Topology and transport of membrane lipids in bacteria

Biochimica et Biophysica Acta 1469 (2000) 43^61 www.elsevier.com/locate/bba

Topology and transport of membrane lipids in bacteria Richard P.H. Huijbregts 1 , Anton I.P.M. de Kroon, Ben de Kruij¡ * Department Biochemistry of Membranes, Center for Biomembranes and Lipid Enzymology, Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands Accepted 12 October 1999

Abstract The last two decades have witnessed a break-through in identifying and understanding the functions of both the proteins and lipids of bacterial membranes. This development was parallelled by increasing insights into the biogenesis, topology, transport and sorting of membrane proteins. However, progress in research on the membrane distribution and transport of lipids in bacteria has been slow in that period. The development of novel biochemical in vitro approaches and recent genetic studies have increased our understanding of these subjects. The aim of this review is to present an overview of the current knowledge of the distribution and transport of lipids in both Gram-positive and Gram-negative bacteria. Special attention is paid to recently obtained results, which are expected to inspire further research to finally unravel these poorly understood phenomena. ß 2000 Elsevier Science B.V. All rights reserved. Keywords: Phospholipid; Lipopolysaccharide ; Bacterial membrane; Lipid transport; Lipid topology

1. Introduction Membranes grow from existing ones. The membrane constituents, proteins and lipids, are multiplied after which the enlarged membrane divides, or mem-

Abbreviations: LPS, lipopolysaccharide; MDO, membranederived oligosaccharides; PA, phosphatidic acid; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PS, phosphatidylserine; CL, cardiolipin; UDP-diC14OH GluN, UDP-2-3-di(3R)-hydroxymyristoylglucosamine ; Kdo, 3-deoxy-D-manno-octulosonic acid; TNBS, trinitrobenzene sulfonic acid; DMDG, dimannosyldiacylglycerol ; RSO, right-side out; PC, phosphatidylcholine; ISO, inside-out; NBD, 7-nitro-2-1,3-benzoxadiazole; Und-PP, undecaprenyl-pyrophosphate; MDR, multidrug resistance; LPP, major outer membrane lipoprotein * Corresponding author. Fax: +31-30-2522478. 1 Present address: Department of Cell Biology, University of Alabama at Birmingham, Birmingham, AL 35294-0005, USA.

brane fragments bud o¡. Prokaryotes contain only one or two membranes in which all membrane functions take place. This relatively simple membrane structure and the possibilities to intervene in cellular processes by genetic manipulation are major advantages for using prokaryotes in studies on the biogenesis of membranes. Most of the speci¢c functions of membranes are conferred by membrane proteins. In recent years, much progress has been made in elucidating the biogenesis of these proteins in bacteria (for reviews see [1,2]). Also the other membrane constituents, the lipids, are important for membrane function. They provide the membrane with its barrier function and furthermore, they play a role in a variety of processes in the bacterial cell [3]. In striking contrast to membrane proteins, knowledge about the mechanisms of transport of newly synthesized lipids and the steadystate distribution of these molecules in the mem-

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brane, is still very limited. Recent developments in this ¢eld, which might lead to new options for future research are the reasons for writing this review on the distribution and transport of lipids in bacteria. The world of the bacteria is large and the diversity of lipids, especially in Gram-positive bacteria, is immense. Most of the research on lipid distribution and transport in both Gram-positive and Gram-negative strains has focussed on the phospholipids. Therefore, this review will for the most part cover this type of lipids. The remaining part will deal with lipopolysaccharide, a lipid present exclusively in the outer membrane of enteric bacteria. Recently, some new developments relating to transport of (precursors of) this lipid have been described. Among the Gram-negative bacteria Escherichia coli has been studied most extensively in lipid synthesis, transport and distribution, while Bacillus megaterium is its Gram-positive counterpart. These two species were chosen to illustrate the topics of this review. Another lipid class present in bacteria, the isoprenoids, will not be discussed here as nothing is known about their membrane distribution and transport, with the exception of undecaprenyl-phosphate, which serves as the membrane anchor of a precursor of LPS, and will be discussed in this context. Phospholipids and LPS will be discussed in parallel throughout this review and brief attention will be paid to transport of protein-linked lipids. First, the membrane structure of bacteria and the localization of the lipids are described. Next, the biosynthesis of the lipids of interest is discussed. This is followed by an overview of the current knowledge on the distribution, the transmembrane movement and the intermembrane transport of phospholipids and LPS in bacteria. 2. Membrane structures of bacteria The schematic representation of the E. coli envelope depicted in Fig. 1 is used to explain the routes of lipid synthesis as well as the transport routes required for the lipids. The outer membrane forms the outermost barrier of the E. coli cell and contains two types of lipids. The outer lea£et contains the lipopolysaccharide (LPS) [4,5]. This lipid is found exclusively in the outer membrane. LPS molecules can be divided in three distinct regions (see Fig. 3). Lipid A

forms the hydrophobic anchor of LPS to which the core region is attached, which consists of a phosphorylated non-repeating oligosaccharide. In wild-type E. coli, the core sugars are usually followed by a third region, an O-antigen repeating oligosaccharide, which is absent in most E. coli K-12 strains [6]. The inner lea£et of the outer membrane contains phospholipids, the other lipid type of E. coli. Phospholipids are also the lipid constituents of the plasma, or inner membrane of E. coli and other enteric bacteria. In most Gram-positive bacteria, phospholipids are the major lipid class in the plasma membrane [7]. Phospholipids consist of a glycerol backbone with two fatty acyl chains esteri¢ed at the 1 and 2 position, respectively. The glycerol backbone contains a phosphate group at the 3 position, to which a hydrophilic group is esteri¢ed. The outer membrane proteins are L-structurebased and include the porins, channel forming L-barrel proteins [8], which allow the rapid passage of small hydrophilic molecules (up to 600 Da) [9]. Membrane-derived oligosaccharides (MDO) are found in the periplasm [10,11]. These polyglucose chains, which are substituted with phosphoethanolamine, phosphoglycerol, and O-succinyl ester residues [12], become very abundant in the periplasm when cells are under hypotonic pressure [13], indicating that they are involved in osmotic regulation. The periplasm also harbors the murein sacculus, a.k.a. the peptidoglycan layer, which is one polymer of muropeptides [14]. The function of this network around the inner membrane is to prevent the cell from lysis in hypotonic medium. Furthermore, it is involved in maintaining the cell shape. The peptidoglycan is bound to the outer membrane by the major outer membrane lipoprotein via a peptide bond [15,16]. Lipoproteins are lipid modi¢ed proteins that are anchored to either the inner membrane or the outer membrane [17]. A structure similar to the periplasm in enteric bacteria surrounds the plasma membrane of Gram-positive bacteria and forms the direct boundary of the cell with the environment. This structure is thicker than the periplasm of Gram-negative bacteria and has a somewhat di¡erent composition (e.g. no lipoproteins) [18]. Besides its role in osmoregulation, it provides the cell with a protective shell, but it does not have the molecular

