Main Concept 1 Microbial Form and Function Chapter 2 of Brock Microbiology text with some examples taken from other chapters Emphasis on prokaryotic microbes-not eukaryotes or subcellular microbes

Thermodesulfobacterium “Universal Tree” of Life-3 Domains BACTERIA ARCHAEA EUKARYA Macroorganisms Animals Slime molds Entamoebae Green nonsulfur bacteria Euryarchaeota Fungi Methanosarcina Mitochondrion Methano- bacterium Gram- positive bacteria Extreme halophiles Plants Crenarchaeota Proteobacteria Thermoproteus Ciliates Chloroplast Pyrodictium Thermoplasma Cyanobacteria Thermococcus Flagellates Nitrosopumilus Pyrolobus Green sulfur bacteria Methanopyrus Trichomonads Thermotoga Figure 1.6b Evolutionary relationships and the phylogenetic tree of life. Thermodesulfobacterium Microsporidia Aquifex Diplomonads Only cellular systems Archaea and Bacteria are prokaryotes Eukarya are eukaryotes 2

Key technique for analyzing cell structure I. A Note on Microscopy Key technique for analyzing cell structure Covered mainly in lab

Most Important Microscopic Test The Gram stain A differential staining technique Most members of the Bacteria can be divided into two major groups called gram-positive and gram-negative Gram-positive bacteria appear purple, and gram-negative bacteria appear red after staining

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 is descriptive but typically does not predict physiology, ecology, phylogeny, etc. of a prokaryotic cell

2.6 Cell Size and the Significance of Being Small Size range for prokaryotes: 0.2 µm to >700 µm Most cultured rod-shaped bacteria are between 0.5 and 4.0 µm wide and < 15 µm long Cellular organisms <0.15 µm in diameter are unlikely Open oceans tend to contain small cells (0.2–0.4 µm in diameter) Most prokaryotes are at the small end of the size range

Why are most prokaryotic cells small? Explained by square-cube rule As cells get larger their internal volume grows faster than their surface area (cube > square) So in larger cells there is relatively less plasma membrane to bring in nutrients for the cytoplasm

Figure 2.13 Surface area and volume relationships in cells.

2.6 Cell Size and the Significance of Being Small Two prokaryotes notable for their large size Epulopiscium fishelsoni (Figure 2.12a and below) Thiomargarita namibiensis (Figure 2.12b)

The largest bacterium. Nature. 1993 Mar 18;362(6417):239-41. Angert ER1, Clements KD, Pace NR. Author information Abstract The large, morphologically peculiar microorganism Epulopiscium fishelsoni inhabits the intestinal tract of Acanthurus nigrofuscus, a brown surgeonfish (family Acanthuridae) from the Red Sea. Similar microorganisms have been found in surgeonfish species from the Great Barrier Reef. As these microorganisms have only been seen in surgeonfish and no free-living forms have been found, they are considered to be specific symbionts of surgeonfish, although the nature of the symbiosis is unclear. Initial reports considered them to be eukaryotic protists, based primarily on their size, with individuals being larger than 600 microns by 80 microns. But their cellular morphology in the electron microscope is more like that of bacterial than eukaryotic cells. To resolve the nature of these symbionts, we have isolated the genes encoding the small subunit ribosomal RNA from two morphotypes and used them in a phylogenetic analysis. In situ hybridization with oligonucleotide probes based on the cloned rRNA sequences confirmed the source of the rRNA genes. Our result identify the symbionts as members of the low-(G+C) Gram-positive group of bacteria. They are therefore the largest bacteria to be described so far. Comment in Microbiology. Giants among the prokaryotes. [Nature. 1993]

How does a large prokaryote such as Thiomargarita survive? https://microbewiki.kenyon.edu/index.php/Thiomargarita_namibiensis Bacteria are generally small which is favourable for rapid growth and efficient nutrient uptake (Jorgensen 2010). Nutrients are able to diffuse faster and are more effectively taken up in smaller cells. The primary mechanism of nutrient uptake in T. namibiensis is through diffusion without usage of special transport systems (Schulz 2002). Despite the large size of the microbe, nutrients are still capable of efficient diffusion throughout the organism due to the large central vacuoles which limit the volume of the effective cytoplasm. The large size of T. namibiensis is a result of its storage compartments for soluble electron donors and acceptors (Schulz 2002). With this adaptation, this bacterium does not need to be in constant contact with nutrients and are still capable of surviving for long periods of time. The occasional exposure to substrates allows T. namibiensis to uptake essential nutrients for storage in the central vacuoles. This adaptation is necessary for its habitat in oceanic sediments where nutrients are only available through occasional sediment re-suspensions (Schulz 2002).

