Biochimica et Biophysica A cta, 737 (1983) 51 - 115 51 Elsevier Biomedical Press

BBA 85241

MOLECULAR ARCHITECTURE AND FUNCTIONING OF THE OUTER MEMBRANE OF AND OTHER GRAM-NEGATIVE BACTERIA

BEN LUGTENBERG a,, and LOEK VAN ALPHEN h " Department of Molecular Cell Biology' and Institute for Molecular Biology', State University, Transitorium 3, Padualaan 8, 3584 CH Utrecht and h Laboratorium voor de Gezondheidsleer, University of Amsterdam, Mauritskade 57, 1092 AD Amsterdam (The Netherlands)

(Received July 26th, 1982)

Contents

Introduction ...... 52 A. Scope of this review ...... 52 B. Ecological considerations relevant to structure and functioning of the outer membrane of Enterobacteriaceae ...... 53 C. General description of the cell envelope of Gram-negative bacteria ...... 53

II. Methods for the isolation of outer membranes ...... 58 A. E. coli and S. typhimurium ...... 58 1. Isolation of peptidoglycan-less outer membranes after spheroplast formation ...... 58 2. Isolation of outer membrane-peptidoglycan complexes ...... 58 3. Differential membrane solubilization using detergents ...... 59 4. Membrane separation based on charge differences of vesicles ...... 59 B. Other organisms ...... 59

II1. Individual constituents of the outer membrane ...... 59 A. Composition of the outer membrane ...... 59 B. Phospholipid ...... 60 C. Lipopolysaccharide ...... 61 1. Introduction ...... 61 2, Methods of isolation and purification ...... 61 3. Chemical structure of lipopolysaccharides ...... 62 4. Effects of polymixin, EDTA and divalent cations ...... 64 D. Enterobacterial common antigen (ECA) ...... 64 E. ...... 64 1. Introductory remarks ...... 64 2. in the outer membrane ...... 67 3. Lipoproteins ...... 68 4. OmpA ...... 70 5. The family of peptidoglycan-associated general diffusion pore proteins ...... 71 a. Introduction ...... 71 b. Function of general pore proteins ...... 72 c. Purification and properties of general diffusion pore proteins ...... 75 d. Characteristics of individual general diffusion pore proteins ...... 76

* To whom correspondence should be addressed. heptose; KDO, 2-keto-3-deoxy-octulosonic acid; LPS, lipopoly- Abbreviations: Abe, abequose; ECA, Enterobacterial Common saccharide; NMR, nuclear magnetic resonance; PAL, pepti- Antigen; ESR, electron spin resonance; GIcN, glucosamine; doglycan-associated lipoprotein; Rha, rhamnose; SDS, sodium GIcNAc, N-acetyl-D-glucosamine; Hep. t-glycero-D-manno dodecyl sulphate.

0304-4157/83/0000-0000/$03.00 © 1983 Elsevier Science Publishers 52

i. OmpC protein and OmpF protein ...... 76 ii. PhoE protein ...... 76

iii. Salmonella pore proteins ...... 77 iv. Other general diffusion pore proteins ...... 77

6, Characteristics of E. coil pore proteins not antigenically related to the family of peptidoglycan-associated general diffusion pore proteins ...... 78 a. T6 receptor protein ...... 78 b. Bacteriophage lambda receptor protein ...... 78 c. Outer involved in the uptake of vitamin B12 ...... 80 d. Outer membrane proteins involved in the uptake of ferric ions ...... 80 Characteristics of E. coil outer membrane proteins without identified function ...... 80 a. Protein a ...... 80 b. Protein II1 ...... 81 c. LPS binding protein ...... 81 d. Outer membrane proteins induced by sulphate limitation ...... 81 e. Phage- and plasmid-coded outer membrane proteins ...... 82

IV. Molecular organization of the outer membrane ...... 82 A. Introduction ...... 82 B. Methods used for studying the localization of individual outer membrane constituents and their interactions ...... 84 1. Localization at the cell surface ...... 84 2. Protein-protein and protein-peptidoglycan nearest neighbour associations ...... 85 3 Interactions between individual proteins and LPS ...... 86 4. The lipid matrix and interactions of proteins with lipids ...... 86 C. Localization of LPS ...... 86 D. Localization of phospholipids ...... 86 E. Localization of ECA ...... 87 F. Localization and topography of outer membrane proteins ...... 87 1. Introduction ...... 87 2. The major lipoprotein ...... 88 3. OmpA protein ...... 89 4. Peptidoglycan-associated pore proteins ...... 90 5. Are matrix (pore) proteins associated with peptidoglycan in vivo? ...... 91 G. The lipid matrix ...... 93 1. Is the outermembrane a lipid bi,layer? ...... 93 2. Nature of OM particles and OM pits on the fracture faces of the outer membrane observed with freeze-fracture electron microscopy ...... 95 3. Physical properties of LPS and phospholipids in the outer membrane ...... 97 4. Interactions of proteins with the lipid matrix ...... 99 5. Distribution of outer membrane constituents over both monolayers ...... 100 H. Molecular organization of the outer membrane of Enterobacteriaceae ...... 101 1. Outer membrane of other Gram-negative bacteria ...... 103

V. Future prospects ...... 103

Acknowledgements ...... 104

References ...... 104

I. Introduction tive and Gram-negative bacteria differ fundamen- tally with respect to the composition of their cell IA. Scope of this review walls. One of the major differences is that Gram- negative cells contain an outer membrane, located The cytosol of a bacterial cell is surrounded by at the outside of a monolayer of peptidoglycan. a complex cell envelope which usually consists of a This outer membrane forms the physical and func- cytoplasmic membrane and a cell wall. Gram-posi- tional barrier between the inside of the cell and its 53

environment. After methods for the isolation of Donor cells of several bacterial species can outer membranes became available, its composi- transfer (part of) their genetic information to tion, structure, function and biogenesis have been acceptor cells, the latter ones usually being related studied extensively in the last decade, especially in to the donor. Conjugative transfer, especially of the enteric bacteria Escherichia coli and Salmonella plasmid DNA, usually is the means by which typhimurium. Recently, a monograph [1] as well as genetic information coding for production of toxins several reviews have been published on the outer and for resistance against antibiotics, heavy metal membrane in general [2,3] or on specific topics like ions or serum is passed from one cell to another, genetics [4,5], functions [6-10] or biogenesis often even from one species to another. Because of [5,7,11-16]. The present review will focus on the the specificity of this conjugation process it is molecular architecture of the outer membrane and likely that again outer membrane receptors are its constituents in relation to the membrane's func- involved. tions. Bacteria which live in the gut must somehow have a mechanism that protects the cytoplasmic IB. Ecological considerations relevant to structure membrane from the detergent-like action of bile and functioning of the outer membrane of Entero- salts, fatty acids and glycerides. Moreover, the gut bacteriaceae content is rich in proteolytic and lipolytic enzymes and in glycosidases. The natural habitat of E. coli and other En- In order to be pathogenic, bacteria must have terobacteriaceae is the colon. From there these properties that enable them to adhere to eukaryotic bacteria can reach the surface water where they cells and/or invade tissue. Also, they must have can survive for quite a long time. Only specific mechanisms to resist the defence mechanism of the classes of these bacteria can colonize or enter into host (for a review, see Ref. 17). Among the most other parts of the human or animal body, like important pathogens are many Gram-negative cerebrospinal fluid, the blood stream and the bacteria e.g. Bordetella bronchiseptica, Bordetella urinary tract. Enterobacteriaceae constitute ap- pertussis, Campylobacter, E. coli, Erwinia amylovora prox. 1% of all bacteria present in the gut, an and Erwinia carotovora, Haemophilis influenzae, anaerobic environment. Generation times are in Klebsiella, Neisseria gonorrhoeae, Neisseria the order of 10-20 h in contrast to only 20-60 rain meningitidis, Salmonella and Shigella. The relative under the usual laboratory conditions. In order to contribution of Enterobacteriaceae as causative be able to compete with other micro-organisms agents of infectious diseases in increasing, espe- (10u-10J2/g faeces) the bacterium should be able cially in elderly patients and in patients treated to take up nutrients effectively in order to remain with broad spectrum antibiotics, corticosteroids in the gut. It also seems likely that the supply of and antimetabolites. It is often not realized by nutrients varies both qualitatively and quantita- molecular biologists that E. coli is the leading tively, and it can be expected that the bacteria cause of Gram-negative bacteremia in adults. adapt continuously to such changes. Bacteria can be attacked by other bacteria, by IC. General description of the cell envelope of bacteriocins (proteins produced by bacteria which Gram-negative bacteria are toxic for other related bacteria) and by which bind to a specific receptor at The cell envelope of Gram-negative bacteria the cell surface of a susceptible bacterium as the has been the subject of a number of quite recent first step of a process that kills the cell. Survivors reviews [18-20]. This envelope (Fig. 1) consists of of such an attack are often mutants which lack the three essential layers, namely the cytoplasmic (or receptor for the bacteriocin or bacteriophage in inner) membrane, the peptidoglycan (or murein) question. As such receptors are often constituents layer and the outer membrane. Although most of the outer membrane, bacteriocins and bacterio- soluble protein species are present in the cytosol, phages are excellent tools for studies on the outer several of such protein species are located between membrane. the two membranes in a compartment known as 54

nating residues of N-acetylglucosamine and N- acetylmuramic acid, are covalently linked to each

c~ ~ta~elk~m other via crosslinks that can be formed between tetrapeptides which are attached to the N- aody acetylmuramic acid residues (see Ref. 21 for a review on peptidoglycan structure). In Gram-nega- tive bacteria a monolayer of peptidoglycan sur- rounds the cytoplasmic membrane. The rigidity of Fig. 1. Features of the cell envelope of Gram-negative bacteria. the peptidoglycan layer enables the cell to with- The cytoplasm usually is surrounded by three layers: the cyto- stand the osmotic pressure of approx. 3.5 atm [22] plasmic membrane, the peptidoglycan layer and the outer of the cytoplasm. Degradation of peptidoglycan membrane. A few lipoprotein (LPP) molecules are drawn in the periplasmic space to indicate that these molecules are involved e.g. by lysozyme, results in lysis of the cell as a in anchoring the outer membrane to the peptidoglycan layer. consequence of heavy swelling caused by the up- The two membranes are connected by zones of adhesion (ZOA). take of water through the cytoplasmic membrane Cells excrete part of their outer membrane into the medium as into the cytosol. Disrupture of the cytoplasmic vesicles or blebs. A few ribosomes, as part of a membrane-bound membrane can be prevented if peptidoglycan is polysome (MBP), are synthesizing proteins from which the exported (periplasmic and outer membrane) proteins are ini- degraded under hypertonic conditions e.g. in the tially synthesized with a N-terminal signal sequence which is presence of 10% sucrose. As the rods then loose cleaved of during biogenesis. An A-layer or a capsular layer are their shape and round up to osmotically fragile sometimes present. The dimensions are not strictly drawn to spheroplasts, peptidoglycan is probably (co-)re- scale. Especially the capsular layer can be much thicker than sponsible for the rod shape as well. Consistent indicated. As the O-antigen (O-AG) and the flagellar body are with this assumption is the observation that iso- very long, their ends have not been indicated. As the way of anchoring of pili is unknown, these structures have not been lated peptidoglycan has the same shape as the cells drawn. The basal body of the flagellum has been drawn sep- from which it has been isolated. arately because the precise sites of connection between flagellar The outer membrane contains, in addition to rings and envelope layers are not known. For further explana- phospholipid and protein, LPS as a major con- lion see text. stituent. The membrane is covalently attached to the peptidoglycan layer via a lipoprotein. The membrane prevents leakage of periplasmic pro- the periplasmic space. The two membranes are teins. Moreover, in Enterobacteriaceae it has to interconnected by so-called zones of adhesion. The protect the content of the cell against the attack by outer membrane is not always the outmost layer of harmful agents like bile salts and enzymes. The the cell envelope as it is often covered with a extent of this protective function is very impressive rather amorphous capsular layer or with a so-called as E. coli can grow in the presence of as much as additional layer or A-layer consisting of a regular 5% of the strong ionic detergent SDS. As phos- pattern of subunits, usually proteineous in nature. pholipid bilayers are very sensitive to this deter- Finally, appendices like flagella, fimbriae and pili gent (erythrocytes lyse in the presence of as little are anchored in the cell envelope. as 0.001% SDS (Bergmans, H.E.N., Overbeeke, N. The cytoplasmic membrane contains phospholip- and Van Scharrenburg, G., personal communica- ids and proteins in about equal amounts. It plays a tion)), this SDS resistance indicates that the outer role in the transport of nutrients, in oxidative membrane either is not a phospholipid bilayer or phosphorylation, in the synthesis of phospholipids, that its bilayer structure is very well shielded from peptidoglycan, lipopolysaccharide and, via mem- the medium. brane-bound polysomes, of periplasmic and mem- The periplasmic space is located between the brane proteins. Moreover, it is probably involved two membranes. It has been reported to comprise in cell division and it serves as an anchor for as much as 20--40% of the total cell volume [22] DNA, at least during replication. although much lower values have also been re- The peptidoglycan layer consists of a network in ported [9]. The space contains proteins [23] and which linear amino sugar chains, containing alter- oligosaccharides [24]. The periplasmic proteins 55

comprise approx. 4% of the total cell protein [23]. cell [29,30]. Secondly, these zones have been im- With respect to their functions three classes of plicated in the translocation of newly synthesized periplasmic proteins can be distinguished [23,25]. LPS [12,35,36] in the synthesis [32,33] and translo- Proteins with a catabolic function (e.g. 5'- cation [34] of (some) outer membrane proteins and and alkaline ) convert so- in the production of sex pili [12]. lutes for which no transport system exists to a Assuming that these 'zones of adhesion' are form that can be transported through the cyto- stable structures several investigators have under- plasmic membrane. Another class of periplasmic taken attempts to purify these domains, but con- proteins are the binding proteins which have affin- sistent data on their composition have not been ity for nutrients like sugars, amino acids or ions. It obtained so far. Recently a fraction, presumed to has been established in a number of cases that consist of these domains, was reported to be en- these proteins are essential for transport of the riched in activities [31]. solute in question. A third class of periplasmic An alternative for permanent 'zones of adhe- proteins is involved in the degradation or modifi- sion' is that these zones exist only temporarily. cation of harmful components such as antibiotics Basically this idea comes from Witholt and co- and heavy metals. workers [32,33], who suggested that these 'zones of Periplasmic membrane derived oligosaccharides adhesion' might be sites of outer membrane pro- [24,26,27], comprising about 1% of the dry weight tein synthesis generated by the fact that the pro- of the cell, are a closely related family of highly tein synthesizing machinery is producing an outer branched molecules, containing about nine re- membrane protein. The consequence would be that sidues of glucose as the sole sugar. They are vari- these zones exist no longer when the protein chain ously substituted with sn-glycerolphosphate and has been completed. phosphatidylethanolamine residues derived from Considering biogenesis of outer membrane pro- the membrane phospholipids. Some species of the teins at the membrane level at specialized tem- oligosaccharides also contain P-succinyl esters, ad- porary domains in the cytoplasmic and/or outer ding to their net negative charge. Membrane-de- membrane one can basically propose two mecha- rived oligosaccharides may play an important role nisms, both of which are based on the presence of in the osmoregulation of the Gram-negative cells non-bilayer lipids. In the first model a protein is as cells grown in medium with low osmolarity exported via a vesicle which blebs off from the synthesize 16 times more of these substances than cytoplasmic membrane and fuses with the outer they do under conditions of high osmolarity [28]. membrane. According to the second mechanism Elegant experiments of Stock et al. [22] have the protein diffuses through a temporary connec- shown that there is a Donnan equilibrium over the tion between the two membranes. The observation outer membrane, and that the periplasm and cyto- that E. coli membrane phospholipids [37] and also plasm are isosmotic. For cells in minimal medium, LPS [162,176] have the tendency to form non-bi- the osmotic strength of the cell interior was esti- layer structures is consistent with these models mated to be approx. 300 mosM [22]. since these non-bilayer phases, especially the 'Zones of adhesion" [ 12] are electron microscopi- hexagonal II phase, are supposed to represent cally visible contact sites between cytoplasmic and intermediate stages in fusion processes [38]. This outer membrane which become apparent only hypothesis is strongly supported by freeze-fracture when the membranes are separated by plasmolysis experiments in model membrane systems [39,40], [29]. About 200-400 of these sites are present per including those containing phosphatidyl- cell, they measure 20-30 nm across and cover ethanolamine [41], the major phospholipid of E. about 5% of the membrane surface [12]. Several coli. For reviews on this subject the reader is lines of evidence indicate a physiological role for referred to Refs. 42, 43 and 44. these 'zones of adhesion'. Firstly, in phage-in- Based on these considerations we propose three fected cells empty phage heads are found opposite models for functional meta-stable connections be- adhesion zones, strongly suggesting that they are tween cytoplasmic and outer membrane (Fig. 2). It involved in the penetration of phage DNA into the should be noted that direct evidence in support of 56

A these models is lacking, but that they are rather Lll:~T.... b c d meant to stimulate investigators to test the attrac- tive possibility of temporary adhesion sites. The observed role of the "zones of adhesion' in the translocation of LPS and of at least some outer membrane proteins has been incorporated in these models. Moreover, the possibility that outer mem- e f l brane proteins and LPS, which often form func- tional complexes (see section IV), are translocated as a complex or are even involved in the induction of fusion, can easily be incorporated in the mod- els. The first model (Fig. 2A) is based on models B published recently to explain translocation of LPS [45] and of outer membrane proteins [15,16[, in OM which it is proposed that a vesicle is released from the cytoplasmic membrane by fusion and subse- quently fuses with the outer membrane. This model can be extended in that the site of translocation is supposed to correspond with a 'zone of adhesion' CM (Fig. 2A). Blebbing starts at a site in the cyto- a ,o, plasmic membrane, which has a special composi- tion (e.g. rich in LPS or certain phospholipids) or C structure (e.g. a strongly asymmetric region due to , Id) l~ the presence of a large number of LPS molecules OM ~ { ~t~ in the inner monolayer or to the presence of or formation of complexes of outer membrane pro- teins and LPS). A vesicle is formed and subse- quently fuses with the outer membrane, perhaps at special sites. Molecular models for fusion and

(allL I(a) to the special structure in the site of fusion, in which a lipidic Fig. 2. Models for connections between cytoplasmic (CM) and particle may be absent (Fig. 2B) or present (Fig. 2C), the LPS outer membrane (OM). A: Vesicle-mediated fusion model. LPS molecule reaches, by a flip-flop mechanism, first the area where (L) and outer membrane proteins (P) are synthesized at the the outer monolayer of the cytoplasmic membrane is connected cytoplasmic membrane (a). Blebbing starts in certain special with the inner monolayer of the outer membrane (c) and domains of the cytoplasmic membrane, possibly due to the subsequently the outer monolayer of the outer membrane (d). presence of a high concentration of LPS molecules or due to It is conceivable that outer membrane proteins either are the presence or formation of protein-LPS complexes. The bleb synthesized at arbitrary sites in the cytoplasmic membrane or pinches off (b) and subsequently fuses with the same mem- at such adhesion zones and that they form complexes with LPS brane (c), e.g. by a mechanism illustrated in Fig. 2B or C. in molecules before, during or after fusion. As no clear choice can which the lipids phosphatidylethanolamine, cardiolipin and/or be made between all these possibilities, outer membrane pro- LPS, which are known to be able to participate in non-bilayer teins have only been drawn at their final location in the outer phases, may be involved. This transient state is followed by the membrane. Finally, it should be noted that all three models release of a vesicle into the periplasm (d). The vesicle fuses with allow, but not require, the translocation of phospholipid to the the outer membrane (e), resulting in the insertion of LPS and outer monolayer of the outer membrane (see Figs. 2B and 2C). protein, or of a complex of these molecules, in the outer It has clearly been established that the outer leaflet of E. coh membrane in the correct orientation (f, g). B and C: Direct contains no or hardly any phospholipid (see section IV), but it fusion models. A LPS molecule in the inner monolayer of the has been argued that fusion of bacterial cells with phospholipid cytoplasmic membrane (a) is synthesized in or diffuses to (b) a vesicles is easier to envisage if some phospholipid is present in special domain in the cytoplasmic membrane (see above). Due the outer monolayer (see subsection IVG-1/. 57

fission of such vesicles are shown in Figs. 2B and incubation of isolated outer membranes with C. The translocation of LPS with its hydrophilic lysozyme at 37°C but not at 0°C [543]. sugar chain is well-explained by this vesicle-medi- It is very likely that both the blebs shown in ated translocation model. However, such a long Fig. 2A and the connections between the two sugar chain will require vesicles with a rather large membranes drawn in Figs. 2B and C are extremely diameter, which can be estimated to be at least 40 unstable and that, unless they are somehow stabi- nm, unless the O-antigen sugar chains are some- lized (e.g. by protein synthesis on membrane-bound how organized close to the bilayer during this ribosomes) they cannot be detected by electron process. This large diameter would require, at least microscopy. Consequently, the detection of 'zones locally, a thick periplasmic space. It can be argued of adhesion' in thin sections could be due to that it also would require rather large gaps in the artificial blebbing or fusion e.g. at sites in the peptidoglycan layer, which have never been de- cytoplasmic (or outer) membrane where a bleb- tected by electron microscopy. However, if growth bing or fusion process is going to start. of the vesicle occurs outside the peptidoglycan Flagella (H-antigen), fimbriae and pili are ap- layer, much smaller - perhaps undetectible - gaps pendages, consisting of protein subunits. Flagella would he sufficient. It should be noted that the are responsible for the cells motility. They are occurrence of gaps in the peptidoglycan is not connected with all three layers of the cell envelope unlikely as the total amount of peptidoglycan in [51]. Fimbriae are smaller and more rigid than the cell is only sufficient to cover half of the cell flagella [52,53]. They occur in many varieties and surface. The model shown in Fig. 2A also implies several types have been implicated in adhesion to a small leakage of cytoplasmic constituents into (often specific) eukaryotic cells [54,55]. A special the medium. Vesicle-mediated excretion of soluble type of fimbriae, called pili, is essential for the constituents of the cell has been described re- adhesion of bacterial donor cells to acceptor cells, peatedly, e.g. during normal growth E. coli releases the first step in bacterial conjugation [6,8]. outer membrane vesicles or blebs, corresponding A capsular layer (K-antigen) usually consisting with approx. 5% of the total outer membrane, into of negatively charged polysaccharides [56,57], can the medium [46-49]. An offensive function has be present outside the outer membrane. The recent been proposed for these vesicles in the delivery of discovery of lipid constituents in the K92 capsule heat-labile enterotoxin by E. coli cells to intestinal lead the authors to suggest that the lipid portion epithelial cells [50]. enables the capsule to anchor in the outer mem- Two other models for hypothetical recta-stable brane [58]. The presence of a capsule sometimes connections between the two membranes are shown enhances the resistance of the cell against phago- in Figs. 2B and 2C, both of which propose a direct cytosis and against the bactericidal action of com- fusion between the membranes. Whereas in Fig. plement. 2B no special structure is proposed within the Additional layers [59,60] or A-layers are surface fusion site, an 'inverted' micelle is proposed in structures consisting of regularly arranged sub- Fig. 2C. Although especially the translocation of units of usually one protein species. They are LPS molecules with their hydrophilic, sometimes located outside the outer membrane and have been very long, sugar chains through flip-flop, even in discovered in several bacterial species like Acin- the special environment of the fusion site is some- etobacter [61,62], Azotobacter vinelandii [63], what hard to imagine, it should be noted that it Campylobacter fetus [64], Spirillurn [65-68], has been established that phospholipid molecules Treponema and, more recently, in Aeromonas do use such structures to cross membranes [41]. salrnonicida [69-71]. It has been proposed that Moreover, evidence that the long O-antigen chains such layers play a role in protecting the cell against of LPS can flip-flop over a membrane comes from a harmful environment [60,72] or that they are the observation that these molecules redistribute involved in adherence to specific tissues [73]. very rapidly over both membrane monolayers upon 58

