The Objectives of This Chapter Are To

The objectives of this chapter are to:

N Describe the steps of membrane synthesis.

N Outline the process of peptidoglycan synthesis.

N Introduce the concepts of protein structure and function.

N Highlight important aspects of protein synthesis and export.

N Describe the structure and function of cell appendages.

© 2007 Sinauer Associates, Inc. This material cannot be copied, reproduced, manufactured, or disseminated in any form without express written permission from the publisher. 11 Assembly of Bacterial Cell Structures

...Topologically, all the layers of the envelope are closed surfaces and must be physically continuous for cell integrity and viability to be maintained. ...All the constituents of the envelope must grow coordinately and with special regard to their location... The overall process is similar in gram-positive and gram-negative bacteria but differs in detail... —Neidhardt, Ingraham and Schaechter, Physiology of the Bacterial Cell: A Molecular Approach, 1990

he generation of precursor metabolites and the reactions that convert these to the monomeric building blocks of a cell were Tdiscussed in Chapter 10. The precursor metabolites originate either from CO2 fixation, gluconeogenesis, the tricarboxylic acid cycle, glycolysis, or allied pathways. Through a limited number of well-integrated reactions, the major monomers—including the amino acids, nucleotides, sugars, and fatty acids—are synthesized from these precursors. Rapid and orderly growth depends on the polymerization or assembly of monomeric building blocks to form macromolecules. Among the essential polymerization reactions are the formation of proteins from amino acids, polysaccharides from sugars, and nucleic acids from nucleotides. Phospholipids are derived from fatty acids. Once formed, macromolecules (DNA, RNA, proteins, and phospholipids) are assembled to generate a cell (Figure 11.1). Note that proteins and RNA make up the major part of a living cell. The actual number of molecules of each macromolecular component present in an Escherichia coli cell is listed in Table 11.1. Replication of DNA, RNA synthesis, and the role of the nucleic acids in protein synthesis will be discussed in Chapter 13. The following is a brief discussion of the synthesis and assembly of cell membranes and protein structures and the assembly and export of constituents to the cell envelope (peptidoglycan layers), outer membrane (in gram-negative bacteria), plus cell appendages.

The precursor …the types of monomers, Further reactions …that interact to metabolites formed in or building blocks, of all polymerize the produce the structures glycolysis, the TCA cellular components. monomers to form that make up the E. coli cycle, and related macromolecules… cell. pathways enter biosynthetic pathways that produce…

Lipids Fatty acids ˜8 Lipopolysaccharides Inclusion 2– Glycogen bodies Glucose-6-phosphate PO4 Sugars ˜25 Fructose-6-phosphate + Peptidoglycan Envelope Pentose-5-phosphate HS– Erythrose-4-phosphate + 2– PO4 Glycerol-3-phosphate NH3 Flagella 3-Phosphoglycerate Glucose Phosphoenolpyruvate Amino acids Fueling Biosynthetic Pyruvate ˜25 Protein Pili reactions Acetyl-CoA reactions α-Ketoglutarate Succinyl-CoA Cytosol Oxaloacetate Poly- Nucleotides 8 ribosomes Figure 11.1 Synthesis of cell structures from glucose ˜ RNA An overview of the anabolic reactions that lead from glu- Nucleoid cose to the structures in an E. coli cell. The numbers of dif- DNA ferent monomer types needed are indicated; the size of Polymerization Assembly each box is proportional to the amount of material required reactions by an E. coli cell.

The overall macromolecular composition of an TABLE 11.1 Escherichia coli cell Percentage of Number of Types of Total Dry Weight Molecules Molecules Molecule of Cell per Cell Possible Protein 55.0 2,360,000 ~4200 RNA 20.5 23S rRNA 18,700 1 16S rRNA 18,700 1 Perry Staley Lory Microbiology 2/e 5S rRNA 18,700 1 Sinauer Associates transfer RNA 205,000 ~60 Elizabeth Morales Illustration Services Figure 11.01.eps Date 02-16-07 messenger RNA Variable ~1,380 DNA 3.1 ~2.1 1 Lipida 9.1 22,000,000 4 Lipopolysaccharide 3.4 1,200,000 1 Peptidoglycan 2.5 1 1 Glycogen 2.5 4,360 1 Soluble organic poolb 2.9 Large ~850 Inorganic poolc 1.0 Large ~20

aThe phospholipids are of four general classes, which may exist in a variety of types based on fatty acid chain. bMetabolites, vitamins, and precursors. cAnions, cations. Adapted from Neidhardt, Ingraham, and Schaechter, 1990.

11.1 Membrane Synthesis able cell may be controlled by proteins in the membrane. This process may require ATP, but that is uncertain at One structure that is present in all Bacteria and Archaea the present time. is the cytoplasmic membrane, also called the cell mem- Modification of bacterial membranes can occur inde- brane. The basic structure of a cytoplasmic membrane pendently of cell growth and division. A constant was described in Chapter 4 (see Figures 4.44 and 4.45); turnover of membrane proteins or phospholipids (or it serves as the primary boundary of the cell’s cytoplasm. both) occurs as organisms adjust to changing environ- The bacterial cytoplasmic membrane is similar in its ba- mental conditions. Cells increase the proportion of mem- sic structure to other membranes—for example, those brane unsaturated fatty acids as a response to a decrease surrounding eukaryotic cells, nuclei, mitochondria, and in growth temperature. This occurs because a functional other organelles. The membranes of Archaea are quite membrane must be fluid and because the double bonds different, as will be discussed in Chapter 18. in unsaturated fatty acids are more fluid at lower temper- A cytoplasmic membrane in a growing cell must as- atures. A cell may also vary the fatty acid chain length. In similate new membrane components (phospholipids and E. coli, C18 fatty acids are more abundant at high growth proteins) and integrate these into the membrane, which temperatures, while C16 predominates at lower temper- is increasing in surface area. During integration of the atures. Newly formed solute transport proteins or respi- newly synthesized phospholipids, the permeability and ratory enzymes may also be inserted into the cytoplasmic barrier functions of the membrane must be maintained. membrane as a microorganism encounters a different substrate or other changes in growth conditions. Synthesis of Lipids Membrane lipids compose about 10% of the cell dry SECTION HIGHLIGHTS weight (see Table 11.1) and thus represent a considerable Assembly of phospholipids occurs at the sur- expenditure of cell energy. Enzymes for lipid synthesis are face of the cytoplasmic membrane and re- generally located in the cytoplasmic membrane with the quires the carrier molecule ACP. The lipid and exception of those needed for precursor biosynthesis (glyc- protein composition of a cell may be adjusted erol-3-phosphate) and fatty acid synthesis; these are dis- independently of cell growth in order to adapt cussed in Chapter 10. Since lipid types differ among bac- to temperature and nutritional changes. teria with respect to fatty acid chain length, saturation, and polar head group, the reader is directed to advanced textbooks and scientific literature for detailed information. As a general plan, long-chain fatty acids (C14–18) are assembled in the cytoplasm from acetyl-CoA precursor mole- 11.2 Peptidoglycan Synthesis cules via small acyl-carrier protein (ACP) intermediates. The degree of fatty acid saturation may vary depending Peptidoglycan (murein) is the main structural compo- on the cell growth condition; the fatty acids are then ester- nent of the bacterial cell wall. It is the source of strength ified with glycerol at the inner surface of the cytoplasmic and provides the characteristic cell size and shape. The membrane. A polar head group is then added to join two composition, structure, and function of peptidoglycan fatty acids, yielding the mature phospholipid (see Figure in both gram-positive and gram-negative bacteria were 4.45). E. coli, for example contains three types of phospho- discussed in Chapter 4. The position beyond the cyto- lipids (phosphatidylglycerol, phosphatidylethanolamine, plasmic membrane (Figure 11.2) presents interesting and cardiolipin (diphosphatidylglycerol), where the polar questions regarding its synthesis and assembly. head groups are glycerol, ethanolamine, and phos- The assembly of peptidoglycan precursor units oc- phatidylglycerol, respectively. Other microbes, for exam- curs in the cytoplasm and the cytoplasmic membrane ple Bacillus subtilis, may contain different head groups: (Figure 11.3). At the inner surface of the cell membrane, glucose and glucose—O—glucose in addition to ethan- both N-acetylglucosamine (NAG) and N-acetylmuramic olamine and glycerol. acid (NAM) are synthesized and then coupled to bac- The newly synthesized phospholipids are incorpo- toprenol (undecaprenol phosphate, Udc; Figure 11.4). rated into the inner leaflet of the cytoplasmic membrane Bactoprenol is a long-chain hydrocarbon that can enter bilayer, and in a relatively short time appear in the outer the hydrophobic core of the cytoplasmic membrane and leaflet. This movement from the inner to the outer mem- facilitate the movement of attached hydrophobic mole- brane leaflet occurs at a more rapid rate in actual cell cules into or through the lipid bilayer. membranes than it does in model bilayers, suggesting In an initial membrane-associated step in peptidogly- that rotation from the inner to the outer leaflet in a vi- can synthesis, bactoprenol displaces the uridine triphos-

(A) Gram-positive cell envelope

Outside of cell

The thick cell wall consists of 20–40 layers of peptidoglycan.

Peptidoglycan

The cytoplasmic membrane consists of two phospholipid leaflets held together by hydrophobic forces.

Cytoplasm

(B) Gram-negative cell envelope

Outside of cell Porin

Lipopolysaccharide

The outer membrane is similar in structure to the cytoplasmic membrane. It contains porins and other proteins, including lipoprotein, and lipopolysaccharide.

Lipoprotein

Peptidoglycan

Periplasm

Cytoplasmic membrane

Figure 11.2 Cell envelopes of bacteria Models of cell envelopes of (A) a gram-positive bacterium, showing the cell membrane and cell wall, and (B) a gram-negative bac- Membrane terium, showing the cell membrane, pepti- Cytoplasm proteins doglycan, and outer membrane.

Periplasm Cytoplasmic Cytoplasm membrane UTP

N-Acetylglucosamine-1- P P P

UDP-N-Acetylglucosamine NADPH Phosphoenol- pyruvate NADP+

UDP-NAM 3 ATP L-Alanine D-Glutamate 3 ADP L-Lysine Cycloserine 6 The Udc is recycled for another round UDP-NAM-Ala-Glu-Lys of synthesis. Bacitracin Pi D-Alanyl-D-alanine

PP Udc Udc P

UDP-NAM—pentapeptide NAG-NAM NAG-NAM UMP

Ala Ala NAG-NAM NAG-NAM— PP Udc Udc PP —NAM—pentapeptide Glu Glu Tunicamycin Ala Ala UDP-NAG 1 The NAM–NAG units Lys Lys (Gly) Glu 5 Glu UDP are synthesized on the inside surface of Ala (Gly) Ala the cytoplasmic 5 Lys Lys (Gly)5 Udc PP—NAM-NAG membrane while Ala Ala pentapeptide attached to undecaprenol phosphate (Udc). Ala 5 glycyl-tRNA 5 Cross-linking between 4 This alanine will peptidoglycan strands be released 5 tRNA occurs here. Release of during Vancomycin the terminal alanine of cross-linking. NAG-NAM PP Udc Udc PP—NAM-NAG the pentapeptide provides the energy for Ala Ala the cross-linking reaction. Glu Glu 3 The existing

peptidoglycan is Lys (Gly)5 (Gly)5 Lys attached to the 2 After synthesis is new NAM–NAG Ala Ala unit here. complete, the NAM–NAG units are transferred Ala from the cytoplasm to Ala the periplasm, where they are added to the peptidoglycan cell wall.

