Energy generation

Energy generation refers to the trapping in a metaboli-cally useful form of the energy absorbed from sunlight or released by reduction of some inorganic molecule or the oxidation or breakdown of an organic molecule serving as energy source. Bacteria can generate energy by many different processes. In simple fermentative pathways, such as the glycolytic breakdown of glucose to lactate, ATP can be formed during several enzymatic steps by the process of substrate level phosphorylation. Important energy-producing processes use electron transport chains to pass electrons from a carrier of high negative redox potential to carriers of successively lower energy states. During respiration, electrons enter these chains following transfer from an organic molecule and are ultimately transferred to an inorganic electron acceptor. During photosynthesis electrons are excited to a higher energy state following absorption of light by a chlorophyll-related molecule. Figure 18.5 summarizes several key steps of microbial energy generation.

During the processes of electron transport to carriers of successively higher redox potential, there is a separation of charge across the membrane. This is ultimately coupled to the movement of protons from one side of the membrane to the other, resulting in the formation of a proton motive force (pmf) which has two aspects. Movement of the positively charged proton creates an electrical charge across the membrane, which is termed A^. In bacteria and mitochondria, in which electron transport results in the release of protons, the electrical charge is negative inside and can be 100-200 mV. Proton pumping also results in a difference in proton concentration, or ApH, across the membrane, such that the exterior is usually more acidic than the interior. Photosynthetic or respiratory electron transport thus results in proton pumping and creation of the pmf.

The proton gradient can be tapped to bring about the formation of ATP, which is the ultimate energy source for most energy-requiring processes in the cytoplasm. Synthesis of ATP is carried out by a family of protein complexes, which include the F1F0 proton-translocating ATPases of mitochondria and bacteria. Related complexes are found in chloroplasts and archaea. These protein complexes contain a membrane-embedded sector, the F0 portion, which includes the pathway to allow the protons to flow back into the cell in response to the chemical and electrical forces acting on them. The F1 portion of the complex contains the sites for conversion of ADP + Pi to ATP, and the energy for this process is coupled to the movement of protons. The stoichiometry of the process is such that entry of three or four protons results in the formation of one molecule of ATP. The individual steps of proton movement through the F1F0-ATPase and ATP synthesis or hydrolysis are tightly coupled under most conditions to prevent wasteful loss of the pmf or of the ATP pool in the cell and consequent heat generation. In bacteria, this ATP synthase can

FIGURE 18.5 Summary of some processes of generation of metabolic energy in bacteria. (Left) Components of respiratory electron transport chains, in which specific substrate dehydrogenases oxidize their substrate, transfer the released electrons to membrane quinones, and in some cases extrude protons to the exterior. The electrons of the reduced quinones are transferred by the terminal oxidase or reductase to the terminal electron acceptor, such as oxygen, with the extrusion of additional protons. (Bottom) Photosynthetic systems whereby absorption of light is converted to a transmembrane gradient of protons. (Right) Two examples in which substrate/product exchange and metabolism result in generation of pmf. (Top) The electrical and chemical components of the proton motive force and a representation of the action of the F1F0-ATP synthase which interconverts the proton gradient and ATP synthesis or hydrolysis.

FIGURE 18.5 Summary of some processes of generation of metabolic energy in bacteria. (Left) Components of respiratory electron transport chains, in which specific substrate dehydrogenases oxidize their substrate, transfer the released electrons to membrane quinones, and in some cases extrude protons to the exterior. The electrons of the reduced quinones are transferred by the terminal oxidase or reductase to the terminal electron acceptor, such as oxygen, with the extrusion of additional protons. (Bottom) Photosynthetic systems whereby absorption of light is converted to a transmembrane gradient of protons. (Right) Two examples in which substrate/product exchange and metabolism result in generation of pmf. (Top) The electrical and chemical components of the proton motive force and a representation of the action of the F1F0-ATP synthase which interconverts the proton gradient and ATP synthesis or hydrolysis.

function in a reversible manner to allow ATP that was generated by substrate-level phosphorylation to drive formation of a proton gradient which can then be used to drive transport systems or motility. Movement of protons causes the F0 sector to rotate within the membrane, like the action of a turbine. The rotation of the F0 sector is coupled to changes in the conformation of the nucleotide-binding sites in the stationary F1 sector, which is linked to interconversion of ADP + Pi to ATP.

