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Molecular Basis of Bacterial Outer Membrane Permeability Revisited

Department of Molecular and Cell Biology, University of California, Berkeley, California

SUMMARY

INTRODUCTION

PROTEIN CHANNELS

Classical Porins

Families of classical porins.

Functional assays.

Crystallographic structure of porins.

Regulation of porin expression.

Porin mutants.

Functional studies with new approaches.

Voltage gating.

Regulation of porin function.

Evolution of porins.

Slow Porins

OprF is the major porin in P. aeruginosa.

The apparent dilemma of low permeability through a large channel.

Other Porins

Other porins in E. coli and Salmonella.

Porins in members of the Enterobacteriaceae other than E. coli and Salmonella.

Porins in {gamma}-proteobacteria outside the Enterobacteriaceae.

Porins in ß-proteobacteria.

Porins in {alpha}-proteobacteria.

Porins in {delta}- and {varepsilon}-proteobacteria.

Porins in the Planctomyces-Chlamydia group.

Porins in the Cytophaga-Flexibacter-Bacteroides group.

Porins in spirochetes.

Porins in cyanobacteria.

Porins in Fusobacterium.

Porins in the Deinococcus-Thermus group.

Porin in Thermotoga.

Porins in the Corynebacterium-Nocardia-Mycobacterium group.

Putative porins in archaea.

Porins and Antibiotic Resistance

Route of antibiotic influx.

Resistance caused by loss or modification of porins.

Specific Channels

LamB or maltose channel.

ScrY or sucrose channel.

''BglH,'' an aryl-ß-D-glucoside channel.

Other specific channels.

Specific channels in bacteria other than the Enterobacteriaceae.

TonB-Dependent Receptors or Gated Channels

Export Channels of the TolC Family

Secretins, Ushers, and Autotransporters Producing Oligomeric Rings

Secretins.

Ushers.

Autotransporters.

Other Export Channels

Type IV secretion pathway.

Two-partner secretion pathway.

Export channels for polysaccharides.

Entry of Colicins and Phage Nucleic Acids

THE LIPID BILAYER AS A DIFFUSION BARRIER

The Asymmetric Bilayer

General Structure of LPS

What Makes the LPS Leaflet an Effective Permeability Barrier?

Low fluidity of the LPS hydrocarbon domain.

Strong lateral interactions between LPS molecules.

Conformation of LPS in bilayers.

Conformation of LPS in the LPS-FhuA complex.

Physiological Adaptation in LPS Structure

Different Lipid A Structures in Various Organisms

Length, number, and position of fatty acyl groups.

Groups at the ends of the disaccharide backbone.

Backbones containing 2,3-diamino-2,3-dideoxyglucose (GlcN3N).

Extractable Lipids Substituting for or Supplementing LPS

Sphingolipids.

Sulfonolipids.

Ornithine lipids.

Alterations of the OM Bilayer Barrier

Effect of energy state.

Perturbation of the OM permeability barrier.

OM Vesicles

EPILOGUE

ADDENDUM IN PROOF

ACKNOWLEDGMENTS

REFERENCES

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INTRODUCTION

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Gram-negative bacteria characteristically are surrounded by an outer membrane (OM). Analysis of prokaryote phylogeny using signature sequences in proteins even led to the notion that a major phylogenetic division exists between organisms with double-membrane envelopes (diderms) and those with only a simple cytoplasmic membrane (monoderms) (240). It is likely that the most important function for the additional membrane layer, the OM, in gram-negative bacteria is to serve as a selective permeation barrier. My earlier review of this subject, written with Martti Vaara, appeared in 1985 (464). It was an opportune time for such a review because the fundamental properties of the Escherichia coli porins and the specific maltodextrin channel, LamB, were characterized a few years earlier (see, for example, references 388 and 460-462) and the asymmetric structure of the OM bilayer, with lipopolysaccharides (LPS) located exclusively in the outer leaflet (316), offered an explanation for the unusually slow influx of lipophilic solutes. In the intervening 18 years, explosive progress has occurred in the field. Diffusion through the nonspecific porin channels and the specific channels are now understood in molecular detail, thanks to the elucidation of the crystal structure of these proteins. Channel proteins were identified in many nonenteric bacteria as a result of the rapid growth of our knowledge of the genome sequences, and in some cases this has offered insights into the physiology of various organisms in their native habitat. Studies of LPS not only showed us the impressive diversity of LPS structures in the bacterial kingdom but also led to the realization that the LPS structure may be modified in response to the conditions prevailing in the environment.

The present review attempts to summarize the development in the field since 1985. The major problem in presenting the results has been the information explosion. For example, more than 650 articles with the word "porin" in the title have been published during this period. If the databases are searched with "porin" or "lipopolysaccharide" as keywords, literally thousands of references are retrieved. Therefore, I had to be severely restrictive with citations in order to keep the review within a reasonable (and perhaps useful) size. I tried to limit the discussion strictly to selective permeability, eliminating most papers dealing with the biosynthesis and assembly of the OM or with the role of the OM in the interaction of bacteria with the environment, including higher animals and plants. I also apologize at the outset for the omission of many references which were not cited because the main message could be found in other articles or reviews or, probably most frequently, because of my oversight.

PROTEIN CHANNELS

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OMs, like other biological membranes, are fundamentally built as a bilayer of lipids. As such, lipid bilayers show little permeability for hydrophilic solutes, including most nutrients. Therefore, they contain channel-forming proteins for the purpose of allowing the influx of nutrients and perhaps for the extrusion of waste products. One such nonspecific channel-forming protein, porin, was discovered in 1976 (438), and the word "porin" was proposed specifically for this class of proteins forming nonspecific diffusion channels. As predicted, porins were found in every species of gram-negative bacteria investigated and even in a group of "gram-positive" bacteria, the Corynebacterium-Nocardia-Mycobacterium complex, which produces a lipid-rich, bilayer-like "cell wall" (see below). (Interestingly, the Corynebacterium-Nocardia-Mycobacterium complex appears to be related to gram-negative bacteria on the basis of signature sequence analysis, since both of these groups have a 12-residue deletion in the S12 ribosomal protein, unlike archaebacteria and other gram-positive bacteria [240]).

At about the same time as bacterial porin was discovered, porins in mitochondria were discovered (584); this was followed a few years later by a study in my own laboratory using different approaches (773). The mitochondrial porins (voltage-dependent anion channels [VDAC]) are not discussed further, except to note that the channels are apparently large (up to 3 nm in diameter), as expected, because mitochondria have no need to exclude toxic molecules in the environment, and that the overall structure is thought to be similar to the ß-barrel structure (to be discussed below) of bacterial porins. Many reviews of mitochondrial porins exist (see, for example, references 65 and 397). Another eukaryotic organelle in which porins were found is peroxisome (540-542).

The word "porin" has suffered from its popularity. As stated above, it was defined to mean only proteins that form nonspecific channels. Workers often concocted fanciful names like "maltoporin" for the LamB channel, which is specific, or "phosphoporin" for the Escherichia coli PhoE porin, which prefers anions in general and has no specificity for phosphate. These abuses of the term cause confusion and misunderstanding, and I present a plea, once again, to limit the word "porin" to references to nonspecific channels only.