Figure 11.3 Synthesis of peptidoglycan Steps in the synthesis of peptidoglycan. Gram-positive peptidoglycan is depicted here; gram-negative peptidoglycan has DAP in place of lysine and pentaglycine (see Figure 4.48) and DAP is directly cross-linked to the D-alanine of the adjacent strand. UTP, uridine triphosphate; UDP, uridine diphosphate; UMP, uridine monophosphate; NAG, N-acetylglucosamine; NAM, N-acetylmuramic acid; Udc, undecaprenol phosphate. Steps blocked by antibiotics are indicated. Perry Staley Lory Microbiology 2/e Sinauer Associates Elizabeth Morales Illustration Services Figure 11.03.eps Date 03-20-07

Figure 11.4 Bactoprenol CH CH CH 3 3 3 Bactoprenol or undecaprenol phosphate with an attached phospho-N-acetylmuramic acid CH3 C CHCH2(CH2 C CH CH2)9 CH2 C CH CH2 and pentapeptide, an intermediate in peptido- O glycan synthesis.

Undecaprenol phosphate O P O–

O P N-acetylmuramic acid (NAM)

O– Pentapeptide phate precursor (see Figure 11.3), which renders the incorporation of NAM–NAG-pentapeptide into the NAM-pentapeptide sufficiently hydrophobic to allow newly forming glycan polymer. passage through the hydrophobic membrane. During this passage, a bridge peptide such as pentaglycine may Forming Gram-Positive Cell Envelopes be attached to the terminal amine of lysine via a peptide bond. This would occur in gram-positive bacteria. The The structural relationship between the cytoplasmic peptidoglycan units then enter the periplasmic space membrane and the peptidoglycan in a gram-positive and are inserted at a growing point in the cell wall. bacterium was described in Chapter 4. During cell The N-acetylmuramic acid (NAM)–N-acetyglu- growth, peptidoglycan subunits are constantly synthe- cosamine (NAG) units are added to the existing NAM– sized and enter the space between the cytoplasmic mem- NAG backbone of the peptidoglycan. The peptide bridge, brane and existing murein layer at points where the sub- in this case pentaglycine, is joined to an alanine molecule units are linked to the existing peptidoglycan layer. Thus on an adjacent NAM–NAG chain. The NAM–NAG back- a growing cell is continually adding a murein layer in bone and the peptide cross-linking are responsible for the the area adjacent to the cytoplasmic membrane. About strength of the peptidoglycan cell wall. 40 layers of murein surround a gram-positive cell. These A battery of periplasmic enzymes is involved in the layers move outward as newly synthesized murein is covalent reactions that result in extension and cross- added to the inner layer. linking between the peptidoglycan strands. The en- Teichoic acids are the second major constituent of zymes are also responsible for the septation of the gram-positive bacterial envelopes (see Chapter 4). Syn- murein cell wall, which occurs during cell division. The thesis of these polymers occurs by assembly from pre- prPerryoteins Staley involved Lory Microbiology in extension 2/e of the peptidoglycan cursor molecules at the inner surface of the cytoplasmic wallSinau erduring Associates growth have a unique ability to bind the membrane and also involves the lipophilic carrier mol- Elizabeth Morales Illustration Services antibioticFigure 11.04.e penicillinps Dateand 02-16-07 some related antibacterials. ecule bactoprenol (undecaprenol). A built-up long-chain The number of these “penicillin-binding” proteins on polymer of ribitol phosphate, as in Staphylococcus aureus, the surface of a bacterium varies with species. Studies for example, is transferred across the lipid bilayer and indicate that the penicillin-binding proteins are in- then covalently attached to the peptidogycan layers by volved with transglycosylation (elongation of glycan a phosphodiester bond. Bactoprenol-phosphate is re- strands), transpeptidation (cross-linking), and the en- cycled for subsequent rounds of polymer assembly. Te- zyme carboxypeptidase, which cuts preexisting cross ichoic acid polymers may be modified by the addition links for the new glycan insertion needed for cell elon- of monosaccharide or oligosaccharide moieties at the gation and division (see Figure 6.1). Binding of peni- –OH group of ribitol by specific glycosyltransferases. cillin to these proteins inhibits murein biosynthesis and There is considerable diversity among the gram-positive can destroy the integrity of the cell. bacteria with respect to the type of polymer (see Chap- A variety of other inhibitors also interfere with pep- ter 4) and side-chain modification. Details of teichoic tidoglycan synthesis and assembly. Bacitracin inhibits acid synthesis as well as production of lipoteichoic acids the removal of phosphate from undecaprenol-pyrophos- are provided in advanced textbooks. phate, thus preventing recycling of undecaprenol-phosphate. Tunicamycin blocks addition of NAM-pentapep- Forming Gram-Negative Cell Envelopes tide to undecaprenol-phosphate, whereas cycloserine inhibits addition of D-alanine to the elongating peptide The cell wall structures of gram-negative bacteria are con- chain on UDP-NAM. Finally, vancomycin inhibits the siderably more complex than those of gram-positives (see

Figure 11.2). The gram-negative bacteria have a complex outer membrane that surrounds the cell envelope. Location of major proteins Growth and expansion of this envelope is consequently TABLE 11.2 associated with a gram- more elaborate. It is apparent that fatty acids, sugars, negative bacterium phospholipids, and proteins must move from the cyto- Location Proteins plasm to the periplasm during wall expansion or during environmental changes in gram-negative bacteria. Cytoplasm Enzymes involved in catabolism of Expansion has been viewed as occurring in minute soluble substrates openings in the cell wall, and these have been termed Enzymes involved in anabolism zones of adhesion because the inner and outer mem- Enzymes involved in DNA replication branes actually make direct contact. The junctions occur Ribosomes and enzymes for protein in the peptidoglycan layer, and all components for the synthesis construction of the outer cell envelope pass through Proteins involved in gene regulation these gaps. However, more recent studies implicate spe- Cytoplasmic Components of electron-transport chain cific protein secretion pathways for cell assembly, very membrane Proton translocating ATP synthase likely not involving these junctions. Solute uptake proteins The components of the lipopolysaccharide (see Chap- Lipid biosynthesis enzymes ter 4) present in the outer membrane are synthesized on Cell wall and outer-envelope the inner surface of the cytoplasmic membrane and are biosynthetic enzymes carried outward with the assistance of bactoprenol. As- Secretory machinery sembly then occurs on the outer surface of the cell by in- Chemotactic receptors teractions between the envelope components. The phos- Periplasm Binding proteins involved in transport pholipid components, including lipid A of the outer and chemotaxis membrane, are likewise synthesized and translocated to Enzymes for peptidoglycan assembly form the outer membrane layer. Certain secretory proteins Methods to characterize the various components that Outer envelope Porin proteins make up the cell envelope are described in Box 11.1. Receptors for bacteriophage, etc. They are routinely applied in the molecular study of model organisms and can be adapted to study of newly External surface Fimbriae/pili isolated strains from the environment. Flagella Extracellular Hydrolases Proteases SECTION HIGHLIGHTS Lipases Synthesis of peptidoglycan, teichoic acids, and Nucleases LPS initiates in the cyptoplasmic membrane Protein toxins and involves the carrier molecule, molecule Capsule assembly proteins bactoprenol. Translocation and insertion of these cell precursor molecules generates the For example, proteins in the cytoplasm are involved mature cell envelope characteristic of a gram with catabolic functions and with the synthesis of DNA, positive or gram negative bacterium. RNA, and cellular components. The electron transport systems in microorganisms capable of respiration are located in the cytoplasmic membranes, as are the proteins of the ATP synthase system. The outer envelopes of 11.3 Protein Assembly, Structure, and gram-negative bacteria contain the protein porins in- Function volved in passage of molecules into the periplasmic space, where binding proteins can retain them. Trans- The term “protein” broadly defines molecules composed port of these molecules to the cytoplasm occurs through of one or more polypeptide chains. A polypeptide is a the action of proteins in the cytoplasmic membrane. Fi- polymer that generally exceeds several dozen amino nally, microorganisms that can digest polysaccharides, acids in length. Some proteins are present in the cyto- fats, or other large molecules secrete enzymes (proteins) plasm; others are associated with the cytoplasmic mem- to the cell exterior that can hydrolyze large molecules to brane, outer envelope, or periplasm of a bacterium low-molecular-weight compounds. These compounds (Table 11.2). can then be transported into the cell.

BOX 11.1 Methods & Techniques

i Cell Fractionation/Separation and Biochemical Analyses of Cell Structures

In order to determine the composi- methods include ultrasound (called Once the cells have been broken, tion of various components of the sonication by biologists), in which the various structural fractions are cell, scientists separate these com- high-frequency sound waves separated, usually by centrifuga- ponents from the rest of the cell, vibrate cells until they break. A son- tion. Two types of centrifugation purify them, and analyze them bio- icator probe is inserted into a cell can be used. In differential or chemically. The initial step is to suspension for this purpose, as velocity centrifugation, fractions break open the cells. Either chemi- shown in the illustration. are separated by the length of time cal or physical procedures can be Alternatively, cells can be broken they are centrifuged at different used to break open small, prokary- by passing thick suspensions of gravitational forces. Denser struc- otic cells. For example, chemical cells through a small orifice tures—such as unbroken cells or procedures include lysis of the cells (“French pressure” cell) at very high bacterial endospores, cell mem- by enzymes or detergents. Physical pressure. branes, or cell walls—sediment at

1 Disrupt cells Cell suspension by sonication. Sonicator causes cell breakage by producing high-frequency sound waves

Approach a: Approach b: Differential Buoyant density centrifugation centrifugation 2b Layer the suspension of disrupted cells on a sucrose density gradient (with 2a Centrifuge the cell suspension Cell increasing density toward at low speed (15,000 × g for suspension the bottom of the tube). 10 minutes), then examine the Supernatant sediment in the electron A density microscope to confirm identity gradient and purity. If pure, analyze Cell walls, of sucrose biochemically. membranes, flagella 3bCentrifuge the 3a Remove the supernatant and tube to equilibrium. centrifuge again at high speed (100,000 × g for 60 minutes).

Supernatant (soluble proteins, 4b Examine the cell fractions enzymes) Cell fractions 4a Examine the sediment in the separated at in the electron microscope electron microscope and, if Ribosomes, different to confirm identity and pure, analyze biochemically. membranes, buoyant purity. If pure, analyze fragments densities biochemically.

