Chapter 26. Applied Microorganisms
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1. Overview
2. Bacteria
3. Yeast
4. Virus
1. Overview
⑴ Various Growth Forms
① Classification Based on Oxygen Requirements and Resistance
Figure 1. Classification Based on Oxygen Requirements and Resistance
○ A. Obligate Aerobe: Can only perform aerobic respiration, cannot perform fermentation or anaerobic respiration.
○ B. Facultative: Microorganisms that can adapt their metabolism to both aerobic and anaerobic conditions.
○ Example: Yeast
○ C. Aerotolerant: Can remove active oxygen like A, B, and E, but cannot perform aerobic respiration.
○ D. Obligate Anaerobe: Suffers lethal damage from oxygen.
○ Mostly methanogens and acetogens.
○ Methanogens and acetogens compete using carbon dioxide and hydrogen, with methanogens having an advantage.
○ E. Microaerophile: Prefers moderate oxygen concentrations.
② Classification of Bacteria Based on Nutritional Methods
○ Auxotrophic Mutant
○ Bacteria, fungi, and cultured cells that have mutated to require specific chemical substances for growth.
○ Example 1: Arginine-requiring mutant: Requires arginine for growth.
③ Diuxic Growth
○ Definition: Growth pattern in microorganisms where one nutrient is consumed first, then another after depletion of the first nutrient.
⑵ Types of Culture Media
① Complete Media: Contains all nutrients such as amino acids, peptone, and triton.
○ Example 1: Agar medium
○ Example 2: LB medium: Contains yeast extract and is specific for E. coli.
○ 1st: Mix 700 ml dH2O, 10 g tryptone, 5 g yeast extract, and 10 g NaCl in a flask.
○ 2nd: Adjust pH to 7.0.
○ 3rd: Add agar to achieve a 1.5% solution and adjust the volume to 1 L with dH2O.
○ 4th: Seal the flask and autoclave.
○ 5th: After cooling to 50-60°C, add ampicillin stock solution to achieve a concentration of 25 μg/ml.
○ 6th: Pour into culture plates and let solidify.
○ 7th: Invert the petri dish with LB medium during E. coli culture to prevent contamination.
② Minimal Media: Contains minimal essential nutrients like vitamin B7, inorganic salts, and glucose.
③ Supplemented Media: Used for nutritional auxotroph confirmation and transformation.
⑷ Counting: Serial Dilution Method, Absorbance Method
2. Bacteria
⑴ Overview
① Unit: Colony
② Size: 0.5 - 5.0 μm
③ Motility using flagella
⑵ Functions of Cell Wall
① Prevents swelling and bursting in hypotonic environments, like plant cells.
② Reminder of the highly variable osmotic concentration of bacterial growth environments.
⑶ Classification Based on Morphology
① Type 1: Spirilla
○ Vibrios (e.g., Vibrio cholerae)
○ Spirilla (e.g., Helicobacter pylori)
○ Spirochaetes (e.g., Treponema pallidum)
② Type 2: Cocci
○ Diplococci (e.g., Streptococcus pneumoniae)
○ Streptococci (e.g., Streptococcus pyogenes)
○ Staphylococci (e.g., Staphylococcus aureus)
○ Sarcina (e.g., Sarcina ventriculi)
③ Type 3: Bacilli
○ Chain of bacilli (e.g., Bacillus anthracis)
○ Flagellate rods (e.g., Salmonella typhi)
○ Spore-formers (e.g., Clostridium botulinum)
⑷ Classification Based on Oxygen Requirement
① Aerobic
② Anaerobic
⑷ Classification Based on Gram Staining: Crystal violet is used first, followed by safranine O.
① 1st: Heat-fix bacteria onto a slide after alcohol lamp treatment.
② 2nd: Apply crystal violet staining reagent to the sample on the slide, let stand for 20 seconds.
○ All cells appear purple at this stage.
