Microbial Genetics, Viral Structure, and Control of Microbial Growth – Study Guide for Exam #2

Microbial Genetics and Gene Regulation

DNA Replication

DNA replication is the process by which a cell duplicates its DNA before cell division, ensuring genetic continuity.

• Key Enzymes: DNA polymerase (synthesizes new DNA), helicase (unwinds DNA), primase (synthesizes RNA primers), ligase (joins Okazaki fragments).

• Directionality: DNA is synthesized in the 5' to 3' direction.

• Leading vs. Lagging Strand: The leading strand is synthesized continuously; the lagging strand is synthesized in short fragments (Okazaki fragments).

• Replication Fork: The Y-shaped region where DNA is actively being unwound and replicated.

Equation:

$\text{DNA}_{\text{template}} + \text{dNTPs} \xrightarrow{\text{DNA polymerase}} \text{DNA}_{\text{new}} + \text{PP}_i$

Transcription

Transcription is the synthesis of RNA from a DNA template, producing messenger RNA (mRNA) for protein synthesis.

• Initiation: RNA polymerase binds to the promoter region.

• Elongation: RNA polymerase synthesizes RNA in the 5' to 3' direction.

• Termination: Transcription ends at a terminator sequence.

Equation:

$\text{DNA}_{\text{template}} + \text{NTPs} \xrightarrow{\text{RNA polymerase}} \text{RNA} + \text{PP}_i$

Enhancers and Silencers

Enhancers and silencers are regulatory DNA sequences that increase or decrease the rate of transcription, respectively.

• Enhancers: Bind activator proteins to increase transcription.

• Silencers: Bind repressor proteins to decrease transcription.

Translation

Translation is the process by which ribosomes synthesize proteins using mRNA as a template.

• Initiation: Ribosome assembles at the start codon (AUG).

• Elongation: tRNAs bring amino acids to the ribosome, matching codons to anticodons.

• Termination: Occurs at a stop codon; the completed polypeptide is released.

Equation:

$\text{mRNA} + \text{tRNA} + \text{ribosome} \rightarrow \text{protein}$

Mutations in DNA, RNA, and Proteins

Mutations are changes in the nucleotide sequence of DNA, which can affect RNA and protein structure and function.

• Types: Point mutations, insertions, deletions, frameshifts.

• Effects: Silent, missense, nonsense, or frameshift mutations.

• Causes: Spontaneous errors, UV light, X-rays, chemicals.

UV Light and X-rays

Physical mutagens that cause DNA damage.

• UV Light: Causes thymine dimers, leading to replication errors.

• X-rays: Cause double-strand breaks and base modifications.

Conjugation

Conjugation is a form of horizontal gene transfer in bacteria involving direct cell-to-cell contact.

• F Plasmid: Carries genes for pilus formation and DNA transfer.

• Significance: Spreads antibiotic resistance and other traits.

Lac Operon

The lac operon is a gene system in Escherichia coli that controls lactose metabolism.

• Inducible System: Activated in the presence of lactose.

• Regulation: Involves repressor, operator, and CAP-cAMP complex.

Trp Operon

The trp operon is a repressible gene system controlling tryptophan synthesis in bacteria.

• Repressible System: Turned off when tryptophan is abundant.

• Attenuation: Additional regulatory mechanism in some bacteria.

CAP + cAMP

Catabolite Activator Protein (CAP) and cyclic AMP (cAMP) regulate operons in response to glucose levels.

• Low Glucose: High cAMP, CAP binds DNA, increases transcription.

• High Glucose: Low cAMP, CAP does not bind, transcription is low.

Viruses and Viral Genetics

Bacteriophage Structure and Infection

Bacteriophages are viruses that infect bacteria, with distinct structures and infection cycles.

• Structure: Head (capsid), tail, tail fibers.

• Virulent Phage: Undergoes lytic cycle, destroys host.

• Temperate Phage: Can integrate into host genome (lysogeny).

Transduction

Transduction is the transfer of bacterial genes by bacteriophages.

• Generalized Transduction: Any bacterial gene can be transferred.

• Specialized Transduction: Only specific genes near prophage insertion site are transferred.

• Importance: Contributes to genetic diversity and horizontal gene transfer.

Viral Structure and Classification

Viruses are classified based on genome type, structure, and clinical features.

• Genome: dsDNA, ssDNA, dsRNA, ssRNA (+ or -).

• Envelope: Enveloped or non-enveloped.

• Shape: Helical, icosahedral, complex.

