MCB3020 Lecture 3 - Cell Structure and Function PDF

LECTURE 3 Cell Structure and Function Copyright © 2023 by W. W. Norton & Company, Inc.  CHAPTER OVERVIEW 3.1 THE BACTERIAL CELL: AN OVERVIEW 3.2 MEMBRANE MOLECULES AND TRANSPORT 3.3 CELL ENVELOPE 3.4 BACTERIAL CYTOSKELETON AND CELL DIVISION 3.5 CELL ASYMMETRY 3.6 S...

LECTURE 3 Cell Structure and Function Copyright © 2023 by W. W. Norton & Company, Inc.  CHAPTER OVERVIEW 3.1 THE BACTERIAL CELL: AN OVERVIEW 3.2 MEMBRANE MOLECULES AND TRANSPORT 3.3 CELL ENVELOPE 3.4 BACTERIAL CYTOSKELETON AND CELL DIVISION 3.5 CELL ASYMMETRY 3.6 SPECIALIZED STRUCTURES The Bacterial Cell ▪ Most bacterial cells share fundamental features: Complex cell envelope Compact genome Tightly coordinated cell functions ▪ Archaea, like bacteria, are prokaryotes Have unique membrane and envelope structures ▪ Eukaryotic cells have a nucleus and extensive membranous organelles The Bacterial Cell ▪ In the early twentieth century, the cell was envisioned as a bag of “soup,” full of floating ribosomes and enzymes ▪ Modern research shows that the cell’s parts fit together in a structure that is ordered, though flexible. Model of a Bacterial Cell ▪ Cytoplasm: consists of a viscous gel- like substance/network ▪ Cell membrane: encases the cytoplasm ▪ Cell wall: covers the cell membrane ▪ Nucleoid: non-membrane-bound area of the cytoplasm that contains the chromosome in the form of looped coils ▪ Flagellum: external helical filament whose rotary motor propels the cell Studying Cell Components ▪ Cell study requires isolation and analysis of cell parts ▪ Cell fractionation Cells must be broken up by techniques that allow subcellular parts to remain intact ▪ Mild detergents ▪ Enzymes ▪ Sonication ▪ Mechanical disruption Subcellular components separated using an ultracentrifuge ▪ high rotation rate produces centrifugal forces strong enough to separate particles by size ▪ Parts are then subjected for structural and biochemical analysis Studying Cell Parts ▪ Genetic analysis is an approach complementary to cell fractionation ▪ Utilizes strains with different genotypes Mutant strains that are selected for loss of a given function Strains that are intentionally mutated as to lose or alter a gene Strains that are constructed with “reporter genes” fused to a gene encoding a protein of interest (e.g. GFP) ▪ The phenotype of the mutant cell may yield clues about the function of the altered part Biochemical Composition of Bacteria ▪ All cells share common chemical components: Water Essential ions (e.g. Mg2+, K+, Cl-) Small organic molecules (e.g. lipids, sugars) Macromolecules (e.g. nucleic acids, proteins, peptidoglycan) ▪ Cell composition varies with species, growth phase, and environmental conditions. Biochemical Composition of Bacteria  CHAPTER OVERVIEW 3.1 THE BACTERIAL CELL: AN OVERVIEW 3.2 MEMBRANE MOLECULES AND TRANSPORT 3.3 CELL ENVELOPE 3.4 BACTERIAL CYTOSKELETON AND CELL DIVISION 3.5 CELL ASYMMETRY 3.6 SPECIALIZED STRUCTURES Cell Membrane ▪ Cell membrane is the structure that defines the existence of a cell ▪ Cell membrane is a two-dimensional fluid comprised of lipids and proteins ▪ Lipids are arranged in a bilayer ▪ Contains the cytoplasm, mediates transport in-and-out of the cell, and carries proteins serving various physiological roles Membrane Lipids ▪ A phospholipid consists of glycerol with ester links to two fatty acids and a phosphoryl head group May have side chain ▪ Phospholipids are amphipathic- polar/charged, hydrophilic “heads” and hydrophobic fatty acid “tails ▪ The two layers of phospholipids in the bilayer are called leaflets  Phospholipid Diversity ▪ Phospholipids vary with respect to their phosphoryl head groups and their fatty acid side chains (Mol. Biol. of the Cell , 2009) Phospholipid Diversity ▪ Cardiolipin or