Pore formation by pho‐controlled outer‐membrane proteins of various Enterobacteriaceae in lipid bilayers

Abstract

The structural genes of the PhoE porins of Klebsiella pneumonia, Enterobacter cloacae and Escherichia coli C, cloned in multicopy plasmids, were transfered into a porin-deficient E. coli K-12 strain, which was constitutive for the pho regulon, and the PhoE porins were isolated and purified. PhoE of Salmonella typhimurium could not be cloned but was isolated from a pho-constitutive strain. Reconstitution experiments with artificial lipid bilayer membranes showed that the different PhoE proteins formed pores exhibiting a single-channel conductance of about 200 pS at 0.1 M KC1. All PhoE porins formed anion-selective channels in KC1 at neutral pH. The degree of the selectivity was dependent on the PhoE species. The different PhoE porins formed general diffusion pores similar to the general porins but exhibited a considerable advantage for the permeation of phosphate through the outer membrane as compared to the constitutive OmpC and OmpF porins of E. coli K-12. [12]. The synthesis of PhoE makes sense because it has been shown that PhoE of E. coli K-12 forms anion-selective pores in vivo [13, 141 and in vitro [15, 161 and that of S . typhimurium in vitro [lo], whereas the general porins (OmpF and OmpC of E. coli K-12 and OmpF, OmpC and OmpD of S. typhimurium) are cation-selective [17].The study of the primary sequence of OmpF, OmpC and PhoE of E. coli K-12 via amino acid [18] or nucleotide sequence analysis [19-221 has shown that these pore-forming proteins have a high homology. The primary sequence of PhoE contains some additional lysines, which are most probably responsible for the anion selectivity of the pore-forming unit [20 -221. Porins form trimers of identical subunits and most of the polypeptide chain is in the form of an antiparallel P-pleated sheet [23]. PhoE genes of different enterobacterial species were cloned and expressed in E. coli K-12 [24]. Their primary sequences exhibit high homology. Their synthesis is regulated by a complicated regulatory system, thepho regulon Correspondence to R. Benz,

Alteration of pore properties of Escherichia coli OmpF induced by mutation of key residues in anti-loop 3 region

“…The ion selectivity of porins is altered by modification of the charge constellation at the pore lining [11,[31][32][33][34]. The K16A and K16D proteins both showed increased cation selectivity compared with the wild-type protein.…”

Cloning and chromosomal localization of a cDNA encoding a mitochondrial porin from Drosophila melanogaster

“…During metabolic stress or in the presence of an unbalanced nutrient condition, other porins forming general diffusion pores (i.e. PhoE porin [33]) or specific pores with small selectivity (i.e. protein P [34]) are inducible.…”

Topology of PhoE porin: the ‘eyelet’ region

“…Single-channel measurements were performed with different salt solutions. The single-channel conductance for the wild-type PhoE and the Gly-114 mutant was 1.5 nS in 1 M KCI, and only a small number of steps had twice this value, indicating simultaneous insertion of two channels (Bauer et aL. 1988b).…”