1. Photosynthesis

Photosynthesis traps the energy of sunlight and converts it into metabolically useful forms such as ATP during cyclic electron transport and NADPH during noncyclic electron transport processes. In some organisms, photosynthetic electron transport is coupled to the formation of oxygen from water—the process that is essential for aerobic life. There are numerous pigments in cells that absorb light energy for use in photosynthesis, but the most important of these are the chlorophylls. Chlorophylls are porphyrin molecules, similar to heme, but containing a magnesium atom instead of iron. Some pigments are carried in protein molecules that serve as antennae or light-harvesting complexes, but the key processes occur in a protein complex called the photosynthetic reaction center, which typically consists of three proteins that spread across the membrane and contain bacteri-ochlorophyll, bacteriopheophytin, menaquinone, and nonheme iron as electron carriers. The light energy is ultimately absorbed by a chlorophyll molecule and this energy excites an electron to a higher energy level. This excited electron passes through a series of electron-carrying prosthetic groups within the reaction center and then out through the pool of membrane-bound quinones, which transfer the electron to a cytochrome-containing electron transport chain. Passage of the electron through the electron transport chain is coupled to pumping of protons across the specialized membranes containing the photosynthetic apparatus. In cyclic phosphorylation, as carried out in the photosynthetic bacteria, the electron ultimately returns to the chlorophyll molecules after transfer to the periplasmic heme protein, cytochrome c. In the process of noncyclic phosphorylation in plants and cyanobacteria, the electron can be transferred ultimately to pyridine nucleotides for use as a reductant in biosynthetic processes. In this process, the electron can be replaced on chlorophyll by another lightabsorption process that removes electrons from water to create oxygen.

A completely different system for conversion of light into metabolic energy is present in Halobacterium salinarum, an extremely halophilic archaeon that thrives in very saline environments such as the Dead Sea or brine evaporation ponds. These bacteria produce patches of membrane that are densely packed with the membrane protein, bacteriorhodopsin, having seven transmembrane helices and covalently bound retinal as in the visual pigment in mammalian retina. Light absorption by the retinal causes the iso-merization of one of the double bonds in the molecule, causing a change in the conformation of the protein which results in the change of the pX of several acidic groups on either side of the membrane. The consequence of these changes is that a proton is released from the bacteriorhodopsin on the outside of the membrane and replaced by one from the cytoplasm. In this way, light is directly converted into a transmembrane proton gradient without the requirement of an electron transport chain.

2. Respiration

Respiration is the process whereby electrons from the metabolism of an energy source are transferred through a proton-pumping electron transport chain to some inorganic molecule. The most familiar form of respiration is aerobic respiration, in which oxygen serves as the ultimate electron acceptor. Owing to the ability of oxygen to accept electrons, aerobic respiration is the most energetically favorable, but some partially reduced forms of oxygen, hydrogen peroxide, superoxide anion, and hydroxyl radical, are extremely reactive and thus toxic to the organism. Many bacteria are capable of carrying out anaerobic respiration, in which the electrons are transferred to alternative acceptors, such as nitrate, nitrite, sulfate, or sulfite. These processes yield less energy but can occur in anoxic environments.

All respiratory metabolism uses electron transport chains, whereby electron transfer from the donor to oxygen or other acceptor is coupled to proton movement across the membrane. In E. coli, electron donors for respiration include NADH, succinate, glycerol 3-P, formate, lactate, pyruvate, hydrogen, and glucose. Electron acceptors include oxygen, nitrate, nitrite, fumarate, dimethylsulfoxide, and trimethylamine-N-oxide. Typical respiratory systems contain two to four transmembrane protein complexes. These include substrate-specific dehydrogenases, which transfer electrons from the donor to quinones in the membrane. The reduced quinones migrate to another protein complex which accepts electrons from them and transfers the electrons to cytochromes and ultimately to the terminal electron acceptor. The transmembrane protein complexes often contain flavin and/or non-heme iron and are arranged in the membrane in such a way that the passage of electron results in the release of proton to the outside or its consumption from the cytoplasm, i.e. the formation of the pmf.