Perry Staley Lory Microbiology 2/e Sinauer Associates Elizabeth Morales Illustration Services Figure Box 11.01.eps Date 02-16-07 Assembly of Bacterial Cell Structures 295

BOX 11.1 Continued

low speeds (15,000 × g for 10 min- centrifuge using different concen- and purified by the procedures out- utes). The supernatant is removed trations of a solute, such as sucrose. lined above, it can be analyzed and centrifuged at higher speed to The sample is layered on the sur- chemically. The electron micro- spin out less dense structures. For face and centrifuged at moderate scope may be used to check the example, ribosomes sediment only speed until the cellular fractions identity and purity of the material after centrifugation at higher equilibrate with the layer in the at each step in the process. speeds (100,000 × g for 60 minutes). gradient that has the same buoyant Proteins may be separated on poly- The remaining material which does density. They can then be removed acrylamide gels (native or denatur- not sediment in the centrifuge tube with a pipette and studied as puri- ing) to characterize those present. contains soluble constituents such fied fractions. For example, the Biochemical assays can be per- as cytoplasmic enzymes. cytoplasmic membranes of E. coli formed using marker enzymes as Alternatively, buoyant density are less dense than the outer mem- an indication of fraction purity ver- or density gradient centrifugation brane because of differences in the sus contamination by other cell can be used to separate the various types of molecules present. fractions. cell fractions. In this procedure, a When the cell fraction of interest density gradient is set up in the to the microbiologist is separated

Protein Structure ary and tertiary structure, are outlined in Figure 11.6. Amino acids are joined together by peptide bond for- The variability in the side chains of the 21 major amino mation on ribosomes through a series of peptidyl trans- acids (see Figure 3.15), the total number incorporated, fer reactions from amino acid-tRNA molecules. When and their sequence in a polypeptide are all factors that a series of amino acids are joined together, the result is contribute to the biochemical characteristics of the pro- a polypeptide chain referred to as the primary structure tein. Amino acids with nonpolar hydrocarbon side (Figure 11.5). The nature and character of a protein is de- chains (valine, leucine, phenylalanine, and tryptophan) termined to a considerable extent by the total number are hydrophobic (do not interact with water). These side and sequence of amino acids in this chain. Functional chains point outward on the external surface of the pro- proteins are generally composed of polypeptides that teins embedded in hydrophobic membrane lipids. In are folded or coiled into a three-dimensional structure; water-soluble proteins present in cytoplasm, these hy- this is the conformation that a functional polypeptide drophobic groups extend inward. Correspondingly, the ultimately assumes. side chains of hydrophilic amino acids (aspartic acid, The folding pattern is determined largely by the glutamic acid, lysine, arginine, and histidine) extend out- amino acid sequence, and the formation of a functional ward to the aqueous environment. protein can potentially occur by unassisted self-assem- Typically, the twisting and coiling of polypeptide bly. However, there is evidence suggesting that random chains results in the formation of a secondary struc- unassisted assembly can result in a structure that is non- ture that is either helical in form, called an a-helix, or a functional (Box 11.2). Consequently there are preexist- flat arrangement, designated a b-sheet. The α-helix is a ing proteins in the growing cell, termed chaperones, linear polypeptide that is wound like a spiral staircase which act to prevent incorrect molecular interactions (Figure 11.7). It is held together by hydrogen bonding that would lead to nonfunctional secondary or tertiary between the amine hydrogen of one amino acid and an structures. These chaperones assist in forming but are oxygen from another amino acid. The hydrogen bond- not a part of the final functional protein. Generally the ing occurs as the polypeptide is formed and leads to a folding of peptides to a functional secondary structure stable helical structure. Glutamic acid, methionine, and is energetically favorable; that is, it occurs without en- alanine are the strong formers of the α-helix. ergy input. β-sheets (see Figure 11.7) are formed when two or more extended polypeptide chains come together side Structural Arrangements of Proteins by side so that regular hydrogen bonding can occur between the amide (NH) and the carbonyl (C==O) of The types of bonding that occur between internal areas an adjacent-chain peptide backbone. Addition of more (domains) of a polypeptide, giving the protein a second- polypeptides results in a multistranded structure. The

The tertiary structure is formed by the folding of the secondary structure. The Histidine flavodoxin molecule (a protein involved in Lysine the N2-fixing reaction) has… HC NH + The primary structure of a peptide is the CH NH3 …four α-helices and… sequence of amino acids in its chain, the H2C N + order in which they are joined together H CH2 during the polymerization reaction. Tyrosine CH2 OH CH2 Hydrogen CH2 Nitrogen CH Carbon 2 CH2 Oxygen

Flavodoxin

…five parallel β-sheets.

Hydrogen bond

Figure 11.5 Formation of a peptide The secondary structure is the way in Formation of the primary, secondary, and which the peptide chain is twisted or coiled tertiary structures of a peptide. Several into shapes held together by hydrogen bonds. Shown here is the α-helix. Another type of amino acid side chains (R groups) are secondary structure is the β-sheet. indicated by shading.

amino acids valine, tyrosine, and isoleucine are common β-sheet formers. In many proteins, the polypeptide chain is bent at specific sites and folded back and forth, resulting in the tertiary structure (see Figure 11.5). Although the α-helices and β-pleated sheets contribute to the tertiary structure, only parts of the macromolecule usually have these secondary structures; large regions consist of structures Perry Staley Lory Microbiology 2/e unique to a particular protein. The protein shown is Sinauer Associates flavodoxin, an electron transport protein in sulfate re- Elizabeth Morales Illustration Services Ionic bonds occur between – + ducing bacteria. The folding of a polypeptide chain to Figure COO11.05.eps H Date3N 02-16-07 charged R groups. form a discrete compact protein molecule requires bends in the chain of amino acids that reverse the direction; Two nonpolar groups interact hydrophobically. Figure 11.6 Types of bonds between areas of a peptide chain Hydrogen bonds form Several types of bonds and interactions form between the H OH between two polar groups. domains of a polypeptide chain. These determine the shape of the protein.

Perry Staley Lory Microbiology 2/e Sinauer Associates Elizabeth Morales Illustration Services Figure 11.06ADJ.eps Date 02-16-07 Assembly of Bacterial Cell Structures 297

α (A) α-helix Subunits Hydrogen bonds between the amine hydrogen and the carbonyl oxygen of the peptide backbone hold together the coils of the helix.

β Subunits Hydrogen bond Heme (B) β-sheet Figure 11.8 Quaternary structure The quaternary structure of hemoglobin is composed of two α− and two β−polypeptide chains or subunits. Note the Hydrogen bonds hold compact symmetry of the structure. together neighboring parallel strands in the sheet. The relative mass (Mr) of a protein is the total molecular mass of the assembled subunits. Some representa- tive proteins, their relative masses, and the number of subunits that form each protein are listed in Table 11.3.

SECTION HIGHLIGHTS The characteristic properties of a protein are dictated in large part by its primary amino acid Figure 11.7 Secondary structures sequence. Folding of the polypeptide chain Hydrogen bonds hold a polypeptide chain in an α-helix or into the native or mature state may proceed β-sheet configuration. These are important determinants of unaided, or may require protein chaperones secondary structure. to aid in forming the correct secondary, tertiary, and quaternary structures. these are called b-bends. Glycine and proline are frequently present in β-bends. A β-bend is a tight loop that results when a carbonyl group of one amino acid forms Perry Staley Lory MicrobiologyRelative 2/e mass and a hydrogen bond with the NH group of another amino Sinauer Associates 2 TABLE 11.3 number of subunits in acid three positions down the polypeptide chain. This Elizabeth Morales Illustration Services Figure 11.08.eps Date 02selected-16-07 proteinsa results in the polypeptide folding back on itself. Many proteins are composed of more than one Protein Relative Mass (Mr) Subunits polypeptide chain. These subunits are adjoined by non- covalent bonding (hydrogen bonding, hydrophobic in- Ferredoxin 6,500 1 teraction,Perry Staley orLory ionic Mic robiologybonding). 2/e The joining of these sub- Ribonuclease 13,700 1 Sinauer Associates units forms what is termed a quaternary structure Lysozyme 14,388 1 Elizabeth Morales Illustration Services Luciferase 80,000 2 (FiguFigurere 11.07.e 11.8ps). The individual Date 02-16-07 polypeptide subunits that make up a quaternary structure can be either the same Hexokinase 100,000 2 or a polypeptide of different composition or size. A clas- Lactate dehydrogenase 223,000 4 sic example of a quaternary structure is the oxygen-car- Urease 483,000 5 rying protein hemoglobin. This protein is composed of NADH dehydrogenase 550,000 13 four polypeptides—of two different kinds—and is aThe average molecular mass of an amino acid residue in a pro- termed an α β tetramer. The individual subunits in a tein is 110 Da, or daltons (average molecular weight less a mol- 2 2 ecule of H O), and an “average” protein molecule has about 330 quaternary structure are proteins which themselves have 2 amino acid residues, or Mr 36,000. A dalton is defined as one- typical secondary and tertiary structures. twelfth of the mass of a carbon atom.

Protein Conformation

Classic experiments in the early agents and exposure to air results in trated in Figure 11.6. This sort of in 1960s by Christian B. Anfinsen led a random reassembly and an inac- vitro study apparently does not to the hypothesis that the sequence tive enzyme. However, exposure of completely reflect the conditions in and nature of the amino acids in a the denatured protein to trace lev- the cytoplasm of a cell. The levels of polypeptide chain were the major els of β-mercaptoethanol promotes polypeptide required for test tube contributing factors to the folding the proper rearrangement of the refolding are much higher than and final conformation of a protein. —S—S— cross links and results in a would occur in vivo, and the phys- This was based on experimentation reformation of the active enzyme. iochemical conditions of the in vitro with bovine pancreatic ribonucle- This and other in vitro experiments experiments do not exist in the liv- ase, a small protein made up of 124 suggest that proper folding might ing cell. In reality, studies in vitro amino acids. The secondary struc- be an inherent property of the pri- that mimic in vivo physiological ture of this protein is maintained by mary structure of polypeptides. conditions result in misfolding or four disulfide bridges. Denaturation Thus folding to secondary and terti- aggregation of the polypeptide. can be accomplished by cleaving ary structures would be promoted This misfolding is uncommon in these covalent disulfide linkages, by the proper spacing of amino vivo except with mutant proteins which reduces the —S—S— link- acids whose side chains could inter- or protein folding at elevated age to —SH HS— as follows: act with counterpart amino acids temperatures.

Heating and treatment with a C If denatured RNase is then chemical reducing reagent to returned to native conditions, 124 break disulfide bonds disrupts it will spontaneously refold to the native conformation, its native conformation. denaturing the protein. N 1 1 1 N N 26 26 110 26 72 72 SS SS SS SS 95 84 84 65 84 65

C C 124 124 95 110 40 95 110 SS SS SS SS 72 40 58 40 58 65 Disulfide bonds

58 Native RNase Denatured RNase Native RNase The secondary structure, and thus the activity, of bovine pancreatic ribonuclease is maintained by disulfide bonds between cysteine residues.

