Supplemental Lecture (98/03/28 update) by Stephen T. Abedon (abedon.1@osu.edu)

  1. Chapter title: Procaryote Cell Walls and Membranes
    1. A list of vocabulary words is found toward the end of this document
    2. "After reduction to elemental parts, there must be resynthesis into complex systems." - John Crabbe
    3. Cellular life conforms to two basic cellular anatomies: (i) cells generally lacking internal, membrane-bound compartments and (ii) cells generally possessing internal, membrane-bound compartments. Anatomically (though certainly not biochemically), cells lacking membrane-bound compartments are simpler and, consequently, are often considered to be a "lower form of life." They are called bacteria.
    4. In fact, in terms of evolutionary variation, numbers of species, numbers of types of environments colonized, biochemical novelty, importance to the survival of all life on planet Earth, photosynthesis, cellular respiration, and, indeed, sheer numbers, bacteria are the dominate organisms on this planet. As noted by the eminent Harvard paleontologist and popular science writer, Stephen Jay Gould, "We live in the Age of bacteria (as it was in the beginning, is now and ever shall be, until the world ends)." (Gould, 1993)
    5. As follows we will discuss the anatomy of bacteria cells, which are also known as procaryotic cells.
  2. Plasma membrane
    1. Cell membrane:
      1. A membrane consisting of phospholipids as well as various proteins which define the edge of the cytoplasm of cells.
      2. Plasma membranes are semipermeable as are most cellular membranes.
      3. "The plasma membrane retains the cytoplasm, particularly in cells without cell walls, and separates (the cell) from the surroundings. The plasma membrane also serves as a selectively permeable barrier: it allows particular ions and molecules to pass, either into or out of the cell, while preventing the movement of others. . . The bacterial plasma membrane also is the location of a variety of crucial metabolic processes: respiration, photosynthesis, and the synthesis of lipids and cell wall constituents. Finally, the membrane contains special receptor molecules that help bacteria detect and respond to chemicals in their surroundings." (p. 44, Prescott et al., 1996)
      4. All bacteria have cell membranes.
    2. May be invaginated:
      1. Some bacteria make up for a lack of membrane bound organelles through an infolding of their plasma membrane.
      2. These infoldings or invaginations of the plasma membrane form interal membranous structures.
      3. While such infolding does not result in additional membrane-bound compartments, it does result in an increased area of plasma membrane which allows the harboring of greater numbers of membrane-associated proteins such as those employed in cellular respiration or photosynthesis.
      4. Note that technically these are not membraned organelles but instead are continuous with the plasma membrane such that one side of these membranes consists of the inside of the cell (cytoplasm) while the other side consists of the outside of the cell (actually, the periplasm).
      5. The classic example of membrane infolding is that seen in the obligate endosymbiotic bacteria, mitochondria (there called cristae).
    3. True membrane-bounding:
      1. Exceptionally, Cloroplasts and their free living cyanobacteria relatives can have infoldings that become autonymous from their parental plasma membrane.
      2. These membrane-bound organelles are called thylakoids.
      3. "Photosynthetic bacteria and cyanobacteria contain internal membrane systems, sometimes known as chromatophores. The membranes of the chromatophores, derived from the cell membrane, contain the pigments used to capture light energy for the synthesis of sugars. Nitrifying bacteria, soil organisms that convert nitrogen compounds into forms usable by green plants, also have internal membranes. They house the enzymes used in deriving energy from the oxidation of nitrogen compounds." (p. 84, Black, 1996)
    4. Permanent breakage of the plasma membrane is, by definition, cell death because it:
      1. results in the leakage of key cell contents
      2. contaminates the cell cytoplasm with compounds normally restricted to the extracellular environments