3. Coupled processes

Some bacteria couple the transport and metabolism of their energy source directly to the production of the pmf. An example of this very simple, but not very energy-rich, process is malo-lactate fermentation in Leuconostoc. The substrate malate is transported into the cell and converted to lactate, which leaves the cell in exchange for a new molecule of malate. The net result of this process is the movement of one negative change into the cell and the consumption of one proton inside the cell, which results in the creation of a pmf that is interior negative and alkaline.

B. Membrane transport

Biological membranes form the permeability barrier separating the cell from its environment. The hydrophobic barrier of the membrane bilayer greatly restricts passage of polar molecules, although non-polar molecules can pass. Transport mechanisms exist to move nutrients and precursors into the cell and metabolic products, surface components, and toxic materials out of the cell. Several types of transport mechanisms and families of transporters have been identified.

1. Types of transport systems

Transport can occur through energy-dependent and energy-independent processes. Several general classes of transport process have been identified. Passive diffusion occurs spontaneously without the involvement of metabolic energy or of transport proteins. It only allows the flow of material down a concentration gradient, and the rate of this process is a linear function of the concentration gradient. The rate of passive diffusion depends on the ability of the permeant to dissolve in the membrane bilayer and thus depends of the polarity of the permeant and its size. These factors are related to the ability of the permeant to fit into transient defects that form in the membrane bilayer. Only water and a few hydrophobic molecules use this mechanism for entry into bacteria.

Facilitated diffusion requires the operation of a membrane protein for passage of the permeant across the membrane. These transporters merely provide a route for diffusion of their substrate down its concentration gradient, and thus the concentration of the substrate on both sides of the membrane will become equal. Transport is not dependent on the polarity of the substrate and usually exhibits stereospecificity, in which isomeric forms of the same compound are transported at very different rates. Because of the involvement of the transporter as a catalyst for movement, the rate of transport can be saturated in the same manner as an enzyme-catalyzed reaction. A possible example is the glycerol facilitator GlpF of E. coli, a transmembrane protein that allows glycerol and other small molecules to diffuse across the cytoplasmic membrane at rates much faster than they cross lipid bilayers. However, this protein may act in the manner of a channel rather than a carrier.

The overwhelming majority of transport systems in bacteria catalyze active transport and expend metabolic energy to allow the accumulation of even very low external concentrations of a nutrient to a much greater concentration inside the cell. Active transport is carried out by a transport protein or complex and thus exhibits substrate stereospecificity and rate saturation. The difference compared to facilitated diffusion is that the substrate can be accumulated at concentrations as much as 1 million times higher than that outside. This accumulation requires the expenditure of energy, and in the absence of energy many but not all transport mechanisms can carry out facilitated diffusion. These active transport systems differ in their molecular complexity and in the mechanism by which metabolic energy is coupled to substrate accumulation. It is important to distinguish active transport, in which the substrate is accumulated in unaltered form, from group translocation, in which the substrate is converted into a different molecule during the process of transport. Some types of transport processes in bacterial cells are summarized in Fig. 18.6.

Group Translocation Bacteria

FIGURE 18.6 Schematic representation of several types of active transport systems in bacteria. Top, transporters linked to the pmf; left, ATP-driven transports; bottom, examples of systems that carry out release of metabolic products of toxic chemicals. (Right) A presentation of all the transport systems known to mediate uptake or release of potassium in E. coli. Some of these transporters are ATP driven; others are coupled to the pmf; and MscL is a channel activated by mechanical stretch of the membrane.

FIGURE 18.6 Schematic representation of several types of active transport systems in bacteria. Top, transporters linked to the pmf; left, ATP-driven transports; bottom, examples of systems that carry out release of metabolic products of toxic chemicals. (Right) A presentation of all the transport systems known to mediate uptake or release of potassium in E. coli. Some of these transporters are ATP driven; others are coupled to the pmf; and MscL is a channel activated by mechanical stretch of the membrane.

