1. Microbial Cell Structure and Function2
Microbial Cell Structure and Function

2. I. Microscopy 2.1 Discovering Cell Structure: Light Microscopy
2.2 Improving Contrast in Light Microscopy

3. 2.1 Some Principles of Light Microscopy
Magnification: the ability to make an object larger
Resolution: the ability to distinguish two adjacent objects as separate and distinct
Resolution is determined by the wavelength of light used and numerical aperture of lens
Limit of resolution for light microscope is about 0.2 μm

4. 2.2 Improving Contrast in Light Microscopy
Improving contrast results in a better final image
Staining improves contrast
Dyes are organic compounds that bind to specific cellular materials
Examples of common stains are methylene blue, safranin, and crystal violet

5. 2.2 Improving Contrast in Light Microscopy
Differential stains: the Gram stain
Differential stains separate bacteria into groups
The Gram stain is widely used in microbiology since 1884 (Figure 2.4a)
Bacteria can be divided into two major groups:
gram-positive and gram-negative
Gram-positive bacteria appear purple, and gram-negative bacteria appear red after staining (Figure 2.4b)

6. Figure 2.4a Procedure Result 1. Flood the heat-fixed
smear with crystal
violet for 1 min
All cells purple
2. Add iodine solution for 1 min
All cells remain purple
3. Decolorize with
alcohol briefly
— about 20 sec
Gram-positive cells are purple; gram-negative cells are colorless
Figure 2.4a The Gram stain.
4. Counterstain with
safranin for 1–2 min G– G+
Gram-positive (G+) cells are purple; gram-negative (G–) cells are pink to red
Figure 2.4a 6

7. Figure 2.4b The Gram stain.
Figure 2.4b 7

8. II. Cells of Bacteria and Archaea
2.5 Cell Morphology
2.6 Cell Size and the Significance of Being Small

9. 2.5 Cell Morphology Morphology = cell shape
Major cell morphologies (Figure 2.11)
Coccus (pl. cocci): spherical or ovoid
Rod: cylindrical shape (bacillus/bacilli)
Spirillum: spiral shape
Cells with unusual shapes
Spirochetes, appendaged bacteria, and filamentous bacteria
Many variations on basic morphological types

10. Figure 2.11 Figure 2.11 Cell morphologies. Coccus Rod Spirillum
Spirochete
Figure 2.11 Cell morphologies.
Stalk
Hypha
Budding and appendaged bacteria
Figure 2.11
Filamentous bacteria

11. 2.5 Cell Morphology
Morphology typically does not predict physiology, ecology, phylogeny, etc. of a prokaryotic cell
May be selective forces involved in setting the morphology
Optimization for nutrient uptake (small cells and those with high surface-to-volume ratio)
Swimming motility in viscous environments or near surfaces (helical or spiral-shaped cells)
Gliding motility (filamentous bacteria)

12. 2.6 Cell Size and the Significance of Being Small
Size range for prokaryotes: 0.2 µm to >700 µm in diameter
Most cultured rod-shaped bacteria are between 0.5 and 4.0 µm wide and < 15 µm long
Examples of very large prokaryotes
Epulopiscium fishelsoni (Figure 2.12a)
Thiomargarita namibiensis (Figure 2.12b)
Size range for eukaryotic cells: 10 to >200 µm in diameter

13. 2.6 Cell Size and the Significance of Being Small
Surface-to-volume ratios, growth rates, and evolution
Advantages to being small (Figure 2.13)
Small cells have more surface area relative to cell volume than large cells (i.e., higher S/V)
Support greater nutrient exchange per unit cell volume
Tend to grow faster than larger cells

14. Figure 2.13 Surface area and volume relationships in cells.

15. 2.6 Cell Size and the Significance of Being Small
Lower limits of cell size
Cellular organisms <0.15 µm in diameter are unlikely
Open oceans tend to contain small cells (0.2–0.4 µm in diameter)

16. III. The Cytoplasmic Membrane and Transport (aka Plasma Membrane)
2.7 Membrane Structure
2.8 Membrane Functions
2.9 Nutrient Transport

17. 2.7 Membrane Structure Cytoplasmic membrane
Thin structure that surrounds the cell
Vital barrier that separates cytoplasm from environment
Highly selective permeable barrier; enables concentration of specific metabolites and excretion of waste products

18. 2.7 Membrane Structure Composition of membranes
General structure is phospholipid bilayer (Figure 2.14)
Contain both hydrophobic and hydrophilic components
Can exist in many different chemical forms as a result of variation in the groups attached to the glycerol backbone
Fatty acids point inward to form hydrophobic environment; hydrophilic portions remain exposed to external environment or the cytoplasm

