INTRODUCTION

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The assembly and targeting of outer membrane proteins (OMPs) in gram-negative bacteria are complex biological processes (for reviews, see references 1 and 5). Part of this complexity originates from the fact that during their biogenesis, OMPs have to interact transiently or permanently with the biochemically distinct environments of the inner membrane, periplasm, and outer membrane. Naturally, compositional changes in any of these three compartments can potentially influence the biogenesis of OMPs. Accordingly, inner membrane components influence protein translocation (for a review, see reference 21), periplasmic components influence the early stages of OMP assembly (2, 14, 19, 23), and outer membrane components such as lipopolysaccharide (LPS) can affect the later stages of OMP assembly (6, 8, 11, 12, 13, 22, 24).

The structural features of assembling molecules, which are governed by their primary sequences, also influence their ability to be correctly targeted and assembled. Thus, alterations in the signal sequence can cause defects in translocation across the inner membrane (1, 21), whereas changes in the mature portion can affect folding and assembly (15, 16, 18, 26, 27). An impressive collection of signal sequence mutants have been obtained, permitting examination of the role of almost every single residue of the signal sequence during protein translocation (9). Such an analysis has not been attempted for the mature region of OMPs because there is no distinct signal sequence-like linear stretch of residues that constitute an outer membrane-sorting signal. It has been suggested that the conserved carboxy-terminal phenylalanine residue of many OMPs may be a part of the outer membrane-sorting signal (4, 16, 26). Indeed, alterations of this residue affect assembly, but this is likely due to misfolding of early assembly intermediates, which are then diverted to a degradation pathway (16). However, it is unlikely that the terminal phenylalanine residue of OMPs or even the last -strand contains all the necessary information for targeting and assembly.

If the critical information for outer membrane targeting and assembly lies within the folded structure, then we must identify residues or regions of mature OMPs that influence folding. Since targeting and assembly are dynamic cellular processes, knowing the atomic structure of a fully assembled protein may not be immediately useful in allowing us to see the role of a residue during these processes. This has been the case with OmpF, for which structural resolution (3) failed to provide meaningful clues as to what residues are critical for targeting and assembly.

Influenced by an elegant piece of work carried out in Jon King's laboratory on the assembly of the P22 tail spike protein (10), we devised a novel genetic scheme which led to the isolation of OmpF mutants with a conditional (temperature-sensitive) assembly defect (15). Reversion analysis of these temperature-sensitive assembly mutants resulted in extragenic suppressor mutations in asm genes, whose products influence the outer membrane lipid environment (6, 11, 12, 17). In this study, we focused our attention on intragenic suppressor mutations in hopes of identifying additional residues within OmpF that play a role during assembly. In addition, we utilized informational suppressors in combination with various ompF amber mutations to effectively carry out in vivo site-directed mutagenesis. A combination of these two genetic approaches allowed us to investigate the role of various OmpF residues in assembly.

	MATERIALS AND METHODS

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Bacterial strains, genetic manipulations, media, and biochemicals. All strains were of MC4100 descent [F araD139 (argF-lac)U139 rpsL150 flbB5301 ptsF25 deoC1 thi-1 rbsR relA]. RAM472 (MC4100 lamB106 ompF205) was used as the maltodextrin-positive (Dex⁺) parental strain from which ompF amber mutations were isolated as described previously (15). RAM496 (MC4100 lamB106 ompC'-lacZ⁺ ompF315) expressed a variant OmpF that shows a temperature-sensitive assembly defect (15). Thus, RAM496 displayed Dex⁺ and Dex phenotypes at 30 and 42°C, respectively. RAM496 was utilized in the isolation of intragenic ompF revertants. Strains containing various suppressor tRNA (Su-tRNA) alleles were obtained from Promega Biochemicals. P1 transductions were performed by the method of Silhavy et al. (25). Minimal medium (M63 salts based) and Luria broth were prepared as described by Silhavy et al. (25). [³⁵S]methionine-cysteine was purchased from Du Pont-New England Nuclear. Heat-killed Staphylococcus aureus cells (Pansorbin) were purchased from Calbiochem. The DNA mutagen 1-methyl-3-nitro-1-nitrosoguanidine (NTG) was purchased from Sigma. Other chemicals were of analytical grade.