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exclusion properties provided by the porins in the outer membrane of Gram-negative bacteria. 2.1. Phospholipid synthesis, turnover and composition In all bacteria, phospholipids are synthesized at the plasma membrane by enzymes which in majority are integral membrane proteins with the catalytic domain residing in the cytoplasm or which are bound to the cytoplasmic lea£et of this membrane [19,20] (Fig. 1). Phosphatidic acid (PA) serves as the precursor for all other phospholipids in bacteria (see Fig. 2 for the synthesis routes, and chemical structures). PA is only found in trace amounts in E. coli (usually less than 1%), because the enzyme CDP-diglyceride synthetase (encoded by the E. coli gene cdsA [21]) e¤ciently converts PA to CDP-diglyceride at the expense of CTP [22]. The product CDP-diglyceride is used by the two routes leading to the main membrane phospholipids of E. coli, the aminophospholipid phosphatidylethanolamine (PE) and the acidic phospholipids phosphatidylglycerol (PG) and cardiolipin (CL). PE is zwitterionic at physiological pH due to the proto-

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nated amino group and the dissociated phosphate group. PG and CL are negatively charged at physiological pH due to the dissociated phosphate group(s). The ¢rst step in the synthesis of PE is the formation of phosphatidylserine (PS) from CDP-diglyceride and serine by the enzyme PS synthetase [23] (pssA [24]). In enteric bacteria, this enzyme is not found in or at the plasma membrane like most other enzymes involved in phospholipid metabolism, instead it cofractionates with the ribosomes [25]. However, in order to be active it has to bind to the membrane [26]. The biosynthetic intermediate PS is a minor membrane constituent of E. coli. After its synthesis, PS is rapidly decarboxylated by the enzyme PS decarboxylase [23] (psd [27]) into PE, the most abundant phospholipid in E. coli [28]. The ¢rst step in the formation of the acidic phospholipids is the synthesis of phosphatidylglycerolphosphate by the enzyme phosphatidylglycerolphosphate synthetase (pgsA [29]) from the substrates CDP-diglyceride and glycerol-3-phosphate [30,31]. The reaction product is rapidly dephosphorylated to PG by the enzyme phosphatidylglycerolphosphate phosphatase (pgp). Two pgp genes have been de-

Fig. 1. Schematic representation of the E. coli envelope (adapted from [75]), showing the localization in and at the inner membrane of the enzymes involved in phospholipid biosynthesis, indicated by the name of the corresponding genes. Only the active, i.e. membranebound form of pssA is shown. LPP, major outer membrane lipoprotein.

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Fig. 2. Biosynthetic routes leading to the major phospholipid classes in E. coli (in rectangles). The genes encoding the enzymes catalyzing the reactions are indicated between parentheses.

scribed [32]. PgpA only exhibits phosphatidylglycerolphosphate phosphatase activity and accounts for about 30% of the total enzyme activity in the cell. In addition to phosphatidylglycerolphosphate phosphatase activity, the enzyme encoded by pgpB also exhibits PA phosphatase and lyso-PA phosphatase

activity. Recently it was observed that pgpB also exhibits diacylglycerolpyrophosphatase activity [33]. When both genes are deleted in E. coli about 50% of the phosphatidylglycerolphosphate phosphatase activity is retained, indicating that a third enzyme might be involved in this step in PG synthesis [34].

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CL is synthesized by a reaction unique for prokaryotes. Two molecules of PG are condensed by the enzyme CL synthetase [35,36] (cls [37]), whereas in eukaryotes, the phosphatidic acid moiety from CDP-diglyceride is transferred to the head group of PG [38]. The overall phospholipid composition of the E. coli cellular envelope was elucidated in the late sixties [39]. PE is the major phospholipid in E. coli constituting 70^80% of the total pool of phospholipids. The remaining phospholipids are PG (15^20%) and CL (5% or less) [39,40]. While PE has a relatively long half life (5% turnover per generation), PG is turned over rather rapidly (25% per generation) [3,28,39]. Besides being the precursor of CL, PG also serves as a substrate for the biosynthesis of other macromolecules. The phosphoglycerol moiety is used in the synthesis of membrane derived oligosaccharides [41] and the diacylglycerol part is used for the synthesis of lipoproteins [42]. The former reaction results in the formation of diacylglycerol which is recycled into the phospholipid metabolic pathway by its conversion into PA by the enzyme diacylglycerol kinase (dgk) [43] (Fig. 2). Apart from PE (35%), PG (48%), and CL (11%), the membrane of the Gram-positive bacterium Bacillus megaterium also contains glucosaminyl phosphatidylglycerol (6%) [7]. Studies on the biosynthesis of the latter phospholipid have not been reported. The other phospholipids are synthesized by the same routes as described for E. coli. In Gram-positive bacteria the enzyme PS synthetase is strongly bound to the plasma membrane, in fact the amino acid sequence predicts several transmembrane helices [44]. For a more extended treatise of the biosynthesis of (phospho)lipids in bacteria, the reader is referred to several recent reviews [3,19,20,45,46]. 2.2. Lipopolysaccharide synthesis The synthesis of lipopolysaccharide (LPS) in E. coli is rather complex and not all the enzymes involved have been identi¢ed and characterized. Only the membrane associated synthesis steps involved in the modi¢cation and extension of LPS intermediates will be discussed. The synthesis of the speci¢c acyl chains and sugar residues present in LPS is not addressed here and for an excellent recent review on all the