2. ATP-driven active transport

Several groups of transport mechanisms use the energy gained during ATP hydrolysis to drive active transport. One of these groups is called the P-type ion-translocating ATPases to indicate the fact that a phospho-enzyme is formed as an intermediate in their reaction cycle. Typically, these transport systems consist of a large polypeptide of approximately 100 kDa which spans the membrane and contains the site for ATP binding and the residue that is phospho-rylated. A smaller subunit usually participates in the activity. The ATP is used to phosphorylate a specific acidic aspartate residue, and this phosphorylation causes a change in conformation of the transporter that is part of the ion pumping process. Several examples of this type of transport system have been extensively studied. The sodium/potassium ATPase in the plasma membrane of higher organisms is responsible for pumping sodium out and potassium into cells, thereby generating the ion gradients that are necessary for many steps of nutrient transport and for neural signal transmission. The electrical potential that exists across the plasma membrane of mammalian and some other cells is based mainly on the difference in sodium ion concentration that is maintained by the action of this transporter. Similarly, the calcium-translocating ATPase located in the sarcoplasmic reticulum acts to lower the intracellular calcium concentrations that accumulate following the processes that initiate muscle contraction. Other P-type ATPases include the proton-translocating ATPase in the plasma membranes of fungi and plants, which establishes the gradient of protons that is used to drive many of these cells' nutrient transport systems, and some of the transport systems for magnesium or potassium ions in bacteria.

Although P-type ATPases function only in the transport of ions, another family of ATP-driven systems transports a wide range of substrates and is involved in the uptake of numerous types of nutrients and in the efflux of both surface and secreted macromolecules (proteins, carbohydrates, and lipids) and of toxic chemicals. This family of transporters is usually the largest family of any set of related genes in those organisms whose genomes have been completely sequenced. Although the subunit composition of these transporters differs, they are called the ABC family to indicate the presence in at least one subunit of a highly conserved ATP-bind-ing cassette, a protein domain that couples ATP binding and hydrolysis to the transport process. A more descriptive name for these proteins is traffic ATPases.

One subset of the family of ABC transporters includes a large group of nutrient uptake mechanisms present only in bacteria and archaea and called periplasmic permeases. These transport systems, such as those for histidine, maltose, oligopeptides, etc., consist of a heterotetramer in which two highly hydrophobic transmembrane proteins with usually five or six membrane-spanning segments are associated with two subunits that contain the ATP-binding cassette and are mainly exposed to the cytoplasm. A fifth protein subunit is responsible for the substrate specificity of these transport systems. This substrate-binding protein usually has very high affinity for the substrate and allows uptake of nutrients even in nanomolar-range concentrations. The structure of these substrate-binding proteins resembles a clam, with two large lobes hinged in the middle. The substrate binds to specific residues in both lobes, which close around the substrate molecule for carriage to the membrane-bound components and entry into the cell. In gram-negative bacteria, the substrate-binding protein floats freely in the periplasmic space between the cytoplasmic and outer membranes. Gram-positive bacteria lack the outer membrane and, to prevent its loss, the binding protein is tethered to the cytoplasmic membrane by a lipoprotein anchor. The mechanism by which ATP hydrolysis is coupled to the release of substrate from the binding protein and its movement across the membrane is currently being studied. A striking feature of these transporters is that they act in a unidirectional manner and only allow nutrient to enter the cell but not to be released.

The basic features of the ABC transport process and homologous transport components also operate in the opposite direction in many processes of macromolecular export, including proteins and surface carbohydrates.

3. Transporters coupled to ion gradients

In addition to ATP-driven active transport systems, bacteria possess many transporters in which the movement of their substrate is obligately coupled to the movement, in the same or opposite direction, of an ion. In this way, accumulation of a substrate is coupled to the expenditure of the gradient of the coupling ion, which is usually a proton or sodium ion. Transmembrane ion gradients are a very convenient source of energy for active transporters. Entry into the cell of at least three protons must occur for synthesis of one molecule of ATP. It is thus very economical for the cell if it can accumulate a molecule of substrate at the expenditure of one proton rather than having to expend one ATP molecule for the same purpose. Symport refers to the process in which the coupling ion moves in the same direction as the substrate, that is, when the downhill movement of a proton into the negative and alkaline interior of the cell is coupled to the uptake of substrate. Antiport is the reverse process, in which the two molecules move in opposite directions. If movement of substrate is coupled to movement of a proton in a 1:1 stoichiometry, then a pmf of — 120mV can achieve a 100-fold accumulation of substrate inside the cell. A pmf of —180 mV can allow a 1000-fold gradient of substrate. Uniporters allow coupling of the movement of a positively charged molecule to the pmf without movement of any other ion. The cationic molecule is drawn into the negatively charged cell interior simply by electrostatic attraction.