X-ray crystallographic analysis showed that porins exist as transmembrane ß-barrels (see below). However, crystal structures are available for only a few porins, and therefore it becomes highly desirable to derive as much information from primary sequence. Unfortunately, comparison of primary sequences of porins is extremely difficult. This is because the external loops between the transmembrane ß-strands undergo very rapid mutational alterations as they interact with elements of the external world, such as antibodies, components of the innate immune system, bacteriocins, and phages. Because of this, simple alignment programs such as BLAST are "fooled" and create gaps at improper places. It is therefore important to first identify the transmembrane ß-strands and compare different sequences solely on the basis of the sequences of these strands. However, none of the simple algorithms proposed for the detection of transmembrane strands work in a satisfactory manner, as mentioned by Ferenci (199). The proposal made by Ferenci was to look at the alignment of related porin sequences and assume that the less variant regions correspond to transmembrane strands. This works very nicely when many sequences from isolates of the same species, for example, exist (362, 482). However, in other cases one has to start from an alignment of sequences of distantly related porins, and this requires the prior identification of transmembrane strands.

One method that seems to work reasonably well for the detection of transmembrane strands is that of Jeanteur et al. (303, 304), which uses the sum of the hydrophobicity and hydrophobic moment of each 9- to 10-residue segments. Their alignment, together with the addition of some newer sequences aligned by myself, is shown in Fig. 1. In this way, we can see that porins from -, ß-, and -protcobacteria are indeed related to each other, a conclusion that BLAST, for example, completely fails to show. Figure 1 also shows some characteristic features of porins from various groups of bacteria. (i) Porins from -proteobacteria show strong similarity to each other and are characterized by the presence of short (10-residue) extensions at their N termini (Vibrio cholerae OmpU is an exception). If the alignment presented is correct, porins from the Vibrio-Photobacterium group are unusual in containing exceptionally long L3 loops. Enterobacterial porins contain the characteristic PEFGGD signature sequence in L3, as noted previously (303). (ii) Porins from ß-proteobacteria have sequences that are similar to each other but quite divergent from those of the -proteobacteria. Although Fig. 1 shows only the sequences of Neisseria meningitidis PorB and Bordetella pertussis porin, several other sequences were aligned earlier (303, 304). (iii) Porins from -proteobacteria again seem to form a group of their own.

View larger version (153K): [in this window] [in a new window]   FIG. 1. Sequence alignment of porins from -, ß-, and -proteobacteria. The classification of the source organisms into -, ß-, and -subdivisions is shown at the beginning. Transmembrane ß-strands are indicated by highlighting, in red, the presence of hydrophobic amino acid residues at alternate positions. The PEFGGD motif of loop 3 in the Enterobacteriaceae and corresponding sequences are colored in blue. The alignment of E. coli OmpF (ECOOMPF), E. coli OmpC (ECOOMPC), E. coli PhoE (ECOPHOE), Haemophilus influenzae Rd P2 porin (HINP2), Neisseria meningitidis PorB (NMEPORB), Bordetella pertussis porin (BPEPOR), Comamonas acidovorans Omp32 (CACPOR), Rhodopseudomonas blastica porin (RBLPOR), and Rhodobacter capsulatus porin (RCAPOR) is basically that of Jeanteur et al. (303, 304), with minor adjustments. The rest of the sequences (Acidithiobacillus ferrooxidans porin [AFEPOR], Pasteurella multocida OmpH [PMUOMPH], Serratia marcescens OmpF [SMAOMPF], Vibrio cholerae OmpU [VCHOMPU], Photobacterium profundus OmpL [PPROMPL], V. cholerae OmpT [VCHOMPT], and Brucella abortus porin [BABPOR]) were aligned by me. The alignment relied mostly on the plot of (average hydrophobicity + average hydrophobic moment) as specified by Jeanteur et al. (303, 304) and took into account the prediction of turns (485). The Gibbs motif sampling program (442) was also utilized (http://bayesweb.wadsworth.org/gibbs/gibbs.html), although this program predicted only the ß-strands facing the lipid bilayer. When multiple sequences were available, deletions and insertions were assumed to have occurred in loops (199); this approach was useful in the analysis of P. multocida OmpH (129, 390) and B. abortus porin (424, 482). The alignment of V. cholerae OmpU, V. cholerae OmpT, and P. profundum OmpL was difficult but was helped by the comparison among these three, as well as with Vibrio fischeri OmpU and VCH1008 from the V. cholerae genome-sequencing project (both sequences retrieved from GenBank). No attempt was made to align the variable-loop sequences carefully.

Functional assays. Nonspecific diffusion of hydrophilic solutes across the OM usually occurs through porin channels, and thus the activity of these channels in intact cells is most conveniently assayed by determining the flux of hydrophilic solutes or ions. For isolated porin proteins, reconstitution into planar bilayer lipids allows the measurement of flux of ions through single channels. Although the single-channel conductance values have been used to calculate the sizes of the channel by assuming that ions within the channel behave identically to those in bulk solutions, this procedure often leads to misleading results (452, 453). A remarkable example is found in some mutants of E. coli OmpF porin, where both crystallography and the proteoliposome swelling assay (see below) showed enlargement of the channel whereas single-channel conductance showed a significant decrease (571). Another problem in the use of single-channel conductance is that it is often a result of insertion of a porin trimer, containing three open channels, rather than that of a "single" channel. Thus, the data that E. coli OmpF gave a single-channel conductance of about 2 nS in 1 M KCl led to the calculated pore diameter of 9 Å (52); this was thought to validate the calculation because of the agreement of calculated diameter with that found later by crystallography. However, the conductance of 2 nS was actually the result of insertion of three channels in a trimer, and the true single-channel conductance of OmpF is around 0.7 nS, which gives an unreasonable prediction for the pore diameter (452). Single-channel conductance is a reasonable indicator of pore size for large channels, but it must be used with the utmost caution for small channels. This problem has also been discussed in a recent review (693). Nevertheless, the planar bilayer study is the only functional assay performed with many porins; therefore, the single-channel conductance value for OmpF, 0.7 nS, is used as the reference in the discussion of many such porins (see "Other porins" below).

The bilayer domain of the OM is asymmetric, at least in Enterobacteriaceae, with the outer and inner leaflets containing nearly exclusively LPS and phospholipid molecules, respectively (see "Lipid bilayer as a diffusion barrier" below). Therefore, there is concern about the use of the phospholipid bilayer, or even nonphysiological barriers such as oxidized cholesterol, for planar film studies. In fact, some authors have argued that porin channels behave quite differently when they are in a natural, asymmetric bilayer of the OM (168, 169). In this connection, it is important that an asymmetric planar bilayer, containing on one side a deep-rough LPS exclusively, was used for experiments involving a trimeric porin of Paracoccus denitrificans (732). There was no difference in single-channel conductance regardless of whether the bilayer was asymmetric or symmetric (i.e., containing phospholipids in both leaflets). However, the spontaneous insertion of porin from the phospholipid side was accelerated more than an order of magnitude if LPS was present on the other side, a result that may have important implications for the mechanism of assembly of OM proteins in intact cells. The voltage-gating behavior was affected somewhat, in a way that was expected if the total potential sensed by the porin molecules was the sum of external voltage and the internal potential arising from the presence of excess negative charges on the LPS-containing outer leaflet.