diphosphatidylglycerol double phospholipid linked by a glycerol concentration increases in bacteria grown to starvation localizes to the cell poles helps to stabilize the curve of the membrane at the poles and supports formation of smaller cells during starvation Phospholipid Diversity ▪ Unsaturated fatty acids - increase membrane fluidity improving function at low temperatures ▪ Saturated fatty acids - decrease fluidity improving function at high temperature ▪ Cyclization - decrease fluidity by forming rigid planar rings Hopanoids ▪ Membranes also include planar molecules that fill gaps between hydrocarbon chains ▪ In bacterial membranes, the reinforcing agents are hopanoids, or hopanes ▪ Hopanoids—pentacyclic lipids that modify membrane fluidity in response to environmental stress Increase membrane rigidity comprise 1-5% total lipids (absent in archaea) ▪ In eukaryotic membranes, the reinforcing agents are sterols, such as cholesterol. Membrane Lipids of Archaea ▪ Archaea have the most extreme variations in phospholipid side-chain structures Ether links between glycerol and fatty acids Hydrocarbon chains can be branched terpenoids (contain isoprene ring) Membrane Proteins ▪ Membrane proteins serve numerous functions: Structural support Detection of environmental signals Secretion of virulence factors and communication Ion transport and energy storage ▪ Have hydrophilic and hydrophobic regions that lock the protein in the membrane  Transport Across the Cell Membrane ▪ The cell membrane acts as a semipermeable barrier ▪ Selective transport is essential for survival Small uncharged molecules, such as O2 and CO2, easily permeate the membrane by diffusion Water tends to diffuse across the membrane in a process called osmosis ▪ Solutes usually move along a concentration gradient, from high concentration to low concentration Mol. Biol. of the Cell , 2009 https://bio.libretexts.org/@go/page/13086?pdf Transport Across the Cell Membrane ▪ Weak acids exist partly in an uncharged (protonated) form and weak bases exist in an uncharged (deprotonated) form that can diffuse across the membrane and change the pH of the cell. Transport Across the Cell Membrane Large polar molecules and charged molecules require transport through specific protein transporters ▪ Passive transport - molecules move along their concentration gradient Facilitated diffusion ▪ Active transport - molecules move against their concentration gradient Requires energy (e.g. ATP hydrolysis) Passive Transport ▪ Facilitated diffusion uses a concentration gradient to transport a molecule across a membrane from high-to-low ▪ Moves molecules that are either too large or too polar for unassisted diffusion ▪ e.g. Aquaporins—transport water and small polar molecules (Slonczewski & Foster) Active Transport: ATP hydrolysis ▪ The energy stored in ATP can drive active transport systems through ATP hydrolysis ▪ ATP-binding cassette (ABC) transporters is the largest family of Permease active transport systems conserved in prokaryotes ATPase comprised of multiple subunits ▪ integral membrane permease ▪ ATP-binding and hydrolysis (ATPase) ▪ substrate-binding subunit Active Transport: Coupled Transport ▪ Coupled transport uses the free energy released from a high-to-low chemical gradient for the transport of a molecule against (low-to-high) its concentration gradient Symporters Antiporters e.g. LacY permease- H+/lactose symporter e.g. Na+/H+ antiporter (Slonczewski & Foster) Active Transport: Group Translocation ▪ Phosphotransferase system (PTS) (aka phosphoenolpyruvate (PEP) group translocation) phosphate transfer energizes active transport of substrates phosphate derived from PEP is used to energize transport of glucose/sugars into the cell (NIH NLM; Gupta et al 2021)  CHAPTER OVERVIEW 3.1 THE BACTERIAL CELL: AN OVERVIEW 