These types of transporters are referred to as secondary active transport systems since they use the pmf that was generated by other means and do not use an immediate source of energy, such as ATP. Ion-coupled transport systems are inhibited by conditions that dissipate or prevent formation of the pmf, such as ionophores which allow ions to distribute across the membrane in response to the electrical and chemical gradients that act on it. An uncoupler or protonophore, such as 2,4-dinitrophenol or carbonylcyanide p-triflu-oro-methoxy phenylhydrazone, is a hydrophobic molecule that can cross the membrane in either its ionized or its neutral form. Its presence allows protons to equilibrate across the membrane, thereby dissipating both the electrical and the chemical gradients of protons and thus the entire pmf. The ionophore valinomycin carries potassium ions and allows them to distribute across the membrane in response to electrical or chemical gradients. The addition of valino-mycin to cells or membrane vesicles that have a pmf allows potassium ions to accumulate inside the negatively charged interior. This accumulation results in dissipation of the electrical potential A^. The ionophore nigericin carries protons and sodium or potassium ions across the membrane, but only in the process of exchange. The action of nigericin thus does not result in any net gain or loss of charge and thus does not dissipate A^. However, if there is a concentration gradient of protons, ApH, nigericin allows the concentration gradient to dissipate, whereas the electrical potential is maintained or even increased.

Ion-coupled transporters typically consist of a single polypeptide chain with 12 transmembrane segments, although examples with 10-14 transmembrane segments are known. It has been proposed that these proteins arose as the result of tandem duplication of a precursor protein with six transmembrane segments.

In the well-studied E. coli lactose permease LacY, several major experiments demonstrated the coupling of lactose accumulation to the pmf. The magnitude of the pmf affects the magnitude of the accumulation ratio of lactose inside the cell in a direct manner indicative of a

1:1 stoichiometry of lactose and proton. When membrane vesicles are energized by provision of a substrate for the electron transport system, a pmf is generated and lactose is accumulated. Even when a final steady-state level of lactose accumulation is reached, the lactose is in continual movement in both directions across the membrane. At the steady state, the rates in and out of the vesicle are equal, although the internal concentration of lactose is much higher than the external concentration. This indicates that the energy has resulted in a decreased affinity of the carrier for lactose on the inside face of the membrane relative to its affinity on the outside. Instead of using the electron transport system, a transmembrane electrical potential, interior negative, can be generated experimentally by diluting vesicles loaded with a high concentration of potassium ions into a medium of low potassium concentration in the presence of valinomycin. The potassium ions flow out down their concentration gradient, carrying positive charge out of the vesicle and leaving behind an interior negative charge. This negative interior can attract protons into the vesicle through the lactose permease, thereby driving lactose accumulation. Finally, if unenergized vesicles are placed in an unbuffered solution that contains a high concentration of lactose, the lactose will flow into the vesicle, bringing along a proton and thereby causing a measurable decrease in the pH of the medium. All these results provide convincing evidence for the coupled movement of proton and lactose.

Transporters are designed to prevent uncoupled movement of substrate without protons or of protons without substrate. If the latter case occurred, it would result in the operation of an uncoupler and allow the futile dissipation of the pmf. How the binding of a proton affects the affinity or binding of the substrate remains an intriguing and central question. It has been shown that the proton must bind before the lactose. If saturating concentrations of lactose are present on both sides of the membrane, the lactose transporter carries out their very rapid exchange independent of the release or re-binding of the proton. This result indicates that the reorientation of the loaded substrate-binding site from facing the interior to facing the exterior does not require changes in proton binding by LacY. If lactose is present on only one side of the membrane, however, its downhill movement is much slower than in the case of exchange and is strongly influenced by the pH. This result indicates that the bound proton must be released from the carrier to allow the empty carrier to re-orient its substrate binding site to pick up another molecule of lactose. This result is in agreement with the model that the binding of a proton is needed to increase the affinity of the carrier for lactose, and that the low concentration of protons inside the cell or vesicle is the factor that slows the rate of release of lactose and hence drives lactose accumulation.

