Cytoplasmic Membrane
Chapter
7
ROBERT J. KADNER
The hallmark morphological feature of the gram-negative bacterial cell is the presence of two concentric membranes enclosing the cytoplasm. The extensive study of the outer membrane, described in chapter 5, was prompted by its unusual lipid composition and asymmetry and its possession of relatively few protein species, all of which have unusual structures and functions. The structure and interactions of the outer membrane components dramatically affect the permeability properties of the cell and its susceptibility to antimicrobial agents, degradative proteins, bile salts, etc. The outer membrane is highly atypical and does not reflect the structure or behavior of most biological membranes.
The cytoplasmic membrane is the boundary between the cytoplasm and the environment and is primarily responsible for regulating the flow of nutrients and metabolic products in and out of the cell. The bacterial cytoplasmic membrane is similiar to membranes in eukaryotic cells, but contains a wider range of protein constituents because it must carry out most of the functions that are partitioned among the numerous eukaryotic organellar membranes. The cytoplasmic membrane is involved in almost every aspect of bacterial growth and metabolism and provides a valuable model for studies of biological membrane function. The presence of the outer membrane complicates some studies of the assembly and organization of the cytoplasmic membrane, but the power of the bacterial genetic system has greatly aided progress in most areas. This review of this topic in the previous edition of this book (75) provided a thorough and critical analysis of the physical properties of membrane lipids and proteins, and that chapter should be consulted for discussion and references to the earlier literature which are not cited here. This chapter focuses on the dramatic advances since the previous edition, particularly in the determination of the sequences, topologies, and phylogenetic relationships of membrane proteins and their complexes, in the development of approaches to the specific functions of the membrane phospholipids, and in the elucidation of the processes of protein translocation, septum formation, and transmembrane signaling.
The outer and cytoplasmic membranes can be isolated following disruption of cells in a French pressure cell or after osmotic lysis of penicillin- or lysozyme-induced spheroplasts (313). Sucrose gradient centrifugation separates Salmonella typhimurium (232) and Escherichia coli (289) membranes into several major fractions which differ in density as a function of their lipopolysaccharide (LPS) content, protein/lipid ratio, and perhaps attachment to other cellular components. The densest fraction (1.22 g/ml) contains the outer membrane (protein inclusion bodies cosediment with this fraction and can lead to erroneous conclusions about the cellular location of overexpressed proteins). The next lighter fraction contains material from both the outer and the cytoplasmic membranes and is likely to represent the membrane adhesion zones, which were first described by Bayer (19) and are thought to be sites of transfer of material between the cytoplasmic and outer membranes (222). Several cytoplasmic membrane fractions (buoyant density, 1.14 to 1.16 g/ml) are obtained which differ in composition and functional properties. Improved fractionation protocols include successive cycles of sedimentation and flotation centrifugation (159). These protocols reveal the existence of minor membrane fractions with unusual sedimentation properties and interesting biological functions, such as oriC DNA binding (55). Fractionation of membranes by other techniques, such as electrophoresis through agarose gels, reveals additional complexity of membrane organization (191).
Widely used methods for the rapid separation of outer membrane proteins from those of the cytoplasmic membrane take advantage of the differential solubility of the cytoplasmic membrane in detergents (Triton X-100 [290] or Sarkosyl [107]) in the presence of divalent cations. Caution is needed in interpretation of studies of the cellular location of proteins because any individual protein or protein complex might have atypical buoyant density or detergent solubility. Immunoelectron microscopy provides a more definitive tool for cellular location, as long as sufficient antigen is available for reliable detection.
The composition, biosynthesis, and regulation of phospholipid levels in E. coli and S. typhimurium membranes are discussed in chapter 37 and references 74, 75, 76, 201, 260, 301, and 331. The cytoplasmic membrane is composed of roughly equal amounts of protein and phospholipid. It contains 65 to 75% of the cellular phospholipids, the remainder being in the inner leaflet of the outer membrane, and 6 to 9% of the cellular protein (about 60% of the protein in the cell envelope). Three major phospholipid species are present, amounting to about 2 107 molecules per cell. There is some variation in their proportions in different strains and growth conditions, but the typical content is 70 to 80% phosphatidylethanolamine (PE), 15 to 25% phosphatidylglycerol (PG), and 5 to 10% cardiolipin (CL, or diphosphatidylglycerol) (reviewed in references 76 and 331). There are small amounts of phosphatidic acid, phosphatidylserine (PS), lysophospholipids, and diacyglycerol, which are biosynthetic intermediates or turnover products. All of the phospholipids contain sn-glycerol-3-phosphate (Gly3P) esterified with fatty acids at the sn-1 and sn-2 positions. The predominant fatty acids present in cytoplasmic membrane lipids are the saturated palmitic acid (16:0) and the unsaturated species palmitoleic acid (cis-Δ 9,10-16:1) and cis-vaccenic acid (cis-Δ 11,12-18:1). The majority of phospholipids contain a saturated fatty acid at the sn-1 position of the glycerol moiety and an unsaturated fatty acid at the sn-2 position, although di-saturated and di-unsaturated species are present (23). Phospholipids of log-phase E. coli grown at 37C contain about 45% 16:0, 35% 16:1, and 18% 18:1 (76). Myristic acid (14:0) and d-3-hydroxymyristic acid comprise 4 to 5% of the total fatty acids, but they occur almost exclusively in the glucosamine-based LPS in the outer membrane.
Fatty acid content (chain length and saturation) can be altered by manipulation of the cell genotype or environmental conditions, including temperature, stage of growth, composition of the growth medium, and presence of membrane perturbants. For example, increase of growth temperature from 17C to 37C results in an increase in 16:0 content from 20% to 31% and a corresponding decrease in 18:1 content from 39% to 27% of total fatty acids (319). Membrane perturbants such as alcohols affect the fatty acid composition similarly to a change in temperature. Cells grown in the presence of ethanol have a less saturated fatty acid composition that is typical of cells grown at a lower temperature, whereas cells grown with hexanol have a more saturated fatty acid composition typical of cells grown at a higher temperature (23, 319).
Several inhibitors of fatty acid synthesis allow manipulation of fatty acid composition. Cerulenin and thiolactomycin at low concentrations preferentially inhibit β-ketoacyl–acyl carrier protein synthase I (fabB gene product) and block unsaturated fatty acid biosynthesis (the pathways of lipid synthesis are presented in chapter 37). 3-Decenoyl-N-acetylcysteamine inactivates 3-hydroxydecanoyl–acyl carrier protein dehydrase (fabA product) and elicits a requirement for unsaturated fatty acids. Mutations in the fabA or fabB gene specifically block synthesis of unsaturated fatty acids, and their combination with mutations that block degradation of unsaturated fatty acids (fad genes [228]) allows the specific incorporation of nutritionally supplied unsaturated fatty acids (see, e.g., reference 177).
Cyclopropane fatty acids are formed by addition of a methylene residue from S-adenosylmethionine across the double bond of the monounsaturated fatty acids in existing phospholipids. The amount of cyclopropane fatty acids increases upon entry into stationary phase. Null mutations in the cfa gene result in complete loss of this modification, but these strains show no obvious changes in their log-phase growth properties or in their susceptibility to oxidizing agents (133). The cfa strain has somewhat reduced survival following repeated cycles of freezing and thawing, indicating that this fatty acid modification, which should reduce the level of oxidation-sensitive membrane components, is not essential, but may be beneficial under certain conditions.
The enzymes and genes involved in nearly every step of phospholipid biosynthesis have been identified (as detailed in chapter 37). Gly3P is esterified, first at the sn-1 position by Gly3P acyltransferase (plsB) and then at the sn-2 position by 1-acyl-Gly3P acyltransferase (plsC) (67), to yield phosphatidic acid. The substrates for the acyltransferases are either endogenously synthesized fatty acids esterified to acyl carrier protein or exogenously provided fatty acids esterified to coenzyme A. The liponucleotide derivative, CDP-diacylglycerol, is formed by the reaction of CTP with phosphatidic acid and is the substrate for the two branches of phospholipid biosynthesis which lead to the zwitterionic (dipolar ionic) lipid PE and the acidic lipids PG and CL, respectively. In the first branch, PS synthase (pssA) combines CDP-diacylglycerol and serine to form PS, which is decarboxylated by PS decarboxylase (psd) to yield PE. In the other branch, PG synthetase (pgsA) combines CDP-diacylglycerol and Gly3P to form PG 3-phosphate, which is converted to PG by various phosphatases. Two molecules of PG are combined to form CL by CL synthase (cls). All of the lipid biosynthetic enzymes are associated with cytoplasmic membrane except Psd, which is recovered with ribosomes upon cell extraction.
The phospholipid head group composition is not appreciably affected by growth conditions, except for an increase in the level of CL at the expense of PG in stationary-phase cells. Information about the effect of altered levels of individual phospholipid species comes from studies with conditional or null mutations in genes for specific biosynthetic steps. The availability of the cloned genes for most of the lipid biosynthetic enzymes allowed the construction of null mutations (where most of the coding sequence was replaced with a selectable marker) and the overexpression of individual genes from regulated promoters or from multicopy plasmids (96). Overexpression of any phospholipid biosynthetic enzyme, even one acting at the CDP-diacylglycerol branchpoint, has relatively little or no effect on the phospholipid distribution, relative to the wild-type cell (reviewed in reference 301). The choice of which phospholipid head group is incorporated during biosynthesis is not determined by the amount of the enzymes, which are normally present in excess of cellular requirements, but at the level of enzyme activity in response to unknown physical parameters of membrane function. A similar conclusion had been reached with respect to the choice of which fatty acid is incorporated by the acyltransferases (primarily PlsC), which in turn determines the proportion of saturated and unsaturated fatty acids that are esterified as appropriate for the current growth conditions (74, 76).
Studies of the consequences of the genetic depletion of individual phospholipid species provide significant insights into the role of individual lipids in membrane function, as described below. The use of null mutations developed by Dowhan and colleagues extends previous observations with potentially leaky conditional-lethal mutants. A null mutation in pssA (blocked in synthesis of both PS and PE) results in almost complete absence of PE (<0.007% of total phospholipids) (89). This Δ pssA mutation prevents growth in normal medium, but partial growth occurs upon supplementation with 5 to 50 mM levels of divalent cations, Ca2+ >Mg2+ > Sr2+, but not with Ba2+ or high concentrations of monovalent cations (268). Stimulation of the growth of the PE-deficient strain by divalent cations is not due only to their effect on medium osmolarity, but also to their effect on lipid structure (see below). Temperature-sensitive mutants affected in the psd gene, whose product is responsible for conversion of PS to PE, accumulate PS in place of PE. Growth of the psd(Ts) mutant stops when PE content falls to about 40% of the total, and can be partially restored by divalent cation supplementation, showing that acidic PS cannot replace dipolar ionic PE for normal membrane function (138). The addition of sucrose allows a partial restoration of growth of PE-deficient cells because increased osmolarity results in decreased synthesis of membrane-derived oligosaccharide (MDO), which thereby reduces the consumption of PE molecules normally used for MDO synthesis (174, 219).
Experimental manipulation of the level of the acidic lipids PG and CL, which are products of the other branch of phospholipid synthesis, can be achieved in a strain carrying a null mutation in the chromosomal pgsA gene and an intact pgsA gene under control of the lac promoter. In this strain, the content of PG+CL is proportional to the level of IPTG (isopropyl-β-d-thiogalactopyranoside), the inducer of the lacP-pgsA gene (141, 142). In the absence of IPTG, growth stops when the PG+CL pool falls to 2 to 4% of the total phospholipid. Blocking expression of the lpp gene for the murein lipoprotein allows growth even under these conditions (10). The amino-terminal residues of lipoproteins are modified during their translocation across the cytoplasmic membrane by addition of a glyceryl moiety from PG. This reaction is a major route of PG turnover and, since the major lipoprotein species is murein lipoprotein, its elimination greatly reduces the cellular PG requirement.
The synthesis of CL is blocked in a cls null mutant defective in CL synthase, which results in a greatly reduced, but not absent, level of CL (226). Although affected in its growth properties, the cls mutant is viable unless combined with a mutation in pssA which blocks PE synthesis (302). The synthetic lethality of pssA and cls mutations suggests that normal membrane function requires the presence of either PE or CL.
Phospholipids not only provide the membrane matrix but are also involved in the synthesis of several types of surface molecules. PE molecules undergo a relatively low degree of turnover (ca. 5% per generation), as a result of the transfer of their phosphoethanolamine head group for attachment to the LPS core sugars and to periplasmic MDO (137, 219). The turnover of PG is more extensive and approaches 30% to 40% per generation. The PG head group is used for the modification of various lipoproteins and MDO, and the entire molecule is used for the formation of CL.
Some proteins are tightly bound to the membrane (integral membrane proteins) with one or more membrane-spanning segments, some are loosely bound (peripheral membrane proteins), and others associate transiently. The protein content of the cytoplasmic membrane is quite varied, with over 100 species seen on two-dimensional electrophoretic analysis (281). This number is certain to be an underestimate, owing to the loss of peripherally bound membrane proteins and the very low concentrations of a substantial number of transmembrane regulatory or transport proteins. The distribution of membrane proteins changes considerably under different growth conditions, which, as described below, elicit the expression of different energy-generating systems and the induction or repression of transport systems.
The familiar fluid mosaic model of membranes portrays the membrane proteins floating freely in the lipid bilayer, where they have restricted vertical travel but are free to diffuse within the plane of the membrane. Although there are 30 to 40 phospholipid molecules (surface area, ca. 0.5 nm2 each) in each monolayer for each membrane protein molecule, the apolar area of typical membrane proteins, such as the photosynthetic reaction center with 11 transmembrane segments, covers about 10 nm2 (266), indicating that the proteins and lipids cover roughly equal amounts of the membrane surface. The protein content of most membranes is so high that protein molecules are separated from one another by only three or four layers of phospholipid molecules.
The lipid bilayer of the cytoplasmic membrane forms a hydrophobic barrier that prevents the uncontrolled movement of polar molecules and allows the accumulation and retention of metabolites and proteins. The lipids also provide a suitable environment or matrix for the proper functioning of the membrane protein complexes involved in bioenergetic and biosynthetic functions. The lipid composition determines the dynamics and interactions of the lipid molecules, which in turn determine the barrier and permeability properties of the membrane and influence the topology, interactions, and functions of the membrane proteins. The structures and dynamic properties of membrane lipids have been extensively reviewed, with particular emphasis on the nature of the thermotropic phase transition, the effect of growth temperature on lipid composition, and the effect of lipid composition on the phase transition and on membrane function (32, 75, 152, 189, 15, 225, 294, 295, 324).