Disruption of the sulfide alters elsewhere in the polypeptide chain. Studies in recent years affirm the conformation of the protein The bonding producing the func- that there are two classes of pro- and leads to loss of enzymatic activ- tional structures would be formed teins associated with the proper ity. Removal of the denaturing by the side chain interactions illus- folding of polypeptides in a cell.

Perry Staley Lory Microbiology 2/e Sinauer Associates Elizabeth Morales Illustration Services Figure Box 11.02.eps Date 02-16-07 Assembly of Bacterial Cell Structures 299

BOX 11.2 Continued

One class consists of the conven- may also interact with individual • They promote the assembly of tional enzymes that catalyze specif- proteins to promote protein-pro- pili and of the carboxysome in ic isomerization reactions that tein arrangements that yield qua- photosynthetic organisms. result in proper polypeptide con- ternary functional structures. • They prevent protein denatura- formations for proper folding. Among the roles that protein chap- tion during environmental stress, The second class of proteins erones play are the following: such as elevated temperature. comprises the chaperones that sta- • They prevent folding of secretory The secondary structure, and thus bilize unfolded or partially folded proteins before translocation. the activity, of bovine pancreatic structures and preclude the forma- • They are involved with the ribonuclease is maintained by tion of inappropriate intra- or inter- assembly of bacterial viruses. disulfide bonds between cysteine chain interactions. A chaperone residues.

11.4 Export of Proteins proteins into or across the cytoplasmic membrane. Sig- Approximately 20% of the polypeptides synthesized by nal peptides are usually cleaved off during or following bacteria are inserted into the cytoplasmic membrane or the membrane translocation process. There are two dis- translocated across the membrane and into the cell en- tinct types of signal sequence found on pre-secretory velope or beyond. In E. coli, this number is estimated to proteins, depending on whether they are recognized by be about 800 distinct proteins. In most cases, the secre- the Sec or the TAT pathway. A somewhat different sig- tion or targeting of a protein to its extracytoplasmic des- nal sequence is found on pre-lipoproteins, a class of tination requires the activity of one of several complex outer membrane proteins that are modified by cova- secretion machineries. Secreted proteins move into or lent lipid attachment during export. through the cytoplasmic membrane via two distinct Each signal sequence consists of three distinct regions: pathways, referred to as the Sec and TAT pathways. the n, h, and c regions. The leading or N-terminal region Each of these pathways will be discussed separately. of the signal sequence is termed “n” and is polar, with a Once transferred to the external side of the cytoplasmic net positive charge (Figure 11.9). The middle region, membrane, the proteins can be released to the cell exte- termed “h,” has at least 10 hydrophobic amino acids and rior, as is the case for secreted proteins of gram-posi- is inserted into the membrane. The “c” region is also hy- tive bacteria, or into the periplasm, in the case of gram- drophobic and is recognized by one of two peptidases, negative bacteria. The presence of a second membrane called signal peptidases, which cleave the signal se- (the outer membrane) in gram-negative bacteria means quences from the secretory proteins. Signal peptidase I that additional machinery is required to move proteins (SPase I) is responsible for cleaving signal sequences from that have been released into the periplasm to the outer the majority of secreted preproteins utilizing the Sec and membrane or exterior of the cell. Alternatively, several TAT pathways, whereas signal peptidase II (SPase II) re- proteins from gram-negative bacteria can be secreted by moves signal sequences from precursors of lipoproteins. a one-step process, in which proteins synthesized in the A significant fraction of bacterial membrane proteins are cytoplasm are translocated to the exterior by machiner- not translocated, but instead are integrated into the cy- ies (types I to V, see subsequent text) that span the inner toplasmic membrane via the Sec pathway. These integral membrane, periplasm, and outer membrane. membrane proteins contain a sequence at their amino terminus that resembles a signal peptide and is not cleaved Signal Sequences off. Once in the membrane these sequences have a second function in anchoring the proteins in the lipid bilayer and The distinguishing characteristic of a subclass of secre- thus preventing further translocation. tory proteins is the presence of a signal sequence (sometimes called a signal peptide) of approximately 17 to 22 The Sec-Dependent Protein Secretion Pathway amino acids at the N-terminus of the pre-secretory protein. The signal peptide functions as a recognition sig- Secretion of most Sec-dependent pathway proteins in nal for the Sec or Tat machinery, directing the secretory bacteria is mediated by membrane-associated translo-

(A) Sec signal peptide Figure 11.9 The tripartite structure The “c” section is also a of signal peptides series of hydrophobic amino Generic structures of signal peptides The “n” section is composed acids. They are recognized by showing the n, h, and c regions. The The “h” section consists of polar, positively charged the signal peptidase that sites cleaved by signal peptidases are amino acids. This net positive of hydrophobic amino eventually cleaves the signal charge enables the leader to acids. It inserts into the peptide from the secretory between –1 and +1. The amino acid at enter the cell membrane. cell membrane. protein. position +1 is the first amino acid of the mature protein. One example is provid- Cleavage site ed from E. coli for each class of secreted n-regionh-region c-region Mature proteins utilizing the Sec (A), TAT (B), and lipoprotein (C) export pathways. Consensus R/K ++ A A Note that the lengths of the n, h, and c regions are variable, so the specific examples don’t necessarily match the –3 –1 +1 consensus as depicted. OmpA, the –1 +1 outer membrane protein A; DmsA, the Example OmpA MKKTAIAIAVALAGFATVAQAAPKD… A subunit of dimethyl sulfoxide reductase; and Lpp, the Braun lipoprotein. For the TAT-secreted DmsA, the twin (B) TAT signal peptide Cleavage site arginines are underlined and the aster- n-regionh-region c-region Mature isk points to the basic amino acid in the c region that is responsible for prevent- Consensus R/K ++ R RxxxK/R A A ing the protein precursor from entering the Sec pathway. –3 –1 +1 * –1 +1 Example DmsA MKTKIPDAVLAAEVSRRGLVKTTAIGGLAMASSALTLPFSRIAHAVDSA…

A R/K ++ L G C Consensus

–3 –1 +1 –1 +1 Example Lpp MKATKLVLGAVILGSTLLAGCSSN…

case complexes. In E. coli, this complex is composed of 11.10A). The mRNA–ribosome–nascent polypeptide–SRP at least three proteins (SecY, SecE, and SecG) that form complex then docks to its receptor, FtsY, and perhaps to a hydrophobic channel spanning the cytoplasmic mem- another, as yet unidentified membrane-bound receptor. brane (Figure 11.10A). The complex also contains a These events facilitate the transfer of the pre-protein to bound protein, SecA, which provides energy for the the translocase complex. As protein synthesis proceeds translocation by hydrolyzing ATP. on the ribosome, the pre-protein is exported though the Depending on the amount of delay following the syn- translocase complex in an ATP-dependent process as- thesis of the secreted protein, two general routes for pro- sisted by the SecA protein. Many of the newly synthe- tein translocation via the Sec-type translocase complex sized proteins remain in the membrane and are not fur- are followed: cotranslational and posttranslational. In- ther processed. However, several types of membrane sertion of integral membrane proteins occurs almost ex- proteins with cleavable signal sequences are routed clusively cotranslationally. During cotranslational protein translocation, the sig- Figure 11.10 Secretion of proteins across L nalPerry sequence Staley Lory (or Mictherobiology N-terminal 2/e anchor sequence) emerg- Sinauer Associates i the cytoplasmic membrane ingElizabeth from Mortheales ribosome Illustrat ionattaches Services to a cytoplasmic chaper- (A) Post- and cotranslational secretion by the oneFigu calledre 11.09.e signalps recognition Date 03-06-07 protein, or SRP (see Figure Sec pathway. (B) The TAT translocation pathway.

(A) Sec secretion pathway

Secreted, folded protein Periplasm

1c Proteins become incorporated 2c SPase I removes the signal into the membrane and, in many peptide prior to completion cases, retain their signal peptides. SecY SPase I of secretion.

Cytoplasmic SecE SecG membrane

SecA 1b This complex is targeted to ATP 2b Secretion is initiated by the membrane, where the interaction with SecA, which nascent peptide–ribosome threads the proteins across a portion is transferred to the channel formed by the translocase complex and ADP SecYEG complex. synthesis of the protein is FtsY + completed. Signal Signal P Srp sequence i sequence Cytoplasm

Ribosome SecB

1a Secretion via the cotranslational pathway begins with association of the signal peptide, as it emerges from the ribosome, Ribosome with the Srp, followed by binding of the FtsY targeting 2a Proteins secreted by the posttranslational protein. pathway remain unfolded because of binding of the chaperone SecB to several sites on the polypeptide. The signal sequence and SecB direct the proteins to the translocase complex. (B) TAT secretion pathway

3 …followed by binding of TatA. TatA 6 Some proteins are 2 The twin-arginine forms a channel, through which the released to the signal binds to the TatC secreted protein traverses the periplasm, whereas component of the cytoplasmic membrane. others remain TatB/TatC membrane anchored to the complex… periplasmic face of H+ the cytoplasmic membrane. Periplasm TatB TatC TatA

Cytoplasmic membrane

Cytoplasm SPase I 4 The proton motive H+ force provides the energy for 5 After completion of translocation. translocation, SPase I cleaves the signal peptide. Perry Staley Lory Microbiology 2/e Additional Sinauer Associates subunit(s) Elizabeth Morales Illustration Services Figure 11.10.eps Date 03-20-07 Cofactor(s)

1 The precursor forms of the secreted proteins are synthesized in the cytoplasm. The proteins are folded and, if needed for their function, cofactors are inserted.

