CLASS: 9:00 – 10:00Scribe: Adam Baird

DATE: October 18, 2010Proof:

PROFESSOR: YotherBacterial Structure, Physiology, and TaxonomyPage1 of 6

  1. BACTERIAL STRUCTURE, PHYSIOLOGY, AND TAXONOMY [S1]
  2. This is an introductory lecture on bacteria that will lay the foundation for upcoming lectures.
  3. See handout that was emailed. (It will cover much of the same material that is discussed in this lecture, but there are some differences.)
  4. DOMAINS (KINGDOMS) [S2]
  5. Living organisms are divided up into domains (kingdoms) based on evolutionary relationships.
  6. These evolutionary relationships distinguish between eubacteria, or “true bacteria” (which will be emphasized throughout the course) and archaebacteria, which aren’t really prokaryotic or eukaryotic (they have some fundamental features of both, which will be described later). Finally, we have the eukaryotic organisms.
  7. NO TITLE [S3]
  8. The simple difference between eukaryotes and prokaryotes: eukaryotes have a nuclear membrane (“true nucleus”) and prokaryotes have no nuclear membrane (“primitive nucleus”).
  9. Eukaryotes have a nuclear membrane (“true nucleus”), meaning that a membrane surrounds its DNA. Types of eukaryotes:
  10. Animals
  11. Plants
  12. Protists - Simple eukaryotes (algae, fungi, and protozoa); many may actually be pathogenic organisms themselves.
  13. Prokaryotes have no nuclear membrane (“primitive nucleus”). They don’t have a nucleus, but they make up for it. We’ll see how later. Types of prokaryotes:
  14. Eubacteria – True bacteria. Includes most bacteria. Includes all of the bacteria that will be discussed in this course.
  15. Archaebacteria – Primitive bacteria. Some characteristics:
  16. Can grow under very extreme conditions.
  17. Methanogens – Produce methane
  18. Halophiles – Grow in high salt
  19. Thermophiles – Grown at high temperatures
  20. Evolutionarily separated from the eubacteria. The differences:
  21. Archaebacteria lack peptidoglycan (PG), eubacteria have peptidoglycan (PG)
  22. Archaebacteria have ether-linked lipid membranes, eubacteria have ester-linked membranes
  23. Archaebacteria have different ribosome components and metabolism than eubacteria
  24. Archaebacteria share some features with eukaryotes (introns, histones, etc.), which are not found in eubacteria.
  25. DISTINCTIVE FEATURES OF PROKARYOTIC AND EUKARYOTIC CELLS [S4]
  26. Prokaryote Characteristics
  27. Nucleus: No membrane. Always has a single chromosome, which is usually circular. Not all prokaryotes have circular chromosomes though; some may a linear chromosome though, like the prokaryote Borreliaburgdorferi, which causes lyme disease.
  28. Extrachromosomal DNA: Often present in the cytoplasm in the form of plasmids or phage (viruses that infect bacteria).
  29. Oranelles in Cytoplasm: None
  30. Cytoplasmic Membrane: Respiration, secretion, macromolecular synthesis
  31. Cell Wall: Peptidoglycan (absent in Mycoplasma; Mycoplasmais the cause of “walking pneumonia”)
  32. Sterols: Absent (absent in Mycoplasma, which acquires sterols from its eukaryotic host, whereas other bacteria do not)
  33. Ribosomes: Functionally, prokaryotic ribosomes are the same as eukaryotic ribosomes (they both perform protein synthesis); prokaryotic ribosomes are different in size than eukaryotic ribosomes though. Prokaryotic ribosomes are 70S (50S + 30S). The “S” components refer to a svedbergcoeffecient (how it sediments in a centrifugation gradient) or the density of the particle. Notice that if you separate the 50S and the 30S, it is actually greater than the total 70S.
  34. Eukaryote Characteristics
  35. Nucleus: Membrane bound; a number of individual chromosomes.
  36. Extrachromosomal DNA: Some present in organelles.
  37. Organelles in Cytoplasm: Mitochondria (and chloroplasts in photosynthetic eukaryotic organisms)
  38. Cytoplasmic Membrane: Lacks functions that prokaryotic organisms possess
  39. Cell Wall: No peptidoglycan (cellulose and chitin in some eukaryotes though, which act like a cell wall)
  40. Sterols: Usually present
  41. Ribosomes:Functionally, eukaryotic ribosomes are the same as prokaryotic ribosomes (they both perform protein synthesis); eukaryotic ribosomes are different in size than prokaryotic ribosomes though. The holoenzyme of eukaryotic cells is 80S (60S + 40S). The “S” components refer to a svedberg coefficient (how it sediments in a centrifugation gradient) or the density of the particle. Notice that if you separate the 60S and the 40S, it is actually greater than the total 80S.