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aspects of LPS synthesis one is referred to [6]. The synthesis of LPS starts with the formation of the socalled lipid IVA (see Fig. 3 for a schematic representation of the various steps and the chemical structure of Kdo2 -lipid A). This intermediate is synthesized from a UDP-2-3-di(3R)-hydroxymyristoylglucosamine (UDP-diC14OH GluN) and a 2-3-di(3R)-hydroxymyristoylglucosamine-1-phosphate (lipid X). The latter is formed by cleavage of the pyrophosphate bond of the former [47,48]. The diC14OH GluN moiety of UDP-diC14OH GluN is transferred to the 6 position of the glucosamine moiety of lipid X resulting in a L1P^6 linkage [49,50]. The enzymes involved in the synthesis of this disaccharide 1-phosphate molecule, encoded in E. coli by the lpx gene cluster, are all located in the cytoplasm. The next steps in the synthesis route lead to the formation of core-Kdo2 -lipid A (Fig. 3). In vitro, all enzymes involved in the synthesis of the intermediate core-Kdo2 -lipid A from lipid IVA require detergent for activity indicating that they are membrane associated. Prior to the linkage of the 3-deoxy-D-mannooctulosonic acid (Kdo) residues, the disaccharide 1phosphate is phosphorylated at the 4P position by the membrane bound 4P kinase (encoded in E. coli by the gene lpxK [51]). Subsequently, two Kdo residues are transferred to the LPS intermediate by the Kdo transferase (kdtA [52]). Not until this point are the remaining lauroyl and myristoyl acyl chains transferred by speci¢c acyltransferases (htrB [53] and msbB [54], respectively) to the 3R-hydroxymyristoyl esters of Kdo2 -lipid IVA , resulting in the acyloxyacyl moieties and completing the hydrophobic anchor which is referred to as Kdo2 -lipid A (Fig. 3). The Kdo residues, together with two heptose residues, form the inner core of the LPS while glucose and galactose residues built up the outer core. The enzymes involved in the synthesis of the complete core domain, encoded in E. coli by the rfa gene cluster, have not been very well investigated. However, the protein sequences predicted from the genes are similar in size and structure and lack putative transmembrane regions. These sugar transferases act on membrane bound LPS precursors. Since no transporters involved in the export of sugar nucleotides to the periplasm have been detected, the sugar transferases are most likely situated at the cytoplasmic lea£et of the inner membrane [55]. For these reasons,

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Fig. 3. Schematic representation of the synthesis of LPS from lipid IVA in the inner membrane of E. coli (adapted from [6]). The transport across the inner membrane of the undecaprenol-linked O-antigen oligosaccharide and of the core-Kdo2 -lipid A is indicated by the arrows 1 and 2, respectively. The inset shows the chemical structure of Kdo2 -lipid A.

the core-Kdo2 -lipid A is shown to be located in that lea£et (Fig. 3). The O-antigen oligosaccharide part of LPS is synthesized independently from the core-Kdo2 -lipid A [56] at the cytoplasmic lea£et of the inner membrane (left side Fig. 3). As laboratory strains of E. coli K12 do not contain this structure and as it only recently became known that E. coli K-12 has the capacity to synthesize O-antigen [57], most of the research on the enzymology of O-antigen synthesis has been conducted in Salmonella strains. The synthesis of the O-antigen unit starts with the transfer of galactose 1-phosphate from UDP-galactose or glucosamine acetate 1-phosphate from UDP-glucosamine acetate in Salmonella [58] and E. coli [57], respectively, to the membrane associated, phosphorylated polyprenol undecaprenyl-phosphate. This is followed by three subsequent glycosylations resulting in a single O-antigen oligosaccharide [59]. Both the synthesis of the speci¢c sugars and their transfer to the grow-

ing O-antigen oligosaccharide unit is regulated by the rfb gene cluster [55]. After polymerization of the unit several modi¢cations as acetylation and glucosylation can occur. Polymerization of the O-antigen units takes place at the periplasmic side of the inner membrane [60], indicating that the undecaprenyl-linked units must £ip across the membrane (step 1 in Fig. 3). This process will be discussed below. The O-antigen polymer is generated by the transfer of a single O-antigen unit from the undecaprenyl linker to the reducing end of a growing chain of O-antigen units linked to another undecaprenyl-phosphate molecule. This reaction is catalyzed by polymerase (rfc [57,61]) an integral membrane protein. The O-antigen repeat is transferred to core-Kdo2 -lipid A by ligase (rfaL [62]). Little is known about this enzyme. In order to ligate the O-antigen repeat to the core-Kdo2 -lipid A, the latter intermediate must also be transported from the cytoplasmic lea£et to the periplasmic lea£et of the inner membrane (step 2 in Fig. 3). A putative

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candidate for the protein involved in this process, called MsbA, has recently been described [63] and will be discussed below. 3. Distribution of lipids in bacterial membranes 3.1. Transverse distribution of lipids in the plasma membrane As it is di¤cult to separate the inner and outer membrane of enteric bacteria quantitatively, the transverse distribution of lipids in the plasma membrane of intact cells has so far only been studied in Gram-positive bacteria. Most of these studies were performed quite some time ago and the published data have been reviewed thoroughly by Rottem in 1982 [64], and will therefore not be discussed in detail here. The distribution of PE has been studied in a variety of Gram-positive bacteria, mainly Bacilli species. Most approaches used amino-reactive chemicals as trinitrobenzene sulfonic acid (TNBS) under conditions of low membrane permeability of the reagent. The distribution of PE as determined with the amino-reactive chemical TNBS varied between the di¡erent bacteria and even between di¡erent strains of the same species, and no clear picture emerged. This contrasts the results obtained for plasma membranes of eukaryotic cells which in general reveal a preferential location of this lipid in the cytosolic lea£et [65]. The di¡erent behavior of the two types of membranes might be related to the relatively high content of PE in the bacterial membranes which can interfere with a complete labeling of the accessible molecules [66]. Moreover, plasma membranes in eukaryotic cells are more rigid and thicker due to the presence of sphingolipids and sterols [67] which reduces the permeability of the amino-reactive chemicals. Phospholipase C and phospholipase A2 have often been used to study the transmembrane distribution of both PG and CL in plasma membranes of bacteria [64]. Bevers and coworkers have shown that the accessibility of PG to phospholipase A2 in Acholeplasma laidlawii is dependent on temperature [68], on the fatty acid source the cells were grown on [69], on the state of membrane energization [70], and on protein^ lipid interactions which prevented the degradation of