Site-directed mutagenesis and cloning of recombinant ompF genes. Site-directed mutagenesis of ompF to alter codon 214 was carried out using GeneEditor in vitro site-directed mutagenesis system from Promega Biochemicals. The mutagenic primer (5'-GCTGAACAGNNNGCTACTGGTCTGAAG-3') was used to randomize codon 214 (NNN) of ompF.

Protein methods. Radioactive labeling of proteins, immunoprecipitation reactions, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis were carried out as described previously (15). Whole-cell envelopes were isolated by the French press lysis procedure (15) and were treated with sodium sarcosyl (1% final concentration) at room temperature for 30 min. A sarcosyl-insoluble pellet was obtained by centrifuging samples at 110,000 × g for 30 min at 4°C in a tabletop ultracentrifuge. Western blot analysis of OmpF was carried out as described by Misra et al. (16).

	RESULTS

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Intragenic suppressors of an OmpF assembly mutant. The isolation of OmpF assembly mutants in which a conditional (temperature sensitive) folding defect within an assembly intermediate prevented the formation of stable OmpF trimers has been described previously (15). Cells that failed to accumulate sufficient amounts of stable OmpF trimers in the outer membrane were unable to grow on a medium with maltodextrins as the sole carbon source and hence expressed a Dex phenotype. In this genetic background, due to a mutation in the lamB gene, an OmpF variant is the sole means of maltodextrin entry (15). By utilizing a severely defective assembly mutant, OmpF315, suppressor mutations conferring a Dex⁺ phenotype were isolated. Further examination revealed that all the suppressor mutations mapped outside the ompF gene (11, 12, 15, 17). A factor contributing to our inability to isolate intragenic suppressor mutations could have been the availability of a large number of potential chromosomal targets other than ompF that can be mutated to yield a Dex⁺ phenotype. In this study, we sought suppressor mutations offsetting the original assembly defect that map within the ompF gene. To enrich the desired class of intragenic suppressor mutations, localized NTG-induced mutagenesis was carried out.

Isolation of suppressors in a surA background. The above analysis showed that all 36 revertants contained a suppressor alteration either at the mutant codon 214 or two codons downstream from it. This suggested that the initial assembly defect imposed by W214E at 42°C could be corrected only by a limited number of suppressor alterations within the protein. This constraint was highlighted by the fact that all nine independently mutagenized cultures repeatedly produced the same mutations.

Characterization of OmpF revertants. Examination of envelope protein profiles showed that the revertants bearing a E214V, T216I, or N230Y suppressor substitution had OmpF levels similar to that of a strain expressing the assembly-proficient OmpF205; OmpF levels in revertants with an E214K substitution were lower than levels of OmpF205. Interestingly, N230Y, which was obtained in a surA background, also elevated OmpF315 levels at 42°C in a surA⁺ background.

View larger version (36K): [in this window] [in a new window]   FIG. 1.   OmpF trimer assays. Cells expressing various OmpF proteins were grown to mid-log phase at 42oC in glycerol minimal medium, labeled for 20 s with [35S]methionine-cysteine, and chased with an excess of nonradioactive methionine. Chase samples were removed after 1, 3, 10, and 30 min. Proteins were extracted, and OmpF was immunoprecipitated using trimer-specific antibodies. Immunoprecipitates were heated at 60°C for 15 min prior to analysis by SDS-PAGE. The gel was dried and subjected to autoradiography at 70°C. Positions of OmpF trimers (T) and denatured monomers (M) are shown. Relevant alterations in various OmpF proteins are shown below the gel. The loading of 1- and 3-min chase samples carrying OmpF with a T216I alteration was inadvertently reversed.