      3. poisons the cells by disrupting its ATP generating machinery
  3. Cell envelope
    1. External to the plasma membrane:
      1. The cell envelope is the outer portion of a bacterial cell.
      2. That is, it is external to the plasma membrane.
    2. Starting from the outside layer and going in the gram-negative cell envelope consists of:
      1. the outer membrane
      2. the cell wall
      3. the periplasmic space
      4. the inner membrane
    3. Starting from the outside layer and going in the gram-positive cell envelope consists of:
      1. the cell wall
      2. the periplasm
      3. the plasma membrane
  4. Cell wall
    1. Continuous, rigid outer layer:
      1. A complex, semirigid, hollow object that surrounds, or nearly surrounds the bacteria cell.
      2. The cell wall is enveloped by the outer membrane in gram-negative bacteria, and envelopes the inner or plasma membrane in both gram-negative and gram-positive bacteria.
    2. Function:
      1. The cell wall has at least three major functions:
        1. constrains the internal volume*
        2. defines the shape of the cell
        3. anchor point to extracellular projections such as flagella.
      2. *Thus protecting against rupturing when exposed to low salt solutions such as distilled water.
      3. In addition, the chemical composition of the cell wall differs among various bacteria. Certain bacteria species, for example, are differentiated on the basis of these differences.
  5. Peptidoglycan [a.k.a., murein]
    1. A lattice formed by repeating disaccharides interconnected by polypeptides that make up the bacterial cell wall.
    2. "Peptidoglycan (pep''ti-do-gly'-kan), also known as murein, is the sinlge most important component of the bacterial cell wall. It is a polymer so large that it can be thought of as one immense, covalently linked molecule. It forms a supporting net around a bacterium that resembles multiple layers of chain-link fence. In the peptidoglycan polymer, molecules of N-acetylglucosamine (gluNAc) alternate with molecules of N-acetylmuramic acid (murNAc). These molecules are cross-linked by tetrapeptides, chains of four amino acids." (p. 78, Black, 1996)
    3. Note that this statement is completely true for eubacteria only, and then only for eubacteria that have cell walls.
  6. Gram-positive cell envelope
    1. The cell wall associated with gram-positive bacteria is thicker than that associated with gram-negative bacteria.
    2. These cells walls consist of multiple layers (unlike gram-negative cell envelope).
    3. In addition, they contain teichoic acids (also unlike gram-negative cell envelope).
    4. See illustration below.
  7. Illustration, gram-positive cell envelope
  8. Teichoic acids
    1. gram positive cell envelope constituents:
    2. Characteristics:
      1. Teichoic acids are phosphate group joined polymers of glycerol or ribitol.
      2. Teichoic acids additionally bind sugars and amino acids and tend to be covalently linked with the peptidoglycan of the cell wall as well as to plasma membrane lipids.
      3. The negative charge of teichoic acids also contributes to the negatively charge of the gram-positive cell envelope.
      4. "Cell walls of gram-positive organisms have an additional molecule, teichoic acid. Techoic acid (tie-ko'ik), which consists of glycerol, phosphates, and the sugar alcohol ribitol, occurs in polymers up to 30 units long. These polymers extend beyond the rest of the cell wall, even beyond the capsule in encapsulated bacteria. Although its exact function is unclear, teichoic acid furnishes attachment sites for bacteriophages (viruses that infect bacteria)." (p. 78, Black, 1996)
    3. Identification/antigenic determinants:
      1. Important antigenic determinants of gram-positive bacteria.
      2. Teichoic acids are useful for distinguishing strains gram-positive bacteria by serological means.
  9. Gram-negative cell envelope
    1. The cell wall associated with gram-negative bacteria is thinner than that associated with gram-positive bacteria), consisting of only a single layer.
    2. Not exposed:
      1. It is not exposed to the extracellular environment due to the presence of an outer membrane surrounding it.
      2. It also bonded to lipoproteins found in the outer membrane.
    3. See illustration below.
  10. Illustration, gram-negative cell envelope
  11. Inner membrane
    1. Another name for plasma membrane in gram-negative bacteria.
  12. Outer membrane
    1. Second membrane:
      1. A membrane consisting of phospholipids, various membrane proteins, lipoproteins, and lipopolysaccharide that is found surrounding the cell wall of gram-negative bacteria.
