Instructor's Manual for Microbiology: an Evolving Science, 3E Slonczewski and Foster © 2014 W

Sample of Instructor’s Manual to accompany

Instructor’s Manual by Robert Carey, Lebanon Valley College

Chapter 3

Cell Structure and Function

SUMMARY

This chapter explores the structure and function of bacterial cells. Comparisons are drawn to archaea and eukaryotes. Discussions of archeal and eukaryotic diversity are found in Chapters 19 and 20, respectively. It is here that we learn how a microbe interacts with its environment, the role of the cell envelope, the nucleoid, and the tightly coordinated mechanisms of bacterial reproduction. We learn how combining microscopic analysis, cell fractionation, and genetic analysis led to the assembly of the cellular puzzle.

3.1 The Bacterial Cell: An Overview

This section introduces cell structure and function in bacteria, including the structure of Gram-negative cell walls and the molecular composition of a typical bacterium. Cell composition varies with species, growth phase, and environmental conditions. This section also discusses methods used when investigating bacterial structure and function. Microscopy unveils the structure of subcellular components, but does not give any clues as to their functions. Cell fractionation can be used to isolate components of interest. Structural analysis of the components can give us an idea about the form of the components, and genetic analysis allows us to address function. This is made possible by the construction of mutants with altered function.

Discussion Points

• Discuss the various methods that can be used to disrupt a cell. • Subcellular fractionation using an ultracentrifuge is a powerful tool for isolation. • Figures 3.2A and 3.2B show the principle of ultracentrifugation. • Genetic analysis of mutants reveals function. Genetic analysis may also include the use of reporter genes such as GFP to study protein function in live cells. • Explain two-dimensional (2D) gels and how they can be used to study an organism. • Figure 3.3 shows a 2D gel of E. coli in which approximately 500 proteins can be distinguished.

3.2 The Cell Membrane and Transport

The cell membrane is a barrier that determines what can get in and out of a cell. Not only does it play a role in all forms of transport, but it affords structural support, detects environmental signals, and plays a role in cell-to-cell signaling.

Description of how bacterial membrane composition is altered depending on the growth phase or environmental conditions leads to an understanding of the structural differences and properties of membranes. It also serves as an introduction to the unique membranes of archaea, which are covered in a later chapter.

Discussion Points

• Figure 3.4 serves as a good jumping-off point for discussion of the membrane and its different components. • The composition of membrane lipids should be discussed. Figure 3.5 shows the chemical structure of a phospholipid. Note the ester links and the two types of phospholipid head groups. Figure 3.19 diagrams various phospholipid side chains. • Discussion of the diffusion of molecules across a biological membrane yields insights into drug design and delivery (see Fig. 3.6)

• Be sure to discuss the different forms of transport. A diagram of how the vitamin B12 transport protein functions can be found in Figure 3.7. Distinguish between different types of transport using this figure. • Figure 3.11 shows the differences in the membrane lipids of the archaea. Mention should be made of what the various ether-linked lipids provide to the archaea.

3.3 The Cell Wall and Outer Layers

The bacterial cell wall, or sacculus, confers shape and rigidity as well as protecting the cell from osmotic lysis. It is highly porous to ions and small molecules, unlike the cell membrane. Peptidoglycan structure is discussed, and differences between Gram-positive and Gram-negative peptidoglycan are illustrated. The thickness of the Gram- positive cell wall, along with the presence of teichoic acids, is mentioned. The S-layer, which is particularly crucial to the structure of some archaea, is also discussed. The periplasm and outer membrane of Gram-negative cells is introduced. This includes discussion of the structural composition of Gram-negative walls as well as their function. Both Gram-positive and Gram-negative cells may have capsules and/or slime layers. Their role in protection from phagocytosis and antibiotics should be mentioned. Mycobacteria have complex cell envelopes that contain mycolic acids, which affect the growth and survival of the organism. The bacterial cytoskeleton is also discussed in light of its importance in regulating cell shape.