Another approach is the reconstitution of porins into multilayered proteoliposomes and the measurement of solute diffusion rates that are reflected in the rates of osmotic swelling of these vesicles in media containing the test solutes (460). Comparison of diffusion rates of solutes of various sizes gave remarkably reliable values of the channel size with respect to the crystallographic structure (see below). However, because swelling occurs in response to the movement of any solute, including components of the buffer, extreme care is needed when this method is used to study the diffusion of charged solutes (461).

Finally, diffusion rates through porin channels can be measured in intact cells by coupling the influx of hydrophilic solutes with a "sink" process. A convenient assay is to examine the influx of cephalosporins by coupling it to their hydrolysis by periplasmic ß-lactamase (462); cephalosporins are especially useful because a diverse collection of cephalosporins has been synthesized and because the hydrolysis can be monitored easily by recording changes in the optical density at 260 nm.

Crystallographic structure of porins. Undoubtedly the most important progress in the study of porins was the elucidation of the three-dimensional structures of trimeric porins by electron diffraction (297-300, 705) and X-ray crystallography. The latter approach had its first success with a Rhodobacter capsulatus trimeric porin (714-718), an achievement that was quickly followed by the elucidation of the structure of the E. coli OmpF and PhoE porins (152). Important conclusions from these studies include the following (there are recent minireviews on the structure of porins [343, 598, 599]).

(i) As predicted from earlier studies, porin monomers were shown to cross the lipid bilayer as a ß-barrel or a series of 16 ß-strands. The strands are tilted rather strongly (by 30 to 60°) in relation to the barrel axis, as shown earlier by Fourier transform infrared spectroscopy (436), and this tilting increases the diameter of the barrel. (For the concept of sheer number, which is related to the degree of tilt, see reference 599.) The length of each transmembrane strand spans the range from only 7 (in strand 5) to 16 (in strand 1) residues in OmpF. Contact among the monomers is stabilized by hydrophobic and polar interactions, and loop 2 tends to bend over the wall of the barrel of the neighboring subunit, playing a significant role in stabilization (Fig. 2A).

View larger version (30K): [in this window] [in a new window]   FIG. 2. Structure of the OmpF porin of E. coli. (A) View of the trimer from the top, that is, in a direction perpendicular to the plane of the membrane. Loop 2, colored blue, plays a role in interaction of the monomer with its neighboring unit. Loop 3, colored orange, narrows the channel. (B) View of the monomeric unit from the side, roughly in the direction of the arrow in panel A. Loops 2 and 3 are colored as in panel A. (C) View of the monomeric unit from the top, showing the "eyelet" or the constricted region of the channel. The eyelet is formed by Glu117 and Asp113 from the L3 loop, as well as four basic residues from the opposing barrel wall, Lys16, Arg42, Arg82, and Arg132, all shown as spheres. The diagrams are based on PDB file 2OMF. This figure and Fig. 4 and 6 were drawn by using the program PyMol (Warren L. DeLano, DeLano Scientific LLC, San Carlos, Calif. [http://www.pymol.org]).

(iii) It was observed, just before the elucidation of the structures of OmpF and PhoE, that porin sequences almost invariably end with a C-terminal phenylalanine (635). This feature was confirmed with practically all OM channel proteins, although in rare cases the C-terminal residue is tryptophan. This C-terminal residue is located at the OM/periplasm interface in OmpF and PhoE (152), and its conservation is at least partially explained by this location.

(iv) Transmembrane strands are connected by short "turns" on the periplasmic side, but the "loops" that connect the strands on the external sides are often long. With both R. capsulatus porin and OmpF, loop 2 folds back outward and contributes to the connection with the neighboring monomer. Loop 3, connecting strand 5 with strand 6, is especially long (33 residues in OmpF) and folds into the barrel to produce the narrowing of the channel (often called the "eyelet") (Fig. 2B and C). It was impossible to predict from simple folding prediction algorithms that the eyelet region, which has the strongest effects on the function of porin, is made in this manner, which falls outside of the regular succession of ß-strands. The size of the eyelet region of OmpF was 7 by 11 Å (152), very close to the estimate of a diameter of 12 Å from sugar diffusion studies (see above).

(v) The nature of the residues lining the channel wall provided a reasonable explanation of the diffusion characteristics through porins. Thus, OmpF and PhoE prefer cations and anions, respectively, despite having a 72% similarity in the mature-protein sequence; this difference in charge preference was shown to be due mainly to the replacement of Gly131 in the eyelet region of OmpF with the positively charged Lys125 in PhoE (42, 152). The electrostatic properties of the OmpF and PhoE channels were calculated and compared (322).

The OmpC channel appears to be slightly smaller than the OmpF channel on the basis of diffusion rates of organic molecules (461, 462). Although the crystal structure of E. coli OmpC is not yet known, the structure of an OmpC homolog from Klebsiella pneumoniae has been determined (187). However, the size of the constriction region and the arrangement of charged residues there are almost exactly the same as in OmpF, and it is not easy to explain the difference in diffusion rates. Schulz has pointed out that more charged residues are pointing toward the pore lumen in OmpC (599). Perhaps this may decrease the functional radius of the solute diffusion pathway. Molecular dynamics simulation (see below) will be valuable in solving this question.

Lipophilicity in the solute molecule strongly retards its diffusion through the porin channel (461, 462). Schulz explained this effect by assuming that the structure of eyelet, in which the cationic and anionic amino acid residues are located in the opposite sides of the channel (Fig. 2C), orients the water molecules in the channels in a highly directional manner, making the disruption of this ordered structure by hydrophobic solutes energetically unfavorable (597).

OmpF, OmpC, and PhoE are so strongly similar in sequence and also in their three-dimensional structure (as shown experimentally for OmpF and PhoE [152]) that they apparently form mixed trimers almost at random (217).

