1. WEEK 3 & 4: PROKARYOTE AND EUKARYOTE CELL 8 & 15 MARCH 2012 Khadijahhanim@unimap.edu.my

2. Prokaryotes- Bacteria and Archaea  Bacteria and Archaea always refer as prokaryotes  Prokaryotes lack a membrane-bound nucleus, a cytoskeleton, membrane-bound organelles and internal membranous structures such as endoplasmic reticulum and Golgi apparatus.  prokaryotes differ from eukaryotes in size and simplicity  most lack internal membrane systems  term prokaryotes is becoming blurred  this text will use Bacteria and Archaea

3. Size, shape and arrangement  The most common shapes in both domain: Cocci, rods  Cocci- roughly spherical cells. - Exist singly - Arrangements; diplococcus- when cocci divide and remain together to form pairs. - In chains; eg: Streptococcus, Enterococcus and Lactococcus. - Grapelike clumps: Staphylococcus - Tetrads: square groups of 4 cells; Micrococcus. - Sarcinae: cubic configurations of eight cells

5.  Rod shapes/bacili  Differ in their length-to-width ratio.  Coccobacili- short and wide, resemble cocci  Shape of rod’s end varies between species- may be flat, rounded, cigar-shaped or bifurcated.  Some occurs singly or can remain together to form pairs/chains.  Eg of chain rods; B. megaterium

6.  Other bacterial cell shapes and arrangements:  Vibrios: closely resemble rods; comma-shaped.  Spirilla are rigid, spiral-shaped cells that usually have tufts of flagella at one or both ends.

7.  Spirochetes are flexible, spiral-shaped bacteria that have a unique, internal flagellar arrangement  These bacteria are distinctive and belong to single phylum, Spirochaetes.  Actinomycetes form long filaments called hyphae. Hyphae may branch to produce mycelium; similar to filamentous fungi

8.  Some bacteria are pleomorphic- variable in shape and lacking a single, characteristic form.  Archaea – curved rods and spiral shapes.  Some has unique shapes such as the branched form of Thermoproteus tenax.  Some are flat, Haloquadratum walsbyi.

9. Size  Bacterial and archaeal cells vary in size as much as in shape.  smallest – 0.3 (Mycoplasma)  average rod – 1.1 - 1.5 x 2 – 6 μ m (E. coli)  very large – 600 x 80 μ m Epulopiscium fishelsoni

11. Size-shape relationship  Being small increases the surface area-to-volume ratio (S/V ratio)  As ratio increases- nutrient uptake and diffusion of molecules within the cell become more efficient, facilitates a rapid growth rate.  Shape also effects the S/V ratio.  A rod with the same volume as coccus has higher S/V ratio.  Rod can have better nutrient flux across its plasma membrane.

12.  Surface area = 4r π 2  Volume = 4/3 π r 2

13. Cell organization  Bacteria and Archaea share a common cell organization

15.  Bacterial and archaeal cells are surrounded by several layers called cell envelope.  Innermost layer- plasma membrane, surrounds the cytoplasm and its contents.  Chemically complex cell wall that covers the plasma membrane.  Many bacteria are surrounded by a capsule or slime layer external to cell wall.  Genetic material is localized in discrete region- nucleoid and not seperated from surrounding cytoplasm.

16.  Ribosomes and inclusions are scattered in the cytoplasm.  Flagella- use for locomotion.

17. Eukaryotic cell structure  Eukaryotic microbes: protists and fungi  Membrane-delimited nuclei  Membranes play a prominent part in the structure of many other organelles.  Organelles are intracellular structures that perform specific functions in cells.

20.  The partitioning of the eukaryotic cell interior by membranes makes possible the placement of different biochemical and physiological functions.  Biochemical reactions can take place simultaneously under independent control and coordination.  The intracytoplasmic membrane complex also serves as a transport system to move materials between different cell locations.

21. Bacterial Cell Envelopes  Cell envelope = plasma membrane and all surrounding layers external to it.  For most bacteria, cell envelopes consist of plasma membrane, cell wall and extra additional layer.

