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Lecture 10: Introduction to Bacteria (Structure, Growth & Physiology) Study Objectives •Explain bacterial classification schemes and nomenclature. •Describe the structural components of bacteria. •Explain cellular and colony morphologies, Gram staining, motility and spore formation. •Give examples of encapsulated microorganisms and discuss their importance to human health. •Describe various structures of the bacterial cell (including the cellular membrane, cell wall, internal and external structures) and how they affect growth, identification and virulence of these organisms. •Describe the metabolic requirements for energy and biosynthesis in bacteria. •Explain the diversity of bacterial metabolism and how metabolic properties of bacteria have impacted classification/identification. •Describe bacterial growth (growth curves, dynamics and sporulation). •Describe the various effects of oxygen, temperature, pH on bacterial growth. •Define aerobes, obligate anaerobes and facultative anaerobes. Feature Prokaryotes Eukaryotes Internal Structure Simple Complex Nucleus No membrane or nucleoli True nucleus: membrane bound and contains nucleoli Organelles (membrane bound) No Yes Cell wall Usually present. Chemically complex If present, chemically simple. Filamentous cytoskeleton No Yes Cell division Binary fission Mitosis and meiosis mRNA processing Little Extensive Ribosomes 70S 80S (70S in mitochondria and chloroplasts) DNA (chromosomes) Usually single. Circular. Naked. Multiple. Linear. Histones. Division rate 20-30 minutes 10-24 hours Bacteria (Prokaryotes)Features •Anucleated; nucleic acid intermingles with cytoplasm – not membrane bound •Lack organelles (membrane-bound compartments) Classification & Nomenclature •Medically important bacteria only account for a fraction of total bacteria •Binomial = Genus and Species •Names should be italicized or underlined •Genus capitalized and species name not capitalized •Can be abbreviate Genus name but not species name •Correct: Bacillus anthracis, B. anthracis •Incorrect: Bacillus Anthracis, Bacillus Anthracis, Bacillus a. Bacterial Cell Structures Pilli- for attachment and locomotion Nucleoid- contains DNA or RNA Bacterial cells are constituted by a cytoplasmic mass where the nucleic acid, ribosomes, storage granules, and other cellular components are found and a protective cell envelope of variable complexity. The bacterial cell envelope consists of a cell wall and an underlying cytoplasmic membrane. The rigid cell wall which provides protection and imparts shape to most bacterial cells is entirely absent in a few unusual bacteria (ex mycoplasmas). o Peptidoglycan (murein) is the principle structural component of the cell wall. This compound is found in both Gram-positive and Gram-negative organisms although it is more abundant in Gram-positive bacteria. Plasma Membrane is selectively permeable phospholipid bi-layer, Bacterial Cell Walls (Gram +: purple; note techoic and lipotechoic acid; Gram -: pink; note more polysaccharides Peptidoglycan Structure & Synthesis Peptidoglycan polymers consist of repeating disaccharides formed by Nacetylglucosamine(NAG) and N-acetylmuramic acid(NAM). Makes a lattice structure NAM and NAG associate in the cytoplasm and then end up in the outer membrane o It does this by attaching to a Bactoprenol which flips the NAM+NAG into the outer membrane o These are things that drugs target Gram-Positive Bacteria Teichoic (TA) & Lipoteichoic Acids (LTA) •TA linked directly to the peptidoglycan layer •LTA possess a transmembrane domain so anchored into the bacterial cell membrane •Expression of both essential for bacterial viability •Provide a channel of negative charges so as to attract positively charged substances to the cell membrane (through the thick peptidoglycan) •Both function in adherence to surfaces Gram-Negative Bacteria Porins •Channels in outer membrane which permit transport of small hydrophilic materials into the periplasm •Antibiotics (Ab) can place selection pressure on the bacteria resulting in mutation of porins to exclude drug passage Gram-Negative Bacteria Lipopolysaccharide (LPS) •~750,000 sepsis cases/y (30% mortality) •~40% of septic patients progress to shock •TNF/IL-1 induce tissue factor release by endothelial cells clots form •Clots lodge in blood vessels lowering profusion of organs organ failure •Clot formation depletes blood of clotting factors (coagulopathy) DIC ***TLR-4 recognizes! LPS is an endotoxin! Variations from Gram + or 1.Acid Fast Organisms 2.Mycoplasmas and Chlamydia Acid-Fast Cell Wall Mycolic Acid (on outer most layer) •Acid-fast microbes contain this waxy material •Prevents Gram staining •Expressed by