The lactose permease has been subjected to extensive genetic and biochemical analysis. Surprisingly few amino acid residues are essential for function, although amino acid substitutions that introduce or affect charged residues in the transmembrane region usually interfere with the stability of the protein or its ability to be stably inserted in the membrane. Models for the folding of the transmembrane segments relative to one another have been proposed from genetic and biophysical assays of regional proximity.

4. Efflux systems

The existence and clinical importance of non-specific drug efflux systems have recently been recognized. An important medical finding was the discovery of an ABC protein that mediates the efflux from cells of nonpolar planar molecules, many of which are used in cancer chemotherapy. Overexpression of this protein, called the P-glycoprotein or multidrug resistance protein-1 (MDR-1), is often associated with failure of chemotherapy and recurrence of the disease after a previous round of treatment. This protein is a single polypeptide, but it resembles the periplasmic perme-ases in having two separate transmembrane domains, each with six membrane-spanning segments followed by a domain with the ABC consensus motifs. It is interesting that a homologous protein in mammalian cells, MDR-2, does not mediate drug efflux but is responsible for the translocation of lipids or their flipping from one side of the membrane to the other. Spontaneous flipping of a lipid across membrane bilayers is extremely slow, and hence an ABC transporter may be assigned this function in cells. Another related protein catalyzes bile acid transport out of liver cells. There are probably many more functions for ABC transporters.

It subsequently has become clear that non-specific efflux systems are widespread in bacteria and account for serious examples of multiple antibiotic resistance. Unlike the ATP-dependence of the MDR carrier, the bacterial efflux systems are coupled to the proton gradient. Most systems possess 4, 12, or 14 transmembrane segments; those with four appear to function as a trimer. In E. coli, the major non-specific efflux system is called the Acr system, indicating its initial discovery as a factor that conferred resistance to acriflavins. It comprises three proteins: AcrB, a proton-coupled cytoplas-mic membrane transporter; AcrA, a lipoprotein than spans the periplasmic space; and TolC, an outer membrane pore-forming protein. Although not essential for growth, the Acr system can very effectively pump many amphiphilic or lipophilic molecules from the cytoplasmic membrane through the outer membrane directly into the medium. This systems is just one example of complex transport systems that act across multiple cellular compartments.

5. Phosphotransferase system

Many bacterial species, but not archaea or eukarya, possess sugar uptake systems that carry out the simultaneous transport and phosphorylation of the sugar. The phosphoenolpyruvate: sugar phospho-transferase system (PTS) uses phosphoenolpyruvate (PEP) as phosphate donor, which is transferred successively to two proteins that act in common in all PTS systems. The enzyme I protein transfers phosphate from PEP to HPr, a small protein that is phosphorylated on a histidine residue. HPr serves as phosphate donor to the sugar-specific components, which comprise three protein domains that can be linked together in various orders and combinations. The IIC domain spans the membrane and catalyzes transport of the sugar, but only under conditions in which it is phosphorylated during its passage. The IIA and IIB domains receive phosphate from HPr-P and transfer it to the sugar molecule that is carried by the corresponding IIC domain. This transport mechanism provides a very economical method to combine the phosphorylation that must occur during sugar metabolism with the transport process. Although the PTS allows substrate accumulation, it is not considered active transport because the substrate is modified during transport, and the metabolic energy has been expended in the substrate modification rather than during the transport.

It is interesting that the PTS system plays a major role in the regulation of cellular metabolism, and the transport of PTS sugars inhibits uptake of potential carbon sources through other types of transport systems. Part of this mechanism involves the inhibitory interaction of the IIA component of the glucose PTS with a variety of transporter proteins for other sugars.