○ Crystal violet, an acidic reagent with a (+) charge, strongly binds to teichoic acids in Gram-positive bacteria.
③ 3rd: Briefly rinse the stained slide with distilled water in a tube and remove excess water.
④ 4th: Apply iodine solution (I2-KI) to the slide with the crystal violet-stained sample, let stand for 1 minute.
○ Iodine forms a complex with crystal violet, preventing decolorization (mordant function).
⑤ 5th: Rinse the slide in a tube with 95% ethanol while removing the iodine solution.
○ Gram-positive bacteria retain the purple color.
○ In Gram-positive bacteria, alcohol shrinks the cell wall, trapping I2-crystal violet in the cytoplasm.
⑥ 6th: Rinse the slide in a tube with distilled water to stop the decolorization.
⑦ 7th: Apply safranine to the slide with the sample, let stand for 1 minute.
○ Gram-positive bacteria maintain the purple color.
○ Gram-negative bacteria show a red color due to safranine.
⑧ 8th: Rinse with water gently for a few seconds, remove excess water.
⑸ Gram-Positive Bacteria
① Structure: Cytoplasm - Cell membrane - Thick peptidoglycan
② Peptidoglycan: Alternating N-acetylglucosamine (N-AG) and N-acetylmuramic acid (N-AM) with β 1→4 linkage.
③ Forms endospores for high-temperature and high-pressure resistance.
④ Teichoic acid protrudes from peptidoglycan and is connected by covalent bonds, only observed in Gram-positive bacteria.
○ Teichoic acid carries gluconic acid, giving it a negative charge.
⑤ Examples: Bacillus subtilis, Bacillus anthracis, Staphylococcus
⑹ Gram-Negative Bacteria
① Structure: Cytoplasm - Cell membrane - Periplasmic space - Thin peptidoglycan - Periplasmic space - LPS, Outer membrane
② LPS (lipopolysaccharide): Toxic and an immune system target.
○ LPS contributes to botulinum toxin toxicity.
○ Penicillin cannot pass through LPS.
○ Blood clotting and fever induction.
③ Outer membrane: More permeable than the cell membrane due to porins.
④ Periplasmic space
○ Separates peptidoglycan layer from the cell and outer membrane.
○ Important for cellular functions like metabolism and transport.
⑤ Examples: Escherichia coli, Helicobacter pylori
⑻ Antibiotics
① Beta-lactam antibiotics
○ Inhibit penicillin-binding proteins (PBPs) irreversibly, crucial for bacterial cell wall synthesis.
Figure 2. Mechanism of Beta-Lactam Antibiotics
○ Initially derived from the fungus Acremonium.
○ Carbapenem
○ Cephalosporin
○ Monobactam
○ Penicillin
○ Clavulanic acid: Irreversibly deactivates beta-lactamase in beta-lactam antibiotics.
② Other Cell Wall Inhibitors
○ Vancomycin: Binds to (D)-Ala-(D)-Ala of pentapeptide. Almost the only drug used against beta-lactam-resistant bacteria.
○ Bacitracin
③ Cell Membrane Damage
○ Polymyxin B: Binds to the outer membrane of Gram-negative bacteria through electrostatic interactions, making it unstable.
④ Folic Acid Inhibition: Bacteria do not easily absorb folate.
○ Sulfonamide: Competes with p-aminobenzoic acid, an essential precursor for folate synthesis.
○ Trimethoprim
Figure 3. Mechanism of Antifolate Agents
⑤ DNA Gyrase Inhibition
○ Fluoroquinolone
○ Quinolone
⑥ RNA Polymerase Inhibition
○ Rifamycin: Includes rifampin, rifapentine, and rifabutin.
⑦ 30S Subunit Inhibition
○ Aminoglycoside
○ Gentamycin
○ Neomycin: Inhibits tRNA-mRNA interactions.
○ Streptomycin: Inhibits initiation of translation.
○ Tetracycline: Inhibits tRNA and ribosome binding.