The cytoplasmic membrane is built upon the familiar lamellar phospholipid bilayer, an extended two-dimensional surface of two opposed monolayers. Isolated membrane lipids spontaneously form this lamellar structure when suspended in aqueous medium. This process is driven by hydrophobic interactions that segregate the nonpolar acyl chains into the interior and leave the polar head groups accessible for hydration (321). Molecular packing is governed by steric and electrostatic repulsion of the polar head groups and by acyl chain interactions. Evidence for the presence of lamellar structure as the principal organization of lipids in the intact membrane is convincingly provided by 2H- and 31P-nuclear magnetic resonance studies, as well as by low-angle X-ray diffraction analysis (reviewed in references 177, 215, 263, and 295). These techniques reveal that the acyl chain at the sn-1 position has an all-trans conformation, whereas the acyl chain at the sn-2 position runs parallel to the membrane surface for two carbon units before turning toward the bilayer center. The two acyl chains are thus staggered relative to one another by about three methylene groups (295). Numerous biophysical probes that report on the rate or extent of acyl chain motions show that biological membranes undergo several structural transitions in response to changes in their physical and chemical environment. In many studies, the behavior of membrane lipids in the intact cell matches well to the properties of the extracted lipids and indicates that there are few or no long-lived interactions (>1 s) between proteins and boundary lipids. Membrane function is affected by each of the following: phase transition between the rigid gel and the fluid liquid-crystalline state; phase separation or the segregation of different lipids into separate fluid and gel regions; and polymorphic transitions or the rearrangement of certain lipids into nonlamellar structures. Some assays of phospholipid dynamics using fluorescent or electron spin resonance probes have given results consistent with the direct measurements, although controversial observations can result from nonuniform distribution of some probes or from the difficulty of relating probe behavior to a macroscopic aspect of membrane fluidity or motion (58, 189).
Phase Transition.
Model membrane bilayers containing a single phospholipid species undergo reversible changes of state in response to temperature (reviewed in reference 295). At low temperature the acyl chains are in the closely packed, ordered array of the rigid gel state in which molecular motion is highly restricted. Upon warming, the membrane undergoes an endothermic transition. Above the phase transition temperature (Tm), the acyl chains have elevated motional freedom around each C-C bond in the acyl chains and the head group region, as well as increased rotation of the entire phospholipid molecule around its long axis and increased translational movement of the molecule in the plane of the membrane. Even in the fluid phase, the motion of the acyl chains is more constrained than in liquid hydrocarbon, and the degree of mobility (assessed by the order parameter deduced by nuclear magnetic resonance techniques from the relaxation rates of fatty acids specifically deuterated at successive methylene groups) remains constant for eight or nine methylene units from the head group and then decreases substantially toward the bilayer center (225). The "melted" lipids in the liquid-crystalline state remain in lamellar structure, but the bilayer is thinner because the acyl chains are less often in their fully extended conformation. The average area occupied by each phospholipid molecule increases upon melting from 0.47 nm2 to around 0.67 nm2, and the distance between polar head groups decreases from 6.5 nm to 5.5 nm (263).
The transition temperature of a lipid bilayer depends on its fatty acid composition and the identity of the polar head group. Unsaturated, branched, and cyclopropane fatty acids confer a lower transition temperature than saturated acyl chains, e.g., replacement of a saturated acyl chain with a monounsaturated fatty acid can lower the Tm by 46C. The kink in the acyl chain caused by the double bond or other discontinuities interferes with the close packing of neighboring acyl chains. Although acyl chain length strongly affects the transition temperature, the narrow range of acyl groups present in the membranes of enteric bacteria means that chain length is not a major factor in the adjustment of a cell’s membrane properties. In model membranes composed of a single phospholipid species, the phase transition occurs in a very narrow temperature range (1 to 3C), indicative of a highly cooperative melting process. In biological membranes containing multiple lipid species, including the E. coli cytoplasmic membrane, this transition occurs over a broad range (10 to 20C).
Membranes from E. coli cells grown at 37C are still completely fluid at 30C (177). The lipid composition is adjusted during growth at different temperatures to maintain the membrane in the fluid, liquid-crystalline state by a considerable margin. The nutritional requirements of mutant strains blocked in steps of fatty acid synthesis indicate that membrane function (defined as cell growth) requires the presence of both saturated and unsaturated fatty acids. Cells lyse when their membranes contain only saturated or only unsaturated fatty acids, suggesting that normal membrane function depends on the coexistence of regions of gel and of liquid-crystalline phase (76). The physical state of the bilayer affects the barrier properties of membranes and the location and activities of their proteins. Fluid membranes have much higher permeability to small molecules than do gel-phase bilayers. The production of an abnormally fluid bilayer, owing to the incorporation of the trans-isomer of 18:1, results in considerable loss of barrier properties. The highest permeability to small molecules in lipid bilayers occurs when membranes pass through the phase transition and at interfacial zones between gel and fluid regions (S. Clerc and T. E. Thompson, in preparation). Most membrane proteins are inactive in or are excluded from ordered gel-phase membranes (324).
Phase Separation.
Phase separation was first described for model membranes composed of two lipids with substantially different gel-to-liquid-crystalline transition temperatures, in which the lipids are fully miscible in the fluid state but only partially miscible in the gel state (215). These bilayers show two distinct phase transitions rather than a single broad transition. The lipid types separate from the mixture during passage through the phase transition and give rise to a mosaic membrane with discrete patches of each lipid type. This separation results from the tendency of the ordered acyl chains of the higher-melting lipid to associate mainly with each other and thereby avoid the energetic cost of ordering the fluid acyl chains of the other lipid type. Examples of phase separation and the exclusion of most proteins from the rigid phase have been seen in electron micrographs of bacterial membranes from cells held below the transition temperature of some of their lipids (20, 332). The rigid, protein-poor fraction can be separated from the fluid, protein-rich patches. Formation of discrete lipid patches can have substantial consequences on membrane function if proteins are excluded from the rigid gel-phase regions (324). When the proportion of the membrane in the protein-containing fluid phase becomes smaller, e.g., upon a decrease in temperature, the fluid regions become disconnected and the ability of proteins to interact with one another or with mobile membrane-embedded substrates, such as quinones, can become increasingly limited (4). These considerations warrant caution about unanticipated changes in membrane properties following changes in temperature or osmolarity (see, e.g., reference 207).
Polymorphic Transitions.
Although some phospholipids preferentially form lamellar bilayer structures, others, PE in particular, have a strong preference for formation of nonlamellar structures under appropriate conditions of temperature, concentration, and degree of hydration. The most common nonlamellar structure is the inverted hexagonal phase (HII), in which the lipids form tubular structures with the polar head groups on the inside, surrounding an aqueous core, and the acyl chains facing outward (reviewed in reference 294). The lipid cylinders form hexagonal arrays that place the acyl chains of one tubule in contact with the acyl chains of surrounding tubules. Possible driving forces for HII phase formation include the geometry of the head group and the ease of its dehydration. Lipids which prefer the lamellar conformation, such as PG, lower the Tm of PE and reduce or prevent its formation of nonlamellar structures, which should be detrimental to biological membrane function. It is likely that PE molecules provide the ability to form transient regions of membrane with a high curvature or different head group interactions, rather than to disrupt the lamellar bilayer completely. These regions may be important for such dynamic processes as membrane fusion, lipid movement between membranes, and the passage of proteins across membranes. A requirement for the presence of phospholipids with the propensity for forming nonlamellar structure is indicated by the fact that high concentrations of divalent cations allow growth of PE-deficient cells (89, 268). The divalent cations (Ca>Mg>Sr) that are effective at restoring growth show the same pattern of effectiveness at inducing the formation of nonlamellar structures by CL. E. coli membranes apparently must contain either PE or CL, but either alone is dispensable (302).
High-resolution three-dimensional structure information is available only for the photosynthetic reaction centers of purple nonsulfur bacteria, several outer membrane porin trimers, and bacteriorhodopsin (54, 73). Despite the difficulty of crystallographic determination of membrane protein structure, there has been considerable progress in the description of these proteins’ hydrophobic character and transmembrane topology.
Hydrophobic Character.
Membrane proteins are significantly different from soluble proteins (265, 321). Cytoplasmic proteins fold into structures that place their hydrophobic segments in the interior to reduce the entropic cost of their exposure to water. Polar residues are generally placed on the protein surface where they can be hydrated. In the case of membrane proteins, the amino acid residues in the interior have similar hydrophobic character as residues in the interior of the soluble proteins, and the residues that are exposed to the aqueous environment have the expected polar character. Residues exposed to the nonpolar lipid acyl chains have even greater aggregate hydrophobic character than residues in the protein interior. The placement of a high proportion of hydrophobic residues on the protein surface probably maintains the correct conformation and penetration of the protein in the nonpolar lipid bilayer. When the protein is placed in an aqueous medium following detergent disruption of the lipid bilayer, this high surface hydrophobicity impels the protein to evert itself and bury its more hydrophobic portions into the interior. To reduce this tendency and retain membrane proteins in their native structure, or at least in a form that retains functional activity when reconstituted into membranes, procedures for solubilization of membrane proteins generally use nonionic detergents to disrupt the forces holding the phospholipids into bilayer structure, additional phospholipids to satisfy the need for a hydrophobic environment for the protein’s surface residues, and an osmolyte to help maintain stability of the polar regions (7).
Transmembrane Topology.
The gross transmembrane orientation and topology of a membrane protein can be deduced using biochemical or genetic approaches. Biochemical studies determine the access of specific sites on the protein to membrane-impermeant probes, including proteases, monospecific antibodies, or chemical reagents. Finding a substantially different degree of reactivity of a site from opposite sides of the membrane provides a convincing test of topology of that site, as long as controls are included to ensure that the protein does not reorient and that the membrane is not disrupted during the analysis. These biochemical tests are often technically challenging owing to the limited number of exposed targets in proteins with short surface-exposed loops, the low solubility of many protein fragments, or the presence of too many or too few reactive sites. Molecular genetic techniques allow the insertion of a short peptide segment, which can be recognized by a monoclonal antibody (8) or other binding protein (70), into specified sites in a membrane protein. It is also possible to substitute any amino acid with a chemically reactive cysteine residue, if the native cysteines can be replaced (exemplified in references 5, 167, 237). These engineered probes can reveal differential access of the site of the insertion to appropriate reagents, as long as it can be independently verified that the altered protein retains the same orientation, topology, and activity as the wild-type protein.
In contrast to the difficulty of obtaining convincing and complete biochemical evidence for the transmembrane topology and orientation of a membrane protein, simple and widely used genetic tests are based on the isolation of fusions of a protein to topological reporters, enzymes whose activity depends on their cellular location. In the approach developed by C. Manoil, D. Boyd, and J. Beckwith (38, 206, 211), a gene of interest is fused upstream of the coding sequence for the mature portion of the periplasmic protein, alkaline phosphatase (PhoA). The mature portion of PhoA lacks the signal sequence needed for its entry into the secretory pathway and is retained in the cytoplasm. The reducing environment of the cytoplasm prevents formation of the disulfide bonds that are required for PhoA stability and enzymatic activity (92). Fusions to cytoplasmic proteins lack significant PhoA enzymatic activity. Fusion to the amino-terminal portion of a periplasmic protein or an outer membrane protein provides the signal sequence that allows translocation of the PhoA moiety across the cytoplasmic membrane, where disulfide bonds can form. PhoA fusions to various positions of a periplasmic protein display similar levels of enzymatic activity, whether the PhoA portion remains attached to the target protein or is released by proteolysis near the fusion joint. Fusions to cytoplasmic membrane proteins, on the other hand, exhibit varying levels of PhoA enzymatic activity. The interpretation is that fusion to a outward-facing portion of a transmembrane protein provides the export signal for translocation of PhoA into the periplasm, whereas fusion to a cytoplasmic portion of the protein retains PhoA in the cytoplasm (Fig. 1). Construction of fusions to successive amino acid residues along a single transmembrane segment indicated that fusion to any residue in a periplasmic loop or in the outer half of the transmembrane segment allows export of PhoA, whereas fusions to residues in cytoplasmic loops or the inner half of a transmembrane segment are retained in the cytoplasm (49).
Manoil and Beckwith (205) constructed a variant of transposon Tn5, called TnphoA, which forms a PhoA fusion protein whenever it inserts into a gene in the proper direction and reading frame. This genetic tool facilitates isolation of large numbers of fusions in chromosomal or cloned genes that encode secreted proteins. The presence of PhoA activity, indicative of an in-frame fusion to a secreted or membrane protein, is readily detected on solid medium containing a chromogenic PhoA substrate. A more specific but expensive method of analysis uses polymerase chain reaction or oligonucleotide-directed techniques to construct fusions of phoA at any desired site. The optimal test for transmembrane topology is to compare the enzymatic activity of fusions placed at the carboxyl end of each extramembranous loop that is predicted from the hydropathy profile (39).
Another useful topological reporter is β-lactamase, which confers resistance to β-lactam antibiotics when exposed to the periplasm but not when retained in the cytoplasm (45, 101). The lacZ gene provides a complementary topological reporter, because β-galactosidase is not normally translocated across the cytoplasmic membrane (190) and exhibits lower enzymatic activity when fused to an external domain of a target protein than when fused to a cytoplasmic segment of the same target protein. A genetic system for the facile interconversion of phoA and lacZ translational fusions has been developed (204).
Topogenic Signals and Membrane Insertion.
The information that targets a protein to its final cellular destination is contained in its primary sequence. Some cytoplasmic membrane proteins use the general protein secretory pathway (see below) for their insertion in the membrane, and most of these are synthesized with a cleavable amino-terminal signal sequence. The size of the external polypeptide segment that must be translocated across the membrane determines whether the insertion of a protein into the membrane requires the general secretory system (9). The degree of dependence on the secretory system can be assessed from the sensitivity of a protein’s export to inactivation of conditional mutations in sec genes or to exposure to azide, a fairly specific inhibitor of the SecA ATPase activity (231).