II. Methods for the isolation of outer membranes from the outer membrane which usually accompa- nies EDTA treatment [85]. More recently Witholt et al. [77] introduced a similar procedure which. IIA. E. coli and S. typhimurium however, has the advantage that it is applicable to laboratory strains of E. coli grown under various IIA-1. Isolation of peptidoglycan-less outer mem- conditions [77]. Also, it turned out to be the proce- branes after spheroplast formation. dure of choice for the isolation of outer mem- If necessary, cells can first be treated in an branes from clinical strains of E. coil (M. Acht- omnimixer in order to remove flagella, pili and man, personal communication). capsular material, a procedure which does not result in cell breakage [74]. One of the most critical llA-2. Isolation of outer membrane-peptidoglycan steps in most procedures used for separating cyto- complexes plasmic and outer membranes consists of convert- Due to the pioneering work of Schnaitman [86], ing cells to spheroplasts. These are then lysed to the outer membrane can be isolated complexed to yield outer and cytoplasmic membrane vesicles the peptidoglycan layer. The method involves cell [75-77]. Outer membranes can be isolated on den- disruption in a French pressure cell followed by sity gradients [75-79], by electrophoretic tech- membrane separation on a sucrose density gradi- niques [80-82] or by aggregation at pH 5, followed ent. Slightly modified versions of this procedure by centrifugation [83]. Lysozyme, which cosep- have been published by Koplow and Goldfine [87] arates with the membranes, can be removed by and Smit et al. [88]. Neither the original procedure washing with 0.2 M KC1 [76]. Outer membranes, nor any of the mentioned modifications could be isolated from spheroplasts, have a buoyant density applied successfully in our laboratory but the in- of 1.22 _+ 0.01. They contain approx. 60% of the troduction of another modification [37] resulted in protein, 50% of the phospholipid and 90% of the a good separation of the two membranes. For LPS of the cell envelope [76]. structural studies the use of outer membrane- Birdsell and Cota-Robles [84] have described peptidoglycan complexes has several advantages the basic procedure for the production of over the use of outer membranes prepared from spheroplasts which can serve as the basis of a good EDTA-lysozyme spheroplasts. (i) The use of membrane separation. Slight modifications of this EDTA, a prerequisite in the latter method as it is procedure have extensively been described by required to make the outer membrane permeable Osborn et al. [76] and Witholt et al. [77]. Basically, to lysozyme, is avoided. (ii) Removal of the the cells are plasmolyzed with 0.5 M sucrose, peptidoglycan-layer by lysozyme results in re- lysozyme is added, and the outer membrane is organization of the LPS component [89]. (iii) Cy- made permeable for this peptidoglycan-degrading toplasmic and outer membranes of heptose-less by treatment with EDTA. The procedure mutants cannot be separated using the EDTA- converts rod-shaped cells into osmotically sensitive lysozyme method [76] but the French press method spheroplasts i.e. the cytosol surrounded by an gives good results [87,88]. The most likely explana- intact cytoplasmic membrane with outer mem- tion for the observed failure in the former case is brane structures attached to it at one or only a few that the buoyant density of the peptidoglycan-less sites [84]. outer membrane of heptose-less mutants is similar Miura and Mizushima [75,78] were the first to that of the cytoplasmic membrane, presumably investigators who successfully separated cyto- due to the loss of sugars as a direct result of the plasmic and outer membranes. In our hands the mutation (see Fig. 3), as well as due to the loss of best results are obtained with the procedure of protein and an increase in phospholipid content as Osborn et al. [76] who slightly modified the proce- an indirect result of the mutation [87,90-96]. The dure of Birdsell and Cota Robles [84] for the fact that the presence of peptidoglycan increases preparation of spheroplasts. Moreover, the Osborn the density of the outer membrane [37,88] is the procedure can completely be carried out at 4°C basis for the success of the separation method and does not result in significant release of LPS developed by Schnaitman. A possible disad- 59

vantage of the latter method is that phospholipids liB. Other organisms are degraded by the action of an outer membrane-bound phospholipase AI (see subsec- Procedures developed for the separation of the tion IIIE-2). This disadvantage can be overcome membranes of E. coli and S. typhimurium have by the use of a mutant lacking this activity [37]. been applied successfully on other Gram-negative bacteria, usually after some modification of the 11,4-3. 'Differential membrane solubilization using procedure. Examples are Acinetobacter sp. [100], detergents Campylobacter fetus [ l01 ], Caulobacter crescentus It is a rather common viewpoint that treatment [102,103], Chlamydia trachomatis [104], Chro- of isolated cell envelopes with certain detergents matium vinosum [105], Haemophilus influenzae can selectively solubilize the cytoplasmic mem- [106,107], Klebsiella aerogenes [108], Moraxella brane while leaving the outer membrane intact. It nonliquefaciens [109], Myxococcus xanthus [ll0], is our view that, at best, the proposed selectivity Neisseria meningitidis [111], Neisseria gonorrhoeae has been proven for the protein component of the [112,113], Proteus mirabilis [114], Pseudomonas membranes. The effect of Triton X-100 on the aeruginosa [ 115-117], Rickettsia prowazeki [ 118], solubilization of membrane fractions has been Selenomonas ruminantium [119], Serratia marces- studied in detail [97]. Extraction of cell envelopes cens [120], Yersinia pest& Y. enterocolitica and Y. with 2% Triton X-100 in the presence of mag- pseudotuberculosis [121]. It should be noted that nesium ions results in complete solubilization of methods based on differential solubilization should the cytoplasmic membrane. Morphologically the be thoroughly checked by the use of methods outer membrane seems to remain intact. However, based on a physical separation as it has been chemical analyses showed that this 'Triton-insolu- found that extraction of H. influenzae membrane ble cell wall' contains only about half of the LPS with Triton X-100-Mg 2÷ results in the extraction and one third of the phospholipid of the outer of some major outer membrane protein species membrane but that all outer membrane proteins (Van Alphen, L., unpublished data). A method are still present [97]. Filip et al. [98] have studied often used for the isolation of certain outer mem- the effect of a large number of detergents on the brane fractions consists of the extraction of cells integrity of the E. coli membranes. From their data with approx. 0.2 M LiC1 (as was used for N. it can be concluded that, in the absence of mag- meningitidis, Ref. 111), Li-acetate (as was used for nesium ions, treatment with 0.5% sodium lauryl N. gonorrhoeae, Refs. 122 and 123) or 0.5 M NaC1 sarcosinate (Sarkosyl) results in solubilization of followed by 0.5 M sucrose (used for Gram-nega- the cytoplasmic membrane proteins and phos- tive marine bacteria, Refs. 124 and 125). This pholipids. With respect to the outer membrane the method is not successful if applied to Enterob- authors do not provide solid quantitative data on acteriaceae or to H. influenzae (Van Alphen, L., the fate of the various constituents. They have not unpublished data). analysed the fate of LPS whereas their data sug- gest that treatment with Sarkosyl results in an IlL Individual constituents of the outer membrane increase of the buoyant density of the outer mem- brane, suggesting loss of phospholipid. IliA. Composition of the outer membranes

11.4-4. Membrane separation based on charge dif- As determined by electron microscopy the outer ferences of vesicles membrane, like the cytoplasmic membrane, has a Using the property that outer membrane vesicles thickness of 7.5 nm [59]. It contains protein (9-12% have a larger negative charge than cytoplasmic of the cellular protein), LPS and phospholipid as membrane vesicles, they can be separated in a its major constituents. Enterobacterial Common preparative particle electropherograph [81,99[ or Antigen (ECA) is a minor component (0.2% of the by sucrose gradient electrophoresis [82]. cellular dry weight). A comparison of the data from various labora- tories on the weight ratio's of the three major 60 constituents [76,88,92,93,114,126-129] shows that phosphatidylglycerol (or cardiolipin) are found. these differ enormously, even if one takes into Excellent reviews on various aspects of phos- account the influence of the length of the sugar pholipids of Gram-negative bacteria have been chain of the LPS (see subsection IIIC). For exam- published [ 131-135]. ple, in the case of E. coli weight ratio's for outer Relatively little attention has been paid to the membrane phospholipid : LPS : protein of approx. distribution of the phospholipids over the two 1 : 1 : 1 [129] and 1 : 1:6 [126] have been reported. individual membranes. Osborn et al. [76,136] re- Another striking example is that, despite the fact ported that the two membranes of S. typhimurium that the groups of both Nikaido and Gmeiner have contain equal amounts of phospholipid and that undertaken extremely serious attempts to quantify the outer membrane is enriched in phosphatidy- the various outer membrane constituents, the lethanolamine whereas the cytoplasmic membrane former group reports that the number of LPS is enriched in the other two major species. The molecules per unit surface area is not influenced lipid composition of the two membranes of E. coli by a deep rough mutation [88], whereas the latter has extensively been studied in our laboratory group claims a 4-fold increase of the relative [137]. Consistent with the results of Osborn et al. amount of LPS in the outer membrane of the [76] we observed that the outer membrane is en- mutant compared with that of the parental strain riched in phosphatidylethanolamine, which -~ in [92,93]. As in both laboratories LPS was de- contrast to the data reported for S. typhimurium termined by assaying the content of 3-hydroxyte- [76] - often represented over 90% of the outer tradecanoic acid, differences in the methods used membrane phospholipid ]137]. Similar high are not a likely explanation in this case. In con- amounts of phosphatidylethanolamine in E. coli trast, the method used for protein assay can in- outer membranes have been found by R.M. Bell fluence the data as it has been shown by Schweizer (cited in Ref. 138). To our knowledge the only et ai. [129] that too high values are obtained for data conflicting with the above mentioned distri- the protein content of outer membrane prepara- bution are those of White et al. [81] who found tions with the method of Lowry et al. [130]. Also, considerably more phosphatidylethanolamine in data from our laboratory show that the method of the cytoplasmic than in the outer membrane. This Lowry et al. yields values that are 2-3-fold higher is probably caused by a preferential degradation than those obtained by scanning of stained poly- of the outer membrane phosphatidylethanolamine acrylamide gels [127]. In addition, it is conceivable by phospholipase and lysophospholipase activities that some of the published differences in the com- located in this membrane (see subsection IIIE-2). position of the outer membrane can partly be Consistent with this explanation is the high per- caused by factors like the bacterial species, the centage of lysophosphatidylethanolamine found by particular strain chosen, the growth conditions these authors in the outer membrane, Only trace and the growth phase. In conclusion, in order to amounts of the latter phospholipid were found in be able to perform detailed calculations on outer other laboratories [76,137]. The relative abundance membrane composition a thorough evaluation of of phosphatidylethanolamine in the outer mem- assay procedures is required to establish which brane was found in various strains under condi- data can be used. Moreover, details on experimen- tions in which the fatty acid composition was tal growth conditions should be standardized care- altered by the growth temperature, by mutation or fully in order to allow the comparison of data by supplementation of the growth medium with from different laboratories. fatty acids [137]. The reason for the enrichment of the outer membrane with phosphatidylethanola- IIIB. Phospholipid mine might be that it forms stable bilayers with LPS [139]. An alternative explanation, namely the Essentially all phospholipids of E. coli are association of major outer membrane proteins with located in the cell envelope [131]. Phosphatidy- this phospholipid, can be excluded as a similar lethanolamine is the major species whereas sub- enrichment is found in mutants lacking the three stantial amounts of phosphatidylglycerol and di- major outer membrane proteins OmpA protein, 61

OmpC protein and OmpF protein (Van Alphen, L. The major fatty acids in E. coli phospholipids and Lugtenberg, B., unpublished data). The en- are palmitic acid (C16 : 0), palmitoleic acid (C16 : 1) richment is remarkable since a fast exchange be- and cis-vaccenic acid (C18:1) or their cyclopro- tween the phospholipids of the two membranes pane derivatives [131]. A slight but significant was reported by Jones and Osborn who fused lipid enrichment of the outer membrane with saturated vesicles with intact cells of S. typhimurium [140]. fatty acids is found whereas the cytoplasmic mem- Vesicle lipids consisting of total Salmonella phos- brane is enriched in unsaturated and cyclopropane pholipid are recovered in both cytoplasmic and fatty acids [81,137]. This enrichment is indepen- outer membranes without clear preference for dent of changes in the fatty acid composition by either of the membranes. Moreover, these authors mutation or by altered growth conditions [137]. showed that, after fusion, phosphatidylserine from Detailed studies have shown that phosphatidyl- vesicles is converted to phosphatidylethanolamine ethanolamine of the outer membrane contains which can be recovered from both membranes. more saturated fatty acids [137] and less molecular Since the enzyme responsible for this conversion is species with two unsaturated fatty acids [141] than located in the cytoplasmic membrane, exchange of phosphatidylethanolamine of the cytoplasmic phospholipids must occur in the membranes in membrane. both directions [136,140]. Application of the fu- sion technique also enabled the authors to see IIIC. Lipopolysaccharide (LPS) whether lipids, which are not normal constituents of Enterobacteriaceae can be incorporated into their IHC-1. Introduction membranes. They could show that phosphati- LPS, which is characteristic for Gram-negative dylcholine was incorporated into both membranes bacteria, is an amphipathic molecule with a hydro- without a significant preference. Similar experi- phobic part, called lipid A, and a hydrophilic, ments using cholesterol oleate suggested that this often branched, sugar chain. The hydrophilic part lipid showed preferential accumulation in the cyto- consists of an oligosaccharide core which usually is plasmic membrane [ 136]. substituted by the O-antigen, the latter being a polymer consisting of repeating carbohydrate units. ~c~ 53--{ co,o --}-t o...... 1 A schematic representation of the general struc- ture of LPS is given in Fig. 3. Colonies of strains with or without the O-antigen often have a S(mooth) or R(ough) appearance respectively. Therefore one often speaks of S and R strains and also of S and R types of LPS. Among Entero- bacteriaceae the structure of the lipid A core re- gion is rather well conserved but that of the O-an- I - Re LPS tigen has been subject to extreme evolutionary I -- RO2LPS -- I RO 1 LPS - I changes. Many reviews have been written on LPS -- Rc LPS I Rb LPS q in general [142,143], on its chemical Ra LPS -- d F S LPS I [56,142,145-150], physical [144] and biological [56,142,145,150,151 ] properties and on its genetics Fig. 3. Schematic representation of the chemical structure of LPS from S. typhimuriurn. Wavy lines represent long chain [ i 52,153] and synthesis [45,154,155]. Therefore we fatty acids (C8-C16). The number of repeating O-antigen units will only briefly outline some general aspects of n is variable (see text). Wild-type cells synthesize the entire LPS. structure (S-LPS) whereas various mutant strains produce the chemotypes Ra through Re LPS. The structures synthesized by so-called deep-rough mutants are rather glycolipids than lipo- IIIC-2. Methods of isolation and purification polysaccharides. Note the large number of charged residues in The standard method for the isolation of pure the lipid A-core region of the molecule. The phosphate residues LPS has long been the phenol-water method, i.e. on the glucosamines are sometimes substituted (see Fig. 4). EA, ,treatment of dried bacteria with 45% phenol for 5 ethanolamine. min at 68°C [156,157]. The water phase obtained 62 after cooling contains nucleic acids as the main of EDTA [117], as well as by the weakening of the contaminants, which can be removed by ultra- hydrophobic interactions, by the addition of deter- centrifugation. Even better results were obtained gents [162,164] or triethylamine [142,158]. when cells as the starting material were replaced by cell envelopes [157a]. The metal ions Na ~ , 11IC-3. Chemical structure of lipopolysaccharides Mg 2+ and Ca 2+ as well as a number of amines The LPS of S. (vphimurium has been studied such as ethanolamine, putrescine, cadaverine, most extensively. Next to chemical methods the spermidine and spermine are often found in LPS availability of mutants has largely contributed to preparations in variable amounts, depending on the elucidation of LPS structures. Lipopolysac- growth and isolation conditions [158]. They have a charides of wild-type and mutant strains have profound influence on the physico-chemical prop- been classified into chemotypes based on the con- erties of LPS and can partly be removed by elec- tent of their sugar constituents (see Fig. 3). trodialysis [158]. Lipid A can be obtained by mild acid hydroly- The phenol-water method is applicable for S- sis of LPS. Lipid A of Enterobacteriaceae is a LPS and Ra and Rb LPS. Since LPS is isolated in glycolipid which contains a/3-(1 ~ 6) linked gluco- low yields when the phenol-water method is ap- samine disaccharide unit which carries a phos- plied to Rc, Rd and Re mutant bacteria (see phate residue in position 1 and a phosphate or Fig. 3) a more hydrophobic solvent was developed pyrophosphate residue in position 4' and also ap- which is adequate for the extraction of R-LPS prox. six fatty acyl chains (Refs. 169 and 165- 168, [159]. This very mild procedure involves extraction see also Fig. 4), two of which are amide-bound. of dried bacteria at room temperature with a mix- Various ligands (glucosamine, ethanolamine, ions) ture of 90% phenol, chloroform and petroleum can be attached non-covalently. Among the fatty ether (PCP method). Other methods include ex- acids /3-hydroxy fatty acids are abundant. Un- traction of bacteria with either EDTA [160,161] or saturated fatty acids are hardly present and most aqueous butanol [160,162] procedures which have of the fatty acids are relatively short (C8-C14) not been extensively tested for other bacteria than compared with the fatty acids found in the phos- E. coli. pholipids. The amide-linked/3-hydroxy fatty acids Isolated LPS forms multimeric states. It can are specific for LPS. In Enterobacteriaceae fi-hy- have the morphological appearance of droplets, droxy tetradecanoic acid is the major amide-linked long rods, unilamellar bilayer vesicles, stacked fatty acid. Like dodecanoic and tetradecanoic acid lamellae, long ribbons, flat sheets or a doughnut it can also be ester-linked. Tetradecanoic acid also [ 144,162,163]. Under certain conditions particles occurs as part of a branched fatty acid when it is and pits can be seen on fracture planes [162]. The esterified to the hydroxyl moiety of the/3-hydroxy morphological appearance depends on the length fatty acids [142,165,168,170,171]. It should be of the sugar chain [144], the degree of purity, the noted that also other types of lipid A backbones composition of the solution used for hydration, the exist for which those of Chromobacterium and presence of Ca 2 + and on whether the structure is Rhodopseudomonas eiridis and standard examples studied at temperatures above or below the phase [142,146]. transition temperature [162]. Van der Waals inter- Lipid A preparations are heterogeneous actions between the fatty acyl chains of lipid A [166,172,173]. It appears that non-equimolar moieties are the basis for the large structures. The amounts of 4-amino arabinose [174] and of the variety of macromolecular organizations are prob- esterbound phosphate residues at C4' [166,167, ably caused by ionic interactions due to salt bridges 175] as well as a disproportionate distribution of between a variety of negatively (phosphate, KDO ester-bound fatty acids [168] contribute to this and uronic sugars) and positively (glucosamine, heterogeneity. Moreover, growth conditions have 4-amino-arabinose, ethanolamine and amino been shown to influence the fatty acid pattern of sugars) charged residues. Therefore, the 'solubility' lipid A [170,596]. of LPS can be increased by both disturbing the It has long been thought that the phosphate salt bridges, by electrodialysis or by the addition residues of lipid A were involved in the formation 63

O-antigen and have a complete core whereas Re < i mutants lack all Hep residues (Fig. 3). Prehm et al. ~ o...', ~ .~.~/o~/<%.~..o\ ? 11 'I"~ [181] have analyzed the core structure of E. coli ./8 ,,8 K-12 LPS in detail (Fig. 5). The core of this strain, HO which lacks the O-antigen, is heterogenous in that 0 R R R several core structures are found with various de- R1 [N-Acyl] R2 O-AcvI ] grees of completion. The structure of the core I r I C|-0 "I ' &o ~.o region of strains of Enterobaceriareae is similar. -0 :=0 However as in quite a few other Gram-negative bacteria KDO or L-glycero-D-mannoheptose or both are lacking, they must have different core structures. Examples are Acinetobacter sp., A na£vs- tis nidulans, Bacteroides spp. [142], Haemophilus a a a b a influenzae [182] Moraxella sp., Pseudomonas spp., Spirillum serpens and Vibrio cholerae [142]. Fig. 4. Proposed structure of lipid A from S. minnesota. From Refs. 165 and 165a. This type of lipid A is probably general for The O-antigen can consist of more than 40 Enterobacteriaceae. Number of fatty acid carbon atoms: a = 14, repeating units containing 3 to 6 sugar residues. b = 12, C = 16. The exact position of amide-bound and ester- The number of repeating units can vary, even in a bound 3-acyloxytetradecanoic and 3-hydroxytetradecanoic culture of one strain [183-185], from none to more acids has not yet been detertnined. It is possible that also than 40, thereby providing the cell with the op- nonacylated 3-OH-14 : 0 as well as trace amounts of 3-O(10 : 0)- 14:0 and 3-O(12:0)-12:0 are amide-bound. The sum of ester- portunity for subtle variations in the molecular bound 3-O(14: 0)- 14 : 0 and 3-O(2-OH- 14 : 0)- 14 : 0 equals ap- make up at different sites at its surface. The struc- prox. 1 mol/mol lipid A. Since three hydroxyl groups are ture of the subunit of the O-antigen shows extreme available at the lipid A backbone and since up to now only two diversity, even within a single genus like E. coli or Ooacyl groups have been detected it is possible that one gluco- S. typhimurium. This property is used in O-sero- samine hydroxyl group is not substituted (R ~ = H). The hy- droxyl group in position C-3' represents the attachment site of typing, an immunological method used to identify the polysaccharide portion. Residues 4-amino-4-deoxy-L- sub-strains of one species in great detail [56]. As arabinose and phosphorylethanolamine are not present in molar thus could be expected, a large diversity of sugars amounts as indicated by dotted bonds. has been found in the repeating units of the O-an- tigen. These include neutral sugars, amino sugars and uronic acids. Moreover sugars can be sub- of cross-links between monomers, thus forming stituted with O-acetyl groups, phosphate, amino trimers [176]. However such cross-links were not acids or even ethanolamine triphosphate. The detected with 31p-NMR by MOhlradt et al. [165]. We therefore assume that the LPS molecule is monomeric. The LPS core of Enterobacteriaceae contains the unique sugars 3-deoxy-D-manno-octulosonic C)4 acid (KDO) and L-glycero-D-mannoheptose, both 0 Cl4 of which are practically LPS-specific. In addition, O~ ' IC Z,~ it contains a number of more common sugars like glucose, galactose and N-acetyl-D-glucosamine G,cNAc-~,c~G,~O,c~H~0_,.. Kt~Cl o. f16 -,:,- f ( e ~'~- ,. [ 142,147,150]. Numerous mutants with defects in Gal Hep Rha EA the structure of LPS have been isolated, usually by ~te'p7 selection for resistance towards a certain bacteriophage or towards the antibiotic polymyxin Fig. 5. Tentative structure of E. coli K-12 LPS composed from [177,178]. Mutants completely missing KDO can data of Refs. 142, 165, 171 and 181. Ions and other non-cova- lently attached constituents are not included. Amide and ester be isolated as conditionally lethal [172,179,180]. bonds are indicated by -N- and -O- respectively. The exact Ra mutants are defective in the biosynthesis of location of the fatty acid residues is not known. 64