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PG at low temperatures from going to completion [71]. These studies did not lead to consistent pictures of the location of the acidic lipids in bacteria and indicated the pitfalls associated with the use of phospholipases in topology studies of these lipids. The success of the use of phospholipases in determining lipid asymmetry in plasma membranes of eukaryotic cells [72] might again be related to the greater stability of these membranes and the lower amount of proteins present. Another method used to establish the distribution of certain lipids is by the speci¢c oxidation of K-diol groups by periodate. Such studies on the Gram-positive bacterium Micrococcus luteus demonstrated that 60% of the PG molecules were localized in the outer lea£et of the plasma membrane [73]. With the same method the glycolipid dimannosyldiacylglycerol (DMDG) was found to be symmetrically distributed in the plasma membrane of this organism. An estimation of the distribution of PG and DMDG could be made because the pro¢le of decrease of these lipids in the plasma membrane during the incubation of protoplasts of M. luteus with periodate indicated a biphasic process in time. Attempts to apply this method for assaying the transmembrane distribution of PG in either isolated membrane vesicles or total cells of E. coli failed [74]. Periodate oxidized the total PG pool with monophasic kinetics indicating that all the PG was equally available to the reaction with periodate. Apparently the plasma membrane of M. luteus is less permeable to periodate than that of E. coli which is most likely due to the markedly di¡erent membrane lipid compositions of the two species [73,75]. Possibly the methyl-branched tetradecanoic acids, which are the most abundant acyl chains present in the plasma membrane of M. luteus [76], cause a tighter lipid packing than that in the E. coli inner membrane. Some studies have been reported on the distribution of glycolipids in archeabacteria. Henderson et al. [77] used the periodate method in combination with a biotin labeling of the oxidized lipids to determine the distribution of the glycolipids triglycosyl diether and glycolipid sulfate in the purple membrane of Halobacterium halobium with electron microscopy. They found that the glycolipids are exclusively present in the extracellular lea£et of the purple membrane, implying that the most abundant lipid in

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this bacterium phosphatidylglycerolphosphate must be primarily located in the cytoplasmic lea£et. Recently, Weik et al. [78], have reported neutron diffraction studies on purple membranes of Halobacterium salinarium that con¢rm the asymmetric distribution of the glycolipids in this membrane. The authors observed that most of the glycolipids are present in the purple membranes, which consist of patches of bacteriorhodopsin and lipids. Interestingly, the sugar moieties of the glycolipids were found to interact with the tryptophan residues located at the exoplasmic membrane interface of the transmembrane helices of the protein. This suggests that these localized interactions are important for anchoring the protein into the membrane. Also the anionic lipids in the E. coli inner membrane were found to localize positively charged membrane protein segments to the cytoplasmic side of the membrane thereby controlling membrane topology [79]. The picture which emerges so far emphasizes the great technical problems associated with the methodologies used to determine membrane phospholipid topology in bacterial plasma membranes. New methods will have to be developed and the use of £uorescent lipid analogs of which the amount in a single lea£et can be measured might be one of them. 3.2. Lateral distribution of lipids in the plasma membrane The lateral distribution of lipids in the plasma membrane, especially (micro)domain formation of either only lipids or of lipid^protein complexes has become of major interest in recent years [80,81]. A few reports have been published on the lateral distribution of phospholipids in bacterial membranes. In M. luteus, the distribution of dimers which originated from the photo-activation of PG and DMDG containing an 9-(2-anthryl)nonanionic acid was determined and compared with the calculated random values [73]. In intact bacteria, the amount of homodimers of PG and DMDG was found to be higher and the amount of PG-DMDG heterodimers lower than the random values. The increased number of homodimers was not due to a highly asymmetric transverse distribution of these lipids, as was determined with the above periodate assay. This indicates that lateral domains of the speci¢c lipids had been

formed in the membrane. A low number of homodomains was present during cell growth, while a high number of homodomains was present during cell division [82]. This indicates that domain formation in this organism is a transient process involved in speci¢c functions of the membrane during the cell cycle. Although the formation of speci¢c membrane domains has been postulated to direct cell events in E. coli [83], no clear proof of this mechanism has been obtained. A study in which dimers of PE, obtained by crosslinking with dimethyl suberimidate, were analyzed revealed no signi¢cant di¡erence in dimeric species from the predicted values based on random cross-linking either in whole cells or in multilamellar vesicles [84]. However, in E. coli, the presence of PE seems to be necessary for the proper formation and constriction of FtsZ rings during cell division [85], suggesting that PE is present at the septum in the dividing cell. This process might involve domain formation, similar to that in M. luteus. 3.3. Transverse distribution of lipids in the outer membrane The outer membrane of Gram-negative bacteria contains two types of lipids, LPS and phospholipid. The composition of the phospholipids slightly di¡ers from that of the inner membrane with the PE content being somewhat higher [86]. Furthermore, the phospholipids in the outer membrane contain higher amounts of saturated fatty acyl chains, particularly palmitoyl, compared to the phospholipids in the inner membrane [86^88]. It is now believed that LPS and phospholipids are completely asymmetrically distributed among the two lea£ets of the outer membrane. The LPS molecules are located in the outer lea£et of the membrane while the phospholipids are con¢ned to the inner lea£et [8]. The unprecedented lipid asymmetry of the outer membrane of Gramnegative bacteria is backed up by a variety of data. With electron microscopy, it was shown that ferritin labeled antibodies against LPS were only present at the outer lea£et of the outer membrane of E. coli [89]. The extent of the oxidation of galactose residues in LPS by galactose peroxidase was the same in both intact cells and isolated membranes of Salmonella, indicating that all the LPS was localized in the outer

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lea£et of the outer membrane [90]. Treatment of intact cells with phospholipase C or A2 did not result in hydrolysis of phospholipids [91]. Furthermore, CNBr activated dextran was unable to react with PE in intact cells, whereas all of the PE was modi¢ed in isolated membranes [91]. These results indicate that phospholipids are con¢ned to the inner lea£et of the outer membrane and absent from the outer lea£et. The general belief is that LPS in the outer lea£et is essential to protect the cells against harmful amphi¢les. In accordance with this view, the outer membrane of mutants disturbed in the assembly of LPS, so-called `deep rough mutants', was found to be more permeable to hydrophobic substances than that of wild-type cells [92]. In the wild-type situation, the hydrophobic barrier is maintained by the tight connections between the heptose residues in the core domain of LPS molecules, which are sustained by divalent cations [93]. The increased permeability in deep rough mutants might be due to the presence of phospholipids in the outer lea£et of the outer membrane [8]. This notion is supported by the susceptibility of phospholipids in intact cells of deep rough mutants phospholipids to degradation by phospholipase and to modi¢cation by CNBr activated dextran [91]. Using CNBr activated dextran, Paul et al. found that a large portion of the phospholipid pool in Vibrio cholerae is present in the outer lea£et of the outer membrane although the LPS is of the normal, smooth, type [94]. The LPS molecules contain considerably less anionic groups compared to the LPS molecules from E. coli. This relatively low negative charge causes a reduced binding and stabilization of LPS by divalent cations. Whether this property is responsible for the reduced phospholipid asymmetry in the outer membrane of these bacteria is not known. 4. Transmembrane movement of lipids across the plasma membrane 4.1. Transmembrane movement of phospholipids across the plasma membrane After synthesis part of the phospholipids must be

