1. Bacterial Cell Structures Stijn van der Veen

2. How do I know what bacterium makes my patient ill?  Bacterial species can be differentiated by:  Morphology (shape)  Composition (cell envelope and other structures)  Metabolism & growth characteristics  Genetics

3. Bacteria are tiny!!!!!  Comparing bacteria with a football is comparing a football with Mount Everest. 10 -6 10 -1 10 4 meter So, what do we need? Microscopes!!!!!!!

4. Optical Methods  The light microscope  Magnification: 1000x (100x obj. + 10x oc.)  Phase contrast microscope  Observe living cells.  Fluorescence microscope  Observe fluorescent dyes or proteins

5. Optical Methods  Confocal microscope  Provide three-dimensional images in multiple layers (z-stack)  Electron microscopes  Transmission electron microscope (TEM) can resolve particles with 1 nm in size  Scanning electron microscope (SEM) can provide three- dimensional images

6. Differentiating bacterial species  Morphology (shape)  Composition (cell envelope and other structures)  Metabolism & growth characteristics  Genetics

7. Shape (morphology) of bacteria  Spherical (coccus)  Rod (bacillus)  Twisted (spiral)

8. Morphology  Bacteria are unicellular… but they can stick together!!

9. Spherical-shaped bacteria Cocci may remain attached after cell division. These group characteristics are often used to help identify certain cocci.  Cocci that remain in pairs after dividing are called diplococci.  Cocci that remain in chains after dividing are called streptococci.  Cocci that divide in two planes and remain in groups of four are called tetrads.  Cocci that divide in three planes and remain in groups cube like groups of eight are called sarcinae.  Cocci that divide in multiple planes and form grape like clusters or sheets are called staphylococci.

10. Rod-shaped bacteria Bacilli only divide across their short axis.  Most bacilli appear as single rods.  Diplobacilli appear in pairs after division.  Streptobacilli appear in chains after division.  Some bacilli are so short and fat that they look like cocci and are referred to as coccobacilli.

11. Twisted bacteria Spiral bacteria have one or more twists.  Vibrios look like curved rods.  Spirilla have a helical shape and fairly rigid bodies.  Spirochetes have a helical shape and flexible bodies.

12. Other shapes…  Some odd types…that you (as doctor) would generally never encounter!!

13. Differentiating bacterial species  Morphology (shape)  Composition (cell envelope and other structures)  Metabolism & growth characteristics  Genetics

14. Prokaryote vs. Eukaryote

15. Bacterial cell structures overview  Basic structures  Cell membrane  Cell wall  Cytoplasm  Chromosome  Ribosome  Specific structures  Plasmid  Flagellum  Pilus  Capsule  Inclusion  Endospores (not shown)

16. Staining methods  Methods to study bacterial morphology and composition:  Common differential staining methods  Gram stain （ Gram-positive vs. Gram-negative)  Acid-fast stain (Mycobacteria)  Special staining methods  The spore staining methods  The flagella staining methods  The capsule staining methods  DNA staining methods

17. Bacterial cell structures overview  Basic structures  Cell membrane  Cell wall  Cytoplasm  Chromosome  Ribosome  Specific structures  Plasmid  Flagellum  Pilus  Capsule  Inclusion  Endospores (not shown) Cell envelope

18.  The cell envelope consists of the cell membrane, cell wall, and associated structures  Bacterial cell envelopes fall into two major categories  Gram positive & Gram negative  This is based on Gram staining characteristics that reflect major structural differences between the two bacterial groups.

19. 1884: Christian Gram: First publication for the Gram stain method Gram-positive cocci Gram-negative bacilli Gram stain

20. Gramm staining procedure  Crystal violet (1 min) => rinse  Iodine (1 min) => rinse  Acetone / Alcohol (10–15 sec) => rinse  Safrinin (1 min) => rinse & dry

21. Simplified diagram and electronic microscopy pictures of the cell envelope of G+ and G- bacteria (murein = peptidoglycan) Cell envelope

22. Gram-positive cell envelope

23. Gram-negative cell envelope

24. Peptidoglycan  The peptidoglycan is a single bag-shaped, highly cross-linked macromolecule that surrounds the bacterial cytoplasmic membrane and provides rigidity (which decides the shape of a bacterium).  It is huge (billions in molecular weight).  Peptidoglycan is found in all eubacteria except Chlamydia and Mycoplasma.

25. Peptidoglycan structure  Glycan (polysaccharide) backbone of alternating residues of N-acetyl muramic acid and N-acetyl glucosamine connected by  -1,4 linkage.  Tetrapeptide side chains usually containing D- and L- amino acid residues, and in some instances diaminopimelic acid (DAP) residues.  The side chains are cross-linked by peptide bridges. These peptide bridges vary in structure among bacterial species (gram-negative bacteria have no peptide bridges).