O-antigen therefore can contribute considerably to unit [197], which therefore also is supposed to be the net surface charge of the bacterial cell. The required for assembly or maintenance of the nor- O-antigen is not present in all strains. For instance mal structural organization of the outer membrane such a structure is lacking in the E. coli laboratory [197]. These affinity sites for divalent cations on strains K-12 [181], B [186] and C [187] and also in the LPS molecule could play an important role in some strains with for instance serotype O14 [56]. the assembly or maintenance of the molecular Also, within a certain strain the presence of the organization of the outer membrane by bringing O-antigen can depend on the growth condition about LPS-protein and/or LPS-LPS interactions. [188] whereas the presence of certain prophages in the chromosome can drastically alter the structure IIID. Enterobacterial common antigen (ECA) and composition of the O-antigen, a phenomenon known as antigenic conversion [189,190]. Also the The enterobacterial common antigen or Kunin presence of plasmids [191,192] can alter the com- antigen is shared by most Enterobacteriaceae [201] position of the LPS. The structures of only a few and is an often forgotten constituent of their outer O-antigens have been established [56]. membranes [202]. ECA represents a polymer of N-acetyl-D-glucosamine and D-mannosaminuronic IIIC-4. Effects of polymyxin, EDTA and divalent acid, partly esterified by palmitic acid [203]. Its cations chemistry and biology have been extensively re- Polymyxin B is a polycationic amphipathic anti- viewed [201,204]. In addition to the free form a biotic which is bacteriacidal to most Gram-nega- few strains contain ECA also in the immunogenic tive bacteria and binds to their membranes form in which it is associated with lipopolysac- [193,194]. The lethal action of polymyxin is attri- charide by an interaction to the core-lipid A part, buted to the cytoplasmic membrane damage as a similar to the way the O-antigen is linked. result of the interaction of polymyxin with the IIIE. Proteins acidic phospholipids [193]. Also the outer mem- brane binds polymyxin [194] and subsequently lllE-1. Introductory remarks looses its permeability barrier function. A clear As the outer membrane is very poor in en- effect of the antibiotic on the structure of the outer zymatic activities [76], the identification of the membrane has been shown [ 195,196]. Initial bind- protein component is mainly dependent on separa- ing to the outer membrane most likely is an inter- tion of proteins in bands using SDS-polyacryla- action with LPS [142,177,197], presumably with its mide gel electrophoresis. Compared with the pre- anionic KDO or phosphate groups [197]. sent systems which give a high resolution [205- EDTA removes about half the LPS of the cell 209] the systems used earlier were rather poor. [85], presumably by complexing divalent cations Also the introduction of slab gels [91,210] has which are involved in LPS-LPS interactions. In largely contributed to the resolution of the outer our hands the procedure does not result in re- membrane proteins. Two-dimensional gels, which moval of significant amounts of outer membrane have given new opportunities for studying com- protein [198]. The presence of Tris ions enhances plex mixtures of soluble proteins [211] have been the removal of LPS from the cell by EDTA [199]. applied to outer membranes in only a few cases Interestingly, a direct interaction between the Tris [212-214] and turned out to give rise to a large ion and the LPS molecule has been reported [197]. number of artefacts due to strong interactions Divalent cations have a high affinity for LPS between outer membrane constituents (see Ref. [85,197,198,200]. Schindler and Osborn [197] have 213). The molecular weight of a protein can be shown that there are two binding sites for Mg 2+ estimated by SDS-polyacrylamide gel electro- and Ca 2+ . The first one, of relatively low affinity, phoresis. As a proven or supposed single amino was attributed to pyrophosphoryl and/or phos- acid substitution can result in a drastically altered phodiester groups of the KDO - lipid A region. electrophoretic mobility [215-219], the apparent The second site, of higher affinity, must probably molecular weight value obtained should be inter- be attributed to the branched KDO trisaccharide preted with care. TABLE I

NOMENCLATURE OF ‘MAJOR OUTER MEMBRANE PROTEINS OF E. COLI K-12

Uniform Bragg Foulds Henning Inouye Lugtenberg Mizushima Rosenbusch Schaitman nomenclature ~2241 [225,226] [227,228] ]2291 [205,218] 12071 ~2301 [230a,23 I] [4,218,223)

? - 011 3b OmpF protein Ia 09 Matrix protein la OmpC protein Ib 08 lb OmpA protein B ToIG II’ 7 010 3a PhoE protein E Ic NmpAB protein ’ a This designation is extremely confusing as it has been shown later on that neither of the nmpA and nmpB is the structural for this protein [218]. 66

Initially, outer membrane protein patterns ob- nism is operative for major proteins and secondly, tained in various laboratories differed drastically, it provides us with a practical standard for cell partly because different solubilization tempera- surface area, which as such is tedious to measure. tures were used during the preparation of the It means for example that our previous data on the sample, and partly because different gel systems amounts of individual proteins relative to the total were applied. Since some interactions between amount of outer membrane protein [95] can be outer membrane constituents are extremely strong, extended to the amount of the individual proteins temperatures above 70°C are required for com- per unit of cell surface (see later on). plete solubilization of the outer membrane pro- The use of different SDS-polyacrylamide gel teins of E. coli and S. typhimurium and the stan- electrophoresis systems in different laboratories dard procedure used in these cases now consists of made a comparison of the early data difficult, boiling the sample in 2% SDS for a few minutes. resulting in several different nomenclature systems In the cases of H. influenzae (Van Alphen, L., (Table I). The use of improved gel systems, the unpublished data) and Pseudomonas aeruginosa availability of mutants lacking one or more pro- [220] complete unfolding of all outer membrane teins and the availability of pure preparations of protein chains even requires boiling for 10 and 30 several proteins are the main factors responsible rain, respectively. Incomplete unfolding of a poly- for resolution of this confusion. In the case of E. peptide chain can result in binding of insufficient coli it was finally agreed upon by most groups to amounts of SDS. (Usually as much as 1.4 g of use a uniform nomenclature system in which the SDS is bound per g protein!). Therefore incom- protein was named after its structural gene [4] plete solubilization can result in an altered electro- (Table I). Thus, a protein previously known as If*, phoretic mobility or even in the absence of a d, 3a, B, TolG protein or O10 is now called OmpA protein from the running gel, a phenomenon which protein as gene ompA is the structural gene for this is often accompanied by the accumulation of pro- protein. (The genetic abbreviation omp stands for tein on top of, or in, the stacking gel. The altered outer membrane protein). Although a generally electrophoretic mobility of a number of incom- accepted nomenclature system certainly is an im- pletely unfolded proteins has resulted in the term provement, its coupling to the genetic code is a heat-modifiable proteins which can often be found severe restriction. For example, as the structural in the earlier literature. gene of the protein known as a, 3b or Oll (see The relatively poor resolution of the earlier gel Table I) is not known with certainty, the protein systems has introduced the notion that one protein cannot be properly named despite the fact that its was supposed to account for 70% of the total outer purification has been described alreay in 1974 membrane protein. Subsequently this 'major outer [230a]. Also, the system cannot easily be used for membrane protein'[86] of E. coil K-12 was resolved other strains than E. coli K-12. For example, cer- into two [221,222] and later on into four [205,207] tain E. coli strains have as many as five proteins proteins (Table I), each of which is still designated which are immunologically related with OmpC as a 'major outer membrane protein' although the protein and OmpF protein of E. coli K-12. It relative abundance of one or two of these proteins would probably take years to find out whether one usually is very moderate [205,232]. Moreover, the of the proteins of the wild strain is indeed coded term is misleading as the lipoprotein, which is by by the presumed ompF gene of this strain, and if far the most abundant protein of the cell, does not so, which one. belong to the 'major outer membrane proteins'! Several outer membrane proteins are designated Finally, the term 'major' is often relative as in as 'peptidoglycan associated' proteins. This term is several cases the growth conditions influence the correct as long as it is used in the operational amount of a given protein. The observation that sense, namely as proteins which under certain the amount of major outer membrane protein per conditions (e.g. in 2% SDS at 60°C) are the only unit of outer membrane surface area is constant proteins which remain strongly but non-covalently under various growth conditions [233] is im- bound to the peptidoglycan fraction. The question portant. Firstly, it shows that a regulation mecha- of whether this is also the case in vivo will be 67

handled in subsection IVF-5. teins. For example, mutations causing a heptose- A comparison of cytoplasmic and outer mem- less LPS result in strongly decreased amounts of brane preparations by SDS-polyacrylamide gel OmpF protein [94], PhoE protein [236], protein III electrophoresis shows relatively few, but often [93] and Lamb protein [237] in the outer mem- heavy, bands in the case of the outer membrane. It brane. Using an ompF-lacZ operon fusion strain it should be stressed that this result not necessarily could be shown that in the case of OmpF protein means that there are only a few outer membrane poor expression was not the result of poor tran- proteins as some proteins might be present in scription (Tommassen, J., Overduin, P. and amounts below the level of detection, e.g. phos- Lugtenberg, B., unpublished data). Therefore it is pholipase A1 and the BtuB protein can hardly or likely that the defects are caused by a poor assem- not at all be detected as bands in crude outer bly or translocation of the proteins. membranes, whereas other proteins may be pre- Another indication for a structurally common sent in the outer membrane in detectible amounts site on various exported proteins comes from work only under certain growth conditions (e.g. induci- with mutants containing a defective perA gene. ble proteins from cells grown under conditions These cells have decreased amounts of alkaline which repress their synthesis, see subsection IIIE, phosphatase, of several other periplasmic proteins 5, 6 and 7). [238], and of the outer membrane proteins OmpF Genetics has played an important role in the protein [238], LamB protein [239], PhoE protein fast increase of knowledge on E. coli outer mem- (Tommassen, J., Beusmans, J. and Lugtenberg, B., brane proteins. As several proteins are used as a unpublished data), protein a, Cir protein, FepA receptor by bacteriophages, mutants resistant to protein and 83 kDa protein [240]. such a phage often lack the receptor protein. Com- The following sections will focus on the struct- parison of properties of mutant and parent has ural and functional characteristics of outer mem- often indicated the function(s) of the protein (see brane proteins. All predominant outer membrane also subsection IVA for interpretation). Mutant proteins have been purified and in many cases cells have been used as the starting material for the biological activity depends on the presence of LPS. purification of proteins as mutants can be con- Outer membrane proteins are usually rich in fl- structed such that an otherwise persistent impurity structure [241]. It appears that certain proteins is lacking or such that they overproduce the re- play a role in the stabilization of the structure of quired protein. Mutants are indispensible for stud- the outer membrane and in anchoring this mem- ies on the regulation of the synthesis of specific brane to the peptidoglycan layer. Other proteins proteins. A recent genetic development is the con- facilitate the permeation of nutrients through the struction of operon and gene fusion strains which outer membrane. Some proteins seem to be pro- can be used as powerful tools for studies on the duced constitutively whereas the synthesis of others regulation of [234,235] and on the is dependent on the growth conditions. subcellular localization of proteins [11,14,16], re- spectively. The availability of mutants is usually IllE-2. Enzymes in the outer membrane also useful for molecular cloning of the corre- The outer membrane is poor in enzymatic activ- sponding wild-type gene. Cloned DNA can be ities [76,242,243]. , M r 28000 used for detailed studies on regulation and expres- [244,245], is the first enzyme detected in the outer sion of the gene . Moreover, cloning not membrane [76,242]. Later on, lysophospholipase, only enables the investigator to determine the lysophosphatidic and UDP-glu- nucleotide sequence of a structural gene and thus cose were found in this membrane [82]. to predict the primary structure of the protein but The first example of an enzyme activity which is it also provides the possibility to determine the clearly present in both membranes comes from signal sequence and to study the regulatory re- Vos et al. [246] who reported that the specific gions and other aspects of the operon structure. activity of monoacylglycerophosphoethanolamine A single mutation sometimes results in de- acylase, which does not require ATP or coenzyme creased amounts of several outer membrane pro- A for activity, is the same in cytoplasmic and outer 68 membrane preparations of E. co#. This location in accompanied by an altered cytoplasmic or outer both membranes makes sense as reacylation of membrane protein pattern [258]. Currently the lysophospholipids formed by the action of endoge- precise role of leader peptidase in the assembly of nous or exogenous phospholipase A provides the M13 precoat is elegantly being studied in vitro organism with the potential of biochemically inex- [259,260]. pensive repair and modification of the envelope Experiments of Wolf-Watz and Normark [261] phospholipids. Moreover, major phospholipids suggest that a peptidoglycan hydrolytic enzyme, hydrolyzed in the outer membrane can be N-acetylmuramyl-L-alanine amidase, is loosely as- resynthesized in the same location, without need sociated with the outer membrane. for transport of the products of hydrolysis to the It has recently been reported that Serratia lipid biosynthetic apparatus in the cytoplasmic marcescens, a Gram-negative bacterium which ex- membrane [246]. cretes , protease and activities The outer membrane was found to contain pro- [120,262], can contain substantial quantities of teolytic activities able to convert the precursor of these enzymes in its outer membrane, presumably to the mature form [247], to an intermediate location before excretion occurs modify the ferric enterobactin receptor [248], to [263]. Even more striking is the observation that in hydrolyze casein [249], to cleave bacteriophage the same organism about 80% of the fl-lactamase M13 precoat protein to coat protein [250], to activity is found in the outer membrane. Finally, solubilize nitrate reductase [251], to cleave outer membranes of Neisseria meningitidis contain Ia [252] and to activate serum plasminogen to tetramethylphenylenediamine oxidase activity active protease plasmin [253]. Presently it is not [264]. known how many enzymes are responsible for these activities but two of them have recently been lllE-3. Lipoproteins purified. Braun and co-workers [265] were the first inves- The casein-hydrolyzing enzyme, designated as tigators who purified an outer membrane protein. protease IV, has been purified by Regnier [254,255 This lipoprotein from E. coli (M r 7200) contains ], It is an endoproteolytic enzyme with a M r of 58 amino acid residues, and is covalently bound to 23 500 which is mainly localized in the outer mem- the carboxylgroup of every tenth to twelfth di- brane. It can be solubilized by deoxycholate or aminopimelic acid residue of the peptidoglycan SDS, it is resistant to thermal denaturation and is layer through the e-NH 2 group of its C-terminal inhibited by EDTA, by various protease inhibitors lysine residue (for a review see Ref. 266). This and by inhibitors of processing enzymes [254,255]. covalently bound form of the lipoprotein is pre- A localization on the inner side of the outer mem- sent in approx. 2.4-105 copies per cell [266]. It brane has been suggested [254]. was subsequently discovered that twice as many The second enzyme which has been purified copies of exactly the same lipoprotein molecule converts M 13 precoat protein to coat protein [256]. exist in the free form i.e. not covalently attached This processing enzyme has been designated as to the peptidoglycan [266a]. The lipoprotein is by leader peptidase or signal peptidase. It has an far the most abundant protein of the cell. Both apparent molecular weight of 39 000 and does not forms of the lipoprotein have been purified and require cofactors for activity [256]. It is the second sequenced [267,268] and the free form has been enzyme which has been found in equal abundance crystallized [268a,268b]. The amino acids histidine, in the cytoplasmic and outer membranes of E. coli tryptophan, glycine, proline and phenylalanine are [257], The enzyme also cleaves precursor forms of lacking. As the amino acid sequence of the lipo- two periplasmic amino acid binding proteins and protein is highly repetitive it was speculated that of the Lamb protein [257]. A 4-6-fold overpro- the lipoprotein is evolved from a 15 amino acids duction of leader peptidase was observed in a long peptide by gene multiplication and subse- strain bearing plasmid pLC7-47, presumably due quent mutations [266]. The N-terminal cysteine to the presence of several copies of the structural residue is substituted both with a diglyceride gene on the plasmid. This overproduction was not moiety in a thioether linkage as well as with an amide-linked fatty acid. 69

The isolation of a lpp mutant lacking the struct- lipoprotein occurred [276]. A similar gene dosage ural gene for both forms of the lipoprotein located effect for the free form, but not for the bound at rain 36.3, shows that the lipoprotein is not form was observed in E. coli cells carrying an F' esential for survival of the cell. However, a lipo- factor containing the Ipp gene [277]. protein deletion mutant has severe defects like With respect to the 3-dimensional structure it increased production of outer membrane vesicles, has been determined that the lipoprotein, in con- increased sensitivity to EDTA and leakage of peri- trast to many other outer membrane proteins, is plasmic enzymes, suggesting a role in the stabili- very rich in a-helix [278,279] which is consistent zation of the outer membrane [269,270]. When in with Inouye's model in which all hydrophobic addition to the lipoprotein also the OmpA protein amino acid residues are regularly arranged in an is missing, cells are unable to grow in the rod form alternating 3 to 4 pattern of repeating hydro- and require high concentrations of Mg 2÷ or Ca 2÷ phobic residues. As 3.6 residues make up one for growth [271]. Moreover abundant blebbing regular right-handed a-helical turn, all the hydro- was observed in the double mutant and the pepti- phobic residues can be alligned as two series on doglycan layer was no longer connected with the one face of the helical rod [266,280]. outer membrane [271] suggesting a role of the The structure of the lipoprotein has been ex- proteins in the determination or maintenance of tremely well conserved in Enterobacteriaceae the rod shape, in stabilization of the outer mem- [274,275,281-284]. Lipoproteins have also been brane structure, and in anchoring the outer mem- found in Aeromonas salmonicida (Evenberg, D., brane to the peptidoglycan. Additional evidence Overbeeke, N. and Lugtenberg, B., unpublished for the latter function comes from recent experi- data), Pseudomonas aeruginosa [285] and in ments of Wensink and Witholt [272] who showed Rhodopseudomonas spheroides [286]. The presence that outer membrane vesicles released by growing of lipoprotein has not been established in other E. coli cells contain only a small amount of free bacteria. For example, it could not be identified in lipoprotein, hardly any bound lipoprotein and re- N. gonorrhoeae, grown at pH = 7, which might duced amounts of OmpA protein. The vesicles also explain the strong blebbing of the outer membrane contained reduced amounts of protein V, a protein which seems only loosely attached to the pepti- assumed to be identical to a newly discovered doglycan layer [287]. lipoprotein [272]. Another class of lipoproteins, peptidoglycan-as- The lpp genes of E. coli K-12 [273], Serratia sociated lipoproteins or PAL proteins, which occur marcescens [274] and Erwinia amylovora [275] have closely but non-covalently associated with pepti- been cloned in lambda phage vectors and their doglycan, has recently been found in various nucleotide sequences have been determined. Al- Gram-negative bacteria like P. mirabilis [288], Ps. though E. coli is only distantly related to the other aeruginosa [288,289] and E. coli [290]. They are not two species, comparison of these sequences sug- immunologically cross-reactive with Braun's lipo- gests that the structure of the lipoprotein has been protein [291]. PAL of P. mirabilis has an M r of highly conserved [275]. Attempts to clone the E. 18 000 and its N-terminus, including the lipid part, coli lpp gene into the high copy number vector in common with Braun's lipoprotein [288]. Very pBR322 have not been successful, presumably due recently several new lipoproteins, which are im- to lethal overproduction of the lipoprotein [276]. munologically different from both Braun's lipo- In contrast, the lpp gene of S. marcescens could be protein and PAL, have been discovered in E. coli cloned into this vector and the resulting plasmid [290]. From these new lipoproteins four were restored the wild-type phenotype of an E. coli lpp located in the outer membrane and two in the mutant. This Serratia lipoprotein was exclusively cytoplasmic membrane, whereas the localization of found in the outer membrane. The effect of multi- one species is uncertain. Thus the total number of ple copies of the structural gene resulted in a biochemically different lipoprotein species in E. 3-fold overproduction of the free form of the coli is at least nine [290]. Interestingly, Inouye and lipoprotein compared with E. coli lpp + cells, co-workers [292] come to a similar conclusion on whereas no overproduction of the bound form the basis of DNA hybridization experiments. 70

llIE-4. OmpA protein lack of OmpA protein is compensated for by in- OmpA protein is present in about 105 copies creased amounts of pore proteins [293,306] and per cell. It is heat-modifiable in that its apparent phospholipid [127] and possibly also of LPS [127]. molecular weight on polyacrylamide gels is higher The mutants do not adsorb the mentioned phages in the heat-modified form (35000) than in the and are defective in F-pilus mediated conjugation non-denatured form (28000) [221,224,241,294]. In [6,305,307]. The suggested receptor or receptor-like two-dimensional gels the protein appears in at activities could be mimicked with purified OmpA least 12 spots, most of which are caused by artefacts protein provided that it was complexed with LPS [2131. [293,308]. The role of OmpA protein in conjuga- A procedure has been described for the purifi- tion was confirmed by independent means by cation of four major proteins from one batch of Havekes and Hoekstra who selected mutants de- cells of E. coli strain B/r [294]. As this procedure fective in the acceptor function in F-pilus media- applied to E. coli K-12 resulted in large losses of ted conjugation and subsequently showed that the OmpA protein in our hands, we have modified the mutation was localized at the ompA locus [307]. Its method in order to purify the E. coli K-12 protein function in conjugation most likely is the stabiliza- [293]. The OmpA protein has a high B-structure tion of mating aggregates [6]. Together with content [241]. Purified OmpA protein is not partic- Braun's lipoprotein OmpA protein is somehow ularly hydrophobic [293,295,296]. Although it has involved in maintaining both the structural integr- been reported previously that OmpA protein con- ity of the outer membrane as well as the rod shape tains glucosamine [297], it was claimed later on of the cell [271]. that non-protein substituents were not detected The structural gene ompA, located at rain 21.5 [295,296]. The complete amino acid sequence of [216] of the E. coil K-12 chromosome, has been OmpA protein of E. coli K-12 has been de- cloned [309,310]. Expression of a few copies of the termined [296]. It consists of 325 residues resulting gene results in a 2-fold overproduction of the in a M r of 35 159 [296]. Computer analyses did protein [309] whereas a further increase in over- neither reveal internal homology nor homology production is lethal for the cell [309,311]. The with OmpF protein [296]. The high electrophoretic overproduction of OmpA protein as a result of mobility of the non-heat modified form is ascribed cloning causes decreased levels of OmpF protein, to the high content of B-structure and excessive OmpC protein, LamB protein and lipoprotein in binding of SDS in the absence of heating in SDS the cell envelope [309]. It is interesting to note that [296,298]. Of the lysine residues of OmpA protein by genetic manipulation of the cloned material a 6-24% are present as allysine (a-amino adipic acid 30 000 dalton fragment, which lacks the 96 [310] or semialdehyde) as a result of an enzymatic [299] 98 [311,312] -COOH terminal residues, is incorpo- post-transcriptional modification process [300]. It rated in the outer membrane. As the presence of has been suggested that a possible function could the fragment makes the cells sensitive to the OmpA be to crosslink the protein to diaminopimelic acid protein specific phages it can be concluded that residues of the peptidoglycan layer since some the carboxyterminal third of the protein is not OmpA protein is covalently linked to this layer in required for incorporation in the outer membrane stationary phase cells [300]. [310,3111. A protein cross-reactive with OmpA protein Although purified OmpA protein is completely was detected in all strains of E. coli [301,302], of degraded by proteolytic enzymes, trypsin [313] other Enterobacteriaceae [302,304] and also in and pronase [312] leave N-terminal parts of M r Aeromonas salmonicida (Evenberg, D. and Lugten- 24000 and 19000, respectively, intact as long as berg, B., unpublished data) and Haemophilus in- the protein is embedded in the membrane. This fluenzae (Van Alphen, L. et al., unpublished data). large fragment is still able to act as a phage OmpA protein deficient mutants of E. coli can receptor whereas it most likely also still functions be obtained by selection for resistance to phages in F-pilus mediated conjugation [312,313]. Results K3 [305] or TuII* [306] or by isolating mutants with proteolytic enzymes [313] and with shortened tolerant to bacteriocin JF246 (colicin L) [225]. The gene fragments [312] indicate that the OmpA pro- 71 tein consists of two domains. The N-terminal tein morphologically appears to contain a hexago- moiety from residues 1 to 180 represents the mem- nal lattice structure, the protein was designated as brane domain whereas the remaining 55 residues 'matrix protein' [230]. Later on the latter term has are proposed to be located in the periplasmic often been used for similar proteins or even for space [312]. mixtures of proteins. However, as the experiments Several investigators have studied the expres- leading to the designation 'matrix protein' have sion of ompA genes in E. coli and other bacteria. only been described in the case mentioned above E. coli K-12 strains harbouring episome F'106 [230], the use of the term 'matrix protein' for these containing the ompA gene, do not produce more of other proteins should be discouraged. We prefer this protein. Transfer of this episome to S. typhi- the term' peptidoglycan-associated protein' as long murium and P. mirabilis resulted in incorporation as it is used in the operational sense (see subsec- of the E. coli OmpA protein in the outer mem- tion IIIE-1). branes of the recipients [314]. Similarly, cloned E. coli strain K-12 contains two peptidoglycan- ornpA genes of Shigella dysenteriae, Enterobacter associated proteins [95,228,318], known as OmpC aerogenes and Serratia marcescens are fully ex- protein and OmpF protein. They are immunologi- pressed in the outer membrane of E. coli K-12 [315 cally related with each other as well as with PhoE ]. These results are hard to reconcile with observa- protein [319], an inducible pore protein in this tions from Schaitman's laboratory which indicate strain [320]. A survey of 45 hospital isolates of E. that ompA genes from a certain E. coli strain are coli has shown that the number of peptidoglycan- not expressed in other E. coli strains unless the associated proteins per strain in the M r range cloned ompA gene has been mutagenized in such a between 30 000 and 42 000 can vary between one way that the affinity of the OmpA mutant protein and four, whereas the electrophoretic pattern of for the LPS of the recipient strain has increased these proteins was OK serotype-specific [301a,b]. [304]. The denatured form of each of these peptidogly- By comparing nucleotide sequences of cloned can-associated proteins reacted with at least one of ompA genes of various bacteria, Henning, Cole the antisera raised against highly purified de- and their co-workers are trying to correlate dif- naturated OmpC protein, OmpF protein or PhoE ferences in structure with differences in biological protein. As none of the other E. coli cell envelope activities [312,316]. proteins reacted, these results suggest that all these pore proteins are derived from a common ancestral IIIE-5. The family of peptidoglycan-associated gen- gene and they show that the structures of these eral diffusion pore proteins genes have been conserved very well during evolu- I11E-5a. Introduction. When whole cells or cell tion [301]. Peptidoglycan-associated proteins of envelopes of E. coli strain B E are incubated at Enterobacteriaceae are very common in nature. 60°C in 2% SDS and subsequently centrifuged, the Three of them have been detected in S. typhimurium pellet consists of peptidoglycan with the lipopro- [90,321] and a large survey has shown that they are tein covalently bound to it and another protein present in all Enterobacteriaceae tested [322-324] with a M r of 36 500 noncovalently bound to it. The and in Ps. aeruginosa [325] and that also these latter protein can be removed from the pepti- proteins are cross-reactive with the E. coli proteins doglycan-lipoprotein complex by incubation in 2% [301-303,326]. The family of peptidoglycan-associ- SDS at a temperature of 70°C or higher [230] or ated proteins is even larger if one takes into account by incubation at 37°C in SDS containing 0.5 M that not all proteins are constitutively present but NaC1 [317]. Heating results in changing of the some are induced under certain growth conditions tertiary structure from B-sheet to a-helix thereby only [320,327] or are coded for by a (pro)phage irreversibly denaturating the protein. Release of [328,329] or by a plasmid [330-332]. It should be the protein by high salt leaves the native confor- noted that the temperature of 60°C is arbitrary. If mation intact. As the peptidoglycan-lipoprotein lower temperatures are used additional proteins complex of E. coli strain B E with, but not without, are found to be associated with peptidoglycan in the non-covalently 'peptidoglycan-associated' pro- E. coli K-12 [333] and Proteus vulgaris [322]. 72