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transported to the outer lea£et of the bacterial plasma membrane. In Gram-negative bacteria, there is also transport to the outer membrane, either directly from the cytoplasmic lea£et or via the periplasmic lea£et of the inner membrane. In vivo pulse labeling studies in B. megaterium showed that the transmembrane equilibration of PE is very rapid with a t1=2 of 3 min at 37³C [95]. The process proceeds independently of protein and lipid synthesis and does not consume metabolic energy [96]. In vitro transmembrane movement of phospholipids across the membrane of right-side out (RSO) vesicles isolated from B. megaterium is aspeci¢c for the head group as was tested for short chain NBDlabeled phospholipid analogs of PE, PG and phospatidylcholine (PC) [97]. The NBD-labeled phospholipids equilibrated across the membrane with a t1=2 of 30 s (37³C) to a 1:1 distribution over the two lea£ets. The di¡erence between the 1:1 distribution of the NBD-labeled lipids and the distribution of endogenous PE and PG, 7:3 and 3:7 cytoplasmic to periplasmic lea£et, respectively [98], is explained by asymmetry being maintained by membrane associated proteins or surface constraints which are absent in the right side out membrane vesicle preparation [97]. The rate of transmembrane movement was decreased two-fold after pretreatment of the vesicles with protease, indicating a protein-mediated process. Recently, C6 -NBD-labeled phospholipid analogs incorporated into the cytoplasmic lea£et of E. coli inside-out inner membrane vesicles (ISO) were shown to move across the membrane with a t1=2 of 7 min at 37³C to reach a distribution of 15^85, periplasmic to cytoplasmic lea£et [99]. This phenomenon is temperature-dependent, independent of ATP and the proton motive force, aspeci¢c for the phospholipid headgroup, and not a¡ected by pretreatment of the inner membrane vesicles with N-ethylmaleimide or proteinase K. Part of the incorporated C6 -NBD-PG is converted into C6 -NBD-CL by CL-synthetase. Upon incorporation of C6 -NBD-PG into ISO vesicles, the pool of newly synthesized NBD-CL protected against reduction by the membrane impermeable probe dithionite increases in time, indicative for transmembrane movement (at a 2.4-fold slower rate than C6 -NBD-PG) from the cytoplasmic lea£et to the periplasmic lea£et [99]. This observation suggests that the site of synthesis and therefore the catalytic

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domain of CL synthetase is localized at the cytoplasmic side of the inner membrane. Consistent with this hypothesis, protease added to the cytosolic side of the inner membrane inhibited the CL-synthetase [99]. In contrast to these studies, Shibuya et al. [100] have reported that the catalytic domain of CL synthetase is located at the periplasmic side of the inner membrane. This was based on the ¢nding that phosphatidylmannitol and diphosphatidylmannitol, sugar alcohol analogs of PG and CL, are synthesized by a mutant E. coli strain de¢cient in mannitol uptake when mannitol was present in the growth medium. Mutants defective in CL synthetase do not synthesize these molecules, whereas phosphatidylmannitol and diphosphatidylmannitol are more abundant in cells overexpressing CL synthetase, indicating that CL synthetase catalyzes the synthesis of these molecules. One way to reconcile the two studies is to postulate that mannitol is able to reach the cytoplasmically localized catalytic domain of CL synthetase without using the mannitol transport system. Recently, a novel in vitro system was devised in order to investigate transmembrane transport of PE in E. coli inner membrane vesicles under conditions more closely mimicking the in vivo situation. The transmembrane movement of endogenously synthesized, radiolabeled PE was monitored using the amino-reactive probe £uorescamine [101]. Due to the high amount of PE present in the inner membrane of wild-type E. coli, steric interference of the £uorescamine derivative of PE is likely to prevent the reaction of the newly synthesized radiolabeled PE with £uorescamine from going to completion [66]. This problem was circumvented by biosynthetically introducing a small amount of PE into inner membrane vesicles of the E. coli strain AD93 which are devoid of PE because of a defect in the enzyme PS synthetase [102]. Addition of a cytosolic lysate from wildtype cells containing PS synthetase [25], together with the appropriate substrates, restores the synthesis of PS in inner membrane vesicles from AD93. Most of the PS synthesized is subsequently converted into PE by the membrane-bound enzyme PS decarboxylase. An additional advantage of using strain AD93 is that about 10% of the phospholipids in its inner membrane consists of PA [102] which conveniently serves as precursor for the synthesis of PS in the reconstituted in vitro system.

The synthesis and subsequent transmembrane movement were measured in both ISO and RSO vesicles (see Fig. 4A for a schematic representation of both systems). The transmembrane movement was measured as the sequestration of the newly synthesized PE molecules from the reaction with £uorescamine. In RSO vesicles synthesis of PE was generated in the lumen of the vesicle, and transmembrane movement was monitored by detecting the appearance of PE in the outer lea£et. Both approaches showed that the newly synthesized PE equilibrates across the membrane with a half time of less than 1 min which is several orders of magnitude faster than spontaneous lipid £ip-£op [103]. In both ISO and RSO vesicles, the newly synthesized PE distributes in a 35 to 65 ratio over the cytoplasmic and periplasmic lea£et, respectively (Fig. 4B). The process was not a¡ected by the presence of ATP or the pmf, nor by a pretreatment of the vesicles with sulfhydryl reagents or proteinase K [101]. The observed transmembrane movement in the in vitro assay is likely to re£ect the process in vivo in wild-type E. coli as all basic components necessary for £ip-£op were present. However, the absence in the in vitro systems of the outer membrane and hence of the transport routes of lipids to this membrane, might restrict the extent of transport. The right-side out vesicle system in conjunction with isolated outer membranes might yield an in vitro system enabling characterization of the more complete transport of newly synthesized lipids. Whether speci¢c £ippases are involved in lipid transport across the inner membrane might be answered in the future by reconstitution studies using e.g. NBD-labeled lipids as marker molecules. Fractionation of the plasma membrane proteins, followed by reconstitution of the protein fractions in proteoliposomes of bacterial lipids might result in a puri¢ed protein or protein complex responsible for the transmembrane movement of NBD-labeled phospholipids. Hrafnsdo¨ttir and Menon took this approach and recently reported the reconstitution of phospholipid £ippase activity in proteoliposomes prepared of the solubilized membrane proteins from Bacillus subtilis [104]. A genetic approach to identify putative £ippases might be cumbersome as no simple screen is available for measuring transmembrane movement of phospholipids. Nevertheless, methods analogous