3.2 MEMBRANE MOLECULES AND TRANSPORT 3.3 CELL ENVELOPE 3.4 BACTERIAL CYTOSKELETON AND CELL DIVISION 3.5 CELL ASYMMETRY 3.6 SPECIALIZED STRUCTURES The Cell Wall is a Single Molecule ▪ The cell wall confers shape and rigidity to the cell, and helps it withstand turgor pressure ▪ The bacterial cell wall, or the sacculus, consists of a single interlinked molecule A. Isolated sacculus from Escherichia coli (TEM). B. Helical movement of the attached red beads shows that peptidoglycan strands extend helically as the cell grows Peptidoglycan Structure ▪ Bacterial cell walls are made up of peptidoglycan (murein) Transglycosylases Transpeptidases (i.e. PBPs) Peptidoglycan Structure ▪ Transpeptidases called penicillin binding proteins (PBPs) cross-link wall peptides ▪ Transglycosylases extends the glycan chains ▪ Peptidoglycan is unique to bacteria ▪ Thus enzymes that mediate peptidoglycan synthesis are excellent antibiotic targets Penicillin inhibits the transpeptidase that cross-links the peptides Vancomycin prevents cross-bridge formation by binding to the terminal D-Ala-D-Ala dipeptide ▪ Unfortunately, the widespread use of such antibiotics selects for evolution of resistant strains Peptidoglycan Structure ▪ Cell wall is synthesized differently in different bacteria ▪ D-Ala is unique to peptidoglycan ▪ Fluorescently labeled D-Ala was used to track regions of peptidoglycan synthesis Pseudomurein of Archaea 1 ▪ Pseudomurein found in Archaea is functionally homologous to bacterial peptidoglycan 2 NAG NAT ▪ Structurally unique NAT instead of NAM NAG-𝛽(1,3)-NAT linkage make it 3 insensitive to lysozyme Unique side-chain peptide cross-links Gram-Positive and Gram-Negative Bacteria ▪ Most bacteria have additional envelope layers that provide structural support and protection ▪ Envelope composition defines: Gram-positive bacteria - thick cell wall ▪ e.g. Firmicutes such as Staphylococcus aureus Gram-negative bacteria - thin cell wall ▪ e.g. Proteobacteria such as Yersinia pestis Gram-Positive Cell Envelope Gram-Positive Cell Envelope: Teichoic Acid ▪ Has multiple layers of peptidoglycan ▪ Threaded by teichoic acids- chains of phosphodiester linked polymers of glycerol or ribitol Wall teichoic acids (WTA)- cross-linked to peptidoglycan Lipoteichoic acids (LTA)- anchored in the membrane Gram-Positive Cell Envelope: S-Layer ▪ An additional protective layer commonly found in free-living bacteria and archaea ▪ Crystalline layer of thick subunits consisting of protein or glycoprotein ▪ May contribute to cell shape and help protect the cell from osmotic stress Mycobacterial Cell Envelope ▪ Mycobacterium tuberculosis and M. leprae have very complex cell envelopes ▪ Include unusual membrane lipids (mycolic acids) and unusual sugars (arabinogalactans) Gram-Negative Cell Envelope ▪ Thin cell wall comprised of 1-2 sheets of peptidoglycan ▪ Cell wall is contained within the periplasm, the space between inner and outer membranes ▪ covered by the outer membrane which confers defensive abilities and toxigenic properties on many pathogens Inward-facing leaflet contains lipoproteins Outward-facing leaflet contains lipopolysaccharides (LPS) and porins Gram-Negative Cell Envelope: LPS and Braun Lipoprotein ▪ Braun lipoprotein is covalently linked to PG and anchors outer membrane to the cell wall ▪ Lipopolysaccharide (LPS) Lipid A (6 fatty acids ester linked to glucosamines) can function as an endotoxin to cause septic shock Core polysaccharide O-antigen varies greatly to evade host immune responses Eukaryotic Microbes ▪ Eukaryotic microbes possess their own structures to avoid osmotic shock- increase in internal pressure due to influx of water into the cell via osmosis Algae form cell walls of cellulose Fungi form cell walls of chitin Diatoms form exoskeletons of silicate Paramecia possess