Most cytoplasmic membrane proteins are not synthesized with a cleavable signal sequence and appear to insert in the membrane in the correct orientation without use of the secretory pathway (see reference 210). Is the topogenic information for correct orientation in the cytoplasmic membrane contained in the primary amino acid sequence of the protein? Is the sequence of each transmembrane segment sufficient to specify its correct orientation? Computer-aided analysis of the sequences of transmembrane segments and their flanking regions from proteins of known or suggested topology revealed that the basic amino acids Arg and Lys were four times more prevalent in cytoplasmic loops than in periplasmic loops, whereas there was no preference for acidic residues on either side of the membrane (338). This finding that cytoplasmic loops tend to have excess positive charge, whereas periplasmic loops have a negative or no net charge, does not apply for large polypeptide loops of more than 60 amino acids. Consideration of the interior-positive charge bias greatly improves the reliability of the prediction of transmembrane segments from hydropathy plots (340). A positively charged polar segment of amino acids acts to stop transfer of its adjacent nonpolar transmembrane segments. Numerous studies support the view that the primary determinant of the orientation of a transmembrane segment is not contained within the sequence of the transmembrane segment, but is the presence of net positive charge in adjacent extramembranous loops, primarily the cytoplasmic loop. The nature of the periplasmic loop has less of an effect and the transmembrane segments themselves appear to have no defined orientation (50, 211). Studies with numerous proteins showed that alteration of the charge on either end of a transmembrane segment, either by addition or removal of positively charged residues, can cause reorientation of that segment (9, 37, 178, 361) (Fig. 2).
Transmembrane segments are generally hydrophobic in character, but many exhibit amphipathic character with polar and even charged residues along one face of the putative helix. In general, transmembrane segments are considered to be α-helices, judging from the high α-helical content of most membrane proteins, shown by circular dichroism spectroscopy, and from the periodicity of exposure of residues to the acyl chains (5, 149). Helix-breaking proline and glycine residues are present in transmembrane segments as frequently as in soluble proteins, although proline residues are less common in inwardly directed transmembrane segments (338). The flexibility or bending that proline and glycine residues confer on transmembrane α-helices may contribute to the function or stability of the protein in the membrane. However, in several proteins most or all of the prolines can be replaced with considerable or full retention of function (339).
The success of topological reporters in defining the orientation of an intact protein requires that fusions containing only part of the membrane protein follow the same route and rules of membrane insertion as the intact protein. Since most fusions reveal the expected or known topology, segments of the protein beyond the site of the fusion junction must not be necessary for correct insertion of the upstream transmembrane segments. Most of the examples of anomalous behavior by PhoA fusions can be explained by the presence of positively charged residues near the fusion junction (e.g., reference 3). Protein insertion into the membrane probably occurs as a cotranslational process, where the orientation is determined sequentially for each separate membrane-spanning segment, rather than through insertion of the completed protein or of helical hairpins or some other large subdomain. There are examples where the insertion or stable maintenance of one protein is affected by the presence of another protein. Analysis of the insertion of MalF, a maltose transport protein with eight transmembrane segments, showed that insertion of the protein into the membrane is independent of the presence of other proteins but that its maturation into a final protease-resistant form requires it to form a complex with MalG (328).
The cytoplasmic membrane carries out a considerable number and variety of important cellular functions, including energy generation and conservation, the regulated transport of nutrients and metabolic products, translocation of envelope macromolecules, and transmembrane signaling. Many of these topics are described in detail in other chapters, and this chapter presents a brief integrated overview of the components and the mechanisms responsible for these processes.
Most biosynthetic and transport processes in E. coli and S. typhimurium are driven either by the hydrolysis of the high-energy phosphate bonds in ATP, GTP, or phosphoenolpyruvate (PEP), or by coupling to transmembrane ion gradients. The topic of bioenergetics addresses the formation and utilization of these two major forms of cellular energy currency. Although these processes in bacteria resemble those in eukaryotic cells, substantial differences exist with respect to the flexibility of the metabolic pathways and their capacity to use many different substrates and electron acceptors. Cells growing in fermentative conditions (absence of oxygen or other inorganic electron acceptors) produce ATP by substrate-level phosphorylation reactions in the glycolytic pathway, the conversion of acetyl-coenzyme A to acetate, or other fermentation pathways (chapter 18). The ATP generated by these reactions can be used to form transmembrane ion gradients, primarily by reversal of the F1F0-proton-translocating ATPase. In respiring cells, passage of electrons through an electron transfer chain to suitable electron acceptors (oxygen, nitrate, nitrite, fumarate, dimethyl sulfoxide [DMSO], trimethylamine N-oxide [TMAO], or hydrogen) is coupled to the extrusion of protons and the creation of a transmembrane electrochemical gradient of protons. This proton motive force (PMF, ) can be used by many transport processes (chapter 19).
The properties of the electron transfer systems are described in detail in chapter 17 and reviewed in references 158, 342, and 357. The composition of bacterial electron transfer chains is quite flexible. Different electron transfer components are produced in response to the presence of particular substrates, and there are considerable differences between the electron transfer chains of aerobic or anaerobic cells. Bacterial respiratory chains act as a series of physically separate protein complexes. Numerous membrane-bound dehydrogenases transfer two electrons or hydrogen atoms from their specific substrates to the pool of quinones, which serve as mobile hydride carriers diffusing through the membrane. Some dehydrogenases are integral transmembrane components (hydrogenase, formate dehydrogenase, and NADH dehydrogenase I), whereas others have a peripheral catalytic subunit(s) which binds to the membrane through a separate transmembrane anchor subunit(s) or by exposing an internal membrane-binding segment. Formate dehydrogenase, NADH dehydrogenase I, hydrogenase, and anaerobic Gly3p dehydrogenase catalyze proton translocation and serve as energy coupling sites, whereas the other dehydrogenases are peripherally bound to the membrane and lack proton-pumping activity. There are usually no c-type cytochromes or cytochrome c oxidases in these bacteria. The reduced quinones are then reoxidized by membrane-bound oxidases or reductases, which transfer electrons to the terminal electron acceptor. Some of the dehydrogenases and terminal oxidases/reductases (e.g., cytochrome oxidases and nitrate reductase) couple electron flow to proton extrusion or charge movement, whereas others are not coupled directly to PMF generation. The existence of multiple isozymes of terminal reductases has been recognized from cloning and sequence analysis, although the function of the multiple isozymes has not been defined yet. Some features of the process of electron transfer are shown in Fig. 3, and the properties of some electron transfer proteins are listed in Table 1.
Table 1Components of several electron transfer complexes |
Respiratory Chain Dehydrogenases.
Membranes from aerobically grown E. coli cells contain dehydrogenases which transfer electrons from NADH, succinate, d-lactate, or pyruvate (pyruvate oxidase) to ubiquinone. Two NADH dehydrogenases (NDH) have been described (51, 139, 209, 357). NDH-2, encoded by the ndh locus at 22.4 min on the E. coli genetic map, is a single 47-kDa polypeptide with bound flavin adenine dinucleotide (FAD) and few if any transmembrane segments (363). It has been purified in active form and acts only on NADH (139). NDH-2 contains no iron and is not coupled to proton extrusion (165). Its function in aerated cells is not obvious, and its absence confers no obvious growth defect (51, 52). Perhaps its ability to bypass the energy-coupled step of electron transport allows energy-replete cells to maintain a high flux of electron flow to the cytochrome oxidases, which lower the dissolved oxygen level and the associated production of toxic reactive oxygen species. The other NADH dehydrogenase, NDH-1, is encoded by the nuo locus at min 49 (51), and its nucleotide sequence reveals the presence of 14 genes, all related to subunits of the mitochondrial NADH:ubiquinone oxidoreductase and other electron transfer components (342, 346). Nine of the 14 NDH-1 proteins are predicted to have a transmembrane orientation. Although this very labile, multisubunit enzyme has not been purified in active form, it contains eight iron-sulfur centers (two of the [2Fe-2S] type and six of the [4Fe-4S] type), can generate a PMF in membrane vesicles, and can oxidize NADH or deamino-NADH (209, 213, 346). A stoichiometry of two protons extruded per electron is suggested by analogy with the bovine mitochondrial counterpart (51). Mutant strains lacking NDH-1 activity grow well (52) but are impaired in their survival in the stationary phase of growth (365). Mutants defective in both nuo and ndh function lack NADH dehydrogenase activity and exhibit poor growth on rich medium and no growth with mannitol as carbon source.
Pyridine nucleotide transhydrogenase catalyzes the exchange of reducing equivalents between NADH and NADPH. The equilibrium constant for the two redox couples is close to 1, and the reduction of NADP+ by NADH is driven by the inward translocation of protons. This enzyme probably serves to form NADPH for anabolic purposes rather than to oxidize NADPH as electron donor, since it is regulated in response to conditions where NADPH production is warranted, such as its induction along with NAD(P)-glutamate dehydrogenase when ammonium ions are the sole nitrogen source (see reference 158). Transhydrogenase is encoded by the pntAB operon and is composed of a 510-residue subunit with four to six transmembrane segments and a 462-residue β subunit with perhaps seven transmembrane segments (62). Like many proton pumps, it is inhibited by DCCD (dicyclohexylcarbodiimide), a reagent for acidic residues in a hydrophobic environment. Transhydrogenase has three DCCD-reactive residues in the α subunit, but they appear to contribute to the NADH-binding site rather than to the proton-transduction pathway (125).
Succinate dehydrogenase is an essential component of the tricarboxylic acid cycle. It catalyzes the conversion of succinate to fumarate, with transfer of the reducing equivalents to ubiquinone. This enzyme is induced by aerobic growth on nonfermentable carbon sources and is subject to catabolite repression by glucose (see reference 158). The sdh genes for this enzyme are organized in an operon at 16.3 min which encodes four polypeptides of 64.3, 26, 14.2, and 12.8 kDa (81, 354). The two larger catalytic subunits are generally polar and contain bound flavin and non-heme iron, respectively, whereas the two smaller membrane-embedded proteins are highly hydrophobic and anchor the larger subunits to the membrane. The large subunits are related in sequence to the corresponding parts of fumarate reductase.
Lactate is a major product of sugar fermentation. The conversion of pyruvate to d- or l-lactate allows reoxidation of NADH formed during glycolysis and is catalyzed by cytoplasmic NADH-dependent dehydrogenases. In addition to these lactate-producing enzymes, cells constitutively produce d-lactate dehydrogenase, an NAD-independent membrane-associated flavoprotein of 571 amino acids (272). This enzyme allows growth on d-lactate as the carbon source by coupling lactate oxidation to ubiquinone reduction. Although its sequence lacks obvious transmembrane segments, the enzyme is associated with the inside surface of the cytoplasmic membrane (208). Since right-side-out cytoplasmic membrane vesicles effectively oxidize d-lactate but not NADH, d-lactate dehydrogenase can probably dissociate and rebind on either side of the membrane following cell disruption. A membrane-bound l-lactate dehydrogenase is also induced by growth on lactate (see reference 158).
Pyruvate oxidase (misnamed in the sense that it does not directly react with oxygen) converts pyruvate to acetate + CO2 with transfer of reducing equivalents to ubiquinone-8. This tetramer of M r 60,000 subunits contains thiamine pyrophosphate and FAD and can be purified as a soluble protein (see reference 158). Its enzymatic activity is stimulated over 20-fold by the addition of membranes or lipids, and the affinity of the protein for membranes is enhanced when the bound FAD is in the reduced state, i.e., when pyruvate is present. No separate membrane anchor protein is needed, and the sequence of the poxB gene does not show obvious hydrophobic transmembrane segments (130). Membrane association apparently occurs when flavin reduction causes a conformational change in the protein, which exposes a membrane-binding amphipathic α-helix near the carboxyl end of the protein (129). Mutants defective in lipid activation also occur outside this region (residues 558 to 568) (56). Similar reversible membrane association occurs with proline oxidase (PutA), a bifunctional protein induced by the addition of proline, following a conformational change in the protein induced by reduction of its bound FAD cofactor by substrate (46). Other inducible membrane-bound dehydrogenases include malate oxidase and dihydroorotate dehydrogenase.
Many strains of E. coli synthesize the membrane-bound apoenzyme of glucose dehydrogenase. This enzyme converts glucose to gluconic acid with transfer of electrons to the quinone pool, but its activity requires the addition of the cofactor pyrroloquinoline quinone (PQQ), which is not made by E. coli (334). The gcd gene at 3.1 min on the genetic map is highly homologous to PQQ-dependent enzymes from other bacteria (63). The gene is expressed from two promoters, one of which is repressed anaerobically and the other is repressed by the cyclic AMP (cAMP)-binding transcription activator protein, CAP (358).
E. coli can grow with glycerol or Gly3P as sole carbon source using oxygen, nitrate, fumarate, etc., as electron acceptor. Aerobic cells produce the membrane-associated glycerol-3-phosphate dehydrogenase (GlpD), a 501-amino-acid flavoprotein (11) whose active site faces the cytoplasm. Anaerobic cells express instead the glpABC operon, which encodes the catalytic dimer formed by the 62-kDa GlpA and 43-kDa GlpB subunits, which contain FAD and flavin mononucleotide binding domains, respectively (66). GlpC (44 kDa) contains FeS centers and is tightly associated with the membrane, where it provides the anchor for the GlpAB complex.
There are at least three formate dehydrogenases, which oxidize formate to CO2 coupled to the reduction of oxygen or, under anaerobic conditions, of nitrate, nitrite, fumarate, or protons. Formate oxidation can be coupled to a hydrogenase for hydrogen production by the formate hydrogenlyase reaction. The formate dehydrogenase that is coupled to hydrogenase-3 in the formate hydrogenlyase reaction is called FDH-H and is encoded by fdhF at 92.4 min. This activity is expressed in the absence of oxygen and nitrate. Another formate dehydrogenase is induced in cells grown anaerobically with nitrate (FDH-N, encoded by fdnGHI at 32 min) and contains the 110-kDa FdnG catalytic subunit, the 32-kDa FdnH electron transfer subunit, and the 20-kDa FdnI cytochrome b (22). The subunit is a selenoprotein and carries the molybdenum cofactor molybdopterin. A third formate dehydrogenase, FDH-O, is similar in size to FDH-N (251) but is expressed aerobically and may participate in transfer of electrons from formate to oxygen (284). The three formate dehydrogenases are unusual proteins in that their primary sequences contain a residue of selenocysteine. This, the twenty-first protein amino acid, is introduced during translation from selenocysteinyl-tRNASec, as directed by an internal UGA codon which lies upstream of a specific stem-loop structure (33).