      2. "The outer membrane . . . is a bilayer membane. It forms the outermost layer of the cell wall (envelope) and is attached to the peptidoglycan by an almost continuous layer of small lipoprotein molecules (proteins combined with a lipid). The lipoproteins are embedded in the outer membrane and covalently bonded to the peptidoglycan." (p. 78, Black, 1996)
    2. Gram-positive bacteria lack an outer membrane.
    3. "Gram-positive bacteria lack both an outer membrane and a perioplasmic space. Therefore, digestive enzymes not retained in the periplasm are released into the environment, where they sometimes become so diluted that the organisms derive no benefit from them." (p. 82, Black, 1996)
    4. Barrier to toxic substances:
      1. The outer membrane provides a barrier to many substances that could otherwise detrimentally affect the cell wall and plasma membrane, as these same substances do readily affect these structures in gram-positive bacteria.
      2. "A most important outer membrane function is to serve as a protective barrier. It prevents or slows the entry of bile salts, antibiotics, and other toxic substances that might kill or injure the bacterium." (p. 51-52, Prescott et al., 1996)
    5. "The outer membrane is more permeable than the plasma membrane and permits the passage of small molecules like glucose and other monosaccharides. This is due to the presence of special porin proteins. Three porin molecules cluster together and span the outer membrane to form a narrow channel through which molecules smaller than about 600 to 700 daltons can pass. Larger molecules such as vitamin B12 must be transported across the outer membrane by specific carriers. The outer membrane also prevents the loss of constituents like periplasmic enzymes." (p. 51-52, Prescott et al., 1996)
  13. Lipopolysaccharide [LPS]
    1. An organic compound found in the outer leaflet of outer membranes, consisting of covalently bound lipid A and O polysaccharide.
    2. Note that since LPS is found in outer membranes, it therefore is, by definition, a characteristic of gram-negative and not gram-positive bacteria.
    3. "Lipopolysaccharide (LPS), also called endotoxin, is an important part of the outer membrane and can be used to identify gram-negative bacteria. It is an integral part of the cell wall and is not released until the cell walls of dead bacteria are broken down. LPS consists of polysaccharides and lipid A. The polysaccharides are found in repeating side chains that extend outward from the organism. It is these repeating units that are used to identify different gram-negative bacteria." (p. 78-79, Black, 1996)
  14. Lipid A
    1. LPS is anchored into the outer membrane by a lipid portion called lipid A.
    2. See endotoxin.
    3. "The lipid A portion (of LPS) is responsible for the toxic properties that make any gram-negative infection a potentially serious medical problem. It causes fever and dilates blood vessels, so the blood pressure drops precipitously. Because bacteria release endotoxin mainly when they are dying, killing them may increase the concentration of this very toxic substance." (p. 79, Black, 1996)
  15. O polysaccharide
    1. The extracellular portion of LPS which is responsible for some aspects of the protective nature of the outer membrane (due, for at least some bacteria, to its strong negative charge).
    2. O polysaccharide is an important antigenic determinant of gram-negative bacteria and is useful for finely distinguishing strains of certain bacteria by serological means.
  16. Periplasmic space
    1. Intra-envelope volume:
      1. The periplasmic space is a volume found between the bacterial plasma membrane and cell wall.
      2. This space is of particular significance in gram-negative bacteria, though is also found between the cell wall and plasma membrane of gram-positive bacteria.