Discussion Points

• The actual chemical structure of peptidoglycan is portrayed in Figure 3.14. A review of L and D enantiomers may be useful to students in understanding this structure. • When discussing Figure 3.14, make it a point to show the sites for involvement with the antibiotics penicillin and vancomycin. • Teichoic acids (chemical structure shown in Fig. 3.16) occur only in Gram-positive organisms, as illustrated in

Figure 3.15. • Figure 3.15 also shows a comparison of the Gram-positive and Gram-negative cell envelopes. • Also use Figure 3.15 to show that the Gram-negative outer membrane is also a bilayer, similar to its inner membrane, and point out that it has porins and lipopolysaccharide (LPS). • Use Figure 3.18 to compare the mycobacterial envelope to Gram-positive and Gram-negative bacterial cell walls. Discuss how these differences affect the function of the cell wall. It may also be useful to discuss laboratory staining techniques and how they are influenced by the type of cell wall found in the subject organism. • Figure 3.19 shows the structure of the outer membrane lipoprotein and LPS. • Figure 3.22 can be used to discuss how eukaryotic microbes protect themselves from osmotic shock. • Figure 3.23 is useful for discussing how the cytoskeleton influences cell shape.

3.4 The Nucleoid, RNA, and Protein Synthesis

In prokaryotes the DNA is organized in a nucleoid, unlike the membrane-bound nucleus of a eukaryote. The genome is organized into looped domains that connect back to the origin of replication (ori). The DNA is condensed by interacting with binding proteins, which also influence gene expression. The DNA opens at the ori and replication proceeds bidirectionally. Since the DNA in a prokaryotic cell is not compartmentalized, transcription and translation are tightly coupled. This results in the formation of polysomes. Secreted and membrane-inserted proteins are managed by binding of a signal recognition particle (SRP).

Discussion Points

• The structure of prokaryotic DNA is illustrated in Figure 3.26. • Figure 3.27 depicts how, as RNA polymerase transcribes DNA producing a single-stranded messenger RNA, ribosomes bind to the mRNA, thereby coupling transcription and translation. • The relationship between transcription and translation in the synthesis of secreted proteins is illustrated in Figure 328.

3.5 Cell Division

Prokaryotic cells constantly make RNA and protein as the DNA is being replicated, and DNA replication has to be coordinated with expansion of the cell wall and ultimately, cell fission. DNA replication proceeds in a bidirectional fashion from the ori. A replisome complex containing DNA polymerase and its accessory components is required for each replication fork; hence two replisomes are required. As the DNA is replicated, the cell wall expands. DNA is still being transcribed during replication and the RNA being produced is still being translated. When the DNA termination site is replicated, this signals septum formation.

Discussion Points

• The fluorescent micrographs in Figures 3.29 and 3.30 reveal the replisome, the ori, and the cell envelope. • Figure 3.32B shows how spatial orientation of septation determines the shape and arrangement of daughter cells. • Figures 3.33 and 3.34 show fluorescent microscopic studies used to help determine the role of specific proteins in determination of cell shape.

3.6 Cell Polarity and Aging

This section discusses the polarity of bacterial cells. Bacterial cells have two poles that differ in age, with the "new" pole arising from the end of the cell closest to the new septum and the "old" pole at the opposite end. These poles may differ in their structure and function. They are also involved in regulating cell growth rates.

Discussion Points

• Figure 3.38 and Special Topics 3.1 Figure 2A illustrate the concept of old and new poles following a cell division. • Figures 3.35–3.37 illustrate how cell polarity directs differential development in Caulobacter. • Special Topic 3.1 figure 2B contains data that elucidate the relationship between pole age and growth rate. • Discussion of how the polar age of Mycobacterium cells can affect antibiotic resistance may be aided by Special Topic 3.1. This concept could be used to illustrate the relationship between basic science research into microbial growth and potential medical applications.