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Fig. 4. (A) Schematic representation of the two vesicle systems derived from E. coli strain AD93 that were used to measure the transmembrane movement of newly synthesized PE, showing the lipid biosynthetic enzymes CDP-DG synthase (cds) and PS decarboxylase (psd). Upon addition to ISO and inclusion in RSO of PS synthetase (pss) and CTP, and upon addition of 14 C-labeled serine, 14 C-labeled PE is synthesized. (B) The accessibility of newly synthesized 14 C-labeled PE to £uorescamine after the indicated periods of lipid synthesis in AD93 ISO vesicles (a), RSO vesicles (b). The sum of the percentages of PE reacted with £uorescamine in ISO and RSO vesicles (E) is also shown.

to the dye-sensitized photokilling approach developed for yeast [105], or the PS decarboxylase assay to detect PSD mutants in yeast [106], may o¡er perspective to pick up mutants defective in phospholipid transport in bacteria. 4.2. Transmembrane movement of LPS intermediates across the plasma membrane As shown in Fig. 3, after its synthesis at the cytoplasmic side of the plasma membrane the O-antigen unit has to be transported to the periplasmic lea£et

in order to serve as a substrate for the polymerase which catalyzes the formation of the O-antigen repeat [60]. The O-antigen unit is bound to undecaprenyl-pyrophosphate (Und-PP) which has to cross the membrane as a whole. The transmembrane movement of the UndPP-linked O-antigen units (Und-PP-O-antigen) has been postulated to be facilitated by the product of the wzx (rfbX) gene, a hydrophobic protein with 12 membrane spanning domains [107]. A wzx3 mutant of Salmonella enterica accumulates UndPP-O-antigen units at the inner membrane to a 50-fold higher level than the control strain con-

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taining the complete unmodi¢ed gene cluster. In spheroplast preparations, both the periplasmic lea£et and the outer membrane were exposed to an antibody raised against O-antigen. A small amount of O-antigen was detected in the wzx3 mutant whereas in the control strain a high amount of O-antigen was detected, which was believed to be located in the outer membrane. The amount of O-antigen in the mutant was hardly higher than that in the Salmonella strain lacking the gene cluster. These results indicated that wzx is responsible for exposure at the periplasmic lea£et, i.e. for the transport of UndPPO-antigen across the plasma membrane. Previously, the enzyme galactosyl-1-phosphate transferase was thought to be involved in the transmembrane movement of the O-antigen units, as a mutation in the gene encoding this enzyme, wbaP (rfbP), resulted in the accumulation of (Und-PP-O-antigen) [108]. However, much less UndPP-O-antigen units accumulated in the wbaP mutant than in the wzx3 mutant. Therefore it was proposed that the mutation in wbaP interfered with the release of the UndPP-O-antigen unit from galactosyl-1-phosphate transferase [109]. The release function of galactosyl-1-phosphate transferase might be involved in the regulation of the rate of UndPP-O-antigen synthesis. After the synthesis of core-lipid A this LPS intermediate has to be transported to the periplasmic leaflet of the plasma membrane where it is ligated with the O-antigen repeat prior to transport to the outer membrane [60] (see Fig. 3). This transport process requires maintenance of the proton motive force [110]. ATP is necessary for the transport of LPS from the inner to the outer membrane [111]. The msbA gene, a suppressor of null mutations in htrB, the gene encoding for the protein that transfers a lauroyl chain from the acyl-carrier protein to Kdo2 lipid IVA , encodes for a protein that is related to the family of ABC transporters [112,113]. One of these ABC transporters in mammalian cells is mdr2 which encodes a phosphatidylcholine translocator [114]. This gave rise to the idea that msbA might be involved in the transmembrane movement of core-lipid A. Recently, Raetz and coworkers have shown that in a thermosensitive msbA mutant grown at the nonpermissive temperature, fully acylated core-lipid A intermediates accumulate at the inner membrane [63]. All other genes involved in LPS synthesis were

present and functional, therefore the accumulation can only be attributed to the defective msbA gene. The authors also reported an accumulation of phospholipids in the inner membrane suggesting that MsbA might also be involved in transmembrane movement of phospholipids. As suggested by the authors, reconstitution studies with this protein will reveal its involvement in both LPS and phospholipid transport across the inner membrane. 5. Intermembrane transport of lipids in bacteria 5.1. Intermembrane transport of phospholipids Transport of phospholipids between the inner and outer membrane in enteric bacteria was ¢rst demonstrated for the Gram-negative bacterium Salmonella typhimurium. Pulse labeling studies in combination with isopycnic sucrose density gradient centrifugation to separate the inner and outer membrane [4] showed that PE was synthesized in the inner membrane and transported to the outer membrane [115]. Radiolabeled PS introduced into the outer membrane of S. typhimurium by vesicle fusion [116] was quickly transported to the inner membrane as was detected by the decarboxylation of 50% of the introduced PS within 5 min [117] by the enzyme PS decarboxylase which is located at the inner membrane [118]. The PE formed could be transported back to the outer membrane. The process of phospholipid transport was relatively non-speci¢c as both the phospholipids present in S. typhimurium (PE, PG and CL) and the foreign lipid PC, were transported to the inner membrane after introduction in the outer membrane by vesicle fusion [117]. The transport of newly synthesized phospholipids in E. coli was studied in pulse labeling studies in combination with separation of the inner and outer membrane by isopycnic sucrose density gradient centrifugation [119,120]. Transport of newly synthesized phospholipid from the inner to the outer membrane was fast with a t1=2 of 2.8 min for PE and a t1=2 of less than 30 s for the anionic phospholipids. Furthermore, the ratio of the doubling time to the t1=2 of PE transport was nearly constant in the temperature range of 23^37³C, indicating that the transport of PE had the same temperature dependence as growth.