© 2007 Sinauer Associates, Inc. This material cannot be copied, reproduced, manufactured, or disseminated in any form without express written permission from the publisher. 302 Chapter Eleven through the Srp/FtsY pathway and SPaseI removes the zyme is transported into the periplasm. Translocation is signal peptide to yield the mature polypeptide. energized by the proton motive force (pmf) while main- During posttranslational protein translocation, the taining a sealed membrane. In a final step, signal pepti- secreted pre-protein (the protein with its signal se- dase cleaves the twin-arginine signal sequence to yield quence) is synthesized in the cytoplasm away from the the mature enzyme. SecYEG translocase complex. Several molecules of the Examples of proteins secreted by the TAT pathway soluble chaperone protein SecB bind to different portions include respiratory enzymes (formate dehydrogenase, of the pre-protein to prevent it from folding prior to ex- hydrogenase, TMAO reductase, DMSO reductase port (see Figure 11.10A). The unfolded protein binds to [DmsABC]) and certain enzymes needed for peptido- the translocase complex, initially by an interaction with glycan assembly. Related TAT secretion pathways are SecA. It is then threaded through a membrane channel found in Archaea and in the thylakoid membranes of formed by SecYEG and is exported. The pre-protein ex- plant chloroplasts. port is driven by ATP hydrolysis via SecA protein con- formational changes. Before secretion is completed, the Protein Targeting to the Outer Membrane signal sequence is removed by SPaseI. Examples of proteins secreted by the Sec pathway in- The outer membrane of gram-negative bacteria contains clude periplasmic binding proteins needed for solute up- a number of proteins, including porins and components take, periplasm-located proteins such as alkaline phos- of various transport systems; most of these span the phatase, cell envelope biosynthetic enzymes, and outer outer membrane bilayer. The outer membrane also con- membrane proteins, including porins and lipoproteins. tains lipoproteins that are anchored in the inner leaflet Given the demand for membrane processing, there are by their attached lipids. All outer membrane proteins an estimated 500 translocase complexes in the cytoplas- utilize the Sec machinery for their translocation across mic membrane of a gram-negative bacterium. the cytoplasmic membrane; however, they also require additional transport machineries to reach their final des- The Twin-Arginine (TAT) Protein Secretion tination in the outer membrane. Pathway Lipoproteins in gram-negative bacteria are transported across the cytoplasmic membrane by the Sec ma- TAT secretion systems export fully assembled protein chinery (Figure 11.11). When the pre-protein is translo- complexes across the cytoplasmic membrane. Such sys- cated to the periplasmic face of the inner membrane, two tems are present in many but not all bacteria and mainly fatty acids are attached via glycerol to the cysteine thiol export enzymes involved in cell respiration. These se- group located at the signal peptide cleavage site (see Fig- creted complexes generally contain three or more ure 11.9), and the signal peptide is cleaved by a lipopro- polypeptide types along with their associated cofactors. tein-specific signal peptidase (SpaseII). The N-terminal For example, the E. coli enzyme, formate dehydroge- amino group of cysteine is further modified by addition nase-N, contains two b-type hemes, four 4Fe-4S clusters, of another fatty acid and the lipoprotein is then exported and a molybdopterin cofactor. Once translocated, the en- to the outer membrane by the so-called LolABCDE ma- zyme complex is anchored in the cytoplasmic membrane chinery. First, the newly formed lipoprotein is removed with the catalytic site exposed to the periplasm. from the inner membrane by the LolBCD complex, and TAT systems are so named by the presence of a cleav- transferred to the periplasmic shuttling protein LolA. able “twin arginine”–containing motif, RRxxxK/R, lo- This is an energy-dependent process and requires ATP cated at the N-terminus of one of the secreted polypep- hydrolysis by LolD. Interaction of the LolA–lipoprotein tides (see Figure 11.9). Like the signal sequence involved complex with LolB at the periplasmic face of the outer in Sec-type secretion, the twin arginine–containing mo- membrane results in the insertion of the lipoprotein into tif provides a recognition signal that exclusively targets the membrane. About half of the lipoproteins become the protein complex to the TAT secretion complex located covalently attached to the peptidoglycan, and contribute in the cytoplasmic membrane (Figure 11.10B). This com- to the integrity of the cell wall (see Figure 4.55). A sub- plex is composed of three proteins, TatA, TatB, and TatC. class of lipoproteins is retained in the cytoplasmic mem- Prior to secretion, specific cytoplasmic chaperone(s) as- brane; these proteins are distinguished by an aspartic sist in enzyme assembly and cofactor insertion following acid residue next to the lipid-modified cysteine in the polypeptide synthesis. The twin-arginine motif targets mature protein (see Figure 11.9). The aspartic acid pre- the assembled multisubunit enzyme to the inner surface vents interaction of the lipoprotein with the LolB, C, and of the cytoplasmic membrane, where it interacts with D components of the transport machinery. TatC and TatB. This pre-secretory enzyme–TatC–TatB The mechanism of assembly of other outer membrane complex then combines with TatA. Following this inter- proteins, many of which function as multimeric chan- action, a channel opens in TatA, through which the en- nels, is not completely understood. The newly synthe-

8 …which inserts the Outside lipoprotein into the of cell 7 LolA shuttles inner leaflet of the the lipoprotein outer membrane. to LolB… Outer

membrane 6 …and is C transferred to LolA. Periplasm Lipoprotein C C LolB LolA Empty Sec YEG SPase II LolA Fatty acids C C C C Cytoplasmic LolC LolE membrane LolD Cytoplasm

Lipoprotein ATP ADP + Pi precursor 2 . . . where it is 3 . . . followed by 4 A third fatty acid is 5 The lipoprotein modified by cleavage of the added to the newly interacts with an 1 The Sec machinery addition of two signal peptide by created amino ABC export complex directs the translocation fatty acids to a SPase II. terminus. (LolCDE). . . of lipoprotein precursor cysteine. . . to the cytoplasmic membrane. . .

Figure 11.11 Transport of lipoproteins to the outer membrane Transport of lipoproteins to the outer membrane involves the Lol machinery and special modifications of the amino terminus.

sized pre-proteins are first translocated through the Sec have evolved specialized machineries of varying com- machinery, followed by removal of their signal peptide plexity to accomplish extracellular secretion. Several dif- by SPase I (Figure 11.12). It is likely that two chaperones, ferent secretion routes can coexist in a single bacterial Skp and SurA, are responsible for preventing aggrega- cell and each protein must be targeted to the correct one. tion of these proteins and shuttling them to the outer For proteins to be recognized by their cognate secre- membrane insertion complex, which consists of YaeT tion machinery they possess a signature (an “ad- (sometimes called Omp85), YfgL, YfiO, and NlpB pro- dress”)—typically a short sequence or a structural mo- teins.Perry StaHereley Lorythe proteins Microbiology assemble 2/e into functional integral tif that is part of the polypeptide chain. outerSinauer membrane Associates proteins. Here we review the major extracellular secretion path- Elizabeth Morales Illustration Services ways of gram-negative bacteria. They have been num- Figure 11.12.eps Date 03-20-07 Extracellular Secretion by Gram-Negative bered type I-V, based on the order of their discovery. Bacteria TYPE I SECRETION PATHWAY This secretion pathway A number of bacterial proteins are not retained in any of utilizes the so called ABC machinery—named after its the cellular compartments but are instead secreted from ABC (for ATP-binding cassette) components (Figure the cell. For gram-positive bacteria, which have a single 11.13). The type I machinery consists of a complex of cytoplasmic membrane, secretion of a protein across this three different proteins, each located in a different com- membrane is sufficient for its release into the surround- partment of the cell. The ABC component is located ing medium, unless the protein is modified such that it within the cytoplasmic membrane, and is linked to an becomes attached to cell wall components. In contrast, outer membrane protein (OMP) through a periplasmic secretion from gram-negative bacteria requires transfer bridge—the so-called membrane fusion protein (MFP). across two membranes. Various gram-negative bacteria The type I secretion complex assembles only after initial

© 2007 Sinauer Associates, Inc. This material cannot be copied, reproduced, manufactured, or disseminated in any form without express written permission from the publisher. 304 Chapter Eleven 3 A complex of YaeT, NlpB, YfgL and YfiO directs the insertion of the proteins Outside into the outer membrane. Figure 11.12 Transport of proteins to the outer of cell membrane YaeT Outer membrane proteins enter the periplasm via SecYEG. Outer membrane Chaperones (SurA, Skp) transfer the newly synthesized pro- NlpBYfgL YfiO tein to an outer membrane protein complex, which inserts the protein into the outer membrane. Periplasm

SurA Skp 2 Transport across the periplasm involves the SecYEG SPase I chaperones SurA and Skp.

Cytoplasmic membrane

Cytoplasm The Sec machinery translocates 1 the secreted proteins across the cytoplasmic membrane with a concomitant cleavage of their Secreted signal sequences. protein

Figure 11.13 The type I, IV, and V secretion pathways Plant cell cytoplasm Three of the five pathways used by gram-negative bacteria to secrete proteins to the outside of the cell. Plant cell membrane

VirB2 Passenger domain Outside VirB5 of cell

Outer OMP membrane

Periplasm VirB7 Perry Staley Lory Microbiology 2/e β-domain Sinauer Associates EVlizabethirB9 Morales Illustration Services MFP Figure 11.11.eps Date 03-06-07 Degradation VirB10 SecYEG β-domain VirB8

Cytoplasmic ABC membrane VirB4 VirD4

Cytoplasm ATP VirD2 VirB11 ADP Passenger Protein + T-DNA domain Pi DNA–protein complex

Type I Type IV Type V

Perry Staley Lory Microbiology 2/e Sinauer Associates Elizabeth Morales Illustration Services Figure 11.13.eps Date 03-06-07 Assembly of Bacterial Cell Structures 305 interaction of the secreted protein with the ABC compo- Among the many secreted proteins that utilize the type nent, which then triggers the assembly of the rest of the I pathway are the hemolytic protein of E. coli, the secreted machinery. An active type I secretion apparatus consists proteases of Pseudomonas aeruginosa and Erwinia chrysan- of a dimer of the ABC component plus two trimers: one themi, the adenylate cyclase toxin of Bordetella pertussis, of Mfp and one of Omp. The energy for protein export and the iron-sequestering protein of Serratia marcescens. is provided by ATP hydrolysis, which is carried out by the ABC component. TYPE II SECRETION PATHWAY Secretion via the type The proteins secreted by this pathway can be as large II secretion pathway is a two-step process. Proteins are as 1,000 amino acids. Because they do not utilize either initially synthesized with an N-terminal signal peptide, the Sec or TAT pathway, they lack a cleavable N-termi- which targets them for secretion via the Sec pathway and nal signal peptide. The signal for routing these proteins results in a transient periplasmic localization (Figure through the type I secretion pathway is usually located 11.14). In the periplasm, these proteins fold into their at their C-terminal end and is not cleaved during secre- final conformation and enter the secretion pathway that tion. Many of the secreted proteins have distinctive will transport them across the outer membrane. The ma- glycine-rich repeats—G-G-x-G-X-D-x-x-x (where x is any chinery of type II secretion is referred to as the general amino acid)—that specifically bind calcium. Given the secretory apparatus (GSP). The precise mechanism of large size of the secreted proteins that utilize this path- protein transport by this apparatus is not well under- way, they are transported in an unfolded state and then stood. One of the proteins of the GSP machinery, GspE, are folded immediately following their exit. It is likely has a nucleotide-binding domain and probably provides that calcium is required for their correct refolding. energy for secretion by hydrolysis of a nucleotide, pos-

PilY

PilA

Protein

Outside of cell PilY

Outer GspD PilQ membrane GspS

Periplasm GspG Protein PilA GspH GspC GspI

GspJ SecYEG PilV PilE GspM Cytoplasmic GspF GspL PilC membrane

Cytoplasm GspE PilB PilT

Type II secretion Type IV pilus

Figure 11.14 The type II secretion pathway and the type IV pilus A comparison of the structure and components of the type II secretion apparatus and the type IV pilus.

