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Bacterial morphology, metabolism and growth Dr Ömer Küçükbasmacı Cell • Fundemental unit of living things (smallest bacterium-largest plants-animals) Bacteria • The smallest cells • Visible only with the aid of a microscope • The smallest bacteria: Chlamydia and Rickettsia-0.1-0.2 micrometer • Larger bacteria: many microns in length A newly described species • Hundred of times larger than the average bacterial cell • Is visible to the naked eye Diversity! Most bacterial cells • Approximately 1 micrometer in diameter • Visible by light microscope • Resolution: 0.2 micrometer Microscopes • Light: – Bright-field – Dark-field (Treponema pallidumSyphilis_Frengi) – Fluorescence – Phase contrast (details of the living cell) • Electron Staining • Simple • Differential: Gram and Acid-fast stain Gram-stain: cell wall Acid fast stain: Mycobacterium • Negative stain: Indian ink (capsule) • Special staining Animal and plant cells • Much larger • Ranging from 7micrometer (red blood cells) • To several feet (certain nerve cell) Each cell • Genetic basis for reproduction (DNA genome) • Biochemical machinery (genetic information is transcribed in mRNA and mRNA translated in proteins) • The machinery for energy production and biosynthesis • This is all packaged by a membrane. Each cell • Replicates by cell division. Cells • Eukaryotic (Greek for true nucleus) • Prokaryotic (Greek for primitive nucleus) Eucaryotes • Animals • Plants • Fungi Procaryotes • Bacteria • Blue-green algae Major characteristics of Eucaryotic and prokaryotic cell Eucaryote >5 μm Prokaryote 0,5-3 μm • Size • Nuclear structure : Nucleus classic membrane no membrane Chromosomes strands of DNA single circular DNA diploid genome haploid genome Major characteristics of Eucaryotic and prokaryotic cell Eucaryote • Cytoplasmic Structures Mitokondria + Golgi bodies + Endoplasmic reticulum + Ribosomes 80S(60S+40S) Cytoplasmic membrane with sterols Prokaryote 70S(50S+30S) no sterol Major characteristics of Eucaryotic and prokaryotic cell Eucaryote • Cell wall -/composed of kitin Prokaryote complex structure (protein, lipits and peptidoglycans) • Reproduction sexual and asexual asexual (binary fission) • Movement complex flagellum (If present) • Respiration via mitokondria simple flagellum (If present) via cytoplasmic membrane Bacteria • Lack nucleus membrane and membrane bound organelles • A smaller ribosome • Peptidoglycan cell wall which protects it from environtment with low osmotic pressure, at temperature extremes (both hot and cold), dryness and with very dilute and diverse energy sources. • They have evolved their structures and functions to adopt these conditions. Differences • Between Eukaryotes and prokaryotes Differences between Prokaryotes • Bacteria differ: -morphology (size, shape, stainig characteristics) -metabolic -antigenic and -genetic characteristics Size • They are diffucult to differentiate by size Shape • Spherical: coccus (Staphyloccus) • Rod-shaped: bacillus (Escherichia) • Snakelike: spirillum (Treponema) • Branched filamentous (Nocardia and Actinomyces) ( Clusters: diplococcus (Neisseria) chains (Streptococcus) grapelike (Staphylococcus) ) Bacterial shape Sperical (coccus) Rod-shaped Spiral Spiral or spirillum Helix or spirochete Bacterial arrangement Chains: streptococcus Clusters: staphylococcus Diplococcus Packets of eight:sarcina Treponema by dark-field microscopy Gram stain • Two major classes of bacteria are distinguished: • Gram-positive and • Gram-negative bacteria • Except: • Mycobacteria (waxy outer shell , distinguished by acid fast stain) • Mycoplasmas(no peptidoglycan) Bacterial Ultrastructure • Internal structure • External structure • Gram-positive and gram-negative bacteria have -Similar internal structure -But different external structure Cytoplasm • • • • • DNA chromosome mRNA Ribosomes Proteins Metabolites Bacterial chromosome • • • • • Unlike eukaryotes A single Double stranded circle Not in a membrane bound nucleus In a discrete area called nucleoid Bacterial chromosome • Unlike eukaryotes • No histons Plasmids • • • • • Smaller Circular Extrachromosomal DNAs Not usually essential for cellular survival Most commonly found in gram-negative bacteria • Often provide a selective advantage: resistance to antibiotics Lack of a nuclear membrane • Simplifies the requirements and • Control mechanisms for the synthesis of proteins Ribosomes • Unlike the eukaryotic 80S(40S+60S)ribosome • Bacterial 70S chromosome (30+50S) • Proteins and RNA of the ribosome are significantly different • Major targets for antibacterial drugs Cytoplasmic membrane • Lipid bilayer • Similar to eukaryotic membranes • But no sterols (cholesterol) Exception: Mycoplasmas Cytoplasmic membrane • Responsible for many functions • Attributable to organelles in eukaryotes: -electron transport -energy production (mitokondria in eukaryotes) Cytoplasmic membrane • Transport proteins: uptake of metabolites release of other substances • Ion pumps: to maintain a membrane potential • Enzymes Mesosome • A coiled cytoplasmic membrane • An anchor to bind and pull apart daughter chromosomes during cell division. Cell wall • Distinguishes gram-positive and gramnegative bacteria The cytoplasmic membrane in most prokaryotes surrounded by • Rigid peptidoglycan (murein) layer • Except: Archaebacteria (pseudoglycan and pseudomurein) and mycoplasmas (no cell wall) • Peptidoglycan provides rigidity and determines the shape of a bacteria • Gram-negative bacteria. + outer membranes Gram positive bacteria • Thick multilayered cell wall • Consisting mainly of peptidoglycan Gram positive bacteria • Peptidoglycan • Sufficiently porous(allows diffusion of metabolites to the plasma membrane) • Essential for structure, replication, for survival Peptidoglycan • • • • During infection İnterferes with phagocytosis Stimulates lymphoctes Pyrogenic activity (induces fever) Peptidoglycan • Degraded by lysozyme • Enzyme in human tears, mucus (produced by bacteria and other organisms) • Degrades the glycan backbone of the peptidoglycan which protects it from osmotic pressure changes Protoplast • Removal of cell wall with lysozyme • Lysis unless it is osmotically stabilized Gram-positive cell wall • • • • Peptidoglycan + Teicoic acid Lipoteichoic acid Complex polisaccarides (C polysaccharides) • M protein of streptococci • R protein of staphylococci Gram-positive bacteria • Teicoic acid : covalently linked to peptidoglycan • Lipoteichoic acid : anchored in the cytoplasmic membrane • Common surface antigens • Distinguish bacterial serotypes • Promote attachment to other bacteria and to spesific receptors on mammalian cell surfaces (adherence) Gram positive-bacteria • Teicoic acid: important virulance factors • Lipoteicoic acid are shed into media and host • Although weaker • Can initiate endotoxic-like activities. Gram-negative bacteria • Cell wall is more complex • Both structurally and chemically Gram-negative bacteria • Two layers external to the cytoplasmic membrane: • -Thin peptidoglycan layer • -Outer mebrane (unique to gram-negative bacteria) • No teicoic acid and lipoteicoic acid • +periplasmic space Periplasmic space • The area between the external surface of rhe cytoplasmic membrane and the internal surface of the outher membrane Periplasmic space • A variety of hydrolytic enzymes • Breakdown of large macromolecules for metabolism • Lytic virulence factors ( collagenases, hyalurodidases, proteases, beta-lactamases) • Components of sugar transport system • Binding proteins for the uptake of different metabolites and of a chemotaxis system Outer membranes • Unique to gram-negative-bacteria • Maintains the bacterial structure • Permeability barrier to large molecules and hydrophobic molecules • Provides protection from adverse environmental conditions (For Enterobactericeae from digestive system of the host) Outer membranes • Asymetric bilayer • The inner leaflet: phospholipits normally found in bacterial membranes • Outer leaflet: amphipathic molecule( having both hydrophobic and hydrophilic ends) contains Lipopolysaccaride (LPS) Outer membranes • LPS molecules are only found in the outer leaflet Lipopolysaccaride • Endotoxin (Lipid A+polysaccharide-O antigen, antigenic variety is great) • Powerful stimulator of immune responses • Causes fever and shock Shwartzman reaction (disseminated intravascular coagulation) follows the release of large amounts of endotoxin. Outer membrane proteins • Porins: transmembrane proteins they form pores allow diffusion of hydrophilic molecules • Structural proteins and receptor molecules for bacteriophages Disruption of the outher membrane • Weakens the bacteria • +lysozyme • Spheroplasts (osmotically sensitive) are formed. External structures • • • • Capsule Pili Flagellum Proteins Capsules • Some bacteria (gram-positive and gramnegative) • Loose polysaccaride or proteinlayer • Slime layer: loosely adherent and nonuniform in density and thickness • Glycocalyx: capsule and slime layer are also called. Capsule • Polypeptide capsule: • Bacillus anthracis (polyglutamic acid) Capsule • Hard to be seen by microscopy • Negative staining: Indian ink Capsule • Unnecessary for growth • Very important for survival Capsule • Poorly antigenic • Antiphagocytic and a major virulence factor (Streptococcus pneumoniae) • Barrier to toxic hydrophobic molecules such as detergents • Promote adherence (Streptococcus mutants: stick the tooth) Quellung reaction • For vizualizing the capsule • Using antibodies • The capsule is swollen Flagella (Kirpik) • • • • Motility Protein (flagellin) Antigenic and strain determinants Anchored in membranes through a hook and basal body • One or several Flagella • • • • Monotrichous Polar: Pseudomonas aeruginosa Peritrichous :Escherichia coli Lophotrichous Fimbriae (Pili) • Protein(pilin) • Different from