The anaerobically expressed hydrogenases catalyze the interconversion of H+ and H2 and allow either the oxidation of H2 as electron donor during anaerobic respiration (uptake hydrogenase) or the production of H2 during fermentation (reviewed in reference 356). E. coli possesses at least three hydrogenase isozymes (283). The six-gene hya operon encodes uptake hydrogenase-1, which is of unknown function although its synthesis is induced by formate and is dependent on the anaerobic transcription activator Fnr. The hydrogenase-1 complex contains three structural subunits: HyaA, which has an unusually long, amphipathic signal sequence; HyaB, which has two nickel-binding Cys-X-X-Cys motifs; and HyaC, which has four to six transmembrane segments and serves as membrane anchor (216). The seven-gene hyb operon encodes uptake hydrogenase-2, which is coupled to fumarate reduction (356). The eight-gene hyc operon encodes the H2-evolving hydrogenase-3 isozyme, which participates in the formate hydrogenlyase pathway (34, 282). Genes in the 58–59 min region of the genetic map affect expression of all three hydrogenases; some members of the hyp operon are involved in the incorporation of nickel ions.
Quinones.
The membrane-embedded quinones are essential for electron transfer because they shuttle reducing equivalents from the dehydrogenases to the terminal reductases or oxidases. Ubiquinone-8 is the predominant species in aerobic cells, and menaquinone-8 is a major species in anaerobic cells (the designation "8" indicates the number of isoprenoid units in their side chain). Their rate of diffusion through or across the membrane is likely to be affected by the state and dynamics of the membrane phospholipids (207). Menaquinone-8 is a poor substitute for ubiquinone-8 as a carrier between electron transport components in aerobically grown cells, but it is absolutely required for the anaerobic reductases acting on TMAO and fumarate (reviewed in reference 75).
Cytochrome Oxidases.
Different terminal oxidases or reductases are made, depending on the available electron acceptors. There are two terminal oxidases in the aerobic E. coli respiratory chain, the cytochrome d complex and the cytochrome o complex (258). Both complexes transfer electrons from ubiquinol-8 to oxygen and have been purified and reconstituted in phospholipid vesicles. Mutants lacking either system grow well on nonfermentable carbon sources (52). Both oxidases couple electron transfer to proton extrusion, although the cytochrome o complex appears to be more efficient (2 H+ extruded per electron) than the cytochrome d complex (1 H+/e– through a scalar reaction, not as a proton pump). Cytochrome d complex (Cyd) predominates when cells are grown at low oxygen tension (as occurs at high cell density in batch cultures or under microaerophilic conditions). Cyd is a transmembrane heterodimer encoded by the cydAB operon at 16.6 min, whose sequence is unrelated to other electron transfer components (131). It contains three heme groups (two are protoporphyrin IX, identified spectrally as b 558 and b 595, and one is heme d). Subunit I (58.3 kDa) contains six or seven transmembrane segments, while subunit II (42.5 kDa) spans the membrane eight times (224). The product of the cydC gene at 19.2 min is required for assembly of the Cyd complex (123).
Sequence analysis of the cyoABCDE operon at 10.2 min, encoding the cytochrome o complex, revealed the presence of five open reading frames (60). The deduced CyoA, CyoB, and CyoC polypeptides are clearly related in sequence to subunits II, I, and III, respectively, of the large superfamily of eukaryotic and prokaryotic aa 3-type cytochrome c oxidases. The major differences are that the E. coli enzyme contains two molecules of heme b (protoporphyrin IX) and one or two copper atoms (154). Topological reporters indicate that all five Cyo proteins span the membrane multiple times (59). As in the other members of the superfamily, electron transfer to O2 occurs at a bimetallic center consisting in Cyo of a high-spin form of heme o and a copper atom. A low-spin heme b 562 is also bound on subunit I, and mutagenesis studies are identifying the ligands for the three prosthetic groups (325). All three metal centers appear to be located on the periplasmic side of the transmembrane complex, suggesting that there must be at least one transmembrane channel to bring protons from the cytoplasm to the bimetallic reduction site. This enzyme consumes four cytoplasmic protons for the four-electron reduction of each molecule of O2, and it also translocates four additional cytoplasmic protons to the periplasmic side, with generation of PMF. Synthesis of both Cyo and Cyd complexes is regulated by the global anaerobic Fnr (72) and Arc (161) sensors. Their differential synthesis and the physiological value of the less efficient Cyd complex at low oxygen tensions may be related to its higher affinity for O2.
Anaerobic Reductases.
Reductases that use nitrate, nitrite, fumarate, TMAO, or DMSO as terminal electron acceptors are expressed only under anaerobic growth conditions through the action of the redox-sensitive transcription activator protein Fnr (195, 312). The presence of nitrate in an anaerobic medium further increases the level of nitrate reductase 10- to 20-fold and represses the synthesis of most other anaerobic reductases. As described in chapter 17 and 95, this nitrate regulation is achieved by the action of an unusual dual two-component system, where the sensor kinases NarX and NarQ control the phosphorylation and action of the NarL and NarP transcription activators (259). This hierarchial pattern of gene expression brings about the synthesis of whichever enzyme and electron transfer pathway provides the highest energy yield, as determined by the midpoint redox potential of each enzyme’s substrate and product (195). Transfer of electrons from NADH (Em,7, –320 mV) to oxygen (Em,7, 800 mV) releases free energy, ΔG0 ', of –196 kJ/mol, whereas electron transfer to nitrate (Em,7, 420 mV) yields ΔG0 '?of –146 kJ/mol, and transfer to fumarate (Em,7, 30 mV) yields only ΔG0 ' of –67 kJ/mol (158). Thus, reduction of nitrate should be preferred over reduction of fumarate, TMAO (Em,7, 130 mV), or DMSO (Em,7, 160 mV).
The structure, topology, and mechanism of nitrate reductase and fumarate reductase have been extensively studied (reviewed in reference 158). There are two nitrate reductases. The major one is encoded by the narGHJI operon at 27.6 min (30), and the minor species is encoded by the narZYV operon at 32.5 min (31). As is the case with most of the reductases, both nitrate reductases are composed of three protein subunits; in the major species, they are called α (NarG), β (NarH), and γ (NarI). The γ subunit (cytochrome b NR) is a hydrophobic polypeptide with multiple membrane-spanning segments, two heme groups, and a menaquinone-binding site. The large 138-kDa catalytic α subunit contains the molybdopterin cofactor, and the 57-kDa electron-transferring β subunit has four non-heme iron [4Fe-4S] centers. Molybdopterin is the carrier of molybdenum ions in all molybdenum-containing oxotransferases (262). This oxygen-labile cofactor contains a reduced pterin moiety with a 6-alkyl adduct that includes the Mo-binding thiolene group. The molybdoenzymes of the enteric bacteria, which include nitrate reductase, DMSO reductase, TMAO reductase, and formate dehydrogenase, contain molybdopterin guanine dinucleotide. The α and β subunits of nitrate reductase together form a hydrophilic heterodimer which binds to the membrane through interaction with the b-type cytochrome (γ subunit). The likely enzymatic mechanism is that ubiquinol is oxidized on the periplasmic side of the membrane by the b-type cytochrome and the electrons are passed to nitrate to form nitrite on the inside of the cell, with the generation of PMF. The NarJ protein is necessary for assembly of the membrane-bound form of nitrate reductase and may act as a chaperone in the maturation of the αβ dimer or in facilitating its interaction with the membrane-bound subunit (99). Formate is the preferred electron donor for nitrate reductase, and synthesis of formate dehydrogenase-N is coregulated with nitrate reductase. The nitrite that accumulates during nitrate respiration can be reduced to ammonium by the formate-linked respiratory system encoded by the nrfABCDEFG operon, or by soluble NADH-nitrite reductase, encoded by the nirBD operon.
Fumarate reductase carries out the reduction of fumarate to succinate and is repressed in the presence of oxygen or nitrate (reviewed in reference 65). This enzyme is very similar to succinate dehydrogenase in structure and sequence. It contains four subunits: catalytic subunit FrdA (66 kDa) with covalently bound FAD, electron-transfer subunit FrdB (27 kDa) with three iron-sulfur clusters, and hydrophobic subunits FrdC (15 kDa) and FrdD (13 kDa). The transmembrane FrdC and FrdD proteins anchor the FrdAB catalytic dimer to the membrane and mediate coupling to the quinol pool. Mutagenesis studies identified the cysteine residues in FrdB that comprise the ferredoxin-like clusters that form the individual FeS centers (203). Analysis of mutations affecting the membrane-associated FrdCD subunits indicated that electrons are transferred from two quinol binding sites in sequential one-electron steps (352). No cytochromes are associated with fumarate reductase, and the reduction of fumarate does not appear to be coupled to PMF generation, as nitrate reductase and the cytochrome oxidases are.
Many bacteria can reduce TMAO, which is widely distributed as an osmoprotectant in fish and invertebrates, or DMSO, which is a primary biogenic source of reduced sulfur in the environment. Two major reductase systems with broad substrate ranges and very different properties have been described. The Dms dimethyl sulfoxide reductase allows use of DMSO, TMAO, and other -S and -N oxides as electron acceptors, coupled to PMF generation (reviewed in reference 349). The dmsABC operon at min 20 is widespread in E. coli isolates, but is not present in most Salmonella strains. The Dms enzyme complex is synthesized constitutively during anaerobic growth and has a similar organization to nitrate reductase (28, 279, 348). Two catalytic subunits face the cytoplasm: DmsA (87 kDa, contains molybdopterin) and DmsB (24 kDa, contains four 4Fe-4S centers). They associate with the membrane through the membrane anchor protein DmsC (30 kDa), which has the menaquinone-binding site and is oriented with eight transmembrane segments with the protein termini facing the periplasm (350). A stoichiometry of 2.9 protons extruded per 2 electrons transferred was measured.
Although the Dms system can reduce TMAO, the major activity (>90%) for this substrate is TMAO reductase, which is encoded by the torCAD operon at 28 min (306) and is common in both E. coli and Salmonella strains. It is induced by TMAO under anaerobic conditions, but is independent of Fnr and nitrate control. Unlike the other anaerobic respiratory molybdoenzymes, the 110-kDa TorA catalytic subunit is located as a homodimer in the periplasm and is synthesized with a 39-residue signal sequence (214). The TorC protein is a c-type cytochrome with perhaps five covalently attached heme moieties; the bulk of TorC is exposed to the periplasm and is anchored to the cytoplasmic membrane through an amino-terminal hydrophobic segment. The TorD protein is unrelated to other proteins in the database, and its function is undefined.
PMF.
The energy released by passage of reducing equivalents along the electron transfer chains is captured in the form of an electrochemical gradient of protons extruded upon quinol oxidation or during action of some of the dehydrogenases. The PMF consists of an electrical potential owing to the separation of charge and a chemical gradient, or pH, when the external pH differs from the internal pH of 7.4 to 7.8. The relative contribution of these two factors to the PMF, Δ p, in millivolts, is given by the relationship, Δ p = /F = Δψ – 2.3RT ΔpH/F, where Δψ is the electrical potential in millivolts and 2.3RT/F (R, gas constant; T, absolute temperature; F, Faraday’s constant) is 59 mV at 25C. The magnitude of the electrical potential, Δψ , is measured from the distribution of permeant ions (12). Useful probes for measurement of interior-negative potentials include tetraphenylphosphonium or potassium (or its homolog rubidium) in the presence of the electrogenic potassium carrier valinomycin. Interior-positive potentials can be calculated from the distribution of permeant anions, such as tetraphenylboron or thiocyanate. The fluorescence of cyanine or oxonol dyes is quenched in a potential-dependent fashion, which provides a convenient but less direct measure of the electrical potential. The pH gradient is measured from the distribution of permeant weak acids or bases, or indirectly from the behavior of pH-indicator dyes, such as pyranine or 9-amino acridine. Aerobic cells typically generate a PMF in the range of –180 mV, interior negative and alkaline.
Various methods allow the experimental alteration of the individual components of the PMF (described in references 118, 134, and 169). Addition of potassium plus the K+ carrier valinomycin results in dissipation of Δψ, while ΔpH increases to keep the total PMF approximately constant. Nigericin mediates the electroneutral exchange of K+ and other cations for protons, and causes the dissipation of pH, while ΔΨ increases and the PMF is unaffected. Protonophores, such as 2,4-dinitrophenol, tetrachlorosalicylide, or CCCP (carbonyl cyanide m-chlorophenylhydrazone), allow protons to equilibrate across the membrane and dissipate all PMF components. There remains some controversy whether electron transfer processes might give rise to a localized PMF, in which protons extruded by the electron transfer chain gain immediate access to proton-utilizing reactions, such as the F1F0-ATPase or transporters, without fully equilibrating with the bulk solution. However, there is no doubt that the bulk-phase PMF is fully competent to carry out all PMF-dependent reactions, and that in most cases, either component of the PMF is equally efficient in driving the reaction.
ATP Synthase.
The F1F0 proton-translocating ATPase, also called ATP synthase, plays a central role in bioenergetics. Its structure and mechanism have been reviewed (40, 108, 115, 245, 298). This multisubunit enzyme carries out the synthesis of ATP at the expense of the transmembrane PMF generated in respiring cells, and in bacteria it can use ATP generated by fermentative substrate-level phosphorylation to create the PMF. The eubacterial enzyme is similar to its homologs in mitochondria and chloroplasts, although only the bacterial one appears to be reversible under physiological conditions. The bacterial enzyme contains eight subunits organized in two distinct complexes, the membrane-embedded F0 complex and the peripherally bound F1 complex. The F0 complex is composed of subunits a, b, and c (in 1:2:6–12 stoichiometry) and forms a transmembrane proton channel(108). Subunit c (79 residues) forms a very hydrophobic helical hairpin across the membrane with its termini facing the periplasm. The hairpin structure is retained even when the protein is dissolved in chloroform-methanol-water (124). Residue aspartate 61, which lies at the level of the middle of the membrane, is necessary for proton conduction and is the site of modification by the ATPase inhibitor DCCD. Subunit b (156 residues) has a single amino-terminal transmembrane segment, and the bulk of the polypeptide faces the cytoplasm. Subunit a (271 residues) probably has six transmembrane segments (336), although models with four to eight membrane spanners have been proposed (193). The F0 complex by itself has proton-translocating activity, although its channel activity is increased by coexpression of some F1 proteins, suggesting a possible role for an F1 protein in the assembly or gating of the channel (242).