19. Figure 2.14 Glycerol Fatty acids Phosphate Ethanolamine Hydrophilic
region
Hydrophobic
region
Fatty acids
Figure 2.14 Phospholipid bilayer membrane.
Hydrophilic
region
Glycerophosphates
Fatty acids
Figure 2.14

20. 2.7 Membrane Structure Cytoplasmic membrane (Figure 2.15) 8–10 nm wide
Embedded proteins
Stabilized by hydrogen bonds and hydrophobic interactions
Mg2+ and Ca2+ help stabilize membrane by forming ionic bonds with negative charges on the phospholipids
Somewhat fluid

21. Figure 2.15 Out In Figure 2.15 Structure of the cytoplasmic membrane.
Phospholipids
Hydrophilic
groups
6–8 nm
Hydrophobic
groups In
Figure 2.15 Structure of the cytoplasmic membrane.
Integral
membrane
proteins
Phospholipid
molecule
Figure 2.15

22. 2.7 Membrane Structure Membrane proteins
Outer surface of cytoplasmic membrane can interact with a variety of proteins that bind substrates or process large molecules for transport
Inner surface of cytoplasmic membrane interacts with proteins involved in energy-yielding reactions and other important cellular functions
Integral membrane proteins
Firmly embedded in the membrane
Peripheral membrane proteins
One portion anchored in the membrane

23. 2.7 Membrane Structure Archaeal membranes
Ether linkages in phospholipids of Archaea (Figure 2.16)
Bacteria and Eukarya that have ester linkages in phospholipids
Archaeal lipids lack fatty acids; isoprenes, phytanyls
Major lipids are glycerol diethers and tetraethers (Figure 2.17a and b)
Can exist as lipid monolayers, bilayers, or mixture (Figure 2.17)

24. Figure 2.16 Ester Ether Bacteria Archaea Eukarya
Figure 2.16 General structure of lipids.
Bacteria
Eukarya
Archaea
Figure 2.16

25. Phytanyl
CH3 groups
Glycerol diether
Isoprene unit
Biphytanyl
Diglycerol tetraethers
Crenarchaeol
Out
Out
Figure 2.17 Major lipids of Archaea and the architecture of archaeal membranes.
Glycerophosphates
Phytanyl
Biphytanyl or
crenarchaeol
Membrane protein In In
Lipid bilayer
Lipid monolayer
Figure 2.17

27. 2.8 Membrane Function Permeability barrier (Figure 2.18)
Polar and charged molecules must be transported
Transport proteins accumulate solutes against the concentration gradient
Protein anchor
Holds transport proteins in place
Energy conservation
Generation of proton motive force

28. Figure 2.18 Functions of the cytoplasmic membrane
Permeability barrier:
Prevents leakage and functions as a
gateway for transport of nutrients into,
and wastes out of, the cell
Protein anchor:
Site of many proteins that participate in
transport, bioenergetics, and chemotaxis
Energy conservation:
Site of generation and dissipation of the
proton motive force
Figure 2.18 The major functions of the cytoplasmic membrane.
Figure 2.18

29. 2.9 Nutrient Transport
Carrier-mediated transport systems bring in nutrients (Figure 2.19)
Show saturation effect
Highly specific

30. Transporter saturated
Figure 2.19 Transport versus diffusion.
Simple diffusion
Figure 2.19

31. 2.9 Nutrient Transport
Three major classes of transport systems in prokaryotes (Figure 2.20)
Simple transport
Group translocation
ABC system
All require energy in some form, usually proton motive force or ATP

32. Figure 2.20 In ATP ADP + Pi Out P P Simple transport:
Driven by the energy
in the proton motive
Force (H+ gradient)
Out In
Transported
substance
Group translocation:
Chemical modification
of the transported
substance driven by
phosphoenolpyruvate P R~ P 1 2
Figure 2.20 The three classes of transport systems.
ABC transporter: Binding
proteins are involved
and energy comes
from ATP. 3
ATP ADP + Pi
Figure 2.20

33. 2.9 Nutrient Transport
Three transport events are possible: uniport, symport, and antiport (Figure 2.21)
Uniporters transport in one direction across the membrane
Symporters function as co-transporters
Antiporters transport a molecule across the membrane while simultaneously transporting another molecule in the opposite direction