      3. "Recent evidence indicates that the periplasmic space may be filled with a loose network of peptidoglycan. Possibly it is more a gel than a fluid-filled space. The substance that occupies the periplasmic space is the periplasm. . . Some recent studies indicate that it may constitute about 20 to 40% of the total cell volume." (p. 51-52, Prescott et al., 1996)
    2. "The periplasmic space of gram-negative bacteria contain many proteins that participate in nutrient acquisition---for example, hydrolytic enzymes attacking nucleic acids and phosphorlyated molecules, and binding proteins involved in transport of materials into the cell. . . The periplasmic space also contains enzymes involved in peptidoglycan synthesis and the modification of toxic compounds that could harm the cell. Gram-positive bacteria may not have a visible periplasmic space and do not appear to have as many periplasmic proteins; rather, they secrete several enzymes that ordinarily would be periplasmic in gram-negative bacteria. Such secreted enzymes are often called exoenzymes." (p. 52, Prescott et al., 1996)
    3. "Periplasmic spaces are rarely observed in gram-positive bacteria. However, such bacteria must accomplish many of the same metabolic and transport functions that gram-negative bacteria do. At present most gram-positive bacteria are thought to have only periplasms---not periplasmic spaces---where metabolic digestion occurs and new cell wall peptidoglycan is attached. The periplasm in gram-positive cells is thus part of the cell wall." (p. 80, Black, 1996)
    4. Niche determiner?
      1. Gram-positive bacteria essentially fill a fungi-like niche with their exoenzymes and absorption of nutrients from the extra-organismal environment.
      2. In contrast, one might speculate the gram-negative bacteria are better adapted to wetter, better mixed environments due to their retention of their digestive enzymes in the periplasm.
      3. However, they are not protozoa-like since they are not engulfers, and, additionally, they are likely limited in the breadth of their niches since the number and types of carbon and energy sources capable of reaching their periplams is likely limited.
  17. Acid-fast cell envelope
    1. "The cell wall of acid-fast bacteria, the mycobacteria, is thick, like that of gram-positive bacteria. It is approximately 60 percent lipid and contains much less peptidoglycan. In the acid-fast staining process, carbolfuchsin binds to cytoplasm and resists removal by an acid-alcohol mixture. The lipids make acid-fast organisms impermeable to most other stains and protect them form acids and alkalis. The organisms grow slowly because the lipids impede entry of nutrients into cells, and the cells must expend large quantities of energy to synthesize lipids." (p. 82, Black, 1996)
  18. Lysozyme
    1. Anti-murein enzyme:
      1. An enzyme produced by a variety of eucaryotic organisms as well as many viruses of bacteria.
      2. Lysozyme hydrolyzes peptidoglycan.
    2. Potent anti-gram-positive:
      1. Extracellular lysozyme is especially active against gram-positive bacteria since these bacteria have their cell walls exposed to the extracellular environment.
      2. Destruction of the cell wall can result in osmotic lysis.
  19. Lysis
    1. When holes are punched in the cell wall (e.g., as following lysozyme digestion), the plasma membrane is extruded and breaks, thus killing the cell.
    2. The cell envelope-destruction associated cell death is called lysis.
  20. Osmotic lysis
    1. The interior of bacteria tends to display a pressure (actually, osmotic pressure) in excess of the extracellular environment (i.e., the inside of cells are more concentrated solutions, generally, than the outside due to the active pumping--concentrating--of substances into cells).
    2. The lysis associated with cell wall destruction and osmotic over-pressure is called osmotic lysis.
    3. See illustration below for a view of osmotic pressure acting on an intact bacterial cell.
  21. Plasmolysis
    1. Essentially the opposite of osmotic lysis. Plasmolysis is the shrinking of the cytoplasmic membrane away from an intact cell wall or just plain shrinkage in the absence of a cell wall (i.e., see protoplast and spheroplast).
    2. See illustration below.
  22. Illustration, movement of water
  23. Protoplast
    1. Osmotic lysis can be avoided even given complete cell wall destruction by suspending such cells in an isotonic solution.
    2. Such not lysed cells lack a cell wall, are alive, capable of replication, and are called protoplasts.
    3. In cellular anatomical terms, the protoplast is all of the cell from the plasma membrane inward (i.e., the plasma membrane plus the cytoplasm).