The F1 complex comprises five subunits (α3β3γ1δ1ε1); it has ATPase activity when it is released from the membranes, and it catalyzes the coupled interconversion of proton translocation and ATP synthesis or hydrolysis when it is complexed to the F0 complex in sealed membranes. The eight ATPase proteins and a nonstructural protein are encoded by the atp or unc operon, whose expression is fairly constant under various growth conditions. The nonstoichiometric expression of proteins from a single mRNA is mediated in part by different translation efficiencies, with only a minor effect of differential rates of mRNA turnover (187).
The extensive analysis of ATPase function supports a model of conformational coupling (40, 298, 322). Passage of protons through the F0 subunit is coupled to a change in conformation of the distant F1 subunits. These changes affect the relative affinities for ATP versus ADP + Pi. On the enzyme surface the adenine nucleotides are nearly isoenergetic owing to the binding energies. There are three catalytic sites on each F1 complex, and it is possible that the three sites cycle sequentially through different conformations favoring binding of ADP+Pi, formation of ATP, and release of ATP.
Specific transport proteins allow hydrophilic molecules to cross the hydrophobic permeability barrier of the lipid bilayer of the cytoplasmic membrane; only a few types of molecules (water, short-chain fatty acids, nonpolar compounds) can enter without a carrier. Unlike higher eukaryotic cells, which generally make a small number of equilibrative transport systems of relatively low substrate affinity and specificity, bacteria produce a remarkable number of active transport systems exhibiting high substrate affinity and specificity and with a broad range of protein organizations and mechanisms of energy coupling. Mechanisms of energy coupling include (i) systems driven by symport or antiport with ion gradients; (ii) periplasmic permeases, which are multiprotein complexes that include a periplasmic substrate-binding protein and are driven by ATP hydrolysis; (iii) ion transport driven by P-type ATPases; (iv) serial transport systems that mediate active transport across both the outer membrane and the cytoplasmic membrane; and (v) group translocation processes that carry out the simultaneous transport and modification of their substrate. Most nutrients are transported by multiple systems (e.g., galactose is transported by five different systems and proline by at least three), usually including a high-affinity, low-capacity one and a lower-affinity, high-capacity one. This use of multiple transport systems may allow the cell to optimize the energetics and affinity of these systems to accommodate particular environmental conditions. Detailed descriptions of uptake mechanisms are presented in chapters 74, 75, and 76 (Fig. 4).
Transport of glycerol via GlpF in E. coli and S. typhimurium has long been cited as an example, perhaps the sole example, of facilitated diffusion in bacteria. The GlpF glycerol facilitator was proposed to act in the manner of a channel to allow increased uptake of glycerol, other polyols, and even unrelated small molecules (143). Sequence analysis showed that the GlpF protein is a 29.7-kDa membrane protein with high homology to the phylogenetically widespread MIP family of integral membrane proteins (239). However, recent experiments show that glycerol equilibrates rapidly into cells with or without GlpF and that the rates of glycerol utilization and accumulation are limited by the rate of its phosphorylation by glycerol kinase, rather than by the rate of its transport (337). GlpF need not be involved directly in transport. Its action as a membrane-associated activator of glycerol kinase activity would give the appearance of increased glycerol uptake owing to the elevated rate of its phosphorylation. This does not explain the reported acceleration of unrelated permeants by GlpF, or the ability of GlpF homologs in the MIP family to accelerate water or solute movement across membranes.
Symport Systems.
Ion symport or cotransport processes use a single integral membrane protein to couple substrate accumulation to the downhill movement of a driving ion in the same direction as the substrate (reviewed in reference 253). These electrogenic transport processes can be driven by proton or sodium gradients. The lactose transporter, LacY, is the best studied ion-driven active transport system (reviewed in reference 168). This 417-residue polypeptide is encoded in the lacZYA operon and is necessary for efficient utilization of lactose as carbon source. The LacY protein has a deduced molecular weight of 46,500, but migrates on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight around 33,000 (47). Anomalously fast migration during electrophoresis is frequently seen with highly hydrophobic polypeptides, perhaps as a result of their partial recovery of ?-helical structure during electrophoresis or their binding of excess molecules of sodium dodecyl sulfate by highly hydrophobic regions of the protein. LacY mediates the accumulation of lactose and other galactosides in response to the electrochemical gradient of protons. Lactose permease has been purified and, when reconstituted in liposomes, displays similar kinetic properties and catalytic activity of lactose transport as the intact cell, indicating that no other components are required for full transport activity (335). The extent of lactose accumulation is directly related to the magnitude of the PMF regardless of the magnitude of the individual PMF components. The PMF can be generated by electron transfer processes in native membranes or by using heterologous PMF-generating systems, such as bacteriorhodopsin or mitochondrial cytochrome c oxidase, in reconstituted membranes (reviewed in references 134, 168, and 253). Equally effective are artificially generated proton gradients, whether in the form of a membrane potential created by a potassium diffusion gradient, or a pH gradient created by acidification of the exterior or by imposition of a diffusion gradient of weak acids. Conversely, a lactose gradient can drive proton movement in the same direction. LacY acts as a reversible transporter, although its behavior is not symmetrical because of its asymmetric orientation in the membrane. A stoichiometry of one proton per lactose molecule is seen under most conditions (44, 366). Analysis of the effect of pH on the rates of lactose efflux and exchange in reconstituted proteoliposomes with purified LacY indicates an ordered process of substrate binding. At least on the external face of the membrane, binding of a proton occurs before high-affinity binding of lactose (169).
Physical studies indicate that LacY has a high content of α-helical structure, and PhoA fusion analysis supports a topological model of 12 transmembrane segments of about 20 amino acids each, with both protein termini facing the cytoplasm (49). Reconstitution at limiting dilution indicates that LacY can function as a monomer, and expression of genetically split genes shows that LacY can function even when in two pieces (26, 355).
LacY has been subjected to extensive mutational analyses to test the effect of substitution of most of its amino acids on transport activity (reviewed in references 49 and 270) or to identify the spectrum of changes that confer a specific functional phenotype. Extensive site-directed mutagenesis by Kaback and colleagues showed that relatively few residues are required for activity. Conservative substitutions could be made with retention of full or partial transport activity for all of the 8 cysteines, all of the 12 prolines, all of the 6 tryptophans, 10 of the 14 tyrosines, and 3 of the 4 histidines (270, 333). This relatively low level of dependence on the identity of each amino acid residue is consistent with the extensive diversity seen in comparison of the sequences of phylogenetically related transport proteins. Only a few residues are critical for substrate recognition or for transport coupling to the proton gradient, although other residues contribute to membrane insertion or stability. Hinkle et al. (149) examined the range of amino acid substitutions along transmembrane helix VIII that could be tolerated without major loss of function. The results are consistent with the helical arrangement of this segment and the exposure of different faces or stripes of the helix to the acyl chains, to other segments of the protein, and to a central polar channel. Residues that comprise the stripe of helix VIII exposed to the lipid acyl chains can be replaced with hydrophobic residues of any size, whereas the stripes that contact other parts of the protein require the presence of residues of similar hydrophobicity and residue volume. The stripe facing the putative central channel was relatively intolerant of substitution.
The topological model of LacY, which is based on extensive analysis with topological reporters (49), predicts that 257 of the 417 amino acids are in membrane-spanning regions and that there are four or five positively charged residues and four negatively charged ones buried in the transmembrane segments. Aspartate 237 in helix VII and lysine 358 in helix XI appear to form a charge pair, since transport function is lost when either residue is changed to another charge type, but function is retained when both charged residues are removed or exchanged (100). Lysine 319 in helix X appears to interact with both aspartate 240 and possibly glutamate 269 (274). The residues in a charge pair are not required for function, but formation of the pair may facilitate protein folding or stability. Other charged residues play direct roles in transport, in particular glutamate 269 and the adjacent pair of histidine 322 and glutamate 325, whose alteration has dramatic effects on coupling of the proton gradient to lactose transport (168).
Mutants with an extended substrate range of LacY can be directly selected and may help identify residues that contribute to the substrate-binding region. Although changes at numerous positions allow transport of maltose, the most striking effect was seen with substitutions for alanine 177, where even conservative replacement with valine allowed transport of maltose, sucrose, or even the monosaccharide arabinose (44, 179). Changes at other positions in combination with A177V (primarily at tyrosine 236, lysine 319, isoleucine 303, or histidine 322) further affected the substrate specificity for lactose, cellobiose, or maltotriose, but the specificity mutants also affected the coupling of substrate movement to proton flux (44).
Other symporters couple substrate accumulation to the sodium ion gradient. In enteric bacteria, a transmembrane sodium gradient is established by the action of sodium/proton antiporters, which extrude Na+ at the expense of the PMF (see below). The experimental demonstration of sodium dependence is complicated by the fact that many transporters have such high affinity for sodium that they are saturated even with the low levels of sodium (<10 mM) that contaminate solutions and glassware. The MelB protein transports melibiose and other α-galactosides coupled to Na+ or H+ gradients, but transport of methyl-thio-β-galactoside is coupled only to the Na+ gradient. Lithium ions can substitute somewhat for sodium ions, and the toxicity of Li+ accumulated during melibiose transport allows selection for mutants defective in MelB-mediated Li+ uptake. Some Li+-resistant melB mutants retain proton-linked melibiose transport and are specifically defective in coupling to the sodium gradient; others are altered in both cation and sugar specificity (36). There are four aspartate residues in the transmembrane region of MelB, and the replacement of any one of them with asparagine or cysteine reduced or eliminated the ability of sodium to activate sugar binding and transport, suggesting that all four aspartate residues contribute to the cation-binding site (256).
Other sodium-dependent symporters mediate uptake of proline (PutP), glutamate (GltS [91]), serine, and pantothenate (PanF [163]). The PutP proline transporter is coupled to the sodium gradient and is also driven by lithium but not protons. It allows cells to utilize proline as sole carbon or nitrogen source, when expressed with the PutA protein. PutA has two separate functions and cellular locations. When proline is present at inducing levels, PutA binds to the cytoplasmic membrane and acts as proline oxidase to transfer electrons from proline to the electron transfer chain (46); at low levels of proline, PutA dissociates from the membrane and binds to a specific DNA operator sequence to repress putP and putA transcription (234).
Numerous other ion-linked symport systems have been defined; some of these are listed in Table 2. Many of these proteins are related in topology (most have 12 or 6+6 transmembrane segments) and share limited regions of common amino acid sequences (15). The phylogenetic relationships of various families of transporters have been deduced (132, 276).
Table 2Partial list of some nutrient transport systems |
Antiporters.
Antiporters or exchange proteins couple the uptake of one compound to the release of a second type of molecule. Some are coupled to the proton gradient. E. coli expresses the potassium/proton exchanger Kha, a calcium/proton exchanger Cha (162), and two sodium/proton exchangers, NhaA and NhaB (reviewed in reference 236). The NhaB protein is a pH-independent electrogenic antiporter with high affinity for Na+ (Km, 0.05 to 0.25 mM). NhaA is also electrogenic, with the stoichiometry of 2 H+ per Na+ measured in proteoliposomes reconstituted with purified protein (320, 323). Unlike NhaB, the activity of NhaA increases about 2,000-fold when the pH is increased from 7.0 to 8.5, owing to a change in the transport rate, not in ion stoichiometry. Both transporters bring about Na+ extrusion, which generates the Na+ gradient that drives the Na+-coupled transporters. Both contribute, but NhaA predominates, to pH homeostasis, by acidifying the cytoplasm in alkaline environments. Mutants lacking both Nha systems have no Na+ extrusion activity at alkaline pH, and their growth is completely blocked by >0.2 M Na+, although they grow well in alkaline medium in the absence of added sodium. The variation of the levels of internal Na+ in cells, as detected by nuclear magnetic resonance, matches the properties expected from the behavior of the isolated transporters (238).
A family of obligate exchange transporters mediates the electroneutral exchange of Pi↔Pi, or organophosphate↔organophosphate, or organophosphate↔Pi. These transporters do not allow net influx of material into the cell, but their action can result in the accumulation of organophosphates at the expense of the Pi gradient (202). The internal pool of Pi is maintained either by action of the PMF-coupled Pit transporter or the ATP-dependent Pst system. Three members of this organophosphate transporter family have been described. UhpT has a broad substrate range, but its synthesis is induced only by external glucose 6-phosphate through the action of the UhpABC regulatory proteins (160). The regulatory components UhpB and UhpC are integral cytoplasmic membrane proteins with 8 and 12 transmembrane segments, respectively. GlpT carries Gly3P for use as a carbon source and is induced by internal glycerol 3-phosphate through the action of the GlpR repressor (102). PgtP carries phosphoglycerates and PEP; it is present in S. typhimurium but not E. coli (126). Synthesis of PgtP is induced by external substrates through the action of the PgtABC regulatory proteins (362). UhpT, GlpT, and possibly PgtP have a similar transmembrane topology of 12 or 6+6 transmembrane segments, like that of the ion symporters (127, 160). They make up a branch of the "major facilitator superfamily" (276), which includes symporters and facilitative carriers. This phylogenetic relationship suggests that their very different mechanism of energy coupling is not reflected in major differences in structure or sequence.
Other antiport systems exchange the substrate for a metabolic process for its structurally similar product (reviewed in reference 252). For example, E. coli growing anaerobically with fumarate as electron acceptor synthesizes a specific C4-dicarboxylate transport system that is different from the Dct uptake system. This transport system exchanges the product, succinate, for a molecule of the substrate, fumarate (103). Other examples include the lysine/cadaverine exchanger CadA and the ornithine/putrescine exchanger PotE.
Other antiporters are used as efflux systems for the removal from the cell of toxic compounds such as tetracycline (3, 359) or ethidium bromide and thiolactomycin (114, 197). Efflux systems comprise diverse families of proteins with 4, 6, or 12 transmembrane segments (192).
Periplasmic Permeases.