Regulation of porin expression. The regulation of expression of nonspecific porins in E. coli is briefly summarized. PhoE is expressed only under phosphate starvation, since the phoE gene is a member of the phosphate regulon (664). The expression of the two major porins, OmpF and OmpC, is exquisitely regulated. The apparent purpose of this regulation became clear when it was discovered that OmpF produces a slightly larger channel than OmpC (461, 462). Thus, noxious agents such as antibiotics and bile acids diffuse far better through the larger OmpF channel, as seen clearly from the observation that low concentrations of antibiotics select for ompF mutants but never for ompC mutants (255) (see "Porins and antibiotic resistance" below). In its natural habitat, the intestinal tract, E. coli encounters 4 to 16 mM bile salts (84), and it is most important to minimize their influx. The conditions prevailing in the intestinal tract, high osmotic strength and high temperature, both favor the production of OmpC (with its narrower channel) and repress the production of OmpF (259). On the other hand, the increased production of OmpF under low-temperature, low-osmolarity conditions (for example, in lake water) will benefit E. coli by facilitating the influx of scarce nutrients. The molecular mechanisms of this regulation have been studied extensively and reviewed in a clear and concise manner (507). Thus, environmental osmotic activity is sensed by the sensor component EnvZ of the archetypal two-component system, EnvZ-OmpR, and high osmolarity results in the phosphorylation of OmpR. The ompF gene, with its high affinity OmpR-binding sites, is transcribed even when the phosphorylated OmpR is scarce (i.e., under low-osmolarity conditions). However, when the concentration of activated OmpR increases, additional binding of these molecules results in increased transcription of ompC and repression of ompF. High temperature, on the other hand, increases the transcription of an antisense RNA, micF (172) (A putative regulatory protein, EnvY, has been reported to affect the temperature regulation process [389], but its effect on micF transcription is not known.) This RNA binds to the 5'-region of the ompF mRNA and inhibits its translation. More recently, another twist was added to this complex regulatory network (383). When E. coli is starved for carbon sources, OmpF production responds strongly to the growth rate (or the concentration of glucose in the medium). Finally, as described below, oxidative stress and the presence of salicylate also increase micF transcription and prevent the production of OmpF porin posttranscriptionally.

The intestinal tract, the normal environment of E. coli, is thought to be mostly anaerobic. Interestingly, anaerobiosis was found to modify the osmoregulation of OmpF and OmpC (406). Thus, under anaerobiosis, OmpC is expressed at a rather high level even in fairly low-osmolarity media, and the repression of OmpF by osmotic activity occurs more strongly than under aerobic conditions. This modification of the regulatory response, which is expected to favor the survival of E. coli in the intestinal tract, occurs through the cross talk activation of OmpR by the ArcB sensor, which senses the anaerobic condition.

Another environmental factor that acts in some cases through the EnvZ-OmpR system is the medium pH (270). At an acidic pH, such as 5.2, the production of OmpF porin becomes strongly repressed and the expression of OmpC becomes increased. This acid induction phenomenon is quite complex (658) and is affected additionally by the nature of the carbon source. A partial explanation of this effect may be the direct phosphorylation of OmpR by acetyl phosphate (269). What advantage would the increased synthesis of OmpC confer to the bacterium trying to survive in an acidic environment? In terms of proton influx, the small difference in the channel size between OmpF and OmpC is unlikely to produce any significant difference in influx. Perhaps a critical factor for survival under acid-stressed conditions is the neutralization of the periplasm, achieved by the decarboxylation of glutamate in the cytoplasm followed by the export of -aminobutyrate into the periplasm, where it may act as a buffer (83). In this scenario, the narrower channel of OmpC may contribute to the retention of these buffer molecules in the periplasm. The closure of porin channels by low pH and by endogenously synthesized polyamines produced under acid stress (see "Regulation of porin function" below) may also help in the same way. The acid response involving OmpR also involves, in Salmonella enterica serovar Typhimurium, the downstream two-component regulatory system SsrAB, encoded by the genes in pathogenicity island 2, and is essential for the survival of this species inside macrophages (34, 368).

Regulation of porin expression also occurs in response to chemicals in the environment. It was found in 1991 that salicylate in the medium decreased OmpF synthesis (560). This is now known to be a part of the global regulation of porins mediated by three XylS-AraC family regulatory proteins, MarA, SoxS, and Rob (9, 174). Thus, the increased production of MarA (caused by some environmental chemicals, such as salicylate, inactivating its cognate repressor, MarR) or SoxS (caused by the inactivation of its repressor SoxR via its oxidation) or the binding of coregulators such as dipyridyl (561) or some bile salts (558) to Rob activates the transcription of micF antisense RNA, decreasing OmpF synthesis. Interestingly, all these environmental signals also result in the increased production of the main multidrug efflux pump, AcrAB. Together, these responses result in prevention of the influx of toxic molecules, a reasonable response for E. coli. The benefit is clear from the observation that resistance to several antibiotics is moderately increased in the presence of bile salts (558), a normal component of the environment of E. coli.

A very interesting possibility is that R plasmids could bring in a new regulatory element that would repress porin synthesis, making the bacteria generally more resistant to drugs. An early example is the finding by Iyer and coworkers (289, 290) that N compatibility group R plasmids decrease drastically the synthesis of OmpF porin when introduced into E. coli. Although Iyer favored the interpretation that the preexisting ompF mutants acted as a better recipient for the plasmids, it seems more likely that the plasmid contains a gene that represses OmpF synthesis. I am not aware of other studies in this potentially important area, except that Rossouw and Rowbury (562) showed that the presence of F compatibility group plasmid R124 resulted in the repression of OmpF, causing increased resistance to many agents.

Porin mutants. Benson et al. (49) used an ingenious approach to isolate porin mutants that allowed the diffusion of large maltodextrins, which cannot diffuse through the wild-type OmpF channel at significant rates. These mutations changed the large, charged residues (Arg82 or Asp113 in the pore constriction [Fig. 2C]) into residues with smaller side chains, such as serine, cysteine, or glycine, or resulted in short, in-frame deletions of the L3 loop (152). The crystal structure of these mutant proteins confirmed the enlargement of the eyelet (387) as expected. Surprisingly, the single-channel conductance in 1 M NaCl was not increased in any of the mutant porins (571). This observation emphasizes the danger of using single-channel conductance uncritically as the indicator for channel size (described in "Functional assays" above). Conductance is affected by the selectivity of the channel for cations versus anions, and the total conductance is the sum of complex factors. Indeed, a liposome-swelling assay with disaccharides unequivocally showed that the mutant channels were larger than the wild-type channels (571). Similar, larger-channel mutants were also isolated in OmpC (419), and the enlargement of the channels was confirmed by biochemical studies (555).

In an interesting case, selection of colicin-N-resistant mutants of E. coli produced a strain in which the Gly119 residue of OmpF was mutated to Asp (305). This change essentially divided the constriction zone of the channel into two smaller channels, and drastically reduced the permeation rates of both ions and sugars.

Site-directed mutagenesis studies were carried out with samples containing residues lining the eyelet region. However, the results are not always easy to interpret. In one example in which the channel assay was accompanied by a structural determination by X-ray crystallography (590), an attempt was made to narrow the eyelet of the Rhodopseudomonas blastica porin (351) by replacing the residues surrounding the eyelet with bulky tryptophan. The introduction of two or three tryptophan residues narrowed the eyelet and indeed decreased single-channel conductance, as expected. However, the introduction of four or six tryptophan residues produced unstable proteins. Similarly, replacement of four eyelet-surrounding residues with alanine produced only a marginal (17%) increase in eyelet cross-section in the X-ray structure, apparently because the remaining arginine side chain assumed a more expanded conformation and narrowed the eyelet. The study by Benson et al. of OmpF mutants (49), mentioned above, was recently complemented by that of site-directed mutants (494). Thus, converting the three arginine residues at the eyelet into a much smaller alanine and converting the glutamate and aspartate residues nearby to uncharged glutamine and asparagine (see Fig. 2C for the location of these residues) produced a 77% enlargement in the eyelet opening, as judged by crystallography. However, the single-channel conductance of this OmpF mutant was decreased by 30% in comparison to that of the wild-type protein (494). Liposome swelling assay with disaccharides, however, showed that the permeation rates of these large sugars was about eight times higher in the mutant.

