A comparative study on the phoE genes of three enterobacterial species

Abstract

The cloned phoE genes from Enterobacter cloacae and Klebsiellu pneumoniae are normally expressed and regulated in Escherichia coli K-12, and their products are correctly assembled into the outer membrane. Differences between the three PhoE proteins were found with binding of two out of ten monoclonal antibodies directed against the cell-surface-exposed part and in pore characteristics, but not in phage receptor function. The DNA sequences of the E. cloacae and K . pneumoniue phoE genes were determined and used to predict the primary structures of the encoded proteins. In the upstream non-coding regions, which showed more variations among the three genes than the coding regions, conserved sequences were identified which might be involved in regulation of phoE gene expression. Comparison of the predicted PhoE primary structures revealed a high degree of homology, with 81 YO of the amino acid residues being identical in all three proteins. Four small variable regions were found where differences are the most pronounced, corresponding to regions which were previously predicted to be exposed at the cell surface. Implications of the sequence comparison for structure-function relationships in PhoE protein are discussed.The outer membrane of gram-negative bacteria functions as a molecular sieve. Pore-forming proteins in this membrane allow the passage of small, hydrophilic solutes up to a defined exclusion limit by a diffusion-like process [l, 21. In Escherichia coli K-12 the family of pore-forming proteins includes three members, the OmpF, OmpC and PhoE proteins. OmpF and OmpC proteins are synthesized constitutively under standard growth conditions. Their relative amounts are determined by the osmolarity of the growth medium [3]. The synthesis of PhoE protein is induced when cells are growing under phosphate limitation [4]. Both in vivo [5] and in vitro [6] experiments have shown that the pores formed by PhoE protein have a preference for anions, in contrast to the cation-selective OmpF and OmpC pores.Nucleotide sequence analysis of the ompF, ompC andphoE genes has revealed an extensive homology of approximately 60% in the primary structures of the three proteins. The porins are unusual among membrane proteins in that they lack hydrophobic sequences long enough to span the membrane. The functional unit is a trimer with a compact structure, lacking large extramembrane domains [lo]. It has been shown that in OmpF protein approximately two-thirds of the polypeptide chain is in the form of an antiparallel P-pleated sheet, with many 10 -12-residue-long membranespanning segments, which run approximately normal to the plane of the membrane [ll]. In view of the close homology between the OmpF, OmpC and PhoE proteins, a similar structure for all three pore proteins is to be expected. A pre- dominant p structure has also been found in two other outer membrane proteins, LamB and OmpA, which are unrelated to the porins [12]. We have studied the topology and structure-function relationships of PhoE protein by two different approac...