⑧ 50S Subunit Inhibition
○ chloramphenicol (trade name : chloromycetin) : Inhibitor of peptide bond formation
○ Derived from streptomycesvenezuelae bacteria, a potent antibiotic
○ Broad-spectrum effectiveness against various bacteria, particularly effective against typhoid
○ clindamycin
○ erythromycin : Inhibits the movement of mRNA along the ribosome
○ linezolid
○ macrolide
○ streptogramin
⑨ Antibiotic Sensitivity : Different bacteria species respond differently to various antibiotics
Figure 4. Antibiotic Sensitivity]
⑩ Antibiotic Resistance : Bacteria start secreting penicillinase, an inhibitor of penicillin, just five years after prescribing penicillin
Figure 5. Antibiotic Resistance]
3. Yeast
⑴ Prefers asexual reproduction, but undergoes sexual reproduction under unfavorable conditions
⑵ Mitochondria inherited from both parents during sexual reproduction
4. Virus
⑴ Overview
① Unit : Plaque
② Burst size : Number of progeny particles produced within a bacterium
③ Biological characteristics
○ Composed of nucleic acids and proteins
○ Can exhibit activity within cells, involving self-replication and genetic transmission
○ Frequent mutations for rapid adaptation to the environment
④ Non-biological characteristics
○ Can exist as protein crystals
○ Existence of auxiliary metabolic enzymes : Enzymes related to DNA replication or transcription exist
⑵ Structure
① Virion : Basic unit of a virus. Particle form when outside the host
② Nucleic acid + Capsid (capsomer) + Cell entry enzyme + (Envelope) + (Reverse transcriptase)
○ Envelope exists only in animal viruses
○ Reverse transcriptase exists only in retroviruses (e.g., retroviruses)
③ Tail fibers : Present in bacteriophages. Recognize specific receptors on the host cell surface for attachment
⑶ Classification
① Presence of Envelope : Naked virus, Enveloped virus
○ Naked virus : No envelope. Genome transport by diffusion. Cell release through lysis
○ Enveloped virus : Envelope present. Genome transport through endocytosis
② Host : Bacteriophages, Plant viruses, Animal viruses
③ Classification by Genetic Material : ds DNA, ss DNA, ds RNA, ss RNA
④ Classification according to Receptor Binding Protein
○ Hemagglutinin
○ Acts during cell entry
○ Binds to sialic acid residues at the carbohydrate terminus on the host cell surface
○ Sialic acid is also known as N-acetylneuraminic acid
○ Neuraminidase
○ Acts upon cell release
○ Temporarily binds hemagglutinin and sialic acid residues on the host cell membrane during animal influenza virus budding
○ Neuraminidase breaks temporary (glycosidic) linkage
○ Differentiation of influenza viruses depends on the types of hemagglutinin and neuraminidase
○ Hemagglutinin exists from H1 to H16
○ Neuraminidase exists from N1 to N9
○ Virus classification follows the pattern H#N#
○ Tamiflu™, oseltamivir, and Relenza™
○ Competitive inhibitors of neuraminidase. Similar to sialic acid
○ Inhibit the replication of influenza viruses
⑤ Classification by Antigenic Variation Mechanism
○ Antigenic drift : Continuous reproduction of a virus results in antigenic variations through mutations
○ Antigenic shift : Two different viruses replicate within the same host, leading to new antigenic variations. Involves recombination of segmented viral genomes
○ Influenza viruses acquire diversity through antigenic shifts
○ Burst size : Number of progeny particles produced within a bacterium
⑷ Classification by Genetic Material : ds DNA, ss DNA, dsRNA, ssRNA
① dsDNA virus
○ Derived from lipid bilayer of the nuclear membrane
○ Examples: AdV, HSV (Herpes virus), Hepatitis B virus, smallpox virus, VACV
② ssDNA virus
○ Derived from lipid bilayer of the nuclear membrane