TABLE il CHARACTERISTICS OF SOME E. COL1 AND S. TYPHIMURIUM PEPT1DOGLYCAN-ASSOCIATEDGENERAL DIFFU- SIONS PORE PROTEINS a

Protein species OmpF protein (E. coil strains K-12 and B) OmpC protein (E. coli K-12) Mr 37 205 [376] 36000 [205] Number of copies/cell Up to 105 [230] Up to l0 s (Part of) receptor for Tula [381], T2 [382], TP1 [383], K20 b, TP2 [384], Tulb [381,385], T4 c [386,3871, phage/bacteriocin TP5 [384], cola [384a] Mel [3881, PA-2 [329], 434 [382], SS1 [389], TP2, TP5, TP6 [384] Structural gene ompF, min 20.7 [392-396] ompC, min 47.1 [217,231,388,396] Purification (Refs.) 230, 322, 396 322, 396 lsoelectric point (pH) 5.9-6.2 [230] n.d. 0 Refs. to amino acid compo- sition and sequence 227, 230, 374, 376, 396 227, 374, 396 Oligomeric form Trimer [356,397] Trimer [397,398] Pore activity (Refs.) 339, 341,342, 343, 344, 358 342, 343, 352, 358 Pore diameter (nm) 1.4 [358] 1.3 [3581 Further characteristics Gene ompF has been cloned [395,402]; it hybri- For effect of osmolarity see under dizes with the phoE gene [395]; synthesis of OmpF protein. Smaller effective diam- OmpF protein is repressed by high osmolarity eter than OmpC pore [342,343,409] [403,404]; synthesis positively controlled by cAMP? [405]; 70% amino acid sequence homology with phoE protein [380]

a Association with peptidoglycan is meant in the operational sense only (see subsections lllE-5a and IVF-5). The group of proteins listed in this table has in common an antigenic relation with OmpF protein and/or OmpC protein of E. coli K-12 [319]. h Manning, P. and Reeves, P,, personal communication.

llIE-5b. Function of general pore proteins. E. bility for nutrients and other solutes with a M r up coli has many high affinity transport systems in its to approx. 600 [335-337]. Although most func- cytoplasmic membrane with K m values around tions of bacterial constituents have been unmasked 1 /~M for most solutes [334]. If such a transport with the help of mutants, the function of pepti- system also has a reasonably high Vma X value the doglycan-associated proteins was discovered by outer membrane will form a serious transport bar- purification of the outer membrane component rier at low solute concentrations unless it contains responsible for the generation of aqueous pores in an extremely high number of channels. Nikaido phospholipid-LPS liposomes through which and Nakae and their co-workers have developed galactose, glucosamine, glucose-l-phosphate, the concept of hydrophilic or water-filled pores in leucine, glutamic acid, lysine, tryptophan, uridine, the outer membranes of Enterobacteriaceae in order UMP, GDP and poly(ethylene glycol) (Mr approx. to explain both the outer membrane's impermea- 600) could pass but which were impermeable for bility for bile salts and its extremely good permea- poly(ethylene glycol) (M r 1540). In the case of S. 73

TABLE 11 (continued) CHARACTERISTICS OF SOME E. COLI AND S. TYPHIMURIUM PEPTIDOGLYCAN-ASSOCIATED GENERAL DIFFU- SIONS PORE PROTEINS a

PhoE protein (E. coli K-12) OmpF protein [4] OmpC protein [4] OmpD protein [4] (35 kDa protein) (36 kDa protein) (34 kDa protein) ( S. typhirnurium ) ( S. typhimurium ) ( S. typhimurium ) 36782 [380] 39300 [321] 39800 [321] 38000 [321] Up to 105 Up to 105 Up to 105 Up to 105

TC23, TC45 [226,390] PH42, PH105, PH221 [391] PH31, PH42, PH51 [391] phoE, min 5.9 [218] ompF, min 21 [4] ompC, min 46 [41 ompD, min 28 [4] 226,236 321 321 321 n.d. 4.77 [321] 4.78 [3211 4.85 [3211

227,236,374,380,396 321 321 321 n.d. Trimer [399] Trimer [357,399] Trimer [357,399] 236,343,345,358,400 340,401 340,401 340,401 1.2 [358] 1.4 [4011 1.4 [4011 1.4 [4011 PhoE protein synthesis is Synthesis repressed by salt derepressed by Pi- [34] limitation [320]; PhoE protein pore is extremely efficient for Pi and organic P [345] due to a recognition site [400]; 70% amino acid sequence homology with OmpF protein [380]; gene phoE has been cloned [406]; over- production of PhoE protein is lethal [406]; phoE gene hybridizes with ompF gene [395]

c Whereas E. coli B LPS alone is sufficient for phage inactivation [379] a complex of LPS and OmpC protein is required in the case of E. coli K-12. d n.d., not determined.

typhimurium the active fraction contained the three [340-344] and enabled one to measure rates of peptidoglycan-associated proteins 34, 35 and 36 penetration which at that time could not be mea- kDa whereas fractions containing the 33 kDa pro- sured with the liposome system. Such in vivo stud- tein (comparable with the OmpA protein of E. ies using carefully designed mutants provided the coli) or the free form of the lipoprotein were first indication for functional differences between inactive [338]. Subsequently Nakae could show the E. coli K-12 pore proteins OmpC protein, that the only peptidoglycan-associated protein of OmpF protein and PhoE protein [342,343,345,346]. E. coli strain B had the same general diffusion In fact, pore protein deficient mutants have an pore activity. He therefore proposed the term increased K m and an unaltered Vma ~ for the up- '' for such proteins [339]. Subsequent in vivo take of solutes [9,341,345-348]. A large improve- work using mutants lacking one or more pepti- ment was the development of rate measurements doglycan-associated proteins confirmed the con- in vitro by the use of black lipid films [349,350], by cept of the pore function for these proteins application [351,352] of the liposome swelling 74

method [353,354] and by the incorporation of a lactam antibiotics whereas many other Gram- specific solute-converting enzyme into liposomes negative bacteria are sensitive. In the latter case it [355]. The biologically active form of the porin is assumed (see section IV) that the antibiotics turned out to be a trimer [356,357]. mainly use the hydrophobic pathway, i.e. they The most important conclusions from the work diffuse through the phospholipid bilayer [9,361]. on the functioning of pores can be summarized as Nikaido [361] has proposed that the hydrophobic follows. pathway does not exist in the outer membrane of (i) The simplest interpretation of a pore is a Enterobacteriaceae and that in this case fi-lactams nonspecific molecular-sieving channel through are dependent on pores for permeation [361]. With which hydrophilic solutes diffuse. The diffusion respect to chemotherapeutic use of fl-lactam anti- rate is determined by the difference in concentra- biotics against Enterobacteriaceae it is interesting tion at the two sides of the membrane. The tem- to note that the permeability coefficient of cepha- perature dependence of the rate of penetration is cetrile and cephaloridine is only 3 4-fold lower low [337,338]. than that of lactose. Thus, Nikaido concluded that (ii) Solutes which are too bulky cannot diffuse the goal of making very hydrophilic, and therefore through a pore. In Enterobacteriaceae the size limit rapidly pore-penetrating, /3-1actams has already for oligosaccharides is 600-700 daltons which cor- been achieved for these compounds [10]. responds with a pore diameter of 1 nm [9]. From (iv) The incorporation of pore proteins into conductivity measurements through black lipid planar lipid bilayer membranes leads to an in- films, and assuming that the pore is a cylinder of crease of the membrane conductance of many 7.5 nm length, the proposed thickness of the outer orders of magnitude [349,350]. At lower protein membrane, pore diameters of 0.9-1.4 nm were concentrations the conductance increases in a calculated [9,350,358]. stepwise fashion. These findings are consistent with (iii) The diffusion rate of solutes through pores the assumption that the protein forms large aque- is influenced by factors like size, charge and ous channels in the membrane. The formation of hydrophobicity, resulting in large differences in channels is induced irreversibly by voltage. The permeability coefficients among solutes [9,10,359]. channels exist in either an open or a closed state, Using the procedure developed by Zimmerman which are in equilibrium with each other. Interest- and Rosselet [360], it is possible to measure rates ingly, among channels was observed of permeation of ,~-lactamase sensitive/3-1actams and the smallest inducible unit presumably corre- through pores in the outer membrane. By applying sponding to a trimer, consists of three channels this procedure on a series of B-lactams with differ- [350,362]. This result, together with structural ent hydrophobicity, Nikaido showed that the per- studies indicating that a functional pore consists meability coefficient increases with decreasing par- of three monomers [356] strongly suggests that tition coefficient in an isobutanol-aqueous buffer each monomer can form a pore and that opening system [9]. Cephaloridine behaved anomalously as of the three channels within a trimer is a highly it penetrated approx. 6-times faster than predicted cooperative phenomenon. [9]. The results certainly show the importance of LPS is required for channel activity [362], a hydrophilicity for penetration through pores. Ap- conclusion not unexpected as a requirement for parent anomalous behaviour must be due to our LPS has been shown for practically all biological too simple picture of pores as just holes. In this activities of pore proteins (see subsection IVB-3). respect it is important to note that it has recently The assumption that LPS is not required in black been shown that the channels formed by LamB lipid films [349] must be due to the misunderstand- protein and PhoE protein have a preference for ing that the used isolation procedure yields com- certain substrates due to the presence of recogni- pletely LPS-free pore protein. tion or binding sites, presumably at the entrance (v) In strains containing more than one pore of the pore (see subsections IIIE-6b and IIIE-5dii, protein species the removal of all but one of these respectively). species (by mutation or by the choice of the right Enterobacteriaceae are relatively resistant to /~- growth condition) leaves the cell with a perfectly 75 functional pore [342,343,363,364] showing that also Secondly, the pore activity per unit weight of in these cases one pore protein species is sufficient. purified pore protein is about 100-fold lower. Al- (vi) The term porin has been introduced to though artefacts cannot be ruled out, the authors indicate general diffusion channels [339]. However, prefer to explain the latter observation by assum- it is known now that some pores, in addition to ing that relatively few pores are open at any given having general pore properties, exert some prefer- time, thus contributing to the antibiotic resistance ence for solutes with respect to the rate of permea- of the organism [358]. Moreover, it has been sug- tion. For example, evidence has been presented gested [9,358] that because of their large pore size, that the LamB protein pore (see subsection IlIE- pore proteins of Pseudomonas sp. are good models 6b) and the PhoE protein pore (see subsection for other pores in nature [9,358] (see for example IIIE-5dii) have recognition sites for certain solutes, Ref. 371). resulting in a faster permeation of these solutes. Although it has been suggested that the in- Next to a built-in recognition site another mecha- creased resistance of Pseudomonas sp. to many nism has been proposed to explain pores which antibiotics is the result of degrading enzymes or of exert a preference. Lo and Bewick [365-368] have reduced rates of transport through the cytoplasmic recently provided strong evidence that an induci- membrane [115,370], recent experiments using in- ble dicarboxylate binding protein is located at the tact cells have shown that the rate of permeation outside of the outer membrane. The observation of several solutes, including fl-lactams, through the that succinate uptake at a concentration of 1.75 outer membrane of Ps. aeruginosa is 100-fold lower /~M is virtually abolished in a mutant lacking both than through the outer membrane of E. coli [378]. OmpC protein and OmpF protein led the authors Pore proteins of Neisseria gonorrhoeae [372] and to suggest that the binding protein is physically of other non-enteric bacteria [10] have been puri- connected with the outside of the pore and serves fied and studied in Nikaido's laboratory. They all as a recognition component [365]. Work had large channel diameters and it was therefore to test this interesting possibility should be suggested that enteric bacteria might be rather pursued. exceptional in producing very narrow channels (vii) A study of the permeability properties of [101. the outer membrane of Pseudomonas sp. is particu- IIIC-5c. Purification and properties of general larly interesting as these organisms are very re- diffusion pore proteins. Most procedures used for sistant towards many antibiotics but are on the the purification of pore proteins are based on the other hand able to utilize a large variety of nutri- one developed by Rosenbusch [230], which is ex- ents. Although it was initially thought that the cellent for the preparation of chemically pure pores of Ps. aeruginosa had the same exclusion monomeric, denatured protein. However, if bio- limit as those of S. typhimurium and E. coli [337], logically active protein is required a high salt later experiments have shown that the exclusion concentration should be used to remove these tri- limit is much larger, approx. 6000 daltons [115, mers from the peptidoglycan [317]. More recently 369], which probably is advantageous for the deoxycholate [349] and fl- [362] or a-octylgluco- organism in that it permits the entry of small side [373] have given equally good or better results. micelles and hydrophobic solutes and of large Denatured pore proteins are acidic poly- peptides into the periplasmic space [115]. peptides with isoelectric points varying from 4.8 Benz and Hancock have extensively studied the [321] to 6.2 [230]. The amino acid composition of behaviour of purified pore protein F [220] in black pore proteins is not hydrophobic [230,374]. The lipid films [358]. Like pore proteins of Enterob- polarity coefficient of OmpF protein is as high as acteriaceae, protein F increases the membrane con- 45% [375]. As the purified trimer form is insoluble ductance of artificial lipid bilayers by many orders in water it is unclear whether the surface of the of magnitude, but protein F differs from the former hydrophilic pore can accommodate the large num- pore proteins in two important respects. Firstly, it ber of polar amino acids and the possibility of causes a relatively high conductance, from which a ion-pair formation on the interior of the protein channel diameter of 2.2 nm can be calculated. should certainly be examined [375]. In contrast to 76

the denatured monomer, the trimer form is rich in high osmolarity results in the disappearance of B-structure [230,357], binds relatively little SDS OmpF protein which is quantitatively com- [230,356] and is resistant to proteases [230,338,357]. pensated for by the synthesis of more OmpC pro- Like in OmpA protein (see subsection IIIE-4), tein [404]. Interestingly, solutes which cannot per- some of the lysine residues of the E. coli K-12 pore meate the outer membrane exert this effect in proteins are present as allysine, although to a substantially lower concentrations than those lesser extent (1-5%) [300]. These modified lysines which pass this membrane [403]. The ompF gene were not detected during the determination of the of E. coli K-12 has recently been cloned [395,402]. complete amino acid sequence of the OmpF pro- Multiple copies of ornpF results in overproduction tein of E. coil B/r [376]. This polypeptide contains of OmpF protein at the expense of OmpC protein 340 amino acid residues resulting in a molecular and PhoE protein [395]. Although transposon in- weight of 37 205. The longest uninterrupted stretch sertions have been obtained over the entire length of nonpolar residues consists of 11 amino acids. of the gene, OmpF protein fragments have never This fact as well as the percentages of polar amino been detected [395]. acids in various regions of the molecule strongly As both OmpA protein and OmpF protein in- suggest that the transmembrane region is not sim- teract with LPS, Mowa et al. [310] have compared ply a single contiguous sequence of hydrophobic the established and predicted sequences respec- amino acids [376] as has been proposed for glyco- tively and found several regions of similarity which phorin [377]. are candidates for sites of interaction with LPS. It lllE-Sd. Characteristics of individual general dif- should be noted, however, that such an idea may fusion pore proteins. The characteristics of the best be too simple as it could well be that different known general diffusion pore proteins of E. coli regions of LPS interact with the two proteins. For and S. typhimuriurn are summarized in Table II. example it has been shown that OmpF protein is Additional comments as well as more recent re- hardly present in the outer membrane of heptose- suits on these and other pore proteins are treated less mutants whereas the amount of OmpA protein below. is hardly affected by the mutation [94]. i. OmpC protein and OrnpF protein (see also ii. PhoE protein (see also Table 11). This pro- Table II). The early suggestion that OmpF protein tein is induced in wild-type cells by phosphate and OmpC protein of E. coli K-12 were products limitation [320] as a component of a series of of the same structural gene [228,231] together with proteins [412] which the cell synthesizes in order to the fact that these proteins are hardly or not at all scavenge the last traces of phosphate or phos- separated in many gel systems [221,297] has led to phate-containing components from the medium the misunderstanding that E. coli K-12 contains (for a review see Ref. 413). Consistent with this one general pore protein. Even in more recent observation is the result that PhoE protein forms a literature it occurs that these proteins are not more efficient pore for both inorganic and organic differentiated (e.g. see Refs. 300, 407, 408). It is phosphate than OmpF protein [345], despite its clear now that these constitutive pore proteins of smaller diameter [358]. This apparent discrepancy E. coli K-12 are coded for by distinct but structur- can be explained by assuming a recognition site ally related structural genes. The polypeptides are for these solutes near the entrance of the pore. similar but differ in several properties [95,343,396]. Strong evidence for this explanation has recently The OmpF protein pore is larger than the OmpC been presented [400]. Because of the structural protein pore [10,358,409]. similarities between PhoE protein and OmpF pro- The relative amounts of the two proteins are tein (see below) it thus is likely that PhoE protein dependent on the composition of the growth has evolved from a general pore and, in order to medium, such that the sum of the amounts is play its role in phosphate uptake, has been pro- almost constant [95]. Some evidence for a positive vided with a weak for certain solutes. control of the synthesis of OmpF protein by cAMP PhoE protein shares many properties with has been published [405]. The osmolarity of the OmpC protein and OmpF protein (Table II). The growth medium has a drastic effect [411] in that a proteins are immunologically related [319] and 77

recent work on the cloned genes ompF [395] and protein, M r approx. 38 000, is produced by some phoE [406] has shown that their DNA's can be colicin-sensitive revertants of porin-defective E. hybridized over the entire length of the genes coli K-12 mutants. The mutation responsible for [395]. Such a homology at the DNA level could the reversion has been localized at min 12. By explain the large number of different pore proteins chemical and immunlogical criteria NmpC protein found in various strains of the same organism was reported to be similar to Lc protein, the [301] as recombination between pore protein genes, former protein 2 [418]. In analogy to PhoE protein in combination with mutation and transposition, [320] it seems likely that NmpC protein is an could generate a large number of different but inducible protein in wild-type cells. similar pores. The nucleotide sequence of the phoE Paakkanen et al. [419] examined the outer mem- gene has recently been established in our labora- brane protein patterns of 47 encapsulated and tory [380]. The predicted amino acid sequence was non-encapsulated E. coli strains. They observed compared with the established sequence of the that a protein band at the electrophoretic position OmpF protein [376,414] and as many as approx. of M r 40 000, designated as K protein, was present 70% of the residues, including several stretches of in all 33 encapsulated strains and absent in all but 15-20 amino acids, were identical [380]. Overpro- one of the 14 non-encapsulated strains. The authors duction of PhoE protein, which is accompanied by therefore suggest that the protein is related to the decreased amounts of OmpF protein and Lamb capsule. The protein has been purified and resem- protein, is lethal to the cells [406]. In contrast to bles the OmpF protein of E. coli B/r in its chemi- the situation with OmpA protein (see subsection cal properties as well as its apparent molecular IIIE-4) expression of PhoE protein in the outer weight [419]. The relation between the occurrence membrane cannot be detected if part of the gene, of the K protein and the capsule is very striking corresponding with the 50 carboxy terminal amino but the designation K (capsule) protein is prema- acid residues, is deleted [380]. ture for two reasons. Firstly, it is based on the Hancock et al. [416] have observed that phos- assumption that proteins with the same electro- phate limitation induces an oligomeric outer mem- phoretic mobility have the same function. Sec- brane protein, designated as P protein in Ps. ondly, Paakkanen et al. have analysed the protein aeruginosa. The observed in vitro pore properties patterns on gradient gels in which the major pro- of protein P show that it is highly specific for teins are present in a relatively narrow region of anions. Although protein P thus has important the gel. It would be interesting to see whether the properties in common with PhoE protein of E. relationship still holds when the patterns are coli, it should be noted that pores formed by PhoE analysed in a series of different gel systems. Using protein are significantly larger [416]. the homology between pore proteins [319,380,395] iii. Salmonella pore proteins (see also Table the structural gene can be cloned and be used to 1I). The Salmonella pores all have the same effec- test the proposed relationship. tive diameter [358] which is of the same size or Lc protein [417], previously designated as pro- slightly larger than those of the E. coli pores. The tein 2, is coded by prophage PA2 [329,420]. When fact that the pores are equally effective makes the strains of E. coli K-12 are lysogenized with phage question why so many pores are needed even more PA2 they replace the existing pore proteins OmpC intriguing. They have been compared with those of protein (the receptor of the phage) and OmpF E. coli K-12 with respect to function, regulation of protein by the new pore protein [329], a form of expression and, in the case of OmpC protein and lysogenic conversion at the protein level. The OmpF protein, equivalence of the genetic loci synthesis of Lc protein is catabolite repressible (Nurminen, M. and M~ikela, P.H., cited in Ref. 4; [329]. By chemical and immunological criteria Lc Ref. 417). The results suggested that pairs of pro- protein was reported to be similar to NmpC pro- teins corresponded (see Table II). The three pro- tein [418]. Recent work of the late Tom Gregg and teins are immunologically [319] and chemically co-workers [420] has shown that hybrid pore pro- (Table II) related to E. coli K-12 pore proteins. teins, consisting of portions of Lc protein and iv. Other general diffusion pore proteins. NmpC OmpC protein, can be constructed. This type of 7~ approach can be very useful for studying struc- product of gene lamB, located at rain 91.0 of the ture-function relationships in pore proteins. E. coli genetic map, which is part of the well lyer [330,331] has observed that E. coli carrying studied maltose regulon, which is used in model an N plasmid produce an altered pore protein. We studies on the mechanisms of protein localization favour the explanation of Nikaido and Nakae [2] [444-447]. Addition of maltose to the growth that this is caused by repression of the synthesis of medium derepresses the synthesis of the structural the host pore protein(s) accompanied by synthesis genes of this regulon. In addition to lambda [425] of new plasmid-coded pore protein. several other phages use the protein as their recep- In Ps. aeruginosa a glucose-inducible pore pro- tor (see Table Ill). tein has been described [421]. The lambda receptor protein is involved in maltose uptake as can be illustrated at low (ap- lIIE-6. Characteristics of E, coli pore proteins not prox. 1 /~M), but not at high (approx. l mM), antigenically related to the family of pep- substrate concentrations [443]. Subsequently it has tidoglycan-associated general diffusion pore proteins been shown that lamB mutants have a 100 to (Table 1II) 500-fold increased K m for maltose transport and lllE-6a. Bacteriophage T6 receptor protein (Ta- that they cannot take up maltotriose [448]. Thus, ble 111). The receptor of phage T6 and colicin K, Szmelcman and Schwartz concluded that the X-re- a protein of M r 26 000 [435,436], has been purified ceptor facilitates the diffusion of maltose and and its amino acid composition has been de- maltotriose through the outer membrane [448]. It termined [436]. Biologically active T6 receptor in- has been thought that the X-receptor pore was variably contains LPS [436] and in vivo experi- specific for maltose and maltodextrins. The first ments suggest that core sugars play a role in a later indication that the X-receptor also facilitates the step of the T6 infection process [422]. Dependence diffusion of other nutrients came from Von on LPS has been confirmed by in vitro experi- Meyenburg and Nikaido [449]. More recently, in ments. The tsx gene is located at rain 9.2 and part vitro experiments have shown that it behaves as a of its has recently been cloned [438]. With respect general pore [351,428,450,451]. The observed bind- to its function, the protein is involved in facilitat- ing of maltose and of certain related compounds ing the diffusion of all nucleosides and deoxynuc- to the X-receptor pore [351,452--455] probably ex- leosides except cytidine and deoxycytidine plains the relatively high permeation rate of these [422,423]. As the former solutes do not compete in solutes through this pore. As maltodextrins too the uptake process and as they do not inhibit T6 large to be transported are also bound by the outer adsorption, it was concluded that the T6 receptor membrane [453] the binding site must be located protein forms a pore to which the diffusing solute at the cell surface. Obviously phage lambda does has only little, it any, binding affinity [422]. As the not interfere with the binding site as maltose does uptake system for nucleosides is catabolite re- not inhibit the rate of adsorption (M. Schwartz, pressible [435,439], the observation that the personal communication). Interestingly, evidence synthesis of the T6 receptor protein is also cata- for a cooperation of the lambda receptor with the bolic repressible [436] suggests that uptake of periplasmic maltose binding protein in the uptake nucleosides is the real function of this protein. of maltose, as first hypothesized by Endermann et Experiments which suggest that the T6 receptor al. [456], has recently been obtained [442,457,458]. protein pore promotes diffusion of serine, glycine This interaction may also contribute to the prefer- and phenylalanine (but not of glucose and arginine ence of the pore for certain solutes. It has been [440]) therefore are best explained as the acciden- shown recently that among lamB missense mutants tal use of this pore by these solutes. strains occur with different in vitro pore properties lllE-6b. Bacteriophage lambda receptor protein and with poor in vitro interaction with maltose (Table I11). The receptor of bacteriophage lambda, binding protein [449]. Such an approach will con- M r 47392 [424], is an outer membrane protein tribute to our understanding of the molecular [425,441] which plays a role in the uptake of functioning of the LamB protein, maltose and maltodextrins [442,443]. It is the The lambda receptor protein has been purified TABLE Ill