The group of transport systems called periplasmic permeases were initially thought to be present only in gram-negative bacteria, but are now seen to be related to a large number of transport systems thoughout the biological world (93, 147). Periplasmic permeases were defined by their susceptibility to osmotic shock, which preferentially releases periplasmic constituents, including the substrate-binding proteins that are essential for the operation of these transport systems. These multisubunit systems also differ from the symporters by virtue of their dependence on the high-energy phosphate pool, rather than on the PMF or other ion gradients. Their transport activity is relatively unaffected by protonophores, which dissipate the PMF and strongly inhibit electrogenic symport systems, as long as the ATP pool remains high (24). Since exposure to protonophores depletes ATP pools in wild-type cells owing to the futile effort of the F1F0-ATPase to restore the PMF, tests of the effect of protonophores or other energy poisons must be carried out in atp strains to uncouple the ATP pool from the PMF. Periplasmic permease function is sometimes inhibited by arsenate and other treatments that deplete the ATP pool. Typically, periplasmic permeases exhibit high affinity for their substrates (Km, 0.1 to 1 μM), which correlates with the affinity of the periplasmic substrate-binding protein. These transporters carry out unidirectional uptake and, unlike symporters, do not mediate efflux.
There are periplasmic permeases for the high-affinity uptake of histidine (HisJQMP [146]), maltose (MalEFGK), oligopeptides (OppABCDE [148]), branched-chain amino acids (LivJKHMGF [1]), glycine betaine and proline (ProU or ProVWX [128]), glycerol phosphoesters (UgpABCE [235]), phosphate (PstSCAB), and others (Table 2). Sequence analyses show that periplasmic permeases are composed of three to five proteins each and are usually encoded in operons. The soluble periplasmic substrate-binding proteins are required for transport. Their crystallographic structures show the presence of two protein lobes connected by a flexible hinge (285, 341). The lobes close together when substrate binds to residues in both lobes. There are usually two hydrophobic integral membrane proteins which possess five to eight transmembrane segments (113, 175, 244); this diversity of topology by similar proteins is surprising. There is a relatively low degree of sequence relatedness among the transmembrane proteins of different transporters, and even the two partners in the same system show considerable diversity. Finally, each permease complex contains a homo- or hetero-dimer of a peripheral membrane protein(s) that contains a region of well-conserved sequence, part of which includes an ATP-binding site with the familiar Walker A and B motifs. The extended sequence motif is present in many proteins, called traffic ATPases or ABC (ATP-binding cassette) proteins (chapter 76; 93, 147). In addition to the periplasmic permeases, ABC proteins include bacterial exporters of proteins and carbohydrates, and eukaryotic transporters, such as the multidrug efflux MDR-1 protein, the cAMP- and ATP-regulated chloride channel CFTR altered in cystic fibrosis, and the yeast mating pheromone transporter STE6 (reviewed in references 93 and 104).
The best characterized periplasmic permeases are those for maltose and histidine. Both systems have been purified and are functional when reconstituted in liposomes (29, 82). Substrate transport is dependent on added binding protein and ATP hydrolysis, but the ATP/substrate stoichiometry is not well defined (84). There is evidence from vectorial labeling experiments that the ABC protein HisJ is exposed to the exterior, even though it lacks an obvious transmembrane segment and must also be exposed to the cytoplasm to obtain its substrate ATP (13, 176). Binding protein-independent mutants have been obtained by selection for transport activity in strains deleted for the binding protein. In the maltose system these mutants are affected in the transmembrane proteins, MalF and MalG, and exhibit a much lower affinity for maltose than does the intact transport system (329). These mutants are blocked by the presence of the wild-type maltose-binding protein (MBP), suggesting that the transmembrane proteins contain a low-affinity maltose-binding site that can normally filled only by maltose that has been delivered by the MBP, but which becomes accessible to free maltose in the mutant proteins (85). The MBP-independent mutants exhibit uncoupled ATP hydrolytic activity, suggesting that a signaling process normally occurs that couples ATP hydrolysis by MalK on the cytoplasmic face of the membrane in response to MBP binding on the periplasmic face (83). Histidine-binding protein-independent mutants carry mutations in the ABC protein HisP and also show uncoupled ATP hydrolysis (246, 311).
Homologs of the periplasmic permeases have been found in gram-positive bacteria, and they are very similar to the gram-negative version in terms of the number, location, and sequence motifs of their component proteins. The major difference is that the substrate-binding protein carries an amino-terminal lipoprotein modification that anchors it to the cytoplasmic membrane (330).
Serial Transport Systems.
Although most nutrients gain access to their transporters in the cytoplasmic membrane by diffusion through the porin channels across the outer membrane, a few substrates are too large and too scarce to effectively enter by this route. Ferric siderophore complexes and vitamin B12 are transported with very high affinity by the serial action of separate active transport systems that act sequentially across the outer and cytoplasmic membrane (170). Active transport across the outer membrane is dependent on specific receptor proteins and is energized by the TonB/ExbB/ExbD protein complex (308), as described in chapters 5 and 71. This energization appears to be dependent on the PMF across the cytoplasmic membrane because dissipation of the PMF blocks vitamin B12 uptake (41). The TonB protein has an unusual proline-rich sequence motif and is anchored in the cytoplasmic membrane by a transmembrane segment near its amino terminus (271). TonB appears to span the periplasmic space and make direct contact with the outer membrane transport proteins (21, 307). The proton gradient across the cytoplasmic membrane may somehow modify the conformation of TonB and thereby transmit its energized state across the periplasmic space to affect the outer membrane transporters. Another possibility is that the PMF is needed to maintain cell turgor, which keeps the two cell membranes closely opposed. Dissipation of the PMF could result in loss of contact between the two membranes and hence prevent the contact of TonB with its client receptors. The ExbB and ExbD proteins are inner membrane proteins that contribute to the stability and energy coupling function of TonB (109). Accumulated substrates in the periplasm are transported across the cytoplasmic membrane by transport systems that are closely related to periplasmic permeases (112, 299, 315).
There are two periplasm-spanning proteins, TonB and TolA. The function of each requires the accessory proteins, ExbBD and TolPQ, respectively, which are related in sequence to each other and can partially substitute for loss of its homolog (42). The TonB and ExbBD proteins are involved in driving a set of outer membrane active transport proteins. The role of TolA and TolPQ is not clear, but they are necessary for the structural integrity of the outer membrane, since their absence confers the phenotype of a leaky outer membrane (345).
P-Type ATPases.
P-type ion-translocating ATPases, also called E1-E2 type ATPases, are typified by the plasma membrane Na/K-ATPases and the sarcoplasmic reticulum Ca-ATPases of mammalian cells. These transporters consist of a large (ca. 100-kDa) protein subunit that is phosphorylated on an aspartate residue by ATP during the transport cycle. A smaller subunit is usually associated with the catalytic subunit. Related transporters carry out ATP-dependent cation transport in bacteria, although their similarity to the eukaryotic homologs can be fairly low. There are multiple transport systems for potassium in E. coli and S. typhimurium, including the constitutively expressed PMF-dependent TrkD, TrkG, and TrkH systems (6, 14, 95). Low external K+ levels impair maintenance of cell turgor and induce expression of the kdpABC operon, which encodes three cytoplasmic membrane proteins that make up the high-affinity Kdp K+-transport system (145). The very hydrophobic KdpA protein (557 amino acids) has up to 12 transmembrane segments and participates in K+ binding. KdpB (682 residues) is phosphorylated during transport and is distantly related in sequence to the eukaryotic P-type ATPases. KdpC (190 residues) resembles in structure the β subunit of eukaryotic Na/K-ATPases. This complex has been purified from E. coli and reconstituted in functional form (6).
There are three independent magnesium transport systems (151). MgtA and MgtBC mediate only Mg2+ influx. MgtB (908 residues) is more similar in size and sequence to the sarcoplasmic reticulum Ca2+-ATPase and other eukaryotic P-type ATPases than it is to other bacterial P-type ATPases (310). Analysis of the behavior of topological reporters fused to MgtB indicates the presence of 10 transmembrane segments. The sequence of MgtA is also related to eukaryotic P-type ATPases (cited in reference 304). The third Mg2+ transport system mediates bidirectional flux of Mg2+ and the uptake of Co2+ and includes the CorA protein (316 residues) (309). The CorA protein possesses a large periplasmic domain and transmembrane segments near its carboxyl terminus. Its sequence is unrelated to other proteins, and its mechanism of energy coupling and transport is as yet unknown.
A novel group of ATP-dependent transporters (called A-type ATPases) mediates resistance in bacteria to toxic oxyanions such as arsenate, arsenite, and antimonite. These plasmid-coded heavy metal resistance determinants are widespread in bacteria. The E. coli plasmid R773 carries the arsRDABC operon (reviewed in references 303, 304, 305). Resistance is mediated by production of an efflux pump for arsenite, composed of the transmembrane protein ArsB (429 residues, 12 transmembrane segments) and the peripherally bound ArsA protein (583 residues), with two ATP-binding sites. ArsB protein can act alone as a uniport transporter of arsenite down the electrochemical gradient, but its activity is increased when it is coupled to the ATP pool upon binding of an ArsA dimer. The ArsC protein confers resistance to arsenate by reducing it to arsenite, which is then transported out of the cell by the ArsAB system. The Fe(II) uptake protein, FeoB, was noted to have an ATP-binding consensus and may also function as a cation-translocating ATPase (172).
Group translocation systems mediate simultaneous transport and modification of their substrate. Best characterized are the PEP:sugar phosphotransferase systems (PTS), which use a cascade of phosphate transfer reactions from PEP via common and sugar-specific proteins to the sugar substrate during transmembrane uptake (reviewed in chapter 75 and reference 255). Sugar accumulation occurs because the sugar-phosphate product is trapped inside the cell. The high-energy phosphoryl group is transferred from PEP to enzyme I (ptsI), a 63.4-kDa dimeric protein, and then to the 9.1-kDa HPr protein (ptsH). Both enzyme I and HPr are cytoplasmic proteins and are phosphorylated on histidine residues (the N-3 position of His-189 of E. coli enzyme I and the N-1 position of His-15 of E. coli HPr). The carbohydrate-specific proteins, which accept the phosphate from P-HPr, are called enzyme II and are made up of three autonomous structural domains called IIA, IIB, and IIC. Each domain may be on a separate polypeptide or may be joined through a flexible linker to other domains in any order. Hydrophilic domain IIA is phosphorylated by P-HPr on a conserved His residue. Hydrophilic domain IIB, also about 100 residues, accepts phosphate from domain IIA onto a cysteine residue. The roughly 350-residue domain IIC is a transmembrane component with six (318) or eight (48) transmembrane segments and a large polar loop; it binds and transports its specific sugar. The best studied PTS system is the mannitol-specific enzyme IImtl, which contains all three domains in one polypeptide of around 60 kDa. The purified protein catalyzes both P-HPr-dependent mannitol phosphorylation and PEP-independent transphosphorylation, in which phosphate is transferred from mannitol 6-phosphate to mannitol. The sequential order of phosphate transfers from P-HPr to the IIA and IIB domains was determined with this protein. Although domain IIC contains an essential glutamate residue which is highly conserved in PTS systems and could be phosphorylated, the stereochemical course of chiral phosphate inversions indicate an even number of phosphate transfers by the sugar-specific enzymes, suggesting that domain IIC is not phosphorylated (221).
In addition to its role in sugar transport, the PTS plays a key role in catabolite repression and inducer exclusion, which coordinate and regulate carbohydrate metabolism (reviewed in references 255 and 275). When E. coli cells are offered multiple carbon sources, the carbohydrate-metabolizing enzymes are expressed in a hierarchical manner. Glucose is the preferred substrate, and its presence blocks the transport of other sugars and the transcription of their catabolic genes. This multifaceted response to glucose includes a decrease in the rate of synthesis of cAMP, which in turn reduces the transcription of many CAP (cAMP-dependent transcription activator protein)-dependent catabolic genes, and a direct inhibition of many non-PTS transporters for potential carbon sources. These processes are controlled by the degree of phosphorylation of the glucose-specific IIA domain, which exists as a separate protein called IIIglc (encoded by crr). Phosphorylated IIIglc activates adenylate cyclase to increase the rate of cAMP synthesis, and the dephosphorylated form of IIIglc inhibits numerous transport systems and glycerol kinase. Sites on several transport proteins, such as MalK and LacY, which are involved in this inhibition by IIIglc, have been identified through mutational analysis and exhibit conserved sequences in the proteins analyzed (e.g., reference 326). The structure of the complex of IIIglc and the soluble protein glycerol kinase has been determined by X-ray crystallography and shows that IIIglc contacts a small portion of the target protein’s surface (157). The relative levels of the phosphorylated and dephosphorylated forms of IIIglc are affected by the presence of glucose in the medium, since glucose transport and phosphorylation via the PTS lower the steady-state level of phospho-IIIglc and increase the level of the unphosphorylated form.
The cytoplasmic membrane must be crossed by proteins and other macromolecules that are destined for the cell surface or for export into the medium. Several macromolecular secretion systems have been described in considerable detail (chapters 61, 64, and 66 and reviewed in references 135, 257, 286, and 353).
Some cytoplasmic membrane proteins and the majority of proteins destined for the periplasm or outer membrane are translocated by the general secretory pathway, which is defined by the requirement for SecA, SecE, and SecY function. The major topogenic determinant required for entry of a protein into the Sec pathway is the amino-terminal signal sequence. Signal sequences typically are 20 to 25 amino acids in length, have no specific sequence requirements, and comprise four segments: a polar, positively charged segment at the amino terminus, a stretch of 7 to 10 nonpolar residues, a polar stretch that often starts with a helix-breaking residue, and the processing/cleavage site for signal/leader peptidase (61, 186). The site cut by the major signal peptidase I is preceded by small amino acid residues at the –1 and –3 positions, and the –1 residue at the cleavage site is almost always an alanine. The positively charged amino terminus of the signal sequence is important for efficient translocation (280) and is thought to associate with acidic phospholipids in the cytoplasmic membrane, in conjunction with the SecA protein (248). The positive charge helps orient the signal sequence by anchoring the amino terminus on the cytoplasmic side of the membrane. The nonpolar segment of the signal sequence has a strong propensity to assume α-helical structure in the nonpolar environment provided by the lipid bilayer or by the transmembrane proteins of the secretory complex. Formation of the α-helix may help draw the polar segment of the signal peptide across the membrane and bring the cleavage site to the leader peptidase, which is anchored on the periplasmic face of the cytoplasmic membrane by two transmembrane segments. Translocation of the remainder of the protein is driven by repeated action of the ATP-dependent peripheral membrane protein SecA and probably occurs through a channel formed by the translocation proteins SecY, SecD, SecE, and SecF, and other proteins (136). Protein translocation is dependent on ATP hydrolysis and is stimulated by the presence of an electrical potential oriented with the positive charge on the side of the membrane toward which the protein will move (97, 98). Addition of a signal sequence to cytoplasmic proteins does not usually result in their efficient export (190), suggesting that features besides the signal sequence identify exported proteins.