34. Figure 2.21 Structure of membrane-spanning transporters and types of transport events.

35. 2.9 Nutrient Transport Simple transport:
Lac permease of Escherichia coli
Lactose is transported into E. coli by the simple transporter lac permease, a symporter
Activity of lac permease is energy-driven by proton motive force

36. 2.9 Nutrient Transport
The phosphotransferase (PTS) system in E. coli (Figure 2.22)
Type of group translocation: substance transported is chemically modified during transport across the membrane
Best-studied system
Moves glucose, fructose, mannose, others
Five cytoplasmic proteins required
Energy derived from phosphoenolpyruvate

37. Glucose
Out
Cytoplasmic
membrane
Nonspecific components
Specific components
Enz
IIc
Direction
of glucose
transport PE P
Enz I
HPr
Enz
IIa
Enz
IIb
Pyruvate P P In
Figure 2.22 Mechanism of the phosphotransferase system of Escherichia coli.
Direction of P transfer P
Glucose 6_P
Figure 2.22

38. 2.9 Nutrient Transport
ABC (ATP-binding cassette) systems (Figure 2.23)
>200 different systems identified in prokaryotes
Often involved in uptake of organic compounds (e.g., sugars, amino acids), inorganic nutrients (e.g., sulfate, phosphate), and trace metals
Typically display high substrate specificity
Gram-negatives employ periplasmic substrate-binding proteins and ATP-driven transport proteins
Gram-positives employ fixed substrate-binding proteins and ATP-driven transport proteins

39. Figure 2.23 Mechanism of an ABC transporter.
Figure 2.23 (Gram -)

40. IV. Cell Walls of Bacteria and Archaea
2.10 Peptidoglycan
2.11 LPS: The Outer Membrane
2.12 Archaeal Cell Walls

41. 2.10 Peptidoglycan-the Wonder Wall
Species of Bacteria separated into two groups based on Gram stain reaction
Gram-positives and gram-negatives have different cell wall structure (Figure 2.24)
Gram-negative cell wall
Two layers: LPS and peptidoglycan
Gram-positive cell wall
One layer: peptidoglycan

42. Peptidoglycan-a layer of sugars and amino acids linked together to form a chain-link type structure outside the
cytoplasmic membrane.
NAG and NAM are the sugars

43. Figure 2.24 Cell walls of Bacteria.

44. 2.10 Peptidoglycan Peptidoglycan (Figure 2.25)
Rigid layer that provides strength to cell wall
Polysaccharide composed of:
N-acetylglucosamine and N-acetylmuramic acid
Amino acids
Lysine or diaminopimelic acid (DAP)
Cross-linked for strength
Different cross-linking in gram-negative bacteria and gram-positive bacteria (Figure 2.26)

45. Figure 2.25 NAG and NAM Non-protein Peptide bond Beta 1-4 Glycoside
N-Acetylglucosamine
( G )
N-Acetylmuramic acid
( M )
NAG and NAM
Non-protein
Peptide bond
Beta 1-4
Glycoside
𝛃(1,4)
𝛃(1,4)
𝛃(1,4)
N-Acetyl
group
Glycan tetrapeptide
Lysozyme-
sensitive
bond
Peptide
cross-links
L-Alanine
Figure 2.25 Structure of the repeating unit in peptidoglycan, the glycan tetrapeptide.
D-Glutamic acid
Diaminopimelic
acid
D-Alanine
Figure 2.25

46. Disambiguation
Lysozyme, antimicrobial enzyme, hydrolyzes B 1-4 glycoside
Penicillin inhibits transpeptidation or linking
of glycan chains

47. 2.10 Peptidoglycan Gram-positive cell walls (Figure 2.27)
Can contain up to 90% peptidoglycan
Common to have teichoic acids (acidic substances) embedded in their cell wall
Lipoteichoic acids: teichoic acids covalently bound to membrane lipids

48. Figure 2.27 Teichoic acid and
Peptidoglycan
cable
Teichoic acid and
Lipoteichoic acid-
Structural-linkers and stiffeners
Wall-associated
protein
Teichoic acid
Peptidoglycan
Lipoteichoic
acid
Wall proteins may be enzymes, receptors or attachment molecules
Figure 2.27 Structure of the gram-positive bacterial cell wall.
Figure 2.27
Cytoplasmic membrane

49. A review article for those who wish more reading:
Microbiol Mol Biol Rev Mar; 63(1): 174–229.
PMCID: PMC98962
Surface Proteins of Gram-Positive Bacteria and Mechanisms of Their Targeting to the Cell Wall Envelope
William Wiley Navarre and Olaf Schneewind

