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 ampli fied by PCR and cloned into different Escherichia coli expression vect ors, The ORF I-encoded protein was similar to 90 kDa and bound H-3-ben zylpenicillin and I-125-cephradine. This high-molecular-weight penicil lin-binding protein (PBP) was also shown to possess transglycosylase a ctivity, indicating that the ORF I product is a bifunctional PBP. The ORF I protein was capable of maintaining the viability of E. coli Delt a ponA ponB::spc(r) cells in transcomplementation experiments, establi shing 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 o f the enzyme not only to recognize the E. coli substrate but also to i nteract with heterologous cell division proteins. The affinity of the ORF I product for H-3-benzylpenicillin and I-125-cephradine, the MIC o f beta-Lactams for E. coli Delta ponA ponB::spc(r) expressing the ORF I gene, and the amino acid alignment of the PBP 1 family of high-molec ular-weight PBPs group the ORF I protein into the PBP 1A family of hig h-molecular-weight PBPs.