E. COLI K-12 PORE PROTEINS NOT ANTIGENICALLY RELATED TO PEPT1DOGLYCAN-ASSOCIATED GENERAL DIFFUSION PORE PROTEINS"

Protein Conditions for op- M r (Part of) receptor for Structural gene Proposed function Further characteristics timal expression phage/colicin

Phage T6 Co-regulated with nu- 26000 T6, Col K tsx, rain 9.2 Uptake Synthesis catabolite repressi- receptor cleotide transport nucleosides and ble [436]; increased amounts syn- [422] deoxynucleosides thesized in (ytR and deoR mu- [422,423] mutants [422]; up to about 4.104 co- pies can be present per cell. Phage 2, Presence of maltose 47392 [424] )~ [425]; KI0 [426]; lamB, rain 91.0 Uptake Synthesis is catabolite repressi- receptor TPI [383]; TP5 [384]; maltodextrins ble [427]; up to 105 copies per SSl [3891 cell can be present; pore diameter 1.5 nm [428] Synthesized in reduced amounts in peril mutants [238,239] and in hep- tose-less LPS mutants [237] ButB protein Vitamin BI2 60000 [430] BF23, E-, btuB, rain 89.0 Uptake Up to 200-300 copies per cell limitation [429] Cola [384] a vitamin BI2 [4311 Cir protein Fe 3 + limitation [432} 74000 Coil, ColV cir, min 44 Uptake Synthesis of Cir, FepA and 83K complexed Fe 3+ ? FhuA protein Fe 3÷ limitation [432] 78000 T1, T5, 1~80, ColM fhuA, min 3.4 Uptake Fe 3+ proteins is reduced in perA mu- ferrichrome tants [240]. Fec protein Presence of citrate 80500 -- fec, min 7 Uptake Fe 3÷- [433,434] citrate FepA protein Fe 3÷ limitation [432] 81000 ColB, ColD fep, rain 13 Uptake Fe 3+- enterochelin 83K protein Fe 3+ limitation 83000 -- Unknown Uptake complexed Fe 3+ ?

" See subsection IIIE-6. 8() to homogeneity [319,351,408,441,450,456,460]. As of ferric ions is extremely low (approx. 10 ~ M) first found in Schnaitman's laboratory (Schnait- and bacteria have developed systems to take up man, C.A., personal communication) it can be these essential ions by first complexing the ferric isolated associated with peptidoglycan. Applica- ions with low molecular weight chelators known as tion of this procedure has been reported to result siderophores of siderochromes. (For excellent re- in large losses [428,456] but in our hands a slight cent reviews see Refs. 468-470), Chelators used by modification of the method used for purification Enterobacteriaceae are aerobactin, enterochelin of general pore proteins yields essentially pure (enterobactin), citrate and ferrichrome. Interest- Lamb protein in a high yield [319]. The active ingly, the antibiotic albomycin is a structural ana- form is a trimer [461]. The protein has neither log of ferrichrome [471,472]. In addition to these chemical [456] nor immunological [319] similarity high affinity uptake systems, ferric iron can be with other major outer membrane proteins. The taken up by a low affinity, chelator-independent observation that both the )~-receptor protein and system [465]. Alternatively, the uptake can be ex- OmpF protein are recognized by the same plained by low constitutive levels of the high affin- bacteriophage, TP1 [383], therefore is most likely ity system which are present irrespective of the explained by assuming that the phage uses differ- cultural conditions [468]. ent regions of its tail for recognition of the two E. coli grown under conditions of limiting ferric proteins. In whole cells the protein is also accessi- iron derepresses the synthesis of siderophores and ble to antibodies [462]. of several outer membrane proteins from which From the recently established nucleotide se- several have been identified as proteins involved in quence of the cloned lamB gene [424] a poly- the uptake of specific iron-chelator complexes (see peptide of 421 amino acids with two cysteine Table III). (For a recent review see Ref. 470). In at residues can be predicted. Many amino acids are least one case it is likely that substrate recognition charged, mostly negatively, and long peptides plays a role in uptake as it has been shown that without charged residues do not occur. Interest- the rate of adsorption of bacteriophage T5 is in- ingly, the residues can be alligned such that many hibited by the presence of ferrichrome [473]. The negative charges are neutralized by positive ones production and uptake of siderophores can play [424]. The first results of the promising study of important ecological roles, e.g. it can be an im- the structure-function relationship of the protein portant virulence factor [474-480] whereas sidero- have recently been published [463,464]. phore production can be beneficial for plant lllE-6c. Outer membrane protein involved in the growth by inhibiting growth of plant pathogens uptake of vitamin BI2 (Table II1). Vitamin B12, M r [481]. Siderophore production is temperature sen- 1327, is too large to pass the outer membrane sitive [482] and it therefore has been suggested that through general pores and it therefore requires a fever might be a host defence mechanism designed specific outer membrane protein for facilitating its to deprive the pathogen of iron [482]. translocation across the outer membrane (for a recent review see Ref. 465). The btuB product, M r IIIE-7. Characteristics of E. coli outer membrane 60 000, has been purified and might be a glycopro- proteins without identified function tein [430] as it was shown to be sensitive to peri- lllE-7a. Protein a. In E. coli K-12 protein a (M r odate treatment. However, as such a treatment can approx. 40000, Ref. 205) usually is present in low also result in protein modification [466] the data amounts compared with other major outer mem- should be interpreted with care. The protein is the brane proteins [95,205]. It is hardly detected after receptor for phage BF23 and the E-colicins. The growth at 30°C but rather high amounts can be vitamin binds to the protein to the extent that it produced at 42°C [95,484]. As protein a was not protects the cell from killing by colicin and phage detected among the membrane proteins in the M r [28,467], Binding in vitro can be obtained in the range of 30000 to 42000 in any of 45 E. coli presence of LPS (C. Bradbeer, cited in Ref. 465). hospital isolates [301], the production of large lllE-6d. Outer membrane proteins involved in amounts might be typical for strain K-12. Strong the uptake of ferric ions (Table III). The solubility support for this idea comes from recent experi- 81 ments which show that interactions between an 81 As the amino acid compositions of protein III kDa ferric enterobactin pore protein and protein a [294,295] and type 1 pilin [53] are very different, (see further on) is confined to strain K-12 of E. the possibility that protein III could be identical to coli [485]. pilin, as suggested by McMichael and Ou [492], Gayda and Markovitz have cloned the struct- can be ruled out. ural gene for protein M2, which they identified as Wu and Heath [493] reported that all LPS of E. protein a [486]. They claim that subsequent studies coli is covalently linked to a protein with a mini- with plasmid mutants demonstrated that this pro- mum M r of about 14000, for which protein III was tein is important in repressing the synthesis of an obvious candidate. Indeed, large amounts of capsular polysaccharide [487]. Earhardt et al. [488] LPS are found in purified preparations of protein have described deletion mutants which lack pro- III but this co-purification is coincidental as it has tein a. Their observation that such strains are been described that both components can be puri- normal with respect to capsule formation [488] is fied free from the other by using mild procedures not consistent with the proposed role [487] of [295]. Similar results were found in other laborato- protein a in repressing capsular synthesis. The ries and it is now generally agreed upon that LPS data of Earhardt et al. suggest that the structural is not covalently bound to any protein. gene for protein a is located at rain 12.5 of the With respect to the regulation of the synthesis chromosomal map [488]. These authors have also of protein III it is interesting to note that it has isolated a phage, LP81, which was claimed to been reported that its synthesis is negatively con- require protein a for infection [488]. However, it trolled by cyclic AMP at the level of transcription has recently been shown that its real receptor is by a mechanism which involves the cAMP recep- not protein a but a protein whose structural gene tor protein [494]. An alternative interpretation is is located close to that of protein a (C.F. Earhardt, that cAMP controls the synthesis of OmpF protein personal communication). and that changes in the level of protein III (and of The outer membrane receptor for ferric enter- OmpC protein) are simply responses to the level of obactin, FepA protein with a M r of 81000, is OmpF protein [405]. converted to a M r 74000 protein by an outer IIIE-7c. LPS binding protein. The purification membrane protease, which has chemical and of a LPS binding protein from Re mutants of S. physical properties ascribed to protein a [248]. minnesota has been described. This M r 15 000 pro- Moreover, as mutants lacking protein a do not tein is surface located and can be considered as a convert the FepA protein [248] the evidence for common antigen in Enterobacteriaceae [495]. Al- protein a being a protease looks rather good. In though several properties suggest a possible iden- fact it resembles the signal peptidase in this en- tity with protein III, such a relation is not very zymatic activity, in its apparent molecular weight likely as Re mutants produce little protein III [93] and its localization in the outer membrane (see and as the amino acid compositions of the two subsection IIIE-2). It seems worthwhile determin- proteins, especially the relative amounts of proline, ing whether these two proteins are identical. glycine and tyrosine, differ too much [295,495]. lllE-7b. Protein IlL This protein (M r 17000, Similarly, the amino acid composition excludes an Refs. 222 and 489), which probably is identical identity with lipoprotein (compare e.g. Refs. 222 with protein G [490,491] is a major outer mem- and 495). brane protein which occurs in l0 4 to 10 5 copies IllE-Td. Outer membrane proteins induced by per cell [294]. The search for the function of sulphate limitation. Growth of E. coli K-12 at protein III is hampered by the fact that no mutants Na2SO 4 concentrations below 50 ~M results in have been found which lack the protein. Outer induction of the synthesis of two cell envelope membranes of E. coli K-12 deep rough LPS proteins of M r 15 000 and 19 000. Preliminary ex- mutants contain decreased amounts of protein III periments indicate that the M r 19000 protein and [93]. A protein band in the electrophoretic position probably also the M r 15 000 proteins are located in of protein III was observed in all Enterob- the outer membrane (Lugtenberg, B. and Over- acteriaceae mentioned in Ref. 322 (Lugtenberg, B., duin, P., unpublished data). unpublished data). 82

lllE-Te. Phage- and plasmid-coded outer mem- with nutrients from the medium in an extremely brane proteins. Phage lambda codes for an outer efficient way. On the other hand, as growth of membrane protein (M r 20 500) for which no func- these bacteria is not affected by the presence of tion has been established so far [328]. Upon infec- high concentrations of bile salts, detergents and tion of E. coil with the virulent phages T4 and T2 many antibiotics, these cells must have an efficient several new proteins of unknown function appear barrier for the latter agents and it has been real- in the outer membrane thereby inhibiting the pro- ized early that the outer membrane constitutes this duction of the major outer membrane proteins of barrier. As detergents and many antibiotics are the host [496,497]. hydrophobic whereas nutrients are hydrophilic, The F-factor, a plasmid present in the best Nikaido has proposed two possible pathways for known E. coli donor strains in conjugation, codes solute permeation through the outer membrane, a for a series of outer membrane proteins [8]. Of hydrophobic one and a hydrophilic one. In order these the, TraT protein has been studied most to explain the discriminatory role of the outer extensively. This protein (M r 25 000) is involved in membrane with respect to the permeation of so- surface exclusion i.e. it prevents a donor cell from lutes, he proposed that the hydrophobic pathway functioning as an acceptor cell by blocking the is non-existent in Enterobacteriaceae but does oc- stabilization of mating aggregates [6]. It has been cur in some other Gram-negative bacteria in the proposed that several specific receptor molecules form of phospholipid bilayer regions [361]. The are involved in the initial stages of conjugation hydrophilic pathways were assumed to consist of and that the action of the TraT protein in surface aqueous or water-filled pores in the membrane exclusion is due to specific binding to one of these through which nutrients permeate, largely by a donor or recipient receptor molecules in the outer diffusion-like process (see subsection IIIE-5). membrane [332]. In a detailed study Manning et The permeability properties of E. coli cells can al. [498] have shown that TraT protein is pepti- be altered by treatment with EDTA under condi- doglycan-associated and exists as multimers in the tions which do not influence the viability of the outer membrane. No pore activity was detected. cells. This results in the release of half of the lts isoelectric point is very basic (pH approx. 9). cellular LPS, in a strong increase in the permeabil- Embedded in the membrane, the protein is re- ity for detergents and hydrophobic antibiotics and sistant to trypsin. In whole cells it is exposed to in accessibility of cellular phospholipids for exoge- the cell surface as it is accessible to CNBr-activated nous [ 127,161 ]. The damage can be dextran and it can strongly be labeled with 125I, restored upon subsequent incubation in fresh using lactoperoxidase [498]. Interestingly, the TraT medium [85]. protein is responsible for the resistance to the The permeability of the cell can also be in- bactericidal activity of normal serum towards E. creased by mutations which lead to a deficiency coli carrying plasmid R6-5 [499,500], R100 [501] or for one or more outer membrane proteins or which R222 [502]. In the latter case the authors suggest reduce the length of the LPS sugar chain. Espe- that the protein, named MRB protein, has an as cially the so-called deep-rough mutants of the Re yet unidentified transport function [502]. and Rd chemotypes (Fig. 3) have a largely in- creased sensitivity towards hydrophobic com- IV. Molecular organization of the outer membrane pounds [410,463,503-505]. LPS mutants as well as an overwhelming number of mutants deficient in I VA. Introduction one of the outer membrane proteins appeared to be very useful in topological studies. It should be Microbiologists have realized already long ago noted, however, that the lack of a constituent that the envelope of Enterobaeteriaceae has very brings about a reorganization in the molecular peculiar permeability properties. On one hand make-up of the membrane. Several examples of these bacteria are among the fastest growing cells such a reorganization are known. The lack of in nature and they therefore must be able to major proteins is compensated for by increased provide the biosynthetic machinery inside the cell amounts of other proteins [96,232,506], of LPS TABLE IV

TOPOGRAPHY OF OUTER MEMBRANE PROTEINS

Protein Presence at the Protein-protein Protein-peptidoglycan interactions Protein-LPS interactions cell surface interactions

Lipoprotein No evidence. Is not known as a Homologous oligomers, mainly Can be chemically cross-linked to No evidence. (free form) receptor for a phage. dimers, are formed upon chemical peptidoglycan [5 131 Does not interact with exogenous cross-linking [S 131. Can also be CNBr-dextran or with anti-lipo- cross-linked with protein III protein serum (5121. [514], and with OmpA protein to a dimeric complex [513]. Evidence for interaction with general pore proteins in vitro 15151. Lipoprotein No evidence. Evidence for interaction with Covalently bound to peptido- No evidence (bound form) general pore proteins in vitro glycan [265.266]; influences pore [516]. protein-peptidoglycan inter- actions in vitro [5 161; such an interactiion is unlikely in viva 12721. OmpA protein Receptor for phages [293.381]; Can be chemically cross-linked to Can be chemically cross-linked to Protein-LPS complex is receptor reaction with antiserum [517]; homologous oligomers [5 13.5141 peptidoglycan 1234.5 14.5 181. for phages K3 [293] and TuII’ the analogous 33 kDa protein of and to a heterologous dimer with [381] and for donor cells in S. typhimurium reacts with the free form of lipoprotein F-factor mediated conjugation bacteriocin 4-59. [513]. [293.308]; complexes also occur in vivo as evidenced by phage adsorption characteristics [170] and freeze-fracture studies [200]. OmpC, OmpD, Receptor for phages (see Table Can be chemically cross-linked to Cannot be cross-linked to pep- Freeze fracture studies indicate OmpF and PhoE II); reaction with antibodies di-. tri-, hexa- and nonamers tidoglycan [513,518]; in vitro protein-LPS complexes [ZOO]. proteins 1341 and CNBr-dextran [519]. [398,399.513,518,520] but not reconstitution of complexes of Heptose-less mutants have to lipoprotein [397,399,513.520, OmpF protein [522,523] and strongly decreased amounts of 5211; biologically active form is OmpC protein [516] with pepti- OmpF protein 190.94) and PhoE trimer [356,357]. doglycan-lipoprotein. protein [236] in their outer mem- brane. Pore activity (3261 and phage receptor activity [217,226, 3811 in vitro are dependent on LPS. LamB protein Receptor for phages (see Table Can be chemically cross-linked to In vitro reconstitution of com- Decreased amounts of lambda III); reaction with antibodies di-, tri-, hexa- and nonamers plexes of LamB protein and pep- receptor are present in heptose- [462]. 14611; trimer is biologically tidoglycan-lipoprotein [525]. less LPS mutants [237]; certain active form 15241. C% particles are probably LamB protein-LPS complexes [200]. 84

[93,127,129] and of phospholipids [88,127,129]. In available data on the molecular organization of the the latter case the mutation most likely results in outer membrane, as mainly studied in E. coli and the insertion of part of these phospholipids into S. tvphimurium. Studies on the surface localization the outer monolayer, thereby making the cells of outer membrane constituents indicate that LPS sensitive towards detergents and exogenous phos- and almost all proteins, but not the phospholipids, pholipases [96,127]. Defects in the structure of have sites exposed at the surface which can act as LPS, especially those caused by deep-rough muta- receptors for phages, colicins and donor cells in tions, can result in decreased amounts of outer conjugation and can react with antibodies, en- membrane proteins [87,90,92,96] and in increased zymes and chemicals. In addition, protein-protein amounts of phospholipids [87,88,92,93] and LPS and protein-peptidoglycan interactions of the [92]. The resulting outer membranes are schemati- major proteins are described. The organization of cally represented in Fig. 6. A more subtle example the phospholipids and LPS in the lipid bilayer and of the mentioned reorganization is that substantial the way the planar structure of this bilayer is amounts of OmpA protein, which cannot be iso- disturbed by protein-LPS complexes, visible in lated complexed with peptidoglycan from E. coli freeze-fracture electron microscopy as particles K-12 wild-type cells, are found associated with with complementary pits, will be treated in subsec- peptidoglycan in pore protein deficient mutant tion IVG-2. Finally, a molecular model is pre- cells (Lugtenberg, B. and Van Boxtel, R., unpub- sented which contains the knowledge collected up lished data). Finally, mutant cells with heptose-less to now. LPS or lacking Braun's lipoprotein excrete outer membrane blebs, which (at least in wild-type cells) 1 VB. Methods used for studying the localization of have an architecture different from their cell-bound individual outer membrane constituents and their membrane in that the relative amounts of several interactions proteins are drastically altered [272]. Thus, it is evident that mutants should be used with care for I VB-I. Localization at the cell surface topological studies. One can conclude that a certain protein has a The cell surface of E. coli was investigated using site exposed at the cell surface if it can be shown freeze etch electron microscopy [507] and found to that the protein is the receptor for a phage or a contain numerous randomly spaced depressions of bacteriocin. This seemingly simple approach is about 4.5 nm in diameter, which could be the hampered by the fact that in vitro experiments entrances of the aqueous pores [200,507]. Freeze- usually show that the receptor activity is lost upon fracture studies were applied to study the interior purification of the protein, often because of a of the outer membrane. It was shown that the complex of both protein and LPS is required for outer membrane can be cleaved into two halves receptor activity (see Table IV and subsection [88,508-510] suggesting a lipid bilayer structure. IVG-4). Therefore the protein is not necessarily The two leaflets clearly differ in that the concave the first recognized, and therefore surface-exposed, or outer fracture face (O~M) is covered with par- part of the receptor. Only in the case of OmpA ticles, 4 to 8 nm in diameter, whereas the O~M, the protein strong evidence has been presented for a convex or inner fracture face, contains pits, proba- primary role of the protein by showing that the bly complementary to the particles. Particles and deeply burried lipid A portion is the active compo- pits are considered to be reflections of protein-LPS nent of the LPS (Refs. 171 and 526). In all other interactions (Refs. 2, 15,200, 511; see also subsec- cases one should be careful not to over-interprete tion IVG-2). As it has been proposed that a sub- the receptor evidence. However, the observation stantial part of the particles represents aqueous that resistant mutants often lack the protein and pores [363], it is interesting to note that EDTA usually do not bind the phage or bacteriocin any- treatment reduces both the number of derepres- more shows that the protein is the most likely sions at the cell surface [507] as well as the number candidate for the primary receptor and that phages of intramembranous particles and pits [200]. and bacteriocins can be used as rather reliable In the following section we will describe the indicators of surface localization. According to 85 this criterium, many outer membrane proteins are the cell surface, as has been used for ECA (see recognized by phages and colicins (see Tables subsection IVE) and LPS (see subsection IVC), in II-IV, and subsection IVG-4). the cases of E. coli LamB protein [430] and OmpA Proteolytic enzymes have been used as tools for protein [517] and peptidoglycan-associated general demonstrating surface localization of proteins. In pore proteins of S. typhimurium [34]. intact cells of E. coli K-12 none of the outer membrane proteins were degraded by proteolytic IVB-2. Protein-protein and protein-peptidoglycan enzymes [527], which is not so surprizing as the nearest neighbour associations natural habitat of Enterobacteriaceae is rich in A variety of bifunctional cleavable cross-linkers such enzymes. with various specificity and distances between the Membrane-impermeable CNBr-activated dex- reactive groups can be used to show interactions tran has been used as a tool to couple surface-ex- between membrane constituents [533]. This ap- posed outer membrane constituents [519]. In the proach has been applied successfully in the case of case of S. typhimurium this approach showed that outer membrane proteins (see Table IV). It should the peptidoglycan-associated general pore proteins be noted that when two molecular species cannot are surface-exposed. Unfortunately, it is not possi- be chemically cross-linked the result not neces- ble for us to interprete the experiment carried out sarily means that these constituents are not with E. coli strain B [519] with certainty. For neighbours as the spacing might not have been example, the authors describe that a M r 31000 correctly chosen or as reactive groups might not be protein is exposed which they presume to corre- present in the right positions (e.g. see subsection spond with protein III [519] although the latter IVF-4). Moreover, it should be realized that pro- protein has a M r of about 17000. teins which meet each other only for a short time Several radio-iodination techniques have been during the experiment can also be cross-linked. applied to whole cells. In the case of the The latter type of cross-linking due to random lactoperoxidase method it has been reported that collisions can be minimized by using photo-sensi- most outer membrane proteins of E. coli K-12, tive reagents which can be activated for a short including both OmpA protein and protein I, can time (approx. 1 ms) with a flash [533]. In general, be labeled [528]. We have obtained similar results one should be careful in interpreting crosslinking but as we calculated that only a few protein mole- data. Selective cross-linking with affinity labels is cules per cell were labeled, the labeling can also be preferable to nonselective cross-linking (for ins- explained by outer membrane damage of a small tance with bis-imido-esters). The latter reagents fraction of the cells (Van Alphen, L. and Lugten- are useful for the detection of interactions, pro- berg, B., unpublished data). Munford and Gotsch- vided that various reagents are applied under lich [529] have clearly shown how careful one must well-defined conditions, such as short reaction be with the interpretation of radio-labeling tech- times, low concentrations of reagents to avoid niques. Using the chloramine T method they found extensive cross-linking due to random collisions that under certain conditions only the lipoprotein and defined temperatures to account for phase was labeled which contains only one tyrosine re- separation of membrane components. The physio- sidue located only three amino acid residues from logical significance of the complexes generated by the covalent linkage of the lipoprotein with the cross-linking can only be established by demon- peptidoglycan [529]. Obviously protein pores are strating that the complexes consisting of the same permeable for chloramine T. Recently promising membrane components as observed after cross-lin- results with non-Enterobacteriaceae have been ob- king are required for biological activity .In this tained with the radio-iodination reagents IodoGen respect the homotrimers of pore proteins seem to (1,3,4,6-tetrachloro-3 a,6 a-diphenyl glycoluril) fullfil these requirements (see subsection IVF-4). [530,531] and DISA (diazotized [35S]sulfanilic acid) Some outer membrane pore proteins can be [103,532]. isolated tightly complexed to the peptidoglycan An obvious method to show surface localization (see Tables II and III). The question of whether is the detection with mono-specific antibodies at such complexes also exist in vivo will be discussed in subsection IVF-5. 86

