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New mutants of Escherichia coli altered in protein export were identified in phoA-lacZ and lamB-lacZ gene fusion strains by searching for mutants that showed an altered lactose phenotype. Several mutations mapped in a new gene, secD. These mutants were, in general, cold sensitive for growth, and the mutations led to an accumulation of precursor of exported proteins. The secD gene is closely linked to tsx on the E. coli chromosome, but separable from another gene proposed to be involved in export, ssaD, which maps nearby. A plasmid carrying secD+ was identified and used to show that the mutations are recessive. The secD gene may code for a component of the cellular export machinery.

OmpF porin is a nonspecific pore protein from the outer membrane of Escherichia coli. Previously, a set of mutants was selected that allow the passage of long maltodextrins that do not translocate through the wildtype pore. Here, we describe the crystal structures of four point mutants and one deletion mutant from this set; their functional characterization is reported in the accompanying paper (Saint, N., Lou, K.-L., Widmer, C., Luckey, M., Schirmer, T., Rosenbusch, J. P. (1996) J. Biol. Chem. 271, 20676 -20680). All mutations have a local effect on the structure of the pore constriction and result in a larger pore cross-section. Substitution of each of the three closely packed arginine residues at the pore constriction (Arg-42, Arg-82, and Arg-132) by shorter uncharged residues causes rearrangement of the adjacent basic residues. This demonstrates mutual stabilization of these residues in the wild-type porin. Deletion of six residues from the internal loop (⌬109 -114) results in disorder of seven adjacent residues but does not alter the structure of the ␤-barrel framework. Thus, the large hollow ␤-barrel motif can be regarded as an autonomous structure.

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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...

Using a genetic selection for mutations which allow large maltodextrins to cross the outer membrane of Escherichia coli in the absence of the LamB maltoporin, we have obtained and characterized two mutations that define a new locus of E. coli. We have designated this locus imp for increased membrane permeability. Mapping studies show that the imp gene resides at approximately 1.2 min on the E. coli chromosome. The mutations alter the permeability of the outer membrane resulting in increased sensitivity to detergents, antibiotics and dyes. The mutations are nonreverting and codominant. Genetic analysis of the mutations suggest that the imp gene is an essential gene. We describe a general cloning strategy that can be used to clone both dominant and recessive alleles. Using this technique, we have cloned the wild-type and mutant imp alleles onto a low copy number plasmid.

The LamB protein is normally required for the uptake of maltodextrins. Starting with a LamB-OmpFstrain, we have isolated mutants that will grow on maltodextrins. The mutation conferring the Dex+ phenotype in the majority of these mutants has been mapped to the ompC locus. These mutants, unlike LamB-OmpFstrains, grew on maltotriose and maltotetraose, but not on maltopentaose, and showed a significantly higher rate of [I4CJmaltose uptake than the parent strain did. In addition, these mutants showed increased sensitivity to certain j3-lactam antibiotics and sodium dodecyl sulfate, but did not exhibit an increase in sensitivity to other antibiotics and detergents. The nucleotide sequence of these mutants has been determined. In all cases, residue 74 (arginine) of the mature OmpC protein was affected. The results suggest that this region of the OmpC protein is involved in the pore domain and that the alterations lead to an increased pore size.

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