Recognition Site for Phosphorus‐Containing Compounds and Other Negatively Charged Solutes on the PhoE Protein Pore of the Outer Membrane of Escherichia coli K12

Abstract

Experiments are described which were designed to obtain insight into the question why Escherichiu coli K12 induces the PhoE protein pore in its outer membrane under conditions of phosphate limitation. Growth experiments showed that the presence of PhoE protein is advantageous for the growth rate of cells growing in a medium with large linear polyphosphates, but not with Pi, as the only phosphorus source. Further experiments, in which rates of permeation of p-lactam antibiotics through the outer membrane were determined, showed that the most likely explanation for this result is that the PhoE protein pore is a relatively efficient channel for negatively charged solutes in general. Comparison of the rates of permeation of the two p-lactam antibiotics cephaloridine and cefsulodin through PhoE protein pores and through OmpF protein pores in vivo suggested that the relatively efficient permeation of cefsulodin through PhoE protein pores must be attributed to the negative charge of a sulphate residue which is present in cefsulodin but absent in cephaloridine. Subsequent experiments showed that the rate of uptake of cefsulodin through PhoE protein pores, but not through OmpF protein pores, was reversibly inhibited by linear polyphosphates whereas phosphorylated compounds and other negatively charged solutes were also found to be inhibitory. The largest inhibition was observed with solutes containing several phosphate residues. Dixon plots showed that inhibition by Pi and linear polyphosphates was of the competitive type. The results are interpreted in terms of a recognition site on the PhoE protein pore for phosphorus-containing solutes and for other negatively charged compounds.

Cloning and expression in Escherichia coli K-12 of the structural gene for outer membrane PhoE protein from Enterobacter cloacae

“…This preference for negatively charged solutes has been explained in terms of a recognition site on the pore for negatively charged compounds (Overbeeke and Lugtenberg, 1982). Proof for this assumption was obt~ned in experi-_ E.coli porins -E.cloacae Pho E OmpA a b c d e f g Fig.…”

“…The rate of permeation of /%lactam antibiotics through the outer membrane and the inhibition of this permeation by polyphosphate were measured using a method originally described by Zimmerman and Rosselet (1977) and modified by Overbeeke and Lugtenberg (1982), except that the final concentrations of the antibiotics cefsulodin and cephaloridine in the suspensions were 0.8 mM instead of 0.7 and 0.9 mM, respectively.…”

“…(r) Pore properties of the E. cfoucue PhoE protein It has been reported that PhoE protein pores of E. coli K-12 have a preference for anionic solutes, in contrast to OmpF and OmpC protein pores (Korteland et al, 1982;Nikaido et al, 1983;Overbeeke and Lugtenberg, 1982). This preference for negatively charged solutes has been explained in terms of a recognition site on the pore for negatively charged compounds (Overbeeke and Lugtenberg, 1982).…”

“…Thus, permeation of /I-lactam antibiotics through PhoE protein pores, but not through OmpF protein pores, is inhibited by negatively charged compounds, e.g., polyphosphates (Overbeeke and Lugtenberg, 1982). To determine whether this recognition site is also present on the E. cloacae PhoE protein pores, the influence of polyphosphate on the rate of permeation of the /?-lactam antibiotics cephaloridine and cefsulodin was measured (Table II).…”

“…The primary structure of these porins, OmpF, OmpC and PhoE is very similar (Mizuno et al, 1983). However, in contrast to the OmpF and OmpC proteins, PhoE protein forms pores with a preference for negatively charged solutes (Korteland et al, 1982;Nikaido et al, 1983;Overbeeke and Lugtenberg, 1982).…”