Functional studies with new approaches. Computer simulation of Brownian movement of cations and anions through channels (589) has become a valuable tool in the study of wild-type and mutant porins. This approach rather accurately predicted the conductance behavior of various mutants with site-directed mutations at the eyelet region of OmpF (494). These analyses do have restrictions, however: only a single ion was usually observed, so that ion-to-ion interaction was neglected, the protein was assumed to have a rigid structure, and the simulation still assumed that each ion behaves as an unhydrated, naked ion. In the most recent studies by Im and Roux (286, 287), most of these limitations were overcome. Thus, a molecular dynamics simulation involving more than 70,000 atoms, including an OmpF trimer, more than 100 phospholipid molecules, 13,470 water molecules, and 231 K⁺ ions, and 201 Cl^- ions was carried out. Observations of great interest from this simulation include the following. (i) The channel is remarkably efficient in taking up monovalent cations even from micromolar solutions. (ii) The constriction "eyelet" of the channel is very stable and shows no sign of closing during simulation. (iii) Water molecules are strongly ordered at the eyelet region, as predicted earlier (597). A recent analysis combining molecular dynamics simulation with planar bilayer studies suggests also that cations bind to an anionic side chain(s) on the eyelet, most importantly Asp113, and that this binding is significant in the cation penetration across the channel (158). Therefore, the diffusion of preferred solutes through the nonspecific porin channels occurs by a mechanism similar to that through the specific channels (see "Specific channels" below), in the sense that both involve solute (ligand) binding to the channel wall. The difference between nonspecific porins and specific channels has now become quantitative rather than qualitative.

Atomic force microscopy has proven to be a useful tool to examine the porin surface (583). Recently, observation in low ionic strength solution detected the electrostatic potential on the surface of porin trimers with very good resolution (496). This may be a valuable method of determining the electrostatic potentials on the surface of various proteins.

Single-channel experiments can now be carried out with much refinement, so that it is possible to observe the clogging caused by the partitioning of polyethylene glycol into the OmpF channel (563). Here polyethylene glycol 1360 was the cutoff size for the OmpF porin, in comparison with polyethylene glycol 2200, which was the cutoff size for a larger, staphylococcal alpha-toxin channel, a result showing excellent agreement with the known diameters of these channels. Even more impressively, transient clogging of the channel accompanying the diffusion of antibiotic ampicillin through the OmpF channel could be observed and analyzed in single-channel experiments (440).

Voltage gating. Planar lipid film reconstitution of porins showed from the earliest days that the channel can be closed ("gated") at high voltage, typically 100 mV or more (see reference 464 and references therein). Much effort has thus been spent in understanding the structural basis of the gating phenomenon. According to one hypothesis (103), transmembrane voltages make the channel narrower by bringing the cationic and anionic amino acid side chains closer to each other within the channel. As mentioned above, the constriction zone, or eyelet, of OmpF and PhoE contain acidic residues on the L3 loop and basic residues on the facing barrel wall (Fig. 2C). Therefore, the movement of the loop containing the acidic residues in response to voltage is in principle possible, as shown by theoretical analysis (710). However, when the loop was fixed to the barrel wall by a disulfide bond, the gating still occurred (29, 194, 492), a result that rules out at least a large-scale movement of the loop against the barrel wall. Small movements in parts of the loop are still possible, and some molecular dynamics simulation studies (628, 659) indeed seem to support this idea. Another possibility is the movement of external loops that are located outside the channel in the crystal structure. This mechanism was supported by an atomic force microscopy study, in which the application of voltage was shown to cause the movement of external loops, resulting in the closure of channel entrance (433). With Haemophilus influenzae porin, which shows significant sequence homology to E. coli porins (Fig. 1), this idea was supported by site-directed mutagenesis as well as chemical modification of basic amino acid residues in the external loops (20, 21, 157).

When the results did not favor the movement of loop 3 in gating, some workers took a broader look and considered the fact that even the channel made by the -toxin of Staphylococcus aureus, which is an empty 14-member ß-barrel with no constrictions or infolding loops, shows typical gating behavior in planar bilayers (28). They refer to the studies in which even holes in a plastic film (polyethylene terephthalate) were shown to exhibit the gating phenomenon, presumably as a result of the fluctuation in the ionization of the charged groups either within or near the channel (347). This is an extreme position, but the possibility that gating can occur in the total absence of conformational changes in channel proteins should be considered more seriously.

Regardless of the mechanism of the gating, we must ask if the phenomenon has any physiological significance. It is difficult to imagine the presence of such a high membrane potential across the OM, and distribution of ions across the OM, which measures directly the potential, show that only low (less than 30 mV) values of Donnan potential exist here (601). This Donnan potential was of the size expected from the presence, in the periplasm, of polyanionic oligosaccharides (membrane-derived oligosaccharides [MDO]) (333) that cannot diffuse through the porin channels. Therefore, clearly there is no large electrical potential across the OM. In the days when the structure of porins was completely unknown, the observation of gating encouraged some workers to propose that porins might serve as "models" of voltage-gated ion channels in nerve cells, for example. However, we now know that real voltage-gated ion channels are constructed in a totally different manner, and the porin gating observed under these high-voltage conditions may be no more than an interesting laboratory artifact. At lower voltages, the open states often last for many seconds (or even minutes) (168), and this also gives an impression that porin channels are normally open at least most of the time. In retrospect, the emphasis on voltage gating could have produced more confusion rather than enlightenment. The most obvious possible benefit of the voltage gating of porins may be to prevent the formation of open channels when porins are misincorporated into the cytoplasmic membrane. A similar benefit may also apply to the (voltage-gated) mitochondrial porin, which apparently becomes inserted occasionally into other membranes of the eukaryotic cell (657).

On the other hand, it was argued that the gating occurs in the OM of living cells. This argument was supported most strongly by early studies involving patch clamping of the membranes of E. coli spheroplasts. The open state was much less stable, and the closure occurred at lower voltages (periplasmic side negative). This behavior is ascribed to the more "natural" environment occupied by the porins here (168, 169). A patch of the size used in these early studies should have contained close to 10⁵ porin monomers if it came from the OM. However, in reports of patch clamping of E. coli "OM," only a dozen or so open channels were observed in a patch (559). These data seemed to imply that an overwhelming majority of porin channels were closed in intact cells and that they opened only under specific conditions such as starvation (168, 169).