Mycobacterium and Nocardia •Use an Acid-fast staining protocol to detect these cell walls •Takes up initial stain (carbol fuchsin) while other bacteria decolorize and take on counterstain (methylene blue) Bacteria Without Cell Walls Mycoplasma •Smallest known bacteria (agent of pneumonia) •Does not take up the Gram stain •Not easily visible under the light microscope Bacterial Replication (Binary Fission) (Make split-pea babies! Only takes 20-30min)!!! The bacterial cell lacks a nuclear membrane; instead, DNA is concentrated in the cytoplasm as a nucleoid (one or multiple copies of a double-stranded, circular closed, supercoiled DNA). In many bacteria, a small portion of the DNA persists as extrachromosomal elements referred to as plasmids, which are also circular but are much smaller than bacterial chromosomes. Plasmids encode variable numbers of genes and often determine virulent behavior. Bacterial Ribosomes Note below: Top is for Prokaryotes; bottom is eukaryotic ribosomes. Description is for Eukaryotes, but applies to prokaryotes! The tetracyclines (tetracycline, doxycycline, demeclocycline, minocycline, etc.) block bacterial translation by binding reversibly to the 16S rRNA in the 30S subunit and distorting it in such a way that the anticodons of the charged tRNAs cannot align properly with the codons of the mRNA. -antibiotics bind to 16s Flagella: motility; made of flagellin; rotary action for movement Flagella Arrangements Peritrichous flagella-distributed over the surface of the bacterium Monotrichous flagellum-some bacteria only have a single flagellum Polar flagella or Lophotrichous-bundled at one or both ends of the bacterium Pili: attachment onto mucosal surfaces F-pili: sex pili for bacterial conjugation Pili are protein fibers cover the entire surface of Gram-negative bacteria. Motility •Some bacteria are motile. •Bacteria may use flagella or other means for motility. •Motility may also be by pili (fimbria) or by gliding. Capsules (protection; made of polysaccharides) (Glycocalyx-Slime Layer) •A polysaccharide material secreted by cells and associated with its surface. •Slime layers are less organized and more loosely attached to surface. •Highly immunogenic (K antigen). Negative staining used to observe. •Considered a virulence factor: –Helps bacteria avoid phagocytosis. –Helps bacteria attach to surfaces. –Capsules aid in adhesion to surfaces. Examples capsule of pathogens: ***Streptococcus pneumoniae ***Pseudomonas aeruginosa ***Bacillus anthracis –Klebsiella pneumoniae –Haemophilus influenzae –Neisseria meningitides –Cryptococcus neoformans Biofilm (community of bacteria in GI tract compete with pathogenic bacteria for surface area) Biofilms formed by aggregation of bacteria secreting protein or carbohydrate coats (capsules) which combine to surround a colony of bacteria. Some bacteria can form aggregates by pili interactions leading to biofilm formation. These biofilms allow bacteria to persist in environments despite body responses such as immunity and chemicals that would remove them. Biofilms are helpful for some environments such as enriching sand or soils but harmful for animal tissues (dental plaque). For animals, if biofilms remain, excess wastes in the form of acids build up and cause tissue loss (cavities). Endospore •Dormant form of certain bacteria that remain viable for decades •Endospores found in soil and tissues/fluids of dead animals but not in tissues/fluids of living animals •Resists heat, desiccation, extremes of pH and radiation (makes hard to kill, like Anthrax) Examples capsule of pathogens: –Bacillus spp. (e.g. B. anthracis, B. subtilis). –Clostridium spp. (e.g. C. tetani, C. difficile ). Endospore Formation: endospore is formed from original cell Bacterial Physiology Chemical/Nutritional Requirements •Carbon, Nitrogen, Sulfur, Phosphorus Carbon is backbone of all living matter Nitrogen, sulfur, phosphorus are required for synthesis of proteins, DNA, RNA and ATP •Oxygen May be needed for energy production through respiration Toxic to some bacteria If required (or if tolerated), bacteria have mechanisms to remove oxygen byproducts Use of oxygen may lead to formation of superoxides, hydrogen peroxide and hydroxyl radicals – enzymes will be needed to protect from these toxins (Respiratory bursts) Oxygen Requirements for Bacterial Growth Bacteria can be divided into five groups on the basis of their oxygen requirements. 1)Obligate aerobes: The growth of aerobic bacteria in a nutrient-rich medium is restricted by limited availability of oxygen. 2)Obligate anaerobes: Conversely, the growth of strict anaerobes may be inhibited by an oxygen tension as low as 10-5 atmospheres (atm). 