22. Plasma membrane  Plasma membranes are an absolute requirement for living organisms because: - Nutrients uptake and elimination of wastes - to maintain their interior in a constant, highly organized state in the face of external changes.  Plasma membrane encompasses the cytoplasm of all cells.

23. Plasma membrane: Functions  separation of cell from its environment  selectively permeable barrier  some molecules are allowed to pass into or out of the cell  transport systems aid in movement of molecules  detection of and response to chemicals in surroundings with the aid of special receptor molecules in the membrane

24.  location of crucial metabolic processes - respiration, photosynthesis and the synthesis of lipids and cell wall constituents.  Bacteria and Archaea do not carry out endocytosis- the nutrients they need and the wastes they dispose must cross the plasma membrane with assistance of transport system for nutrient uptake, waste secretion and protein secretion.

25. Fluid Mosaic model of Bacterial Membrane Structure

26. Membrane structure  Fluid mosaic model: Singer and Nicholson  They proposes that membranes are lipid bilayers within which proteins float.  Most membrane-associated lipids (e.g phospholipid) are amphipathic- structurally asymmetric with polar and nonpolar ends  The polar ends interact with water and are hydrophilic.  The nonpolar hydrophobic ends are insoluble in water and tend to associate with one another.  In aqueous: amphiphatic lipids interact- lipid bilayer  Hydrophobic ends are buried in the interior away from water.

27.  Two types of membrane proteins: - Peripheral proteins: loosely connected to the membrane and can be easily removed. Soluble in aqueous and make 20-30% of total membrane protein. - Integral proteins: embedded within the membrane and not easily removed. Amphiphatic; their hydrophobic region are buried in lipid while hydrophilic portions project from membrane surface.

28. The structure of polar membrane lipid

29. Bacterial Lipids  Bacterial membranes are similar to eukaryotes- lipid bilayers and their amphiphatic lipids are phospholipids.  saturation levels of membrane lipids reflect the environmental conditions such as temperature.  For eg: bacteria growing at lower temp have more unsaturated fatty acids in their membrane phospholipids; 1 or more double bond in the long hydrocarbon chain.  Other factors that effect lipid composition: some pathogens change the lipids in plasma membrane in order to protect themselves from antimicrobial peptides produced by immune system.

30.  Bacterial membrane: - Usually lacking sterols such as cholesterol. - But contain sterol-like molecules- hopanoids. - hopanoids- stabilize the membrane - Hopanoids- formation of petroleum.

31. Membrane steroids and hopanoids

32. Bacterial Cell Walls  Cell wall is the fairly rigid layer that lies just outside the plasma membrane  It help to determine the shape of cell, protect the cell from osmotic lysis, protect cell from toxic substances and in pathogen- can contribute to pathogenicity.  2 types of cell wall based on Gram Stain. - Gram-positive bacteria stained purple - Gram-negative bacteria stained pink/red

33.  Gram-positive cell walls consists of thick homogeneous layer of peptidoglycan (murein) lying outside the plasma membrane.  Gram-negative cell wall is quite complex. Thinner layer of peptidoglycan covered by thick outer membrane. - The walls of gram-positive are more resistant to osmotic pressure- thicker peptidoglycan layer.

35. Peptidoglycan structure  Peptidoglycan- enormous, meshlike polymer composed of many identical subunits.  Each subunit: N-acetyl- glucosamine (NAG) and N- acetylmuramic acid (NAM), and several different amino acids.  Amino acids form a short peptide consisting of 4 alternating D- and L-amino acids

36.  The meshlike peptidoglycan polymer is formed by linking subunits together to form peptidoglycan strand.  These strands are cross- linked to each other.  The backbone: composed of alternating NAG and NAM residues.  Each peptidoglycan strand is helical, and peptides extend out from the backbone at right angles to each other.

37.  Thus, each strand can become crosslinked to strands at each side, above and below.  Many bacteria cross-linked peptidoglycan by connecting the C group of terminal D-alanine (position 4) directly to the amino group og diaminopimelic acid (position 3).  Some, use peptide interbridge instead  Cross-linking results in enormous cross-linking sac.  These sacs- strong enough to retain shape and integrity and yet relatively porous and elastic. Figure 3.13

38.  most gram-negative have amino acid composition and cross-linking as shown in Figure 3.13a.  Gram-positive genera such as Bacillus, Clostridia, Mycobacterium and Nocardia also have composition in Figure 3.13a.  Other gram-positive bacteria substitute the diamino acid lysine for mesodiaminopimelic acid and cross- link chain via interpeptide bridges.