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Transport of phospholipids from the site of synthesis to the outer membrane was independent of ATP and of the ongoing synthesis of proteins and lipids [119]. Depletion of the proton motive force by either proton ionophores or an inhibitor of the respiratory chain had a strong inhibitory e¡ect on the transport of phospholipids. With the use of a thermosensitive PS decarboxylase mutant, the transport of PS between inner and outer membrane was studied [120]. It was shown that at the semipermissive temperature PS molecules that were not decarboxylated were transported to the outer membrane with a t1=2 of about 12 min. During the chase at the semipermissive temperature, these PS molecules were decarboxylated to PE and hence transported back to the cytoplasmic lea£et of the inner membrane, indicating that phospholipid transport between the inner and outer membrane as well as the transmembrane movement across the inner membrane in E. coli is bidirectional [120]. The molecular mechanisms underlying these transport processes, both transport from inner to outer membrane (and back) and the transmembrane movement (£ip-£op) of phospholipids across the inner membrane are not known. Transport of phospholipids between the inner and outer membrane is unlikely to occur by aqueous diffusion since phospholipids have a very low critical micelle concentration [121,122]. For instance, upon incubating vesicles consisting of PC and containing 15% of spin-labeled PC with unlabeled PC vesicles, no redistribution of the spin-labeled probe between the two vesicle populations was observed [123]. Several mechanisms have been postulated for the transport of phospholipids from the inner to the outer membrane. Transient contact between the inner and the outer membrane might be required for transport of phospholipids. Membrane adhesion zones (Bayer's bridges), have been demonstrated by electron microscopy [124], but it is not yet clear whether these structures are physiologically relevant or an artefact of the ¢xation technique [125,126]. Vesicle transport, a major transport route for lipids in eukaryotic cells [127] is not likely to play a role in E. coli. Vesicles have never been observed with electron microscopy, and furthermore, the periplasm contains the peptidoglycan layer, a gel-like network around the inner membrane [128] which is incompatible with such a transport mechanism.

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Another possibility is that a phospholipid transfer protein shuttles between the inner and outer membrane and hence delivers phospholipids to their ¢nal destination. Although many phospholipid transfer proteins have been identi¢ed in eukaryotic cells (for review see [129]), phospholipid transfer activity has not been reported in E. coli or Salmonella yet. A protein with phospholipid transfer activity has been described for the Gram-positive strain Bacillus subtilis, and was found to facilitate the transport of phospholipids between mesosomes (membrane vesicles packed in an invagination of the plasma membrane [130]) and protoplasts isolated from this bacterium [131]. In the Gram-negative photosynthetic bacterium Rhodopseudomonas sphaeroides two proteins with phospholipid transfer activity were characterized [132]. One was preferentially situated in the periplasm displaying higher transfer activity for PE than for PG in vitro, while the second one was situated in the cytoplasm displaying an a¤nity opposite to its periplasmic counterpart. Highest levels of transfer activity were found in cells with extensive invaginations of the cytoplasmic membrane. The functions of both transfer proteins have not been elucidated. Protein complexes spanning the periplasm from the inner to the outer membrane might be involved in lipid transport to the outer membrane similar to the complexes responsible for the secretion of extracellular proteins, e.g. pullulanase [1]. The enrichment of PE in the outer membrane might indicate that sorting occurs in the transport of phospholipids to the outer membrane. However, it should be taken into consideration that the reduced PG content of the outer membrane might also be due to its use as substrate for the periplasmic enzymes involved in the synthesis of MDO and lipoproteins. 5.2. Transport of LPS from the inner to the outer membrane Using pulse-chase studies in combination with isopycnic sucrose gradients, Osborn et al. [20] found that in Salmonella, most of the LPS synthesized in the inner membrane during a 1-min pulse was chased to the outer membrane within 2 min. In contrast to the transport of phospholipids, transport of LPS to

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the outer membrane is irreversible, as was shown in mutants which depend on a speci¢c nutrient for synthesis of either the core or the O-antigen. Incomplete LPS synthesized under non-permissive growth conditions is not completed upon shifting the cells to permissive growth conditions where newly synthesized LPS was complete. As the enzymes involved in LPS synthesis are located at the plasma membrane these results indicated that the incomplete LPS molecules did not return to the inner membrane once arrived in the outer membrane. Pulse-chase studies conducted by Marino et al. [111] showed that newly synthesized LPS was transported from the inner to the outer membrane of S. typhimurium with a t1=2 of 1.2 min at 32³C. The process was dependent on the proton motive force and ATP. Both these requirements can be attributed to the transmembrane movement of LPS across the plasma membrane. The uncoupler 2,4-dinitrophenol prevented the ligation of O-antigen to core-lipid A, although both the synthesis and the ligase activity were not inhibited by the uncoupler [110]. Furthermore, core-lipopolysaccharide synthesized in the presence of the uncoupler is a functional acceptor of O-antigen in vitro when the membranes are solubilized with detergent. This indicates that the uncoupler inhibits the transport of the core-lipopolysaccharide from the cytoplasmic to the periplasmic lea£et of the membrane. However, this does not rule out the involvement of the proton motive force in the transport of LPS from the inner to the outer membrane. The fact that an ATP-binding cassette (ABC) transporter (MsbA) is involved in the transmembrane movement of LPS [63] indicates that it is this step which requires ATP. Using mutants defective in synthesizing complete LPS when grown at the non-permissive conditions in combination with a ferritin-labeled antibody against complete LPS, Mu«hlradt et al. found in electron microscopy studies that newly synthesized LPS is located at the outer lea£et of the outer membrane in patches around the zones of adhesion (Bayer's bridges) [133]. These patches then di¡use by lateral movement of the LPS molecules in the outer lea£et of the outer membrane [134]. Although lipid synthesis, transport and distribution are usually investigated independently of one

another, this certainly does not mean that these processes are not interconnected, or linked to that of other cell envelope components. Recently, reports have appeared in which interactions between membrane transport routes have been indicated. The assembly of the outer membrane protein OmpF was in£uenced by the phospholipid levels and/or the phospholipid to LPS ratio [135]. LPS is involved in the formation of folded monomers of the outer membrane protein PhoE [136]. Inhibition of phospholipid synthesis results in an inhibition of the synthesis of murein and O-antigen, suggesting that the common step in these biosynthetic routes, the translocation of undecaprenyl-linked precursors from the cytoplasmic to the periplasmic lea£et of the inner membrane, is in£uenced by the inhibition of phospholipid synthesis [137]. 5.3. Transport of lipid containing proteins from the inner to the outer membrane Lipoproteins contain a lipid anchor that consists of a diacylglycerol moiety attached to the N-terminal cysteine residue that keeps the protein a¤xed to either the inner or outer membrane [138]. Recently, a periplasmic protein has been isolated which is involved in the sorting and transport of the major outer membrane lipoprotein in E. coli [139]. This protein, formerly known as p20 and now renamed LolA, forms a soluble complex with the major outer membrane lipoprotein as well as with other lipoproteins destined for the outer membrane. In reconstitution studies, the outer membrane lipoprotein LolB was identi¢ed as the docking receptor for the LolA^lipoprotein complex [140]. The molecular details of the transfer of the lipoprotein from LolA to LolB and from LolB into the outer membrane are not yet known. Some proteins that are secreted by bacteria contain fatty acyl chains. These fatty acids exert functions either in the lytic activity of the protein as is the case for hemolysin (HlyA) or in binding the protein to the cell wall as is the case for pullulanase (PulA). The fatty acyl chains of PulA were suggested to play a role in the transport of the protein by improving the e¤ciency of secretion [141].