1 VB-3. Interactions between individual proteins and unequivalently attributing to the :~P-NMR spec- LPS (see also subsection 1 VG-4) tra. LPS is often required for in vitro biological 2H-NMR and electron spin resonance (ESR) activities of outer membrane proteins, e.g. receptor spectroscopy are both useful for the study of inter- activity for phages, for donor cells in conjugation, actions of the specifically labeled compounds with for a tight association with peptidoglycan and for their environment. Since the time scale of the pore activity. Strong evidence that OmpA protein- measurement is relatively long with NMR ( l0 4 l0 t LPS interactions also occur in vivo has been pro- cm 2. s -l ) in comparison with ESR (10 v- 10 ~ cm ~- vided [170]. Some proteins are hardly or not at all s ~), long-term and short-term interactions can be found in the outer membrane of heptose-less LPS discriminated. mutants. The application of freeze-fracture and biochemical techniques on appropriate mutants 1 VC. Localization of LPS has indicated the existence of protein-LPS interac- tions for many major proteins (see subsection Studies in which ferritin-labeled antibodies di- IVG-2). Finally, recent experiments have shown rected against the O-antigenic part of the lipopoly- that passive immunization of animals with anti- saccharide of S. typhimurium were used, have bodies raised against protein-LPS complexes, from clearly shown that LPS is located exclusively at the wich the anti-LPS activity had subsequently been outside of outer membranes [36]. This conclusion removed, results in a much better protection was confirmed recently, since it appeared that the against experimental infection than the use of sugar chains of the LPS molecules in whole cells of antibodies raised against highly purified protein S. typhimurium are oxidized by exogenous galac- (Refs. 534 and 535; Dankert, J., Hofstra, H. and rose oxidase [536]. LPS occurs in various forms in Veninga, T.S. (1980) in FEMS Symposium on the outer membrane. Half of it can be extracted Microbial Envelopes, Saimaanranta, Finland, Ab- with EDTA [199]. Newly synthesized LPS does stract 51). not mix with old LPS [537], indicating different domains. Finally, outer membrane proteins form 1 VB-4. The lipid matrix and interactions of proteins strong complexes with a substantial portion of the with lipids LPS molecules (see subsection IVG-4). X-ray diffraction, 31p-nuclear magnetic reso- nance spectroscopy (31P-NMR) and freeze-frac- 1 VD. Localization ofphospholipids ture electron microscopy are standard methods for studying the organization of membranes, espe- Phospholipids are part of the lipid bilayer of cially of its phospholipids, since extensive data are the outer membrane (see subsection IVG). As it available from model studies. With X-ray diffrac- has been calculated that the outer membrane con- tion the presence of regularly arranged fatty acyl tains hardly enough phospholipids to cover one chains in regions of a fair size can be detected, monolayer [88] and as it has been established that such that the occurrence of phospholipid and LPS LPS is located exclusively at the outside of the in bilayers can be detected. Using 31p-NMR spec- outer membrane (see subsection IVC), it seems troscopy, which measures the ways of motion of likely that phospholipids are mainly or completely phosphorus-containing components (including located in the inner monolayer. The fact that, LPS), lamellar, isotropic and hexagonal phases despite serious attempts, investigators have not were detected. This technique is especially power- succeeded in showing accessibility of phospholi- ful in combination with freeze-fracture electron pids by exogenous agents in intact cells of E. coli microscopy, which reveals the morphology of the strains K-12 and B, and of S. typhimurium, sup- fracture faces. These faces are usually the sites ports this idea. Perhaps even more convincing is where the ends of the fatty acyl chains approach the simple observation that wild-type cells and Rc each other. A special problem for the 31P-NMR mutants can grow in the presence of bile salts and study of outer membranes is the presence of LPS, SDS, whereas Re mutants and pore protein defi- which contains several phosphorus atoms, thereby cient mutants are sensitive to these and other 87 hydrophobic agents [503-505,538]. Cells of the phospholipid molecules of the outer membrane are sensitive strains have increased amounts of phos- localized in its inner monolayer. Consistent with pholipid [88,92,93,127], which are probably located this conclusion is the result of our own work [ 170], in the outer leaflet (Refs. 88 and 127; subsection which shows that wild type cells of E. col] K- 12 the IVA). Such areas of phospholipid bilayer are likely receptor for phage K3, consisting of a complex of to be susceptible to detergents. Similarly, chemical LPS with OmpA protein [293], does not contain labeling of S. typhimurium cells with membrane- phospholipid molecules in its immediate impermeable CNBr-activated dextran revealed that neighbourhood, but that in pore protein-deficient phospholipids are not accessible in wild-type cells mutants the presence of phospholipid in these and Rc LPS mutants but that phosphatidyl- domains can clearly be shown [170]. ethanolamine in cells of Rd and Re strains, which The results of studies on the localization of LPS lack most of the core sugars of LPS [539] and and phospholipids and the changes in chemical therefore contain decreased amounts of protein composition resulting from mutations and EDTA (see subsection IVA), could be coupled to this treatment enable us to draw models of outer mem- agent. Using conjugated dansylchloride, Schindler branes of the cells discussed in the previous sec- and Tauber [540] showed that phospholipids in Rc tions. Schematic representations of outer mem- mutant cells of S. typhimurium are not accessible branes of wild-type cells, protein deficient mutants, for this agent unless the cells had been pretreated heptose-less LPS mutants and of EDTA-treated with chelating agents like EDTA and Tris. The use cells are shown in Fig. 6. of various exogenous phospholipases shows that the phospholipids of intact cells of E. col] strains IVE. Localization of ECA K-12 [127] and B [541] and of S. typhimurium Rc mutants [539] are resistant to degradation by phos- The ease with which anti-ECA antibodies can pholipases A 2 and C, irrespective of the source of be adsorbed by S- and R-form bacteria suggests a the enzyme, thereby excluding that lack of de- localization of ECA at the cell surface [544,545]. gradation is due to influences of lateral surface The recent chemical purification of ECA in pressure [127]. Tris-EDTA treatment sensitized Mayer's laboratory enabled the investigators to cells of E. col] B and S. typhimurium, but not those localize ECA reliably. Using either ferritin-labeled of E. col] K-12 [127,539,541]. Degradation of or fluorescent monospecific antibodies, Rinno et phospholipids was also observed in cells of al. [202] demonstrated the cell surface location of deep-rough LPS mutants [ 127,539] and of mutants ECA. Interestingly, more ECA was detected in deficient in several outer membrane proteins [ 127]. immunogenic than in non-immunogenic strains These experiments convincingly show that phos- [202]. pholipids are not accessible in intact wild-type Nothing is known about protein-ECA interac- cells. They strongly suggest a localization of the tions except that the level of ECA in E. col] K-12 phospholipids in the inner monolayer, but do not is not altered by the absence of one or more of the strictly exclude a localization outside this layer in proteins OmpA-, OmpC- and OmpF-protein such a way that the phospholipid molecules are (Mayer, H., unpublished data). shielded against the used agents by endogenous molecules. Indeed, it has been shown that phos- I VF. Localization and topography of outer mem- pholipids can be protected by LPS against solu- brane proteins bilization by detergents [163,324]. Moreover, phos- phatidylethanolamine and LPS have been shown 1VF-1. Introduction to form close complexes [139]. However, electron Most proteins of the outer membrane are rich spin resonance studies have clearly shown, both in fl-structure [230,295,317]. They are not solubi- for outer membranes [542] and for model bilayers lized from the membrane by extraction with buffers [542,543], that phospholipids and LPS are segre- with high ionic strength or with EDTA [97, 199, gated into separate domains. These data clearly 222,294], but solubilization requires the presence indicate that the vast majority, if not all, of the of detergents or strong chaotropic agents. Ionic 88

interactions seem to be important for their anchor- wild type K-12 ing in the membrane since either an ionic deter- PL~,~,~,~ ~ ~~ ~,~~ ~,~ ~ ,L gent or a combination of a non-ionic detergent (e.g. Triton X-100, Sarkosyl) with EDTA is re- pore protein quired to solubilize the proteins [97,294,317]. Stud- deficientmutant ies on the localization of outer membrane proteins are merely restricted to three types of major pro- ~ ~I~t~ 1~ ~ll'~ mutant missing teins, namely lipoprotein, OmpA protein and c 1~ ~ 11~~11I~ ~I~I~ m.A peptidoglycan-associated pore proteins. The pre- sent knowledge on their localization will be de- scribed in detail. d ,~ ~ ~11I~I~1~1 ~111~t~ hept o s e_le s s ~J~l~,~~l~ ~l~q~,~ LPS mutant I VF-2. The major lipoprotein (see also Table 1 V) One third of the total amount of lipoprotein is covalently bound to peptidoglycan, whereas the EDTAtreated wild type remainder exists in the free form. Evidence for a cell surface localization of either form is lacking. Fig. 6. Schematic models of the bilayer organization of the Bacteriophages using the lipoproteins as (part of) outer membrane of cells of (a) wild-type E. coli K-12 and Rc their receptor or coupling of the lipoprotein from mutants of S. typhimurium, (b) a pore protein deficient strain, intact cells to exogenous CNBr-activated dextran (c) a strain lacking the three major proteins OmpA, OmpC and have never been described. Moreover, antiserum OmpF protein, (d) a deep rough LPS mutant and (e) wild type E. coli K-12 treated with EDTA. The LPS molecules (L) are directed against the lipoprotein does not react with present exclusivelyin the outer leaflet (OL). In wild type E. coil intact wild-type cells [512]. K-12 (a) all phospholipid (PL) is drawn in the inner leaflet (IL) From X-ray analysis of paracrystals of the lipo- whereas for reasons of simplicity only transmembrane proteins protein, this protein appears to consist of a super- (P) are shown. Outer membranes of cells with complete LPS helix or a coiled coil structure [280]. Since a high are supposed to have the same structure except that the sugar chains are longer. In pore protein deficient mutants (b) part of content of a-helix and lack of #-structure was the missing protein is replaced by molecules of another protein deduced from infra-red and circular dichroism whereas the remainder is compensated for by additional spectra, long stretched a-helices are the most likely amounts of LPS and phospholipid [127]. The latter compensa- conformations of the lipoprotein [278,546,547]. tion is assumed to lead to insertion of phospholipid in the outer Treatment of peptidoglycan-outer membrane monolayer. The resulting phospholipid bilayer regions explain complexes with chemical cross-linkers results in the observed sensitivity of such cells to SDS [236] but are obviously too small to be attacked by exogenous phospholi- dimerization of the free form of the lipoprotein. pases [127]. Sensitivity to phospholipase is observed when, in Moreover, free lipoprotein can be linked to pepti- addition to the two pore proteins, also OmpA protein is doglycan and also to OmpA protein in a dimeric lacking. The outer membrane of such a strain (c) is very poor in complex [513]. Evidence for another interaction of transmembrane proteins, the lack of which is compensated for lipoprotein, namely with pore proteins, comes from by an increase in both LPS and phospholipid content. Deep rough Re mutants (d) have outer membranes with extremely work which shows that the in vitro interaction short LPS sugar chains (see Fig. 3). Substantial amounts of between peptidoglycan and pore protein is outer membrane proteins are lacking [90,92-94] which is com- weakened in a mutant lacking lipoprotein and by pensated for by increased amounts of LPS and phospholipid, trypsin treatment which cleaves the bound form of resulting in sensitivity to detergents [503,504], hydrophobic the lipoprotein from the peptidoglycan [515]. Simi- antibiotics [538] and phospholipases [127]. EDTA-treated E. coli K-12 cells (e) have lost half of their cellular LPS [85] but no larly, experiments designed to study the interac- protein [200]. The resulting lesions in the outer leaflet of the tions between peptidoglycan and pore proteins outer membrane are presumed to be rapidly filled with phos- have shown that the presence of bound lipoprotein pholipid molecules derived from the inner leaflet and from the is required to obtain an ordered lattice structure cytoplasmic membrane, The resulting cells are transiently sensi- [375,398,516]. Also spin labeling studies provide tive to hydrophobic antibiotics [85] and phospholipases [127,541]. For reasons of simplicity ECA and divalent cations evidence for an interaction between lipoprotein have not been indicated. and pore proteins [548]. Therefore it initially was 89 surprising that no cross-linking was found between Lugtenberg, B., unpublished data). The models of lipoprotein and OmpC or OmpF proteins Braun [266] and McLachlan [552] propose that the [397,513,520,521] or between lipoprotein and acyl chains are anchored in the inner leaflet, Salmonella pore proteins [399]. More recent data whereas the protein traverses the periplasmic space. indicate that the vast majority of the amino groups The observation that the protein is still integrated are located within the pore [550,551] and therefore in the membrane of mutant cells lacking some or are not available for cross-linking. Thus, the pre- all of the acyl chains [554-557] seemingly con- sent results certainly do not exclude a direct inter- tradicts these models. However, these results can action between free lipoprotein and pore proteins. be explained by an association of the protein part For a discussion on this subject the reader is of the lipoprotein with OmpA protein or with pore referred to subsection IVF-5. proteins. In conclusion, the localization of the Three different models for the localization of lipoprotein is far from certain. On the basis of the the lipoprotein have been proposed. Inouye [280] available experimental evidence we tend to favour and McLachlan [552] propose an uninterrupted the model of McLachlan. The outside surface of a-helix with a width of 7.6 nm and Braun [266] McLachlan's dimeric lipoprotein structure is hy- proposes two anti-parallel a-helical segments inter- drophilic, consistent with a location in the peri- rupted by a bend near the middle of the molecule. plasmic space [552]. Recently, Inouye and co-workers have proposed a new model consisting of transmembrane assem- IVF-3. OmpA protein (see also Table IV) blies of three monomers of the pore protein, each OmpA protein is an oligomeric transmembrane of which is associated with a trimer of lipoprotein protein. It has sites located at the cell surface (see [7]. Table IV). Its presence prevents cleavage of the Inouye and McLachlan propose interactions be- two leaflets in freeze-fracture experiments tween at least six, and two lipoprotein molecules, [198,200,508]. It can be cross-linked to the free respectively. The proposed existence of lipoprotein form of the lipoprotein and to peptidoglycan. The dimers has been confirmed by cross-linking studies observation that ompA, lpp double mutants, but [513,514] supporting McLachlan's model but not not the two single mutants, grow in spheres and rejecting the others. Inouye's first model proposes lose part of their outer membrane by 'blebbing', a pore function for the lipoprotein [280]. Evidence strongly suggests that both the OmpA protein and for such a function could neither be obtained in the lipoprotein interact with peptidoglycan [271]. vitro [2,338] nor in vivo [553]. Whereas in Inouye's As intact subunits of OmpA protein (but not its models [7,280] the protein part spans the thickness pronase resistant fragments) could be cross-linked of the outer membrane, it is located outside the [513], it is likely that the interaction between indi- lipid bilayer in the periplasmic space according to vidual OmpA protein molecules takes place near the outer models [266,552]. The latter models have the C-termini in or near the periplasmic space. the advantage that they leave some space between Evidence for a localization of the -COOH part of the outer membrane bilayer and the peptidoglycan the OmpA protein molecule at the periplasmic side layer. Under the usual physiological conditions of the membrane comes from the observation that this periplasmic space must be present between this part is only degraded by trypsin after damag- these two layers as it should be expected that due ing the cell such that the enzyme can reach the to turgor pressure no substantial space is left be- periplasmic side of the outer membrane (Refs. 213, tween the cytoplasmic membrane and the pepti- 526, 527 and Van Alphen, L. and Lugtenberg, B., doglycan layer. Moreover, Inouye's models which unpublished data). These experiments suggest a propose large transmembrane assemblies [7,280] localization of the N-terminal part at the cell are not supported by freeze-fracture electron mi- surface or in the membrane, whereas the C-termi- croscopy experiments of wild-type cells and lpp nal part could span the periplasmic space and even mutants as no differences could be detected in the interact with the peptidoglycan. Consistent with morphology of either of the outer membrane frac- this idea are the results on the localization of ture faces (Van Alphen, L., Verkleij, A. and OmpA protein fragments lacking the C-terminal 90 third of the molecule [310,311]. The transmem- sheet structure with strands nearly parallel to the brane part is located between residues 1 and 177 normal of the membrane plane [560]. It can be and the hydrophobic segment of 27 residues is a expected that this approach will yield considerably good candidate to span the membrane ([269], see more structural information in the near future. subsection IIIE-4). A beginning of a study of the structure-function relationship of OmpF protein was made in Nakae's I VF-4. Peptidoglycan-associated pore proteins (see laboratory. Isolated porins were chemically mod- also Table IV) ified, incorporated into liposomes and tested for These proteins include those listed in Table II activity. The results suggest that hydrophilic amino as well as the LamB protein. They have sites acids play an important role in the diffusion of exposed at the cell surface as they interact with charged solutes through the pore [551]. More re- phages, antibodies and CNBr-dextran (Table IV). cently a similar approach has been undertaken in Experiments with chemical cross-linkers indicate Rosenbusch's laboratory [550]. In the latter study that the proteins occur as trimers. Functional pore electrophoretic mobility and several other parame- proteins of strains producing one pore protein ters, but not pore activity, were used as criteria for species consist of three identical monomers [356, native pores. The results support those of Nakae's 357], which act in highly cooperative fashion (sub- laboratory [551] and seem to indicate that all but section IIIE-5). Despite attempts in several labora- one of the lysine residues are contained within the tories to convert inactive monomers into func- pore [550]. Results with insertion and deletion tional trimers renaturation has never been re- mutations in the cloned genes ompF and phoE ported (see for instance Refs. 356 and 357). indicate that protein fragments are not easily ob- Most of the advanced work on the structure of tained (see subsections IllE-5di and ii). The pore proteins has been carried out in Rosenbusch's carboxy-terminal end of PhoE protein seems to be laboratory using the matrix proteins of E. coli essential for the presence of the protein in the strain B E [230,373,375,558]. Electron microscopic outer membrane [415], suggesting a situation dif- examination of protein-peptidoglycan complexes ferent from that of OmpA protein from which the of E. coli B ~ shows that the protein is arranged in carboxy-terminus can be deleted without strong a hexagonal lattice structure [230] with a repeat of functional losses [310,311]. 7.7 nm, which covers the outer surface of the The question of whether heterologous or mixed peptidoglycan [558]. The periodic structure of such pores, containing monomers of different pore pro- a two-dimensional crystal is maintained and even teins, exist in strains with more than one pore improved [375] in the absence of peptidoglycan, protein was approached in three different labora- and is therefore based on strong protein-protein tories. Whereas the results of Palva and Randell interactions [558]. A unit cell probably contains [397] and of Ishii and Nakae [561] with E. coli three protein molecules and a triplet of identations K-12 and S. typhimurium, respectively, strongly which may represent channels which span the pro- suggest that mixed pores do not exist or only to a tein monolayer [558]. Based on these and other minor extent, results of Mizushima's laboratory observations a model of a pore trimer is proposed indicate that when cells synthesize OmpC protein in Ref. 375. The thickness of the protein layer and OmpF protein simultaneously, heterotrimers (4-5 nm) [375] is the same as that of the apolar are formed at random. In contrast, when the two part of a lipid bilayer (approx. 4.5 nm) suggesting proteins are synthesized in separate periods het- that, if the structure still represents the in vivo erotrimers were not found [562]. situation, the porin is practically completely em- The possible interaction of pore proteins with bedded in the membrane. Recently, three-dimen- lipoprotein has already been discussed in subsec- sional crystals of matrix protein have been ob- tion IVF-2. tained [373,559,560]. Successful association into Evidence for an interaction of pore proteins large crystals depended on the use of ~-octylg- with LPS is summarized in Table IV and described lucoside [373]. The first results show that a large in subsection IVG-4. It should be noted that only fraction of the polypeptide is present in fi-pleated very little LPS can be cross-linked to pore proteins 91