39. Gram-positive cell walls  Composed primarily of peptidoglycan.  Their cell walls usually contain: large amount of secondary polymers including teichoic acids.  Teichoic acid: polymers of glycerol/ribitol joined by phosphate groups.  The teichoic acids are covalently connected to peptidoglycan or to plasma membrane lipids- lipoteichoic acids.  Teichoic acids extent to the surface of peptidoglycan- they give gram +ve cell wall its -ve charge.  Teichoic acid- do not exist in gram –ve.

40. Teichoic acid structure

41.  Functions of teichoic acids: - Create and maintain the structure of the cell envelope - To protect the cell from harmful substances in the environment (eg antibiotics) - Some help pathogenic species to host tissues.

42. Periplasmic space of gram +ve bacteria  lies between plasma membrane and cell wall and is smaller than that of gram-negative bacteria  periplasm has relatively few proteins  enzymes secreted by gram-positive bacteria are called exoenzymes  aid in degradation of large nutrients- to large for transport across the plasma membrane.

43. Gram-negative cell walls  more complex than gram positive  consist of a thin layer of peptidoglycan surrounded by an outer membrane  outer membrane composed of lipids, lipoproteins, and lipopolysaccharide (LPS)  no teichoic acids

44.  peptidoglycan is ~5-10% of cell wall weight  periplasmic space differs from that in gram-positive cells  may constitute 20–40% of cell volume  many enzymes present in periplasm hydrolytic enzymes, transport proteins and other proteins

45.  outer membrane lies outside the thin peptidoglycan layer and is thought to be linked to the cell by Braun’s lipoprotein.  Braun’s lipoproteins connect outer membrane to peptidoglycan.  The small lipoprotein is covalently joined to the underlying peptidoglycan and is embedded in the outer membrane by its hydrophobic end.

47. Lypopolysaccharides (LPSs)  Constituents of the outer membrane  Large and complex molecules contain both lipid and carbohydrate and consists of 3 parts - Lipid A - Core polysaccharide - The O side chain  Lipid A region contain 2 glucosamine sugar derivatives, each with 3 fatty acids and phosphate/pyrophosphate attached.  Lipid A are embedded in outer membrane.

48.  The remainder of LPS molecule projects from the surface  Core polysaccharide- joined to lipid A  The O side chain/ O antigen: polysaccharide chain extending outward from the core.

50. LPSs important functions  contributes to negative charge on cell surface, since the core polysaccaride contains charged sugars and phosphate.  helps stabilize outer membrane structure because lipid A is a major constituent of exterior of outer membrane.  may contribute to attachment to surfaces and biofilm formation  creates a permeability barrier. LPS molecules are thought to restrict entry of bile salts, antibiotics and other toxic substances that might kill the bacterium.  protection from host defenses (O antigen). It elicits an immune response by an infected host. However many gram –ve bacteria can rapidly change their antigenic nature of their O side chain, thus thwarting host defenses.  can act as an endotoxin (lipid A). If lipid A/LPS enters the bloodstream, a form of septic shock develops.

51. Mechanisms of Gram Staining  Gram stain reaction due to nature of cell wall  During the procedure- bacteria are 1 st stained with crystal violet and treated with iodine to promote dye retention.  When bacteria are treated with ethanol in decolorization step, the alcohol shrinkage of the pores of peptidoglycan layer of gram-positive cells  constriction prevents loss of crystal violet during decolorization step  thinner peptidoglycan layer and larger pores of gram-negative bacteria does not prevent loss of crystal violet

52. Osmotic Protection  Microbes have several mechanisms for responding to changes in osmotic pressure.  hypotonic environments  solute concentration outside the cell is less than inside the cell  water moves into cell and cell swells  cell wall protects from lysis  hypertonic environments  solute concentration outside the cell is greater than inside  water leaves the cell  plasmolysis occurs