a contractile vacuole to pump water out of the cell Eukaryotic Microbes: Contractile Vacuole  CHAPTER OVERVIEW 3.1 THE BACTERIAL CELL: AN OVERVIEW 3.2 MEMBRANE MOLECULES AND TRANSPORT 3.3 CELL ENVELOPE 3.4 BACTERIAL CYTOSKELETON AND CELL DIVISION 3.5 CELL ASYMMETRY 3.6 SPECIALIZED STRUCTURES Bacterial Cytoskeleton ▪ Shape-determining proteins: FtsZ = forms a “Z-ring” in spherical cells, initiates cell division by assigning the division plane MreB = forms a coil inside rod- shaped cells, together with other proteins of elongasome supports cell elongation CreS “crescentin” = forms a polymer along the inner side of crescent-shaped bacteria Bacterial Cell Division ▪ Prokaryotes divide by binary fission 1 mother cell splits to form two daughter cells ▪ Requires highly coordinated growth and expansion of all the cell’s parts ▪ Unlike eukaryotes, prokaryotes synthesize RNA and proteins continually while the cell’s DNA undergoes replication. ▪ Bacterial DNA replication is coordinated with the cell wall expansion and ultimately the separation of the two daughter cells. Bacterial Cell Division: Chromosome Replication ▪ Circular chromosome must be replicated prior to cell division ▪ Replication begins at the origin of replication aka ori site ▪ Two replication forks proceed outward in both directions (bidirectional replication) At each fork, DNA is synthesized by DNA polymerase with the help of accessory proteins (replisome) As the termination site is replicated, the two forks separate from the DNA Bacterial Cell Division: Divisome ▪ FtsZ initiates the formation of the Z-ring, as the replication forks near the termination site ▪ Divisome assembles to coordinate the synthesis of peptidoglycan and lipid membrane with the cell division and chromosome segregation Bacterial Cell Division: Septation ▪ Division septum, forms at the mid-cell Z-ring and the site of divisome activity ▪ Daughter cells physically separate by septation ▪ Septation is the inward growth of the division septum that constricts and seals off the two daughter cells to form two new cells, completing cell division Bacterial Cell Division: Septation The Bacillus subtilis septum grows from the outer ring inward. Cells were pulse-labeled with different-colored fluorescent d- alanine molecules that are incorporated into peptidoglycan, catalyzed by penicillin-binding proteins. The d-alanine fluorophores are: HADA, blue (first 60 minutes); BADA, green (next 5 minutes); TADA, red (final 30 seconds). Bacterial Cell Division: Septal Planes ▪ The spatial orientation of septation has a key role in determining the shape and arrangement of cocci Parallel planes ▪ Streptococcus pyogenes Random planes ▪ Staphylococcus aureus Perpendicular planes ▪ Micrococcus tetragenus  CHAPTER OVERVIEW 3.1 THE BACTERIAL CELL: AN OVERVIEW 3.2 MEMBRANE MOLECULES AND TRANSPORT 3.3 CELL ENVELOPE 3.4 BACTERIAL CYTOSKELETON AND CELL DIVISION 3.5 CELL ASYMMETRY 3.6 SPECIALIZED STRUCTURES Cell Asymmetry and Aging ▪ Bacterial cell poles differ in their origin and “age” This phenomenon is called polar aging ▪ In bacteria that appear superficially symmetrical, polar differences may appear at cell division Bacillus species can undergo an asymmetrical cell division to form an endospore at one end Some bacteria expand their cells by extending one pole only Cell Asymmetry ▪ Some bacteria generate two kinds of daughter cells: one stationary and the other mobile. Example: the flagellum-to-stalk transition of the bacterium Caulobacter crescentus Polar Aging ▪ Turns out that not only is a bacterial cell asymmetrical, but the actual process of cell division itself determines that the poles of each daughter cell differ chemically from each other ▪ Variable polar aging might result in variable antibiotic susceptibility This was observed in M. tuberculosis  