  42. These characteristics are helpful in distinguishing between prokaryotes and eukaryotes, but they are also important when treating them with antibiotics and using antibacterials. Most of the characteristics that distinguish the prokaryote from a eukaryote are actually the target characteristics for antibiotics and antibacterials.
  43. The ribosome differences, for example, are critical for antibiotics to work.
  44. The peptidoglycan difference (present in prokaryotes, absent in eukaryotes), for example, is another major target for antibacterials. The reason antibacterials work is because they differ between the prokaryote and the eukaryote.
  45. Another slide added. All information can be found in the handout that was emailed. Nomenclature:
  46. Eubacteria
  47. Kingdom Eubacteria
  48. Division
  49. Class
  50. Family
  51. Genus
  52. Species
  53. The further down this list, the more specific the organism is and the more closely related organisms will be.
  54. For example, borelliaburgdorferi is the bacetium that causes lyme disease. It is a member of the genus borellia, it’s species is burgdorferi, and it’s a member of the order and family that belong to the spirochetes.
  55. BACTERIA [S5]
  56. Prokaryotes lack nuclear membrane (unlike eukaryotes).
  57. Single-celled organisms.
  58. For example: Streptococci grow in a chain, but are still considered single cell organisms, because all of the cells are identical; there is only one kind of cell for a given bacteria.
  59. Reproduction occurs by simple division (binary fission, which means that they basically “divide in half”).
  60. For example (drawn on chalkboard): Streptococci were grown in a culture and reacted with a fluorescently labeled antibody (which would react with the surface). So when the antibody reacts with the surface, fluorescents of all of the cells shows. Then the bacteria are removed from the label, washed, and allowed to grow. At the initial microscopic observation, all of the bacteria were fluorescently labeled. As the bacteria start to grow, the new cell wall that is put in is not labeled, so the dark spots are where the bacteria are not labeled. As time goes on, the labeled parts grow further and further apart as more cell divisions occur (the bacteria grown, divided, and inserted new cell walls).
  61. Small, ~1 µm (mycoplasmas as small as 0.2 µm; bacillus as large as 10 µm). Microscope needed for viewing.
  62. When you are in the laboratory and you are sterilizing something with a liquid filter, most of the time, you will use a filter 45 microns in size. The reason: the .45-micron filter will trap things that are larger (and most bacteria are larger than .45 microns.)
  63. Sometimes a .20 or a .18 micron filter will be used.
  64. Various shapes (cocci, rods, coccobacillius, vibrio, spiral, spirochete) and arrangements (chains, clusters)
  65. Example: Streptococci – Spherical organisms that grow in chains
  66. Tetrads – Groups of four
  67. Clusters – Grape-like
  68. All of the properties are important in identification of organisms.
  69. Most are free-living, meaning that they don’t have to have a cell to grow inside of. A few, like rickettsiae or chlamydiae, are obligate intracellular parasites, meaning that when they grow in the host, they will always be found in the cell; they take special conditions in order to grow in the laboratory, requiring cells to help actually culture them.
  70. FIGURE: PROKARYOTIC CELL MORPHOLOGY [S6]
  71. FIGURE: NO TITLE [S7]
  72. FIGURE: NO TITLE [S8]
  73. BACTERIAL CELL [S9]
  74. 50% protein
  75. 20% nucleic acids (10x more RNA than DNA)
  76. Note: when you isolate plasma DNA from the cell, one of the methods may be using ethanol precipitate (to precipitate down the plasma DNA). Most of what precipitates down, however, is RNA, because that’s what is primarily in the cell.
  77. RNA is important for protein synthesis, which occurs often for cell viability.
  78. 10% polysaccharides (seen usually on the cell surface)
  79. 10% lipids
  80. Cell membrane and other structures are made of lipids
  81. Major component of cell
  82. BACTERIAL CHROMOSOMES [S10]
  83. We’re going to start looking more in-depth at the inside of the cell, working outwards.
  84. Singular, usually circular (except for borrelia, which has a linear chromosome), and double-stranded DNA
  85. Haploid
  86. One, single chromosome; they can have more than one copy of that chromosome, but all copies are the same chromosome; the reason that chromosomes are replicated is because of growth preparation and growth rate. The faster the growth rate, the more chromosome copies are needed. They have to have enough chromosomes to be sure that each daughter cell gets a chromosome, and they do that by having multiple copies of chromosome present in the cell during division.