53. Evidence of Protective Nature of the Cell Wall  Lysozyme attacks peptidoglycan by breaking the bond between N-acetyl glucosamine and N- acetylmuramic acid  penicillin inhibits enzyme transpeptidase which is responsible for making the cross-links between peptidoglycan chain.  if cells are treated with either of the above they will lyse if they are in a hypotonic solution

54. Loss of Cell Wall May Survive in Isotonic Environments  Protoplasts: if gram +ve bacteria are treated with lysozyme/penicillin- complete loss of cell wall. The bacteria become protoplasts.  Spheroplasts: when gram –ve bacteria are exposed to lysozyme/penicillin. The peptidoglycan is lost but the outer membrane remains.  Both protoplast and spheroplast are osmotically sensitive.  Mycoplasma  does not produce a cell wall  plasma membrane more resistant to osmotic pressure

56. Components Outside of the Cell Wall  outermost layer in the cell envelope  Glycocalyx- refers to a layer consisting of a network of polysaccharides extending from the surface of the cell.  capsules and slime layers  S layers  Glycocalyx aid in attachment to solid surfaces  e.g., biofilms in plants and animals

57. Capsules  usually composed of polysaccharides  well organized and not easily removed from cell  visible in light microscope  Capsules are not required for growth and reproduction  protective advantages  Help pathogenic bacteria resistant to phagocytosis. Eg S. pneumoniae. When it lacks a capsule, can be destroyed easily and does not cause disease.  protect from dessication- contain water.  exclude viruses and detergents

59. Slime Layer  similar to capsules except diffuse, unorganized and easily removed  Composed of polysaccharide but not easily observed by light microscopy.  slime may aid in motility

60. S-layers  regularly structured layers of protein or glycoprotein that self-assemble.  in gram-negative bacteria the S layer adheres to outer membrane  in gram-positive bacteria it is associated with the peptidoglycan surface  Their roles: - Protecting cell against ion and pH fluctuations, osmotic stress, enzymes or predacious bacteria - Helps maintain the shape and envelope rigidity - Promote cell adhesion to surfaces - Protect bacterial pathogens against host defense, contribute to their virulence.

62. Archaeal Cell Envelopes  differ from bacterial envelopes in the molecular makeup and organization  S layer may be only component outside plasma membrane  some lack cell wall but have glycocalyx lying outside the cell membrane.  capsules and slime layers are rare

63. 63 Figure 3.28

64. Archaeal Plasma Membranes  composed of unique lipids  isoprene units (five carbon, branched)  ether linkages rather than ester linkages to glycerol  some have a monolayer structure instead of a bilayer structure

66. Eukaryotic cell envelopes  Eukaryotic differ greatly from Bacteria and Archeae in structures they have external to plasma membrane.  Many are lack of cell wall.  If the cell wall is present, their composition is different.  The plasma membrane of eukaryotes is lipid bilayer composed of high proportion of sphingolipids and sterols  This contribute to the strength of the plasma membrane despite the lack of cell wall.

67.  Chemical composition of cell wall are varies  Cell wall of photosynthetic protists, commonly algae: layered appearance and contain large quantities of polysaccharides such as cellulose and pectin  Fungal cell wall normally rigid.  Composition: normally cellulose, chitin, glucan.

68. Cytoplasmic Matrix  The plasma membrane and everything within is called the protoplast  Cytoplasm is the material bounded by the plasma membrane.  is a major part of the protoplasm  substance in which nucleoid, ribosomes and inclusion bodies are suspended  lacks organelles bound by unit membranes  composed largely of water

69. Inclusions  granules of organic or inorganic material that are stockpiled by the cell for future use  Inclusions- form of granules, crystals or globules  Common storage inclusions: glycogen inclusion, PHA granules, sulfur globules and polyphosphate granules.  some are enclosed by a single-layered membrane  membranes vary in composition- proteins or protein and phospholipids  may be referred to as microcompartments

70. Storage Inclusions  storage of nutrients, metabolic end products, energy, building blocks  glycogen storage  carbon storage  poly- β -hydroxybutyrate (PHB)  phosphate - Polyphosphate (Volutin)  amino acids - cyanophycin granules

72. Ribosomes  Site of protein synthesis and large no. of them are found in nearly all cells.  The cytoplasmic ribosomes synthesize protein destined to remain within cell  Plasma membrane-associated ribosomes- proteins that reside in the cell envelope/transport outside.

