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Three types of prokaryotes possess outer membranes with distinct compositions and structures, suggesting they evolved independently Milton H. Saier, Jr. acterial cytoplasmic membranes consist primarily of amphipathic phospholipids with lesser amounts of glycolipids, providing a degree of protection from deleterious, toxic substances that is lacking in organisms having a single membrane. Almost all bacterial membranes are assembled in bilayers with embedded integral and associated peripheral membrane proteins. In the case of gram-negative bacterial outer membranes, phospholipids are present in the inner leaflet of the bilayer, while lipopolysaccharides predominate in the outer leaflet. Carbohydrates in lipopolysaccharides and in glycolipids and glycoproteins usually extend outwards. Meanwhile, many gram-positive bacteria, particularly firmicutes and mollicutes with genomes of low or moderate G⫹C content, lack an outer B Summary • Prokaryotes show far greater diversity at the molecular level than eukaryotes—particularly in terms of the structures of their membranes. • The protective outer membranes of many prokaryotes have very different compositions from those of their inner membranes. • With respect to lipid and protein constituents, the outer membranes of gram-negative bacteria differ drastically from those of high G⫹C grampositive bacteria and of archaea. • The outer membranes, along with associated components for transport and their assembly, of gram negatives, gram positives, and archaea appear to have evolved independently of one another. membrane. However, acid-fast, high G⫹C gram-positive bacteria such as mycobacteria and corynebacteria have mycolic acid-containing outer membranes that are structurally very different from the outer membranes of gramnegative bacteria. Some archaea contain membranes of very different protein and lipid constituents, typically consisting of hydrophobic tails linked by ether rather than ester bonds to the glycerol-containing lipid backbone. A few archaea have complex envelopes which, like those in some bacteria, consist of inner and outer membranes that differ in their lipid and protein constituents. These prokaryotic outer membranes are so different as to suggest independent evolutionary origins. Perspective Structure and Evolution of Prokaryotic Cell Envelopes Bacterial Cytoplasmic Membranes The cytoplasmic membrane (CM) of any bacterium is about 70 Å (7 nm) thick, separating the interior of the cell from the external environment and preventing diffusion of most substances in and out of the cytoplasm. Thus, the CM acts as a selective barrier, concentrating metabolites and nutrients within the cell while secreting waste products and toxins. Bacterial CMs typically consist of nearly equal amounts of phospholipid and protein, accounting for about 70% of cellular phospholipids and 25% of cellular proteins. The phospholipids are amphipathic, having hydrophobic tails and hydrophilic heads. They contain a glycerol backbone to which is attached two fatty acid molecules and a phosphoryl head group. The membrane is stabilized by hydrophobic interactions, hy- Milton H. Saier, Jr., is Professor of Molecular Biology in the Division of Biological Sciences, University of California, San Diego. Volume 3, Number 7, 2008 / Microbe Y 323 Bacterial Compartments and Organelles Two-membrane prokaryotic cells are divided into at least five compartments: the cytoplasm, the inner membrane, the periplasm between the two membranes, the outer membrane, and the extracellular milieu. Moreover, the inner and outer leaflets of both membrane bilayers can be considered to be distinct compartments. Numerous dissimilar and evolutionarily distinct protein insertion complexes are responsible for integrating inner and outer membrane proteins into the envelope. In addition to these membranes, some bacteria have membrane-bound organelles, including magnetosomes, which allow bacteria to orient in the Earth’s magnetic field, chromatophores where photosynthesis occurs, gas vacuoles that provide for flotation, and sulfur granuoles that house elemental sulfur. While magnetosome and chromatophore membranes are lipid bilayer based, gas vacuole and sulfur granuole membranes consist of two-dimensional arrays of proteins. FIGURE 1 Integral membrane proteins in the CM are also amphipathic, anchored into the membrane with one or more hydrophobic transmembrane segments. However, peripheral membrane proteins are loosely bound and interact transiently, often due to ionic attractive forces. The largest known functional class of CM proteins includes transport proteins that account for about 10% of the cell proteome. Cytoplasmic