Micro 201 Exam 2

Unit 5: Microbial Growth & Controls

5.1: Cell Division and Population Growth

• Binary Fission (asexual reproduction)

  • Cell elongation: Cells increase size through cellular structures accumulation (anabolism).

  • Septum formation: A partition that forms between dividing cells.

  • Generation: One cell divides into two; generation time is the duration of this process.

• Exponential growth: Cell numbers double at regular time intervals.

• Growth curve: Measurement of microbial population over time includes:

  • Number of cells.

  • Number of viable cells.

  • Turbidity (cloudiness indicating growth).

• Growth Phases:

  • Lag phase: Little to no growth as cells acclimate.

  • Exponential phase: Fast growth occurs, population increases rapidly.

  • Stationary phase: Long generation time, little to no growth, stable population size.

  • Decline phase: Cell numbers decrease as resources become scarce.

5.2: Culturing Microbes and Measuring Their Growth

• Quantification of Growth allows:

  • Determining metabolic capacity.

  • Calculating growth rate.

  • Standardizing experimental protocols.

• Spread plate method:

  • Spread sample evenly on a plate, incubate, count number of colonies.

  • Colony forming unit (CFU): Each colony represents the growth of an individual cell.

• Limits: Serial dilution may be needed for countable colonies.

Unit 5: Environmental Effects on Growth - Temp/Oxygen

5.3: Environmental Effects on Growth

• Cardinal temperatures: Minimum, optimum, and maximum temperature for growth.

• Psychrophiles:

  • Thrive in freezing temperatures.

  • Structural adaptations: More alpha helices, fewer beta sheets result in increased flexibility.

  • Cytoplasmic membranes have more unsaturated fatty acids, increasing fluidity.

• Thermophiles:

  • Prefer higher temperatures, such as those found in hydrothermal vents.

  • Have adjusted ionic bonds due to acidic/basic amino acids to withstand high temperatures.

  • Cytoplasmic membranes contain more saturated fatty acids.

• Oxygen:

  • Benefits: Excellent electron acceptor enabling aerobic respiration.

  • Costs: Toxic by-products can cause cellular damage.

  • Reactive oxygen species (ROS): Include superoxide (O2-), hydrogen peroxide (H2O2), hydroxyl radicals (OH•), and water (H2O).

• Types of Microorganisms:

  • Aerobes (use oxygen):

    • Obligate aerobes: Require normal atmospheric O2 (21%).

    • Facultative aerobes: Can grow in O2 but do not need it.

    • Microaerophilic: Need O2 at levels lower than 21%.

  • Anaerobes (cannot use oxygen):

    • Aerotolerant: Can survive with or without oxygen.

    • Obligate anaerobes: Oxygen is lethal.

5.4: Controlling Microbial Growth

• Antimicrobial agents: Chemicals to control microbial growth.

  • Bacteriostatic: Stops growth when the agent is added.

  • Bactericidal: Decreases viable cells, total cells remain constant.

  • Bacteriolytic: Decreases both viable and total cell counts.

• Minimum Inhibitory Concentration (MIC): Minimum amount of antimicrobial agent needed to inhibit growth.

• Zone of Growth Inhibition: Size and presence of inhibition zones indicate susceptibility.

Unit 6: Microbial Regulatory Systems

6.1: DNA-Binding Proteins and Transcriptional Regulation

• DNA-binding proteins:

  • Transcription factors: Affect gene expression by binding to DNA.

  • Operator: Genetic element recognized by DNA-binding proteins; sequence/spacing is crucial.

• Regulation Types:

  • Negative control: Repression occurs when a compound decreases enzyme activity.

    • Co-repressors bind to the repressor, blocking transcription.

  • Positive control: Induction occurs when a compound increases enzyme activity.

    • Co-repressor binding promotes RNA polymerase action.

• Regulon: All operons controlled by a single transcription factor.

6.2: Sensing and Signal Transduction

• Two-component system:

  • Kinase: Enzyme that phosphorylates compounds.

  • Response regulator: Function varies based on phosphorylation site.

