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110 CHAPTER 5 • Amino acids: Proteins are made up from 21 α-amino acids (Chapter 3), and some Bacteria and Archaea cannot synthesize one or more of these. They would therefore require that they be present in the growth medium. For example, most strains of Staphylococcus epidermidis, a normal inhab...

110 CHAPTER 5 • Amino acids: Proteins are made up from 21 α-amino acids (Chapter 3), and some Bacteria and Archaea cannot synthesize one or more of these. They would therefore require that they be present in the growth medium. For example, most strains of Staphylococcus epidermidis, a normal inhabitant of human skin, require six amino acids: proline, arginine, valine, tryptophan, histidine, and leucine. The lactic acid bacteria require a greater complement, as indicated in Table 5.3. The level required is proportional to the amount of that amino acid in the cellular protein. This can be calculated as follows: a cell is about 50% protein, and the aromatic amino acid phenylalanine present in protein averages about 5%. Therefore, to grow one gram of an organism that required this amino acid, one should add at least 25 mg phenylalanine to the growth medium (1 gram cell = 500 mg protein, 500 × 0.05 = 25 mg). • Purines and pyrimidines: Requirements for the nu– cleic bases are most often observed in lactic acid bacteria (Table 5.3) and other fastidious organisms with many growth factor requirements. The need for added nucleic acid bases is rare in free-living soil microbes. UPTAKE OF NUTRIENTS INTO CELLS The cytoplasmic membrane of a bacterial or archaeal cell forms a highly selective barrier between the external environment and the cytoplasm. It permits or facilitates the entry of essential nutrients inward and rejects many of those that are not necessary. The hydrophobic nature of the lipid bilayer is responsible for the high degree of selective permeability inherent in cytoplasmic membranes. Polar solutes such as amino acids or sugars cannot traverse unaided across the cytoplasmic membrane. Water can pass freely across the membrane; alcohols, fatty acids, or other fat-soluble compounds may also pass through at varying rates. Microorganisms in nature generally live in environments where virtually all nutrients are available at quite low concentrations. Effective growth can occur only if nutrients can be accumulated inside the cell at levels that far exceed those present externally. Microorganisms have evolved with mechanisms that permit them to concentrate nutrients such as sugars or required cations to levels inside the cell that are thousandfold or more higher than the level in the environment. To accomplish this uptake of nutrients essential to growth and reproduction, a microorganism utilizes a number of distinct transport mechanisms. Among the transport mechanisms most utilized are facilitated diffusion, active transport, and group translocation. These carrier-mediated processes are necessary because simple diffusion would permit an internal concentration that is only equal to that present externally. Under most environmental conditions, this would not support balanced growth. The following is a discussion of the mechanisms that are important in concentration of nutrient solutes inside a bacterial cell. Diffusion Two types of diffusion occur across cytoplasmic membranes: passive diffusion and facilitated diffusion. Diffusion, both passive and facilitated, requires a concentration gradient, and molecules will flow across the membrane from an area of high concentration to one of low concentration until equilibrium is established. Generally, passive diffusion occurs with fat-soluble compounds. Glycerol is a compound that may enter a cell by passive diffusion, and the rate of glycerol uptake is solely dependent on the external concentration. Facilitated diffusion is a carrier-mediated transport process using transmembrane proteins termed permeases. One end of the transport protein protrudes to the outside of the membrane, and the other end extends to the interior. Some of these permeases are highly selective in that they transport only a single type of molecule but most are active with classes of substrates such as a group of similar amino acids or a series of related sugars. Facilitated transport does not require energy, but depends on a conformational change in the transport protein. The solute to be transported binds to the external portion of the transport protein, then a change in conformation of the protein moves the solute inward, and it is released to the inside of the cell (Figure 5.2). Although facilitated diffusion speeds the entry of solutes into a cell, the total internal concentration will not exceed the level in the immediate external environment. Facilitated diffusion is effective because it “speeds up” the diffusion process, and generally the transported material is metabolized on entry. This maintains a constant internal concentration and promotes continued uptake. Because facilitated diffusion will not function against a concentration gradient, a microbe in nature requires a better mechanism for concentrating a nutrient that is present at a low external concentration. Active Transport Active transport is the direct utilization of energy to move a solute from the side of a membrane where the concentration is low to establish a higher concentration on the other side. This accumulates a solute against a concentration gradient. As with facilitated diffusion, solute-specific transmembrane proteins are involved in active transport, and the solute transported is not chemically altered as it passes into the cytoplasm. For a single solute, an organism may have multiple transport systems that may differ in the energy source required and their affinity for the solute that is transported. 