membrane proteins also mediate transmembrane electron flow, energy generation and interconversion, biosynthesis of lipids and cell envelope precursors, and translocation of these precursors to an extracytoplasmic locale where they are assembled. Owing to their hydrophobic characters, membrane proteins are difficult to study, and consequently, they account for fewer than 1% of the known high-resolution protein structures. Gram-Negative Bacterial Outer Membranes The outer membranes (OMs) of typical gram-negative bacteria are asymmetric lipid bilayers where the inner leaflet consists of phospholipids and the outer leaflet contains a preponderance of lipopolysaccharide (Fig. 1). Gram-negative bacteria lacking lipopolysaccharide may instead have sphingolipids and/or various glycolipids. Because these bilayers show low permeabilities to many solutes, nutrients of less than 600 Da in size cross the outer membrane by diffusion through porin channels. These channel proteins form Schematic view of the E. coli cell envelope, typical of those of most gram-negative -barrel structures with transmembacteria. Lipopolysaccharide (LPS), embedded within and extending from the outer brane-spanning segments consisting of surface of the OM, consists of three moieties: lipid A, core oligosaccharide, and the O-antigen repeat polysaccharide side chains as indicated. A trimeric porin in the OM, and amphipathic antiparallel -strands. integral membrane proteins in the CM, are depicted schematically. The peptidoglycan ␣-Helical proteins in the OMs of these cell wall in the periplasm separates the two membranes. Modified from a figure on the organisms are rare, just as -structured website http://www.microbialcellfactories.com/content/figures/1475–2859-5–13-1.jpg. proteins in the CMs are rare. Other compounds such as vitamin B12 and drogen bonds, and divalent cations such as iron siderophore complexes cross the OM via Mg2⫹ and Ca2⫹. The asymmetric bilayer, with substrate-specific, high-affinity, active transdifferent lipid and protein compositions for the porters. These OMs also contain structural litwo apposed monolayers, is thus a stable strucpoproteins, membrane-anchored enzymes, and ture that serves as an encapsulating “bubble” multicomponent surface structures such as fimwith the cell cytoplasm inside. briae (organelles of adhesion), pili (organelles of 324 Y Microbe / Volume 3, Number 7, 2008 conjugation), and flagella (organelles of motility). Lipopolysaccharides (Fig. 1) consist of three parts: the proximal, hydrophobic lipid A region that is embedded in the outer leaflet of the OM; the distal, hydrophilic O-antigen polysaccharide region that protrudes into the medium; and the core oligosaccharide region that connects lipid A to the O-antigen repeat units. Lipid A is a polar lipid of unusual structure in which a backbone of glucosaminyl--(136)-glucosamine is esterified with six or seven saturated fatty acids. FIGURE 2 Outer Membranes of Acid-Fast, Gram-Positive Bacteria Acid-fast bacteria belong to a distinctive actinomycete taxon that includes mycobacteria, corynebacteria, nocarThe mycobacterial cell envelope showing the most important structural components. Passage of small hydrophilic molecules through the OM requires the involvement of dia, rhodococci, and several other genporins (top). The figure illustrates the covalent linkages between cell wall peptidoglycan, era. All these organisms have an unarabinogalactan and mycolic acids. The width of the CM is about 7 nm (bottom), but that usual cell envelope composition and of the outer membrane is about 10 nm. architecture (Fig. 2). The envelope layers consist of typical CMs of phospholipid and protein, a characteristic wall The OMs of these bacteria may be the thickest of unusual structure, and a complex outer layer. of all biological membranes yet identified. Their Although more detail is available for mycobaccell walls are formed by thick peptidoglycan teria, the envelopes of these related bacteria are layers, covalently linked to arabinogalactan via similar, especially in terms of ultrastructure and phosphodiester linkages. The arabinogalactan, cell wall composition. External to the mycobacin turn, is esterified with mycolic acids (Fig. 3). terial CM is the cell wall, the arabinogalactan/ They possess very long chains (C60 –90) that may arabinomannan polysaccharide layer, the OM, contain various branches, unsaturations, and and sometimes an external proteinaceous suroxygen functions such as hydroxyl, methylated face (S)-layer. However, the permeability barrihydroxyl, and keto groups. Mycolic acids present ers in these bacteria depend on both covalently in other actinomycetes are similar in structure wall-linked long-chain ␣-alkyl, -hydroxy fatty but contain shorter chains. acids, the mycolates, and noncovalently bound In addition to OM lipoproteins and enzymes, lipids. Extractable lipids include mannosylated mycobacteria possesses several porins, one inositol and phenolic glycolipids, glycopeptidolipids, and trehalose-based lipooligosaccharides whose properties have intrigued lipid chemists for decades. These lipids and mycoOuter Membrane Vesicle-Mediated Communication lates account in large measure for the remarkable drug resistance of mycobacteria, Gram-negative bacteria sometimes release OM blebs or vesicles of 0.5– rendering treatment of mycobacterial dis1.0 m in diameter into the culture medium. Typically, they contain enzymes and signalling molecules that may be delivered to other bacteria eases difficult. This difficulty is particularly when the vesicles fuse to the OM of the recipient cell, providing a important to human health because onemechanism for prokaryotic communication. In other circumstances, third of the global human population is inthese vesicles deliver bacterial protein toxins to mammalian cells. fected with mycobacteria, and millions die from mycobacterial diseases every year. Volume 3, Number 7, 2008 / Microbe Y 325 FIGURE 3 entirely different from the typical trimeric porins of gram-negative bacteria that seem to be lacking in acid-fast bacteria. The approximately 50-fold-lower numbers of porins in acid-fast bacteria compared to gram-negative bacteria, and the increased lengths of mycobacterial pores, are two primary determinants of the low permeabilities of outer mycobacterial membranes to small hydrophilic solutes. Archaeal Membranes Although similar in many respects, the lipid compositions of archaeal and bacterial membranes differ radically. In particular, archaeal membranes contain L-glycerol ether-linked lipids rather than ester-linked D-glycerol lipids found in bacteria and eukaryotes. Additionally, bacterial-type peptidoglycan cell walls are altogether lacking in archaea, which instead contain cell wall surface layer Structures of mycolic acids (mycolates) in M. tuberculosis. ␣-Mycolates: the meromyproteins. OMs are not found in the colate chains contain two cis-cyclopropanes. Methoxymycolates: the meromycolate better-characterized archaea, although chains contain an ␣-methyl-ether moiety in the distal position and a cis-cyclopropane or they have been identified in one class of an ␣-methyl trans-cyclopropane in the proximal position. Ketomycolates: the meromycolate chains contain an ␣-methyl ketone moiety in the distal position and proximal these organisms and are probably functionalities as in the methoxy series. Unsaturations are present in some meromycopresent in others. late chains (not shown). Polar ether lipids (Fig. 4) account for 80 –90% of the total membrane lipids in archaea. The remainder consists of of which is the low-activity channel protein neutral squalenes and other isoprenoids. While OmpATb, which enables such cells to adapt to some archaeal species have only the standard low pH and survive in macrophages. In contrast diether core lipids, certain sulfur-dependent arto other acid-fast bacterial porins, it shows sechaea also contain tetraether lipids. Polar headquence similarity to outer membrane porin A groups in glycosidic or phosphodiester linkage (OmpA) homologues of gram-negative bacteria. to glycerol consist of polyols, other carbohyAnother mycobacterial porin, MspA, forms a drates, and amino compounds. Ether-containcone-like tetrameric complex with a single cening lipids are more stable than the ester-containtral pore 10 nm in length. This structure is ing lipids of bacteria and eukaryotes, allowing archaea carrying them to live in extreme environments such as strongly acidic lakes and in hot springs and thermal vents at temProtein Secretion and Mycobacterial Disease peratures exceeding 100°C. Mycobacteria secrete proteins that contribute to the pathogenesis of Ignicoccus is a hyperthermophilic archaeon many human and animal diseases. Information about how the secreted belonging to the Desulfurococcales subdiviproteins and the polysaccharides of the capsule cross the outer lipid sion of the Crenarchaeota. This chemolithobarrier is fragmentary. In any case, the secretion and construction of the OM depends on very different components from those that are found in autotrophic organism obtains its energy by gram-negative bacteria. It is possible that additional knowledge of reducuing elemental sulfur with molecular mycobacterial envelope assembly and permeability will lead to new hydrogen. Cells of Ignicoccus have yet anapproaches to treating mycobacterial diseases. other unusual cell envelope consisting of cytoplasmic and outer membranes