  • Phosphorylation steps:

    1. Sensor detects environmental signals and phosphorylates histidine.

    2. Sensor phosphorylates response regulator (aspartate).

    3. Phosphorylation alters function, leading to a cellular response.

    4. Sensor is dephosphorylated.

• Quorum sensing: Need sufficient individuals for coordinated behavior.

6.3: RNA-Based Regulation

• Noncoding RNA (ncRNA):

  • Includes rRNA, tRNA, sRNA (small): regulates post-transcriptional gene expression.

  • Responds to various stresses, quorum sensing, and biofilm formation.

  • Mechanism: Pairing with mRNA can inhibit translation or stability.

• Riboswitches:

  • Bind with mRNA or small molecules affecting transcription/translation.

  • Located upstream of coding sequences, forming secondary structures.

Unit 7: Molecular Biology of Microbial Growth

7.1: Bacterial Cell Division

• Initiation of chromosomal replication: Begins at the origin site with dnaA in active form (DNA-ATP).

• Inhibition mechanisms:

  • dnaA-ADP (inactive).

  • DnaA-ATP binding site depletion.

  • Binding prevention by seqA.

  • Repression of dnaA expression by seqA.

• Cell division and Fts proteins:

  • FtsZ ring: Forms at the septum within the cytoplasmic membrane, influencing division.

• Peptidoglycan biosynthesis: Provides structural integrity; holes in PGN can lead to lysis.

7.2: Regulation of Development in Model Bacteria

• Bacterial biofilms:

  • Polysaccharide matrix that houses bacterial cells.

  • Represents typical bacterial states in environments.

  • This formation has medical implications when present on medical devices.

• Techniques for observation:

  • GFP (Green Fluorescent Protein): Used for visualizing.

  • Confocal microscopy: Laser scanning creates 3D images of samples across multiple planes.

• Stages of biofilm development:

  • Attachment: Initial adhesion of motile cells to solid surfaces.

  • Colonization: Intercellular communication initiates further growth and polysaccharide formation.

  • Development: Continued growth and polysaccharide accumulation.

  • Active dispersal: Triggered by environmental factors like nutrient availability.

7.3: Antibiotics and Microbial Growth

• Antibiotics: Antimicrobial agents naturally produced by microorganisms that target essential molecular processes.

• Mechanisms of antibiotic resistance:

  • Spontaneous mutations lead to enzyme adaptations.

  • Antibiotic modifications via specific enzymes altering antibiotic functionality.

  • Efflux pumps: Transport antibiotics out of cells reducing interaction frequency.

Unit 8: Phage and Their Replication

8.1: The Nature of Viruses

• Virus: A genetic element capable of replication only within a living host cell.

• Host cell functions: Provides energy, metabolic intermediates, protein synthesis; viruses are obligate intracellular parasites.

• Phage virion structure:

  • Bacteriophage: Viruses that infect bacteria (e.g., T4 bacteriophage infecting E. coli).

• Replication cycle (lytic cycle):

  1. Attachment

  2. Penetration of viral nucleic acids

  3. Synthesis

  4. Assembly and packaging of new virions

  5. Cell lysis and release of new virions.

• Lysogenic cycle: Phage DNA integrates into the host genome (prophage) contributing to genome evolution.

• Culturing methods:

  • Titer: Number of infectious virions per volume.

  • Plaque: Zone of cell lysis among a lawn of host cells on solid media.

8.2: Viral Defense Mechanisms in Bacteria

• CRISPR: Regularly interspaced short palindromic repeats that protect bacteria from phage infections.

• CAS proteins:

  • Processes transcription into crRNA for targeting foreign DNA; endonuclease activity cleaves these complexes.

• Immunization process:

  • PAM (protospacer adjacent motif): Recognized by CAS; can release and incorporate foreign DNA into CRISPR regions.

Unit 9: Microbial Genome

9.1: Microbial Genomic

• Genome: All genetic information defining an organism predicting aspects regarding:

  • Energy and nutrient sources (catabolism).

  • Growth products (biosynthesis).

  • Fitness strategies (ecology).

  • Molecular adaptations (evolution).

• Genomics: The study of mapping, sequencing, analyzing, and comparing genomes.