Although a microscopic observation suggested that the OM was intact in the spheroplasts (107), the same paper reported that the patches also contained stretch-activated channels (107), now known to be a component of the inner membrane (642). Therefore, it seems possible, even likely, that these early patch clamp studies using spheroplasts observed the behavior of the inner membrane, containing perhaps a very few porin channels misincorporated into the wrong membrane, or that patches were made on mixed membrane fragments. This view is now shared by one of the authors of these early papers (A. Delcour, personal communication). Consistent with this interpretation, we can calculate the permeability coefficient of the OM in intact cells to uncharged or zwitterionic solutes of a known size on the basis of the cross-section of the always-open porin channels in the OM (460). This calculation yields the predicted permeability coefficient, P, of 2.5 µm/s for lactose (342 Da), a value quite close to the measured value of P of 1 µm/s (601) for a zwitterionic antibiotic of a comparable size (415 Da), cephaloridine. This agreement again reinforces the notion that most of the porin channels are open in intact E. coli cells.

Indeed, the porin channels were open most of the time, even when investigated by the patch clamp method, if OM fragments were diluted into a large excess of phospholipid bilayers (171). Using this latter system, functional studies were carried out. The addition of MDO produced prolonged closure of E. coli porin channels (170). The physiological significance of this observation is not clear. First, the closure required about 10 mM MDO. This is not an excessively high concentration in cells grown in low-osmolarity media, but once the cells are grown in more physiologically relevant media of moderate osmolarity (for example, containing 0.3 M NaCl) the MDO concentration decreases almost 20-fold (601). Second, the closure could be seen only when the voltage across the membrane was periplasmic-side positive, opposite of the direction of the Donnan potential. Delcour argues that inside-positive potential could be created by the influx of cations when bacteria are diluted into a high-salt medium (168), but this has not been demonstrated experimentally. Perhaps the most interesting observation of this group was that polyamines increased the closure of porin channels in patch clamp experiments; since it seems likely that this represents closure, rather than intensified voltage gating, these data are discussed in the next section.

Regulation of porin function. Delcour's group observed that polyamines affected the permeability of porin channels not only in artificial membranes (166) but also in intact cells when measured with cephalorididine influx (167). The effect was modest when the polyamines were added to the medium; a 40% decrease in OmpF permeability required the addition of 100 mM putrescine, 60% inhibition required 30 mM spermidine, and 70% required 1 mM spermine. These polyamine concentrations can be compared to the total intracellular concentrations of putrescine and spermidine, 20 and 6 mM, respectively (which are mostly tied up by polyanionic macromoles such as ribosomes and LPS), and the inability of E. coli to synthesize spermine (643). However, Samartzidou and Delcour (574) made an important observation that when E. coli is synthesizing and excreting cadaverine, the OM permeability decreases to about 30% of the normal level. In an experiment in which acid stress was used to induce cadaverine synthesis, the decrease in OM permeability could be interpreted as the result of increase in OmpC and decrease in OmpF, a part of the acid pH response (see "Regulation of porin expression" above). However, in the experiment where cadaverine synthesis was controlled by a lactose promoter, the decrease is unlikely to be the result of anything other than the cadaverine synthesis and excretion. The concentration of cadaverine in the medium, after 1 h of induction, was only 0.2 mM. Thus, surprisingly low concentrations of endogenously produced cadaverine exerted this profound effect in intact cells, although cadaverine needed to be present at 300 mM for 50 to 60% inhibition of porin activity when added to the external medium. Perhaps the constant efflux of cadaverine from the periplasm tends to close the porin channel more effectively (574). (It should be noted that the channel closing is not the result of voltage gating but appears to be the result of direct interaction between the polyamines and the interior of the channel [291]). It is also likely that polyamine synthesis and export, as a part of the acid stress response, are meant to lead to the neutralization of the periplasm and that closing of the porin channels tends to build up high concentrations of polyamines in the periplasm by retarding the release of polyamines into the medium (see "Regulation of porin expression" above). Thus, the polyamine effect seems to be truly significant in bacterial physiology. Indeed, this point was proven in a recent study in which a strain producing polyamine-resistant OmpC (with the Gly195 changed to Asp) was shown to be more susceptible to acid shock (575).

A single-channel conductance assay of OmpF and OmpC by Todt et al. (662) showed that the conductance value was lowered to almost one-half by going from pH 8.1 to 5.4. A liposome-swelling assay also was reported to show a similar decrease in permeability. However, the authors' finding of very high permeation rate for maltose (about 60% of the rate for glucose at pH 9.4) is quite unexpected, because in our hands disaccharides usually penetrate through the OmpF channel with rates that are about 2 orders of magnitude lower than that of glucose (460, 461). The same group suspected the participation of the sole histidine residue of OmpF or OmpC in this switching of channel size and reported that diethylpyrocarbonate modification of His21 abolished this pH-induced alteration of pore function (661). However, another laboratory could not reproduce the acidic-pH-induced alteration of pore size and saw no effect of a His21-to-Thr substitution (572). The latter authors suggest that the different results were possibly obtained because of the presence of LPS in the porin preparations used by the earlier workers. This explanation seems reasonable because Todt et al. (660) showed that the alteration of channel size can be seen in whole E. coli cells. This important issue needs renewed attention.

Evolution of porins. In the real world, the exposed external loops of porins interact with antibodies, phages, and colicins. For example, OmpF (together with LPS) is the receptor for phage K20 (616, 666) and for colicins A and N (210). The specific maltodextrin channel LamB was initially known only as the receptor for phage lambda. Therefore, it is understandable that these loops underwent rapid evolutionary alterations in structure, as seen clearly by comparisons of sequences of the same (orthologous) porin from different strains of the same species or from strains belonging to the related species (the latter is seen in Fig. 1). In contrast, comparison of porins of Rhodobacter capsulatus strains kept for more than 30 years in separate laboratories (that is, in the absence of external selective agents) revealed that changes occurred nearly exclusively in the transmembrane strands (677).

"Evolution" experiments can be carried out in the laboratory. When E. coli was grown for a few hundred generations in chemostat under glucose-limited conditions, mutations that resulted in the overproduction of the specific LamB channel (which also facilitates the diffusion of glucose [see "Specific channels" below]) were regularly observed (465). In contrast, when a disaccharide lactose was the sole carbon source in a minimal medium, "evolved" strains overproduced the larger-channel porin OmpF over OmpC and, moreover, contained ompF mutations that altered the residues in the constriction region (Arg82 and Asp113) (Fig. 2C) or resulted in a short deletion in the channel-constricting loop L3 (780). These are precisely the mutations found by Benson et al. (49), who looked for larger channel mutants of OmpF; indeed, the presence of the larger channel in the evolved strains was confirmed by their hypersusceptibility to large antibiotics such as cloxacillin. This experiment shows that the porin channel can become limiting for the diffusion of even disaccharides when their concentration is low. Furthermore, the experiment is important in two other areas. (i) Such "evolution" obviously does not take place in nature, in spite of the obvious advantage of the larger channel. This is consistent with the idea that in its natural habitat, E. coli must balance the desirability of more efficient uptake of nutrients against the danger of more rapid influx of toxic compounds, especially bile salts. It would be interesting to repeat the in vitro evolution experiment in the presence of bile salts. (ii) If the porin channels are mostly closed, as claimed by some workers, the mutations will occur in the regions of porin protein that affect the voltage-induced closing. The constriction zone is not such an area, as described above. Therefore, the results do not favor the idea of normally closed porin channels.