of S. typhimurium [399], although it is known that Mizushima's laboratory. Initially it was shown that pore proteins of E. coli interact with LPS isolated OmpC protein and OmpF protein can be [200,217,226,381]. physically reassociated with peptidoglycan in the Tight associations between pore proteins and presence of 5 mM Mg 2+ [318]. Added LPS stimu- peptidoglycan have been found in vitro. The ques- lates the binding, especially of OmpF protein [522]. tion of whether such interactions also occur in vivo Binding is not altered when the covalently bound will be discussed in subsection IVF-5. lipoprotein has been removed from the pepti- The LamB protein has many properties in com- doglycan by pretreatment with pronase [318,522]. mon with the general pore proteins, but seems not Electron microscopic examination showed that an to be related to these proteins [319]. It is surface- ordered hexagonal lattice structure consisting of located (Table IV) and the native form of the OmpC protein and LPS can be obtained over the lambda receptor is a trimer [461,563], which prob- entire surface of lipoprotein-bearing peptidoglycan ably interacts with LPS [237,525], peptidoglycan [516], resembling that obtained after treatment of [319,441,460,525] and with the bound form of the cells in 2% SDS at 60°C [230,558]. Omission of lipoprotein [525]. The lambda receptor activity is either protein or LPS resulted in failure of forma- decreased in heptose-less LPS mutants [237], sug- tion of lattice structure. No ordered lattice struc- gesting that the amount of this protein in the outer ture was formed when peptidoglycan without membrane of such mutants is decreased as has bound lipoprotein was used and OmpC protein been described for other proteins [90,94-96,236]. and LPS assembled in vesicles [516]. Essentially Two-dimensional sheets recently obtained will al- the same results were obtained with OmpF pro- low structural studies [564]. tein. The role of LPS was studied in more detail Since anti-B-galactosidase reacts with intact cells and it was observed that when LPS was replaced containing hybrid proteins of the lambda receptor by lipid A or even by fatty acid, an ordered lattice protein and fl-galactosidase [565], it has been pro- structure was also formed although the lattice con- posed that the carboxy-terminus of LamB protein stant was smaller [523,566]. The intriguing ques- is located near the cell surface. As models for tion of whether these structures are functional was protein translocation are based on this result [5], it studied using adsorption of phage T4 as a tool. should be noted that the evidence for this assump- Indeed, T4 phage adsorption onto reconstituted tions is insufficient [5]. The localization of the cell surfaces containing OrnpC protein as the pro- hydrophilic LacZ protein at the cell surface will be tein component was observed. Both the protein thermodynamically favourable but this not neces- and LPS were essential for this interaction. The sarily implies that the carboxy-terminus of LamB needle even penetrated the cell surface [567] and, protein, without the extremely bulky LacZ protein, when phospholipid liposomes had been included behaves in the same way. in the system, the phage also ejected its DNA [567a]. In this case wild-type LPS could not be IVF-5. Are matrix (pore)proteins associated with replaced by either heptose-less LPS or lipid A. A peptidoglycan in vivo? function is also attributed to the peptidoglycan in Rosenbusch described that the insoluble frac- generating a flat surface large enough to interact tion obtained after heating cells or cell envelopes with all tail fibres of a single phage particle [567]. of E. coli B E in 2% SDS at 60°C consists of The in vitro results described above raise the peptidoglycan-lipoprotein complexes with non-co- questions of whether the hexagonal (matrix) pat- valently attached to it the OmpF protein organized tern is also present in vivo and even whether the in a regular so-called 'matrix' pattern [230]. This observed association between pore proteins and periodic structure is even improved upon removal peptidoglycan as well as the proposed association of peptidoglycan [375]. Evidence has been pre- between pore proteins and lipoprotein mimicks the sented that in vitro binding of the pore protein to in vivo situation. the peptidoglycan occurs partly through the lipo- As argued by Rosenbusch et al. [375] it is protein [515]. unlikely that the regular array of pore proteins 'Matrix' structures have been reconstituted in also exists in the native outer membrane but the 92 hexagonal structure rather is a consequence of the that pore protein cannot [513] or hardly [518] be extraction procedure [375]. The tendency of pore cross-linked to peptidoglycan. However, the ob- protein trimers to form regular aggregates on the servation that most charged amino acid residues peptidoglycan [230] and the improvement of the seem to be located within the pore [550,551] is still long-range order by peptidoglycan degradation consistent with a native complex as the pore pro- [375] suggests the possibility that the protein-pro- tein probably has no reactive amino acid residues tein interaction between pore protein trimers might in the correct position for efficient cross-linking. be an artefact which is easily obtained in a strain Crystallographic data from Rosenbusch's labora- like E. coli B E with a number of monomers of as tory [375] suggest that native pore protein only many as 105 per cell [230] or in reconstituted spans the apolar part of the membrane. This apolar systems with highly purified constituents, but part is probably at a distance of about 9 nm from which is more difficult to obtain in strains contain- the peptidoglycan due to the presence of the lipo- ing several pore proteins or containing less pore protein in the periplasmic space (see subsection protein. We have approached the question of the IVF-2). It is hard to imagine that pore proteins are in vivo occurrence of regular patterns with the spanning the periplasmic space since no space freeze-fracture technique which is so fast and sim- would than be left for the periplasmic binding ple that it is unlikely to give artefacts. Cleava,a,a~e proteins which transport nutrients from the pore planes through the outer membrane show OM to the cytoplasmic membrane (see subsections IA particles which, as they are the reflections of pro- and IIIE-5). The only experimental evidence for tein-LPS aggregates (see subsection IVG-2), indi- the absence of an association in vivo between pore cate the position of proteins. If the matrix pattern proteins and peptidoglycan-bound lipoprotein of the OmpF protein of the outer membrane E. complexes comes from analyses in Witholt's group coli strain B E described by Rosenbusch [230] also of vesicles excreted in the medium by growing occurs in vivo, it should be observed by freeze cells. In comparison with the corresponding cellu- fracturing of wild-type cells of E. coli B E, and even lar outer membranes these medium vesicles con- more striking in cells of an ornpA mutant of this tain only 35% free lipoprotein and almost none of strain. The choice of this mutant has the ad- the bound lipoprotein. Medium vesicles also have vantages that cleavage is preferential through the reduced amounts of OmpA protein, which proba- outer membrane in such mutant cells and that the bly interacts with peptidoglycan, and of the bound OmpF protein represents over 90% of the outer form of the lipoprotein (Table IV), while they membrane protein in this mutant. The results (Fig. contain large amounts of pore forming protein 1 7A) show that, although the OM particles seem to and LamB protein [272], suggesting that interac- be very homogeneous, no crystalline network is tions between pore forming proteins and the present. We consider this as the best evidence that peptidoglycan-lipoprotein complex - if existing the regular pattern is an in vitro artefact. are rather weak [272]. Summarizing, conclusive The answer to the question whether the associa- evidence for a direct interaction between pepti- tion between pore protein with peptidoglycan, or doglycan and pore proteins in vivo is lacking. An in a trimeric complex which also contains the indirect interaction through lipoprotein molecules lipoprotein, is an artefact is much more difficult to is conceivable but, if occurring, is certainly not give. The observation that the complex resists operative for all molecules as discussed above rather extreme conditions (2% SDS, 60°C) seems [272]. Moreover, a space between peptidoglycan to be a strong indication for a natural interaction. and pore proteins has the advantage that the func- Also the observation that the presence of LPS tioning of periplasmic proteins is easier to imag- which is required for many biological activities of ine. outer membrane proteins (see Tables II, III and IV A schematic drawing of the topography of outer and subsection IVG-4) increases the binding, sug- membrane constituents, as discussed so far in sec- gests that it concerns a native situation. An indica- tion IV, is presented in Fig. 8. tion of the opposite was the surprizing observation 93

Fig. 7. Freeze fracture morphology of the outer membrane of an ompA mutant of E. coli strain BE . The concave (outer) fracture face of the outer membrane (O"~I) is almost completely covered with particles (A) whereas the convex (inner) fracture face (OM) contains numerous pits (B), presumably complementary to the particles. In this case the particles are very homogeneous in size which presumably is caused by the fact that the vast majority of the particles and pits represent complexes of LPS with only one protein species, namely OmpF protein. Cells were slowly cooled from 37°C to 4°C in order to optimize conditions for the formation of twodimensional crystals. Subsequently the sample was quenched from 4°C. No signs of crystallization can be detected ( × 100,000). (From Verkleij, A., Leunissen-Bijvelt, J., Van Boxtel, R. and Lugtenberg, B., unpublished data).

IVG. The lipid matrix weakest part of membranes [568,569]. Outer mem- branes of many Gram-negative bacteria are frac- 1VG-1. Is the outer membrane a lipid bilayer? tured indeed [88,200,308,508,510,51 1, This question was approached using three dif- 570-573], indicating that this membrane is basi- ferent techniques, namely X-ray analysis, freeze- cally a bilayer [569]. Remarkable is that only small fracture electron microscopy and 31p-NMR. Al- fracture faces were obtained in wild-type cells. ready in 1975 it was reported that fatty acyl chains This is obviously due to the nature of the outer of E. coli outer membranes occur in bilayer con- membrane, since this phenomenon was also ob- formation as X-ray analysis of isolated membranes served with isolated E. coli outer membranes [574]. showed lamellar reflections [128]. Freeze-fracture The high content of transmembrane proteins, electron microscopy is widely used to study the especially the OmpA protein [198,200], is likely to inside of membranes since fracturing occurs be- be responsible for this phenomenon. The outer tween the fatty acyl ends of the lipids, which is the fracture face of the outer membrane (OM) is '~4 densely covered with particles, which consist of aggregates of protein-LPS complexes (see subsec- tion IVG-2), thereby perturbing the planar lipid bilayer structure and making impressions (pits) in the inner fracture face (O~M) complementary to the particles (cf. subsection IVG-2). More recently, ~P-NMR was used to study the orientation of the phospholipid and LPS in the lipid matrix. The question was of special interest since at the growth temperature the isolated phospholipids yield 3Lp_ NMR spectra and freeze-fracture morphology which are characteristic for a superposition of isotropic, hexagonal and lamellar phase [575], probably due to the presence of phosphatidylg- lycerol and diphosphatidylglycerol next to phos- phatidylethanolamine [575]. The 31P-NMR spectra of the outer membrane (linked to peptidoglycan) turned out to be the superposition of lamellar phospholipids and lamellar IPS [162,574], identi- P6 cal to the spectra obtained from phospholipid-LPS liposomes, which only showed bilayer freeze-frac- CM ture morphology [162]. Moreover, using X-ray dif- fraction on purified LPS, lamellar reflections were Fig. 8. Molecular organization of the outer membrane of Enter- observed, indicating that LPS itself is able to obacteriaceae. The most likely positions of outer membrane maintain the bilayer configuration [577]. In addi- constituents are schematically indicated. LPS and phospholipid tion to the lamellar phase a small amount of molecules are the major constituents of an asymmetric bilayer. Divalent cations (not indicated) are supposed to play important non-bilayer phase was observed in ~LP-NMR spec- roles in interactions of LPS. Only three types of proteins have tra of outer membranes at and especially above been drawn namely pore proteins including LamB protein (PP), the growth temperature [574], which may be re- OmpA protein (A) and lipoprotein (LP). Pore proteins have lated to translocation of proteins, LPS and phos- been drawn without interactions with peptidoglycan and lipo- pholipids from the cytoplasmic membrane to the protein, although such interactions cannot be excluded (see subsections IVF-5 and IVF-1, respectively). Several O-antigen outer membrane (compare with subsection IC and chains are much longer than visualized. ECA has not been Fig. 2). Although the amount of lipid participating drawn for reasons of simplicity. Other aspects of the cell in non-bilayer phases is small (less than 1%) in the envelope like the periplasmic space (PPS) with a nutrient time scale of the measurement, it is possible that a binding protein (BP), the peptidoglycan layer (PG} and the substantial amount of lipid is participating for a cytoplasmic membrane (CM) with a carrier protein (('P) in- volved in transport, have also been drawn. For further explana- very short period of time, which would be con- tion, see test and Table IV. sistent with the idea of temporary adhesion zones involved in the biogenesis of the outer membrane (Fig. 2). Four physico-chemical characteristics of freeze-fracture electron microscopic observations the outer membrane might favour non-bilayer of the fracture faces of the outer membrane, are phases, since they facilitate transbilayer move- caused by LPS (see Fig. 9, for evidence see subsec- ments in model systems, namely gel to liquid tion IVG-2). However, the latter perturbations are crystalline phase transitions [578,579], the presence not responsible for the isotropic signal in the ~P- of integral membrane proteins [580,581], the pres- NMR spectra or for any other spectral feature, ence of large amounts of phosphatidylethanola- since the spectra of outer membranes of wild-type mine [582] and, finally, the presence of LPS which cells (rich in particles) of a mutant lacking OmpA is also supposed to disturb the lipid bilayer, since protein, OmpF protein and OmpC protein (and OM-particles and OM-pits, which are visible in therefore deficient in particles) and of LPS-phos- 95 pholipid liposomes are indistinguishable. This in- nature of the freeze-fracture particles have been dicates that LPS has probably little diffusional misinterpreted in an important review [2]. motion around the curved surfaces of the particles, Several E. coli K-12 mutants lacking major probably due to immobilization by the protein in outer membrane proteins have reduced amounts of the particle [574]. In conclusion, the data of X-ray both particles and pits especially those lacking two diffraction, freeze-fracture electron microscopy and or three proteins. This reduction roughly correlates 3~p-NMR spectroscopy indicate that the outer with the total reduction in amount of major outer membrane is largely a planar lipid bilayer with membrane protein, although the effect of the perturbations visible as O'M-particles and (~M-pits. OmpA protein is always stronger than that of the OmpF and OmpC proteins [198]. A special prob- IVG-2. Nature of O~M particles and OM pits on the lem in these experiments was that the lack of one fracture faces of the outer membrane observed with protein is compensated for by an increase in the freeze-fracture electron microscopy amounts of one or more other proteins (Refs. 96, In the Gram-negative bacteria E. coli 129, 306 and 586; see also Fig. 6B). Since these [200,308,508,510,570], S. typhimurium [88,571,572], compensatory effects are absent in strains lacking Ps. aeruginosa [511] and Acinetobacter [57~ the all three of these major proteins (Refs. 96 and 306; outer fracture face of the outer membrane (OM) is see also Fig. 6C), the role of various outer mem- densely packed with particles. In E. coli K-12 the brane proteins in particle formation was studied particles are 4-8 nm in diameter and occur in by comparing mutants lacking the three proteins estimated numbers of 6000-10 000/~m 2 [198,508] OmpA, OmpC and OmpF protein with strains (Fig. 7A). The opposite inner fracture face of the which contain large amounts of one of the proteins outer membrane (O~M) contains 3000-5000 pits/ OmpA, OmpF, OmpC, LamB or PhoE protein t~m2 (Fig. 7B). The latter numbers are probably [200]. Whereas only about 25% of the surface area underestimated [198] and, as the numbers of both of the OM of the former strains was covered with particles and pits are decreased in various mutants particles (and of the (~M with pits), the presence [~8,200] it is very likely that O~M-particles and of each one of the latter five proteins, which OM-pits are complementary [200,570,584]. Around resulted roughly in an increase of the protein 1974 freeze-fracture particles were generally con- content of the outer membrane to the wild-type sidered as reflection of membrane proteins. The level, lead to an increase to 90-100%. These availability of mutants deficient in outer mem- experiments convincingly showed the capacity of brane proteins therefore prompted several investi- these five proteins in the formation of O~M-par- gators to study the freeze-fracture morphology of ticles and OM-pits [200]. the outer membrane of E. coli in order to elucidate The first indication for a crucial role of LPS which proton(s) was (were) involved in the forma- came from the observation that treatment of E. tion of OM-particles [88,198, 200, 308, 508~ 510, coli K-12 cells with EDTA, which causes the re- 571,572]. In the course of this work it became lease of half of the cellular LPS but not of protein, clear that protein could not be solely responsible resulted in the loss of about half of the OM-par- [198,200] and that particles with complementary ticles thereby causing smooth patches on the OM pits must be considered as perturbations of the [198]. These results lead to the hypothesis that LPS lipid bilayer by lipids [569,584,585]. By now there complexes form the basis of O'M particles [198]. is a general agreement that the O~M particles and Reasoning that, if this hypothesis were correct, it O~M pits in outer membranes of Gram-negative might be possible to generate LPS-particles in bacteria are reflections of protein-LPS complexes particle-poor cells, the strain missing OmpA, [2,15,200,511]. It is likely that in the cell protein is OmpC and OmpF proteins was incubated with required to generate such complexes from which Ca 2+ in the presence of chloramphenicol. Indeed, the morphological appearance is determined by this Ca 2+ treatment resulted in an increase of the the molecular organization of LPS [198,200,363]. particulated area from 25-30% to 90-100% [200], In the following we will explain the evidence for without affecting the protein or LPS content of the this notion in detail as the experiments on the cells. That these newly generated particles and 96 pits, which are morphologically indistinguishable pit, which explains all published data, is given in from those in wild type cells, are indeed caused by Fig. 9. It should be emphasized that in addition to LPS aggregation, was shown by three types of lipidic intramembranous particles, which have experiments. Firstly, EDTA treatment of the complementary pits and which occur in bacterial Ca"~4 -treated cells resulted in particle-poor outer outer membranes but also in many other systems membranes accompanied by the loss of 60% of the including phospholipid model membranes [585], cellular LPS without loss of protein. This result many other intramembranous particles exist which stressed the role of LPS by showing that particles do not have complementary pits and which reflect and pits with the same morphology as those occur- proteinous structures e.g. those in red blood cell ring in the outer membrane of wild-type cells, can membranes. be generated by LPS-Ca 2+ complexes. Secondly, O~M particles of Ps. aeruginosa have been when the mutant which lacks three proteins and studied by Eagon, Gilleland, Stinnett and co- therefore is particle-poor was replaced by another workers. Initially we were unaware of their results, strain which, in addition, also has shorter, which despite the very different approach, lead to heptose-less, LPS, Ca 2+ treatment did not result in a similar hypothesis. Their results can be sum- an increase of the number of particles [200]. The marized as follows. Also in Ps. aeruginosa EDTA most likely explanation for this result is that the treatment results in disappearance of particles. LPS-Ca 2 + interactions occur via the heptose-bound The treatment is accompanied by the release of phosphate residues of the LPS [200]. Thirdly, LPS and, in contrast to E. coli, also of protein. purified LPS itself is able to form particles of the Moreover, small cylinders could be detected in the same size as those found in whole cells [162]. extract with the diameter of OM particles [511]. In conclusion, it has been established that both The damage to the cells can be restored by incuba- LPS and at least five different major outer mem- tion in fresh medium and this process requires brane proteins play a role in the generation of protein synthesis and energy [587]. particles and pits. The protein component inter- The only results containing evidence against acts with LPS, resulting in the generation of par- our model (Fig. 9) are those of Smit and co-workers ticles and pits. As the LPS component is responsi- [88]. Using LPS mutants of S. t)~phirnurium, they ble for the morphology of these structures, it is determined the number of particles per ~m 2 and likely that only a small portion of the protein is calculated the density of various outer membrane located within the fracture plane [569]. All experi- constituents per unit cell surface• They found a mental data are explained by a model in which perfect correlation between the number of par- complexes of various LPS molecules and one or ticles and the amount of protein per unit of cell more protein molecules of one protein species are surface [88]. With the present knowledge several assumed to form a complex, which is visible as an comments must be made with respect to their O~M particle with a complementary O~M pit. Thus data. (a) The amount of protein was determined the population of particles in a wild type outer with the method described by Lowry et al. [130], membrane is a collection of protein-LPS com- which is known to overestimate the amount of plexes from which in 75-80% of the cases the protein in some outer membrane mutants [127, protein component is one of the proteins OmpA, 129]. (b). Smitet al. [88] conclude that the number OmpC or OmpF protein [200]. As LPS without of LPS molecules per unit of surface area is not protein can generate such a particle and as influenced by LPS-mutations. Data from other EDTA-treated cells with their normal outer mem- laboratories on similar mutants of E. coli and S. brane protein content have lost about half of the typhimurium indicate that deep-rough mutations number of particles [198], LPS is likely to play a result in considerable [94,96] or even dramatic more direct role in the morphogenesis of the par- [92,93] increases in the number of LPS molecules• ticle than the protein component. The presence of (c). As LPS is now known to form the basis of the protein component is assumed to be a prere- (~M-particles (Fig. 9), it is dangerous to use LPS quisite for LPS aggregation [200]. A molecular mutants for quantifying the number of particles. It model of an (~M particle with complementary OM is conceivable that such mutations influence fac- 97

tors like size, number and stability of OM par- Boxtel, R., and Lugtenberg, B., unpublished data). ticles. Even slow cooling from 37°C to 4°C did not yield Some additional comments to the model of O~M regular patterns. (iv) As many proteins involved in particles and OM pits are the following. (i) The the generation of particles are pore proteins, it was presence of units consisting of only one protein hypothesized that most of the particles were the species has subsequently been supported by data morphological reflections of the intramembranous from stt/dies with chemical cross-linkers (see Table part of the pores [200]. Indeed, a correlation be- IV). It therefore is likely that the protein compo- tween the number of particles and the activity of nent of the particle is a trimer in the case of a pore various pores has been reported [363]. (v) In protein. (ii) As mutants lacking both forms of the mutants with a decreased number of OM particles lipoprotein have a morphology indistinguishable it clearly can be shown that both particles and pits from that of wild-type cells (Van Alphen, L., are laterally mobile. Slow cooling of the prepara- Verkleij, A. and Lugtenberg, B., unpublished data) tions before quenching showed, in addition to it is likely that the lipoprotein is not involved in large particulate areas, large smooth areas on the the formation or stabilization of particles and pits. O~M [198,200]. In wild-type E. coli K-12 the same (iii) several attempts have been undertaken in our effect can be observed except that the smooth laboratory to study particles and pits in more areas are much smaller [508]. The reported lack of detail. All biochemical isolation procedures used smooth areas on the O~M of a S. typhimurium Rc were unsuccessful. Also attempts to generate mutant [88] is probably caused by quenching from two-dimensional crystals by forcing high amounts a temperature above the phase transition. of only one protein species into the outer mem- brane, have failed. This was accomplished by either IVG-3. Physical properties of LPS and phospholi- using an ompA mutant of E. coli B E in which over pids in the outer membrane 90% of the remaining outer membrane protein The outer membrane undergoes a relatively consists of OmpF protein (Fig. 7) or by introduc- broad thermotropic order-disorder phase transi- ing a multicopy plasmid harbouring the cloned tion over a temperature range of about 20°C as phoE gene in an otherwise particle-poor back- measured with X-ray diffraction [128], 2H-NMR ground (Verkleij, A., Leunissen-Bijvelt, J., Van [588-590], differential scanning calorimetry [575,591] and fluorescence spectroscopy [578]. The temperature of this transition range is about 7°C higher than that of the cytoplasmic membrane [590], which can be explained by the higher degree of saturation of the fatty acids of the phospholi- pids [137] and the lower phosphatidylglycerol con- tent of the outer membrane [590]. These results were obtained using fatty acid auxotrophic mutants fed with various fatty acids. Using wild-type cells of E. coli K-12 the thermotropic phase transition temperature range of the phospholipids appeared to be dependent on the growth temperature of the cells, such that the cytoplasmic membranes are in Fig. 9. Schematic model of a transversal section through the a mixed gel plus liquid-crystalline state or liquid outer membrane with a LPS-protein complex in the middle, state at the growth temperature [578]. Wild type E. which - upon cleavage of the lip~id bilayer - results in an O'~M coli K-12 cells are unable to grow below 8°C, particle with corresponding OM pit. Interactions of an when all the phospholipids are in the gel state oligomeric (in this case a pore protein) with LPS, divalent cations and possibly polyamines are sup- [578]. The shift in phase transition temperature posed to result in a wedge-shaped organization of the LPS range is most likely caused by changes in the fatty molecules and therefore in the perturbation of the lipid matrix. acid composition of the phospholipids as a conse- See also Ref. 200. quence of differences in the growth temperature 98