Viral Reproduction

Viruses reproduce by infecting host cells and hijacking their machinery.

• Lytic Cycle: Virus replicates and lyses host cell.

• Lysogenic Cycle: Viral genome integrates into host DNA (provirus/prophage).

Retrovirus Infection (HIV)

Retroviruses, such as HIV, use reverse transcriptase to convert RNA into DNA, integrating into the host genome.

• Steps: Entry, reverse transcription, integration, transcription, assembly, release.

Types of Viral Infections

• Acute: Rapid onset, short duration (e.g., influenza).

• Chronic: Persistent, long-term (e.g., hepatitis B).

• Latent: Dormant phase with possible reactivation (e.g., herpes simplex).

Viral Isolation and Detection

• Proper Pore Size: Filters with 0.2 μm pores retain bacteria but allow viruses to pass.

• Cytopathic Effects: Visible changes in host cells due to viral infection.

• Hemagglutination Assay: Detects viruses that agglutinate red blood cells.

• RT-PCR vs. PCR: RT-PCR detects RNA viruses by reverse transcription; PCR amplifies DNA.

• Enzyme Immunoassay: Detects viral antigens or antibodies (e.g., ELISA).

Prions and Prion Diseases

Prions are infectious proteins causing neurodegenerative diseases.

• Diseases: Creutzfeldt-Jakob Disease, Mad Cow Disease (BSE).

• Mechanism: Misfolded proteins induce misfolding in normal proteins.

COVID-19

COVID-19 is caused by SARS-CoV-2, a novel coronavirus.

• Genome: ssRNA (+) virus.

• Transmission: Respiratory droplets, aerosols.

• Clinical Features: Fever, cough, respiratory distress.

• Prevention: Vaccination, masks, social distancing.

Control of Microbial Growth

Biosafety Levels (BSL 1-4)

Biosafety levels define containment precautions for handling microbes.

BSL	Description
1	Non-pathogenic microbes; basic precautions
2	Moderate risk; gloves, lab coats, biosafety cabinets
3	Serious pathogens; controlled access, negative pressure
4	High-risk, life-threatening agents; full-body suits, specialized facilities

Levels of Sterilization

• Sterilization: Destroys all forms of microbial life, including spores.

• Disinfection: Destroys most pathogens, not spores.

• Antisepsis: Disinfection of living tissue.

• Sanitization: Reduces microbial load to safe levels.

Decimal Reduction Time (DRT) and Thermal Death Time (TDT)

• DRT (D-value): Time to reduce microbial population by 90% at a given temperature.

• TDT: Minimum time to kill all microbes at a set temperature.

Physical Methods of Control

• Autoclave: Uses steam under pressure (121°C, 15 psi, 15 min) to sterilize.

• Pasteurization: Reduces pathogens in liquids (e.g., 72°C for 15 sec).

• Refrigeration/Freezing: Slows or halts microbial growth.

• Desiccation/Salting: Removes water or creates hypertonic environment to inhibit growth.

• Radiation: UV causes DNA damage; gamma rays sterilize by ionization.

• HEPA Filters: Remove microbes from air (0.3 μm pores).

• Membrane Filtration: Physically removes microbes from liquids.

Chemical Methods of Control

• Phenols: Disrupt cell membranes and proteins; structure is a benzene ring with a hydroxyl group.

• Triclosan: Antimicrobial banned by FDA due to resistance concerns.

• Betadine: Iodine-based antiseptic.

• Alcohols: Denature proteins and disrupt membranes (e.g., ethanol, isopropanol).

• Soaps: Remove microbes by emulsification; not antimicrobial.

• Quats: Quaternary ammonium compounds; disrupt membranes.

• Hydrogen Peroxide: Produces free radicals that damage cells.

Testing Effectiveness of Antimicrobials

• Disk Diffusion: Measures zone of inhibition around antimicrobial disks.

• In Use Test: Assesses effectiveness of disinfectants in real-world conditions.

Other Important Topics

• Clostridium botulinum: Produces botulinum toxin; causes botulism.

• CRE (Carbapenem-resistant Enterobacteriaceae): Hospital-acquired, highly resistant bacteria.

Essay Topics for Exam Preparation

• DNA Replication

• Lac Operon

• Trp Operon

• HIV Viral Life Cycle

• Transcription/Translation (be prepared to transcribe and translate a DNA sequence)

Additional info: Students should be able to apply these concepts to practical and clinical scenarios, such as interpreting genetic regulation, understanding viral diagnostics, and evaluating methods for controlling microbial growth.