Lecture 5 - Microbial Growth

Lecture 5: Microbial Growth

Genome Replication and Cell Growth Regulation

  • Genome Replication: Essential before initiating cell growth.

  • Cell Cycle Phases:

    • G1 Phase (analogous): Precedes initiation of genome replication in prokaryotes.

    • S Phase (analogous): DnaA binds to origin of replication (oriC), recruits machinery.

  • Mechanisms:

    • SeqA prevents genome reinitiation after synthesis initiation.

    • Chromosome migration occurs to opposite poles.

    • Formation of Z-ring signifies cell division.

Regulation of Initiation of Genome Replication

  • Key Components:

    • oriC has multiple GATC sequences with DnaA boxes bound by DnaA (needs methylation on A).

  • Replication Process:

    • DnaA binding leads to synthesis machinery recruitment.

    • Newly synthesized strands become unmethylated; now they are hemimethylated.

    • SeqA binds hemimethylated DnaA boxes, prohibiting further initiation.

  • Transcription Regulation:

    • DnaA binding permits transcription while SeqA binding inhibits it.

DnaA as ATPase

  • Role of DnaA:

    • Requires ATP binding for activity.

    • Replisome recruitment activates ATPase activity for DnaA.

    • ATP hydrolysis leads to DnaA inactivation.

  • Site of Action:

    • GATC in oriC methylated by DNA adenine methylase (Dam methylase) allowing reinitiation.

Initiation in Rapidly Dividing Cells

  • Replication Timing:

    • DNA synthesis takes approximately 40 minutes to replicate chromosome.

    • In nutrient-rich media, cells double in 20 minutes, quicker than replication.

    • Dam methylase ensures oriC reaches a fully methylated state 10 minutes post-initiation, allowing for another DNA synthesis round.

Regulation of Cell Growth

  • Inhibition Mechanism:

    • Cell growth is inhibited until genome replication is completed, ensuring daughter cells receive copies.

    • Min Proteins Function:

      • MinD forms spiral along plasma membrane, oscillates to prevent FtsZ recruitment.

      • MinE oscillates at the cell midpoint, pushing MinCD to poles, allowing FtsZ to bind at the midpoint and promoting growth.

Cell Growth and DNA Synthesis

  • MinE's Role:

    • Acts as an inhibitor of MinCD, promoting cell growth by facilitating FtsZ recruitment to cell midpoint.

    • Leads to the recruitment of enzymes for peptidoglycan and lipid biosynthesis.

    • Ensures production of all components necessary for cell wall growth and overall cellular growth.

Peptidoglycan Synthesis and Cell Growth

  • Process Initiation:

    • Anchoring of ZipA in membrane recruits FtsA, an ATPase liberating energy for further recruitment of divisome machinery.

    • Continued ATP hydrolysis by FtsA is crucial for recruiting FtsI and FtsK for peptidoglycan incorporation.

Chromosome Partitioning and Separation

  • Necessary Separation:

    • Each daughter cell must receive a chromosome copy.

    • FtsK facilitates chromosome migration to the cell poles.

    • Completion of migration allows for daughter cell separation, driven by FtsZ GTP hydrolysis promoting depolymerization.

Partitioning of Chromosomes to Daughter Cells

  • Mechanism:

    • Asymmetric partitioning in Caulobacter during budding.

    • ParB and parS sequence interaction ensures one chromosome copy is tethered to the stalked pole and the other is moved to the opposite pole.

Peptidoglycan Synthesis and Cell Division

  • Chemical Processes:

    • Begins with NAG-NAM precursors which cannot cross the plasma membrane without modification.

    • Complexed to bactoprenol and facilitated by flippase, leading to incorporation into existing layers.

Laboratory Media for Microbial Growth

  • Medium Types:

    • Chemically Defined: Precise known composition.

    • Chemically Complex: Rich media, often with extracts, unknown composition.

    • Selective Media: Promotes growth of desired organisms, suppressing undesirables.

    • Differential Media: Most organisms grow, unique phenotypes for desired organisms.

Total Count vs. Viable Count

  • Count Definitions:

    • Total count may not represent the number of viable cells; varies by species.

    • Viable count shows growth potential under specific conditions.

  • Measurement Techniques:

    • Hemacytometer for direct measurements, counting in a grid.

Serial Dilution for Viable Count

  • Process:

    • Create dilutions (e.g., 1:10), then plate via spread or pour plate methods, counting colonies formed (30-300 ideal).

Indirect Counts

  • Turbidity Measurements:

    • Assess bacterial numbers through light absorption.

Phases of Growth

  • Lag Phase:

    • No division, no change in viable cell count, varies based on inoculation and media specifics.

  • Exponential Phase:

    • Cell count doubles, growth dependent on nutrient availability.

  • Stationary Phase:

    • Nutrient limitation and toxic metabolite accumulation inhibit further growth.

  • Death Phase:

    • Viable count decreases from nutrient depletion and toxic accumulation.

Factors Affecting Microbial Growth - Temperature

  • Growth Rate: Influenced by temperature ranges.

    • Psychrophile: Example - Polaromonas vacuolata (optimally grows at 4°C).

    • Mesophile: Example - Escherichia coli (optimal growth at around 37°C).

    • Thermophile: Example - Geobacillus stearothermophilus (ties to higher temperatures).

Factors Affecting Microbial Growth - Temperature Effects

  • Membrane and Enzyme Kinetics:

    • Temperature affects membrane fluidity and enzyme function, leading to stability or denaturation.

    • Low-temperature changes increase membrane fluidity with unsaturated fatty acids, while high temperatures favor saturated fatty acids for membrane integrity.

Factors Affecting Microbial Growth - pH

  • Optimal pH:

    • Bacteria thrive at neutral pH (6.5-7.5), fungi prefer mildly acidic conditions (pH 5-6).

    • Acidophiles and alkaliphiles have unique adaptations to maintain stability in extreme environments.

Factors Affecting Microbial Growth - Osmotic Pressure

  • Concentration Effects:

    • Importance of isotonic, hypotonic, hypertonic environments; osmotic pressure impacts cell integrity and survival.

  • Water Activity (aw): The interrelation between solute concentration and water availability affects growth capabilities.

Factors Affecting Microbial Growth - Oxygen Requirements

  • Classes of Organisms:

    • Obligate aerobes and anaerobes differ in vital oxygen needs, affecting metabolic pathways and survival.

Problems with Oxygen Based Metabolism

  • Toxic Byproducts:

    • Oxygen metabolism generates superoxide anion (O2-) and hydrogen peroxide (H2O2), both toxic and necessitating enzyme detoxification.

  • Detoxification Mechanisms:

    • Superoxide dismutase and catalase work to convert harmful byproducts into less toxic forms.