3)Facultative anaerobes are able to use molecular oxygen, organic and inorganic compounds as terminal electron acceptors. 4)Microaerophilic bacteria grow best under decreased oxygen tension (generally 2-5%-air is 20%) as obtained in a candle jar. 5)Aerotolerant bacteria can survive (but not grow) for a short period of time in the presence of atmospheric oxygen. The tolerance to oxygen is related to the ability of the bacterium to detoxify superoxide and hydrogen peroxide, produced as by-products of aerobic respiration. Two key enzymes are involved in this detoxification: 1)Superoxide dismutase: which converts superoxide (the most toxic metabolite) into hydrogen peroxide, is present in aerobic and aerotolerant bacteria. 2)Catalase: which converts hydrogen peroxide into water and oxygen is also present in all aerobic bacteria but is lacking in aerotolerant organisms. Strict anaerobes lack both enzymes. •Energy metabolism –3 pathways: 1.Fermentative metabolism 2.Respiratory metabolism 3.Autotrophic metabolism 1.Fermentative •Uses organic compounds as both electron donors and acceptors •Includes the –Glycolytic (Embden-Meyerhof pathway)-is the major glucose utilization pathway used by most aerobic and anaerobic bacteria Pathway divided into 2 phases o Phase I glycolysis o Phase II glycolysis – Enter-Doudoroff pathway (alternative pathway; doesn’t make enough ATP – would only make 1 ATP)-is the major hexose-degrading pathway in organisms that lack phosphofructokinase (ex. Members of Pseudomonas genus). –Pentose-Phosphate pathway Autotrophic Metabolism •Anaerobes can use a variety of inorganic sources for energy and reducing power •Can use the ETC but they just need to find an alternative terminal electron acceptor; instead of O2 •Examples of alternates: –Nitrate/Nitrite –Sulfate/Sulfite –Carbon Dioxide -Iron Iron Acquisition (Lactoferrin takes up free iron) •Iron is an essential element for bacterial growth and pathogenesis •Require free iron •Have evolved mechanisms to acquire iron –Siderophores –Iron reductase-reduces ferrrin iron (insoluble) to ferrous iron (soluble) –Hemolysins-lyse red blood cells thereby releasing hemoglobin –Exotoxins-may lyse eukaryotic cells Bacterial Growth Physical Requirements •Temperature −Required for normal enzymatic function −Most medically important are mesophiles −Food spoilage usually occurs by psychrotrophs •pH −Range from 6.5 to 7.5 −Preserved food with high pH prevents bacterial growth −Acidophiles may survive in high acidity −Bacterial products result in change in pH oBreakdown of carbohydrates produce acids −Buffers are added to growth media Generation Time •Interval of time between successive replication events •Highly variable for different species: −Staphylococcus aureus generation time = 30 min −Treponema pallidum generation time = 33 h •Uncontrolled growth prevented only by depletion of food, buildup of waste or some other limiting physical requirement Phases •Lag Phase −First few hours of growth −No cell division occurs −Bacteria adapting to the environment •Log Phase −Active stage of exponential growth •Stationary Phase −Reproduction = Cell death •Decline Phase −Death > Reproduction Bacterial Identification •Staining Properties: Gram Pos: Purple; Gram Neg: Pink/Red •Morphological: SHAPE: Spirilla, bacilli, cocci; ARRANGEMENT: diplo, strept, tetrad, staph, sarcina •Metabolic Activity. •Serological. •Molecular Examples of morphological: •Macroscopic: –Colony size and shape. –Colony color and texture. –Colony elevation and margin. •Microscopic: –Cell shape. –Arrangement. –Differential staining –Motility. –Spore formation. Classification by Metabolic Activity 1.Oxygen requirements: –Aerobic –Anaerobic –Facultative 2.Carbohydrate utilization –Identified by the variety of carbohydrates they use as energy sources 3.Enzyme Production Carbohydrate Utilization •Identification of microbes based upon pathways involved in ATP synthesis (specific enzymes used or metabolic products) Examples of tests for Enzyme Production Identification by Serologic Reactivity •Determined by using specific antisera that are directed against antigenic determinants of the bacterial cell •Carried out by agglutination techniques – test blood types Serotyping is usually carried out by agglutination techniques that entail mixing a drop of antiserum with a drop of bacterial suspension. A positive reaction results in clumping of the bacterial suspension. Genetic relatedness established by: –The ability to exchange genetic information (i.e. via transformation or conjugation) which is •only possible between related organisms –Nucleotide base composition •G-C:A-T ratio –Nucleic acid homology among bacteria •Determined by hybridization studies –Nucleic acid sequences –Homology of 16s rRNA sequences