Perry Staley Lory Microbiology 2/e Sinauer Associates Elizabeth Morales Illustration Services Figure 11.14.eps Date 03-06-07 306 Chapter Eleven sibly ATP. GspE is found in the cytoplasmic membrane, machinery to target the secreted proteins directly into where it associates with two other membrane proteins, eukaryotic cells. Typically, bacteria adhere to the target namely GspL and GspF. A filamentous structure—con- cells, bringing their surfaces into close proximity. The sisting largely of GspG plus a few molecules of GspJ, external tip of the secretion apparatus is inserted into the GspI, and GspH—spans both the inner and outer mem- host plasma membrane, allowing the secreted protein to branes and connects the cytoplasmic membrane compo- be delivered directly into the eukaryotic cell without nents with a ring-like structure of assembled GspD ever being released into the surrounding medium. The monomers in the outer membrane. This filament is re- proteins are “injected” into the target cell cytoplasm, ferred to as pseudopilin, because it resembles the type with the bacterium being the syringe and the secretion IV class of pili (see subsequent text). apparatus the needle. The complete type III secretion The secreted proteins fold into their final conforma- machinery, sometimes referred to as an “injectosome,” tion in the periplasm, and in some cases, such as cholera consists of 20 components that span the cytoplasmic toxin, they assemble into their final multimeric com- membrane, the periplasm, and the outer membrane. The plexes prior to release from the cell. Because the type II secretion signals that target proteins to the type III secre- secretion machinery must differentiate between proteins tion pathway are approximately 20 amino acids long that are destined to remain in the periplasm and those and are located at the N-termini of the secreted proteins. that are to be secreted, the latter proteins must contain They consist of an equal mix of hydrophobic and specific extracellular secretion signals. However, the pre- charged amino acids and include several serines. The cise sequence or structural motifs that define these sig- signals are not cleaved during secretion. nals have not yet been identified. Several components of the type III secretion appara- The type II secretion pathway is one of the major tus are similar to components of a bacterial flagellum, mechanisms utilized by bacterial pathogens to secrete and the flagellum plays a limited role in secretion. The protein toxins, which can affect organs that are located flagellum monomers (FliC) that are added to the tip of far from the site of colonization (see Chapter 26). the filament during flagellum assembly and the regula- One class of appendages, the type IV pili on the sur- tory protein FlgM (see Chapter 13) are secreted through face of certain gram-negative bacteria, resembles the as- the channel formed by the basal body. By an analogous sembled type II secretion apparatus (see Figure 11.14). mechanism, the type III secretion machinery of a Yersinia The type IV pili are used primarily as adhesins by cer- species exports its toxins to a targeted cell. tain pathogenic bacteria to attach themselves to solid surfaces, including tissues of infected humans (see TYPE IV SECRETION PATHWAY The type IV protein Chapter 26). The main feature of these pili is the pres- secretion system is evolutionarily related to the bacter- ence of a long filament—a multisubunit structure—an- ial conjugation machinery. It resembles the type III se- chored in the cell envelope. Many of the components cretion pathway in its ability to transfer proteins directly of type IV pili are similar in amino acid sequence to into another bacterium or into a eukaryotic cell. The type those of the type II secretion apparatus, and therefore IV pathway is also used by a handful of proteins, includ- they probably function in an analogous manner (see Fig- ing the Bordetella pertussis toxin, which are secreted into ure 11.14). The type IV pili do not secrete proteins but the medium instead of being transferred into the cyto- instead direct the export and polymerization of the pilus plasm of another cell. subunit (the PilA protein). In contrast, the protein ho- One of the most extensively studied type IV secretion molog to PilA in the type II secretion system, GspG, systems is in Agrobacterium tumefaciens, a plant pathogen forms the pseudopilin structure and serves as a channel that transfers several proteins into the cytosol of cells for the secretion of a number of soluble proteins. of the infected plant. One of the transferred proteins, VirD2, carries a covalently attached single-stranded TYPE III SECRETION PATHWAY Although this pathway DNA (T-DNA) from A. tumefaciens into the plant cell, is common among mammalian and plant pathogens, a where the T-DNA induces tumor formation (see Chap- closely related mechanism that directs the assembly of ter 16). The assembled type IV apparatus of A. tumefi- bacterial flagella has a much wider distribution among ciens (see Figure 11.13) is a multisubunit structure span- gram-negative bacteria. Type III secretion systems have ning the inner and outer membranes and consisting of been identified in Yersinia spp., Pseudomonas aeruginosa, a secretion channel and a pilus filament. The VirB2 pro- Shigella flexneri, Salmonella typhimurium, Chlamydia spp., tein is the major component of the pilus structure, along and certain pathogenic E. coli, and in the plant pathogens with a few alternating VirB5 proteins. The secretion or symbionts Pseudomonas syringae, Erwinia, Xanthomonas channel is formed from a group of cytoplasmic mem- spp., and rhizobia. brane proteins, VirB4, VirB6, VirB8, and VirB11, and two The unique feature of the type III pathway for additional proteins, VirB7 and VirB9, which form an an- host/pathogen interactions is the ability of the secretion chor for the pilus in the outer membrane and connect it

© 2007 Sinauer Associates, Inc. This material cannot be copied, reproduced, manufactured, or disseminated in any form without express written permission from the publisher. Assembly of Bacterial Cell Structures 307 to the cytoplasmic components of the apparatus. VirB4 proteins are employed, as are TAT-like systems, in certain and VirB11 are inner membrane proteins that are AT- eukaryotic processes. The origin of these processes is ar- Pases and provide the energy for the transfer process. guably prokaryotic. In addition to A. tumefaciens and B. pertussis, a number of intracellular parasites (bacteria that grow inside infected host cells), including Legionella, Brucella, and SECTION HIGHLIGHTS Bartonella species, utilize the type IV secretion pathway The synthesis and folding of cytoplasmic pro- to deliver proteins into the cytoplasm of their host cell. teins generally occurs spontaneously. How- These proteins facilitate bacterial survival and growth ever, the synthesis of proteins destined to the within the host cell. cell envelope often requires chaperone proteins. Movement of these proteins into or TYPE V SECRETION PATHWAY This is the simplest across the cytoplasmic membrane generally of the gram-negative extracellular secretion pathways and does not use any energy for protein transport. The requires the assistance of specialized protein type V secretion pathway is sometimes referred to as secretion machinery, usually the Sec or TAT autotransporter secretion. As can be inferred from this apparatus. Proteins destined for the outer name, the proteins promote their own transfer across membrane or beyond require additional spe- the outer membrane. Proteins secreted by this mech- cialized machinery; these are termed type I to anism are relatively large, and share a similar overall V secretion pathways. Regardless of the mech- domain organization. They are synthesized with an N- anism used, protein secretion involves the terminal signal peptide followed by the passenger do- recognition of specific targeting signals on the main, which specifies the protein’s extracellular func- secreted protein and energy in the form of tion. At their carboxy-termini, these proteins contain ATP or ion gradients to drive the movement. another domain, the b-domain, which forms a pore in the outer membrane. The secretion of these proteins is initiated by the Sec pathway, which cleaves the signal peptide and releases the protein into the periplasm. 11.5 Structure and Function of Cell During the next stage of export, the β-domain inserts Appendages into the outer membrane and forms a barrel-like structure, through which the passenger domain is then Flagella and pili are composed of multiple protein sub- threaded. Some proteins secreted by this pathway re- units assembled into macromolecular structures outside main associated with the outer surface of the cells, the cytoplasmic membrane. Their synthesis, assembly, whereas others are released following cleavage of the and function are described below. bond between the passenger and β-domains. Several important secreted proteins of pathogenic Bacterial Flagella gram-negative organisms utilize the type IV secretion pathway. These include the antibody-specific protease Flagella are specialized cellular appendages that allow of N. gonnorhoeae, the protease of H. influenzae, and the directed cell movement under appropriate environmen- vacuolating toxin produced by H. pylori. A number of tal conditions. They are multiprotein assemblies that surface adhesins—proteins that mediate binding of bac- span the cell envelope and extend several cell lengths teria to tissues, such as the protractin of B. pertussis and beyond the cell surface. Some types of microbes possess the BabA adhesin of H. pylori—are also autotransporters. a single flagellum located at one pole of the cell (Pseudomonas, for instance), while others, E. coli, for ex- Protein Secretion in Lower Eukaryotes ample, possess multiple flagella distributed uniformly about the cell (see Chapter 4). Flagellar movement is con- In fungi, the production and secretion of enzymes and trolled by the cell in response to environmental stimuli other proteins is fundamentally different from that in bac- that allow the cell to seek out nutrients and to avoid toxic teria. Fungi, of course, are eukaryotes, and fungal proteins materials that create unfavorable conditions for survival. are synthesized at the rough endoplasmic reticulum; they are then transported in vesicles and enter the Golgi appa- STRUCTURE A flagellum consists of three major parts: ratus. Vesicles then bud from the Golgi and are trans- • A long filament, which extends into the surround- ported to the inner surface of the cell membrane. At the ing environment site of secretion, the membrane of the protein-containing vesicle fuses with the cytoplasmic membrane and the • A hook, a curved section connecting the filament to protein is released to the outside. Interestingly, SRP-type the cell surface

Figure 11.15 Flagellar structure (A) (B) (A) Model of a flagellar cross sec- Filament tion showing the location of the rings and the Mot proteins in the cell envelope of a gram-negative bacterium. (B) Electron micrograph showing the basal ring structure of Hook a gram-negative flagellum. B, cour- Outside of cell tesy of David DeRosier, Brandeis L-ring University. Outer membrane Drive shaft

Peptidoglycan P-ring Cytoplasmic MS-ring membrane Motor C-ring subunits Protein Inside 45 nm export of cell apparatus The flagellum is rotated by complex motor proteins secured in the plasma membrane.