  24. Spheroplast
    1. The gram-negative equivalent of protoplasts.
    2. Spheroplasts are called something other than protoplast because the cell wall tends not to be destroyed by lysozyme treatment (because it is better protected from lysozyme; aside, this is true unless the outer membrane is first disrupted as it can be via EDTA treatment) and, additionally, the outer membrane remains intact.
  25. Links
    1. Life (Molecular Biology for Beginners)
  26. Cell theory [cell doctrine]
    1. Living things are composed of cells or the products of cells.
    2. Cells are the smallest and least complex units which life is composed which possess all of the characteristics attributed to modern life forms.
    3. All modern cells arise only from the replication (or transformation) of preexisting cells.
  27. Cells
    1. What is a cell?
      1. A cell is
        1. is membrane enclosed
        2. is metabolically active
        3. contains some form of hereditary material which is capable of replication*
        4. conforms to the additional basic tenets of cell theory
      2. *In all instances known, that hereditary material is DNA.
  28. Vocabulary
    1. Acid-fast cell envelope
    2. Cell envelope
    3. Cell wall
    4. Gram-negative
    5. Gram-negative cell envelope, illustration
    6. Gram-positive
    7. Gram-positive cell envelope, illustration
    8. Inner membrane
    9. Lipid A
    10. Lipopolysaccharide
    11. LPS
    12. Lysis
    13. Lysozyme
    14. Movement of water, illustration
    15. O polysaccharide
    16. Osmotic lysis
    17. Outer membrane
    18. Peptidoglycan
    19. Periplasmic space
    20. Plasma membrane
    21. Plasmolysis
    22. Protoplast
  29. Practice questions
    1. Permanent breakage of a bacterial plasma membrane leads to ________________? [PEEK]
      1. Plasmolysis
      2. cell death
      3. binary fission
      4. cell wall damage
      5. all of the above
      6. none of the above
    2. Gram-positive bacteria have thick ones, Gram-negative bacteria have relatively thin ones which are covered by a second lipid bilayer, and both consist predominantly of a substance which is not the same as that which makes up these structures in fungi and plants. I am describing __________. (circle one correct answer) [PEEK]
    3. nuclear membranes.[PEEK]
      1. cell envelopes.
      2. cell walls.
      3. cytoplasmic membranes.
      4. all of the above.
      5. none of the above.
    4. We have discussed a number of types of Eubacteria in class, specifically formal or informal taxonomic categories which group bacteria evolutionarily above the level of genera. Name three such categories. (note that Archaeobacteria, Eubacteria, bacteria, any binomial or individual genera, and cell shapes are all not answers to this question---and if you think that this question is difficult, you may be reading too much into it)? [PEEK]
    5. Other than that one strain is gram-negative and the other gram-positive by crystal violet, iodine, and alcohol destaining, name three significant differences between the gram-positive and gram-negative cell envelopes. Be sure to tell me which aspect goes with which organism, and then describe the feature as it appears (or does not) in the other organism. [PEEK]
    6. Name a functional reason for the existence of outer membranes? [PEEK]
    7. Mycoplasma spp. lack cell walls. How might lacking a cell wall limit the environmental range of a microorganism (short answer) [PEEK]
    8. Lipid A is found anchored to what cell structure (LPS is not the answer)? (choose best answer) [PEEK]
      1. plasma membrane
      2. cell wall
      3. periplasm
      4. outer membrane
      5. all of the above
      6. none of the above
    9. Name a difference between gram-negative and gram-positive bacteria which accounts for the latter being more susceptible to inhibitors of cell wall synthesis such as penicillin. (choose best answer) [PEEK]
      1. plasma membrane
      2. cell wall
      3. periplasm
      4. outer membrane
      5. all of the above
      6. none of the above
    10. Which tends not to be a characteristic of gram-positive bacteria treated with lysozyme? (circle correct answer) [PEEK]
      1. spheroplast
      2. protoplast
      3. osmotic lysis
      4. cell death
      5. all of the above
      6. none of the above
    11. Show where LPS is located by drawing and labeling a cross section of an appropriate bacteria plasma membrane and cell envelope. Be sure to distinguish the cytoplasm from the extracellular volume (one word answer) [PEEK]
    12. Another name for murein is? [PEEK]
    13. Describe what would happen to a gram-positive cell which is treated with lysozyme in a hyperosmotic environment. [PEEK]
    14. Name a cell envelope component completely lacking in gram-negative cells but typically present in gram-positive cells (hint: periplasm is not the answer; I'm looking for qualitative difference, it's either there or it's not, not just a quantitative difference). [PEEK]
    15. Distinguish protoplast from spheroplast. [PEEK]
    16. What type of organisms possess an outer membrane as well as an inner membrane?. [PEEK]
    17. Another name for plasma membrane in gram-negative bacteria is ________. [PEEK]
    18. Name the two things to which the gram-negative bacteria Lipid A is typically found attached to, adhered to, or as an integral part of. [PEEK]
  30. Practice question answers
    1. ii, cell death
    2. iii, cell walls.
