Sample of Instructor’s Manual to accompany Instructor’s Manual by Robert Carey, Lebanon Valley College © 2014 W. W. Norton & Company, Inc. 1 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. Instructor's Manual for Microbiology: An Evolving Science, 3e Slonczewski and Foster © 2014 W. W. Norton & Company, Inc. 2 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 Instructor's Manual for Microbiology: An Evolving Science, 3e Slonczewski and Foster © 2014 W. W. Norton & Company, Inc. 3 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. Instructor's Manual for Microbiology: An Evolving Science, 3e Slonczewski and Foster © 2014 W. W. Norton & Company, Inc. 4 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.