  87. Some bacteria, like E. Coli, can double every 20 minutes. It takes a chromosome 40 minutes to replicate, so they have to start replicating their plasmid long before the time when they will actually divide.
  88. Most bacterial chromosomes are 600 to 4500 kb in size (~1 kb/gene), The size of the chromosome depends on the host that it’s in. The smaller the bacteria chromosome, the more dependent the bacterium is on host/environment.
  89. Recall that mycoplasma does not have a cell and requires sterols from its host. It also requires a lot of things to be grown in the laboratory.
  90. So, bacteria that have these very large chromosomes can actually synthesize most of the things they need from very simple carbon and energy source. Bacteria that have smaller chromosomes don’t have the ability to do that, so you have to give them what they need in order to grow (serum, vitamins, etc.)
  91. Question: Do animal pathogens have smaller genomes in general? No necessarily; more determined by where they can live. E. Coli, for example, can cause urinary tract infections, can cause GI infections, and it normally resides in the gut. E. Coli can also live in dirt, or hamburger meat, or other environments that are not as favorable as the host. So, the ones with the larger chromosomes tend to be able to live in many different environments, and the ones with smaller chromosomes, like mycoplasma, are more restricted to their animal host.
  92. Up to 1mm in length. Recall that the size of the average cell is 1 micron. The reason that such a large chromosome can be fit into such a small bacteria is because of supercoiling (similar to taking a rubber band, stretching it, and then twisting it up into a little ball).
  93. Bacteria chromosomes are contained in the nucleoid
  94. BACTERIAL NUCLEOIDS [S11]
  95. Bacterial nucleoids contain chromosomal DNA (60%), RNA (30%), and Protein (10%)
  96. No nuclear membrane, but the chromosome isn’t just totally loose in the cytoplasm; it’s contained by the nucleoid.
  97. No histones; ~6 chromosome-associated basic proteins involved in determining chromosomal structure
  98. They do have a number of basic proteins that will associate with the chromosome and help determine its structure.
  99. Polyamines, like spermidine and putrescine, are highly charged molecules that will neutralize negative charges on phosphates, allowing a folded position.
  100. Haploid chromosome in cytoplasm (1 -4 nuclear bodies/cell, number depends on growth rate; faster = more)
  101. The DNA can be membrane associated, but that doesn’t mean that a membrane surrounds it; it is never completely bound by a membrane though.
  102. EXTRACHROMOSOMAL DNA[S12]
  103. Main content of the DNA is in the chromosome. Everything essential for its survival is found there. There can be accessory DNA (which will talked about later).
  104. Plasmids replicate in cytoplasm, independent of chromosome.
  105. Usually circular chromosomes (except for borrelia, which has a linear chromosome)
  106. Can be very small. The only thing that is contained there is what is necessary to replicate themselves.
  107. The size indicates the functions that take place; up to several hundred kb. The size indicates the functions that go on.
  108. Many plasmids are important in antibiotic resistance. Ones that are very small may simply code the antibiotic resistance and what’s necessary to replicate itself. Ones that are very large (up to several hundred kbs) may actually encode the genes necessary to transfer themselves (transfer the plasmid from one cell to another cell; known as conjugation; will be talked about later).
  109. Conjugative (F,R), antibiotic resistance, metabolic virulence may all be carried on plasmids.
  110. Bacteriophage (phage) are viruses that infect bacteria
  111. Replicates in cytoplasm or integrates into chromosome
  112. Have many functions
  113. Can contribute to virulence
  114. Neither plasmids nor bacteriophages are required for the bacteria to grow. If they are cultured in a laboratory or a controlled environment, they can grow just fine without their plasmids or their phage. However, because both plasmids and bacteriophages are essential to virulence determinance, the bacteria may not be a very good pathogen if they lack plasmids or bacteriophages.
  115. Many of the toxins (caused by scarlet fever, for example) are actually because a bacteriophage is present in the bacterium. In the absence of the bacteriophage, that same bacterium would not cause disease. So, many toxins are encoded by phage.
  116. NEWLY ADDED SLIDE
  117. See update.
  118. Double stranded, circular, single, supercoiled DNA is located inside the cytoplasm
  119. DNA Polymerase and other enzymes mediate DNA replication.
  120. When the DNA is in the cell, it can be transcribed into mRNA and ultimately translated to protein.
  121. The mRNA, different from the DNA, is single stranded and linear.
  122. DNA transcription is mediated by RNA Polymerase, which recognizes sequences (called promoters) in the DNA. The ribosome, then, binds to the single stranded, linear mRNA, recognizes ribosome binding sites in the mRNA, which allows it to initiate DNA Translation, and catalyzes amino acid additions to ultimately result in protein. If plasmids or phage in the cell that replicate in the cytoplasm, they basically do the same thing as this. They look just like the DNA, only smaller, and are replicated by similar mechanisms.