Suppression specificity of N230Y. The N230Y mutation suppressed an assembly defect caused by W214E. We tested N230Y's ability to suppress other alterations at position 214 and whether its presence is detrimental for assembly when the wild-type tryptophan residue is present at position 214. Through site-directed mutagenesis, position 214 was altered to introduce A, D, and T substitutions, all of which conferred a severe assembly defect (Fig. 2). Recombinant ompF genes were constructed (see Material and Methods) so as to express 10 different OmpF proteins carrying one of five substitutions at position 214 (A, D, E, T, or W) and either N (wild type) or Y (suppressor) at position 230. The results showed that the N230Y suppressor had no effect when combined with the wild-type W214 residue (Fig. 2). Furthermore, although N230Y was able to suppress to various degrees the assembly defect caused by the replacement of W214 by A, D, or E, it had the greatest effect on the E214 mutation against which it was isolated and was unable to suppress the T214-mediated assembly defect.

View larger version (23K): [in this window] [in a new window]   FIG. 2.   SDS-PAGE analysis of sarcosyl-extracted cell envelopes obtained from strains producing either the parental OmpF205 protein (lane 1) or various mutant OmpF proteins with indicated alterations at position 214 (lanes 3 to 10). The suppressor alteration N230Y was present in proteins analyzed in even lanes.

Informational suppression of ompF amber mutations. In the original mutant isolation strategy, various ompF(Dex) amber mutations were genetically reverted in a conditional manner, and only two of the four amber sites resulted in assembly mutants (15). In this scheme, the reversion to Dex⁺ at 30°C required that OmpF(Dex) be properly synthesized, translocated across the inner membrane, and inserted and assembled into functional trimers in the outer membrane. It is conceivable that this demand for functional trimers may have precluded us from examining other OmpF residues important for assembly but also involved in channel function or -barrel stability. Thus, an approach involving informational suppression of amber mutations was exploited to overcome this limitation. In this scheme, ompF amber mutations were suppressed without demanding functionality of the suppressed protein. To achieve this, we introduced various amber suppressor tRNA (Su-tRNA) alleles into genetic backgrounds harboring different ompF(Dex) amber mutations. In all, four different ompF(Dex) amber mutations, affecting residue Y40, Q66, W214, or Y231 of mature OmpF, were suppressed with four different Su-tRNAs having the capacity to insert an S, Y, Q, or L residue at the respective amber sites, thus generating 16 different variant OmpF(Dex) proteins. It may be noted that three such combinations produced the parental protein, while two variant OmpF(Dex) proteins, bearing W214Q and Y231Q substitutions, were identical to those synthesized when amber mutations at these sites were genetically reverted (15). The Su-tRNAs chosen in this study produced efficient suppression of OmpF amber codons.

Characteristics of tRNA-suppressed OmpFs. Strains carrying different combinations of ompF(Dex) amber mutations and Su-tRNAs were tested for sensitivity to an OmpF-specific bacteriophage, K20, and growth on maltodextrin medium. Phage plaquing efficiencies of all 16 mutants were similar to that of a strain expressing the parental OmpF protein, showing that (i) the phage receptor activity of the mutant OmpF proteins was unaffected and (ii) sufficient levels of the variant proteins were assembled in the outer membrane under steady-state growth conditions. In contrast to the phage sensitivity phenotype, mutants showed different growth patterns on maltodextrin minimal medium (data not shown), which could be due either to alterations in structure or to the level of OmpF trimers. On glucose or glycerol minimal medium, all mutants formed similar-sized colonies, showing that the argE amber mutation was efficiently suppressed by the Su-tRNAs.