    3. possible categories include cyanobacteria, mitochondria, Cloroplasts, gram-positive bacteria, gram-negative bacteria, acid-fast bacteria, etc.
    4. gram-negatives: outer membrane, thin cell wall, significant periplasmic space, no teichoic acids, periplasmic enzymes, LPS, endotoxin, lipid A, O-polysaccharide; gram-positive: no outer membrane, thick cell wall, insignificant periplasm, teichoic acids, no periplasmic enzymes, no LPS, generally no endotoxin, no lipid A, no O-polysaccharide.
    5. Protection from toxins and degradants, retention of digestive enzymes.
    6. Unless they come up with an alternative means of preventing osmotic lysis (as have, by the way, Mycoplasma spp.) they will have to avoid hyperosmotic environments.
    7. iv, outer membrane
    8. iv, outer membrane
    9. i, spheroplast, it's a characteristic of gram-negatives.
    10. Draw a gram-negative inner membrane, followed by periplasmic space, followed by thin cell wall, followed by outer membrane (both membranes lipid bilayers). The LPS is found in the outer leaflet of the outer membrane with the Lipid A portion found in the membrane and the O polysaccharide found external to the cell, attached to Lipid A.
    11. peptidoglycan.
    12. The cell wall would be digested away and the protoplast would shrivel, just as a red blood cell would under similar conditions.
    13. teichoic acids
    14. protoplasts are formed from gram-positive cells while spheroplasts are formed from gram-negative cells
    15. outer membranes are a characteristic of gram-negative eubacteria
    16. inner membrane, cytoplasmic membrane
    17. O polysaccharide, the outer membrane, LPS
  31. References
    1. Black, J.G. (1996). Microbiology. Principles and Applications. Third Edition. Prentice Hall. Upper Saddle River, New Jersey. pp. 78-82, 84-90.
    2. Campbell, N.A. (1996). Biology. Fourth Edition. Benjamin/Cummings Pub. Co. Menlo Park, CA. p. 498-499.
    3. Crabbe, J. (1996). After the genome. Science 274:???
    4. Gould, S.J. (1993). Prophet for the Earth. Nature 361:311-312.
    5. Prescott, L.M., Harley, J.P., Klein, D.A. (1996). Microbiology. Third Edition. Wm. C. Brown Publishers, Dubuque, IA. pp. 40-72.
    6. Raven, P.H., Johnson, G.B. (1995). Biology (updated version). Third Edition. Wm. C. Brown publishers, Dubuque, Iowa. pp. 78-85.
    7. Stryer, L. (1981). Biochemistry. Second Edition. W.H. Freeman and Company, San Fransisco, CA, p. 662.
    8. Tortora, G.J., Funke, B.R., Case, C.L. (1995). Microbiology. An Introduction. Fifth Edition. The Benjamin/Cummings Publishing, Co., Inc., Redwood City, CA, pp. 70-90.