C. Export of surface molecules

All bacteria have specialized processes for the secretion or export of proteins and polysaccharides across the cytoplasmic membrane to the periplasm, cell wall, outer membrane, or external medium. Exported components can comprise 10% or more of the weight of the cell, indicating the magnitude and variety of macromolecular transport activities that occur. For peptidoglycan, lipopolysaccharide, and capsular polysaccharide synthesis, the precursor subunits are translocated across the cytoplasmic membrane and assembled on its outer surface. Some of these precursors are flipped across the membrane after they are coupled to a lipid membrane carrier.

There are several specialized mechanisms for secretion of proteins. To be secreted, a protein must carry a suitable secretion signal. The most common of these is the signal peptide, an amino-terminal extension that is removed during secretion. The typical signal pep-tides in all cell types comprise 20-30 amino acids with positively charged residues at the amino terminus and then a stretch of hydrophobic residues, followed by a more polar stretch and a peptidase cleavage site. Variants of this structure are used to target proteins to specific cellular organelles in eukaryotic cells. The Sec system acts on the majority of translocated proteins in bacteria. It operates along with several cytoplasmic chaperone proteins, mainly one called SecB, that slow the folding of the precursor protein into its stable final structure, which would prevent its movement across the membrane. The SecYEG protein complex in the cytoplasmic membrane forms a channel through which the polypeptide chain can move. The SecA protein plays a crucial role by binding to the precursor in the cytoplasm, bringing it to the SecYEG translocation complex, and sequentially inserting approximately 30 amino acid segments to the other side of the membrane. The insertion process requires ATP hydrolysis. The SecA and SecB proteins are found only in bacteria and not eukaryotic cells. Once the signal peptide has appeared on the other side of the membrane, it is cleaved off by the action of a leader peptidase enzyme.

Other secretory systems operate in bacteria. The Tat system is named for the twin-arginine motif present in the signal sequences of its substrates, and can move even folded proteins across the cytoplasmic membrane. The SRP-dependent system is related to the process that predominates in eukaryotic cells and involves a signal recognition particle. This system also uses the SecYEG complex for transmembrane movement of its substrate proteins during their synthesis on the ribosome, and appears to be used mainly for proteins destined to the cytoplasmic membrane.

At least five types of transport systems allow export of specific proteins out of the cell. These proteins employ special targeting signals to specify their entry into the appropriate secretory pathway. One of these, the type II secretion system, uses the Sec pathway for initial movement of the precursor into the periplasmic space, where a very complex protein assemblage recognizes the exported protein and moves it across the outer membrane. The type I secretion system resem bles the Acr multidrug efflux system in the simplicity and location of its protein components, except that the cytoplasmic membrane component uses the energy of ATP hydrolysis for its action. The type III and type IV secretion systems are of particular interest because they can secrete their substrate proteins, or DNAprotein complexes carried by some type IV systems, directly into a eukaryotic cell. Other specialized systems exist for the assembly of flagella and fimbrial adhesins (pili and fimbriae) on the cell surface. The number, mechanism, and regulation of the many specialized export systems have only recently begun to be understood.

D. Cell growth

1. Cell division

The cytoplasmic membrane plays numerous important roles in the processes of cell growth and division. The rate of growth of a cell is determined by the rates of synthesis of its macromolecules, RNA and protein. Their continued production produces pressure that drives the expansion of the cytoplasmic membrane, with the insertion of additional membrane proteins and of the phospholipids to maintain a set protein: lipid ratio. Somehow, the pressure of the expanding cytoplasm triggers insertion of additional cell wall material to allow the increase in cell volume. Once the cell volume has reached a critical point, or the concentration of a specific protein has reached a certain concentration, or a certain time has passed since the chromosome was replicated, the process of cell division is initiated. A critical step in cell division is the assembly of a ring of tubulin-like FtsZ proteins from the cytoplasm onto the membrane at the division site. This assembled complex contracts to pull the membrane together at the division septum to close off the two progeny cells.

A poorly understood process of cell division that may require membrane action is the separation and equal partitioning of the bacterial chromosomes into each progeny cell. In one model, the chromosomes attach to a membrane protein and are pulled apart by the growth of the membrane. This model is unlikely to account for the rapidity with which chromosome separation can occur under certain conditions, but no convincing evidence for cytoskeletal components that might pull the chromosomes apart has been presented.