  123. Because bacteria do not have a nuclear membrane surrounding their DNA, they can do something that eukaryotic organisms cannot do, called coupled transcription/translation. DNA is transcribed into the mRNA by the RNA polymerase and is a start codon for initiation of translation. Ribosomes grow polypeptides along the chain. This allows transcription and translation to occur at the same time, so bacteria can actually start translating before transcription is complete. The reason: there is no nucleus to separate the process. In a eukaryotic cell, because the nuclear membrane is present, transcription occurs in the nucleus, and then the mRNA is translated outside of the nucleus. Question: Can replication and transcription/translation all occur at the same time? In different parts of the chromosome, yes, possibly.
  124. FIGURE [S13]
  125. BACTERIAL STRUCTURE [S14]
  126. CYTOPLASMIC MEMBRANE [S15]
  127. Relatively similar in most bacteria
  128. Lipid Bilayer Characteristics
  129. Permeability barrier
  130. Active transport
  131. Electron transport
  132. Oxidative phosphorylation
  133. Photosynthesis(with all of the accompanying structures also found in the cytoplasmic membrane)
  134. Affected by Antibacterials
  135. Many things that we use as antibacterials will disrupt the membrane. (Antibiotics in hand soap is an “overkill”.)
  136. Detergents
  137. Polymyxins (damaged PE-containing membranes)
  138. Ionophores (disrupt membrane potential)
  139. CELL WALL [S16]
  140. Surrounding the cytoplasmic membrane is the cell wall
  141. The cell wall is entirely responsible for the shape of the bacteria
  142. Example: If you remove the cell wall from bacteria, all the bacteria would be the same shape.
  143. Circular in shape
  144. Serves as a osmotic resistant barrier. If you take bacteria and suspend them in water, they’ll be okay. If you remove the cell wall (enzymatically), and suspend them in water, the cells will immediately lyse. Osmotic resistance is provided by the cell wall.
  145. Comprised of highly crosslinked peptidoglycan
  146. Affected by antibacterials
  147. Basis for gram-stain
  148. PEPTIDOGLYCAN [S17]
  149. Backbone of N-acetyl glucosamine and N-acetyl muramic acid.
  150. Repeating linear structure of these two sugars.
  151. Off the N-acetyl muramic acid is a peptide side chain. These peptides can differ depending on the bacteria, but they are relatively common. There is linkage from the side chain of one muramic acid to the side chain of another muramic acid.
  152. Cross-linked by peptide bridges at MurNAc (from the third amino acid of one to the fourth amino acid of the other, which is important for the overall structure).
  153. If cross-links are broken or they are prevented from forming, lysis of the cell occurs due to osmotic pressure build up (because the cell wall is not structurally in tact).
  154. PEPTIDOGLYCAN[S18]
  155. Gram positive bacteria peptidoglycan
  156. PEPTIDOGLYCANS [S19]
  157. The Glc-NAc – MurNAc makes up the repeating units of the backbone structure
  158. Gram positive and Gram negative bacteria don’t always have the same peptidoglycans
  159. Gram negative cross links are from the third to the fourth amino acid are direct (no bridge between it)
  160. Gram positive cross links have multiple amino acids serving as a cross bridge between amino acids
  161. Enzymes involved:
  162. Transglycosylases (TG) are the enzymes ink the backbone together and form the Glc-NAc – MurNAc structure
  163. Transpeptidases (TP) are the enzymes that link across the peptide side chains
  164. Hydrolases are the enzymes that cleave the backbone (like lysosyme or mutanolysin)
  165. Amidases are the enzymes that cleave the peptide side chain and also help in the building of the cell wall (which must be cleaved or broken to put in new pieces).
  166. Note: Lysozyme, which is present in secretions and tears and other forms of first defenses against bacterial infection, needs to recognize the acetylated group on the backbone. Many bacteria, after synthesizing the cell wall, remove the acetyl group, making it resistant to lysozyme.
  167. Antibiotics to note:
  168. Beta-lactams (like penicillin) resemble the transpeptidase substrates; they ultimately block the cross linking of the growing chain (it is only effective on growing cells). So, penicillin is only effective on growing chains.
  169. Lysosyme can work on both growing and non-growing cells. Lysozyme cleaves the peptidoglycan of growing or non-growing cells, removing the cell wall, leading to cell death.