flagella: smaller in diameter and not coiled in structure. • Promote adherence to other bacteria or to th host (adhesins, lectins, evasins, aggresins) Fimbriae (Pili) • Fimbriae are important virulance factors as an adhesin in E. Coli (urinary tract), Neisseria gonorrhoeae • The tips of fimbriae may contain proteinslectins that bind to spesific sugarsmannose Fimbriae (Pili) • F pili (Sex) • Promote transfer of large segments of bacterial chromosome between bacteria • Encoded by a F plasmid. Bacterial exceptions • • • • Mycobacteria Corynebacterium Nocardia Mycoplasmas Mycobacteria • • • • • • • Peptidoglycan (slightly different) Waxlikelipit coat of mycolic acid Cord factor Wax D Sulfolipids Acid-fast staining The coat responsible for virulence and antiphagocytic. • Corynebacterium • Nocardia Produce mycolic acid lipids. Mycoplasmas • No peptidoglycan cell wall Structure of Bacterial Cell Wall • The components are large structures • They are made up of polymers of subunits • Synthesis of it occurs outside the bacteria Peptidoglycan (Mucopeptide, Murein) • Linear polysaccaride chain: -repeating disaccarides: N-acetylglucosamine N-acetylmuramic acid • Cross-linked by peptides Cell wall synthesis • Cross-linking reaction is catalyzed by: -membrane bound transpeptidases -DD-carboxypeptidasespenicillin-binding proteins (PBPs) Cell wall synthesis • Penicillin-binding proteins (PBPs): -remove extra terminal D-alanines -These terminal D-alanines limit the extent of cross-linking -They are targets for penicillin and betalactam antibiotics Cell wall • Peptidoglycan is constantly being synthesized and degraded. • Autolysins such as lysozyme are important for determining the shape of bacteria. Cell wall • During starvation: -New peptidoglycan synthesis does not occur -Peptidoglycan is weakened -Gram-staining property changes (old cultures) Biosynthesis of peptidoglycan • Unique to bacterial cells • İnhibited with no adverse effect of human cells • An important target for antibiotics (selective toxicity) Lipopolysaccaride • Lipid A • Core polysaccaride • O antigen Lipoppolysaccaride • Lipid A: basic component essential for bacterial viability endotoxin activity • O antigen: antigenic part (serotypes) Inclusion granules • • • • Storage of energy Poly-beta-hydroxybutyric acid (PHB) Glycogen Polyphosphate Inclusion granules • Polymetaphosphate: Corynebacterium -Babes-Ernst bodies Spores • Resistant to harsh conditions • (loss of nutritional requirement, dessication, intense heat, radiation and attack by most enzymes and chemical agents) Spores • Exist for centuries • Diffucult to decontaminate with standart disinfectants Spores formers: • Some gram-positive • Never gram-negative Spore formers • Bacillus • Clostridium Kinds of spores • Endospore: Bacillus subtilis • Terminal endospore: Clostridium tetani ‘drumstick’ • Subterminal: Clostridium botulinum Under harsh conditions • Vegetative state is converted to dormant state (spore) Localisation of the spore within a cell • Characteristic of bacteria • Can assist in identification of the bacterium. Spore • Dehydrated • Multishelled structure • A complete copy of chromosome • Minimum concentration of proteins and Ribosomes + High concentration of calcium bound dipicolinic acid Spore • Outside the core: -inner membrane -spore wall -cortex -outher membrane -keratin-like protein coat -exosporium Sporulation • 6-8 hours Germination • Spor__vegetative state: disruption of the outher coat by mechanical stress, pH, heat or another stressor It takes about 90 minutes Bacterial metabolism • Bacterial growth: -a source of energy -raw materials *To build the proteins, structures and membranes *That make up the structure and biochemical machines of the cell Bacterial metabolism • Bacteria should obtain or synthesize: -aminoacids -carbohydrates -lipids as building blocks of the cell The minimum requirement for growth • • • • • Carbon Nitrogen Energy source Water Various ions • Chemotrophs: derive energy from inorganic material • Chemoorganotrophs: Most bacterial Heat • Cardinal temperatures: -minimum temperature -optimum temperature -maximum temperature Temperature • • • • Psychrophiles Mesophiles Thermophiles Hyperthermophiles ph • Acidophiles: Below pH 5 (many fungi) • Alkaliphiles: Above pH 9 (Vibrio) • Neutral pH: most pathogens Oxygen requirement • Obligate anaerobes : Clostridium perfringens • Obligate aerobes • Facultative anaerobes • Microaerophilic Incubation for growth • Aerobic • Anaerobic • Capneic (%5 Carbon dioxide) Methabolism • Catabolism: substrate breakdown and conversation into usable energy • Anabolism: synthesis of cellular constituents (cell wall, proteins,fatty acids, nucleic acids Bacterial growth • Bacterial replication • Two equivalent daughter cells • Binary fission (Escherichia coli: 20 minutes Mycobacterium much slower: 12-24h) Bacterial growth curve • • • • Lag phase Exponential phase Stationary phase Death phase