• A basal structure, which anchors the flagellum and force (pmf; see Chapter 8). Some bacterial species use hook into the cell wall and membrane by special protons, while others, especially marine microorgan- disk-shaped structures called plates or rings isms, use sodium ions to drive rotation. This energy is imparted to the Mot proteins (MotA and MotB), which The basal structure for a typical gram-negative bac- are embedded in the cell membrane and act as a motor terium contains a rod structure plus L, P, MS, and C rings that drives flagellar rotation. The rate of rotation varies that associate with cell envelope components (Figure among species but is in the range of 200 to more than 11.15) to anchor it to the cell. Several of the rings (MS 1000 rpm. Motility requires a major expenditure of en- and C) function as parts of a molecular motor that, along ergy, but the result is immensely useful because it allows with the motor shaft protein (FlgG), spins or rotates the the cell to “swim” to a place where conditions for growth hook and filament assembly in a clockwise or counter- are more favorable. clockwise direction. This action propels the cell in three- The flagella of enteric bacteria have been studied in- dimensional space in response to external signals includ- tensively. More than 50 genes involved in motility have ing chemicals, light, and magnetism. The motion, been identified in Salmonella typhimurium and E. coli. The sometimes called “swimming,” confers selective advan- assembly and function of these structures include the tage to motile cells in seeking out environmental niches favorable for growth/survival. The hook is composed of a single protein type (FlgE) that transmits the rotary movement of the motor shaft FliD to the filament (see Figure 11.15). Several additional proteins are involved in joining it to the filament. FliC flagellar filament Perry Staley Lory Microbiology 2/e The is composedSi ofnau a erprotein Associates (FliC) called flagellin. With exceptions, bacteriaElizabeth generally Morales have Illustration Services a single type of flagellin subunit (CaulobacterFigure 11.16.eandps some Date 02-19-07 Vibrio spp. have two subunit types). Repeating subunits of flagellin are joined together to form the filament, which is helically wound and has a hollow core that is capped by FliD (Figure 11.16). The filament (and thus the flagellum) is about 20 nm in diameter. The filaments are not straight, and each has a characteristic sinusoidal curvature that varies with each species. Figure 11.16 Flagellar filament and flagellin subunits Top (left) and side (right) views of the flagellar filament MOVEMENT Flagellar rotation, and hence cell move- with flagellin subunits and cap structure. Courtesy of ment, requires energy; it is fueled by the proton motive Keiichi Namba.

genes responsible for producing the flagellin subunit, 2 A capillary containing a maltose hook, basal body structure with associated motor pro- solution is dipped into the suspension of cells, creating a teins, and additional genes that regulate flagellar bio- 1 An empty capillary is dipped nutrient gradient. The cells are into a suspension of bacterial genesis. Another set of genes encode proteins making attracted to the maltose by cells. The cells (dots) remain positive chemotaxis, as shown by up the sensory system that detects environmental sig- evenly distributed. nals and, in response, directs the flagellar motor to an increased turbidity near the opening of the capillary. change the direction of rotation. The functions of these structures in signal gathering and processing are described below.

ASSEMBLY The synthesis and assembly of flagella is controlled temporally (time of event) and spatially (location in the cell); these processes are initiated by the induction of gene expression in response to environmental conditions. The 50 or so flagellar (fla) genes needed for this to occur are divided into three groups called the “early,” “middle,” and “late” genes based on the timing of their expression. The early genes encode key regula- tory proteins which, upon induction, direct transcrip- tion of the middle genes, followed by the late genes. Control of gene expression allows for the synthesis of certain proteins before others, so that the assembly of Figure 11.17 Chemotactic capillary assay polypeptides into a mature flagellum occurs in the An illustration of the capillary chemotactic effect. proper sequence. The middle genes encode parts of the basal body structure and hook. The late genes encode additional proteins needed for motor assembly, filament to propel themselves into areas where organic nutrients assembly, and chemosensory proteins that control fla- are concentrated. This phenomenon, termed chemotaxis, gellar movement. Several additional fla genes encode can be illustrated with a capillary technique. If a glass chaperone proteins needed to assist the structural pro- capillary containing an organic energy source such as teins to their destinations. maltose is placed in a suspension of heterotrophic bac- Assembly of a new flagellum occurs in several highly teria, the bacteria will accumulate at the tip of the cap- regulated stages. The process begins with synthesis and illary from which the nutrient is diffusing (Figure 11.17). assembly of the MS-ring proteins at the cytoplasmic Chemotaxis has been thoroughly studied in enteric membrane surface (see Figure 11.15). This is followed by bacteria such as E. coli. These peritrichously flagellated the ordered synthesis and insertion of other proteins bacteria orient their flagella in a polar bundle during composing the basal body, including the motor shaft motility. The bundle is rotated counterclockwise during proteins; the C-, P-, and L-ring proteins; and the hook. forward movement, called running or smooth swim- Products of the late genes are then synthesized and as- ming. Running is disrupted when the flagella reverse to sembled. These include the addition of motor stator pro- the clockwise direction: the cell stops and then somer- teins (MotA and B) and motor switch proteins (FliM, N), saults—a movement referred to as tumbling. After a mo- and the buildup of the filament (FliC). A specialized ment, the flagella reverse again to rotate in a counter- transport apparatus composed of at least eight proteins clockwisePerry Staley direction,Lory Microbiology and the2/e organism begins another Sinauer Associates directs protein secretion and assembly of the mature runElizabeth for an Mor extendedales Illustrat period.ion Servi However,ces the direction of structure; this occurs by type III secretion. To reach their cellFigu movementre 11.18.eps is completely Date 02-16-07 random. When the organ- final destination at the growing tip of the filament, FliC ism is in the gradient of increasing nutrient, fewer tum- subunits are transferred through the hollow core of the bling events occur, allowing the organism to spend more basal body shaft, hook, and growing filament, where time in the favorable area (Figure 11.18). This phenom- they self-assemble into the helically wound structure. By enon, in which an organism effectively becomes directed a mechanism that is not well understood, filament toward a utilizable nutrient—an attractant—is positive growth, once it extends to several cell lengths, is ulti- chemotaxis. Negative chemotaxis—movement away mately terminated by the action of another filament pro- from toxic materials, called repellants—is also com- tein (FliD), which caps the tip (see Figure 11.16). monly exhibited by bacteria. Many utilizable organic nutrients serve as attractant TACTIC RESPONSES: CHEMOTAXIS, AEROTAXIS, AND molecules, causing positive chemotaxis. In E. coli, these PHOTOTAXIS Heterotrophic bacteria use their flagella would be various sugars and amino acids (Figure 11.19).

In a solution without a nutrient Figure 11.18 Runs and tumbles in chemotaxis gradient, cells tumble more This type of cell movement depends on the presence or frequently and have shorter runs. absence of a nutrient gradient. Tumble event Starting point

Run This type of movement is called a “random walk.”

In a nutrient gradient, cells exhibit positive chemotaxis, so runs are longer and tumbles less frequent. Tumble event Cells suppress tumbling when they sense a higher Run concentration of attractant. Starting point

High Low Concentration of nutrient

1 The first step in 2 The incoming signals are chemotaxis is signal then processed and 3 Phosphorylated reception and integrated by a series of CheY interacts with transduction. phosphorylation reactions. the motor switch...

Receptor demethylation 4 ...resulting in motor by CheB switching to change the Periplasmic Signals direction of flagellum binding proteins Receptors rotation. Dipeptides DppA Tap CheB Signal Maltose MalE Tar ATP ADP processing Asparate proteins

Perry StaRleyibose Lory MicrobiologyRbsB 2/e Trg CheA P Sinauer Associates ElizabethGala Morctoseales IllustratMglBion Services P CheW Y Y Figure 11.19.eSerineps Date 02-16-07 Tsr P Z Z O2 Aer

CheR Pi

Receptor methylation by CheR Receptor methylation Figure 11.19 Components of chemotaxis by CheR and demethylation by CheB modulates the An illustration of the chemotactic components signal response. and their locations in the cell. Their general oper- ation in signal reception and processing to control the direction of motor rotation is indicated.

Perry Staley Lory Microbiology 2/e Sinauer Associates Elizabeth Morales Illustration Services Figure 11.20.eps Date 02-19-07 Assembly of Bacterial Cell Structures 311

The molecules are bound to specific receptor proteins lo- was described in Chapter 4 (see Figure 4.68); it allows a cated either in the periplasmic space or the cell mem- cell to position itself at the specific wavelengths used for brane. For the amino acids serine and aspartate, binding photosynthesis. occurs at the specific membrane-bound chemoreceptors called Tsr and Tar, respectively. Other attractants are Bacterial Pili bound to specific binding proteins, located in the periplasmic space, that interact at the chemoreceptor (for Pili are hair-like appendages on many gram-negative example the maltose-MalE complex binds to Tar). E. coli bacteria and are involved in attachment, motility, and ge- possesses five different chemoreceptors; each exhibits netic exchange (see Chapter 4). Structures with similar specificity toward a subset of the attractants and repel- appearance can also be found as components of a num- lants. In the presence of their signal molecules, each re- ber of extracellular secretion machineries (see Figure ceptor transfers a message across the membrane to the 11.14). The various classes of pili have somewhat differ- CheA-CheW proteins, which, in turn, process and inte- ent structures, roles, and routes of assembly. Many of the grate all the newly received information from outside functions of pili involve interactions with solid surfaces the cell (i.e., the periplasm). Based on the information in a cell’s environment, including other cells. These in- accumulated, the flagella are then instructed to either ro- clude conjugation pili involved in DNA transfer between tate counterclockwise or clockwise (motor switching), cells (see Chapter 15), in cell adhesion, or in cell motil- thus directing cell movement toward attractants and ity, where the pilin subunits form a hair-like structure away from repellants. For E. coli, repellant molecules that pulls the bacteria along a solid surface or towards include ethanol, phenol, and certain heavy metals. The other bacteria. Regardless of type, pilus synthesis is an chemical signaling events that lead to directed cell move- energy consuming process and involves chaperone pro- ment are described in Box 11.3. Note that each chemore- teins to prevent misfolding of subunits prior to translo- ceptor molecule exists in the cell as a dimer complexed cation and assembly. Several examples follow. with two molecules each of CheA and CheW. Depend- One type of pilus involved in bacterial adhesion— ing on growth conditions, there may be over 10,000 re- termed the type I pilus—was described in Chapter 4 ceptor complexes per cell! (see Figure 4.71). E. coli strains that infect the human uri- It is noteworthy that in chemotaxis, both spatial gra- nary track use pili to attach to the surface of eukary- dients and temporal changes in the chemical environ- otic cells in order to colonize this niche (see Chapter 26). ment have an effect on cell movement. Adaptation of the These pili are assembled as shown in Figure 11.20. Al- organism to its environment also occurs in chemotaxis. though the P pili and type I pili (as well as related pili In the prolonged presence of the attractant substrate, the on E. coli and other Enterobacteriaceae) are similar in their rate of tumbling is less than it would be in the absence structure and assembly mechanisms, the primary de- of the attractant (see Box 11.3). This may be a mechanism terminants of their specificity for different human tis- whereby the organism can conserve energy, when it is sues are the unique proteins located on the tip of the fil- not needed, that would be needlessly spent on chang- ament (PapG in Figure 11.20), which recognize specific ing direction. receptors on the surface of the mammalian cells (see The simple behavior exhibited by bacteria during Chapter 26). motility is of great interest to biologists, because an un- Certain types of pili are involved in cell movement derstanding of this behavior at the molecular level may by a mechanism termed gliding. This process is distinct lead to a fuller understanding of more complex behav- from chemotaxis because the cell employs a retractable ioral responses of eukaryotic organisms, such as the pilus to propel itself across a solid surface (Figure 11.21). sense of smell. Bacteria like E. coli and S. typhimurium Myxobacter xanthus, for example, assembles multiple continue to provide excellent models for unraveling the type IV pili at the poles of the cell. The extended pili at highly sophisticated mechanisms needed for protein se- one pole attach to the solid surface in front of the cell via cretion, assembly, and cell behavior. an adhesion protein at the tip. Retraction of the pili then Flagella are also involved in the phenomena of aero- pulls the cell forward. This retraction occurs by depoly- taxis and phototaxis. The former process involves a spe- merization and removal of the pilin subunits that make cial receptor protein called Aer (see Figure 11.19), which up each pilus filament. Pilus extension sets the stage for detects oxygen and transmits a signal through the another cycle of cell movement. The cell can reverse di- chemotactic machinery to control flagellar motor rota- rection using the pili at the opposite pole, although the tion. Depending on the bacterial species, it may seek control of these gliding events is not understood. Type oxygen at low, intermediate, or high levels or avoid it IV pili are related to the type II secretion apparatus (see completely. Phototaxis occurs in many types of photo- Figure 11.14) and are used by many bacterial pathogens synthetic bacteria. This light-associated phenomenon to attach to mammalian cells during infection.