[ 137]. The transition temperature range of the outer membrane. However, only Nikaido et al. used membrane was less sensitive to changes in the outer membranes still connected to the pepti- growth temperature than that of the cytoplasmic doglycan layer while the other data [593] were membrane, presumably due to the presence of LPS obtained with outer membranes from spheroplasts. [592]. The result is a mixed gel plus liquid crystal- The latter isolation procedure is known to result in line state for the outer membrane at all growth randomization of LPS, and therefore probably also temperatures (8 46°C) [578]. X-ray diffraction of phospholipid, over both monolayers of the outer data indicate that only 25-40% of the outer mem- membrane [89]. Such a randomization could ex- brane lipids is participating in the phase transi- plain the strong influence of LPS on the motional tion. This percentage is twice as high in the cyto- freedom of the phospholipids. It would be interest- plasmic membrane [128]. On the other hand, 2H- ing to repeat the experiments of Takeuchi et al. NMR spectroscopy experiments indicate that all [594,595] with outer membranes connected to the phospholipids are participating in the order- peptidoglycan. disorder transition [590]. The phospholipids of the LPS is not only able to participate in phase outer membrane certainly are not immobilized transition, but shows a phase transition itself. X-ray [589,590], although they have less motional free- diffraction [577,592]~ light scattering [170] and a dom than those of the cytoplasmic membrane in di fferential scanning calorimetry [ 170] experiments the phase transition [590]. Restricted motion of E. with E. coli LPS revealed a rather broad transition coli outer membrane lipids has also been reported [170,577] in which the midpoint temperature and by Rottem and Leive [593] from electron spin the transition range were dependent on the growth resonance spectroscopy, using spin-labeled fatty temperature of the cells from which LPS was acids as a probe. The motional freedom increased isolated [170]. A lower growth temperature was strongly when the cells used for membrane isola- correlated with the occurrence of more un- tion had previously been extracted with EDTA, saturated fatty acids and a lower transition tem- suggesting that LPS plays an important role in the perature in light scattering, the disappearance of a restricted mobility of the lipids in the membrane transition observed with X-ray diffraction, and an [593]. Nikaido et al. [542], however, could not increase in the fluidity of lipid A as measured with detect any restricted motion for the same spin- electron spin resonance spectroscopy [ 170,592,596]. labeled fatty acids. Spin-labeled fatty acids as Using freeze-fracture electron microscopy on probes have the disadvantage that the distribution purified LPS several transitions were observed be- of the probe over the various lipid domains in the tween 22 and 37°C at temperatures which were membrane is unknown. Therefore, Takeuchi et al strongly dependent on the hydration conditions [594,595] used a much more 'natural' approach by and the degree of purity of the LPS preparation labeling the membranes of E. coli biosynthetically [144,162,597]. The broad range of transition might in situ with spin-labeled phospholipids. Spin- be explained by the heterogeneity in the composi- labeled phosphatidylglycerol was found to be much tion of LPS, both with respect to the various more restricted in its motional freedom than phos- substituents [165,599] and to the length of the phatidylethanolamine. The authors explain their sugar chain [ 142,183-185,597,598]. result by assuming that phosphatidylglycerol inter- The diffusion constant of the phospholipids in acts strongly with proteins, while phosphatidy- the outer membrane as determined with ESR spec- lethanolamine probably forms the matrix of the troscopy is as high as in the cytoplasmic mem- outer membrane. When part of the LPS was re- brane and in model membranes (D = 2.5-10 s moved by EDTA treatment, the mobility of both cm2/s) [542], implying that a phospholipid mole- phospholipids was found to be increased, con- cule moves over the length of the E. coli cell in 1 s sistent with the data of Rottem and Leive [593] [138]. The diffusion constant for LPS is about with spin-labeled fatty acids. All these experi- 3 • 10 13 cm2/s [600], determined by following the ments, except those of Nikaido et al. [542], indi- rate of distribution of newly synthesized LPS over cate that the outer membrane phospholipids are the cell surface. In these experiments a galE mutant more immobilized than those of the cytoplasmic of S. (vphimurium was used and a pulse of corn- 99 plete LPS was synthesized by shifting the cells PhoE protein and protein III were found. The from galactose-less to galactose-containing me- existence of interactions of LPS with the proteins dium. This LPS was detected using ferritin-labeled OmpA, OmpC, OmpF, Tsx, LamB and PhoE pro- antibodies specific for the O-antigen [600]. With tein was clearly established by using the property similar experiments it was shown that LPS diffuses that LPS was required for in vivo and/or in vitro from its 50 sites of insertion in the outer mem- phage receptor activity ([ 171,217,236, 237,293,422, brane over the surface of the cell [35,600,601] into 526]; see also Table IV). Additional evidence for domains [537]. This LPS can be isolated and does an interaction of pore proteins with LPS comes not mix with 'old' LPS for at least one generation from the observation that the presence of LPS [537]. In E. coli and S. typhimurium two classes of stimulates the binding of these proteins to pepti- LPS have been recognized, one of which is extrac- doglycan [522,606]. Moreover, the presence of LPS table with EDTA [85]. The latter LPS fraction is is required for in vitro pore activity of OmpF not identical to the fraction of newly synthesized protein [362]. In the case of OmpA protein, several LPS described above, since extraction of cells with properties like resistance against protease and heat EDTA [85] resulted in extraction of both newly denaturation [526], electrophoretic mobility, recep- synthesized and 'old' LPS [537]. tor activity for phages and for donor cells in Lateral diffusion constants of phopsholipids, conjugation [217,308,381] are dependent on the LPS and pore proteins were determined very interaction with LPS. Lipid A is essential for these elegantly with the 'fluorescence redistribution after properties, although the core sugars may also play photobleaching' technique by Schindler et al. a role as the rate of adsorption of phage K3 to [602,603]. They found that the diffusion coefficient vesicles containing lipid A and OmpA protein is for both LPS and phospholipids in LPS-phos- strongly reduced compared to the rate measured in pholipid (1 : 1, w/w) liposomes is about 10 -9 cm 2 the same system containing complete LPS [171]. • s ~ at 24°C. For phospholipids this value is an Freeze-fracture electron microscopic studies order of magnitude lower than in pure phospholi- strongly suggest that the majority of the particles pid liposomes. The mobility of the phospholipids on the outer fracture face with corresponding pits is hardly influenced by the incorporation of pore on the inner fracture face consist of complexes of protein in these mixed bilayers, whereas the mobil- LPS with either OmpA, OmpC, OmpF, PhoE or ity of LPS decreases ten-fold when liposomes con- Lamb protein (see subsection IVG-2). taining 60% protein were used. The diffusion con- In order to obtain a better understanding of the stant for the protein was < 10 12 cm 2 . s- ~. After interaction between OmpA protein and LPS, the fusion of cells with liposomes containing spin- interaction of phage K3 both with its receptor in labeled LPS, Schindler et al. determined the diffu- whole cells and with its reconstituted receptor was sion coefficient of LPS in outer membranes as examined in detail [170]. The temperature depen- 2.10 l0 cm 2" s-l, indicating more restricted mo- dence of the adsorption rate of phage K3 to OmpA tion than in liposomes. This value is much higher protein-LPS complexes (containing LPS isolated than that obtained by M~ihlradt et al. [600]• from cells grown at either 12°C or 37°C) is similar to that of the adsorption rate of the phage to the IVG -It. Interactions of proteins with the lipid matrix corresponding cells, showing that the reconstituted (see also Table IV) complex imitates the in vivo situation very well. To our knowledge the first indications for Therefore it was concluded that in wild type cells LPS-protein interactions for E. coli, at least in OmpA protein-LPS complexes do indeed occur. In vitro, were published in the early seventies an Arrhenius plot of the rate of phage adsorption [604,605]. The first indications for in vivo interac- to the OmpA protein-LPS complexes, two inflec- tions came from analyses of the proteins and LPS tion points were detected [170] at temperatures in mutants with incomplete structures. In deep- which fall in the transition range of the outer rough mutants of E. coli [87,93,94,96,236] and S. membrane (subsection IVG-3). The inflection point typhimurium [88,90,92] decreased amounts of outer temperatures are dependent on the fatty acid com- membrane proteins, especially of OmpF protein, positions of LPS [170]. Moreover, since the higher lOt) inflection point temperature, both for LPS from with a model in which wild-type cells harbour no cells grown at 12°C and at 37°C, was also found phospholipid in the outer monolayer of their outer in pure LPS in both light scattering and differen- membrane (Fig. 8). tial scanning calorimetry measurements, it was concluded that this transition is a thermal one. IVG-5. Distribution of outer membrane constituents ~ P-NMR measurements showed that at the higher over both monolayers transition temperature LPS is more mobile [162]. Nikaido and co-workers were the first who This increase in fluidity could be correlated with calculated the distribution of the various outer differences in the fatty acid composition of LPS membrane constituents over both monolayers isolated from cells grown at 37°C and at 12°C [88,607]. These calculations are based on the [170]. A similar increase in the fluidity of LPS was knowledge of the surface covered by the individual observed in electron spin resonance spectroscopy molecules, of the amounts of the various outer when lipid A from Pr. mirabilis cells grown at membrane constituents relative to the mass of the 43°C was compared with that of cells grown at cell and of the surface to mass ratio of the cells 15°C [596]. The transition of purified LPS from [88]. However, the surface of the cell is very dif- small bilayer ribbons to large vesicles, observed by ficult to determine since in each culture cells of freeze-fracture electron microscoppy [162], might various sizes occur, and since artefacts may be be related to this thermal phase transition. Since introduced during the preparation of samples for the lower inflection point temperature could not electron microscopic observation. The surface area be correlated with a transition in isolated LPS, but occupied by phospholipids and lipopolysaccharide as it is influenced by the type of LPS which is can be estimated from measurements in model incorporated in the protein-LPS complexes, it is systems, but this is impossible for proteins for likely that the interaction between OmpA protein which the tertiary structure is unknown. Smit et al. and LPS is involved in this transition. This transi- [88] therefore determined the surface occupied by tion may be detected with ESR, using spin-labeled the proteins by subtracting the surface of LPS and LPS or with 2H-NMR measurements, using de- phospholipids from the measured total cell surface. uterated LPS. In order to investigate whether the In order to circumvent the surface measure- phase transitions found for the OmpA protein-LPS ments we have used another approach to de- complexes generally occur in protein-LPS com- termine the distribution of outer membrane con- plexes, it would be interesting, but extremely tedi- stituents over both monolayers. It is based on the ous, to study the phase transition characteristics of observation that the lack of the proteins OmpA, other phage adsorption processes, in which outer OmpC and OmpF protein in E. coli K-12 strain membrane proteins are involved. P692tut2dl is compensated for by increased The phase transition behaviour of the OmpA amounts of phospholipid and LPS [127]. We there- protein-LPS complexes was strongly influenced by fore reasoned that the bilayer space occupied by the presence of phospholipids. The phase transi- these three proteins in the parent strain P400 is tion characteristics of the inactivation of phage K3 equal to that occupied by the increased amounts by OmpA protein-LPS-phospholipids differed of phospholipids and LPS in the mutant strain from those of OmpA protein-LPS complexes and P692tut2dI. Furthermore, as these three proteins the characteristics of the adsorption of phage K3 together with the lipoprotein account for 95% of to cells of a mutant containing wild-type LPS, but total outer membrane protein (see legend of increased amounts of phospholipids in its outer Table V), we assumed that the bilayer surface oc- membrane, part of which is present in the outer cupied by the total outer membrane proteins was monolayer (see Fig. 6B), differed from the phase equal to this area plus the area occupied by the transition characteristics of the adsorption to lipidic part of the lipoprotein, probably resulting wild-type cells in a similar way [170]. These results in a small underestimation of the space occupied indicate that in wild-type cells phospholipids do by the outer membrane proteins. The results of not influence the receptor domain of OmpA pro- our calculations are summarized in Table V. The tein-LPS complexes [170], a conclusion consistent surface area occupied by a lipoprotein molecule 101 can be estimated as about 0.8 nm 2 since according layers. Smit et al. [88] calculated that approxi- to the models of McLachlan [552] and Braun [266] mately half of the outer monolayer is occupied only the fatty acids of the lipoprotein are em- with lipopolysaccharide and three quarter of the bedded in the bilayer region and since these fatty inner monolayer with phospholipids. The dif- acids are linked to a glycerol residue as in tri- ferences between their values and ours cannot be glycerides. This results in an almost negligible ascribed to inaccuracies in the surface determina- contribution of the lipoprotein to the bilayer area tions alone. Other factors may be (i) most recent (see footnote g of Table V). The surface area oc- value for the surface area occupied by a LPS cupied by a phospholipid molecule is 0.59 nm 2 as molecule is three times higher than the value de- determined with monolayer experiments on total termined previously [176], and (ii) differences in E. coli phospholipid at maximal surface pressure protein : LPS : phospholipid ratio, probably caused [608]. This is in good agreement with the value by an overestimation of the amount of protein as a found for S. typhimurium [176]. From recent de- consequence of the use of the method developed terminations of the surface area of a monomer of by Lowry et a1.[127,129] and by differences in LPS in monolayer experiments a value of 2.50 nm 2 growth conditions and strains (compare subsection can be deduced [139]. The results of these calcula- IIIA). tions (Table V) indicate that about one half of the surface area of the inner monolayer and about one IVH. Molecular organization of the outer membrane third of surface of the outer monolayer is occupied of Enterobacteriaceae by protein, whereas phospholipids and LPS, re- Based on the data of the previous sections of spectively, cover the remainder of these mono- this chapter, a model (Fig. 8) can be constructed

TABLE V CALCULATED CONTRIBUTIONS OF PHOSPHOLIPID, LPS AND PROTEINS TO THE SURFACE AREA OF THE OUTER MEMBRANE OF ESCHERICH1A COLI K-12 STRAIN P400

Component Composition Average Molar Surface area Fraction of surface area in % g (wt%) a molecular ratio per molecule weight (nm2 ) Outer Inner monolayer monolayer

Phospholipid 19.5 700 27.9 0.59 c 0 46 LPS 43.7 4500 b 9.7 2.50 d 68 0 Major protein (OmpF, C, A) 19.8 36000 0.55 _ e 32 50 Lipoprotein 15.2 7 200 2.1 0.8 f 0 4

a Derived from Ref. 127. b From unpublished observations and data from Ref. 181. LPS is considered to be a monomer [165]. c From monolayer studies by Haest et al. [608] on total phospholipid of E. coli. d From monolayer studies by Fried and Rothfield [139] on LPS of S. typhimurium strain G30(galE), which has a structure similar to that of E. coli K-12. c The surface area occupied by individual protein molecules is not known. The amount of bilayer space occupied by OmpA, OmpC and OmpF proteins in E. coli K-12 wild-type cells was calculated by assuming that the bilayer space which is not occupied by these proteins in cells of the OmpA, OmpC and OmpF protein deficient strain P692tut2dl is filled with LPS and phospholipid. f Only the fatty acids are assumed to occupy space in the bilayer [552], predicted to correspond with 1.5 times the surface of a phospholipid molecule (see text). g The outer membrane of strain P400 is considered to be asymmetric, in that LPS is exclusively located in the outer monolayer and phospholipid and the lipoprotein exclusively in the inner monolayer (see text). The sum of the amounts of OmpA, OmpC and OmpF protein and the lipoprotein is assumed to be equal to total outer membrane protein for the calculations of the surface areas occupied by the proteins (c.f. note e), since the total amount of OmpA, OmpC and OmpF protein is 54% and the amount of the lipoprotein is 40% of the total outer membrane protein. The latter value is obtained from the weight ratio of the sum of OmpA, OmpC and OmpF protein (about 2.105 copies per cell with an average molecule weight of 36000) over total lipoprotein (about 7.5.103 copies per cell, molecular weight 7200). 102 for the outer membrane of wild-type cells of E. coli fusion with liposomes [136,140] and blebbing and probably also for thai of other Enterob- [47,272] are easier to understand. acteriaceae. Only the pore proteins, OmpA pro- 3. The outer membrane of an E. coli K-12 tein, lipoprotein, LPS and phospholipids are in- mutant in the envA gene, contains a decreased cluded in this picture, since only their localization amount of LPS and has an increased influx of and interactions have been established reasonably hydrophobic drugs. This phenotype could be sup- well. The most important properties of the outer pressed by a second mutation which brings about membrane will be discussed here using this model. an increase in the amount of outer membrane 1. Diffusion of hydrophilic solutes occurs via protein [612]. Considering the model of Fig. 8, we general pores which consist basically of trimers of favour the following explanation. The decreased pore proteins complexed with LPS. The specificity amount of LPS due to the envA mutation is partly of the pore is determined more or less by the replaced by phospholipid. The resulting phos- properties of the pore protein and, at least in the pholipid bilayer explains the sensitivity to the anti- case of the LamB protein, by the periplasmic biotics. This phenotype can be suppressed by a maltose-binding protein. second mutation, sefA, which causes an increase of 2. The outer membrane is hardly or not at all the protein content and/or a decrease in the phos- permeable for hydrophobic substances. Permea- pholipid content resulting in the virtual disap- tion of hydrophobic substances between LPS pearance of phospholipid from the outer mono- molecules is unlikely since these molecules are layer. In the light of the often occurring protein- strongly negatively charged in the core-lipid A LPS interactions it should be noted that an in- region. The LPS molecules are held together by crease in outer membrane protein content, accom- positively charged divalent ligands (especially panied by a still lower LPS content [612], seems Ca 2+) [158,197,199], thereby complexing LPS in only possible if not all LPS present was already various ways [158,162]. The impermeability of the involved in LPS-protein interactions. In other outer membrane for hydrophobic substances has words, the results are consistent with the presence been attributed to the absence of phospholipid of substantial amounts of LPS in interactions bilayer regions in the outer membrane [361]. In- which differ from protein-LPS interactions. LPS- deed it has been shown that small hydrophobic LPS interactions seem to be the only possible molecules dissolve in a lipid bilayer [609,610]. We alternative and this explanation is consistent with have already described that pore protein deficient both the model of Fig. 8 and with the observation mutants have increased amounts of phospholipid, that LPS is present in various domains [537]. part of which is located in the outer monolayer. 4. During bacteriophage infection and bacterial This is also considered to be the explanation for conjugation specific processes, of which most de- the sensitivity of these mutant cells for SDS [236]. tails are still unknown, are involved in the penetra- A similar effect may be caused by the treatment of tion of large DNA molecules into the cell. Some P. mirabilis cells with the antibiotic cerulenin in phages push their injection needle into the cell the presence of exogenous fatty acids. Such a after recognition of the receptor, likely in a mecha- treatment makes the cells sensitive to the antibio- nical way [613]. Since zones of adhesion are likely tics vancomycin and rifampicin and results in a involved, non-bilayer conformations of the mem- decrease of the LPS content by 30-50% [611]. brane might be important for this process (see Although the outer monolayer is certainly poor in Fig. 2). phospholipids it may be an exaggeration to say Naked DNA can be brought into E. coli cells in that they are absent in that monolayer. Nixdorff et a very inefficient way by a process called transfor- al. [163] have shown that the presence of LPS mation. This can only occur in competent cells, i.e. protects phospholipids in liposomes against solu- cells which have been treated with high concentra- bilization by detergents. Therefore such shielding tions of Ca 2+ and which subsequently have been effects might have prevented the detection of small given a temperature shock [614,615]. The two com- phospholipid-containing areas in the outer leaflet. ponents of this treatment, the effect of Ca z~ and With the presence of such areas processes like the passage through the phase transition, are 103

known to promote non-bilayer organization of the near future progress can be expected in our lipids. DNA thus may enter, in a very inefficient knowledge of the standard laboratory strains as way, through such temporary perturbations in the well as in that of strains which are more relevant membrane. A decrease of the outer membrane to men, animals or plants. barrier can also be induced by Ca 2÷ treatment Since it is clear that LPS is a heterogeneous alone [616]. This causes a less drastic effect as it is molecular species (see subsection IIIC-3) there is a not sufficient for obtaining transformation-com- need for application of better separation methods, petent cells (Van Die, I.M., Bergmans, J.E.N. and for instance high pressure liquid chromatography. Hoekstra, W.P.M., unpublished data). The significance of the separated species for the 5. The outer membrane is a dynamic structure, various functions of lipopolysaccharide can subse- in which newly synthesized material is inserted quently be investigated in vitro. The physico- and from which certain parts are excreted as blebs. chemical behaviour of LPS is largely unknown Most of these mechanisms are unknown. Essential [ 162]. Extensive NMR-studies (both 31P-NMR and is that the structure allows such changes. It seems 2H-NMR) will be required on complete LPS as likely that non-bilayer phases in zones of adhesion well as on partial degradation products in order to play an important role (see Fig. 2). Moreover, understand its complex properties as a membrane since experiments in which vesicles have been fused component. with cells indicate that the lipids are exchanged Detailed information on the structure of the between the two membranes [136,140], a similar pore proteins will be available when crystals of the process might be involved in the biogenesis of the purified proteins are large enough for crystallo- outer membrane. graphic analysis of the tertiary and quaternary structure of the pore proteins [373]. The structure- 1VI. Outer membrane of other Gram-negative function relationship of outer membrane proteins bacteria will be studied using chemically modified proteins. Site-specific mutations in cloned DNA will be Very little is known about the structure of the used to bring about any desired mutation. Also the outer membrane of other Gram-negative bacteria. use of monoclonal antibodies will be a great help The occurrence of proteins similar to lipoprotein, in elucidating the topology of surface-exposed sites. OmpA protein and pore proteins has been Furthermore, the availability of cloned DNA will described in various species. Since several Gram- enable us to study the regulation of the synthesis negatives like Neisseriaceae and H. influenzae are of outer membrane constituents in detail. It seems very sensitive to hydrophobic antibiotics, it seems likely that many details of the exiting area of reasonable to predict that their outer membranes biogenesis of outer membrane proteins will be contain phospholipid bilayer regions. Indeed, in a known within a few years as cloning and site- recent paper evidence for this idea is presented in specific mutation enable us to study the role of the the case of N. gonorrhoeae [617]. The presence of signal peptide and of other important features of such phospholipid bilayer areas can be a reason the molecule. why certain Gram-negative bacteria shed off Gram-negative bacteria occur in a wide variety numerous outer membrane blebs. A low content of of environmental circumstances. For example, those outer membrane proteins involved in Chlamydia lives intracellular [618], Pseudomonas in anchoring the membrane to,the peptidoglycan layer surface water and soil, E. coli in the digestive tract, is also likely to contribute to this phenomenon. N. meningitidis in the upper respiratory tract and Agrobacterium in plants. This implies that these V. Future prospects organisms have totally different requirements for growth and that their outer membranes meet vari- The knowledge of the architecture and function ous environmental conditions. Comparison of of the outer membrane of E. coli and S. membranes of various organisms has just started. typhimurium has reached an advanced state, al- Moreover, within certain bacterial species large though many questions are still unanswered. For differences have been observed. For example E. 104 coli is a harmless inhibitant of the intestine of other hand, it has been shown that expression of many animal species, but some strains are patho- OmpA protein of various Gram-negative bacteria genic causing severe diarrhea whereas other strains into E. coli did not meet serious problems [315]. It can cause urinary tract infections, they are able to can also be expected that the near future will be invade the host organism and to grow in the blood important for the development of peptide vaccines stream of in the cerebrospinal fluid causing the [632]. The second decade of outer membrane stud- very severe infectious diseases sepsis and meningi- ies will be as competative and laborious as the first tis. Complex mechanisms are involved in ad- one and certainly even more exciting. herence, invasion of the tissue, and in coping with the many defense mechanisms of the host ]17]. Acknowledgements Surface structures of the cell are involved and data are beginning to appear showing that fimbriae, The authors greatly enjoyed the many fruitful outer membrane proteins, capsular polysac- discussions with Arie Verkleij. They thank V. charides and LPS are involved in several aspects. Braun, S.T. Cole, C.F. Earhart, R.E.W. Hancock, Mechanisms like antigenic variation, immunosup- U. Henning, M. Inouye, P.M. M~ikel~i, S. pression, molecular mimicry of host components Mizushima, T. Nakae, J.B. Neilands, H. Nikaido, J.P. Rosenbusch, C.A. Schnaitman, B. Withoh and and toleragenicity are major mechanisms used by H.C. Wu for providing them with unpublished bacteria [619]. An enormous field of research is results. We thank Lia Claessens, Winny Geelen open to elucidate these mechanisms, in which outer and Joy Zantkuyl for typing the manuscript and membranes certainly play a role [619]. For the Dick Smit for excellent art work. development of vaccines (Refs. 534, 535 and 620, and Dankert J., Hofstra, H. and Veninga, T.S. References (1980) in FEMS Symposium on Microbial En- velopes, Saimaanranka, Finland, Abstract 51) and I lnouye, M. (ed.) (1979) Bacterial Outer Membranes, Bio- diagnostics [621] important features like surface genesis and Functions, Wiley-lnterscience, New York localization and immunogenicity have to be de- 2 Nikaido, H. and Nakae, T. (1979) Adv. Microbial Physiol- ogy (Rose, A.H. and Morris, J.G., eds.), pp. 163-250. termined. It also has to be established whether Academic Press, London antigenic determinants are common or specific. 3 Lugtenberg, B. (1981) Trends Biochem. Sci. 6, 262-266 This field is rapidly developing and vaccines for 4 Reeves, P. (1979) in Bacterial Outer Membranes, Biogene- Neisseriaceae and H. influenzae are already in field sis and Functions (Inouye, M., ed.), pp. 255-29l. trial [622-626]. Important combinations of bio- Wiley-lnterscience, New York 5 Hall, M.N. and Silhavy, T.J. (1981) Annu. Rev. Genet. 15, chemical and immunological techniques have re- 91-142 cently been developed [531,627-629] including the 6 Achtman, M. and Skurray, R. (1977) in Microbiological construction of cell lines producing monoclonal Interactions (Receptors and Recognition, Series B., Vol. 3) antibodies [630,631]. Molecular genetics can be (Reissig, J.L, ed.), pp. 233-279, Chapman and Hall, used to clone DNA coding for important con- London 7 DiRienzo, J.M., Nakamura, K. and lnouye, M. (1978) stituents in E. coli K-12. The cloning of genes for Annu. Rev. Biochem. 47, 481-532 E. coli K1 capsule formation is a recent example 8 Manning, P.A. and Achtman, M. (1979) in Bacterial Outer [632]. The recently constructed colony bank of Membranes, Biogenesis and Functions (lnouye. M., ed,), cloned DNA of virulent Treponema pallidum [633] pp. 409-447, Wiley-Interscience, New York might enable the investigator to express and pro- 9 Nikaido, H. (1979) in Bacterial Outer Membranes, Biogen- esis and Functions (Inouye, M., ed.), pp. 361-407. duce antigens of this important pathogen in E. coli Wiley-lnterscience, New York K-12. As T. pallidum cannot be grown on labora- 10 Nikaido, H. (1981) in /3-Lactam antibiotics (Salton. M. tory media, such an approach would be the only and Shockman, G.D., eds.), pp. 249-260, Academic Press, possibility to develop a vaccine. However, troubles New York might arise in the expression of foreign outer 11 Emr, S.D., Hall, M.N. and Silhavy, T.J. (1980) J. Cell Biol. 86, 701-711 membrane constituents in E. coli K-12, e.g. be- 12 Bayer, M.E. (1979) in Bacterial Outer Membranes, Biogen- cause of the possibly strict requirements of pro- esis and Functions (Inouye, M., ed.), pp. 167-202, tein-LPS interactions [304,393,535,634]. On the Wiley-lnterscience, New York 105

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