Genetic identification of the pore domain of the OmpC porin of Escherichia coli K-12

Abstract

We have isolated and characterized 31 mutations in the ompC gene which allow Escherichia coli to grow on maltotriose (Dex+) in the absence of the LamB and OmpF porins. These ompC(Dex) mutations include single-base-pair substitutions, small deletions, and small insertions. DNA sequence analysis shows that all of the alterations occur within the coding region for the first 110 amino acids of mature OmpC. The 26 independent point mutations repeatedly and exclusively alter residues R37, R74, and D105 of mature OmpC. In each case, a charged amino acid is changed to an uncharged residue. Biochemical and physiological tests suggest that these alterations increase the size of the pore channel. Starting with three different ompC(Dex) strains with alterations affecting R74, we isolated mutants that could grow on maltohexose (Hex'). These mutants each contained a second alteration in the ompC gene involving residues R37, D105, or R124. The combined effects on pore function of the two mutations appear to be additive. These experiments suggest that we have identified the important residues of OmpC peptide involved in pore function. On the basis of these mutations and general rules for membrane protein folding, a model for the topology of the OmpC protein is proposed.The outer membrane of the gram-negative bacterium Escherichia coli K-12 provides the first permeability barrier of the cell. Small hydrophilic molecules cross this structure by diffusing through water-filled channels formed by the two major porins, OmpF and OmpC (18). These proteins also serve as receptors for various bacteriophages, facilitate transport of colicins, and play a role in membrane integrity through their interaction with other membrane components such as lipopolysaccharides and peptidoglycans (for a review, see reference 21).The DNA sequences for the ompF and ompC genes are known (9, 15), and the primary amino acid sequences for the proteins are available. These proteins show a high degree of homology to each other and to other porin proteins both at the DNA level and at the protein level (3, 15).An extensive structural analysis of the porins, predominantly OmpF, has been done by using biochemical approaches such as circular dichroism, infrared spectroscopy, X-ray diffraction, and high-resolution electron microscopy (25). The following facts are generally accepted with regard tp the porin structure. (i) The porins exist as trimers. (ii) They are rich in P-sheet structures which are oriented roughly perpendicular to the plane of the membrane. (iii) The porins protrude a little on both sides of the membrane. (iv) They interact with both the peptidoglycan and the lipopolysaccharide moiety of the outer membrane. These studies provide a general model for the anatomy of the porins but do not provide information regarding the locations of functional domains.One means of defining functional domains of porin proteins is the use of mutants with specific defects. This approach has been very effectively used to study the topology of LamB (6, 8), a maltoporin wh...

Cloning and nucleotide sequence of the Pseudomonas aeruginosa glucose‐selective OprB porin gene and distribution of OprB within the family Pseudomonadaceae

“…A group of phenylalanine and tryptophan residues occurs in the N-terminal portion of OprB. Several investigations have consistently linked residues in the N-terminal third of porins to channel size and ion selectivity Misra and Benson, 1988;Tommassen et al, 1985) and formation, of a binding site in substrate-selective porins (Werts et al, 1992). In Rhodobacter capsulatus porin and OmpF and PhoE of E. coli, crystal-structure analysis has demonstrated that the third external loop in the N-terminal portion of these proteins folds into the p barrel (Cowan et al, 1992;Weiss and Schulz, 1992).…”

“…In Rhodobacter capsulatus porin and OmpF and PhoE of E. coli, crystal-structure analysis has demonstrated that the third external loop in the N-terminal portion of these proteins folds into the p barrel (Cowan et al, 1992;Weiss and Schulz, 1992). Epitope insertion and deletion mutants demonstrate that this loop affects channel size in PhoE, OmpF and OmpC and ion selectivity of PhoE (Struyvi et al 1993Misra and Benson, 1988). Given the location of the phenyalanine and tryptophan clusters in OprB, some of these residues could potentially be involved in the formation of the constriction in the OprB channel and/or formation of the carbohydrate-binding site.…”

Identification of a segment of the Escherichia coli Tsx protein that functions as a bacteriophage receptor area

“…Strains HF1(pHS11; tsx+) and HF1(pGP15; es (Fig. 4, lane 6 proteins (1,12,16,19,20,22,28,(30)(31)(32)46 (27). The number of strains producing Tsx was fairly high among the phage-resistant mutants selected against phage H3 (10 of 18 cultures) and phage Oxi (11 of 31 cultures) but was very low (7 of 70 cultures) among those selected against phage T6.…”

T4-Like Genome Organization of the Escherichia coli O157:H7 Lytic Phage AR1

“…6B). Previous studies (46,83) indicated that eight point mutations of OmpC can make K-12 resistant to T4-like phage infection (Fig. 6B, asterisks).…”