3.7 Specialized Structures

Specialized structures are introduced here and will be discussed in greater detail in later chapters. These include thylakoid membrane organelles, storage granules, magnetosomes, and adherence structures. There is also a discussion of flagellar rotation and chemical signaling during chemotaxis.

Discussion Points

• Photosynthetic, magnetotactic, and attachment structures are discussed here. The various specialized structures can be observed in Figures 3.39–3.43. • Figure 3.44 should be used to illustrate the various placements of flagella and the associated designations. This can be important in identifying an organism. • Note that chemotaxis was one of the first molecular regulatory circuits elucidated in living organisms. • Chemotaxis is illustrated in Figure 3.46. Things to note are the direction of flagellar rotation and the overall pattern of swimming in the direction of the attractant. • Something for the students to think about is whether the organism senses a difference in concentration from one

end of itself to the other, or over time.

Once again, emphasize that all of the previously described techniques can be combined to provide a more complete model of a system. Also, students now have become aware of antibiotic interactions with cell walls and ribosomes. More will be said about these interactions in later chapters.

PROCESS ANIMATIONS

The following Process Animation for Chapter 3 can be viewed through the ebook, by using the QR codes in the printed book, and in the coursepacks, which are provided for download on the instructor’s resource site, wwnorton.com/instructors.

• Replisome Movement in a Dividing Cell • Chemotaxis: Molecular Events

eTOPICS

eTopics are supplementary, stand-alone sections that explore additional material in depth. They are available to students within the ebook and within the coursepacks, which are provided for download on the instructor’s resource site, wwnorton.com/instructors.

3.1 Isolation and Analysis of the Ribosome eTopic 3.1 discusses cell fractionation (including a discussion of sedimentation rate), X-ray crystallography, and analysis of genetic mutants. All of these techniques are discussed in the context of the discovery of the bacterial ribosome and the elucidation of its function.

3.2 How Antibiotics Cross the Outer Membrane eTopic 3.2 discusses how antibiotics are able to get into a bacterial cell. This includes a discussion of porins and the effect of pH (and charge) on antibiotic passage through these channels.

3.3 Outer Membrane Proteins: Isolation for Vaccines eTopic 3.3 covers the use of bacterial outer membrane proteins as potential vaccines.

3.4 Experiments that Reveal the Bacterial Cytoskeleton eTopic 3.4 discusses experiments that helped elucidate the structure of the bacterial cytoskeleton. The discussion covers the study of various cytoskeletal mutants and a model of our current understanding of the bacterial cytoskeleton.

RECOMMENDED READINGS

The following readings are presented at the end of the textbook chapter as resources for further exploration of the topics discussed in Chapter 3.