26. Function of the cell wall  Maintain the bacterial characteristic shape.  Provide resistance to osmotic changes.  Provide anchoring for surface appendages such as flagella and pili.  Assist in cell division

27. The effect of lysozyme on the cell wall  Lysozyme can cut the  -1,4 linkage.  So lysozyme can kill G+ and G- bacteria by destroying their glycan backbone.

28. Effect of penicillin on the cell wall  Penicillin can block the linkage of tetrapeptide side chains and peptide bridges.  So penicillin can kill bacteria by inhibiting their peptidoglycan synthesis.  But…only replicating/growing bacteria are killed.

29. Characteristics of gram-positive cell wall  G ram-positive cell wall is typically 20-80 nm thick.  It contains 15-50 peptidoglycan layers.  It may contain additional components such as teichoic acids and proteins  Teichoic acids are water-soluble polymers of polyphosphates.  Wall teichoic acids are linked to the peptidoglyacan.  Lipoteichoic acids are anchored in the cytoplasmic membrane.

30.  Gram-positive cell wall is thin: 10-15 nm.  It only contains 1-2 peptidoglycan layers  No teichoic acids. Characteristics of gram-negative cell wall

31. phospholipids Gram negative outer membrane  Outer membrane consists of lipopolysaccharide (LPS) and phospholipids.  It contains lipoproteins such as porins.  Porins form channels to allow passage of small hydrophilic nutrients (such as sugars, amino acids and certain ions) through the outer membrane.

32. Lipopolysaccharide (LPS)  LPS is an endotoxin because it is poisonous to mammalian cells.  LPS consists of 3 regions  O antigen: highly variable polysaccharide region composed of repeating units of specific monosaccharides.  Core polysaccharide: conserved within a genus.  Lipid A: contains β-hydroxy fatty acids (bacteria specific), which display endotoxin activity.  Free lipid A may trigger fever, inflammation, and septic shock

33. Summary PropertyGram positiveGram negative Peptidoglycan layers15-501-2 Peptidoglycan content>50%10-20% Teichoic acids+- Outer membrane-+ lipopolysaccharide-+ Sensitive to penicillinyesLess sensitive Digested by lysozymeyesweakly

34. Mycobacterial cell envelope 1.Outer lipids 2.Mycolic acid 3.Polysaccharides  The Mycobacterial cell envelope is waxy.  This enables Mycobacteria to survive exposure to:  acids  alkalis  detergents  oxidative bursts  lysis by immune system  many antibiotics 5.Molecules involved in evading host immune cells & function. 4.Peptidoglycan 5.Plasma membrane 6.6 &

35. Acid-fast (Mycobacterial) staining procedure  Ziehls carbol fuchsin (3 – 5 min heat) => rinse  Acid Alcohol (10 – 15 sec) => rinse  Crystal violet (1 min) => rinse & dry

36. Cell membrane  Separates the cell from its environment  Consists of a phospholipid bilayer  Semipermeable (important for osmosis)  Flexible

37. Cell membrane and osmosis  Osmosis is the diffusion of water across a semi-permeable membrane.  Changes in the bacterial environment such as the amount of dissolved molecules results in changes of the osmotic pressure.  Water will move in or out of the cell.  Cells need water, which is important for many metabolic reactions.  The cell wall will protect the bacteria from exploding when too much water moves into the cell, but bacteria are sensitive to conditions when to much water moves out of the cell (dehydration/desiccation).

38. Cell membrane proteins  Transmembrane proteins, porins, membrane anchored proteins, etc.  Important for many processes  Sensing the environment  Provide active transport across the cell membrane  Proteins  Solutes  Lipids  Cell wall polymers  Generation of energy

39. Bacterial cell structures overview  Basic structures  Cell membrane  Cell wall  Cytoplasm  Chromosome  Ribosome  Specific structures  Plasmid  Flagellum  Pilus  Capsule  Inclusion  Endospores (not shown)

40. Cytoplasm  Jelly intracellular environment composed largely of water (80%), proteins, nucleic acids, lipids, salts, sugars, and various low molecular weight molecules.  The cytoplasm also harbors:  Chromosome  Ribosomes  Inclusions  Plasmids

41. Bacterial cell structures overview  Basic structures  Cell membrane  Cell wall  Cytoplasm  Chromosome  Ribosome  Specific structures  Plasmid  Flagellum  Pilus  Capsule  Inclusion  Endospores (not shown)

42. Chromosome Bacterial chromosome (nucleoid)  Freely floating double stranded DNA (not covered by membrane)  Haploid  More efficient => grows quicker  Mutations allow adaptation to environment  Circular

43. Bacterial cell structures overview  Basic structures  Cell membrane  Cell wall  Cytoplasm  Chromosome  Ribosome  Specific structures  Plasmid  Flagellum  Pilus  Capsule  Inclusion  Endospores (not shown)