4 The subunits are added in 5 The growth of the the order of their location filament continues by on the filament: PapG first, polymerization of a followed by PapF, E, and K. large number of PapA The PapD chaperone is subunits and it released back to the terminates with the periplasm. addition of PapH. 3 …the outer membrane, where PapC serves as a PapG platform for the assembly PapF PapA of the filament. Outside PapE of cell PapK PapH

Outer PapC membrane

Periplasm Unfolded PapG, F, E, K, A, H 1 The various subunits of PapG PapF PapE PapK PapA PapH the filament are secreted as monomers by the Sec apparatus. Sec PapD

Cytoplasmic membrane 2 The periplasmic chaperone PapD facilitates folding of Cytoplasm SecYEG the subunits and brings them to…

(A) Figure 11.21 Myxococcus xanthus type IV retraction pilus involved in cell motility. (A) Atomic force micrograph of Myxococcus xanthus. Note the polar location of retraction pili needed for gliding. (B) Diagram showing the cycle of extension and retraction of pili during gliding. A, courtesy of J. K. Gimzewski.

(B) 1 The pili extend by lengthening of the filaments. 2 The pili attach to the solid surface in front of Pili Adhesion the cell. protein

Perry Staley Lory Microbiology 2/e Solid surface Sinauer Associates Elizabeth Morales Illustration Services Glide Figure 11.21.e4 psThe pili somehow Date 03-06-07 detach 3 Depolymerization of the from the surface and begin filaments shortens the pili, another round of pulling the cell forward. extension and retraction.

Perry Staley Lory Microbiology 2/e Sinauer Associates Elizabeth Morales Illustration Services Figure 11.22B.eps Date 03-06-07 Assembly of Bacterial Cell Structures 313

L Figure 11.20 Assembly of the P pilus The P pilus is assembled from subunits secreted into the periplasm. PapC, the outer membrane component of the pilus, is assembled first and it serves as a platform for polymerization of the remaining subunits that make up the filament. Components that are at the tip of the filament are added first, followed by the components that are closest to the cell surface. A periplasmic chaperone, PapD, plays an important role in the assembly process; it prevents misfolding and premature aggregation of the major (PapA) and minor (PapG, F, K, and H) subunits of the filament.

BOX 11.3 Milestones

Flagellar Chemotaxis Signaling (A) Run Flagellum bundle

How does the chemotaxis signaling (A) Arrangement and rotation of process operate at the molecular flagella during running. (B) The level? If the concentration of an (B) Tumble duration of running versus tum- attractant molecule is lower than a bling affects the speed and direc- prior cell signal determination, the Ru tion of movement. chemoreceptors are unable to effi- n ciently interact with the CheA–CheW proteins. This leads to CheA autophosphorylation, where ATP is the phosphate donor. CheA- Tumble phosphate then donates this phosphate to the CheY messenger pro- Run tein to give the activated form, CheY-phosphate. Since CheY-phosphate (and CheY) is a soluble cytoplasmic protein, it is free to diffuse throughout the cell. If CheY-phos- signal determination. The chemore- processing proteins and results in phate levels are sufficiently high, it ceptors report the current signal the ability of the cell to momentarily binds to the CheY-phosphate recep- level to CheA–CheW as before. remember attractant and repellant tor protein complex (FliM–FliN) However, the elevated attractant conditions. This chemical memory located at the base of the flagellar signal suppresses CheA autophos- device allows temporal (time) and motor (see Figure 11.15). This causes phorylation, and this in turn results spatial (space) comparisons. Methyl- the motor to reverse direction to in a low CheY-phosphate level that ation is accomplished by a methyl- clockwise rotation (see the figure), is insufficient to trigger motor rever- transferase (CheR), an enzyme that thus inducing a cell tumble event. sal. This permits a longer period of uses S-adenosylmethionine as a However, the flagella quickly reverse smooth swimming (i.e., suppresses substrate. The process of chemore- direction again (counterclockwise) cell tumbling). For some sugars ceptor binding and subsequent and initiate a period of smooth serving as attractant molecules, methylation of the methyl-accept- swimming (see the figure). Since the positive chemotaxis can occur at ing chemoreceptor (termed cell is continually sampling its envi- concentrations as low as 10–8 M. methyl-accepting chemotaxis pro- ronment, the length of the smooth Recall that a reverse signaling rela- tein, or MCP) results in an attenuat- run is dependent on signal intensity. tionship applies for repellants. ed signal. Demethylation is accom- Short runs report decreasing levels An additional level of chemotaxis plished by a methylesterase (CheB), of attractant, whereas longer swim- control involves chemoreceptor an enzyme that relieves the attenu- ming runs report increasing attrac- methylation and demethylation. ated signaling by the chemorecep- tant level(s). This highly complex process allows tor, thus increasing signal response. Let us examine the chemosenso- for the modulation of signal transfer Aer signaling is independent of ry process when the attractant con- from the Tap, Tar, Trg, and Tsr CheB and CheR. centration is higher than a prior cell chemoreceptors to the CheA–CheW

Perry Staley Lory Microbiology 2/e © 2007 Sinauer Associates, Inc. ThisSinau materialer Associates cannot be copied, reproduced, manufactured, or disseminated in any form without express writtenElizabeth permission Morales Illusfromtrat theion publisher Services . Figure Box 11.03.eps Date 02-19-07 314 Chapter Eleven

• The structural component of a bacterial cell wall is SECTION HIGHLIGHTS peptidoglycan. It is composed of sugar amines that Formation of cell appendages requires spe- are complexed with peptides. Synthesis of peptido- cialized protein secretion and assembly ma- glycan units occurs in the cytoplasm and within the chinery. Flagella and many pili are generated cytoplasmic membrane. Final assembly occurs out- using a type III secretion process. The func- side the cytoplasmic membrane. tioning of these assemblies differs consider- • Five different extracellular secretion pathways ably, and range from taxis to cell attachment, function in bacteria to deliver proteins to their DNA transfer, and toxin delivery. exterior. • Gram-positive microorganisms have a thick peptidoglycan layer whereas a gram-negative bacterium has a thinner peptidoglycan layer. The gram-nega- SUMMARY tive bacteria have an elaborate cell envelope outside the peptidoglycan layer that requires special- • The monomers that are essential for the synthesis of ized protein export machines for its construction. cell matter are polymerized to form macromole- • Flagella are highly specialized appendages that cules, and these are the functional components of confer on the cell the ability “swim.” Their forma- the cell. Major macromolecules in a cell are proteins, tion requires the synthesis of numerous proteins phospholipids, DNA, RNA, and polysaccharides. that are assembled in a temporal and spatial fash- • Over one-half of the dry weight of a bacterial cell is ion. When instructed by the associated chemosen- protein. About 20% is RNA and 10% is lipid. sory proteins, the cell exhibits the ability to seek attractants and avoid repellants. • A polypeptide may become a functional protein by assuming a tertiary configuration. The tertiary con- • Pili perform varied roles and, as a result, contain dif- figuration results from the joining of secondary ferent structural proteins and are assembled by dif- structures such as the a-helix and b-sheets. Joining ferent means, aided by distinct chaperone proteins. to two or more tertiary structures yields a quaternary structure. i Find more at www.sinauer.com/microbial-life • Cytoplasmic membranes are formed from bilayers of phospholipid. REVIEW QUESTIONS • About 20% of the polypeptides synthesized by bacteria are integrated into the cytoplasmic membrane 1. Proteins make up over half of the dry weight of a translocated or across the membrane. cell. What are the diverse roles that proteins play • Most proteins are translocated across the mem- and where are the various types located? brane as they are translated on a ribosome. This 2. Define primary, secondary, tertiary, and quater- process is termed cotranslational translocation. nary protein structural information. Draw a pep- • A chaperone is a protein that assists in the folding tide bond of a peptide composed of several of a polypeptide chain to assume the proper func- amino acids. tional configuration. 3. What are the two major secondary structures that a protein may assume? How do they differ, and • The Sec-dependent secretion pathway proteins what is our shorthand for depicting them? What identify a specific protein export signal, termed a is the major bonding that holds polypeptides in signal sequence, located at the N-terminus of a these configurations? secreted protein. Several alternative pathways exist 4. What are some structures (see question 3) that that allow membrane targeting. are involved in the functional configuration of • The TAT-dependent protein secretion pathway tar- proteins? gets fully folded and assembled protein complexes 5. Draw a phospholipid. Which is the hydrophobic to the cytoplasmic membrane. This secretion sys- and hydrophilic end of the molecule? What hap- tem recognizes a distinct targeting signal located on pens when a cell is shifted to grow at an accept- one of the secreted polypeptides. ably higher temperature?

6. How can a peptide pass through the hydrophobic SUGGESTED READING cytoplasmic membrane? What is the difference between cotranslational insertion versus post- Berg, H. C. 2003. “The rotary motor of bacterial flagella.” translational insertion? What role might chaper- Annu Rev Biochem 72:19–54. ones play in secretory protein translocation? Garrett, R. H. and C. M. Grisham. 1999. Biochemistry. 2nd 7. What types of proteins are exported by the TAT ed. Fort Worth, TX: Saunders College/Harcourt Brace secretory pathway and why? Publishing. Gerhardt, P. R., G. E. Murray, W. A. Wood and N. R. Krieg. 8. Where are the major components of a gram-nega- 2007. Methods for General and Molecular Bacteriology. 3rd tive outer envelope assembled? Discuss the ed. Washington, DC: ASM Press. processes involved. Neidhardt, F. C., J. L. Ingraham and M. Schaechter. 1990. 9. Outline several distinct routes for export of pro- Physiology of the Bacterial Cell: A Molecular Approach. teins to the cytoplasmic membrane, and to the Sunderland, MA: Sinauer Associates Inc. Neidhardt, F. C., R. Curtiss III, J. L. Ingraham, C. C. Lin, K. outer membrane. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M. 10. How do pili types differ in structure and function? Schaechter and H. E. Umbarger. 1996. Escherichia coli and Salmonella: Cellular and Molecular Biology. Washington, DC: ASM Press. Purves, W. K., D. Sadava, G. H. Orians and H. C. Heller. 2007. Life: The Science of Biology. 8th ed. Sunderland, MA: Sinauer Associates Inc. Saier, M. H. 2006. “Protein secretion systems in gram-negative bacteria.” Microbe 1:414–419.