○ Examples: H1-PV, Parvovirus
③ dsRNA virus : (+) is sense, (-) is antisense for RNA synthesis
○ Derived from circular membranes of the nuclear envelope
○ 1st. Double-stranded RNA separates into (-) ssRNA and (+) ssRNA
○ 2nd. (-) ssRNA uses its enzyme (RNA dependent RNA pol) to create dsRNA
○ 3rd. (+) ssRNA functions as mRNA, translating viral proteins
○ Examples: reovirus, rotavirus
④ ssRNA virus : (-) negative-sense ssRNA virus, (+) positive-sense ssRNA virus, classified under retroviruses
○ Derived from circular membranes of the nuclear envelope
⑤ (+) positive-sense ssRNA virus : (+) is sense, (-) is antisense for RNA synthesis
○ Positive strand carries fundamental genetic information
○ Functions as mRNA for translation of viral proteins, with 5’ cap and poly A tail
○ 1st. (-) ssRNA synthesized using its RNA pol
○ 2nd. (-) ssRNA used by its enzyme (RNA dependent RNA pol) to create (+) ssRNA
○ 3rd. (+) ssRNA used for genome assembly and protein translation
○ High mutation rate as the genome is used for both translation and replication
○ Examples: Hepatitis C virus, SARS, Coronaviruses, poliovirus, coxsackievirus, NDV
⑥ (-) negative-sense ssRNA virus : (+) is sense, (-) is antisense for RNA synthesis
○ Negative strand complements the positive strand. Negative strand’s genetic information used mainly as a template for replication
○ 1st. Uses its RNA dependent RNA pol to create (+) ssRNA
○ 2nd. (+) ssRNA used by its enzyme (RNA dependent RNA pol) to create (-) ssRNA
○ 3rd. (-) ssRNA used for genome assembly, (+) ssRNA used for protein translation
○ Examples: Influenza virus, Ebola virus
, MV, NDV, VSV
○ Memory tip: Influenza… Unfluenza…
⑦ Retroviruses : Contain reverse transcriptase (RT), a type of (+) positive-sense ssRNA virus
○ Reverse transcriptase virus : Contains reverse transcriptase (RT) enzyme
○ Reverse transcriptase viruses classified into retroviruses and non-retroviruses (e.g., Hepatitis B virus, a dsDNA virus)
○ HIV virus
○ Contains two identical ssRNA strands forming the genome. Note: These strands are not complementary
○ HIV RNA has a 5’-cap and poly-A tail
○ Key enzymes in HIV virus: Reverse transcriptase (RT), integrase, protease
○ (Note) Influenza virus genome is RNA and can replicate RNA directly from it
○ However, in the case of avian influenza, a reverse transcription process is present to synthesize cDNA
Table 1. Types of DNA viruses
Table 2. Types of RNA viruses
Figure 6. Types of viruses
⑸ Bacteriophage : Has a life cycle of about 30 minutes
① Lytic Cycle : Pathogenic viruses like T1, T2, T4 phages (DNA viruses)
○ 1st. Attachment : Tail attaches to the host cell surface
○ 2nd. Entry : Phage action immediately halts host replication, transcription, and translation
○ 3rd. Replication : Early genes expressed → Induction of late gene transcription → Expression of late genes
○ Early genes : Determine the life cycle, use host chromosome for self-DNA synthesis
○ Late genes : Capsid genes, lysis genes
○ 4th. Assembly : Virus DNA replication using host enzymes. Produced virus proteins cut and degrade host DNA
○ 5th. Release : Cell membrane lytic enzyme produced by phage leads to release
② Lysogenic Cycle : Temperate phages like λ phage (DNA viruses)
○ 1st. Attachment
○ 2nd. Entry : Upon insertion into bacteria, linear DNA becomes circular
○ 3rd. Replication : Unlike the lytic cycle, phage DNA integrates into host DNA
○ Prophage : Phage DNA integrated into host DNA
○ 4th. Assembly and Release : Original phage DNA remains in the host
○ 5th. Switches to lytic cycle if host replication is hindered
○ More cI factor results in lysogenic cycle, more cro factor results in lytic cycle
○ Memory tip: I.. in.. O.. out..