111 ISOLATION, NUTRITION, AND CULTIVATION OF MICROORGANISMS Outside of cell A substance is more concentrated on the outside than on the inside of the cell. A molecule binds to the transport protein, which then undergoes a change in conformation. Transport protein The polar substance can diffuse across the membrane. Cytoplasm Figure 5.2 Facilitated diffusion In facilitated diffusion, the substance passes through a transporter protein, or permease. No energy source is required. If the internal concentration exceeds the external concentration, the process can be reversed. The nutrients that are taken into the cell by active transport include some of the sugars, amino acids, organic acids, and inorganic ions. The energy for active transport generally comes from ATP or from a proton gradient established across the membrane. The proton gradient can result from the metabolism of inorganic or organic substrates, or it can be generated by light energy (in photosynthetics). Transport of molecules by ATP-dependent systems involves ATP-Binding Casette transporters (ABC transporters). These transporters are present in bacterial, archaeal, and eukaryotic cells. An ABC transporter consists of hydrophobic transporter proteins that span and form a pore through the cytoplasmic membrane. On the cytoplasmic end of the transporter are nucleotide-binding sites where ATP is bound and hydrolyzed to promote uptake (Figure 5.3). The ABC transporter system depends on substrate binding proteins that are present in the periplasmic space in gram-negative bacteria and attached to the membrane near the outer end of the transporter in gram-positive bacteria. These binding proteins specifically bind the molecules to be transported and pass them on to the transporter protein. Conformational changes in the transporter protein promoted by energy from ATP-hydrolysis move the mole- cule into the cell. In gram-positive microorganisms, the molecules to be transported would diffuse through the cell wall to the membrane surface where they would be available to the binding proteins. In gram-negatives, the molecules would pass through porins in the outer membrane (see Figure 4.48) and come in contact with the binding proteins in the periplasmic space. Bacteria also transport solutes by utilizing the energy inherent in an electrical charge separation across the membrane. There are no periplasmic binding proteins involved in this transport. The charge separation, termed a proton motive force, is established by a proton gradient that is generated during electron transport. The proton force drives ATP synthesis (see Chapter 8) and can be used to drive the uptake of substances through the transport proteins. There are three types of these transport systems: uniporters, symporters, and antiporters. Uniporters are transport proteins that take in cations such as K+ through the electrochemical gradient established by a high positive charge on the outside of the membrane and negativity on the inside (Figure 5.4). Symporters carry sugars, anions, or amino acids into the cell when accompanied by a proton. The binding of a proton to the transport protein and a change in conformation transports the solute inward. Outside of cell Substrate molecule A substrate molecule attaches to a binding protein, and the binding protein releases the substrate to the ABC transporter complex. The substrate molecule is moved through the transporter by conformational changes in the transporter protein… Substrate binding protein ABC transporter complex Cytoplasm Energy transducer protein ATP ADP + P …the energy for which is supplied by the hydrolysis of ATP. Figure 5.3 ABC transporter Active transport requires energy. In this transport system energy is supplied by hydrolysis of ATP. 112 CHAPTER 5 Outside of cell H+ H+ H+ H+ H+ K+ H+ H+ H+ H+ H+ H+ Na+ Glucose Na+ Glucose H+ Na+ H+ Cell membrane K+ Cytoplasm Uniporters transport one substance in one direction, such as K+ into the cell. Symporters transport two different substances in the same direction, such as glucose and Na+ into the cell. H+ Na+ Antiporters transport two different substances in opposite directions, such as H+ into and Na+ out of the cell. Antiporters use the energy of a proton moving inward through the transport protein to drive a cation such as Na+ outward, creating a sodium concentration gradient. This sodium gradient can drive the uptake of an amino acid or other molecule. The sodium ion apparently effects a change in shape of the transport protein driving the solute inward. All of these mechanisms depend on the generation of a proton gradient across the membrane to drive the transport processes. Group Translocation Group translocation is a transport process utilized by bacteria where the transported compound is chemically altered. The best definition of the group translocation is the Figure 5.4 Proton-driven transport phosphotransferase system (PTS) that is Three types of active transport that use the proton gradient across the meminvolved in transport of sugars into the cell brane as energy source. An electrochemical charge is generated across the including glucose, fructose, and α-glucomembrane by electron transport, during which protons are “pumped” to sides. The PTS system is quite complex and the outside of the cell. The energy in this proton gradient is used to transport substances across the membrane. involves a high-energy phosphate from phosphoenopyruvate and the direct participation of at least four enzymes to transport one sugar (Figure 5.5). The first two enzymes, Enzyme I and a heat-stable protein (HPr), are soluble and present in the cytoplasm. Enzymes IIa and b are peripheral proteins attached 1 The high-energy phosphate of to the inner surface of the phosphoenolpyruvate is transferred Cytoplasm Outside of cell membrane at the site of Enthrough enzyme I and a heat-stable protein in the cytoplasm to… zyme IIc. Cell membrane The transmembrane proPhosphoenolHPr- P Enzyme I tein Enzyme IIc is an integral pyruvate part of the cytoplasmic mem3 …enzyme IIc, a Enzyme transporter protein brane and the final recipient of IIa in the cytoplasmic the high-energy phosphate, membrane. which it passes on to phosPyruvate Enzyme I- P HPr Enzyme IIa- P phorylate the sugar in the transport process. 2 …enzymes IIa and IIb, Enzyme IIb- P These enzymes are quite Glucose attached to the inside of the cell membrane, to… specific and are involved Enzyme IIb in the transport of a single sugar, such as glucose. HPr c and Enzyme I are nonspecifzyme II 4 As the phosphate is transferred n E P from enzyme IIc to glucose inside ic and function in the PTS the protein pore, the glucose is system for various substrates. transported into the cell. Glucose- P The PTS transport system is (Glucose-6-phosphate) energy conserving in that the high-energy phosphate from Figure 5.5 The phosphotransferase system Perry / Staley Lory phosphoenolpyruvate ultiActive transport of Sinauer glucoseAssociates into the cell via the phosphotransferase Microbiology 2/e, mately becomes a part of the system. HPr is a heat-stable protein; the other components of the Figure 05.05 Date 12/10/01 transported sugar. system are enzymes.