• Sanger Sequencing: A specific DNA sequencing technique based on DNA synthesis via DNA polymerase with dNTPs and ddNTPs.

• Next Generation Sequencing:

  • Shotgun sequencing: Genomic DNA fragments prepared without user specification (untargeted).

  • Massively parallel methods: Allows simultaneous sequencing of multiple samples (up to 10^6 individual reactions).

  • Computing Power: Handles and stores extensive DNA sequences (>10^9 base pairs), enabling automated identification.

• Genome Assembly: Involves connecting sequence fragments in the correct order and eliminating overlaps.

• Genomic Annotations: Identifies genes and functional regions.

Unit 10: Genetics of Bacteria

10.1: Mutations

• Mutation: Heritable changes in the nucleotide sequence of the genome.

• Genotype vs. Phenotype:

  • Genotype: Nucleotide sequence of the genome.

  • Phenotype: Observable properties driven by that genotype.

• MacConkey Agar:

  • Pink colonies signify acid production.

• Selection vs. Screening:

  • Selection: Isolation of mutants with a fitness advantage under given conditions (e.g. antibiotic resistance).

  • Screening: Identifying mutants with specific non-selectable traits.

• Mutation Types:

  • Point mutation: Single nucleotide substitution; impact depends on context.

  • Frameshift mutations: Insertions or deletions that disrupt reading frames, altering codon sequences.

10.2: Gene Transfer in Bacteria

• Horizontal Gene Transfer: Acquisition of genetic information from another cell:

  • Transformation: Uptake of free DNA from the environment.

  • Transduction: Viral particles carrying non-viral DNA facilitate transfer.

  • Conjugation: Plasmid-mediated DNA transfer between cells.

• DNA outcome in host cells:

  • Degraded by cell defenses (catabolism).

  • Replicate independently as plasmids.

  • Incorporated into chromosomes via homologous recombination.

10.3: Homologous Recombination and Natural Transformation

• Recombination: Physical DNA exchange between genetic elements.

• Homologous Recombination:

  • Depends on sequence homology between donor and recipient DNA.

  • Donor DNA yields single strands, facilitated by RecA.

• Transformation: Uptake of free DNA incorporated into recipient genomes.

  • Natural transformation: Some species can take up DNA naturally when competent.

10.4: Transduction and Conjugation

• Transduction: Phage-mediated horizontal gene transfer.

  • Example: P1 phage utilizing core polysaccharide of LPS for genetic exchange.

  • Accidental formation of transducing particles occurs during the lytic cycle.

• Conjugation: Plasmid-mediated DNA transfer.

  • oriT: Origin of replication transfer.

  • oriR: Origin of replication for vegetative growth.

  • Tra region: Genes encoding transfer factors (e.g. sex pilus formation).

Unit 11: Biotechnology

11.1: Polymerase Chain Reaction (PCR)

• PCR: Amplifies specific DNA regions in vitro.

  • Components: Template DNA, DNA polymerase, and DNA primers (with dNTPs).

  • Process: DNA separates; primers extend strands (exponential amplification: 1→ 2 → 4 → 8).

• Reverse Transcription PCR: Identifies specific transcripts and synthesizes DNA from RNA (cDNA).

• Gel Electrophoresis: Separates DNA fragments by size; smaller fragments migrate faster towards the positive electrode.

11.2: Cloning

• Cloning: Introduction of target DNA into a vector (plasmid tool).

• Restriction Endonucleases: Enzymes that cleave double-stranded DNA to facilitate cloning.

  • Positive identification: Inserted target appears white; cells without inserts appear blue.

11.3: Genome Editing

• CRISPR/Cas9-mediated insertion: Uses synthetic guide RNA (sgRNA) for specificity and cleavage depends on PAM (protospacer adjacent motif).

• Cas9 protein: Recognizes sgRNA-DNA complex and exhibits endonuclease activity.

  • Insert DNA uses homologous recombination.

• CRISPR/Cas9-mediated deletion: Involves two distinct sgRNA for specificity; PAM sites essential for DNA deletion.

• DNA Repair Mechanism:

  • Involves degradation of excised DNA and activation of double-stranded break repair pathways.