III. The Cytoplasmic Membrane and Transport (aka Plasma Membrane) Three relevant sections of Chapter 2 2.7 Membrane Structure 2.8 Membrane Functions 2.9 Nutrient Transport

2.7 Membrane Structure Cytoplasmic membrane or plasma 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 Generic 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 or other lipids 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 (integral/peripheral) 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 depart from “standard” model Ether linkages in phospholipids of Archaea (Figure 2.16) Bacteria and Eukarya that have ester linkages in phospholipids Archaeal lipids lack fatty acids; have isoprenes instead 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 and other 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 based on mechanics (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 types of transport events are possible based on directions: 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

2.9 Nutrient Transport Simple transport-example: 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 Example of Group Transport: 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 (PEP)

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 Gram-positives and gram-negatives have different cell wall structure (Figure 2.24) Gram-negative cell wall Two layers: lipopolysaccharide (LPS) and peptidoglycan Gram-positive cell wall One layer: peptidoglycan

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-vary Cross-linked for strength Different cross-linking in gram-negative bacteria and gram-positive bacteria (Figure 2.26)

Peptidoglycan-(aka murein) 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 2.25 NAG and NAM Non-protein Peptide bond Beta 1-4 Glycoside in N-Acetylglucosamine ( G ) N-Acetylmuramic acid ( M ) NAG and NAM Non-protein Peptide bond Beta 1-4 Glycoside in Gram -) 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

Unusual amino acids Targets for antibiotics: Penicillin, Vancomycin: block cross-bridge formation

2.10 Peptidoglycan Gram-positive cell walls (Figure 2.27) Can contain up to 90% peptidoglycan Common to have teichoic acids (charged carbohydrates) embedded in their cell wall for rigidity Wall teichoic acids-embedded in peptidoglycan only Lipoteichoic acids: extend to cytoplasmic membrane and covalently bound to membrane lipids

Wall-associated protein Teichoic acid Peptidoglycan Lipoteichoic acid Figure 2.27 Structure of the gram-positive bacterial cell wall. Figure 2.27 Cytoplasmic membrane

2.10 Peptidoglycan-not present in all microbes Some Bacteria have cell walls with a different chemical structure that is not really Gram + or – (Mycobacteria) they are called “acid-fast” Some prokaryotes lack cell walls Mycoplasmas Group of pathogenic bacteria Thermoplasma Species of Archaea

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 and a membrane anchor (Lipid “A”)(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 In O-specific polysaccharide Core polysaccharide Lipid A Protein Out Lipopolysaccharide (LPS) Porin Porin Outer membrane 8 nm Cell wall 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)

2.12 Archaeal Cell Walls Most Archaea have cell walls, some do not Same functions as in Bacteria When present, Archaeal walls are built differently than Bacterial cell wall Highly variable but two main types Pseudopeptidoglycan (pseudomurein) or “S-layers”

2.12 Archaeal Cell Walls Pseudomurein aka pseudopeptidoglycan Polysaccharide similar to peptidoglycan (Figure 2.30) Composed of N-acetylglucosamine and N-acetylalosaminuronic acid (NAG and NAS) Beta 1-3 glycosides Found in cell walls of some Archaea including Methanobacteria (methane producers)

2.12 Archaeal Cell Walls S-Layers (Surface Layer) “Most common” cell wall type among Archaea Consist of protein, glycoprotein or sugar Paracrystalline structure (Figure 2.31) Network of proteins/glycoproteins attached to outer surface of plasma membrane Has openings or “pores”

Diagrams of wall structure http://www.nature.com/nrmicro/journal/v13/n10/fig_tab/nrmicro3480_F1.html Diagrams of wall structure http://faculty.ccbcmd.edu/courses/bio141/lecguide/unit1/prostruct/cw.html More information on cell walls http://www.ucmp.berkeley.edu/archaea/archaea.html More on the Archaea Further reading if you want it

V. Other Cell Surface Structures and Inclusions 2.13 Cell Surface Structures (Glycocalyx) 2.14 Cell Inclusions 2.15 Gas Vesicles 2.16 Endospores

2.13 Cell Surface Structures Glycocalyx = capsules and slime layers Polysaccharide layers, glycolipids, glycoproteins (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

