Expression and characterization of the ponA (ORF I) gene of Haemophilus influenzae: functional complementation in a heterologous system.
J Bacteriol
; 177(23): 6745-50, 1995 Dec.
Article
em En
| MEDLINE
| ID: mdl-7592463
ABSTRACT
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 ponBspcr 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 ponBspcr 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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Proteínas de Bactérias
/
Muramilpentapeptídeo Carboxipeptidase
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Proteínas de Transporte
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Peptidil Transferases
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Haemophilus influenzae
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Genes Bacterianos
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Hexosiltransferases
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Complexos Multienzimáticos
Idioma:
En
Revista:
J Bacteriol
Ano de publicação:
1995
Tipo de documento:
Article
País de afiliação:
Índia