Aldridge, Bree B., Marta Fernandez-Suarez, Danielle Heller, Vijay Ambravaneswaran, Daniel Irimia, et al. 2012. Asymmetry and aging of mycobacterial cells lead to variable growth and antibiotic susceptibility. Science 335:100–103. Bardy, Sonia L., and Janine R. Maddock. 2007. Polar explorations: Recent insights into the polarity of bacterial proteins. Current Opinion in Microbiology 10:617–623. Celler, Katherine, Roman I. Koning, Abraham J. Koster, and Gilles P. van Wezel. 2013. Multidimensional view of the bacterial cytoskeleton. Journal of Bacteriology 195:1627–1636. Feucht, Andrea, and Jeff Errington. 2005. ftsZ mutations affecting cell division frequency, placement and morphology in Bacillus subtilis. Microbiology 151:2053–2064. Lam, Hubert, Whitman B. Schofield, and Christine Jacobs-Wagner. 2006. A landmark protein essential for establishing and perpetuating the polarity of a bacterial cell. Cell 124:1011–1023. Lenz, Peter, and Lotte Søgaard-Andersen. 2011. Temporal and spatial oscillations in bacteria. Nature Reviews. Microbiology 9:565–577. Lele, Uttara N., Ulfat I. Baig, Milind G. Watve. 2011. Phenotypic plasticity and effects of selection on cell division symmetry in Escherichia coli. PLoS ONE 6:e14516. Libby, Elizabeth A., Manuela Roggiani, and Mark Gouliana. 2012. Membrane protein expression triggers chromosomal locus repositioning in bacteria. Proceedings of the National Academy of Sciences USA. [Epub ahead of print.] doi:10.1073/pnas.1109479109. Nilsen, Trine, Arthur W. Yan, Gregory Gale, and Marcia B. Goldberg. 2005. Presence of multiple sites containing polar material in spherical Escherichia coli cells that lack MreB. Journal of Bacteriology 187:6187– 6196. Noji, Hiroyuki, Ryohei Yasuda, Masasuke Yoshida, and Kazuhiko Kinoshita, Jr. 1997. Direct observation of the rotation of F1-ATPase. Nature 386:299–302. Pagès, Jean-Marie M., Chloë E. James, and Mathias Winterhalter. 2008. The porin and the permeating antibiotic: A selective diffusion barrier in Gram-negative bacteria. Nature Reviews. Microbiology 6:893–903. Renner, Lars D., and Douglas B. Weibel. 2011. Cardiolipin microdomains localize to negatively curved regions of Escherichia coli membranes. Proceedings of the National Academy of Sciences USA 108:6264–6269. Ruiz, Natividad, Daniel Kahne, and Thomas J. Silhavy. 2006. Advances in understanding bacterial outermembrane biogenesis. Nature Reviews. Microbiology 4:57–66. Saier, Milton H., Jr. 2008. Structure and evolution of prokaryotic cell envelopes. Microbe 3:323–328. Stewart, Eric J., Richard Madden, Gregory Paul, and François Taddei. 2005. Aging and death in an organism that reproduces by morphologically symmetric division. PloS Biology 3:e45.

ANSWERS TO REVIEW QUESTIONS (p. 111)

1. What are the major features of a bacterial cell, and how do they fit together for cell function as a whole? ANS: The bacterial cell has a relatively small genome that is condensed within a region called the nucleoid. It contains very little, if any, noncoding or extraneous DNA. The cytoplasm is packed with 70S ribosomes. Depending on the genus, there are various subcellular components that coordinate cell function. The outer envelope of the cell consists of the cell membrane, which is surrounded by a cell wall in most bacteria. Together they protect the cell, regulate exchange with its surroundings, and are involved in communication with other cells.

2. What fundamental traits do most prokaryotes have in common with eukaryotic microbes? What traits are different? ANS: Overall, they are very similar. The traits stated in question 1 could also be stated for eukaryotes, except the eukaryotes have more noncoding DNA and the genetic material is housed in a membrane-bounded nucleus. Whereas the cell membranes have similar structures and functions, some of their chemical makeup is very different. Cell walls, if present, are also chemically distinct. Eukaryotes also contain other organelles, which are membrane-bound, highly specialized entities.

3. Give examples of how our views of ribosome structure and function have emerged from microscopy, cell fractionation, X-ray diffraction crystallography, and genetic analysis. Explain the advantages and limitations of each technique. ANS: Microscopy gave us a general view of the ribosome, but no chemical or physical specifics. Cell fractionation enabled us to learn that the ribosome consists of a small and a large subunit. By analyzing fractions, we detected polysomes. We can also use fractions in cell-free expression systems. This technique cannot address processes requiring cellular integrity. X-ray diffraction crystallography was used to determine the structure of the 30S ribosome with all of its ribosomal RNA (rRNA) and protein components. It also enabled discovery of the three transfer RNA (tRNA) binding sites, A, P, and E. Again, this only works on isolated entities whose full function cannot be observed. The actual function of cellular components can be dissected by genetic analysis. Genetic analysis, in conjunction with X-ray crystallography, enabled discovery of how the antibiotic streptomycin specifically inhibits protein synthesis.