Many of the components of the general secretory pathway were identified by genetic approaches pioneered by Beckwith and colleagues (286). Expression of a hybrid protein in which a signal sequence is attached to the cytoplasmic protein β-galactosidase is toxic, owing to the apparent jamming of the protein translocation machinery by the rapidly folded portions of β-galactosidase. Mutations that confer resistance to expression of MalE-LacZ fusion proteins affect either the signal sequence, so that the fusion protein can no longer enter the secretory pathway, or sec genes that encode early components in the secretory pathway. Most sec mutations are conditionally lethal, since protein translocation is essential. A second mutant selection took advantage of the finding that expression of the secA gene increases when protein secretion activity is impaired by jamming with a precursor hybrid or by mutations in other components of the secretory pathway (230, 269). Selection for increased expression of a secA-lacZ fusion yielded mutations in sec genes (269). These two methods identified the secA, secB, secD, secE, secF, and secY genes.
Another genetic approach involved selection for strains that acquire the ability to secrete a protein with a defective signal sequence. The genes that were identified by this approach were named prl (protein localization). They occurred primarily in the sec genes: PrlA alleles are in secY, PrlD in secA, and PrlG in secE, showing that alterations of components of the normal secretory pathway can allow recognition and translocation of a protein with an aberrant or even with no signal sequence. The PrlC suppressor of the export defect of some lamB signal sequence mutations resulted from loss of an oligopeptidase (69), indicative of kinetic competition between the relative rates of precursor export, folding, and degradation.
The SecD, SecE, SecF, and SecY proteins are integral transmembrane components, whereas SecA associates reversibly with the membrane when carrying a precursor to a secreted protein. SecB is a cytoplasmic chaperone that binds to and maintains some precursor proteins in an unfolded, export-competent state (183, 264). SecB function is required for the export of some proteins (MBP, OmpA, OmpF, LamB), but not for many others (ribose-binding protein, PhoA, β-lactamase). The factors that determine the dependence on SecB are located in the mature portion of the protein, although no conserved sequence elements have been identified (68). There is evidence that SecB binds to unfolded proteins and retards the folding process; the presence of a signal sequence also retards the rate of folding, perhaps by embedding itself in the hydrophobic core (117, 196, 264). Although SecB binds to many unfolded proteins in vitro, its in vivo binding is highly restricted (184, 264). There may be other chaperones for other proteins, and the involvement of several homologs of components of the eukaryotic signal recognition particle or docking complex has been suggested (247, 254).
All of the sec proteins have been purified and found to be essential (SecAYE) or stimulatory for in vitro protein translocation. A few polypeptides that had not been identified genetically were found to be important in vitro, and their genes are being identified by reverse genetic approaches (227). The SecY protein (12 transmembrane segments) plays a key role in recognition of the signal sequence, since it is the site of the PrlA mutations, which have the strongest effect on recognition of altered signal sequences (233). SecY also appears to contribute to formation of a transmembrane channel through which the translocating polypeptide might pass (166). SecE has three transmembrane segments, while SecD and SecF each have six (119, 250). There are 300 to 500 molecules per cell of SecE and SecY, but only about 30 molecules of SecD and SecF, suggesting that the four proteins do not make up a fixed translocation complex (250).
Current models for the mechanism and energetics of protein translocation by the Sec pathway suggest that repeated cycles of binding and release of SecA protein occur at the membrane SecY-SecE complex. This binding and rebinding may mediate stepwise transfer of short segments (about 30 residues) of the translocated protein across the membrane (17, 18, 27). SecA protein has ATPase activity that is stimulated by the presence of a precursor protein and acidic phospholipids or of membranes containing them (43, 144, 194; see below). Protein translocation in vitro is dependent on ATP hydrolysis and is stimulated by a PMF of appropriate direction (98, 287). The dependence on the PMF is related to the charge distribution on the regions flanking the signal sequence (120). The translocation process is accompanied by opening of an ion channel for halides or protons (173, 288), and this may be the cause of the effect of the PMF on the translocation of the mature portion of the exported protein. The orientation of cytoplasmic membrane proteins is determined in large part by the action of the stop-transfer signal given by positively charged residues at the end of a hydrophobic segment. The response to these signals is dependent on the function of the FtsH protein (2).
A separate secretory system is involved in the export of lipoproteins, which are located in the outer or cytoplasmic membrane and are modified at their mature amino terminus during their translocation. A glyceryl moiety is added to the amino-terminal cysteine from PG, and fatty acyl groups are esterified to the two hydroxyl groups on the glycerol and the α-amino group of the cysteine (140). Lipoprotein processing uses a separate signal peptidase, SPII, which cleaves the signal sequence to leave an amino-terminal cysteine on the mature portion of the protein. Processing is inhibited by the antibiotic globomycin, and both processing and translocation are dependent on all of the sec proteins except SecB (182). Amino acid residues early in the sequence of the mature portion influence the final destination of the lipoprotein (360), but not in an absolute manner (121).
The subsequent steps of protein targeting after removal of the signal sequence and translocation of the mature sequence across the cytoplasmic membrane are not known. Periplasmic proteins may fold into their stable conformation following extrusion. Proteins destined for the outer membrane could move across the periplasmic space in membrane vesicles or through specialized structures (Bayer junction) or even in soluble form, either alone or complexed with other proteins. Outer membrane porin proteins must pass through several intermediates before achieving their final trimeric structure (314), and the step of trimerization appears to require the presence of LPS (267, 297). More work is needed to define the later steps of export.
The Sec pathway mediates the translocation of most periplasmic and outer membrane proteins, but specialized pathways operate for a few other proteins, especially those that are secreted across the outer membrane to form an organelle on the cell surface or to be released into the medium. Assembly of the flagellum employs a special system (reviewed in reference 199 and chapter 10). Only two of the protein components of the flagellar basal body (FlgI and FlgH) are synthesized with a cleavable signal sequence and are substrates for the Sec-dependent pathway. All the other flagellar proteins lack a signal sequence and are translocated and assembled by unknown processes. The flagellar filament grows at the tip, but the flagellin subunits do not pass through the medium. It is likely that the flagellin is secreted through the flagellar filament itself, and that the basal body contains a flagella-specific secretory apparatus.
Special systems operate for assembly of other surface organelles, particularly the adherence fimbriae (chapters 11, 150). Best studied is the assembly of the Pap fimbriae by strains of E. coli associated with urinary tract infections (156). The pap operon encodes five proteins that will be assembled into the fimbrial filament, along with two assembly and two regulatory proteins. The five structural proteins are synthesized with a typical signal sequence and are translocated into the periplasm, where they are bound by the periplasmic chaperone PapD, which has an immunoglobulin-like peptide-binding region (155). The outer membrane usher protein PapC selects, from the pool of subunit-chaperone complexes, the subunit needed for proper assembly of the structure, taking first the three subunits for the tip of the filament, then multiple copies of the major shaft protein PapA, and finally a subunit whose incorporation at the base of the fimbria halts growth of the shaft and thus determines the length of the filament. Similar processes are likely to operate for the other types of fimbriae, although some differences in detail are likely.
Secretion of E. coli α-hemolysin provided the first example of an export process in bacteria that uses members of the ABC family of traffic ATPases (reviewed in references 104, 257, and 278 and chapter 153). The 1,023-residue HlyA hemolysin protein lacks an amino-terminal signal sequence (105), and its secretion signal, which is located within the 70 carboxyl-terminal residues, is not removed during export (106). Three other proteins are necessary for its export. HlyB (707 residues) and HlyD (477 residues) are cytoplasmic membrane proteins encoded by the hly determinant. HlyB has an amino-terminal domain of about 150 residues, a central domain of about 275 residues that spans the membrane six times, and a carboxyl-terminal region of 275 residues that contains the ABC, although there are conflicting results about the transmembrane topology of HlyB (122, 344). HlyD is in the periplasm and anchored to the cytoplasmic membrane by a single transmembrane segment (344). Outer membrane protein TolC is necessary for hemolysin export across the outer membrane (343). Other protein export systems involve even more protein components, although most include an ABC protein for energization and an outer membrane warden protein (278).
Protein Maturation.
Several modifications of translocated proteins occur. Numerous periplasmic proteins or fimbrial proteins possess disulfide bonds that contribute to their stability or enzymatic activity. The disulfide bonds form only after the protein has been translocated out of the reducing environment of the cytoplasm (92). Although oxidation to form disulfide bonds occurs spontaneously, it is greatly accelerated by the action of periplasmic disulfide bond isomerase enzymes. The DsbA protein in the periplasm (16, 171) and DsbB in the cytoplasmic membrane (220) carry out the oxidation of periplasmic proteins and transfer of the reducing equivalents back into the cell, respectively. This system is necessary for timely synthesis of alkaline phosphatase, flagella, and fimbriae.
Several polysaccharides are assembled outside the cytoplasmic membrane, including peptidoglycan (chapter 68) and MDO (membrane-derived oligosaccharide) (chapter 70) in the periplasm, and the core and O-specific side chains of LPS (chapter 69), enterobacterial common antigen polysaccharide (chapter 9), and multiple capsular polysaccharides on the cell surface. Each of these requires specialized and different processes and sites for polymerization of the precursors and for translocation across the cell membranes. Translocation of precursor units for peptidoglycan and LPS on polyprenyl phosphate carrier lipid is described in chapters 68 and 69. Many of the penicillin-binding proteins that polymerize, cross-link, and modify the peptidoglycan are exposed to their site of action in the periplasm but anchored to the cytoplasmic membrane through a hydrophobic segment.
MDOs are periplasmic, branched (β-1,2 and β-1,6) glucose oligomers which are substituted with phosphoglycerol, phosphoethanolamine, and succinyl esters. A membrane glucosyltransferase forms the β-1,2 glucan backbone by transferring glucose units from UDP-glucose. This enzyme is encoded by mdoA at 23 min and requires acyl carrier protein and polyprenyl phosphate for maximal activity. Another membrane-bound enzyme, encoded by mdoB at 99 min, transfers sn-1-glycerol-phosphate from PG to the glucan. MDO synthesis is regulated by osmolarity (273).
Strains of E. coli from clinical isolates often produce polysaccharide capsules that are important for their virulence traits. Colanic acid capsule is produced by many strains and may provide protection against desiccation or osmotic stress. Analyses of the genes encoding proteins for synthesis of various capsules, such as the K1 and K5 polysaccharides, have identified components that are involved in capsule export across both membranes and which resemble members of the ABC family of ATP-dependent translocators. In these systems, KpsM has the membrane-spanning domains and KpsT the ABC motif (104, 243).
Bacteria respond to many environmental signals through transmembrane signal transduction systems, as described in chapter 80. A large and continually growing number of these regulatory systems act through protein phosphorylation cascades which are widespread in bacteria and have been identified recently in eukaryotic systems. These systems are termed "two-component regulatory systems" since they comprise, at a minimum, a protein called a sensor kinase, which phosphorylates itself on a histidine residue, and another protein called a response regulator, which accepts the phosphate from the sensor kinase onto an aspartate residue (reviewed in references 240, 241, 316, and 317). Most of the phosphorylated response regulators activate transcription of specific genes. Over 100 two-component systems have been described in bacteria, about a quarter of them from E. coli or S. typhimurium. Two-component regulators are typically constructed in modular form (180, 241). The roughly 250-residue kinase portion of sensor kinase proteins is called the transmitter module, and the 100- to 120-amino-acid portion of the response regulator that is phosphorylated is called the receiver module. These modules can be arranged in various positions on one or more polypeptide chains. For example, the chemotaxis regulator CheY consists only of a receiver module, while ArcB and RcsC contain both a transmitter and receiver module. Typically, the sensor kinase protein contains a transmitter module at its carboxyl end and an input module, which recognizes the effector molecule, at its amino end. Response-regulator proteins have the receiver module at their amino end, followed by an output module which contains a DNA-binding site. Three major subfamilies (the NtrC, OmpR, and UhpA families) of transcription-activating response regulators can be distinguished on the basis of the nature of their output modules, including the structure of the DNA-binding region and their mechanism of transcription activation. Some sensor kinases, such as NtrB, are cytoplasmic and respond to an intracellular signal, such as the ratio of glutamine to α-ketoglutarate. For most sensor kinases (e.g., EnvZ, DctB, PgtB, NarQ, NarX, and RcsC), the input module contains two transmembrane segments that anchor the protein to the cytoplasmic membrane and expose a large periplasmic domain containing the effector binding site. Some exceptions to this pattern are PhoR, which has two closely spaced transmembrane segments but no external domain (291), and UhpB, which has a very nonpolar amino-terminal half with probably eight transmembrane segments (160). Thus, the cytoplasmic membrane is the site of numerous (at least 30) transmembrane regulatory proteins that are expressed at a low constitutive level and control gene expression in response to environmental changes.
Bacteria elicit specific and rapid changes in metabolic activities and gene expression in response to environmental stresses, such as heat shock, high or low osmolarity, acidic or alkaline pH, uncouplers, oxidizing agents, UV light, DNA alkylating agents, organic solvents, hydrostatic pressure, and many others. Some of the induced homeostatic mechanisms act at the cytoplasmic membrane to alter transport activities. Shift to medium of higher osmolarity results in increased potassium transport by the Kdp and other transport systems and subsequently increased synthesis and activation of transport systems for osmoprotectants, including trehalose, glycine betaine, and proline (reviewed in references 78 and 79 and chapter 77). Conditions of low osmolarity result in increased synthesis of periplasmic MDO and its increased decoration with phosphoethanolamine, phosphoglycerol, and succinate adducts (chapter 70). Synthesis of highly charged MDO presumably increases the number of fixed charges in the periplasmic space and helps spread over both cellular membranes the osmotic stress caused by water influx at low osmolarity. The mechanism by which the cell senses changes in osmolarity remains unclear.
