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Chapter 26. Applied Microorganisms

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1. Overview

2. Bacteria

3. Yeast

4. Virus

5. Viroids and Prions

6. Comparative Microbiology

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

○ Heterotrophic Bacteria

○ Lack chlorophyll

○ Contribute to the material cycle in ecosystems by decomposing the remains and excretions of animals and plants

○ e.g., Escherichia coli

○ Chemosynthetic Bacteria: Autotrophic Bacteria

○ Lack chlorophyll

○ Synthesize organic compounds using chemical energy obtained by oxidizing inorganic substances

○ e.g., nitrite bacteria

○ Photosynthetic Bacteria: Autotrophic Bacteria

○ Contain chlorophyll a (green)

○ Synthesize organic compounds using light energy

○ e.g., Cyanobacteria

③ Auxotrophic Mutant

○ A mutant strain of bacteria, fungi, or cultured cells that has undergone mutation and now requires a specific chemical substance for growth

○ Example 1: Arginine-requiring strain – requires arginine for growth

④ Diauxic Growth

○ Definition: A growth pattern in which microorganisms, when provided with two types of nutrients, consume one nutrient first, and after it is depleted, begin to consume the second 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.

⑶ Cell Culture

⑷ Counting: Serial Dilution Method (Plaque Assay 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

⑸ 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 2-3 times.

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, a basic 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 crystal violet - iodine complex, preventing decolorization.

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.

⑹ 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

⑺ 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 Bacterial Examples

⑼ 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 as a beta-lactam antibiotic.

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. The first antibiotic developed by humans.

○ 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

○ A powerful antibiotic derived from streptomycesvenezuelae bacteria

○ 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 are inherited from both parents during sexual reproduction

4. Virus

⑴ Overview

① Unit: Plaque

② Burst size: Number of phages 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; however, 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. Transported into the nucleus via dynein. Cell release through lysozyme.

○ Enveloped virus: Envelope present. Transported into the nucleus via endosomal system.

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

○ Hemagglutinin temporarily binds to sialic acid residues on the host cell membrane during animal influenza virus budding

○ Neuraminidase breaks the 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 phages 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 cell membranes.

○ 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: Classified as (-) negative-sense ssRNA virus, (+) positive-sense ssRNA virus, and retroviruses

○ Derived from cell membranes.

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, thus exists with 5’ cap and poly A tail

○ 1st. (-) Synthesis of multiple ssRNA strands using its RNA pol

○ 2nd. (-) ssRNA is used by its enzyme (RNA dependent RNA pol) to create (+) ssRNA

○ 3rd. (+) ssRNA is 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 is used mainly as a template for replication, not genetic information

○ 1st. Uses its RNA dependent RNA pol to create multiple (+) ssRNA strands

○ 2nd. (+) ssRNA is used by its enzyme (RNA dependent RNA pol) to create multiple (-) ssRNA strands.

○ 3rd. (-) ssRNA is used for genome assembly, and (+) ssRNA is 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 are 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 that 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: genes that determine the phage lifecycle and degrade the host DNA by hydrolyzing the host chromosome entirely to utilize it for viral 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, while 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 are activated 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 is inhibited instead

○ 4th. As a result, P_RM promoter is activated during the determination of 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

○ The cI gene is transcribed from the PRE promoter during the establishment of lysogeny, and from the PRM promoter to maintain lysogeny.

○ λ repressor binding to OR1 and OR2 also promotes the establishment of lysogeny

○ PR and cII influence the establishment of lysogeny

○ PL, which regulates lytic genes, also influences the establishment of lysogeny

⑤ Induction of Lysogeny

○ The λ repressor protein is shaped like a dumbbell, with its N-terminal and C-terminal regions located at opposite ends.

○ 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: Following endocytosis, the virion disrupts the endocytic vesicle membrane to release its contents.

○ 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

○ 1^st. Spike glycoprotein on the viral envelope binds to cell membrane receptors

○ 2^nd. Fusion of envelope and endosomal membrane → release of virion into the cytoplasm

○ 3^rd. RNA-dependent RNA polymerase activity

○ 4^th. Viral protein synthesis → budding

③ HIV: ssRNA Virus

○ Capsid contains reverse transcriptase, protease, integrase, and two identical (literally; not complementary) ssRNAs

○ 1^st. 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

○ 2^nd. Fusion with plasma membrane, penetration into the cell, and capsid removal by enzymes

○ Capsid P24: Primary marker for diagnosing HIV virus infection

○ Secondary marker for diagnosing virus infection is antibodies

○ 3^rd. Action of reverse transcriptase

○ 3^rd-1^st. RNA-dependent DNA polymerase activity: Reverse transcription of mRNA using poly T as a primer. ssRNA → ss cDNA

○ 3^rd-2^nd. RNAaseH activity

○ 3^rd-3^rd. DNA-dependent DNA polymerase activity: Functions like DNA pol Ⅰ. ss cDNA → ds cDNA

○ 4^th. Double-stranded DNA (ds cDNA) becomes provirus and remains latent in the nucleus

○ 5^th. RNA transcription: Formation of capsid and nucleic acid after alternative splicing

○ 6^th. Virus is enveloped by the cell 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

○ Etymology: 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. Comparison of Viruses, Bacteria, and Fungi

Input: September 9, 2017, 19:00

Last Modified: January 22, 2019, 15:56