CHAPTER OVERVIEW 3.1 THE BACTERIAL CELL: AN OVERVIEW 3.2 MEMBRANE MOLECULES AND TRANSPORT 3.3 CELL ENVELOPE 3.4 BACTERIAL CYTOSKELETON AND CELL DIVISION 3.5 CELL ASYMMETRY 3.6 SPECIALIZED STRUCTURES Specialized Structures: Membrane Vesicles ▪ Concept of the cell assumes a defined boundary of membrane that encloses the cell’s contents ▪ Some microbes export bits of cytoplasm in membrane vesicles Carry proteins, nucleic acids, toxins, immunogenic molecules Function? Phage decoys, DNA transfer, attraction of other heterotrophs Specialized Structures: Thylakoids, Carboxysome, Gas Vesicles ▪ Thylakoids - extensively folded intracellular membranes for photon absorption and photosynthesis ▪ Carboxysomes - polyhedral protein covered bodies packed with the enzyme Rubisco for CO2 fixation ▪ Gas vesicles - to increase buoyancy and stay at the top of the water column e.g. Photosynthetic Cyanobacteria Specialized Structures: Storage Granules and Magnetosomes ▪ Storage granules Glycogen for energy Sulfur for oxidation ▪ Magnetosomes Membrane-embedded crystals of magnetite, Fe3O4 Orient the swimming of magnetotactic bacteria (Frank 2012) Specialized Structures: Pili and Stalks ▪ Pili or fimbriae – straight filaments of pilin N. Gonorrhoeae Type IV pili protein attachment and/or motility E.g. Neisseria gonorrhoeae attachment to mucous membranes of the reproductive tract ▪ Sex pili – specialized pili for conjugation transfer of DNA from donor to recipient cell ▪ Stalks are membrane-embedded extensions B. subtillis nanotubes of the cytoplasm (e.g. C. crescentus) Tips secrete adhesion factors called holdfasts ▪ Nanotubes are intercellular connections that pass material from one cell to the next Specialized Structures: Novel Structures Observed by Cryo-ET Functions unknown! Specialized Structures: Rotary Flagella ▪ Motile prokaryotes generally use flagella for swimming ▪ Flagella number and location can vary: Peritrichous – flagella distributed around the cell and rotate in a bundle Lophotrichous – flagella in a bundle located at one pole ▪ E.g. Salmonella enterica Amphitrichous – single flagellum at each pole Monotrichous – single flagellum Specialized Structures: Rotary Flagella ▪ Each flagellum is a spiral filament of protein monomers called flagellin (FliC) ▪ The filament is rotated by a motor driven by the proton motive force ▪ Flagella rotate either clockwise (CW) or counterclockwise (CCW) relative to the cell Chemotaxis Receptor array detects chemotactic ▪ Chemotaxis is the movement of a signals for motility bacterium in response to chemical gradients ▪ Attractants cause CCW rotation Flagella bundle together “Run” - Push cells forward ▪ Repellents cause CW rotation Flagellar bundle falls apart “Tumble” - bacterium briefly stops, then changes direction ▪ “random walk” - alternating runs and tumbles Chemotaxis Chapter Summary ▪ While prokaryotes are diverse, they share certain fundamental traits and biochemistry ▪ The study of cells employs various methods including subcellular fractionation, structural analysis, and genetic analysis ▪ The cell membrane consists of a phospholipid bilayer containing proteins ▪ Bacterial phospholipids are ester linked, while those of archaea contain ether linkages ▪ The Gram-negative cell envelope is much more complex than that of Gram-positive cells. Chapter Summary ▪ Most bacteria divide by binary fission ▪ Cell growth and DNA replication are coordinated ▪ Bacteria may have specialized structures, including thylakoids, storage granules, and magnetosomes ▪ Pili and stalks are used for attachment ▪ Flagella are rotary appendages used for movement and chemotaxis Suggested Reading ▪ Microbiology: An Evolving Science, 6th Edition – CHAPTERS 3 & 4 ▪ The Atlas of Bacterial & Archaeal Cell Structure https://www.cellstructureatlas.org