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6. Summarizing discussion The knowledge about the distribution and transport of lipids in bacteria has slowly increased during the last few decades with a few recent developments which could further open the ¢eld. A number of aspects remain obscure. The phospholipid distribution in the plasma membrane has not yet been unraveled for Gram-negative bacteria, while the diversity in distribution found among the Gram-positive species is not understood. The asymmetric lipid distribution found in the outer membrane of enteric bacteria was established quite some time ago and serves a clear function. The LPS in the outer lea£et forms a barrier for hydrophobic components that might otherwise destabilize the cell. The transport of lipids from the site of synthesis to the outer membrane has been studied, but so far nothing is known about the mechanism(s) of this process. On the other hand, newly devised in vitro systems as well as genetic approaches have recently provided new information on the transmembrane movement of lipids and lipid intermediates across the plasma membrane of both Gram-positive (B. megaterium) and Gramnegative (E. coli) bacteria. Here, the characteristics of phospholipid transport across bacterial plasma membranes will be compared to several mechanisms proposed for transmembrane lipid movement which have been depicted in Fig. 5. Spontaneous £ip-£op of phospholipids in proteinfree bilayers is an extremely slow process [103,142], which cannot account for the fast rates of £ip-£op

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found in both bacterial systems. Therefore the existence of proteinaceous lipid £ippases is postulated which catalyze the process. The process of lipid transmembrane movement across the bacterial plasma membrane is reminiscent of that found in the ER membrane of eukaryotes (recently reviewed in [143]). This process also lacks headgroup speci¢city as a variety of spin-labeled lipid analogs showed transmembrane movement across the membrane of microsomes [144]. The rate of transmembrane movement was reduced when microsomes were preincubated with sulfhydryl reagents, indicative for a protein mediated process. Like in the bacterial systems, there is no obvious energy source for the transport of lipids across the ER membrane [143]. Direct proof for the existence of the putative £ippases awaits their isolation or genetic identi¢cation. The driving force for the transmembrane movement of phospholipids in bacteria might be the imbalance in surface pressure resulting from the insertion of newly synthesized lipids in the cytoplasmic lea£et of the plasma membrane. The imbalance would induce the transmembrane movement of the newly synthesized phospholipids from the cytoplasmic lea£et to the periplasmic lea£et via a transversal di¡usion mechanism. Such an imbalance in surface pressure might be maintained by a coupled transport of lipids to the outer membrane in the case of Gramnegative bacteria. Proteins could facilitate this process and analogous to the process of facilitated diffusion one could designate this process as facilitated

Fig. 5. Schematic representation of possible mechanisms by which transmembrane movement of phospholipids could occur in the plasma membrane of bacteria as discussed in the text.

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£ip-£op (Fig. 5). Facilitated £ip-£op may be catalyzed by speci¢c proteins. Alternatively, it could proceed randomly via any protein^lipid interface. Multidrug resistance (MDR) P-glycoproteins, which are part of a larger family of ABC membrane transporters have been shown to be lipid translocators. In an mdr2 knock-out mouse, no PC is secreted in the bile of the animals, leading to the development of a liver disease [114]. When the mdr2 gene is expressed in yeast, secretory vesicles containing the protein show Mg2‡ -ATP dependent, PC-speci¢c phospholipid £ippase activity as was tested with short chain NBD-labeled lipids [145]. Studies with both human MDR1 and MDR3, the latter being the analog of the mouse mdr2, overexpressed in epithelial cells, con¢rmed the speci¢c PC translocase activity of the latter and showed a broad lipid translocase activity of MDR1 for short-chain lipid analogs only, in an energy-dependent manner [146]. Many multidrug resistance proteins are present in E. coli [147] which either belong to the ATP binding cassette superfamily analogous to the mammalian MDR P-glycoproteins, or are drug/H‡ antiporters (see Fig. 5). In several bacteria, both Gram-positive and Gram-negative, multidrug resistance proteins belonging to the family of ABC-transporters have been discovered [148]. In E. coli, transmembrane movement of both NBD-labeled lipid and in vitro synthesized PE is not dependent on the presence of either ATP or the proton motive force [99,101], indicating that phospholipid £ip-£op is not the main function of the multidrug resistance proteins in bacteria. However, the ABC-transporter MsbA has been found to mediate £ip-£op of the LPS precursor core-lipid A in E. coli [63]. Sequence analysis shows a high homology to the mammalian MDR proteins which are capable of transporting lipid analogs. Further studies will have to establish the ATP dependence of LPS transport as well as the suggested role of MsbA in phospholipid transport [63]. The latter would contradict the observation that metabolic energy is not necessary for the transmembrane movement of phospholipids across the inner membrane of E. coli. The aminophospholipid translocator was the ¢rst £ippase identi¢ed [149], of which the occurrence so far appears to be con¢ned to eukaryotic cells. This translocator £ips the aminophospholipids PS and PE

from the extracellular lea£et of the plasma membrane to the cytoplasmic lea£et at the expense of ATP. Pretreatment of membranes with sulfhydryl reagents or orthovanadate resulted in a decreased activity of aminophospholipid translocation. The transmembrane movement of phospholipids in bacteria does not have the characteristics of the aminophospholipid translocator, as it lacks its speci¢city and energy dependence. The transport of phospholipids in bacteria is a highly dynamic process consisting of an interplay between synthesis, transmembrane movement and intermembrane transport, of which some of the secrets have been revealed recently. The challenge for the future will be to identify and characterize the proteins involved in the transmembrane movement and the intermembrane transport of lipids in these organisms. Acknowledgements The authors wish to thank A.K. Menon for providing data prior to publication.

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