Nakae and coworkers criticized almost every aspect of the concept of OprF as the major P. aeruginosa porin (references are cited in reference 459). The argument was based on results that purportedly showed that the porin channels in the intact P. aeruginosa OM was much narrower, instead of wider, than the E. coli porin channels and that the purified OprF lacked pore-forming activity. This group also claimed that the major porins in P. aeruginosa were OM proteins other than OprF. Careful reexamination of the data published by this group showed, however, that their claims could not be substantiated (459). The claim for the narrower channel was based on an improper and arbitrary way of plotting the permeation rate versus the solute size. Purification of OprF following their procedure produced an active pore-forming protein, with a wide channel and low penetration rates. Finally comparison of various P. aeruginosa OM proteins for nonspecific porin activity showed that OprF was the major porin in this organism.

Even more decisive was the study from the laboratory of Hancock (46). They showed, by expressing genes for the metabolism of raffinose (MW 505) in P. aeruginosa, that oprF⁺ cells, but not an oprF mutant, can grow readily by utilizing this sugar. Since raffinose cannot diffuse through E. coli general porin channels at sufficient rates, this is the ultimate proof that the larger pore size of OprF channel is not an in vitro artifact and that the major porin of P. aeruginosa is indeed OprF.

The apparent dilemma of low permeability through a large channel. Although it has been established that OprF is the major porin, it was difficult to explain why solutes penetrated the P. aeruginosa OM at rates about 2 orders of magnitude lower than the rates at which they penetrated the E. coli OM (19, 766), because the number of OprF molecules per cell was similar to that of the major porins in E. coli and because the larger channel in OprF porin was expected to result in a faster, not slower, diffusion of solutes. The answer to this question came through the studies of E. coli OmpA, a homolog of OprF. Like OprF, OmpA also produces permeability on reconstitution into proteoliposomes, but the diffusion rates of solutes are about 2 orders of magnitude lower than in proteoliposomes containing comparable amounts of classical porins (638). (The channel-forming activity of OmpA was also confirmed by a planar lipid bilayer assay [24, 570]). When unilamellar proteoliposomes each containing only a few molecules of OmpA were fractionated on the basis of permeability to sucrose, only a small percentage of the OmpA molecules were found to have the ability to form open channels (636). A similar experiment using OprF also showed that the OprF population was heterogeneous, with only a minority containing the open channel (E. Sugawara, E. K. Nestrovich, S. M. Bezrukov, and H. Nikaido, unpublished data).

These data explain the slow permeation of solutes mediated by OmpA and OprF but leave open the question of the nature of the difference between the "open" and "closed" proteins. OmpA has always been hypothesized to fold as a two-domain protein (545), with its N-terminal half spanning the OM as an eight-strand ß-barrel and its C-terminal segment located in the periplasm and interacting with the peptidoglycan layer (173, 342) (Fig. 3, Majority Conformer). This folding model is supported by a large amount of convincing data, beginning with the location of phage resistance mutations in the predicted external loops (429) and culminating in the determination of the crystal structure of the N-terminal domain of OmpA as an eight-stranded ß-barrel (488, 489). Since the eight-stranded ß-barrel has little room inside for the passage of even small molecules, this conformer cannot serve as an open channel for the passage of organic solutes. (However, N-terminal domains of both OmpA and OprF were shown to produce very small conductance steps [24, 100], and these observations have been rationalized by the recent molecular dynamics simulation study [81]).

View larger version (29K): [in this window] [in a new window]   FIG. 3. Folding model of OmpA-OprF family slow porins. The major fraction of the population folds as a two-domain protein (left) and is important in binding the OM to the underlying peptidoglycan, since the C-terminal globular domain contains a peptidoglycan-binding motif (165, 342). A minor fraction of the population, however, folds differently to produce an open ß-barrel (right). In E. coli, which produces trimeric, high-permeability porins, the presence of this fraction has no functional consequence. However, in fluorescent pseudomonads, which lack the high-permeability porin, this fraction functions as the major nonspecific porin. This fraction also tends to form a loosely associated oligomeric structure, as shown. The oligomer is shown as a trimer only for illustrative purposes. Modified from reference 455a with permission of the publisher.

Recently, the current model of OmpA and OprF (Fig. 3) has received strong experimental support. The model predicts that the N-terminal fragment (alone) of these proteins will not produce significant permeability because it contains only the eight-stranded ß-barrel and that the entire protein sequence is needed to produce an approximately 16-stranded ß-barrel that would allow the permeation of large molecules. Indeed, Arora et al. (24) obtained such a result by using our OmpA preparation that was enriched for open conformers (see below), and this was soon followed by similar papers dealing with OprF (100, 190).

The two-conformer model of OmpA-OprF is thus quite useful and is likely to be correct. The model has received further support. The C-terminal domain of OmpA (as well as that of OprF) contains the immunodominant epitope, which is present on the surface of intact bacteria on the basis of studies using fluorescent-labeled antibodies. However, these data appeared to be inconsistent with the two-domain model of these proteins. A recent study showed that the reaction, with S. enterica serovar Typhimurium cells, of monoclonal antibodies directed to the C-terminal domain of OmpA was dramatically enhanced when the OM was made permeable to these antibodies, confirming that the C-terminal domain was exposed only in minority conformers (621). If residues in the C-terminal domain of OprF are exposed on the cell surface only in the minority conformer containing open channels, one should be able to enrich for this conformer by inserting an additional cysteine residue in one of the predicted external loops, by labeling the residue with a biotinylation reagent in intact cells, and by capturing this conformer (but not the majority conformer, in which the cysteine residue is hidden in the periplasm) via binding to avidin. We have indeed been able to enrich for the biotin-labeled species and to show that this species has higher specific activity in terms of pore formation (E. Sugawara, E. K. Nestrovich, S. M. Bezrukov, and H. Nikaido, unpublished data). Even more intriguing is the observation that the open-channel conformers tend to have a larger size (637), presumably in an oligomeric form. We have again been able to utilize this property and enrich the OmpA and OprF preparation for their open conformers (Sugawara et al., unpublished data).

There is therefore no longer any controversy about the porin functions of OprF and OmpA, in spite of several reviews that argue that their porin function is controversial or unsubstantiated (see, for example, reference 343). Presumably the C-terminal portion of their majority conformer stabilizes the cell envelope structure through their interaction with peptidoglycan (173, 342, 533), and this is their primary function. However, in organisms that lack the classical trimeric porin, such as fluorescent pseudomonads, the protein of this family functions as the major porin and contributes to the high levels of intrinsic resistance to toxic agents through their low permeability. At least in one strain of P. fluorescens, the deletion of oprF gene is followed by compensatory suppressor mutations resulting in the overexpression of OprD family channel proteins (described in "Specific channels in bacteria other than the Enterobacteriaceae" below), a result that convincingly demonstrates that OprF functions as the major porin (130). It is remarkable that OprF-OmpA family proteins are found in almost every gram-negative bacterial genome sequenced; they may serve as the major porin also in some organisms other than the pseudomonads.