4. Outline the structure of the peptidoglycan sacculus, and explain how it expands during growth. Cite two different kinds of experimental data that support our current views of the sacculus. ANS: The peptidoglycan sacculus is a single interlinked molecule that encloses the entire cell. It consists of parallel polymers of disaccharides called glycan chains cross-linked with peptide side chains. The layers are - alternating units of N-acetylglucosamine and N-acetylmuramic acid, forming a large sheet. Layers of these sheets are held together by peptide side chains. Peptide extensions can form cross-bridges connecting parallel strands of glycans. In essence, the sacculus is a huge mesh bag, holding the cell together. Cell wall expansion and septation can be observed by EM and further dissected by using fluorescence microscopy to follow specific proteins during the growth and septation process. Genetic mutants have been used

in conjunction with fluorescence microscopy to reveal an even greater understanding of the process.

5. Compare and contrast the structure of Gram-positive and Gram-negative cell envelopes. Explain the strengths and weaknesses of each kind of envelope. ANS: The Gram-positive cell has only two layers: the cell membrane and the cell wall. The cell wall contains multiple layers of peptidoglycan. It also contains teichoic acids, which are not found in Gram-negative cells. Gram-positive cells have much more structural integrity than Gram-negative cells because of the thick, highly cross-linked peptidoglycan. A Gram-negative cell can be considered to have four components. It has a cell membrane or inner membrane, peptidoglycan, periplasm, and outer membrane. The cell membranes of Gram-positive and Gram-negative cells are very similar. The Gram-negative cell wall has a very thin peptidoglycan layer that does not contain teichoic acids. The periplasm is the region bounded by the inner and outer membranes. It contains specialized proteins and enzymes. The outer membrane is a bilayer composed of phospholipids and LPS. It also contains transport proteins called porins. The outer membrane prevents uptake of certain toxic molecules and allows growth in harsher environments. The LPS acts as an endotoxin, and the O-polysaccharide component helps the bacteria resist phagocytosis by white blood cells. Gram-positive and Gram-negative cells are quite often covered by a slippery capsule composed of polysaccharides. Its presence may be observed by negative staining. Organisms freshly isolated from their natural environment may also possess a protein or glycoprotein surface layer, called an S-layer.

6. Outline the process of DNA replication, and explain how it is coordinated with cell wall septation. ANS: DNA replication begins at the origin of replication and proceeds bidirectionally, forming two copies, one for each progeny. A replication fork is propagated by unwinding the DNA. DNA polymerase then begins the synthetic process. DNA polymerase, with its accessory proteins, forms a replisome, which includes two DNA polymerases. One polymerase replicates the leading strand and the other the lagging strand. Two replisomes are required since replication is bidirectional. One cell becomes two daughter cells, each containing a complete copy of the chromosome. This entails coordination of septation and replication. As the replication termination site is replicated, septum growth is triggered.

7. Explain how DNA transcription to RNA is integrated with translation and protein processing and secretion. ANS: Since there is no nucleus in a prokaryotic cell, replication, transcription, and translation all occur within the same compartment, the cytoplasm. As soon as a ribosome binding site is transcribed, a ribosome binds and begins the translation process. Multiple ribosomes may translate one message. This is referred to as a polysome. Proteins destined to be secreted bear a signal recognition sequence. Signal recognition particles (SRPs) bind to these proteins as they are being synthesized and ensure correct processing and secretion.

8. What kinds of subcellular structures are found in certain cells with different functions, such as magnetotaxis or photosynthesis? ANS: Magnetotactic organisms contain magnetosomes. These are magnetite-containing sacs found in anaerobic

aquatic organisms. The magnetosomes orient the organism along the magnetic field. This directs them to the bottom of the water source, where less oxygen is present. Photosynthetic organisms contain phycobilisomes and thylakoids. Phycobilisomes are the light-harvesting complexes. The thylakoids are folded sheets packed with photosynthetic proteins and electron carriers. It is here that the light reaction of photosynthesis occurs. Carboxysomes are present for carbon dioxide fixation, and gas vesicles keep the organisms higher in the water, closer to the needed light.

