Microbial Cell Structure and Function 2 Microbial Cell Structure and Function

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

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

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

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)

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

Figure 2.4b The Gram stain. Figure 2.4b 7

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

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

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

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)

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

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

Figure 2.13 Surface area and volume relationships in cells.

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)

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

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

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

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

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

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

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

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)

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Figure 2.24 Cell walls of Bacteria.

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)

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

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

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

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

A review article for those who wish more reading: Microbiol Mol Biol Rev. 1999 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

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

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

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

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

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

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)

A link to a course site on the Gram Negative Wall http://faculty.ccbcmd.edu/courses/bio141/lecguide/unit1/prostruct/gncw.html

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

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”

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

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

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

Cell Capsule Figure 2.32 Bacterial capsules. Figure 2.32

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

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)

Virus- covered pilus Figure 2.34 Pili. Figure 2.34

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)

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

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

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

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

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

Figure 2.42 The bacterial endospore.

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

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

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

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

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

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

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

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

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

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

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

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

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

Figure 2.58 Measuring chemotaxis using a capillary tube assay.