○ In healthy E. coli, phages first synthesize cro, later influenced by cI protein, leading to lysogenic cycle
③ Competitive Expression of cro and cI
Figure 7. Gene Expression in Lytic and Lysogenic Cycles]
○ Gene expression in lytic and lysogenic cycles regulated by two genes (cI, cro) and three promoters (PR, PL, PRM)
○ Gene expression pattern in lytic cycle
○ 1st. Protein from cro inhibits transcription of cI by binding to OR3 within PRM
○ 2nd. Resultantly, PL and PR promoters activate during the determination of lytic growth
○ Gene expression pattern in lysogenic cycle
○ 1st. λ repressor generated by cI gene
○ 2nd. λ repressor binds to OR1 within PR and spans OR2 across PRM and PR, activating cI expression
○ 3rd. cro expression inhibited instead
○ 4th. PRM promoter activated with increased cI expression due to competitive gene expression of cro and cI, leading to lysogenic growth
○ Induction of Lysogeny
○ 1st. Lysogeny induction
○ 1st - 1st. Increased cII synthesis under poor growth conditions
○ 1st - 2nd. cII protein binds to the top of the PRE promoter
○ 1st - 3rd. Forming DNA loop between OR1-OL1, OR2-OL2, OR3-OL3 with a repressor in between
○ 1st - 4th. DNA loop promotes transcription of cI gene
○ 1st - 5th. As cI expression increases due to competitive gene expression of cro and cI, lysogenic growth is induced
○ 2nd. Establishment of Lysogeny
○ cI gene transcribed from PRE and maintains transcription if cII gene remains
○ λ repressor binding to OR1 and OR2 also promotes lysogeny
○ PR and cII influence lysogeny
○ PL, which regulates lytic genes, also influences lysogeny
⑤ Induction of Lysogeny
○ λ repressor appears as a loop-shaped structure with N and C termini
○ λ repressor monomers form dimers, and dimers combine to form tetramers
○ UV light induces monomer formation of λ repressor, inhibiting cI expression
○ As cro expression increases relatively due to competitive gene expression of cro and cI, lytic growth is induced
⑥ Antitermination : Involves N and Q proteins in lambda phage
⑦ Retroregulation : Hairpin structure in int expression is resistant to nucleic acid digestion
⑹ Animal Viruses
① Penetration Mechanisms of Animal Viruses
○ Exposed Viruses : After absorption by endocytosis, the viral envelope destroys the endosomal membrane.
○ Enveloped Viruses
○ First Mechanism : Fusion of the envelope and endosomal membrane after endocytosis
○ Second Mechanism : Fusion of the host cell plasma membrane and virus envelope
② Influenza Virus : ssRNA Virus
○ Composed of a total of 8 RNA segments
○ RNA polymerase directly attached to the capsid
○ Classification of Influenza : Classification based on types of hemagglutinin and neuraminidase
○ 1st. Spike glycoprotein on the viral envelope binds to cell membrane receptors
○ 2nd. Fusion of envelope and endosomal membrane → release of virion into the cytoplasm
○ 3rd. RNA-dependent RNA polymerase activity
○ 4th. Viral protein synthesis → budding
③ HIV : ssRNA Virus
○ Capsid contains reverse transcriptase, protease, integrase, and two identical complementary ssRNAs
○ 1st. Glycoprotein gp120 on the envelope binds to CD4 on helper T-cells
○ Envelope consists of glycoproteins gp120 and gp41
○ Glycoprotein gp120 recognizes CD4
○ Glycoprotein gp41 recognizes CCR5
○ Individuals lacking CCR5 exhibit HIV resistance
○ 2nd. Fusion with plasma membrane, penetration into the cell, and capsid removal by enzymes