73. Ribosomes  complex structures  consisting of protein and RNA  sites of protein synthesis  entire ribosome  bacterial and archaea ribosome = 70S (50S and 30S)  eukaryotic (80S) S = Svedburg unit  bacterial and archaeal ribosomal RNA  16S small subunit  23S and 5S in large subunit  archaea has additional 5.8S (also seen in eukaryotic large subunit)  proteins vary (bacteria has 55 proteins, archeae has 68 and eukaryotic has 78 proteins)  archaea more similar to eukarya than to bacteria

75. Nucleoid  irregularly shaped region in bacteria and archaea  usually not membrane bound (few exceptions)  location of chromosome and associated proteins  usually 1  a closed circular, double-stranded DNA molecule  Bacterial and archeael chromosomes are longer than the length of the cell  supercoiling and nucleoid proteins (HU) probably aid in folding

77. Plasmids  extrachromosomal DNA  found in bacteria, archaea, some fungi  usually small, closed circular DNA molecules  exist and replicate independently of chromosome  episomes – may integrate into chromosome  contain few genes that are non-essential  confer selective advantage to host (e.g., drug resistance)

78.  may exist in many copies in cell- may present at concentrations of 40 or more per cell.  Single-copy plasmid produce 1 copy per cell.  inherited stably during cell division but they are not normally apportioned into daughter cells and sometimes are lost.  curing is the loss of a plasmid- occur spontaneous or induced by treatment that inhibit plasmid replication.  classification of plasmids based on mode of existence, spread, and function

79.  Conjugative plasmid- can transfer copies of themselves to other bacteria during conjugation.  Bacteriocin-encoding plasmid (Col Plasmids): bacteria that harbors them can destroy other, closely related bacteria.  Col plasmids contain genes for the synthesis of bacteriocins known as colicins.  Virulence plasmid: encode factors that make their hosts more pathogenic. Eg enterotoxigenic E. coli srains that causes diarrhea.  Metabolic plasmid: carry genes for enzymes that degrade substances such as aromatic compounds, pesticides and sugars

81. External Structures  extend beyond the cell envelop in bacteria and archaea  function  protection, attachment to surfaces, horizontal gene transfer, cell movement  pili and fimbriae  flagella

82. Pili and Fimbriae  fimbriae (s., fimbria); pili (s., pillus)  short, thin, hairlike, proteinaceous appendages (up to 1,000/cell) but only visible under EM  mediate attachment to surfaces ie host tissues  some (type IV fimbriae) required for motility or DNA uptake during bacterial transformation  sex pili (s., pilus)  similar to fimbriae except longer, thicker, and less numerous (1-10/cell)  genes for formation found on plasmids  Are required for conjugation

83. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 83 Figure 3.40

84. Flagella  threadlike, locomotor appendages extending outward from plasma membrane and cell wall  functions  motility and swarming behavior  attachment to surfaces  may be virulence factors

85. Bacterial Flagella  thin, rigid protein structures that cannot be observed with bright-field microscope unless specially stained. The detailed structure can be observed using EM.  ultrastructure composed of three parts: - Filament - Basal body - hook  pattern of flagellation varies

86. Patterns of Flagella Distribution  monotrichous – one flagellum  polar flagellum – flagellum at end of cell  amphitrichous – one flagellum at each end of cell  lophotrichous – cluster of flagella at one or both ends  peritrichous – spread over evenly entire surface of cell

87. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 87 Figure 3.41

88. Three Parts of Flagella  Filament  longest and most obvious  extends from cell surface to the tip  hollow, rigid cylinder  composed of the protein flagellin  some bacteria have a sheath around filament, V. cholerae- lipopolysaccharide sheath  hook  A short, curved segment and wider than filament  Made of different protein subunits  links filament to basal body

89.  basal body  Embedded in the cell  series of rings that drive flagellar motor  Gram –ve has MS ring, C ring, L ring and P ring  Gram +ve has 2 rings, an inner ring connected to plasma membrane and outer ring attached to peptidoglycan