2.13 Cell Surface Structures Capsules and slime layers Slime layer = unorganized, loosely attached, aka “slime wall” Capsule = more organized, tighter, harder to remove Exact composition, function, appearance is species-specific

Cell Capsule Figure 2.32 Bacterial capsules. Figure 2.32

2.13 Cell Surface Structures Fimbriae Filamentous protein structures for attachment curlin, adhesin Enable organisms to stick to surfaces or form pellicles Relatively thin and short May enhance pathogenicity

2.13 Cell Surface Structures Pilus (pili) Filamentous protein structures Pilin Not much different from fimbriae but typically longer and thicker-term usually reserved for appendage with specific known function Such as sex pilus of E. coli. Facilitate genetic exchange between cells (conjugation) Type IV pili involved in twitching motility (grappling hook motility)

2.13 Cell Surface Structures Sex pilus facilitates genetic exchange between cells (conjugation) Type IV pili involved in twitching motility (grappling hook motility)

2.14 Cell Inclusions Inclusions visible in many prokaryotic cells Solid or semi-solid aggregates Mostly used for storage of vital nutrients Minerals, carbohydrates Not membrane-bound Aka storage granule

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 (Figure 2.36b) Carbonate minerals: composed of barium, strontium, and magnesium (Figure 2.37)

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

2.14 Cell Inclusions Magnetosomes: magnetic storage inclusions (Figure 2.38) These inclusions do have a membrane to enclose iron magnetite crystals Magnetosomes orient cell in Earth’s magnetic field Helps organisms know up from down

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

Photosynthetic bacteria like this cyanobacterium usually have internal membranes Figure 14.4 Thylakoids in cyanobacteria. Figure 14.4

Others-like this green sulfur bacterium-utilize membrane-bound Figure 14.15 The thermophilic green sulfur bacterium Chlorobaculum tepidum. Others-like this green sulfur bacterium-utilize membrane-bound Vesicles to house photosynthetic apparatus Figure 14.15

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.

Endospore Formation Endospore forms inside mother cell Core is dormant, inert, minimal but alive Several tough layers form a resistant shell around core Chemical makeup of each layer is different Small acid-soluble spore proteins (SASP) contribute to resistance

YouTube version: https://www. youtube. com/watch YouTube version: https://www.youtube.com/watch?v=7zCQLITFEb0 Note: dipicolinic acid

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 Flagellum/flagella: extracellular structure that assists in swimming Tend to be found on rods or spirals Cell may have 0, 1 or more Different arrangements: (a) peritrichous, (b) polar, (c) lophotrichous (Figure 2.48) also amphitrichous Structurally very different from a eukaryotic flagellum

2.17 Flagella and Swimming Motility Flagellar structure of Bacteria Consists of several components (Figure 2.51) Hollow filament composed of flagellin Move by rotation Hook region allows propeller action Powered by proton motive force

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

2.17 Flagella and Swimming Motility Flagellar structure of Archaea Archaellum More similar to bacterial pilus than flagellum Thinner, not hollow Probably powered by ATP No hook region Motility, recognition, attachment

14.20 Spirochetes and atypical flagella Treponema pallidum and Borrelia burgdorferii are notable Spirochaetes Gram-negative, motile, and coiled (Figure 14.49) Widespread in aquatic environments and in animals Have endoflagella: located in the periplasm of the cell (Figure 14.50)

Figure 14.50 Endoflagellum (rigid, rotates, attached to one end of protoplasmic cylinder) Outer sheath (flexible) Endoflagellum Protoplasmic cylinder Outer sheath Figure 14.50 Motility in spirochetes. Protoplasmic cylinder (rigid, generally helical) Figure 14.50

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 Mechanisms (not well understood) Excretion of polysaccharide slime Gliding-specific proteins Pili

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 “Run and tumble” behavior (Figure 2.57) Attractants sensed by chemoreceptors http://web.biosci.utexas.edu/psaxena/MicrobiologyAnimations/Animations/BacterialMotility/PLAY_motility.html

Flagella rotate clockwise = tumble Flagella rotate counterclockwise = straight line run Controlled by simple molecular clock system in cell With no chemotaxis tumbles and runs alternate so that no net movement occurs: random walk

With chemotaxis, timing of clock is adjusted Runs are longer Produces net movement Chemotaxis works by controlling the rate of switching between run and tumble-it re-sets clock to favor run over tumble

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 walk Attractant present: Directed movement Figure 2.57