44. Ribosome  Translates messenger RNA’s (mRNA) into proteins  Bacterial cell contains multiple copies (usually thousands).  Free floating or attached to cell membrane.  Composed of ribosomal RNA (rRNA) and proteins.  Typically composed of two subunits (large and small)

45. Bacterial cell structures overview  Basic structures  Cell membrane  Cell wall  Cytoplasm  Chromosome  Ribosome  Specific structures  Plasmid  Flagellum  Pilus  Capsule  Inclusion  Endospores (not shown)

46. Plasmids  Small extra-chromosomal double stranded DNA.  Generally circular (in few instances linear).  Usually present in multiple copies.  Capable of self-replication.  Often encode antibiotic resistance markers and virulence factor.  Generally not essential for survival.

47. Bacterial cell structures overview  Basic structures  Cell membrane  Cell wall  Cytoplasm  Chromosome  Ribosome  Specific structures  Plasmid  Flagellum  Pilus  Capsule  Inclusion  Endospores (not shown)

48. Flagellum  Hair-like appendage on the bacterial surface that is responsible for movement (motility).  Consist of various different proteins  The main protein of the filament (flagellin) can be used for identification (H-antigen).  Flagellum-dependent motility is important for virulence and chemotaxis (movement towards food and away from toxics).  Only visible with light microscopy after specific flagellum staining

49. Flagellum structure

50. Flagellum arrangement  Monotrichous: single polar flagellum  Lophotrichous: multiple flagella at single pole  Amphitrichous: flagella at both poles  Peritrichous: flagella distributed all around

51. Flagellum-dependent movement

52. Bacterial cell structures overview  Basic structures  Cell membrane  Cell wall  Cytoplasm  Chromosome  Ribosome  Specific structures  Plasmid  Flagellum  Pilus  Capsule  Inclusion  Endospores (not shown)

53. Pilus (plural pili)  Hair-like appendage on the bacterial surface that is involved in adhesion to host cells, surfaces, and other bacteria.  Composed of several different proteins and the structural protein of the filament is pilin.  Important for virulence.  Two major types can be distinguished:  Common pilus (frequently referred to as fimbria).  Shorter, thinner, and numerous present per bacterium.  Major role in adhering to host cells.  Sex (F) pilus  Longer, broader, and only 1-4 per bacterium  Important for bacterial conjugation (sex).

54. Conjugation Donor Recipient

55. Bacterial cell structures overview  Basic structures  Cell membrane  Cell wall  Cytoplasm  Chromosome  Ribosome  Specific structures  Plasmid  Flagellum  Pilus  Capsule  Inclusion  Endospores (not shown)

56. Capsule and slime layer (glycocalyx)  Layer surrounding the outside of the cell envelope.  Usually composed of polysaccharides, and less frequently of polypeptides, glycoproteins, or glycolipids  Not present in all bacteria and even variable within capsule containing species.  Capsule contributes to virulence of pathogens and protects against phagocytosis and antimicrobial compounds secreted by host cells.

57. Capsule and slime layer  Helps in surface attachment and nutrient absorption, and prevents dehydration.  Not essential for viability.  Capsule: firmly attached and structured layer surrounding cells.  Slime layer: loosely attached unorganized layer surrounding cells

58. Bacterial cell structures overview  Basic structures  Cell membrane  Cell wall  Cytoplasm  Chromosome  Ribosome  Specific structures  Plasmid  Flagellum  Pilus  Capsule  Inclusion  Endospores (not shown)

59. Inclusion  Aggregates of storage molecules, found as small bodies in the cytoplasm.  Consist of organic molecules (such as glycogen or PHB), or inorganic molecules (such as sulfur or polyphosphate).  Inclusions accumulate in conditions of excess nutrients.

60. Bacterial cell structures overview  Basic structures  Cell membrane  Cell wall  Cytoplasm  Chromosome  Ribosome  Specific structures  Plasmid  Flagellum  Pilus  Capsule  Inclusion  Endospores (not shown)

61. Endospore  Highly specialized bacterial cell that is very resistant to extreme conditions such as heat, cold, desiccation, radiation, starvation, etc.  It is produced under unfavorable conditions and enables survival of the species.  Dormant endospores can survive for many years.  Endospores are unable to replicate in this form.  Under favorable conditions, endospores germinate (change) into vegetative (standard) cells again and are able to replicate.  Commonly found in the soil.

62. Sporulation (endospore formation)

63. Endospore structure

64. Serological identification  Serological detection and identification of bacterial cell surface antigens.  Binding of specific antibodies to the cell surface antigens.  Agglutination assays (clumping of bacteria due to antibody binding.  Serotyping (capsule, O/H antigens)  Lancefield grouping (Streptococcus)  Etc.

65. Next lecture  Bacterial Metabolism & Growth Characteristics