The regulation of cytoplasmic pH is a complex and essential process (reviewed in reference 35). Exposure of S. typhimurium or E. coli to low pH (ca. pH 5.8) induces tolerance to subsequent exposure to even lower pH values (pH 3.3), as might be encountered following phagocytosis (111). Acidification tolerance requires protein synthesis and changes in the level of at least 18 proteins. The F1F0-ATPase is essential for acid tolerance, presumably by pumping proteins out of the cytoplasm. Mutants in the atp (unc) operon are extremely acid sensitive and unable to induce the acid tolerance response (110). The sodium/proton antiporters NhaA or NhaB (236) participate in acidification of the cytoplasm in alkaline environments, but are not required for survival under those conditions, indicating the role of multiple transport systems for maintenance of constant internal pH.
It is now recognized that cells of E. coli undergo substantial changes in gene expression and metabolic capabilities upon entering into stationary phase (chapters 93 and 106). Gradual depletion of nutrients or increase in toxic products induces entry into stationary phase (181). Stationary-phase cells exhibit spherical morphology and increased resistance to many environmental insults, including temperature, oxidizing agents, chemical agents, etc. Transcription of many stationary phase-specific genes is controlled by the stationary phase-specific sigma factor RpoS (also called KatF). One genetic component found to be important for recovery from stationary phase, SurB, is homologous to the ABC-type transport systems, although its substrate has not been identified.
Cell motility and bacterial taxis are complex sensory and behavioral responses that are dependent on cytoplasmic membrane constituents and are related to transport and transmembrane signaling. As described in chapter10, bacterial motility occurs by rotation of helical flagellar filaments, which is driven by proton flux through the MotAB proteins of the flagellar basal body. The direction of flagellar rotation is controlled by the chemotaxis system, which senses temporal changes in the concentration of chemicals that are sensed as chemoattractants or chemorepellents. As described in chapter 73, there are multiple processes whereby environmental signals affect flagellar rotation, and there are several types of chemotactic signaling as well as osmotaxis. The best studied chemotactic systems employ protein phosphate transfer reactions by cytoplasmic proteins, in which the phosphorylation reactions are controlled by occupancy of membrane-embedded signal transducer molecules, or receptors. These transducers (tar, tsr, trg, and tap products) possess two transmembrane segments so placed that about one-third of the molecule is exposed to the periplasm. External ligands (aspartate/maltose, serine, ribose/galactose, and oligopeptides, respectively) bind to the periplasmic portions of these transducers, either directly or in complex with the periplasmic substrate-binding proteins that are also components of the periplasmic permeases. The structure of the periplasmic domain of Tar reveals that the ligands (aspartate and the complex of maltose and MBP) bind to it at one end of the domain, about 60 (ca. 6 nm) from the membrane surface (217). Receptor occupancy by an attractant alters the conformation of the cytoplasmic portion of the transducers, which indirectly decreases the kinase activity of the cytoplasmic CheA protein kinase. Phosphorylated CheA can donate its phosphate to CheY, and P-CheY shows increased binding to the FliM switch protein in the flagellar basal body (292, 351). Binding of P-CheY directly or indirectly results in clockwise rotation of the flagellum, leading to tumbling of the cell and reorientation of its direction of swimming. The transducer proteins are also methylated on some glutamate residues during the process of adaptation, which continually resets the transducer to reflect the current degree of receptor occupancy.
Synthesis and modification of peptidoglycan by the various penicillin-binding proteins anchored on the cytoplasmic membrane are described in chapters 6 and 68. Several important features of this process rely directly on the cytoplasmic membrane. All of the penicillin-binding proteins, which carry out the incorporation and processing of the peptidoglycan precursors, are anchored in the cytoplasmic membrane with their active site exposed to the peptidoglycan in the periplasm. There are two separate modes of peptidoglycan synthesis: diffuse growth of the cylindrical side wall of the cell by incorporation over the entire surface, and localized growth of the polar cap at the division septum. The differences in the peptidoglycan cross-linking that confer the different shapes of the wall are not known and are likely to be subtle (90). In both growth modes, the peptidoglycan precursor, a disaccharide-peptapeptide, is synthesized in the cytoplasm and translocated across the cytoplasmic membrane coupled to the polyprenyl phosphate carrier. The same lipid carrier is used for the translocation of precursors for LPS sugar chains and for synthesis of enterobacterial common antigen.
Constriction of the cell surface occurs at the division septum (reviewed in references 86 and 94). Penicillin-binding protein 3 is associated specifically with septum formation, and it seems likely that it and other wall-modifying or synthesizing activities must be localized to the nascent septum. One mechanism that might contribute to this localization is the formation of periseptal annuli, or adhesion zones that join the cytoplasmic membrane to the outer membrane in two rings that gird the cell on either side of the septum (64). These annular rings may separate septum proteins from other periplasmic contents, and vice versa. It remains to be seen how these adhesion zones or the ones that occur elsewhere over the cell surface are formed and whether specific proteins are necessary for their formation or function.
A second aspect of septum formation is the condensation of the FtsZ protein onto the membrane at the site of the septum (chapter 101). At the onset of septation, FtsZ molecules move from the cytoplasm to the inner surface of the cytoplasmic membrane, where they form a narrow ring around the circumference of the cell (25, 198). This FtsZ ring shrinks in diameter while remaining associated with the ingrowing septum. The constriction of the FtsZ ring is probably dependent on its intrinsic GTPase activity and may be the process that drives the constriction and division of the cell (88, 198). Another membrane-related process is the determination of the site of septum formation, in which the MinCDE proteins cooperate in specifying that the septum forms at the midpoint of the cell and not at other potential sites, such as the septum of the precursor cell, which has become a cell pole (reviewed in references 86 and 87).
A major role for the cytoplasmic membrane in the processes of chromosome replication and segregation has long been postulated. As described below and in chapter 99, the DnaA protein initiates the process of DNA replication by unwinding the origin of chromosomal DNA replication, oriC. The in vitro activity of the DnaA protein is strongly stimulated by acidic phospholipids or membranes containing them. Perhaps a complex of the origin and DnaA and accessory proteins must assemble on the cytoplasmic membrane for initiation of replication.
The early replicon model proposed that replicating chromosomes were attached to specific membrane sites and were pulled apart from one another by membrane growth between those two sites (164). High-resolution cell fractionation procedures indicate that some proteins that specifically bind to DNA sequences in the oriC region of the bacterial chromosomal origin of replication are associated with the cytoplasmic membrane, but exhibit anomalous buoyant density (55). The ability of membrane attachment sites to serve for chromosome segregation is discussed in chapters 104 and 105 and critically reviewed in reference 71.
The potential role of cytoskeletal elements in chromatin attachment and movement is now drawing attention (150). Chromosomes remain at the midpoint of the cell when the function of the cytoskeletal protein MukB is blocked in a conditional mutant. Restoration to permissive conditions resulted in very rapid separation of the progeny chromosomes to their proper place at the midpoint of each nascent progeny cell. This redistribution occurred more rapidly than could be explained by the very limited amount of membrane growth that could have occurred during that time. Other interesting bacterial cytoskeletal structures, such as the Cfa filament (229), have been found under unusual conditions.
The physical state of the membrane lipids has a strong influence on the properties of embedded proteins. Most or all transporters and other membrane proteins are dependent on the presence of the fluid, liquid-crystalline phase of the fatty acyl chains, and some processes may occur preferentially at boundary regions between fluid and solid phases. Many solubilized or reconstituted membrane enzymes are specifically stimulated by addition of particular phospholipids (e.g., reference 223). However, verification of the requirement for specific phospholipid head groups in these processes in vivo was refractory to analysis until the development of mutant strains blocked in specific steps of phospholipid biosynthesis (96). As described above, strains with null mutations in the pss and psd genes are blocked in synthesis of the dipolar ionic phospholipid PE, and a strain containing a single copy of the pgsA gene under control of the lac promoter allows manipulation of the level of the acidic phospholipids PG and CL.
Protein Translocation.
Depletion of the cellular content of the acidic phospholipids, PG and CL, affects two major processes of macromolecular synthesis. During translocation of proteins across the cytoplasmic membrane, the cytoplasmic SecA protein undergoes repeated cycles of association with the membrane SecY/SecE translocation complex (353). The low ATPase activity of SecA protein is increased by association with membrane vesicles containing the acidic phospholipids PG or CL (194). Similar stimulation of SecA ATPase activity is seen with cytoplasmic membranes from cells expressing the pgsA gene and containing the acidic lipids, but not with membranes from the uninduced strain lacking those lipids. Binding of SecA, its insertion into the membrane bilayer, and the activation of its ATPase activity occurred in the absence of cytoplasmic factors but were maximal when a complex was formed containing the SecY/SecE protein complex in inverted membrane vesicles containing normal levels of PG, SecA protein, and a precursor protein (43, 144). Translocation of the precursor protein occurred upon addition of ATP to the complexes on membranes containing the acidic phospholipids, but not on PG-depleted membranes. The translocation function of the depleted membranes could be restored by supplementation with acidic phospholipids, including some unnatural lipids, which indicates that the lipid dependency was for the negative surface charge rather than for the interaction with a particular type of lipid head group (185, 248).
Chromosome Replication.
Initiation of chromosomal DNA replication at oriC requires assembly of a multienzyme complex of soluble proteins, including the DnaA initiator protein to promote opening of the DNA double helix (296). DnaA binds to oriC DNA when it is liganded with either ADP or ATP, but only the DnaA-ATP complex activates replication. Exchange of ADP for ATP on DnaA in complex with oriC DNA is facilitated by fluid acidic phospholipids (e.g., CL or PG containing an unsaturated acyl chain) in the lamellar phase (53, 364). Exposure of free DnaA to acidic phospholipids blocks subsequent binding to DNA, although complex formation can be restored by addition of the chaperone protein DnaK or phospholipase (77). An in vivo correlate of this behavior is indicated by the preliminary finding that replication from oriC is blocked by repression of acidic phospholipid synthesis. Replication from cryptic DnaA-independent origins that are active in the absence of RNase H is not affected by the lipid content (356a). Removal of IPTG also results in rapid loss of oriC-based plasmids, but not of ColE1-based plasmids that do not require DnaA function. Thus, acidic phospholipids play important roles for the membrane association and action of cytoplasmic proteins that are involved in protein translocation and chromosome replication.
Although the pss and psd mutants lacking PE are conditionally viable, several membrane-related functions are impaired. These strains are nonmotile, owing to decreased expression of the genes encoding flagellar proteins (300). The reconstitution of the lactose (57) and proline (58) transporters into proteoliposomes exhibits an absolute dependence on the presence of PE. However, PE-deficient strains incorporate normal amounts of LacY protein in the cytoplasmic membrane, although the Vm for lactose transport is reduced 10- to 20-fold (33a). There may be a defect in energy coupling by LacY, but recent evidence using lacY-phoA fusions indicates that several transmembrane segments are incorrectly oriented in PE-deficient cells. It is reasonable to suggest that the presence of PE might be important for proper decoding of the topological signals in an integral membrane protein.
PE-deficient cells possess normal levels of most of the electron transport components, including NADH and succinate dehydrogenases, cytochrome oxidases, and quinones (218). They also have normal generation and utilization of the PMF when driven by succinate or lactate oxidation, but only about 20% of wild-type levels of electron transfer from NADH via NADH dehydrogenase 2 (NDH-2) to oxygen. The level and activity of NDH-2 assayed with artificial electron acceptors were normal, and the activity in the complete reaction was restored by addition of short-chain analogs of ubiquinone. These results are interpreted as indicating a requirement for PE for proper interaction of NDH-2 with the mobile quinone pool, which could be consistent with the requirement for a propensity of the boundary lipids surrounding membrane proteins to adopt a nonlamellar orientation.
Finally, PE-deficient strains exhibit hypersensitivity to many antibiotics (261). A possible basis for this response can be alterations in the packing or synthesis of LPS. PE-deficient strains could lack the decoration of the core sugars with phospho-ethanolamine adducts, which are transferred to the LPS from PE. Furthermore, some of the sugar transferases in the cytoplasmic membrane that are involved in synthesis of the LPS sugar core show a preference for the presence of PE (223).
The development of topological reporters and the elucidation of the Sec pathway of protein translocation have led to considerable insight into the processes of insertion of membrane protein (277). There is no evidence for the requirement for specific chaperones or other insertion factors for the majority of the cytoplasmic membrane proteins which are Sec independent, and their insertion is likely to be cotranslational. The role of FtsH and other export components in controlling membrane protein orientation remains to be demonstrated.
The process of lipid insertion into bacterial membranes has received less attention recently. The small cell size, the rapid kinetics of lipid synthesis, and the presence of the outer membrane have impeded many types of studies of membrane assembly, even the determination of the existence of asymmetric lipid distribution between the inner and outer leaflets of the cytoplasmic membrane. Lipid asymmetry (excess of amino-phospholipids in the inner leaflet) is common in eukaryotic cell membranes. So far, little is known about the mechanism of lipid movement from the inner to the outer leaflet of the cytoplasmic membrane (188).
An emerging topic of study is the coordination of lipid synthesis with other macromolecular synthetic processes. There is an optimal protein/lipid ratio, since cell growth stops when that ratio has doubled following inhibition of phospholipid synthesis in a strain in which the rate of phospholipid acylation is dependent on addition of glycerol (212). Lipid synthesis, like many other processes, appears to be regulated by ppGpp and the associated stringent response. The importance of this system in control of lipid synthesis during normal growth remains to be proven. In addition to the sensing of protein/lipid ratio, there is the question of coordination of lipid and membrane synthesis with the cell cycle. The kinetics of precursor incorporation during the cell cycle suggests that the growth of the membrane is determined by the available surface area, which is itself determined by the rate of growth of the peptidoglycan sacculus (116, 249).
There are conditions where abnormally high levels of expression of particular membrane proteins result in the formation of intracellular membrane structures, as if the pressure caused by increased production of membrane proteins distorts the protein/lipid ratio and induces increased membrane synthesis (347). Formation of intracellular tubules is not seen with other overexpressed membrane proteins, and it is difficult to draw general conclusions.
Another topic of current interest concerns the supermolecular organization of the membrane and the polarized distribution of surface structures (200). There is convincing immunoelectron microscopic evidence that the transmembrane chemotaxis receptors and their associated protein kinase, CheA, are preferentially located at the cell poles. There is no genetic analysis of the potential cellular organization yet in this system, nor of the preferential placement of other proteins at specific sites in the cell envelope. The placement of the division septum in the midpoint of the cell may be an analogous system, and the products of the minCDE locus play prominent roles in this fascinating process (86, 87). Further attention to the molecular details of supermolecular assembly and localization can be expected in the future.
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