One remaining question, though, is whether the two folding pathways of these proteins are regulated. In this connection, P. fluorescens was found to produce OprF of lower single-channel conductance when grown at low temperature (163). Although low ionic conductance cannot be immediately equated with smaller channels (see "Classical porins" above), the low-temperature conformer is more susceptible to protease hydrolysis (190), as expected for the two-domain conformer, and it seems likely that the growth temperature affects the folding of OprF, at least in this organism.

A recent survey of the K-12 genome using a program detecting ß-barrel proteins indicated that the product of gene b1377 is a homolog of OmpC belonging to the classical porin family (778). Actually this trimeric porin, called OmpN, was originally found in E. coli B and then in K-12 and was expressed and purified (510). Its channel property was also reported to be very similar to that of OmpC. This protein is apparently expressed at a very low level in wild-type strains; I am not aware of any report on the effect of environmental conditions on the production of this porin.

S. enterica serovar Typhimurium produces OmpD, an additional member of the classical porin family. It has channel properties comparable to those of OmpF-OmpC as judged from single-channel conductance data (51). Liposome-swelling studies apparently have not been carried out with this porin. Interestingly, the synthesis of this porin is dependent on cyclic AMP (B. Rotman and G. F.-L. Ames, personal communication). The gene ompD is located downstream of the putative transporter gene yddG, and recently the inactivation of either of these genes was reported to cause hypersusceptibility to methyl viologen (577). The authors propose that the OmpD porin forms a multiprotein complex with the YddG pump, playing the role of the exit channel like TolC. However, OmpD does not contain the periplasmic "tunnel" found in TolC (see "export channels of the TolC family" below). Furthermore, this reviewer found that homologs of yddG gene in other organisms occur without the neighboring ompD-homologous gene. A porin not corresponding to OmpF and OmpC was reported in S. enterica serovar Enteritidis and was called OmpE (126). The nomenclature suggests that the authors were aware of the presence of OmpD, yet they did not compare their porin with OmpD. The production pattern of this porin on complex agar media and in synthetic liquid media seems to fit with the idea that it is actually OmpD that is synthesized in a cyclic AMP-dependent manner.

Mutants expressing OmpG porin were isolated in E. coli K-12 by Misra and Benson (420) by using a selection procedure that favors mutant cells capable of taking up large nutrients. OmpG is a porin with unusual properties (197). First, it appears to lack, on the basis of its sequence, the large loop 3 that is ubiquitous in classical trimeric porins. Second, it produces an unusually large channel, as expected from this structure. Third, it appears to exist as a monomer, unlike members of the classical porin family. The large channel size and the monomeric nature of OmpG were confirmed by single-channel conductance and folding studies in another laboratory (143, 144). The protein was made into a two-dimensional crystal, and its study also confirms the monomeric nature of this porin (45). The ompG gene appears to be the last gene in a putative 11-gene operon, which contains genes needed for the ATP-binding cassette (ABC) transporter-catalyzed uptake of oligosaccharides, as well as various genes presumably involved in the degradation of such compounds (197). Therefore, it seems likely that it is a large-channel porin needed for the uptake of larger oligosaccharides. OmpG is expressed, at a low level, in Salmonella and Shigella, but only trace levels are seen in wild-type E. coli K-12.

The ompL gene of K-12 was identified by transposon insertion mutagenesis as a gene whose inactivation made a dsbA mutant more dithiothreitol resistant (159). Its sequence suggests that it may exist as a ß-barrel in the OM, and the purified OmpL showed porin activity. One could explain the phenotype of ompL mutants if it were assumed that OmpL facilitated the outward diffusion of hypothetical, low-molecular-weight oxidizing agents in the periplasm, but recent attempts to reproduce the reported phenotypes of ompL and ompL dsbA mutant strains have failed (578). OmpL is not expressed at a high level in wild-type cells and presumably plays only a minor role in the flux of nutrients in general. Recently, OmpL was found to be a homolog of the Erwinia chrysanthemi oligogalacturonate channel protein, KdgM (490) (see "Other specific channels" below).

E. coli OmpW, a small (191 residues in the mature form) OM protein, is used by a colicin as a receptor (498); otherwise, its function is unknown. Interestingly, this protein belongs to a homology group that includes P. putida AlkL, which is hypothesized to be an alkane channel (690), and NahQ (SwissProt accession no. Q51498), which is part of the cluster coding for naphthalene utilization and is also likely to code for an OM channel. This AlkL-OmpW group also includes Omp21 of Comamonas acidovorans, which was studied as a two-dimensional crystal (31) and showed patterns similar to other eight-strand ß-barrel proteins. With slow porins like OmpA and OprF, there was a possibility of alternative folding to produce large channels. However, it is currently unclear how these small proteins of the AlkL-OmpW group would produce sizable channels, if they indeed function as channels.

Many other small OM proteins are known (701) or suspected (31) to produce eight-stranded ß-barrels. These include OmpX of E. coli (701), opacity proteins of Neisseria, and Ail protein of Yersinia. More recently, PagP, the enzyme that transfers a palmitoyl residue from phospholipid to lipid A (discussed in "Physiological adaptation in LPS structure" below), was shown to be an eight-stranded ß-barrel protein by solution nuclear magnetic resonance spectroscopy (NMR) (285). One example of the opacity protein family protein was actually shown by X-ray crystallography to have a 10-strand ß-barrel structure (511), and another example from this family, NspA, was recently characterized to be an 8-strand ß-barrel protein (691). As Baldermann and Engelhardt argue (31), what is important in these proteins are the external loops that may be needed for interaction with the external structure, such as the surface of the host cell membrane or the substrates of enzyme reaction, and the 8- or 10-strand ß-barrel may simply serve as the OM anchor. None of these proteins has been shown to form channels, as far as I am aware. Other examples where OM-spanning ß-barrels are thought to function as simple anchors include OmpT and OmpLA. The crystal structure of OmpT, an OM-associated protease, shows that its 10-strand ß-barrel protrudes far beyond the plane of the OM bilayer and that the membrane-external portion of the barrel and the loops apparently function in proteolysis (692). In OmpLA, the OM-associated phospholipase A, the structure shows a 12-strand ß-barrel (625), yet the interior of the barrel appears to be occluded by a dense hydrogen-bonding network, and the protein is not thought to function as a channel.

Most E. coli strains, including K-12 and B, are incapable of metabolizing raffinose, a trisaccharide. However, some strains contain plasmids that contain genes for degradation and transport of this sugar. One of the genes is rafY, which produces an OM channel (353). When RafY, a fairly large (50.7-kDa) protein that exists as a trimer, was inserted into a planar lipid bilayer, it produced a high single-channel conductance of 2.9 nS in 1 M KCl (16). Interestingly, however, there was no blocking of the channel by several sugars, including raffinose. Although negative results must be interpreted with caution, it seems likely that RafY, unlike such specific channels as LamB or ScrY (which shows low single-channel conductance that is blocked by sugars), simply produces a pore large enough for the influx of trisaccharides. RafY does not appear to be strongly similar to other known porins or channel proteins.

Porins in members of the Enterobacteriaceae other than E. coli an