9. Compare and contrast bacterial structures for attachment and motility. Explain the molecular basis of chemotaxis. ANS: Flagella are used for motility. They tend to be longer than appendages for attachment, and they rotate like a propeller, at the expense of a proton gradient, to afford movement. They are also a quite complex structure composed of many different proteins. The most common structures for attachment are pili, composed of protein monomers of pilin. There are pili for attachment and pili for exchange of genetic information. Chemotaxis is a mechanism that affords an organism the ability to swim toward attractants and away from repellents. Simplistically, receptors sense a compound, and the receptor is either phosphorylated or dephosphorylated, which sends a signal to the flagella to rotate in either one direction or the other. This leads to swimming or tumbling, allowing the organism to react to chemicals in its environment.

ANSWERS TO END-OF-CHAPTER THOUGHT QUESTIONS (p. 111)

1. The aquatic bacterium Caulobacter crescentus alternates between two cell forms: a cell with a flagellum that swims, and a stalked cell that adheres to particulate matter. The flagellar cell can discard its flagellum to grow a stalk and adhere, then the stalked cell divides to give one stalked cell and one flagellated cell. What would be the adaptive advantage of this alternating morphology? ANS: The advantage of the stalk is that it enables a cell to remain attached in a habitat providing a good supply of nutrients. However, the nutrients will eventually be depleted. If the stalked cell generates motile progeny, they will swim away and avoid competing for nutrients; also, they may find new habitats after that of the stalked cell is depleted. Once a flagellated cell finds a rich habitat, it is favorable to form a stalk and attach, then continue the cycle of generating flagellated cells.

2. Suppose that one cell out of a million has a mutant gene blocking S-layer synthesis, and suppose that the mutant strain can grow twice as fast as the S-layered parent. How many generations would it take for the mutant strain to constitute 90% of the population? ANS: In one parental generation, the parent strain increases twofold, whereas the mutant numbers increase by a factor of four. The mutant fraction is equal to (4N)/[4N + (106 × 2N)], where N is the number of mutant cells after a given parental generation. Plotting this out on an Excel spreadsheet or graphic calculator shows that by the twenty-third generation, the mutant fraction approximates 90%.

3. Explain two ways that an aquatic phototroph might use its subcellular structures to maximize its access to light. Instructor's Manual for Microbiology: An Evolving Science, 3e Slonczewski and Foster © 2014 W. W. Norton & Company, Inc. 10

Explain how an aerobe (an organism requiring molecular oxygen for growth) might remain close to the surface, with access to air. ANS: The phototrophic microbe needs to avoid being shaded by other organisms, or by the water column. The microbe can possess phototaxis, that is, a sensory response to light which signals its flagella to rotate smoothly when the cell is moving in a direction of increasing light intensity. Alternatively, the microbe may possess gas vesicles, which increase buoyancy. By floating near the surface, the cell will maximize light exposure. By similar means, an aerobic organism can maximize oxygen exposure either by aerotaxis (swimming up a gradient of oxygen) or by gas vesicles causing the cell to float near the water surface.

4. How do pathogenic bacteria avoid engulfment by phagocytes of the human blood stream? How do you think various aspects of the cell structure can prevent phagocytosis? ANS: The most important feature that enables pathogenic bacteria to evade phagocytosis is the cell envelope. A capsule of polysaccharide chains can coat the cell, making it difficult for phagocytes to attach and covering up surface proteins that may stimulate the immune system. Other cell surface components that can inhibit phagocytosis include the thick, waxy envelope of Mycobacteria, the LPS layer of Gram-negative enteric bacteria, and the S-layer. Another way to evade host defenses is through flagellar motility, which enables pathogens to disperse widely from their original infection site. Finally, through gene expression and protein secretion bacteria can produce toxins that disable phagocytic cells.