50. 2.10 Peptidoglycan Prokaryotes that lack cell walls Mycoplasmas
Group of pathogenic bacteria
Thermoplasma
Species of Archaea

51. 2.10 Peptidoglycan Summary
The cell wall protects the cell
Bacteria that have cell walls have peptidoglycan
Peptidoglycan structure differs from species to species
NAG and NAM are present but peptide part varies

52. 2.11 LPS: The Outer Membrane
Total cell wall contains ~10% peptidoglycan
Most of cell wall composed of outer membrane, aka lipopolysaccharide (LPS) layer
LPS consists of core polysaccharide and O-polysaccharide (Figure 2.28)
LPS replaces most of phospholipids in outer half of outer membrane (Figure 2.29)
Endotoxin: the toxic component of LPS

53. Figure 2.28 Structure of the lipopolysaccharide of gram-negative Bacteria.

54. Figure 2.29 Figure 2.29 The gram-negative cell wall. Out Cell Wall or
O-specific
polysaccharide
Core polysaccharide
Lipid A
Protein
Out
Lipopolysaccharide
(LPS)
Porin
Porin
Outer
membrane
8 nm
Cell Wall or
Envelope
Phospholipid
Peptidoglycan
Peptidoglycan
Periplasm
Lipoprotein
Cytoplasmic
membrane In
Figure 2.29 The gram-negative cell wall.
Outer membrane
Periplasm
Cytoplasmic
membrane
Figure 2.29

55. 2.11 LPS: The Outer Membrane
Periplasm: space located between cytoplasmic and outer membranes (Figure 2.29)
~15 nm wide
Contents have gel-like consistency
Houses many proteins
Porins: channels for movement of hydrophilic low-molecular-weight substances (Figure 2.29c)

56. A link to a course site on the Gram Negative Wall

57. 2.12 Archaeal Cell Walls Some Archaea have cell walls, some do not
When present, walls are different from Bacterial cell walls

58. 2.12 Archaeal Cell Walls S-Layers
Most common cell wall type among Archaea but may also be found in Bacteria
Consist of protein or glycoprotein
Paracrystalline structure (Figure 2.31) or shell
Network of proteins/glycoproteins attached to outer surface of plasma membrane
Has openings or “pores”

59. 2.12 Archaeal Cell Walls No peptidoglycan (no Wonder Wall)
Typically no outer membrane
Pseudomurein aka pseudopeptidoglycan
Polysaccharide similar to peptidoglycan (Figure 2.30)
Composed of N-acetylglucosamine and N-acetylalosaminuronic acid, with cross-links
Found in cell walls of some Archaea

60. V. Other Cell Surface Structures and Inclusions
2.14 Cell Inclusions
2.15 Gas Vesicles
2.16 Endospores

61. 2.13 Cell Surface Structures
Capsules and slime layers
Polysaccharide layers (Figure 2.32)
May be thick or thin, rigid or flexible
Assist in attachment to surfaces
Protect against phagocytosis
Resist desiccation
External to cell
In Gram + and –
Not part of LPS outer membrane

62. Cell
Capsule
Figure 2.32 Bacterial capsules.
Figure 2.32

63. 2.13 Cell Surface Structures
Fimbriae
Filamentous protein (curlin, adhesin) structures for attachment
Enable organisms to stick to surfaces or form pellicles

64. 2.13 Cell Surface Structures
Pili
Filamentous protein (pilin) structures
Typically longer than fimbriae
Facilitate genetic exchange between cells (conjugation)
Type IV pili involved in twitching motility (grappling hook motility)

65. Virus-
covered
pilus
Figure 2.34 Pili.
Figure 2.34

66. 2.14 Cell Inclusions Carbon storage polymers
Poly-β-hydroxybutyric acid (PHB): lipid (Figure 2.35)
Glycogen: glucose polymer
Polyphosphates: accumulations of inorganic phosphate (Figure 2.36a)
Sulfur globules: composed of elemental sulfur or hydrogen sulfide (Figure 2.36b)
Carbonate minerals: composed of barium, strontium, and magnesium (Figure 2.37)
Magnetosomes: magnetic inclusions (Figure 2.38)

67. Figure 2.35 Figure 2.35 Poly-β-hydroxyalkanoates. β-carbon

68. Figure 2.36 Sulfur Polyphosphate
Figure 2.36 Polyphosphate and sulfur storage products.
Figure 2.36

69. 2.15 Gas Vesicles Gas vesicles
Confer buoyancy in planktonic cells (Figure 2.39)
Spindle-shaped, gas-filled structures made of protein (Figure 2.40)
Impermeable to water

