
D001426:Bacterial Proteins; D001483:Base Sequence; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics*; D002511:Cephalosporins / Q000378:metabolism; D002515:Cephradine / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D006193:Haemophilus influenzae / Q000235:genetics*; D006602:Hexosyltransferases / Q000096:biosynthesis / Q000235:genetics*; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000096:biosynthesis / Q000235:genetics*; D002267:Muramoylpentapeptide Carboxypeptidase / Q000096:biosynthesis / Q000235:genetics*; D016366:Open Reading Frames; D046915:Penicillin-Binding Proteins; D010457:Peptidoglycan / Q000096:biosynthesis; D010458:Peptidyl Transferases / Q000096:biosynthesis / Q000235:genetics*; D016133:Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
D001426:Bacterial Proteins; D002352:Carrier Proteins; D002511:Cephalosporins; D009097:Multienzyme Complexes; D046915:Penicillin-Binding Proteins; D010457:Peptidoglycan; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D002267:Muramoylpentapeptide Carboxypeptidase; D002515:Cephradine