90. Figure 3.42

91. Differences of Archaeal Flagella  flagella thinner  more than one type of flagellin protein  flagellum are not hollow  hook and basal body difficult to distinguish  more related to Type IV secretions systems- type IV bacterial pili  growth occurs at the base, not the end

92. Figure 3.44

93. Motility  Bacteria and Archaea have directed movement  4 major methods of movement in bacteria: - Swimming movement conferred by flagella - The corkscrew movement of spirochetes - The twitching motility associated with type IV pili - Gliding motility  They do not move aimlessly; they move towards nutrients  chemotaxis  move toward chemical attractants such as nutrients, away from harmful substances  move in response to temperature, light, oxygen, osmotic pressure, and gravity

94. Bacterial Flagellar Movement  flagellum rotates like a propeller  very rapid rotation up to 1100 revolutions/sec  The direction of flagellar rotation determines the nature of bacterial movement  in general, counterclockwise (CCW) rotation causes forward motion (run)  in general, clockwise rotation (CW) disrupts run causing cell to stop and tumble

95. 95 Figure 3.45

96. Spirochete Motility  multiple flagella form axial fibril which winds around the cell  Flagella do not extend outside the cell wall but flagella remain in periplasmic space inside outer sheath  Thought to rotate like the external flagella causing corkscrew shape exhibits flexing and spinning movements  Flagellar rotation may also flex and bend the cell- creeping and crawling movement observed when spirochetes in contact with solid surface.

97. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 97 Figure 3.47

98. Twitching and Gliding Motility  may involve Type IV pili and the production of slime  twitching  pili at ends of cell involve in twitching  Characterized by short, intermittent, jerky motions  cells are in contact with each other on a very moist surface  gliding  smooth movements

99. Myxococcus xanthus Movement  social  Type IV pili move together in large groups of cells  adventurous (Gliding)  Observed when single cells move independently  Hypothesis 1:cells contain pores through which slime is released and moves cell forward  Hypothesis 2: adhesion complexes are located along the length of cell and that these attach the cell to surface. adhesion complexes move in track provided by cytoskeleton.

100. Figure 3.48

101. Chemotaxis  movement toward a chemical attractant or away from a chemical repellent  If bacteria are placed in the centre of a dish of semisolid agar containing an attractant, the bacteria will exhaust the local supply of the nutrient and swim outward following the attractant gradient they have created.  Results in expanding ring of bacteria

102.  When a disk of repellent is placed in a petri dish of semisolid agar and bacteria, the bacteria will swim away from the repellent, creating a clear zone around the disk The bright disks are plugs of concentrated agar containing acetate that have been placed in diluted agar inoculated with E. coli. Acetate conc increases from 0 at top right to 3M at top left.

103.  Attractants and repellents are detected by chemoreceptors.  Chemoreceptors; protein that bind chemicals and transmit signals to other components of the chemosensing system  In an absence of chemical gradient- bacteria move randomly switching back and forth between a run and tumble.  During a run, the bacterium travels in a straight or curvy line.  After a few seconds, flagella ‘fly apart’ and the bacterium stops and tumbles  Tumbles change the orientation of bacterium and facing different direction

104.  When bacterium is exposed to attractant, it tumbles less frequently-having longer run when travelling towards attractant.  Over time, bacterium get closer to the attractant.

105. Figure 3.50

106. The Bacterial Endospore  complex, dormant structure formed by some bacteria, survival mechanism  various locations within the cell  resistant to numerous environmental conditions  heat  radiation  chemicals  desiccation

107. Figure 3.51

108. Endospore Structure  spore surrounded by thin covering called exosporium  A coat lies beneath the exosporium. Composed of several protein layers and fairly thick.  cortex, beneath the coat, thick peptidoglycan  core has nucleoid and ribosomes but very low water content and metabolically inactive

109. Figure 3.52

110. Sporulation  process of endospore formation  occurs in a hours (up to 10 hours)  normally commences when growth ceases because of lack of nutrients  complex multistage process

111. Figure 3.53

112. Comparison of Prokaryotic and Eukaryotic Cells