70. 2.15 Gas Vesicles Molecular structure of gas vesicles
Gas vesicles are composed of two proteins, GvpA and GvpC (Figure 2.41)
Function by decreasing cell density

71. 2.16 Endospores Endospores
Highly differentiated cells resistant to heat, harsh chemicals, and radiation (Figure 2.42 )
“Dormant” stage of bacterial life cycle (Figure 2.43) may persist for decades in environment (soil)
Ideal for dispersal via wind, water, or animal gut
Present only in some gram-positive bacteria

72. Figure 2.42 The bacterial endospore.

73. 2.16 Endospores Endospore structure (Figure 2.45) Structurally complex
Contains dipicolinic acid
Enriched in Ca2+
Core contains small acid-soluble spore proteins (SASP)

74. Figure 2.47 Figure 2.47 Stages in endospore formation. Vegetative
Coat
Spore coat, Ca2+
uptake, SASPs,
dipicolinic acid
Free endospore
Maturation,
cell lysis
Stage VI, VII
Growth
Stage V
Germination
Cortex
Vegetative
cycle
Sporulation
stages
Cell wall
Cytoplasmic
membrane
Cell
division
Asymmetric
cell division;
commitment
to sporulation,
Stage I
Stage IV
Cortex
formation
Figure 2.47 Stages in endospore formation.
Prespore
Septum
Engulfment
Mother cell
Stage II
Stage III
Figure 2.47

75. Some Important Spore-formers
Bacillus anthracis-anthrax
Clostridium difficile- C. diff.
Clostridium botulinum-botulinum toxin or botox
Clostridium tetani-tetanus or lockjaw

76. VI. Microbial Locomotion
2.17 Flagella and Swimming Motility
2.18 Gliding Motility
2.19 Chemotaxis and Other Taxes

77. 2.17 Flagella and Swimming Motility
Flagella: structures that assists in swimming
Different arrangements: (a) peritrichous, (b) polar, (c) lophotrichous (Figure 2.48)
Helical organization
Very different from a eukaryotic flagellum

78. Figure 2.48 Peritrichous Polar Lophotrichous
Figure 2.48 Bacterial flagella.
Peritrichous Polar Lophotrichous
Figure 2.48

79. 2.17 Flagella and Swimming Motility
Flagellar structure of Bacteria
Consists of several components (Figure 2.51)
Filament composed of flagellin
Move by rotation
Flagellar structure of Archaea
Half the diameter of bacterial flagella
Composed of several different proteins

80. 15–20 nm L
Filament P
Flagellin MS
Outer
membrane
(LPS)
Hook
L Ring
Rod
P Ring
Periplasm
Peptidoglycan + + + + + + + +
MS Ring
Basal
body − − − −
C Ring − − − −
Cytoplasmic
membrane
Mot protein
Fli proteins
(motor switch)
Mot protein
Figure 2.51 Structure and function of the flagellum in gram-negative Bacteria.
45 nm
Rod
MS Ring
Mot protein
C Ring
Figure 2.51

81. 2.18 Gliding Motility Gliding motility
Flagella-independent motility (Figure 2.56)
Slower and smoother than swimming
Movement typically occurs along long axis of cell
Requires surface contact
Gliding proteins

82. 2.19 Chemotaxis and Other Taxes
Taxis: directed movement in response to chemical or physical gradients
Chemotaxis: response to chemicals
Phototaxis: response to light
Aerotaxis: response to oxygen
Osmotaxis: response to ionic strength
Hydrotaxis: response to water

83. 2.19 Chemotaxis and Other Taxes
Best studied in E. coli
Bacteria respond to temporal, not spatial, difference in chemical concentration
“Run and tumble” behavior (Figure 2.57)
Attractants and receptors sensed by chemoreceptors

84. Figure 2.57 Tumble Attractant Tumble Run Run
Figure 2.57 Chemotaxis in a peritrichously flagellated bacterium such as Escherichia coli.
No attractant present: Random
movement
Attractant present: Directed
movement
Figure 2.57

85. 2.19 Chemotaxis and Other Taxes
Measuring chemotaxis (Figure 2.58)
Measured by inserting a capillary tube containing an attractant or a repellent in a medium of motile bacteria
Can also be seen under a microscope

86. Figure 2.58 Measuring chemotaxis using a capillary tube assay.