View larger version (72K): [in this window] [in a new window]   FIG. 3.   Trimer assays of various OmpF proteins generated through the informational suppression of amber codons located at various positions in ompF (shown at the bottom). Various amino acids introduced through the suppressor tRNAs are shown on top. Strains were grown to mid-log phase at 37°C (A) or 40°C (B) in glycerol minimal medium. Labeling and immunoprecipitation conditions were similar to those described for Fig. 1 except that only 3-min chase samples were analyzed. C, control (assembly-proficient OmpF205 protein); T, OmpF trimers; M, denatured monomers.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Suppression with or without requiring porin function. We took two fundamentally different genetic approaches to examine the roles of various residues within OmpF in trimer assembly. In the first approach, we asked for the correction of an assembly defect, imposed by a W214E substitution, with a second alteration within OmpF. Because the starting strain produces an assembly-defective OmpF, all revertants are expected to possess an alteration that compensates for the assembly defect. However, since revertants were selected for their abilities to form functional OmpF channels, isolates in which a substitution simultaneously reduced channel activity were lost. In the second approach, amber codons located at various positions within ompF were suppressed by utilizing various Su-tRNAs and the resulting proteins were analyzed for assembly proficiencies. So, unlike with the previous strategy, active channels were not sought, and thus various substitutions were introduced without a functional bias.

Reversion at 42°C. The strategy for isolating revertants at high growth temperatures revealed that the negative effect of the W214E substitution on OmpF assembly was correctable by only two alterations located at or near the original substitution. One such alteration affected residue E214 itself, changing it to a K. While in the second group of revertants, the W214E substitution was maintained but a change of T216 to I was introduced. A T216I substitution may contribute positively by increasing regional hydrophobicity within the -strand, thus offsetting the effect of W214E. The results showed that although W214 is not absolutely required for OmpF assembly, its replacement by lysine is preferred over polar glutamate. It is worth pointing out that revertants bearing a true reversion substitution of E214W were not obtained, as this would have required two simultaneous base pair changes.

Overlapping assembly constraints of temperature and surA on OmpF315 assembly. SurA is a major folding factor whose activity in the periplasm is needed for proper OMP assembly (14, 23). The phenotypic defect of OmpF315 at 37°C in a surA background is identical to that observed at 42°C in a surA⁺ background. Thus, SurA's removal from the cell exaggerates the assembly defect of OmpF315 and hence permits the isolation of suppressors at 37°C. Moreover, these two physiologically different environments must produce overlapping assembly constraints because identical suppressors of OmpF315 were obtained under these conditions. It is noteworthy that the N230Y suppressor, which was obtained only at 37°C in a surA background, also corrected the assembly defect of OmpF315 at 42^oC in a surA⁺ background. As none of the suppressors obtained affected OmpF315's proline residues, it is conceivable that it is the absence of the general chaperone function rather than the loss of the peptidyl prolyl isomerase activity of SurA that conferred an assembly defect.

Suppression specificity. All intragenic suppressors acted by fully or partially restoring the assembly defect. The N230Y suppressor showed specificity towards alterations at position 214. In the folded OmpF molecule, residues 214 and 230 are present in two consecutive -strands of the monomer. The side chain of W214 projects outward in the exterior lipid environment of the membrane, but N230's side chain is oriented inward in the barrel. Thus, if suppression involves a specific side chain interaction, it can occur only transiently and must occur within partially folded structures in which the two side chains have not yet assumed the opposite orientation with respect to each other. Alternatively, suppression may involve compensation for a conformational defect. As the conformation may vary depending on the nature of the side chain, the ability of N230Y to suppress will change. Regardless of the mechanism of suppression, our data showed that the residues or regions encompassing 214 and 230 of OmpF have a role in assembly. This is further substantiated by our earlier finding that changes at position 231 of OmpF can also confer an assembly defect (15).

Informational suppression. Amber suppressor analysis provided useful information regarding the acceptability of various substitutions at particular sites. Of the four amber sites analyzed, two were located at residues 214 and 231, where assembly mutants have previously been isolated (15; also, see above). In the wild-type OmpF trimer molecule, the side chain of Y40 extends inwards within the channel, and substitutions at this position would therefore be expected to alter the interior of the channel in the folded molecule. The data presented here showed that alterations at Y40 can also influence OmpF trimerization, indicating an additional role of Y40 during OmpF biogenesis. Two quite different amino acids, Q and L, can replace Y40 at 37°C, but the Y40Q substitution was temperature sensitive for trimerization, indicating a preference for a hydrophobic or an aromatic residue at this position.