Sequence of the ponA gene and characterization of the penicillin-binding protein 1A of Pseudomonas aeruginosa PAO1

The nucleotide sequence of the ponA gene encoding the high molecular-mass penicillin-binding protein 1A (PBP1A) of Pseudomonas aeruginosa ( Pa) PAO1 was determined and characterized. The predicted PBP1A protein of 822 amino acids (aa) has a calculated molecular mass of 91.2 kDa corresponding to the size of the protein expressed in vitro and in vivo. A penicillin-binding (PB) assay showed that the Pa ponA gene product covalently binds penicillin. The deduced PBP1A aa sequence has features typical of class-A high-molecular-mass PBPs: a highly hydrophobic N-terminus portion containing a potential transmembrane segment which might anchor the protein to the cytoplasmic membrane; an N-terminal module with the conserved boxes 1 (E 86D(DN)F(AN)H(Y)G), 2 (G 117GS(T)I(TM)Q), 3 (R 139K(IN)E(ILL)AL) and 4 (R 221R(NW)IL); a PB module with the conserved boxes 5 (S 461SFK), (S 520RN) and (K 695TG); an internal extension at aa 297–407 between the N-terminal and PB modules; and a C-extension at the end of the PB module at aa 742 to 822. The highest percentage of similarity (62.8%) was found with the class A high-molecular-mass PBP1A of Escherichia coli ( Ec) and Haemophilus influenzae. The observed extensive homology in the modular design of the Pa PBP1A with the bifunctional Ec PBP1A suggests structural and functional relationships between these proteins and refutes the proposed correspondence between Pa PBP1A and Ec PBP1B.