2. Signal transduction

Bacterial gene expression is very responsive to changes in the cell's environment, and many types of regulatory systems allow specific genes to respond to the presence of or need for a wide range of pathway substrates or products. Some regulatory systems are controlled by the binding of the effector molecule to a specific DNA-binding protein that controls the level of expression of the controlled gene. Other systems are controlled by effector molecules that remain outside the cell. Many of these are controlled by two-component regulatory systems, which are widespread in bacteria and archaea and even occur in eukarya. One component of these systems is a transmembrane protein that recognizes its effector molecule in the medium and responds to its binding by a transmembrane signaling event that changes its ability to phosphorylate or to transfer that phosphate. Phosphorylation of the sensor kinase protein can result in transfer of that phosphate to the second component, which is a response regulator protein. The ability of the response regulator protein to bind to a target DNA sequence or to activate transcription of that gene is directly related to the level of its phosphoryla-tion. This mode of transmembrane signaling by regulation of protein phosphorylation is reminiscent of the myriad signaling processes in eukaryotic cells, although the mechanisms and components are not related.

3. Cell movement

Bacteria exhibit several types of motility. Many rod-shaped bacteria and a few cocci can swim through liquid medium through the use of flagella. Flagella are long, helical filaments that extend from the poles or the periphery of the cell body and are assembled from a single protein subunit, called flagellin. Motility is initiated by the rotation of the flagellar filament, which in the case of bacteria with multiple filaments results in their coalescence into a bundle that acts similar to a propeller. Their rotation is driven by the downhill entry of protons through the flagellar basal body, a complex structure embedded in the cytoplas-mic membrane and driven by the pmf.

Spirochetes are spiral-shaped bacteria in which the cell body is wrapped around the flagellar filaments which grow from the cell poles. Their flagella do not extend into the medium but are retained in the periplasmic space. These bacteria exhibit a characteristic corkscrew motility that is thought to result from rotation of the endo-flagella.

Several types of bacteria are capable of movement on solid surfaces or in very viscous solutions, in which flagella are ineffective. In some types of these bacteria the mechanism of movement is unknown, whereas in some enteric bacteria, such as Proteus, this swarming motility is related to differentiation of the cell into very long forms that are covered with a profusion of lateral flagellar filaments.

Motility is a regulated process that allows bacteria to swim toward or away from gradients of nutrients or repellents or physical conditions, such as oxygen or light. Response to chemical attractants or repellents is called chemotaxis and this senses whether a bacteria is moving in a direction which increases or decreases the concentrations of the chemical signal. In the absence of a signal, bacteria exhibit periods of swimming in a straight line followed by short periods in which they tumble aimlessly before setting off in a new direction. Straight-line swimming is associated with counterclockwise (CCW) rotation of the flagel-lum (viewed toward the cell), and tumbling is associated with clockwise (CW) flagellar rotation. The process of chemotaxis controls the direction of rotation so that when cells are swimming in a favorable direction, their period of straight swimming (CCW rotation) is extended. Movement in an unfavorable direction results in an increased frequency of tumbling (CW rotation). This process is controlled by several chemoreceptors in the cytoplasmic membrane, whose occupancy by substrates indicates the level of the chemical signals. Occupancy of these receptors by their ligands results in changes in the activity of a protein kinase that is related to the two-component regulatory systems described previously. Changes in the activity of the kinase result in changes in the level of phosphorylation of a small cytoplasmic protein, CheY, whose phosphorylated form signals CW rotation of the flagella. Another form of covalent modification is the methylation of certain glutamate residues on the chemoreceptors which serves to adjust their signaling properties and allow the receptors to respond to changes in the level of the chemicals rather than to their static concentration. This adaptive response allows the bacterium to resume its normal behavior once it finds itself in a steady supply of the chemical attractant and to prepare to set off in response to new signals. Chemotaxis thus provides a well-studied example of transmembrane signaling and communication between protein complexes in the cytoplasmic membrane. Of particular interest is the localization of the chemoreceptors and associated components at the poles of the cell.

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