○ (Note) Capsid P24 : Primary marker for diagnosing HIV virus infection
○ (Note) Secondary marker for diagnosing virus infection is antibodies
○ 3rd. Action of reverse transcriptase
○ 3rd-1st. RNA-dependent DNA polymerase activity : Reverse transcription of mRNA using poly T as a primer. ssRNA → ss cDNA
○ 3rd-2nd. RNAaseH activity
○ 3rd-3rd. DNA-dependent DNA polymerase activity : Functions like DNA pol Ⅰ. ss cDNA → ds cDNA
○ 4th. Double-stranded DNA (ds cDNA) becomes provirus and remains latent in the nucleus
○ 5th. RNA transcription : Formation of capsid and nucleic acid after alternative splicing
○ 6th. Virus is enveloped by the viral membrane and is released
⑺ Plant Viruses
① Horizontal Infection : Virus moves to adjacent cells through plasmodesmata
② Vertical Infection : Plant viruses infect generations through reproduction
⑻ Major Viral Diseases
① HBV (Hepatitis B Virus) : Hepatocellular carcinoma, increased expression of IGF-2 (Insulin-like Growth Factor)
② SV40 (Simian Virus 40) : Simian lymphoma, inhibition of pRb and p53 by SV40 T-antigen
③ Herpes Simplex Virus (Causing Chickenpox) : Lymphoma, Kaposi’s sarcoma, inhibition of antigen presentation on MHC I of tumor cells
④ HTLV (Human T-leukemia Virus) : T-cell lymphoma, excessive secretion of cytokines due to T-cell infection
⑤ HIV Virus
5. Viroids and Prions
⑴ Viroids
① Single-stranded circular RNA composed of 200 to 300 nucleotides
② Replicates in plant cells without encoding proteins
⑵ Prions
① Overview
○ Prions can be eliminated by heating up to 135°C
○ Origin : protein + infection
② Mechanism : Normal prpc in the brain undergoes structural transformation to prpsc
○ prpc exhibits an α-helical structure
○ prpsc exhibits a β-sheet structure
○ β-sheet structure can compactly stack, allowing abnormal prion proteins to form crystals
○ This compact structure is called an amyloid structure
○ Such compact structures are not easily degraded by enzymes
○ Replication : prpc + prpsc → prpsc + prpsc
③ Diseases
○ Symptoms : Causes transmissible spongiform encephalopathy in animals and humans, may even lead to death
○ Examples : Scrapie (sheep), Bovine Spongiform Encephalopathy (BSE, mad cow disease), Creutzfeldt-Jakob Disease (CJD) in humans
○ Currently, there is no cure for prion diseases
④ Detection
○ For years, BSE testing was possible only after the animal’s death
○ Initial tests took relatively long time : About a week to three years
○ Recent tests provide results within hours, but it’s not always possible
⑤ History
○ BSE outbreak in the UK in 1980
○ Mid-2005, 2 confirmed cases and 1 suspected case in the US
Table. 3. BSE and vCJD cases by country
6. Comparative Microbiology
⑴ Comparison of Animal Cells, BEVS (Baculovirus Expression Vector System)/Insect Cells, Yeast, and Bacteria
① Generally, Escherichia coli (E. coli), Saccharomyces cerevisiae (yeast), Chinese hamster ovary (CHO) cells are used
② Growth rate : Bacteria > Yeast > BEVS/Insect Cells > Animal Cells
③ Cost : Bacteria < Yeast < BEVS/Insect Cells < Animal Cells
④ Production yield : Yeast > Bacteria > BEVS/Insect Cells > Animal Cells
⑤ FDA approval priority : Animal Cells > Bacteria > Yeast > BEVS/Insect Cells
⑵ Comparison of Viruses, Bacteria, and Fungi
Table. 4. Comparison of Viruses, Bacteria, and Fungi
Input: September 9, 2017, 19:00
Last Modified: January 22, 2019, 15:56