  170. -LACTAMS AND PEPTIDOGLYCAN CROSSLINKING [S20]
  171. Notice that there are only four amino acids in the cross-linked side chain (the fifth amino acid is cleaved during the cross linking)
  172. Terminal D-ala-D-ala is recognized by transpeptidase and allows for linkage to another side chain
  173. Benzylpenicillin has a beta-lactam ring (as all penecillins do). The beta-lactam ring is similar to the terminal D-ala-D-ala, and so the transpeptidase binds to the beta-lactam ring, so cross linking doesn’t occr
  174. Beta-lactams work because they block cross-linking from occurring. If cross-linking doesn’t occur, the structure is not stable and will ultimately result in lysis of the cell.
  175. The R-group varies in different beta-lactam drugs. Notice that the beta-lactam ring looks very similar to the transpeptidase.
  176. So if a beta-lactam is present, the transpeptidase will recognize and bind to the “wrong thing”; it is bound up by the beta-lactam antibiotic and ends up not being cross-linked to the peptide side chains.
  177. GRAM STAIN [S21]
  178. Peptidoglycan is the basis for gram staining
  179. Shows that gram-positive and gram-negative bacteria are evolutionarily different
  180. Bacteria are placed on slide and then heated, making the bacteria stick to the slide
  181. First stain: Gram’s crystal violet (CV)
  182. Second stain: Potassium-iodide (KI)
  183. The CV and the KI form complexes
  184. Ethanol is added, causing dehydration of the cell wall
  185. The complexes are then washed. (CV-I complexes get trapped in thick cell walls and won’t wash out; if the cells remain purple, the bacteria is Gram positive)
  186. Safranin (red) is then added; counter stain. (Thin cell walls don’t retain CV-I complexes; if the cells remain red, the bacteria is Gram positive)
  187. Notice the difference in between the gram-positive picture and the gram-negative picture. The difference is in the thickness in the cell wall.
  188. EXCEPTIONS TO GRAM-POSITIVE / GRAM-NEGATIVE STAINING [S22]
  189. There are a couple of exceptions to gram-positive / gram-negative staining
  190. Mycoplasmas have no cell wall, so gram-staining won’t work
  191. Mycobacteria (which causes tuberculosis or leprosy) have a highly-lipidated outer surface, which interferes with gram staining. Mycobacteria are actually genetically related to gram-positive bacteria.The bacteria can still be tested with acid fast stain (carbolfuschin retained following decolorization with HCl/EtOH)
  192. Both are related to gram positive bacteria, based on genetic analyses (rRNA sequence)
  193. GRAM POSITIVES [S23]
  194. See Figure
  195. Gram positive organism
  196. Black – Cytoplasmic Membrane surrounded by cell wall
  197. Blue – Cell wall (peptidoglycan); will actually be a lot thicker than shown
  198. Green – Lipoteichoic Acid
  199. Orange – Teichoic Acid
  200. Black – Protiens
  201. GRAM POSITIVES [S24]
  202. Cell Wall
  203. Thick peptidolycan (10 – 100 nm)
  204. Within that cell wall arlteichoic acids (WTA), which are repeating units of phosphodiester-linked glycerol or ribitol backbone + side chains (D-ala, glucose); covalently linked to peptidoglycan structure; probably linked to the mur-NAc
  205. Lipoteichoic acids (LTA) are membrane-anchored, not linked to the petidoglycan; structure may differ from WTA in a typical gram-positive cell.
  206. GRAM POSITIVES [S25]
  207. LTA and WTA lead to ion binding, charge maintenance, membrane integrity, adherence, anchor proteins
  208. Both the LTA and the WTA are highly negatively charged (to maintain surface structure and bind ions, specifically calcium, which is important for protein folding and the structure of the cell). They are also important in adherence and in anchoring proteins.
  209. In gram-positive bacteria, the cell wall is important in inflammatory responses. Example: Cell wall can be injected into meninges of an experimental animal and can develop something that looks like meningitis. So it’s the cell wall that carries the inflammatory characteristics of the cell.
  210. FIGURE: GRAM POSITIVES [S26]
  211. The Mur-NAc repeating structure (Peptidoglycan = Green)
  212. Linkage of the teichoic acids, which are covalently linked to the peptidoglycan. These teichoic acids can have proteins that are linked to the positively charged phosphocholine residues. The peptidoglycan backbone can also have another structure linked to it, called the polysaccharide capsule (linked the Glc-NAc of the backbone).
  213. When you isolate the peptidoglycan, when you isolate the cell wall of a bacterium, it has many things that are present on the cell wall. It’s not just peptidoglycan that can cause infection; it’s everything else that is linked to the peptidoglycan as well.
  214. (Teichoic Acid = Red)
  215. Gram positives may have polysaccharide capsule covalently linked to PG

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