separated 326 Y Microbe / Volume 3, Number 7, 2008 by a periplasmic space of variable widths containing membrane-bound vesicles. The OM, approximately 9 nm wide, contains three types of particles: numerous irregularly packed single particles, about 8 nm in diameter, putative pores with a diameter of 24 nm, and particles arranged in rings surrounding the pores with a diameter of 130 nm. FIGURE 4 Unique Archaeal Symbioses Ignicoccus lives in symbiosis with another archaeon, a very small, singlecelled organism called Nanoarchaeum equitans (Fig. 5). This cell has one of the smallest genomes yet sequenced (fewer than 500,000 bp). In fact, too few genes are present to code for all the biological functions that are essential for life, meaning it can live only by being together with Ignicoccus. Among the missing functions are the enzymes that catalyze lipid biosynthesis. If these enzymes are really absent from this organism, how does Nanoarchaeum construct its CM? Ultrastructural analyses reveal not only the two-membrane envelope of Ignicoccus, but also the presence of intraperiplasmic vesicles. Based on analyses showing that the lipid and protein compositions of the inner and outer membranes are different, it appears that these vesicles derive from the CM of Ignicoccus. Moreover, the lipids in the nanoarchaeal membrane resemble those in the CM of Ignicoccus. These observations suggest that one organism makes the lipids for both members of this pair, and that the vesicles mediate passage from one to the other. Moreover, some transport proteins in the nanoarchaeal membrane, as well as essential cytoplasmic enzymes, apparently also derive from its symbiotic partner, Ignicoccus. Thus, these two symbiotic archaea apparently developed unusual mechanisms for intercellular communication and molecular transfers involving periplasmic vesicles. Structure of a typical archaeal ether lipid, based on L-glycerol (top), compared to that of a typical bacterial ester lipid, based on D-glycerol (bottom). Primary structural differences are illustrated. (Modified from the website http://www.ucmp.berkeley.edu/archaea /archaeamm.html.) FIGURE 5 An electron microscopic depiction of an Ignicoccus cell (bottom), showing the inner and outer membranes, in symbiotic association with two Nanoarchaeum cells (top). (Reproduced from H. Huber, M. Hohn, R. Rachel, T. Fuchs, V. Wimer, and K. Stetter, A new phylum of Archaea represented by a nanosized hyperthermophilic symbiont. Nature 417:63– 67, 2002). Volume 3, Number 7, 2008 / Microbe Y 327 Conclusions Prokaryotes possess cell envelopes of extremely varied composition and structure. In both prokaryotic domains, as in organelles of eukaryotes, the envelopes may have one or two membranes, which always possess different combinations of lipids and proteins. We are now coming to appreciate the complexities of the assembly machineries that function to construct these envelopes. Many are present in specific membranes while others span the entire cell envelope structure. Moreover, completely different transport components are found in the inner and outer membranes of organisms that have both. The structural data available for the OMs of gram-negative bacteria, high G⫹C gram-positive bacteria, and archaea suggest that outer prokaryotic membranes probably evolved independently in these three organismal types. Further research should help to clarify other important but poorly understood issues dealing with basic aspects of the functions, structures, biogenesis, and evolution of these membranes. Moreover, because fewer than 1% of prokaryotic life forms have been characterized, it seems likely that other envelope types will be discovered, making this feature a snapshot of progress rather than a definitive report. SUGGESTED READING Brennan, P. J. 2003. Structure, function, and biogenesis of the cell wall of Mycobacterium tuberculosis. Tuberculosis 83:91–97. Dowhan, W. 1997. Molecular basis for membrane phospholipids diversity: why are there so many lipids? Annu. Rev. Biochem. 66:199 –232. Kartmann, B., S. Stengler, and M. Niederweis. 1999. Porins in the cell wall of Mycobacterium tuberculosis. J. Bacteriol. 181:6543– 6546. Koga, Y., and H. Morii. 2005. Recent advances in structural research on ether lipids from archaea including comparative and physiological aspects. Biosci. Biotechnol. Biochem. 69:2019 –2034. Rachel, R., I. Wyschkony, S. Riehl, and H. Huber. 2002. The ultrastructure of Ignicoccus: evidence for a novel outer membrane and for intracellular vesicle budding in an archaeon. Archaea 1:9 –18. Saier, M. H., Jr. 2006. Protein secretion and membrane insertion systems in gram-negative bacteria. Microbe 1:414 – 419. 328 Y Microbe / Volume 3, Number 7, 2008